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UBC Theses and Dissertations

Organ decellularization used as a novel approach to engineer three-dimensional urogenital tumor models Tan, Zheng 2019

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 ORGAN DECELLULARIZATION USED AS A NOVEL APPROACH TO ENGINEER THREE-DIMENSIONAL UROGENITAL TUMOR MODELS by  Zheng Tan  B.Sc., Simon Fraser University, 2016  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2019   © Zheng Tan, 2019    ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: ORGAN DECELLULARIZATION USED AS A NOVEL APPROACH TO ENGINEER THREE-DIMENSIONAL UROGENITAL TUMOR MODELS  submitted by Zheng Tan in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine  Examining Committee: Alan So, Urologic Sciences Supervisor  Michael Cox, Urologic Sciences Supervisory Committee Member  Karen Cheung, Electrical and Computer Engineering Supervisory Committee Member Vincent Duronio, Respiratory Medicine University Examiner     Additional Supervisory Committee Members: Claudia Chávez-Muñoz, Urologic Sciences Supervisory Committee Member  Supervisory Committee Member     iii Abstract  Cancer is currently the leading cause of death in Canada and is responsible for 30% of all deaths (1). With various treatments available nowadays, it is necessary to develop a three-dimensional (3D) model to mimic in vivo condition of patients’ disease in order to test drug efficacy prior to any actual treatments. My study is focusing specifically on kidney and bladder cancer. Therefore, a 3D, acellular organ-specific extracellular matrix (ECM) can be established as bioactive substrates with the long-range goal of bioengineering tumors for drug testing. Specifically, decellularization is the process by which cells are removed from the organ to produce the aforementioned matrix. It is crucial to achieve a tight balance between effective cell removal and retention of native ECM architecture. Henceforth, the decellularized matrix can be used as a template and be repopulated with normal human organ-specific cells or cancerous cells. With these approaches, a bio-matrix that ultimately serves as a cancer model for drug testing can be developed. In this master’s thesis project, I hypothesized that decellularization preserves organ microarchitecture and retains various matrix-bound growth factors necessary for cell homing. Afterward, tumor models can be established through recellularization of human cells onto the decellularized ECMs as a proof-of-principle drug-testing platform to predict treatment response.    iv Two objectives were pursued to test this hypothesis. Under objective 1, I optimized and established decellularization protocols for both kidney and bladder. The protocols ensure complete cell removal and preservation of ECM microarchitectures. In addition, comprehensive protein-profiling of the decellularized kidney and bladder’s ECM was completed by performing proteomic analysis with liquid chromatography–mass spectrometry (LC-MS/MS). Under objective 2, recellularization protocols for both decellularized kidney and bladder ECM were established and optimized. As the decellularized kidney and bladder’s ECM was repopulated with normal human cells and cancerous cells, the proposed in vitro urogenital cancer model can be developed.  In conclusion, a 3D in vitro urogenital cancer model can be developed from kidneys and bladders using protocols optimized under objective 1 & 2. This work establishes the model as proof –of principal and sets the foundation for the developments of personalized cancer treatments.   v Lay Summary  Current and future management of kidney and bladder cancers ideally will be based on choosing the optimal treatments for each patient, potentially from many options. It is necessary to develop cancer models to mimic the condition of patients’ disease in order to test drug efficacy prior to any actual treatment. To obtain these models, we obtained kidneys and bladders from animals, removed all cellular components from each organ while the organ’s inside structure remained intact. Afterward, these cell-free organs can be repopulated with normal human organ-specific cells or cancerous cells. The repopulated organs could ultimately serve the purpose of mimicking patients’ disease. Therefore, building these models may allow healthcare providers to use patients’ own cells to create replicas of their urogenital tumors. Hence, multiple drug tests can be performed on this clinical platform instead of on the patient to prevent unnecessary side effects.    vi Preface    The content presented in this thesis is the original and unpublished work written by me with extensive review by Dr. Alan So, Dr. Claudia Chávez-Muñoz, Dr. Michael Cox, and Dr. Karen Cheung.   All experiments were designed and supervised by Dr. Alan So and Dr. Claudia Chávez-Muñoz and performed by me, with assistance from Dr. Gethin Owen, Dr. Igor Moskalev and Mr. Hans Adomat for Scanning Electron Microscope (SEM), animal organ retrieval and LC-MS/MS. All animal procedures were conducted according to the guidelines and principles of the Canadian Council on Animal Care (CCAC). The organ retrieving protocol was approved by the Animal Care Committee of the University of British Columbia (Protocol No. A18-0203).    vii Table of Contents Abstract ........................................................................................................................ iii Lay Summary ............................................................................................................... v Preface ......................................................................................................................... vi Table of Contents ...................................................................................................... vii List of Tables ............................................................................................................... xi List of Figures ............................................................................................................ xii List of Abbreviations ................................................................................................xiii Acknowledgements ................................................................................................... xv Dedication .................................................................................................................xvii Chapter 1: Introduction ............................................................................................... 1 1.1 Urogenital cancers .......................................................................................... 1 1.1.1 Kidney cancer .............................................................................................. 1 1.1.2 Bladder cancer ............................................................................................ 1 1.1.3 Clinical limitations ........................................................................................ 2 1.2 Current available models ................................................................................ 3 1.2.1 Two-dimensional tissue models .................................................................. 3 1.2.2 Three-dimensional tissue models ............................................................... 4 a) Co-culture models ....................................................................................... 4 b) Spheroid models ......................................................................................... 5 1.2.3 Animal models ............................................................................................. 6 1.3 Tissue engineering as an alternative study model ......................................... 7 1.4 Decellularization as a method for tissue engineering .................................... 8   viii 1.5 Recellularization as a method for tissue engineering .................................... 9 1.6 Hypotheses and objectives ................................................................................ 9 Chapter 2: Materials and Methods........................................................................... 11 2.1 Organ retrieval from animals ........................................................................ 11 a) Kidney retrieval .......................................................................................... 11 b) Bladder retrieval ........................................................................................ 13 2.2 Decellularization protocols ............................................................................ 14 2.2.1   Background ............................................................................................... 14 2.2.2   Bioreactor .................................................................................................. 16 2.2.3 Decellularization by perfusion - kidney ..................................................... 18 2.2.4 Decellularization by immersion - bladder ................................................. 22 2.3 ECM evaluation ............................................................................................. 23 2.3.1 DNA quantificiation .................................................................................... 24 2.3.2 Histological assessments .......................................................................... 24  a) H&E ....................................................................................................... 24  b) SEM ....................................................................................................... 26 2.4 ECM characterization.................................................................................... 27 a) ECM protein extraction and enrichment ................................................... 28 b) In-solution digestion of ECM-enriched proteins ....................................... 30 c) Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) ........ 31 2.5 Recellularization protocols ............................................................................ 33 2.5.1 Cell lines .................................................................................................... 34 2.5.2 Kidney ECM recellularization protocol ...................................................... 35   ix 2.5.3  Bladder ECM recellularization protocol .......................................................... 36 2.6 Evaluation of recellularization protocols ....................................................... 38 a) IHC ............................................................................................................. 38 b) Penetration rate of fibroblasts ................................................................... 40 Chapter 3: Results ..................................................................................................... 41 3.1 Optimized kidney decellularization protocol ................................................. 41 3.1.1 Residual DNA quantification of decellularized kidney proved complete cell removal........................................................................................................... 42 3.1.2 Evaluation of decellularized kidney reveals complete cell removal and preservation of ECM microarchitectures ............................................................. 44 3.1.3 Structural protein and ECM-bound growth factors are preserved after decellularization of kidney .................................................................................... 47 3.2 Successful Bladder decellularization protocol.............................................. 55 3.2.1 Residual DNA quantification of decellularized bladder proved complete cell removal........................................................................................................... 55 3.2.2 Evaluation of decellularized bladder reveals complete cell removal and preservation of ECM microarchitectures ............................................................. 58 3.2.3 Structural protein and ECM-bound growth factors are preserved after decellularization of bladder .................................................................................. 60 3.3 Optimization of kidney ECM recellularization ............................................... 68 3.4 Optimization of bladder ECM recellularization ............................................. 72 a) Single cell-type recellularization ............................................................... 72 b) Multi cell-type recellularization .................................................................. 75   x 3.5 Human fibroblast demonstrated ECM penetrating capacity ........................ 77 Chapter 4: Discussion ............................................................................................... 79 Chapter 5: Conclusions and Future Directions ..................................................... 86 5.1 Summary ....................................................................................................... 86 5.2 Future directions ........................................................................................... 86 Bibliography ............................................................................................................... 88 Appendices ................................................................................................................. 93 Appendix A ............................................................................................................... 93 Appendix B ............................................................................................................... 94 Appendix C ............................................................................................................... 95 Appendix D ............................................................................................................... 96 Appendix E ............................................................................................................... 97 Appendix F ............................................................................................................... 98            xi List of Tables Table 1. Proteomic analysis of decellularized (Rattus norvegicus) kidney ................ 49 Table 2. Proteomic analysis of native (Rattus norvegicus) kidney............................. 51 Table 3. Proteomic analysis of decellularized (Rattus norvegicus) bladder .............. 62 Table 4. Proteomic analysis of native (Rattus norvegicus) bladder ........................... 65                    xii List of Figures Figure 1. Decellularizatio protocols ............................................................................. 15 Figure 2. The bioreactor used for kidney decellularization (perfusion) ...................... 17 Figure 3. The bioreactor-peristaltic pump set up for perfusion decellularization ....... 20 Figure 4. The schematic diagram of the bioreactor for perfusion decellularization ... 21 Figure 5. Macroscopic assessment of native and decellularied kidney ..................... 42 Figure 6. DNA Quantification of native and decellularized kidneys. .......................... 43 Figure 7. Macroscopic visualization and histological examination of native and decellularized kidneys. ................................................................................................ 45 Figure 8. Structural analysis of native and decellularized kidneys. ........................... 46 Figure 9. DNA Quantification of native and decellularized bladders .......................... 57 Figure 10. Macroscopic visualization and histological examination of native and decellularized bladders ................................................................................................ 58 Figure 11. Structural analysis of native and decellularized bladders ......................... 59 Figure 12. Macroscopic visualization and histological examination of recellularized kidney (HUVEC)........................................................................................................... 70 Figure 13. Macroscopic visualization and histological examination of recellularized kidney (Caki-1) ............................................................................................................. 71 Figure 14. Macroscopic visualization and histological examination recellularized bladder (singel cell-type) ............................................................................................. 74 Figure 15. Macroscopic visualization and histological examination of decellularized and recellularized bladders (multi cell-type) ............................................................... 76    xiii List of Abbreviations ACN – Acetonitrile ccRCC – Clear cell renal cell carcinoma DAB – 3,3’- diaminobenzidine dH2O – Distilled Water ddH2O – Double Distilled Water DMEM - Dulbecco Modified Eagle Medium DNA - Deoxyribonucleic Acid DNase I - Deoxyribonuclease I DTT - 1,4-dithiothreitol EBMTM- Plus - Endothelial Cell Growth Basal Medium-Plus ECM - Extracellular Matrix EnGS – Endothelial Growth Supplement FBS - Fetal Bovine Serum HCD – higher-energy collisional dissociation H&E - Hematoxylin and Eosin HRP – Horseradish Peroxidase IHC – Immunohistochemitry KSFM - Keratinocyte Serum Free Medium LC-MS/MS - Liquid chromatography–Mass spectrometry MEM - Minimum Essential Medium Eagle MIBC - Muscle Invasive Bladder Cancer m/z – mass/charge ratio   xiv PBS - Phosphate-buffered saline PDX - Patient-derived tumor xenografts PNGase F - Peptide-N-Glycosidase F rhEGF - Recombinant human epidermal growth factor RNAse A - Ribonuclease A SDS – Sodium Dodecyl Sulphate  SEM - Scanning Electron Microscope TBS – Tris-buffer Saline TFA – Trifluoroacetic acid TURBT - Transurethral Resection of Bladder Tumor USP - United States Pharmacopeia unit 2D – Two Dimensional 3D – Three Dimensional              xv Acknowledgements  First and foremost, I would like to express my sincere gratitude to my supervisor Dr. Alan So for giving me this precious opportunity to start my career in research. I would not have developed this much passion and determination in this field without his learned guidance and tireless support. Even I am a student with many flaws, he tolerated me and guided me to the right directions. I would also like to thank Dr. Claudia Chávez-Muñoz for guiding me through every steps of my study with her extensive knowledge and experiences. She is always a phone call away when I have questions, concerns, or need assistance and even for pep talks. It is a privilege to have her with me for the past three years. I appreciate her intelligence and admire her as an omnipotent sister.  I am very grateful to my supervisory committee member, Dr. Michael Cox. He teaches me how to think critically in both research and life. I appreciate him being straightforward and I am impressed by his sense of humor.  He never knows how his voice cheers me up when I am in the doldrums. Dr. Karen Cheung, my other committee member, I want to thank her for being there monitoring my research progress and I sincerely appreciate her technical support even before I started my master’s study. I appreciate that she introduced me to her lab and provided me a valuable opportunity to learn from her students. That was how I get to know biomedical engineering at the first place. I would also like to thank Dr. Caigan Du, who is a mentor   xvi to me for the past three years. I appreciate that although I am not one of his students, he still spent so much time encouraging and enlightening me with his big heart and formidable vision. He influenced me in aspects that even himself didn’t notice. Lastly, I would like to express my deepest gratitude to my parents, grandparents and aunt for their unconditional love and trust.  I am fortunate enough to have everyone mentioned above during my study. I would never have learned the beauty and fascination of scientific research without them being around.                 xvii Dedication  To my dearest parents.     1 Chapter 1: Introduction  1.1 Urogenital cancers  1.1.1 Kidney cancer  Kidney cancer is the tenth most commonly diagnosed cancer in Canada. The incidence of kidney cancer in Canada has increased by approximately 1.3% per year for both genders since the late 1990s (2). In 2017, there were approximately 6,600 Canadians were newly diagnosed and 1,900 died from this malignancy (1) (3). Patients presenting with localized kidney cancer are usually initially treated with partial/radical nephrectomy. Resected tumors are assessed pathologically for both grade and stage and this information provides prognostic information and directs further management. Medical management options in addition to surgery include targeted therapy, immunotherapy, radiation therapy or a combination of these (2). However, identification of the optimal medical procedures is considered challenging and inappropriate decision can cause additional side effects to patients.   1.1.2 Bladder cancer  Bladder cancer is the fifth most commonly occurring cancer in Canada. Due to its high recurrence rate (60-70%), bladder cancer is the most expensive cancer to treat (4). In   2 2017, approximately 8,900 Canadians were newly diagnosed and 2,400 died from this malignancy (1). Patients presenting with bladder cancer are initially treated with transurethral resection of bladder tumor (TURBT). Tumors are assessed pathologically for both grade and stage and this information provides prognostic information and directs further management. In bladder cancer, chemotherapy is frequently administered neoadjuvantly in patients undergoing radical cystectomy for pT2 (patient with stage II, invasion of the muscle) bladder cancer, but identification of those with chemo-sensitive cancer is challenging.   1.1.3 Clinical limitations  Current treatment limitations in both kidney and bladder cancer center on the inability of predicting optimal treatments from a large myriad of possible options; hence, identifying drug sensitivities prior to treatments would allow for a more efficacious management with minimal delay that may be caused by using therapies in which the tumors have inherent treatment resistance.   Therefore, current and future management of kidney and bladder cancers ideally will be based on choosing the optimal and treatment-sensitive approach for each patient potentially from a vast array of options. In order to study the variable response to treatment secondary to interpatient heterogeneity and, more importantly, translate this knowledge into true patient benefit, predictive tumor models provide an opportunity to guide individual treatment and identify tumor-specific optimal therapies. However,   3 many existing models do not necessarily fulfill these requirements due to many limitations that restrict their predictive power (5).  1.2 Current available models  1.2.1 Two-dimensional tissue models   Currently, many tumor models play important roles in cancer research and serve as screening platforms for drug testing. For both in vitro and in vivo tumor models, there are needs for improved understanding of not only cancer progression but also prediction of the cancer’s response to therapies.   The typical representative in vitro model uses an adherent two-dimensional (2D) monolayer cell culture. In this model, cancer cells are continuously cultured and adapted to in vitro cell culture conditions. Consequently, this model is well established and has plenty of previous experimental data to compare with. The model is cost effective and also easy to visualize and analyze. However, the 2D model is not a representative of actual cell microenvironment due to low physiological relevance, such as the lack of true tissue architecture and cell-cell communication. Cancer cells cultured in 2D monolayer exhibit different cellular morphology, metabolism, and gene expression compared to those in vivo (6) (7) (8). Hence, 2D cell models do not mimic the complex cellular interactions and as such studying either normal or malignant cells using this model may not be true representations of either canonical or malignant   4 biologic pathways. In other words, 2D models are not optimal tools as a preclinical cancer model.  1.2.2 Three-dimensional tissue models   a) Co-culture models Three-dimensional (3D) in vitro models such as co-cultured tumor models and spheroid tumor models attempt to emulate the complexities of cell-cell interactions in vitro.  3D cultures can, in contrast to 2D monolayers, have higher degree of structural complexity and retain cell-cell and cell-matrix interactions that better reflect the characteristics of normal and malignant tissue (7). Therefore, 3D models better simulate true biologic conditions.   Co-culture tumor models are developed to have cancer cells cultured with a variety of stromal cells, with which the model can apply stromal-mediated effects that are found within in vivo cancer tissue (9) (10). Unlike 2D monolayer models wherein different cell types are mixed and cultured on the same monolayer, the co-culture model system allows different cells to be cultured in different compartments or matrix layers. Stromal cells and cancer cells can be co-cultured in separate 3D layers to mimic different components in the tumor microenvironment. However, the models are complex to grow and lack sufficient extracellular matrix (ECM) as that of in vivo conditions; they are also challenging to be used in a high-throughput manner and cells in co-culture models are difficult to detect and quantify at the end of the study (11). In   5 addition, the absence of ECM inhibits cancer cells from invading or metastasizing to surrounding tissues due to lack of cell-ECM interactions (12). Hence, co-culture model is not an optimal tool to study cancer cell behaviors.  b) Spheroid models Spheroid tumor model aggregates cells grown in suspension or embedded in a 3D matrix using 3D culture methods such as suspension in culture, non-adherent surface methods, hanging drop methods, and microfluidic methods (13) (14) (15) (16). Larger spheroids sustain nutrient and oxygen level differently throughout the spheroid that usually result in the formation of a necrotic core (17), which is similar to poorly vascularized in vivo tumors. Moreover, protein and gene expression profiles of cancer cells in spheroids also more closely resemble those found in clinically and in vivo models better than 2D models (13). These features make the spheroid tumor models more biomimetic than the co-culture tumor models. However, spheroid tumor models produced with the aforementioned four methods have different limitations. Spheroids maybe developed with uniformity in size but this is not usually done in a high-throughput manner (i.e. non-adherent surface methods and hanging drop methods). Conversely, other protocols do produce spheroids in a high-throughput manner with little control of sizes and uniformity (i.e. suspension in culture method) (13) (14) (15) (16). Other spheroid tumor models may be produced with extrusion 3D bioprinters. Normal and cancerous cells as well as matrix can be positioned one by one within a 3D architecture to drive the formation of tumor spheroids. This model is considered a heterogeneous tumor model and it mimics the true cellular density of target tissues   6 and organs (18).  However, shear-stress-induced cell deformation can occur during bioprinting and potentially cause apoptosis afterward (19). Similar to that of the co-culture models, the major limitation of the spheroids models is the matrix. Even though cells in spheroids are in a matrix instead of co-cultured or cells attached to plastic, the matrices are usually synthetic or only collagen, fibrin or hyaluronic acid (20) (21). They do not have the complexity of natural mammalian ECM. In this case, the “less sophisticated” ECM could inhibit cancer cells from invading or metastasizing to surrounding tissues due to lack of cell-ECM interactions (12), which could lead to partial or false translation of certain cancer cell behaviors.   1.2.3 Animal models   Animal or in vivo tumor models may be the ideal model to assess clinical efficacy of various treatments. Despite differences between human and animal biology, in vivo models capture the high degree of complexity of tumor biology in a living system, but have limited utility in imaging and high-throughput studies. A wide range of animal-based tumor models have been developed of tumors, including environmentally induced models, patient-derived tumor xenografts (PDX) models and genetically engineered mouse models (22) (23). The most studied predictive tumor model is the PDX model, in which cancer cells obtained from patients are implanted into immunocompromised mice and grown. These tumors can then be tested using various therapeutics to identify optimal treatments for the specific patient’s tumor (24) (25) (26). Although usually grown in rodents, the in vivo model best mimics the original   7 tumor environment. However, this model is very expensive and requires tremendous resources. These limitations have also restricted these models to research study and have prevented their practical clinical use in high-throughput drug screens (27). Therefore, a better more suitable tumor model is required, capable of 1) predicting response to therapies; 2) being cost-effective and reproducible; and 3) having high-throughput approach.   1.3 Tissue engineering as an alternative study model  Overall, my aim in this study is to establish an economical and reliable high-throughput drug-screening platform. My study proposed to develop the foundation of a 3D in vitro patient-derived urogenital tumor model as a potential preclinical platform for drug testing. Therefore, in order to develop such tumor models that are potentially capable of predicting response to therapies, rodent/porcine kidneys and bladders were collected from animals being euthanized for other research projects that did not compromise the structural and functional integrity of either organs. With collection of these organs, it is essential to decellularize and recellularize them into ready-to-use models that are capable of mimicking the patient’s diseases. The rationale of engineering an animal organ into a patient’s disease model required techniques of tissue engineering.  In general, tissue engineering is a combination of the principles and methods of life sciences with that of engineering, to develop material and methods to replace   8 damaged or diseased tissues, and create entire tissue replacements (28) (29). The triad of tissue engineering are biomaterial/scaffold, cells and growth factors/bioreactor. The scaffold in tissue engineering provides 3D structure for cells to grow, proliferate and delivery biomolecules such as growth factors, cytokines, etc. (30). Growth factors are soluble proteins that stimulate cells in order to proliferate, migrate and differentiate (31) (32). Moreover, a bioreactor provides physical stimulation and condition for cell proliferation.  In my study, I aim to transform an animal organ/tissue into a tissue engineered scaffold to home normal cells and cancer cell lines in later procedures to simulate a certain disease. In order to home these new cells, cells from the animal organ are required to be completely eliminated first. Compared to synthetic matrices, the animal organ scaffolds are already biocompatible, biodegradable, vascularized and have mechanical properties that are suitable for cell growth. In summary, the principle is to remove previous resident cells and leave the ECM with preserved micro-architectures and composition.  1.4 Decellularization as a method for tissue engineering  In order to achieve the tight balance between complete cell removal and the integrity of ECM micro-architecture, a methodology called decellularization can be applied. Decellularization is the process used in biomedical engineering to isolate the ECM of a tissue from its inhabiting cells, leaving the ECM scaffold of the original tissue, which   9 can be used in artificial organ and tissue regeneration (33) (34) (35). Decellularization process can be accomplished through perfusion or immersion. As long as the tissue/organ is completely decellularized, other procedures for creating the tumor model can be applied.   1.5 Recellularization as a method for tissue engineering  With the completion of decellularization, resident cells are eliminated and the ECM microarchitecture was preserved. In order to engineer a model that mimics human tissue base on this scaffold, human cells can be reseeded into the decellularized ECM at correct position according to the nature of different cells. Recellularization is achieved as cells proliferating and growing in the matrices under appropriate culturing conditions.  1.6 Hypotheses and objectives  With the aforementioned principle and methodologies, I hypothesized that my decellularized ECM model has more clinical relevance to represent in vivo conditions compared to other models and it is able to retain various matrix-bound growth factors that may participate in cell homing. That being said, organ decellularization can be used to develop a high throughput urogenital tumor model resembling patient’s tumor environment.    10 There are two objectives in my study: 1) To establish kidney and bladder decellularization methodology including characterization and evaluation of the decellularized ECMs. 2) To recellularize the decellularized ECMs with different human normal cells or cancerous cells to create a tumor model. With these two objectives, I aim to establish a novel 3D clinical platform that can potentially predict treatment outcomes of urogenital cancers. This would be the first test-model that utilizes human cell lines to study the accessibility and cellular behavior of urogenital cancers in an environment that mostly mimics that of in vivo conditions. Overall, I am hoping that this study will set the foundation of a new preclinical tumor model that will significantly impact the way clinicians treat patients with kidney and bladder cancers. This study could potentially give a clue to researchers who want to generate engineered 3D patient-derived models that accurately recreate the urogenital tumor of an individual patient. Developing this 3D in vitro cancer model may allow us to potentially use patient’s own cells to create a replica of the patient’s tumor; this model may then provide the ability to perform multiple drug tests on this clinical platform and thus potentially allow healthcare providers to simultaneously screen multiple anti-cancer drugs in an individualized manner ex vivo. This model potentially avoids the need to sequentially use ineffective therapies in a patient to prevent unnecessary side effects and costs. If successful, the engineered 3D patient-derived urogenital cancer model will allow healthcare providers to identify the most efficacious therapy and will lead to the improvement of the patient’s overall survival and quality of life.   11 Chapter 2: Materials and Methods  2.1  Organ retrieval from animals  a) Kidney retrieval Prior to organ collection from rodents, intracardiac injection of heparin (200 USP in 800 µL of Phosphate-buffered saline) (Fresenius Kabi, sodium injection, C504730, Bad Homburg, Germany) was performed to introduce systemic distribution of anticoagulant to prevent thrombosis in organs. To perform intracardiac injection of heparin, 1 mL syringes were loaded with heparin (1000 USP units/mL) and set aside for later use. Under overdose of anesthesia, rodents were placed in dorsal recumbency and pedal reflexes were checked to ensure that they were deeply anesthetized. After the needle tip was through the skin, syringe plunger was pulled slightly to create negative pressure and then advanced until blood was drawn into the syringe. Heparin is then infused over 1 minute to allow circulation throughout the body. Once heparin injection was completed, euthanasia was initiated. Euthanasia consisted of two steps: a) overdose of isoflurane (5% flow rate concentration or greater) followed by b) cervical dislocation.  For kidney retrieval: immediately after euthanizing the rodents, the abdominal cavity was opened and the aorta was dissected to the renal artery. After that, the celiac, splenic and mesenteric arteries as well as renal vein were ligated with 5-0 Ethilon   12 nylon suture (Ethicon, GA, USA) leaving patent the renal artery and the ureter. A 24-gauge catheter (Becton, Dickinson and Company, 382512, NJ, USA) was placed in the aorta toward the renal artery with 1000 units of heparin perfused through the catheter (perfusion time was around 1 minute). As fading of the red-brown color of the kidney confirmed proper placement of the catheter, ligature was tightened to hold the catheter in place. All other connected vessels were ligated and removed. Once the kidney was detached, it was placed in 20 mL of phosphate-buffered saline (PBS) with 100 units of heparin and a 10 mL syringe containing the same solution was attached to the catheter. Small amount of solution was drop-wisely perfused every 2 - 3 minutes to wash out the blood from the organ. The kidney obtained and processed to this point would be ready to use for decellularization. The kidney – catheter system was kept on ice.   Important aspects of organ harvesting should be mentioned: 1) Perfusion of heparin should be very slow and gentle to prevent the flimsy renal capillaries from bursting. 2) Saline should be instilled in the abdominal cavity when kidney is exposed to air to prevent desiccation. If working with one kidney at a time, the contralateral side should be wrapped with saline moist gauze to prevent damage of kidney during exposure in air. 3) Placement of the catheter must be done carefully as this step may damage the arterial lumen to prevent infusion. Minimal attempts of catheter placement should be performed. 4) Infusion of heparin must be started quickly to prevent thrombosis in the small caliber vessels, which would make them unable to achieve complete decellularization and thus not a good candidate for future experiments. Therefore, the   13 aforementioned precautions during dissections are vital for us to obtain eligible starting materials for the upcoming decellularization procedures.  b) Bladder retrieval. The rodent bladder retrieval was performed subsequent to kidney retrieval. A midline incision from the xiphoid process to the pubic symphysis was made and the urinary bladder would usually be located in the lower center of the abdomen close to the pelvic bone. Both ureters and the urethra were ligated with silk suture and then divided and then the bladder was dissected from the animal.   For porcine bladder retrieval, animals were euthanized under corresponding primary research protocol, which can be an overdose of injectable anesthetic. Immediately after euthanizing the pig, the abdominal cavity was opened with a midline incision and then the bladder was identified. The porcine bladder, which is long and flat, can be found between the blood vessels in the umbilical cord. The bladder was then cut into multiple 1 cm × 1 cm × 0.5 cm pieces to obtain greater surface area during decellularization and were placed in PBS solution (on ice) immediately. The size of each pieces is similar and thus could be used as experimental replicates in future procedures.         14 2.2    Decellularization protocols  2.2.1 Background  Since the success of decellularization were mainly relied on four aspects 1) the original conditions of the organs 2) performance during organ dissections 3) parameter settings of the peristaltic pump during kidney perfusion/cycle controls during bladder immersion and 4) the sterilization of the experimental organs during decellularization. Any inconsistencies in between would cause failure or inefficiency of decellularization and ended up with wasted organs, of which consequences deviate from our original intention to reduce animal usage. In worse cases, the failure or inefficiency during decellularization was not prominent enough to observe and the organs were proceeded to following recellularization procedures. It could end up with flawed tumor models that are not representatives of human conditions and could possibly provide false therapy suggestions to patients. Gender was not considered an issue for my study and diseases/treatments applied to the animals needed to be evaluated individually, since some may or may not alter the physiological condition of the organs. Perfusion and immersion decellularizations were both performed under sterile condition in this study. Whole kidney decellularization was performed with perfusion and decellularization of bladder pieces were performed with immersion (Figure 1).   15  Figure 1. Decellularization protocols. Two decellularization strategies: decellularization by perfusion in a bioreactor with a peristaltic pump (kidney) and decellularization by immersion (bladder).       16 2.2.2 Bioreactor  An in-house developed perfusion-based bioreactor was constructed with an airtight polycarbonate food container which is BPA-free, has a volume of 3-quart with silicone gasket. Three holes were drilled on the container (Figure 2) and they were insulated with three lure-lock connecters. The top two connecters were attached with a needle-free IV connectors & catheter extension set, respectively. Inside of the bioreactor, a stainless-steel rod was inserted and glued onto the upper inside wall and two hooks were hanged on it. This system would be able to suspend the organ with attached catheter and could keep balance of the organ during perfusion. A peristaltic pump was connected to the set and could perfuse solutions into the bioreactor. The bottom connecters were attached to a needle-free IV connecter only and could release used solutions.    17   Figure 2. The bioreactor used for kidney decellularization (perfusion).               18 2.2.3 Decellularization by perfusion – kidney.  At room temperature, the rodent kidney was placed inside of the bioreactor described in section 2.2.2 and the catheter previously inserted in the renal artery was connected to a peristaltic pump (Braintree Scientific, BS-900 programmable peristaltic pump with 4 roller pumping head & 1/16 ID tubing, MA, USA) to allow continuous passing through of different solutions from the inserted catheter (Figure 3). The whole system of the peristaltic pump including all tubes and the bioreactor were sterilized with 70% ethanol beforehand or the kidneys would end up with bacterial or fungal contamination. The kidneys need to be securely placed in the bioreactor because peristalsis could possibly push the kidneys down and disconnect the catheter during perfusion.   Since one of the 1% Triton X-100 solution perfusion cycles was overnight, it is crucial to immobilize the kidneys with an extra ligature tied with the hook hanged on the stainless rod to prevent them from falling down (Figure 4). On the other hand, the peristaltic pump needs calibration for each usage since it was found that the flow rate could vary to some extent from time to time. The ideal way to minimize this mechanical error is to measure the actual flow rate before perfusion of the kidneys. The pump could be turned on for five to ten minutes beforehand and the amount of solution being passaged was measured. With this approach, the actual flow rate could be calculated and thus certain adjustments could be applied to the pump parameter settings.    19 Distilled water (dH2O) was perfused at a flow rate of approximately 5 mL/min for 200 minutes in total to eliminate remaining blood cells. 1% Triton X-100 solution (Fisher Scientific, BP151, PA, USA) was perfused at the same rate for 400 minutes in total. The same solution would then be perfused at a rate of approximately 1 mL/min for 1000 minutes in total. Finally, 0.1% sodium dodecyl sulphate (SDS) solution (Fisher Scientific, BP1311, PA, USA) was delivered at a flow rate of approximately 5 mL/min for 200 minutes in total. The above cycle can be repeated for 2 – 3 time depends on the size and volume of the organ. At the end of the last cycle, the kidney was perfused with dH2O for 24 hours followed with perfusion of Deoxyribonuclease I (DNase I, 2,000 units) (Sigma-Aldrich, D4513, MO, USA) for 120 minutes. Afterward, additional perfusion with dH2O was performed for 4 - 5 days at a flow rate of approximately 1 mL/min (5760 to 7200 mL in total) to eliminate any remaining detergents/chemicals in the kidney.  During perfusion, it is observed that the kidneys were not blanching much (still have brown color) within the 1% Triton X-100 cycles but turned almost translucent during the short 0.1% SDS cycle. It is because Triton X-100 solution disrupts DNA-protein, lipid-lipid and lipid-protein interaction (35) whilst maintaining native protein structures. SDS solutions completely solubilize cell and nucleic membranes and fully denature proteins (36). Structural proteins of kidneys were not supposed to be disrupted in this study and that is the reason why 0.1% SDS cycle is much shorter than the 1% triton X-100 cycle. On the other hand, the original size of the dissected kidneys is also   20 crucial. Larger animals usually have larger kidneys and larger kidneys always require more perfusion cycles to ensure complete decellularization.  Other earlier tested protocols can be found in appendix A & C.    Figure 3. The bioreactor-peristaltic pump set up for kidney decellularization (perfusion).    21   Figure 4. The schematic diagram of the bioreactor used for kidney decellularization (perfusion). (A) Sealed top of the bioreactor. (B) Kidneys during decellularization. (C) Peristaltic pump connecting portals. (D) Waste portal. (E) stainless-steel rod glued to the inside wall of the bioreactor to support kidneys.     22 2.2.4 Decellularization by immersion – bladder  Bladders were collected and processed as described in section 2.1 (b). Immediately after collection, bladders were immersed in Dulbecco Modified Eagle Medium (DMEM)  (Fisher Scientific, 13345364, PA, USA) with 50 nM Latranculin B (Cayman Chemical, CAS 76343-94-7, Michigan, USA) and incubated for 120 minutes at 37ºC. After incubation, tissues were transferred into sterile dH2O at room temperature and they were agitated on a shaker for 15 minutes, making one change of sterile dH2O and agitating for another 15 minutes. Afterward, tissues were immersed in sterile 0.6 M potassium chloride solution (Fisher Scientific, AC424090010, PA, USA) at room temperature and were agitated on a shaker for 120 minutes. Then, tissues were immersed in sterile 1 M potassium Iodide solution (Sigma-Aldrich, 03124, Darmstadt, Germany) at room temperature and were agitated on a shaker for 120 minutes. Finally, tissues were immersed in sterile dH2O at room temperature overnight. The above described steps were considered one full cycle. Each cycle was repeated 10 - 12 times depending on size and volume of the tissue to ensure proper decellularization. Tissues were then incubated in DNase I (1 kU/mL; Sigma-Aldrich, D4513, MO, USA) for 120 minutes. Finally, tissues were washed in dH2O for 2 days with daily water changes to remove remaining reagents. 150 mm x 25 mm tissue-culture treated culture dishes (Sigma-Aldrich, CLS430599, Darmstadt, Germany) were used for bladder immersion decellularization.    23 Each solution used for bladder immersion decellularization are prepared in sterile conditions (including dH2O) and solution changes between each immersion cycles should also be performed in a sterile biosafety cabinet to reduce the risk of bacterial or fungal contamination. Larger petri dishes (150 mm x 25 mm in size) were used for agitation in order to obtain stronger centrifugal forces. Also, there were no more than five bladder pieces in each petri dish to ensure sufficient movement when agitated. In general, porcine bladder immersion involved fewer complex procedures compared to that of rodent kidney perfusion. Porcine bladders are less delicate than kidneys and thus are easier to handle.   Other earlier tested protocols and rationales can be found in appendix B.   2.3 ECM evaluation  Decellularization protocols were evaluated from several aspects. Since the success of each protocol was determined on the extent of cell removal and the preservation of ECM microarchitecture, I evaluated the ECMs with three approaches: macroscopic visualization, Deoxyribonucleic acid (DNA) quantification and histological assessments including hematoxylin and eosin (H&E) stains and visualization with scanning electron microscope (SEM).      24 2.3.1 DNA quantification  Both native and decellularized organ/tissue were cut up to 25 mg pieces prior to DNA extraction procedures. The small pieces were digested individually using Proteinase K (QIAGEN, 19133, Hilden, Germany) at 56ºC with agitation until they were completely lysed. DNA was distracted and purified using the DNeasy Blood & Tissue Kit (QIAGEN, 69504, Hilden, Germany) according to the manufacturer’s instructions. Briefly, upon removal of RNA, DNA samples were isolated by spin column-based nucleic acid purification procedures. Afterward, both native and decellularized tissue’s DNA extracts were quantified spectrophotometrically using NanoDrop technology (Thermo Scientific, ND-2000, MA, USA). This approach of DNA quantification was to determine how thorough the decellularization protocol was and according to previous publications (37), it is proved with a destructive method that less than 5% of DNA remnant/less than 50 ng of double strand DNA in 100 mg of decellularized tissues compare to that of native tissue considered successful decellularization (38). The unpaired (Two Sample) t test was performed to assess speculated changes.  2.3.2 Histological assessments  a) H&E To assess cell removal and the preservation of ECM microarchitecture of the decellularized tissues, H&E stain was performed to both native and decellularized tissues. All tissues were fixed overnight in 1:10 dilution neutral buffered formalin (10%   25 formalin) (Fisher Scientific, 22-046-361, PA, USA) and then transferred to 70% ethanol prior to paraffin embedding, sectioning and staining. First, tissues were serially dehydrated in 70% (30 minutes), 80% (120 minutes, two changes), 95% (120 minutes, two changes) and 100% ethanol (90 minutes, three changes). Following dehydration, tissues were immersed in two xylene solutions to for clearing: a) Immersed in 100% ethanol and 100% xylene solution (1:1) (Sigma Aldrich, 534056, Darmstadt, Germany) for 30 minutes then b) immersed in 100% xylene for 30 minutes. With completion of these steps, all tissues were embedded into paraffin blocks and sectioned into 5 µm thickness. Sections were baked at 37 °C overnight prior to deparaffinization and rehydration. For deparaffinization, the sections were immersed in CitriSolv hybrid solvent and clearing agent (Fisher Scientific, 22-143-975, PA, USA) for 5 minutes (repeated three times). Sections were then serially rehydrated in 100%, 95%, 80% and 70% ethanol solution for 3 minutes each (with two repeats for each step) and were immersed in dH2O for 5 minutes with three repeats. Afterward, the sections were stained with hematoxylin solution, Gill No.2 (Sigma Aldrich, GHS232, Darmstadt, Germany) for 3.5 minutes and rinsed with tap water followed with immersion in Shandon bluing reagent (Thermo Fisher, 6769001, MA, USA) for 30 seconds. The sections were again rinsed in tap water and stained with Eosin Y-solution (Millipore Sigma, 1098441000, Darmstadt, Germany) for 30 seconds. Another tap water rinse was performed prior to the serial dehydration afterward. The sections were dipped 10 times in 70%, 80% and 95% ethanol each and followed with two 1-minute immersion in 100% ethanol. At the end, sections were immersed in xylene for 3 minutes with three repeats and mounting solution was added prior to   26 cover-slipped visualization. The slides were then scanned with Leica SCN400 slide scanner (Meyer Instruments).  b) SEM A segment of both native and decellularized kidney was primarily fixed in 4% paraformaldehyde (Electron Microscopy Sciences, 15710, PA, USA) in 0.1 M PIPES (Sigma-Aldrich, P6757, Darmstadt, Germany) buffer at room temperature for 10 hours, the pH was titrated down to 7.4. The tissue was briefly rinsed with the same buffer prior to the fixation in 2.5% glutaldehyde (Electron Microscopy Sciences, 16020, PA, USA) in 0.1 M PIPES buffer at room temperature for 20 hours, the pH was titrated down to 7.4. Tissue was then rinsed three times in the same buffer for 1 hour each, it was fixed in 1% Osmium Tetroxide (OsO4) (Electron Microscopy Sciences, 19150, PA, USA) in 0.1M PIPES buffer, pH 6.8 at room temperature for 5 hours. Alter fixation, tissues were rinsed three times with double distilled water (ddH2O) for 1 hour each. After rinsing, the specimen was dehydrated at room temperature in a graded ethanol-water series: 50%, 60%, 70%, 80%, and 90% solution for 1 hour each, followed with three times 100% ethanol (Electron Microscopy Sciences, 15056, PA, USA) for 1 hour each. Critical point drying using CO2 (Tousimis Samdri®-795 critical point dryer) was applied for 1 hour to the specimen to ensure absolute dehydration and the specimen was finally mounted on an aluminum stub using sticky carbon tapes. The specimen on the stub was then coated with a thin layer of Au/Pd coating (2 nm thickness) using the Leica EM MED020 Coating System. Same sample preparation strategies were   27 performed to native and decellularized bladders. Images were recorded with a Helios NanoLab 650 Focused Ion Beam SEM.   2.4 ECM characterization  Other than the evaluation of complete cell removal, proteomics analyses were performed as a novel approach to determine the full protein profile of the decellularized organs (kidney and bladder) in this study. Current methods evaluating the completeness of organ decellularization are based mainly on the elimination of nucleic acids since they are easy to quantify. However, other structural and cellular materials, such as proteins are also essential in indicating the preservation of ECM. Therefore, additional characterization, such as proteomic analyses, will provide molecular readouts of the composition of decellularized kidney and bladder ECM scaffold.   A compartment protein extraction kit (Millipore Sigma, 2145, Darmstadt, Germany) was used to isolate subcellular and structural proteomes from the decellularized organs. This kit was also used to perform enrichment of some low-abundant proteins for further proteomic analysis using liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The procedures were followed according to manufacturer’s instructions.    28 a) ECM protein extraction and enrichment  Before starting, a cocktail of protease inhibitor (provided with the compartment protein extraction kit) was added to all reagents/buffers from the kit to make it a 1X solution. All reagents and tissue samples were kept on ice for the duration of the processing and the only exception was the buffer CS, which was kept at room temperature to prevent SDS (used in decellularization) precipitation. According to the manufacturer’s manual, the composition of buffers used in this experiment was provided:  100 mg of decellularized and native tissues (kidney or bladder) were homogenized in 500 µL of Buffer C containing protease inhibitor until the tissues were completely disrupted and a homogeneous suspension was obtained. The homogenate was incubated on a rotator for 20 minutes at 4°C. After incubation, it was centrifuged at 16,000 x g for 20 minutes at 4 °C and the supernatant was discarded. This step was to extract cytosolic proteins. The pellet was washed in 400 µL of Buffer W containing protease inhibitor and the suspension solution was incubated on a rotator for 20 Buffer C HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium OrthoVanadate Buffer W HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium OrthoVanadate Buffer N HEPES (pH 7.9), MgCl2, NaCl, EDTA, Glycerol, Sodium OrthoVanadate Buffer M HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium deoxycholate, NP-40, Sodium OrthoVanadate Buffer CS PIPES (pH 6.8), MgCl2, NaCl, EDTA, Sucrose, SDS, Sodium OrthoVanadate   29 minutes at 4 °C. The suspension solution was centrifuged at 16,000 x g for 20 minutes at 4 °C and the supernatant was discarded. In order to extract nuclear proteins, the pellet was re-suspended in 150 µL of Buffer N containing protease inhibitor, deoxyribonuclease I (DNase I, New England Biolabs, M0303S, MA, USA) and ribonuclease A (RNase A, QIAGEN, 19101, Hilden, Germany). The final concentration of DNase I and RNase A in Buffer N was 200 µg/ml and 20 µg/ml, respectively. The sample was incubated on a rotator for 30 minutes at 4 °C and later on centrifuged at 16,000 x g for 30 minutes at the same temperature. The supernatant obtained was discarded. Furthermore, to extract membrane protein, the pellet was re-suspended in 100 µL of Buffer M containing protease inhibitor and the sample was incubated on a rotator for 30 minutes at 4 °C followed by centrifugation at 16,000 x g for 30 minutes at the same temperature. The supernatant obtained was discarded. Afterward, to extract cytoskeletal proteins, the pellet was re-suspended in 200 µL of Buffer CS containing protease inhibitor and the sample was incubated on a rotator for 30 minutes at room temperature followed by centrifugation at 16,000 x g for 30 minutes also at room temperature. The supernatant obtained from this step was discarded. To perform additional wash of the pellet, it was re-suspended in 500 µL of PBS containing protease inhibitor and the suspension was incubated on a rotator for 5 minutes at 4 °C. Similarly, the suspension was centrifuged at 16,000 x g for 5 minutes at the same temperature. The supernatant obtained was discarded and this wash procedure was repeated for three times. At this point, the pellet remaining was the extraction of insoluble ECM-enriched proteins of the tissue. For further proteomic analyses, these proteins were digested into soluble peptides.   30 b) In-solution digestion of ECM-enriched proteins The ECM-enriched pellet was re-suspended in 8 M urea (100 µL) and was agitated at 1,400 rpm for 2 hours at 37 °C. The sample was cooled to room temperature and was added with 2 µL of 500 mM 1, 4-Dithiothreitol (DTT, Sigma-Aldrich, 10197777001, Darmstadt, Germany) to achieve a final concentration of 10 mM. The mixture solution was incubated with continuous agitation at 1,400 rpm for 2 hours at 37 °C. 5 µL of 500 mM iodoacetamide (Thermo Fisher, 90034, MA, USA) was added to prevent the formation of disulfide bonds and ended up with final concentration of 25 mM. The mixture solution was incubated in the dark for 30 minutes at room temperature to complete alkylation. The mixture was then diluted to a 2 M urea concentration with 100 mM ammonium bicarbonate pH 8.0 (Sigma-Aldrich, 09830, Darmstadt, Germany) prepared with ddH2O. Afterward, 4 µL of peptide-N-glycosidase F (2000 U) (PNGase F, New England Biolabs, P0704S, MA, USA) used to remove N-linked oligosaccharides from glycoproteins was added and followed with continuous agitation at 1,400 rpm for 2 hours at 37 °C. In addition, 4 µL of mass spectrometry grade endoproteinase LysC (2 µg) (New England Biolabs, P8109S, MA, USA) was pipetted to the mixture followed with continuous agitation at 1,400 rpm for 2 hours at 37 °C to ensure peptide bonds at the carboxyl side of lysine were cleaved. An aliquot of 10 µL of mass spectrometry grade trypsin (5 µg) (Thermo Fisher, 90057, MA, USA) was added to the mixture and was incubated with continuous agitation at 1,400 rpm overnight at 37 °C, the cloudy solution turned cleared overnight. Another 6 µL (3 µg) of mass spectrometry grade trypsin was added to the mixture followed with continuous agitation at 1,400 rpm for 2 hours at 37 °C. To inactivate the trypsin, the mixture was   31 acidified with freshly prepared LC-MS grade 50% trifluoroacetic acid (TFA, Thermo Fisher, 85183, MA, USA) pipetted in 1 µL increment (4 µL used in total) until the pH was less than 2.0. Finally, the acidified solution was centrifuged at 20,000 x g for 5 minutes at room temperature and the supernatant was collected. The peptides were desalted by solid phase extraction with C18 Sep-Pak cartridges (Thermo Fisher, 60108, MA, USA) and it was used according to the instructions from the manufacturer and the mixture was eluted with freshly prepared 60% LC-MS grade acetonitrile (ACN) (Sigma-Aldrich, 51101, Darmstadt, Germany) and 0.1% TFA, followed by concentration in a vacuum concentrator (vacuum dried to about 25 µL). Protein concentration was measured spectrophotometrically using NanoDrop technology and peptide solutions were diluted to a final concentration of 0.5 µg/µL with 0.2% formic acid. The samples were stored at -80 °C until LC-MS/MS analysis.   c) Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Approximately 1 µg protein digest (in loading buffer made of 5% ACN and 0.1 % TFA) from decellularized and native kidney or bladder tissues were analyzed using an EASY-nLC 1200 chromatography system (Thermo Fisher, LC140, MA, USA) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher, IQLAAEGAAPFADBMBHQ, MA, USA). The EASY-nLC 1200 chromatography system was set up with the acclaim PepMap ESLC C18 LC 75 µm × 50 cm; 3 µm columns (Thermo Fisher,164570, MA, USA) at 50 °C using the following gradient: 0 – 5 minutes 2% ACN; 5-140 minutes, 2 – 28% ACN;140 – 150 minutes,28 – 40% ACN; 150 – 160 minutes, 40 – 95% ACN; 160 – 170 minutes, 95% ACN. The washout step was   32 performed at a flow rate of 0.5 µL/min at 80% ACN for 12 minutes prior to column equilibration. The total run time was about 3 hours.  A full mass scan (375 – 1500 m/z, 2400V) was performed in the Orbitrap Fusion Lumos mass spectrometer at a resolution of 1,200,00. Data was collected with peptide monoisotopic selection (5 × 104 threshold, charge states 2 – 7, 60-second dynamic exclusion, quad isolation m/z of 0.8). The most intense peaks were selected for fragmentation, and the higher-energy collisional dissociation (HCD) was automatically determined by the instrument based on the m/z values and charged stated of the peptides. The fragmentation spectra were analyzed and the MS raw files were downloaded from the PeptideAtlas data repository. Data was processed using Thermo Proteome Discoverer 2.1 with Sequest and the Rattus norvegicus dataset (SwissProt TaxID=10116, 2017-10-25) with the following parameters: precursor mass 350-5000 Da; enzyme, trypsin; missed cleavage, 2; min/max peptide length 6/144; precursor/fragment tolerance, 10 ppm/0.02 Da; all neutral losses; b,y ions only; static modification, carbamidomethyl; dynamic modifications, oxidation (K, M, P), protein terminus acetyl; Cn max 0.05; PSM, peptide and decoy DB target FDR’s, 0.01 strict, 0.05 relaxed, based on q-value. Filtering was set for peptides at least medium confidence. However, only high confidence data was selected with final post process filtering with Proteome Discoverer software (Thermo Scientific, OPTON-30795). Protein and Protein Group were exported to Excel for further sorting and analyses. The full-protein profiling described above is a qualitative analysis to identify existence of different proteins in both native and decellularized tissues. However, it also   33 indirectly provides quantitative information based on the relative peak intensity and peak width of the individual peptides which means the coverage number could potentially represent relative abundance of each protein. Relative ion intensity was measured by the area under the curve of each peak and was compared to the extracted ion chromatogram standard.  2.5 Recellularization protocols  Decellularized organ-ECMs went through series of evaluation and characterization procedures as described in section 2.3 and 2.4 to ensure their eligibility as starting materials for ECM recellularization. In this study, recellularization is to reseed corresponding human normal or cancerous cells to the decellularized kidney or bladder ECMs. Reseeded cells were expected to proliferate, migrate and even produce their own matrices within the decellularized ECMs (39). However, inconsistencies between different batches of recellularization were observed. Considering the completion/success of recellularization mainly relied on three aspects: 1) Viability of the cells, 2) reseeding technique, and 3) control of cell culture conditions (particularly in close watch of mycoplasma contamination since it is undetectable with just a microscope). The aim of this study was to observe different cells proliferating in a certain location of the ECMs, in other words, to observe similar cell-distribution compare to that observed in a native organ. Nevertheless, any experimental inconsistencies in between would cause failure or inefficiency of   34 recellularization and ended up with random cell-penetration/adhesion to the ECM, which is to the contrary of the research hypotheses.   2.5.1   Cell lines  Human bladder cancer cell line UM-UC-3 and immortalized epithelial cell line SV-HUC were kindly provided by Dr. Peter Black (Vancouver Prostate Centre, Vancouver, BC, Canada). Human embryonic kidney 293 cell line HEK293 was generously provided by Dr. Amina Zoubeidi (Vancouver Prostate Centre, Vancouver, BC, Canada). Human umbilical vein endothelial cells (HUVEC) were attained from Lonza (CC-2953, Basel, Switzerland). Human dermal fibroblast cells were isolated from neonatal foreskin undergoing circumcision and kindly provided by Dr. Aziz Ghahary. Human metastatic clear cell renal cell carcinoma (ccRCC) cell line (Caki-1) was purchased from American Type Culture Collection (Manassas, Virginia, USA). All media used in this study except EBM TM-Plus was supplemented with 1% Antibiotic-Antimycotic (Gibco, 15240062, ON, Canada) to prevent bacterial and fungal comtamination. Fibroblasts and HEK293 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 11995-065, ON, Canada) with 10% fetal bovine serum (FBS) (Gibco, 10437028, ON, Canada). SV-HUC were cultured in Keratinocyte Serum-Free Medium (KSFM) (Gibco, 17005042, Carlsbad, CA) supplemented with bovine pituitary extract (50 µg/ml) and recombinant human epidermal growth factor (rhEGF) (0.2 µg/ml). Caki-1 cells were cultured in McCoy’s 5A medium (ATCC, 30-2007, Virginia, USA) supplemented with 10% FBS. UM-UC-3 were cultured in Minimal Essential Medium   35 (MEM) (Gibco,11090-073, ON, Canada) supplemented with 10% FBS, 1% L-glutamine (GlutaMax, 35050061), 1% non-essential amino acids (Gibco, 11140050, Burlington, ON, Canada) and 1% sodium pyruvate (Gibco, 11360070, Burlington, ON, Canada). Reagents used for UM-UC-3 were obtained from Thermo Fisher Scientific (Gibco), Burlington, ON, Canada. HUVEC cells were cultured in Endothelial Cell Growth Basal Medium-Plus (EBM TM-Plus) (Lonza, CC-5036, Basel, Switzerland) supplemented with 2% FBS, 0.1% hydrocortisone, 5% L-glutamine, 0.1% ascorbic acid, 0.1% Heparin, 0.2% Endothelial Growth Supplement (EnGS), 0.1% rhEGF and 0.1% Gentamicin Sulfate-Amphotericin (GA-1000) (Lonza, CC-4542, Basel, Switzerland). All cells used were cultured at 37 °C in a 5% CO2 incubator and mycoplasma contamination was tested at regular intervals for each cell-line/primary cell. When cells were confluent they were passaged by incubation at 37 °C for 3 - 4 minutes with 0.25% trypsin (Gibco, 25200056, ON, Canada), centrifuged at 1,500 RPM for 5 minutes and resuspension in medium. Cells were stored long term in Bambanker (NIPPON Genetics, BB01, Düren, Germany) at a concentration of one million cells per mL and in liquid nitrogen.  2.5.2 Kidney ECM recellularization protocol  Decellularized kidneys were cut into thin pieces with sterile blades, washed with repeated agitation in 10% Antibiotic-Antimycotic and placed in a 24-well plate (well diameter: 15.6 mm; Well volume: 3.4 mL) (Corning, CL-S3524, ME, USA) with sterile forceps. Tissues were cultured in a mixture of different growth media (for 24 hours)   36 depending on the different cell types the tissue would be recellularized with. Afterward, all cultured cells were dissociated into single cells using 0.25% trypsin for 4 minutes at 37 °C and centrifuged at 1500 rpm for 5 minutes. Cell pellets were re-suspended with Matrigel Matrix (Corning, 354234, ME, USA) (4,000 cells in 10 µL of Matrigel Matrix). Each 10 µL of the cell-Matrigel mixture were loaded onto the tissue surface. The 24-well plate were incubated in 37 °C with 5% CO2 for 2 hours to ensure the mixture loaded were completely solidified while cells could maintain their viability. Tissues were again immersed with corresponding growth media (1 mL of each well) after incubation and the cell-loading steps were repeated 4 – 5 times (from day 10) until reached 21 days of incubation at 37°C. Within the 21-day timespan, media were replaced on a daily basis. At day 21, the recellularized tissues would be fixed in 10% formalin for H&E and immunohistochemistry (IHC) evaluations.  Other earlier tested protocols can be found in appendix D.  2.5.3 Bladder ECM recellularization protocol.  Decellularized bladders were cut approximately into 1 cm × 1 cm × 0.5 cm pieces and placed in a 6-well plate (well diameter: 34.8 mm; Well volume: 16.8 mL) (Corning, CL-S3516, ME, USA). Tissue was placed on cell culture inserts (Fisher Scientific, 08-771, PA, USA) using sterile forceps and the orientation of the bladder’s placement was crucial in this study. After decellularization, the bladder matrix clearly shows two sides, one surface is visibly shiny (Adventitia) and the other gives the appearance of a rough   37 surface (Lumen side). Starting at the lumen and moving outward through the bladder wall, the urothelium (mucosa) consists of three layers: umbrella cells, intermediate cells and basal cells. The lamina propria (basement membrane) underlies the basal cells and separates the urothelium from stromal cells layer (submucosa) and muscles. In this study, muscularis propria of the bladder was removed to some extent in order to expose stroma for recellularization of fibroblasts and HUVECs while SV-HUC and UM-UC-3 were recellularized on the urothelium.  For either single cell-type or multi cell-type recellularization, bladder tissues were immersed in different growth media according to cell types being reseeded. Tissues for single cell-type recellularization: HUVECs were with EBM TM-Plus, fibroblasts were with DMEM supplemented with 10% FBS, SV-HUCs were cultured with KSFM kit and UM-UC-3 were with MEM supplemented with 10% FBS, 1% L-glutamine, 1% non-essential amino acids and 1% sodium pyruvate. Bladders recellularized with HUVEC: fibroblast: SV-HUC (ratio of 2: 1: 2) required a combination of all corresponding media described above at 1:1:1 ratio. Prior to recellularization of the aforementioned cells, decellularized ECMs were immersed in media with corresponding growth factor for 24 hours. Cells grown in 2D culture were trypsinized and counted. For single cell type recellularization, cell concentration of 2106 cells/cm3 was applied. For multi cell-type recellularization, cell concentration of 2106 cells/cm3 (HUVEC, SV-HUC, respectively) was reseeded while 1106 cells/cm3 of fibroblasts was reseeded. All cells were dissociated into single cells using 0.25% trypsin for 4 minutes at 37 °C and centrifuged at 1,500 rpm for 5 minutes. Each cell types were re-suspended with   38 Matrigel Matrix (1106 cells in 50 µL of Matrigel Matrix). Each 50 µL of the cell-Matrigel mixture were loaded onto the tissue surface. For multi cell-type recellularization, bladder tissues were flipped in order to load cells into their corresponding environment (SV-HUC and UM-UC-3 on urothelium; fibroblast and HUVEC on the stromal layer). The 6-well plate were incubated in 37 °C with 5% CO2 for 2 hours to ensure the mixture loaded were completely solidified while cells could maintain their viability. Tissues were then immersed with corresponding growth media (3 mL of each well) after incubation and the cell-loading step were repeated with 4 – 5 times (from day 10) until reached 21 days of incubation at 37 °C. Within the 21-day timespan, media were replaced on a daily basis. Disturbing of the tissues during incubation was minimized. At day 21, the recellularized tissues were fixed in 10% formalin for H&E and IHC evaluations.  Other earlier tested protocols can be found in appendix E & F.  2.6 Evaluation of recellularization protocols  a) IHC To evaluate cells’ position, distribution and penetration after being reseeded, both kidney and bladder tissues were fixed in 10% formalin for 24 hours followed by immersing in 70% prior to H&E. H&E was performed as described in section 2.3.2 (a). For single cell-type recellularization of kidney and bladder, H&E only was sufficient for the evaluating purposes. However, for bladder multi cell-type recellularization, IHC   39 staining would be essential to identify cell types and their distributions. Formalin-fixed, paraffin-embedded tissue sections were prepared as described in section 2.3.2 (a) and before proceeding to IHC staining, the section slides were deparaffinized and rehydrated. The slides were placed in a rack and were immersed in 100% xylene for 10 minutes; this step was repeated for three times. The slides were then immersed in 100% ethanol for 5 minutes and this step was repeated for two times. Next, the slides were immersed in 95% for 5 minutes and this step was repeated for two times followed by immersion in 80% and 70% ethanol for 5 minutes, respectively. After rehydration, the slides were rinsed with tap water until ready to perform antigen retrieval. Antigen retrieval was performed by boiling the slides (850W, microwave) in Tris/EDTA (pH 9.0) for 10 minutes followed with immersion in the Antigen Unmasking Solution (1:1 with H2O) (Vector Laboratory, H-3301, CA, USA). The slides were cooled down to room temperature on ice and then rinsed briefly with dH2O and PBS. Endogenous peroxidase was blocked by 0.3% H2O2 in tris-buffer saline (TBS) for 10 minutes and then rinsed with PBS for 5 minutes, three times in total. Non-specific binding sites were blocked by 4% goat serum (Abcam, ab7481, ON, Canada) in PBS and left in room temperature for 1 hour. Afterward, the slides were incubated with pre-diluted Anti-CD31 antibody (specifically react with human CD31) (Abcam, ab76533, ON, Canada) and pre-diluted Anti-Vimentin antibody (specifically react with human vimentin) (Abcam, ab16700, ON, Canada) overnight at 4 °C. After incubation, the slides were rinsed in PSB with agitating three times for 15 minutes. The slides were then incubated with streptavidin HRP (horseradish peroxidase) at 37 °C for 30 minutes (Abcam, ab7403, ON, Canada) followed by PBS rinsing, five times for 5 minutes.   40 Finally, the slides were incubated with 3,3’- diaminobenzidine (DAB) (Abcam, ab64238, ON, Canada) for 5 minutes at room temperature. After rinsing with running water, the slides were counterstained in Mayer’s hematoxylin solution (Sigma Aldrich, MHS1, Darmstadt, Germany), dehydrated and mounted prior to cover-slipped visualization. The slides were then scanned with Leica SCN400 slide scanner (Meyer Instruments).  b) Penetration rate of fibroblasts Fibroblasts reseeded to the bladder tissues were expected to penetrate and eventually randomly distributed in the lamina propria due to its cell-nature (40). In this study, we calculated fibroblasts’ penetration rate into the decellularized ECM (distance vs. time). The penetration distance was measured from the H&E slide (tissue cross section) using digital ruler; multiple measurements were applied from the surface of stroma to the location where the cells were visualized. Mean distance was then calculated based on these measurements. In this case, with the tissue’s thickness being measured, the penetration rate of fibroblasts was calculated based on a 21-day timespan. Student’s t-test was performed to ensure the statistical significance of this calculation.         41 Chapter 3: Results  3.1  Optimized kidney decellularization protocol  Kidneys, obtained from rodents, were decellularized by perfusion through the abdominal aorta using the protocol described in chapter 2. This process led to an almost translucent appearance with gross preservation of ECM microarchitecture upon visual inspection (Figure 5-D). In this study, the completeness of decellularization is confirmed when there are no visible nuclear materials in histological assessment with H&E, and less than 5% residual DNA content or less than 50 nanogram of double-stranded DNA per milligram dry weight (37). These criteria are widely accepted for organ decellularization based on previous endpoint analysis by destructive methods (41). Other attempts using different protocols described in the appendix section did not completely decellularize the organs and caused significant damage to the ECM.       42  Figure 5. Macroscopic assessment of native (A) and decellularized (B, C, D) kidney. (A) The native kidney under decellularization at timepoint 0. (B) The same kidney under decellularization after 24 hours. (C) The same kidney completed decellularization after 36 hours. (D) A piece of decellularized kidney from (C).    3.1.1  Residual DNA quantification of decellularized kidney proved complete cell removal  To evaluate cellular contents within the ECMs, the residual DNA content in both native and decellularized kidney was measured spectrophotometrically using NanoDrop technology. The level of residual DNA in native kidney was compared to those of decellularized ones (110.10 ng/l  4.87 ng/l; n=5 vs. 4.92 ng/l  0.30 ng/l SD; n=5. p=0.0049) (Figure 6). The measurement of residual DNA in native and decellularized kidneys was repeated for five times, respectively (native kidney: 113.8 ng/l, 113.2 ng/l, 113.1 ng/l, 107.9 ng/l and 102.5 ng/l; decellularized kidney: 4.91 ng/l, 4.59 ng/l, 4.68 ng/l, 5.12 ng/l and 5.30 ng/l). Statistical analysis was performed using the unpaired (Two Sample) t test. H0: the DNA concentration of decellularized kidneys equals to 5% of native kidneys’ DNA concentration. H: the   43 DNA concentration of decellularized kidneys is less than 5% of native kidneys’ DNA concentration. p<0.05 rejected the null hypothesis and approved the alternative hypothesis that DNA concentration of decellularized kidneys is less than 5% of native kidneys’ DNA concentration with decellularization protocols developed in this study.     Figure 6. DNA Quantification of native and decellularized kidneys. Native kidneys have mean DNA concentration of 110.10 ng/µL  4.87 ng/µL; n=5 and decellularized kidneys have mean DNA concentration of 4.92 ng/µL  0.30 ng/µL; n=5. Statistical analysis was performed using the unpaired (Two Sample) t test, p=0.0049. Data obtained from the R platform.   44 3.1.2 Evaluation of decellularized kidney reveals complete cell removal and preservation of ECM microarchitectures  Microscopic assessment by H&E staining (in various renal regions) of decellularized kidney mirrored the macroscopic evaluation that gross preservation of ECM microarchitecture was achieved (Figure 7). The almost translucent appearance (Figure 7 - B) was suggestive of complete cell removal, which was confirmed by H&E. Panel C, E and G showed medulla, artery and glomerulus of a native kidney that is occupied with cells while panel D, F and H showed there are no cells observed in that of decellularized kidney.   This protocol confirms the preservation of the ECM microarchitecture including delicate structural/functional components such as the glomerulus. They were well preserved and similar in appearance to the native kidney as observed with the SEM. The glomerular tubules in the decellularized kidney coiled less tightly than in that of the native kidney (Figure 8, panel C & D), which, could possibly be caused by the complete loss of cellular components during decellularization. A honeycomb-like appearance of renal medulla was well preserved after decellularization and cells were completely removed compared to that of native kidney (Figure 8, panel A & B). Individual tubules in a glomerulus were visualized at 20X magnifications for native and decellularized kidney (Figure 8, panel E & F). It proved complete cell removal from the glomerulus after decellularization and the preservation of glomerular basement membrane’s microarchitecture.   45  Figure 7. Macroscopic visualization and histological examination of native (A, C, E, G) and decellularized (B, D, F, H) kidney. (A) The entire native kidney with ureter. (B) The entire decellularized kidney with ureter. (C, D, E, F, G, H) Hematoxylin & Eosin stains (20X): renal medulla of native (C) and decellularized (D) kidney. Renal artery of native (E) and decellularized (F) kidney. Glomerulus of native (G) and decellularized (H) kidney.  Scale bars in panel C, D, E, F, G and H represent 100 m.               46   Figure 8. Structural analysis of native (A, C, E) and decellularized (B, D, F) kidneys. SEM images: (A) Renal medulla of native kidney (1200X). Cells are observed inside of the renal medulla. (B) Renal medulla of decellularized kidney (350 X). No cells are observed. (C) Glomerulus of native kidney (1000X). Cells are observed in the tubules next to the glomerulus. (D) Glomerulus of decellularized kidney (1000X). No cells are observed and the structure of the glomerulus is preserved. (E) Glomerular basement membrane & epithelial podocytes of native kidney (10,000X). Cells are clearly showed on the tubule. (F) Glomerular basement membrane of decellularized kidney (5000X), epithelial podocytes were unseen.  Scale bars in panel A, B, C and D represent 50 m; scale bars in panel E and F represent 5 m.              47 3.1.3 Structural protein and ECM-bound growth factors are preserved after decellularization of kidney.  Once kidneys were decellularized and the remaining DNA concentration was less than 5% compare to that of native tissue, I wanted to evaluate the remaining full-protein profile of the decellularize kidney ECM. Both native and decellularized kidneys were evaluated using LC-MS/MS (Table 1 and 2). The most abundant components of the rodent kidney ECM are collagen IV and laminin which are important to the formation of glomerular basement membrane that supports tubule formation and renal development (42) (43) (44). Structural proteins such as collagen IV and laminin were preserved with the optimized decellularization protocol. Synthesized by glomerular cells, fibronectin is a major structural and functional component of the ECM that plays important roles in cell adhesion, growth, migration and differentiation (45). Proteins that interact or bind to fibronectin such as decorin, biglycan and gelsolin that are crucial in matrix assembly (46) (47) (48) was also preserved after decellularization. Another type of structural proteins in the ECM preserved are fubulins. They associate with basement membrane and elastic fibers and interact with many extracellular matrix constituents including fibronectin and laminins (49). Other functional proteins such as galectin, which has broad functions including mediation of cell-cell interaction, cell-matrix adhesion and transmembrane signaling in the ECM (50) were seen in both native and decellularized kidneys. Therefore, the existence of galectin may ensure the recellularization potency in future experiments. Overall, proteins that play key roles in maintaining ECM functionality were preserved after decellularization.   48 Growth factors such as Transforming growth factor is responsible in epithelial cell differentiation (51). The existence of this growth factor was indirectly proved by finding the protein it secreted (Transforming growth factor beta-1 induced transcript 1 protein) in both native and decellularized kidneys under the optimized decellularization protocol developed in this study.                        49 Table 1. Proteomic analysis of decellularized (Rattus norvegicus) kidney Accession Decellularized Kidney Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] Q4FZU4 ADAMTS-like protein 4 1.941747573 2 2 112.7 O35889-1 Afadin 1.202843084 2 2 207.5 P47853 Biglycan 38.21138211 10 9 41.7 P47854 Cathepsin B 7.374631268 2 2 37.4 P47855 Ceruloplasmin 6.987724268 7 7 120.8 P47856 Clusterin 25.05592841 10 10 51.3 P47857 Collagen alpha-1(I) chain 64.34962147 93 90 137.9 P47858 Collagen alpha-1(II) chain 20.71881607 20 18 134.5 P47859 Collagen alpha-1(III) chain 50.10252905 82 80 138.9 P47860 Collagen alpha-1(V) chain 14.07608696 21 17 183.9 P47861 Collagen alpha-1(XI) chain 10.75388027 16 13 180.9 P47862 Collagen alpha-1(XII) chain 18.92744479 5 5 32 P47863 Collagen alpha-2(I) chain 66.39941691 81 81 129.5 P47864 Decorin 16.10169492 6 5 39.8 P47865 Dystrobrevin beta 3.669724771 3 3 73.8 P47866 Dystrophin 1.087843351 5 3 425.6 P47867 Elastin 6.551724138 2 2 73.3 P47868 Extracellular matrix protein 1 10.3202847 6 6 63.2 P47869 Fibrinogen alpha chain 21.35549872 16 16 86.6 P47870 Fibrinogen beta chain 43.42379958 18 18 54.2 P47871 Fibrinogen gamma chain 36.85393258 19 19 50.6 P47872 Fibronectin 29.10779168 55 55 272.3 P47873 Fibulin-5 19.41964286 7 7 50.1 P47874 Fibulin-1 6.268656716 2 2 36.6 P47875 Galectin-1 34.07407407 4 4 14.8 P47876 Gelsolin 15.12820513 10 10 86 P47877 Isoform 2 of Alpha-1B-glycoprotein 3.382663848 2 2 112.7 P47878 Isoform 2 of Collagen alpha-1(XI) chain 10.68281938 16 13 207.5 P47879 Isoform 2 of Elastin 6.909090909 2 2 41.7 P47880 Isoform 2 of Fibrinogen alpha chain 30.36363636 16 16 37.4 P47881 Isoform 2 of Fibrinogen beta chain 39.76143141 17 17 120.8 P47882 Isoform 2 of Fibronectin 29.66066192 54 54 51.3 P47883 Isoform 2 of Gelsolin 16.14227086 10 10 137.9 P47884 Isoform 2 of Nidogen-2 53.97727273 11 11 134.5 P47885 Isoform 3 of Collagen alpha-1(XI) chain 10.99150142 16 13 138.9 P47886 Isoform 3 of Elastin 6.559263521 2 2 183.9   50 Accession Decellularized Kidney Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] P47888 Isoform 4 of Collagen alpha-1(XI) chain 11.28563118 16 13 32 P47889 Isoform 4 of Elastin 6.666666667 2 2 129.5 P47890 Isoform 4 of Fibronectin 30.57675997 55 55 39.8 P47891 Isoform 5 of Collagen alpha-1(XI) chain 11.20739457 16 13 73.8 P47892 Isoform 5 of Elastin 6.917475728 2 2 425.6 P47893 Isoform 6 of Collagen alpha-1(XI) chain 11.54761905 16 13 73.3 P47894 Isoform 6 of Elastin 6.674473068 2 2 63.2 P47895 Isoform 7 of Elastin 7.037037037 2 2 86.6 P47896 Isoform 8 of Elastin 7.045735476 2 2 54.2 P47897 Isoform Gamma-A of Fibrinogen gamma chain 37.52860412 19 19 50.6 P47898 Isoform Short of Collagen alpha-1(XII) chain 22.90076336 93 5 272.3 P47899 Laminin subunit beta-2 59.68906163 8 93 50.1 P47900 Lumican 26.33136095 7 8 36.6 P47901 Metalloproteinase inhibitor 3 31.27962085 3 7 14.8 P47902 Nidogen-1 8.333333333 35 3 86 P47903 Nidogen-2 33.5243553 4 35 52.5 P47904 Plasminogen 7.266009852 2 4 182.1 P47905 Syntenin-1 7 2 2 112.7 P47906 Uromodulin 2.639751553 5 16 207.5 P47907 EGF-containing fibulin-like extracellular matrix protein 1 16.02434077 5 17 41.7 P47908 Hepatocyte growth factor-regulated tyrosine kinase substrate 5.154639175 5 54 37.4 P47909 Isoform 2 of Hepatocyte growth factor-regulated tyrosine kinase substrate 5.188067445 9 10 120.8 P47910 Latent-transforming growth factor beta-binding protein 1 6.83411215 6 11 51.3 P47911 Transforming growth factor beta-1-induced transcript 1 protein 15.18438178 6 13 137.9        51 Table 2. Proteomic analysis of native (Rattus norvegicus) kidney Accession Native Kidney Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] A2RUV9 Adipocyte enhancer-binding protein 1 8.333333333 6 6 128 O35889-1 Afadin 3.280481137 5 5 207.5 P36953 Afamin 3.125 2 2 69.3 P25304-1 Agrin 12.86370597 18 18 208.5 P14046 Alpha-1-inhibitor 3 22.74881517 29 9 163.7 Q63041 Alpha-1-macroglobulin 38 46 46 167 P24090 Alpha-2-HS-glycoprotein 43.18181818 10 10 38 P01015 Angiotensinogen 22.43186583 8 8 51.9 P04639 Apolipoprotein A-I 16.21621622 5 5 30 P02651 Apolipoprotein A-IV 38.87468031 12 12 44.4 Q7TMA5 apolipoprotein B-100 12.14421252 43 43 535.7 Q9ESS6 Basal cell adhesion molecule 21.79487179 10 10 67.5 P26644 Beta-2-glycoprotein 1 29.96632997 9 9 33.2 P47853 biglycan 24.66124661 6 5 41.7 Q63514 C4b-binding protein alpha chain 19.35483871 10 9 62.2 P00787 Cathepsin B 36.87315634 11 11 37.4 P24268 Cathepsin D 21.62162162 6 6 44.7 P07154 Cathepsin L1 14.97005988 4 4 37.6 P13635 Ceruloplasmin 5.571293673 4 4 120.8 Q00657 Chondroitin sulfate proteoglycan 4 1.891659501 3 3 251.8 P05371 Clusterin 16.55480984 6 6 51.3 P02454 Collagen alpha-1(I) chain 48.7267722 54 53 137.9 P05539 Collagen alpha-1(II) chain 3.03030303 3 1 134.5 P13941 Collagen alpha-1(III) chain 33.15105947 42 41 138.9 Q9JI03 Collagen alpha-1(V) chain 3.532608696 4 3 183.9 P20909-1 Collagen alpha-1(XI) chain 1.884700665 3 2 180.9 P70560-1 Collagen alpha-1(XII) chain 7.255520505 2 2 32 P02466 Collagen alpha-2(I) chain 45.40816327 44 44 129.5 P31720 Complement C1q subcomponent subunit A 10.20408163 2 2 25.9 P01026 Complement C3 34.69633193 48 48 186.3 P08649 Complement C4 18.88313184 25 25 192 O35796 Complement component 1 Q subcomponent-binding protein, mitochondrial 26.52329749 4 4 31 Q811M5 Complement component c6 5.139186296 3 3 105 P55314 Complement component C8 beta chain 3.904923599 2 2 66.6 Q62930 complement component C9 28.33935018 15 15 62.2 Q63135-1 Complement component receptor 1-like protein 10.19677996 4 4 61.6   52 Accession Native Kidney Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] Q9WUW3 Complement factor I 7.450331126 3 3 67.3 P31211 corticosteroid-binding globulin 7.828282828 3 3 44.6 Q01129 decorin 15.53672316 6 5 39.8 Q7M0E3 Destrin 60.60606061 13 12 18.5 Q9QX79 Fetuin-B 38.88888889 9 9 41.5 P06399 Fibrinogen alpha chain 34.65473146 23 23 86.6 P14480 Fibrinogen beta chain 65.55323591 32 32 54.2 P02680-1 Fibrinogen gamma chain 58.20224719 26 26 50.6 P04937-1 fibronectin 27.89664917 44 44 272.3 Q9WVH8 Fibulin-5 8.705357143 4 4 50.1 Q9WUH4 Four and a half LIM domains protein 1 26.42857143 6 6 31.9 O35115 Four and a half LIM domains protein 2 60.93189964 14 14 32.1 P11762 Galectin-1 46.66666667 6 6 14.8 P08699 Galectin-3 14.1221374 4 4 27.2 Q68FP1 Gelsolin 40.12820513 23 23 86 P23785 Granulins 5.102040816 2 2 63.3 P06866-1 Haptoglobin 15.56195965 5 5 38.5 P20059 Hemopexin 15.65217391 5 5 51.3 Q64268 Heparin cofactor 2 6.054279749 2 2 54.5 P63159 High mobility group protein B1 30.69767442 7 7 24.9 Q5XIE8 Integral membrane protein 2B 21.42857143 4 4 30.3 P20909-2 Isoform 2 of Collagen alpha-1(XI) chain 1.872246696 3 2 182.1 P06399-2 Isoform 2 of Fibrinogen alpha chain 45.45454545 22 22 60.5 P14480-2 Isoform 2 of Fibrinogen beta chain 59.44333996 30 30 56.6 P04937-2 Isoform 2 of Fibronectin 28.94847088 44 44 262.6 Q68FP1-2 Isoform 2 of Gelsolin 40.35567715 22 22 80.9 P06866-2 Isoform 2 of Haptoglobin 14.17322835 5 5 42.4 Q03626-2 Isoform 2 of Murinoglobulin-1 22.88993923 29 9 164.5 B5DFC9-2 Isoform 2 of Nidogen-2 28.40909091 5 5 20.1 P12346-2 Isoform 2 of Serotransferrin 16.33064516 7 7 54.5 P20909-3 Isoform 3 of Collagen alpha-1(XI) chain 1.926345609 3 2 176.4 P04937-3 Isoform 3 of Fibronectin 28.18107667 44 44 269.6 P20909-4 Isoform 4 of Collagen alpha-1(XI) chain 1.977894124 3 2 171.3 P04937-4 Isoform 4 of Fibronectin 29.30449534 44 44 259.2 P20909-5 Isoform 5 of Collagen alpha-1(XI) chain 1.964182553 3 2 172.5 P20909-6 Isoform 6 of Collagen alpha-1(XI) chain 2.023809524 3 2 166.7 P02680-2 Isoform Gamma-A of Fibrinogen gamma chain 59.26773455 26 26 49.6 P08934-2 Isoform LMW of Kininogen-1 14.54965358 6 3 47.9   53 Accession Native Kidney Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] P70560-2 Isoform Short of Collagen alpha-1(XII) chain 8.778625954 2 2 25.4 Q9ERB4-2 Isoform V3 of Versican core protein 6.41221374 4 4 74.4 Q9ERB4-3 Isoform Vint of Versican core protein 0.848082596 2 2 296.7 P08934-1 kininogen-1 9.85915493 6 3 70.9 P15800 laminin subunit beta-2 15.32481954 23 23 196.3 P51886 Lumican 32.24852071 10 10 38.3 P30904 Macrophage Migration inhibitory factor 38.26086957 4 4 12.5 P02761 Major urinary protein 62.98342541 12 12 20.7 P48032 Metalloproteinase inhibitor 3 19.43127962 4 4 24.2 Q03626-1 Murinoglobulin-1 22.79757902 29 9 165.2 Q6IE52 Murinoglobulin-2 16.98895028 20 0 161.5 B5DFC9 Nidogen-2 13.18051576 13 13 152.9 Q66H86 Olfactomedin-like protein 1 4.975124378 2 2 45.6 B0BNI5 Olfactomedin-like protein 3 5.911330049 2 2 45.9 P14272 Plasma kallikrein 7.993730408 5 5 71.2 Q6P734 Plasma protease C1 inhibitor 5.158730159 2 2 55.6 Q01177 Plasminogen 49.87684729 30 30 90.5 Q9EQP5 prolargin 21.48541114 6 6 43.2 Q64240 Protein AMBP 28.93982808 9 9 38.8 P18292 Prothrombin 32.90113452 15 15 70.4 P08424 Renin 6.965174129 2 2 44.2 P25236 Selenoprotein P 5.194805195 2 2 43.1 P12346-1 Serotransferrin 16.04584527 10 10 76.3 P02770 Serum albumin 27.63157895 16 16 68.7 P35446 Spondin-1 13.25898389 7 7 90.7 P01048 T-kininogen 1 19.06976744 6 3 47.7 P08932 T-kininogen 2 19.53488372 7 1 47.7 P31232 transgelin 48.25870647 10 10 22.6 Q5XFX0 Transgelin-2 58.79396985 13 13 22.4 P02767 Transthyretin 21.76870748 3 3 15.7 P27590 uromodulin 18.01242236 10 10 71 Q9ERB4 Versican core protein 1.533966399 4 4 299.8 P04276 vitamin D-binding protein 16.80672269 8 8 53.5 P62994-1 Growth factor receptor-bound protein 2 19.35483871 4 4 25.2 Q9JJ50 Hepatocyte growth factor-regulated tyrosine kinase substrate 11.21134021 6 6 86.2 Q8VHK7 hepatoma-derived growth factor 19.83122363 5 5 26.5  P62994-2 Isoform 2 of Growth factor receptor-bound protein 2 20.68965517 4 4 23.5   54 Accession Native Kidney Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] Q9JJ50-2 Isoform 2 of Hepatocyte growth factor-regulated tyrosine kinase substrate 11.28404669 6 6 85.7 P62994-3 Isoform 3 of Growth factor receptor-bound protein 2 37.28813559 2 2 6.9 Q6P686 osteoclast-stimulating factor 1 25.70093458 4 4 23.7 Q99PD6 Transforming growth factor beta-1-induced transcript 1 protein 10.62906725 4 4 50.1                     55 3.2  Optimized bladder decellularization protocol.  Pig bladders were harvested and opened through a mid-line incision to form a flat butterfly-like shape. Once the epithelium is exposed, the bladder is sectioned into multiple 1 cm × 1 cm × 0.5 cm squares (Figure 10 - A) and decellularized by immersion using the protocol described in section 2.2.4 (Figure 10 - B). After decellularization, the bladders started to show clear white color with contracted appearances. It was proved in this study that immersion decellularization can be completed in fewer cycles with increased centrifugal forces (larger petri dishes used). Other attempts using different protocols described in the appendix section did not achieve complete decellularization since the tissues failed to show homogenous clear white appearance.   3.2.1  Residual DNA quantification of decellularized bladder proved complete cell removal  To evaluate cell removal using our protocol, the residual DNA content in both native and decellularized bladder ECM was measured spectrophotometrically using NanoDrop technology. The level of residual DNA in native bladders was compared to those of decellularized bladders (55.9 ng/l  3.41 ng/l; n=5 vs. 2.61 ng/l  0.42 ng/l; n=5. p=0.013) (Figure 9). The measurements of residual DNA in native and decellularized bladder were repeated for five times, respectively (native bladders: 60.4 ng/l, 54.3 ng/l, 51.3 ng/l, 57.4 ng/l and 55.8 ng/l; decellularized bladders: 2.79   56 ng/l,1.96 ng/l, 2.41 ng/l, 3.01 ng/l and 2.87 ng/l). Statistical analysis was performed using the unpaired (Two Sample) t test. H0: the DNA concentration of decellularized bladders equals to 5% of native bladders’ DNA concentration. H: the DNA concentration of decellularized bladders is less than 5% of native bladders’ DNA concentration. p<0.05 rejected the null hypothesis and approved the alternative hypothesis that DNA concentration of decellularized bladders is less than 5% of native bladders’ DNA concentration.       57   Figure 9. DNA Quantification of native and decellularized bladders.  Native bladders have mean DNA concentration of 55.9 ng/L  3.41 ng/L n=5 and decellularized bladders have mean DNA concentration of 2.61 ng/L  0.42 ng/L; n=5. Statistical analysis was performed using the unpaired (Two Sample) t test, p=0.013. Data obtained from the R platform.         58 3.2.2 Evaluation of decellularized bladder reveals complete cell removal and preservation of ECM microarchitectures  H&E stains of decellularized bladders (4X and 20X magnification) illustrated that the urothelial and stromal cells were completely removed after decellularization as compared to those of native tissues (Figure 10, panel E & F) and the clear border between stroma and muscle layer was observed (Figure 10, panel C & D). The ECM microarchitecture was further evaluated using SEM. Panel B and D from Figure 11, show a surface image of the basement membrane of the decellularized bladder showing no cells left from the urothelium. Panel A and C clearly show the hexagonal cells characteristic of the umbrella cells from the urothelium in a native bladder.   Figure 10. Macroscopic visualization and histological examination of native (A, C, E,) and decellularized (B, D, F) bladders. (A) Native bladder in a 1 cm × 1 cm × 0.5 cm square. (B) Decellularized pig bladder in a 1 cm × 1 cm × 0.5 cm square. (C, D, E, F) H&E stains: native (C) and decellularized (D) bladder with 4X magnification. (E) Urothelium, lamina propria and submucosa of native bladder square with 20X magnification. (F) Lamina propria and submucosa of decellularized bladder square with 20X magnification.  Scale bars in panel C, D, E and F represent 100 m.    59   Figure 11. Structural analysis of native (A, C) and decellularized (B, D) bladders. SEM images: urothelium of native bladder (A) (250X) and (C) (500X). Hexagonal cell characteristic of the umbrella cells from the urothelium was clearly showed. Basement membrane (lamina propria) of decellularized bladder (B) (250X) and (D) (500X). No hexagonal cell characteristic was seen indicates the complete removal of umbrella cells. Microarchitecture of the bladder ECM was preserved (no significant structural changes were observed on the basement membrane) comparing between native (A) (C) and decellularized bladder (B) (D). All scale bars represent 100 m.          60 3.2.3 Structural protein and ECM-bound growth factors are preserved after decellularization of bladder  Once bladders were decellularized and remaining DNA was within accepted ranges, I wanted to evaluate the remaining full-protein profile of the decellularization bladder ECM. Bother native and decellularized bladders were evaluated using LC-MS/MS (Table 3 and 4).  Specific structural proteins that maintain the integrity of the impermeable bladder surface (formation of basal membrane and lamina propria) such as elastin, collagen, laminin and fibronectin (52) (53) (54) were preserved in decellularizated bladders compare to native bladders. Biglycan and agrin, proteins that are responsible for cell-matrix interactions (55) (56) were consistently found in both native and decellularized bladders. Cohesive proteins that link actin filaments and other support proteins: destrin and dystrophin (57) (58) were preserved during decellularization. Lumican, the protein that is responsible of epithelial cell migration and potential tissue repair was seen in both native and decellularized bladder matrix (59). Similar to previous proteomic evaluation of kidneys, ECM structural proteins – fubulins associate with basement membrane and elastic fibers and interact with many extracellular matrix constituents including fibronectin and laminins (49). Functional proteins - galectins, which has broad functions including mediation of cell-cell interaction, cell-matrix adhesion and transmembrane signaling in the ECM (50) were seen in both native and   61 decellularized bladders. Again, the existence of galectin may ensure the recellularization potency in future experiments.  Various growth factors such as latent-transforming growth factors, which are critical to tissue repairs (60) (61) and transforming growth factors that are responsible for epithelial cell differentiation (51) were well preserved using the optimized decellularization protocol developed in this study.                        62 Table 3. Proteomic analysis of decellularized (Rattus norvegicus) bladder Accession Decellularized Bladder Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] Q99PD6 Agrin 1.020929045 3 3 208.5 O35568 alpha-internexin 3.366336634 2 0 56.1 Q6P686 Basal cell adhesion molecule 7.852564103 2 2 67.5 Q00918 biglycan 24.11924119 7 6 41.7 P31232 Calponin-1 46.12794613 15 13 33.3 P02454 Clusterin 30.20134228 15 15 51.3 Q9WUH4 Collagen alpha-1(I) chain 70.88781831 130 124 137.9 P02466 Collagen alpha-1(II) chain 11.69837914 12 6 134.5 P85972 Collagen alpha-1(III) chain 56.32262474 106 104 138.9 Q63532 Collagen alpha-1(V) chain 19.07608696 26 21 183.9 P13941 Collagen alpha-1(XI) chain 16.40798226 14 9 180.9 Q08290 Collagen alpha-2(I) chain 66.6909621 111 110 129.5 Q7M0E3 Cornifin-A 58.55263158 3 3 16.7 P09656 decorin 33.05084746 12 11 39.8 Q01129 Destrin 41.81818182 11 10 18.5 Q99J82 Dystrophin 11.36796301 29 29 425.6 P05371 EGF-containing fibulin-like extracellular matrix protein 1 22.10953347 6 6 54.6 P15800 Elastin 21.14942529 5 5 73.3 Q68FP1-2 Extracellular matrix protein 1 2.846975089 2 2 63.2 Q68FP1 Fibrinogen alpha chain 8.823529412 8 8 86.6 P47853 Fibrinogen beta chain 14.61377871 5 5 54.2 Q99372-8 Fibrinogen gamma chain 11.23595506 5 5 50.6 Q99372-7 fibronectin 14.4529673 25 25 272.3 Q99372-5 Fibulin-5 15.17857143 6 6 50.1 Q99372-2 Four and a half LIM domains protein 1 68.21428571 22 22 31.9 Q01177 Galectin-1 13.33333333 2 2 14.8 Q99372-6 Galectin-3 3.053435115 2 2 27.2 Q99372-4 Gelsolin 24.35897436 15 15 86 Q99372-3 Glycogen phosphorylase, brain form 13.96181384 13 11 96.1 Q99372 Glycogen phosphorylase, muscle form 11.28266033 9 7 97.2 P51886 Hemopexin 9.130434783 3 3 51.3 Q9JI03 Integrin alpha-1 5.338983051 7 7 130.7   63 Accession Decellularized Bladder Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] B5DFC9 integrin alpha-7 4.933920705 5 5 124.1 P20909-6 Integrin beta-1 15.01877347 12 12 88.4 B5DFC9-2 Integrin beta-4 1.881571666 2 2 200.5 P20909-4 Integrin-linked protein kinase 31.4159292 15 15 51.3 P20909-5 Isoform 1 of Neuroplastin 16.72597865 5 5 31.3 P97546-3 Isoform 2 of Agrin 1.025641026 3 3 207.4 P20909-3 Isoform 2 of Collagen alpha-1(XI) chain 16.29955947 14 9 182.1 P97546-1 Isoform 2 of Elastin 22.3030303 5 5 69.1 P20909-1 Isoform 2 of Fibrinogen alpha chain 12.54545455 8 8 60.5 P20909-2 Isoform 2 of Fibrinogen beta chain 12.32604374 4 4 56.6 P21807 Isoform 2 of Fibronectin 14.45328865 24 24 262.6 Q9JI92 Isoform 2 of Gelsolin 25.99179207 15 15 80.9 P04937-4 Isoform 2 of Nidogen-2 17.61363636 4 4 20.1 Q9WVH8 Isoform 3 of Agrin 1.030927835 3 3 206.4 P49134 Isoform 3 of Collagen alpha-1(XI) chain 16.77053824 14 9 176.4 P14480 Isoform 3 of Elastin 21.17376295 5 5 73.2 P04937-3 Isoform 3 of Fibronectin 14.60032626 25 25 269.6 P04937-2 Isoform 3 of Neuroplastin 16.96750903 5 5 30.8 P04937-1 Isoform 4 of Agrin 1.026694045 3 3 207.3 Q642A6 Isoform 4 of Collagen alpha-1(XI) chain 17.21931355 14 9 171.3 P53534 Isoform 4 of Elastin 21.52046784 5 5 71.8 P11762 Isoform 4 of Fibronectin 15.18235793 25 25 259.2 P06399-2 Isoform 5 of Agrin 1.025115325 3 3 207.6 P14480-2 Isoform 5 of Collagen alpha-1(XI) chain 17.09994223 14 9 172.5 P97546-2 Isoform 5 of Elastin 22.33009709 5 5 69 P05539 Isoform 6 of Agrin 1.023017903 3 3 208 P02680-2 Isoform 6 of Collagen alpha-1(XI) chain 17.61904762 14 9 166.7 P11530 Isoform 6 of Elastin 21.54566745 5 5 71.7 P09812 Isoform 7 of Elastin 22.71604938 5 5 67.6 P02680-1 Isoform 8 of Elastin 22.74412855 5 5 67.6 P20059 Isoform Alpha-7X1A of Integrin alpha-7 5.017921147 5 5 121.6   64 Accession Decellularized Bladder Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] P06399 Isoform Alpha-7X1C of Integrin alpha-7 5.204460967 5 5 117.4  Q9ESS6 Isoform Gamma-A of Fibrinogen gamma chain 11.4416476 5 5 49.6 Q9EQP5 Lamin-B1 6.473594549 4 3 66.6 P70615 laminin subunit beta-2 29.65019434 39 39 196.3 P08460 Latent-transforming growth factor beta-binding protein 1 4.497663551 6 6 186.5 P35446 Lumican 20.71005917 5 5 38.3 P18614 Neuroplastin 11.95928753 5 5 43.9 Q63258-3 Nidogen-1 5.864197531 2 2 35.6 Q63258-2 Nidogen-2 18.26647564 15 15 152.9 Q63258 osteoclast-stimulating factor 1 11.68224299 2 2 23.7 Q63321 Peripherin 15.38461538 9 5 53.5 P23565 Plasminogen 21.79802956 13 13 90.5 P08699 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 3.983516484 2 2 83.6 Q62894 prolargin 7.692307692 2 2 43.2 Q64632 Serine protease inhibitor Kazal-type 1-like 34.17721519 4 4 8.6 P25304-3 Spondin-1 5.576208178 2 2 90.7 P25304-4 Syntenin-1 15.33333333 2 2 32.4 P25304-2 Transforming growth factor beta-1-induced transcript 1 protein 24.94577007 8 8 50.1 P25304-5 transgelin 73.6318408 19 19 22.6 P25304-6 Vinculin 61.35084428 65 65 116.5 P25304-1 von Willebrand factor A domain-containing protein 1 13.97590361 3 3 44.8                             65 Table 4. Proteomic analysis of native (Rattus norvegicus) bladder Accession Decellularized native Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] O35568 Agrin 2.603369066 4 4 208.5 Q99PD6 Angiotensinogen 9.853249476 4 4 51.9 Q00918 Basal cell adhesion molecule 7.051282051 4 4 67.5 P02454 biglycan 26.82926829 7 6 41.7 P02466 Bone marrow proteoglycan 8.370044053 2 2 25.1 P31232 Calponin-1 42.08754209 12 10 33.3 P13941 Cathepsin D 16.70761671 5 5 44.7 Q7M0E3 Chondroitin sulfate proteoglycan 4 1.418744626 2 2 251.8 P85972 Collagen alpha-1(I) chain 73.98485891 115 112 137.9 P11762 Collagen alpha-1(II) chain 15.08104299 16 11 134.5 Q01129 Collagen alpha-1(III) chain 64.38824334 107 105 138.9 Q08290 Collagen alpha-1(V) chain 25.43478261 35 29 183.9 Q9WUH4 Collagen alpha-1(XI) chain 9.201773836 10 5 180.9 P15800 Collagen alpha-1(XII) chain 6.624605678 2 2 32 B5DFC9-2 Collagen alpha-2(I) chain 70.91836735 90 88 129.5 P14480 decorin 43.22033898 16 15 39.8 Q68FP1-2 Destrin 63.03030303 11 10 18.5 P51886 Dystrophin 11.09600218 34 34 425.6 Q68FP1 EGF-containing fibulin-like extracellular matrix protein 1 29.00608519 8 8 54.6 P14480-2 Elastin 20.8045977 5 5 73.3 Q9QX79 Extracellular matrix protein 1 10.3202847 5 5 63.2 P06399 Fetuin-B 32.01058201 6 6 41.5 P02680-2 Fibrinogen alpha chain 27.87723785 20 20 86.6 P02680-1 Fibrinogen beta chain 35.6993737 14 14 54.2 P47853  27.19101124 11 11 50.6 P04937-4 fibronectin 25.3532499 49 49 272.3 P04937-2 Fibulin-5 20.3125 9 9 50.1 P04937-3 Four and a half LIM domains protein 1 38.57142857 11 11 31.9 Q9JI03 Galectin-1 51.11111111 6 6 14.8 P04937-1 Galectin-3 7.633587786 2 2 27.2 Q99J82 Gelsolin 33.33333333 21 21 86 Q9EQP5 Hemopexin 15 6 6 51.3 Q5XFX0 Integrin alpha-1 9.830508475 11 11 130.7 P08932 integrin alpha-7 2.907488987 3 3 124.1 Q99372-5 Integrin beta-1 13.89236546 11 11 88.4 Q99372-2 Integrin-linked protein kinase 25 12 12 51.3   66 Accession Decellularized native Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] Q99372-6 Isoform 2 of Agrin 2.615384615 4 4 207.4 Q99372-4 Isoform 2 of Collagen alpha-1(XI) chain 9.140969163 10 5 182.1 Q99372-3 Isoform 2 of Elastin 21.93939394 5 5 69.1 Q99372 Isoform 2 of Fibrinogen beta chain 32.60437376 13 13 56.6 Q9WVH8 Isoform 2 of Fibronectin 25.76455802 48 48 262.6 Q2LAP6 Isoform 2 of Gelsolin 35.56771546 21 21 80.9 P01048 Isoform 2 of Nidogen-2 38.06818182 6 6 20.1 P24268 Isoform 3 of Agrin 2.628865979 4 4 206.4 B5DFC9 Isoform 3 of Collagen alpha-1(XI) chain 9.40509915 10 5 176.4 P05539 Isoform 3 of Elastin 20.82853855 5 5 73.2 P20059 Isoform 3 of Fibronectin 25.61174551 49 49 269.6 P49134 Isoform 4 of Agrin 2.618069815 4 4 207.3 Q01177 Isoform 4 of Collagen alpha-1(XI) chain 9.656777196 10 5 171.3 P18292 Isoform 4 of Elastin 21.16959064 5 5 71.8 P11530 Isoform 4 of Fibronectin 26.63273961 49 49 259.2 Q62894 Isoform 5 of Agrin 2.61404408 4 4 207.6 P20909-6 Isoform 5 of Collagen alpha-1(XI) chain 9.589832467 10 5 172.5 P01015 Isoform 5 of Elastin 21.96601942 5 5 69 P18614 Isoform 6 of Agrin 2.608695652 4 4 208 P20909-4 Isoform 6 of Collagen alpha-1(XI) chain 9.880952381 10 5 166.7 P20909-5 Isoform 6 of Elastin 21.19437939 5 5 71.7 P20909-3 Isoform Gamma-A of Fibrinogen gamma chain 27.68878719 11 11 49.6 P20909-1 Isoform Short of Collagen alpha-1(XII) chain 8.015267176 2 2 25.4 P20909-2 kininogen-1 5.320813772 3 1 70.9 Q63189 laminin subunit beta-2 [ 38.14547474 55 55 196.3 P70560-2 Latent-transforming growth factor beta-binding protein 1 1.752336449 3 3 186.5 P08699 Lumican 33.72781065 10 10 38.3 P37805 Nidogen-1 5.864197531 2 2 35.6 Q9ESS6 Nidogen-2 16.26074499 17 17 152.9 Q566D0 Peripherin 6.623931624 4 1 53.5 P70560-1 Plasminogen 13.1773399 8 8 90.5 P21807 Procollagen C-endopeptidase enhancer 1 5.555555556 2 2 50.2 Q75WE7 prolargin 24.40318302 10 10 43.2 P08460 Prothrombin 12.96596434 7 7 70.4 O08628 Testin 20.0477327 9 9 47.6 P08934-1 T-kininogen 1 18.60465116 5 5 47.7   67 Accession Decellularized native Protein Coverage Number of Peptides Number of Unique peptides MW [kDa] Q63258 T-kininogen 2 23.25581395 7 5 47.7 P25304-3 Tenascin 27.76572668 10 10 50.1 P25304-4 transgelin 66.16915423 16 16 22.6 P25304-2 Transgelin-2 24.12060302 6 6 22.4 P25304-5 Transgelin-3 7.537688442 2 0 22.5 P25304-6 Uroplakin-1b 6.923076923 2 2 29.8 P25304-1 Vinculin 55.15947467 53 53 116.5 Q00657 von Willebrand factor A domain-containing protein 5A 5.96107056 4 4 91.4                         68 3.3  Optimization of kidney ECM recellularization  In order to test viabilities of different cells within the decellularized kidney ECM and reduce experimental uncertainties, we decided to perform single cell-type recellularization to each decellularized kidney ECM sections. Human endothelial cells (HUVEC) were reseeded onto the sectioned decellularized rodent kidney ECM (Figure 12 - A) as described in section 2.5.2. As seen in Figure 12 - B, the H&E staining of a cross section of panel - A showed that endothelial cells were only positioned around a vascular structure and no cellular distributions were seen in other areas (20X magnification).To ensure these cells were human endothelial cells but not pre-existed animal cells, the section of the same area was stained with Anti-CD31 and the positive stains demonstrated successful recellularization of human endothelial cells around the tubule-like structure (Figure 12 – C, 10X magnification). I considered the key point of success owes to the viability of the cells; the cells being reseeded were of early passage (P3). It is recorded that previous late passage (P12 and P15) HUVEC-reseedings were performed under the same conditions (total cell number, incubation time and cell culture condition) but they were not successful. Therefore, I assume that the success of HUVEC-reseeding to kidney ECMs is highly associated with cell passage, the earlier the passage, the more likely a successfully reseeding can be performed. Researchers should always make account of cell viability for future recellularization.  It is also noteworthy that more areas were stained in panel C than in panel B, possible reason of this observation could be the slide obtained in panel C was sectioned deeper than in that of panel B. Different sections of the tissue could   69 show different cellular distribution since cell penetration after reseeding may not occur at the same time or with the same rate.   Human clear cell carcinoma cells (Caki-1) were reseeded onto the sectioned decellularized rodent kidney ECM (Figure 13 - A) as described in section 2.5.2. As seen in Figure 13 - B, the H&E staining of a cross section of panel - A showed cancer cells were penetrating and forming aggregates in the decellularized ECM (Figure 13 – B, 10X magnification). In panel – B and C (30X magnification), there are more gaps between cancer cells and the adjacent decellularized ECM compared to areas with less cancer cells being reseeded. Also, the cancer cells were randomly aggregating into clumps at different areas of the tissue, which mirrored the characteristic of cancers. Comparing between Figure 12 – B and Figure 13 – B, the appearances of ECM were different when reseeded with different cells. More organized structures were seen in the ECM recellularized with normal human cells (Figure 12 – B) and more irregular structures were seen in the ECM reseeded with cancer cell. This phenomenon was observed in concurrent experimental duplicates (not shown) and indicated that different cells affect the decellularized ECM differently. Human embryonic cells (HEK293) were also reseeded onto the decellularized kidney ECM as described in section 2.5.2 but unfortunately after trying twelve times, no viable cells were observed on the decellularized tissues (evaluation using H&E) (result not shown).     70   Figure 12. Macroscopic visualization and histological examination of recellularized kidney. (A) Longitudinal section of a recellularized rodent kidney (recellularized with HUVEC). (B) Haematoxylin & Eosin stains of the recellularized rodent kidney (20X). (C) Immunohistochemistry: Anti-CD31 staining of the same area as in (B) with a 10X magnification Scale bars in panel B and C represent 100 m.            71   Figure 13. Macroscopic visualization and histological examination of recellularized kidney. (A) Cross section of a recellularized rodent kidney (recellularized with Caki-1 cell lines). (B) Hematoxylin & Eosin stains of the recellularized rodent kidney (10X magnification). (C) magnified area of (B) (30X magnification). Scale bars in Panel B and C represent 100 m.                72 3.4  Optimization of bladder ECM recellularization  a) Single cell-type recellularization In order to test viabilities of different cells within the decellularized bladder ECM and reduce experimental uncertainties, we decided to perform single cell-type recellularization to each decellularized bladder ECM squares. Human urothelial cells (SV-HUC) were reseeded onto the urothelium of the sectioned decellularized pig bladder ECM (Figure 14 – A) as described in section 2.5.3. The H&E staining showed cells were uniformly positioned on the surface of the urothelium showing few layers (Figure 14 – D, 10X magnification). It was observed that the reseeded urothelial cells were aligning at the urothelium in an organized manner and were not penetrating the basement membrane. It showed in both panel D and G (20X magnification) that the recellularization of human urothelial cells did not jeopardize the integrity of the basement membrane. Human UM-UC-3 cancer cells were reseeded onto the urothelium of the sectioned decellularized pig bladder ECM (Figure 14 – B) as described in section 2.5.3. The H&E staining showed cells were aligning at the urothelium and some were penetrating into the ECM (Figure 14 – E, 10X magnification). Also, the cancer cells were changing the decellularized ECM while entering the stroma (Figure 14 – H, 20X magnification). Different from the organized positioning pattern of human urothelial cells, bladder cancer cells were randomly aligning (cells are layering up) at the urothelium, which mirrors the characteristics of invasive bladder cancer. Human fibroblasts were reseeded onto the decellularized pig bladder ECM squares (Figure 14 – C) as described in section 2.5.3. The H&E staining   73 showed cells were scattered throughout almost all the stromal layer (Figure 14 – F, 10X magnification).   Overall, single cell-type recellularization of bladder ECM was successfully generated with human urothelial cells (SV-HUC), fibroblasts and bladder cancer cells (UM-UC-3), respectively. According to several tested protocols described in Appendix E, cell number, media used when reseeding and time of incubation (days) all matter to the outcomes of the recellularization. It clearly shows in Figure 13 that comparing between reseeding SV-HUC and UM-UC-3, human urothelial cells were uniformly aligning on the urothelium without penetrating the basement membrane while bladder cancer cells were unevenly aggregating on the urothelium with formation of thick layers and trying to penetrate more into the basement membrane. As for fibroblasts recellularization, it was observed that cells were randomly penetrating the stroma and it demonstrated the low density of the bladder ECM. It was speculated that fibroblasts were consuming the decellularized bladder ECM to make rooms for their own penetration. These phenomena explain the nature/property of different cells and more importantly, illustrated the possible cell-guiding feature of the decellularized bladder ECM. Interestingly, morphological changes were observed only on the bladder ECM that was reseeded with fibroblasts. The edges of the ECM curled up (Figure 14 - C) thus created a small cave in the center, which mirrors the fact that fibroblasts tend to have cell traction during proliferation (62). Human endothelial cells (HUVEC) were also reseeded on the stroma of the decellularized bladder ECM as described in   74 section 2.5.3 but unfortunately after trying eight times, no viable cells were observed on the tissue when reseeded alone (evaluation using H&E) (result not shown).   Figure 14. Macroscopic visualization (A, B, C) and histological examination (D, E, F) of recellularized bladder. Recellularized pig bladder with surface area of 1cm2. (A) Recellularized with SV-HUC. (B) Recellularized with UM-UC3. (C) Recellularized with primary fibroblast. Hematoxylin & Eosin stains of the recellularized pig bladder (10X): (D) Cross section of (A), (E) Cross section of (B), and (F) Cross section of (C). Hematoxylin & Eosin stains of the recellularized pig bladder (30X): (G) magnified image of (D), (H) magnified image of (E), and (I) magnified image of (F).  Scale bars in panel D, E, F, G, H and I represent 100 m.      75 b) Multi cell-type recellularization Human endothelial, fibroblasts, and urothelial cells were reseeded together onto a decellularized pig bladder ECM square (Figure 15 – B) (endothelial and fibroblast cells onto the stroma while urothelial cells onto the urothelium, as described in section 2.5.3. As seen in Figure 15 – D, multi cell-type recellularization of bladder ECM was successfully generated with human endothelial cells (HUVEC), human urothelial cells (SV-HUC) and primary fibroblasts. According to several tested protocols described in Appendix F, cell ratio (HUVEC: Fibroblast: SV-HUC), media used when reseeding and time of incubation (days) all matter to the outcomes of the recellularization. Similar to single cell-type recellularization, the edges of the bladder ECM also curled up (Figure 15 – B) due to existence of fibroblasts. It clearly shows in Figure 15 - D that cells are reseeded/distributed on both sides of the basement membrane and showed different patterns. This observation illustrated that the previous decellularization protocol did not compromise the integrity of the basement membrane and proved its effectiveness. In order to identify cell type and ensure whether or not cellular distributions were related to cell types. Positive IHC staining of Anti-CD31 antibody (specifically react with human CD31) confirmed that HUVECs but not porcine cells were distributed around multiple vessel-like structures (Figure 15 – E, 20X magnification). Similarly, positive IHC staining of Anti-Vimentin antibody (specifically react with human vimentin) confirmed that human fibroblasts but not porcine cells were uniformly distributed in the stromal layer (Figure 15 – F, 20X magnification). Although SV-HUC was not stained with its corresponding antibody, they were the only reseeded cells on the urothelium side (HUVEC and fibroblast were reseeded on the stromal side). In this   76 case, the unstained cells could only be SV-HUC (Figure 15 – D & F, 20X magnification). Overall, it was clear that HUVECs, fibroblasts and SV-HUCs were growing independently without interfering with each other. This finding indirectly proved the bladder immersion protocol is valid.  Figure 15. Macroscopic visualization and histological examination of decellularized (A, C) and recellularized (B, D, E, F) bladders. (A) Decellularized pig bladder with surface area of 1cm2. (B) Recellularized pig bladder with surface area of 1cm2. (C) Hematoxylin & Eosin stains of the decellularized pig bladder (20X). (D) Hematoxylin & Eosin stains of the recellularized pig bladder (20X, recellularized with HUVECs, primary fibroblasts and SV-HUCs). (E, F) Immunohistochemistry: (E) Anti-CD31 staining of recellularized pig bladder with a 20X magnification and (F) Anti-Vimentin staining of recellularized pig bladder with a 20X magnification. Scale bars in panel C, D, E and F represent 100 m.           77 3.5  Human fibroblast demonstrated ECM penetrating capacity   As described in section 2.6 (b) and 3.4 (a), cell penetration of fibroblast cells was measured using digital ruler from the H&E slides (cross section of the tissue) and the penetration rate was calculated according to each tissue’s thickness. With measurement of four duplicate recellularizations, human fibroblast cells had 1) penetrated 332.7 µm in a tissue of 3500 µm thickness (mean penetration rate is 0.011 µm/min); 2) penetrated 453.6 µm in a tissue of 2400 µm thickness (mean penetration rate is 0.015 µm/min); 3) penetrated 423.4 µm in a tissue of 4600 µm thickness (mean penetration rate is 0.014 µm/min) and 4) penetrated 302.4 µm in a tissue of 3800 µm thickness (mean penetration rate is 0.010 µm/min). Therefore, fibroblasts had a mean penetration rate in decellularized bladder ECM of 0.013 µm/min with a standard deviation of 0.0064 µm/min, n=4.  Since the mean penetration rate of fibroblast in decellularized bladder ECM is 0.013 µm/min  0.0064 µm/min, statistical analysis was performed using the Student’s t-test. H0: the fibroblasts’ penetration rate in decellularized bladder ECM is zero. H: the fibroblasts’ penetration rate in decellularized bladder ECM is bigger than zero. Under null hypothesis, T=4.063 and p=0.013<0.05. Therefore, p<0.05 rejects H0. It approved the alternative hypothesis that the calculated fibroblasts’ penetration rate in decellularized bladder ECM was statistically significant.    78 However, the fibroblasts’ mean penetration rate was calculated (µm/min) based on current recellularization duplicates. With this information, researchers would have an estimation for future fibroblasts’ reseedings under the same conditions. Since corresponding media was replaced on a daily basis and frequent movements could possibly have some interference to cell growth in the ECMs, therefore, extra cautious was required when handling and disturbing during incubation should be minimized.                       79 Chapter 4: Discussion  Currently, little information is available regarding organ/tissue derived ECM in the application of 3D in vitro urogenital tumor models. However, other 3D tumor models were recently described and could potentially share similar principles. Previous studies showed that decellularized ECM produced from decellularized rodent lungs appeared to be a promising 3D metastatic breast tumor model with correct spatial arrangement, biomechanical properties and biocompatibilities (63). Altered expression of biomarkers was observed in this model compared to in a 2D system. Another study showed that a decellularized patient tissue-derived (brain) ECM were used to generate research upon invasion route of glioblastoma (64). Glioblastoma cells exhibited different cell behaviors in the 3D environment compare to that of in 2D condition. These studies illustrated that 3D tumor models are essential since they bridge the gap between 2D models and corresponding “real-life” in vivo scenarios. Therefore, superior physiologic/biologic and clinical relevance can be achieved with the development of 3D tumor models for urogenital cancers. Rodent kidneys and porcine bladder squares were chosen as starting materials of the 3D scaffolds, as they share many anatomical, histological and physiological similarities with human beings (65) (66).    In this study, a protocol with combined physical and chemical approaches for the perfusion decellularization of whole rat kidney was established, representing a preliminary step toward the building of a bioengineered scaffold for kidney tumor   80 models. The kidney was secured in an in-house developed bioreactor as described in section 2.2.1 during decellularization. After testing, the actual time used per decellularization cycle is 20% longer than that described in a similar study (38), which has longer exposure of Triton X-100 than ours, and with no disclosure of the cycle numbers needed. However, although complete cell removal was revealed, our study demonstrated more orderly distributed renal microarchitecture with intact tubules and uniform ECM density in the H&E evaluations. Our study suggests that slower decellularization process (less peristaltic force introduced) may apply less disruption to the fine ECM microstructures. On the other hand, slower peristalsis results in longer exposure of renal ECM to detergents. It has been reported that prolonged exposure of ionic detergents may denaturize structural and functional ECM proteins as well as alter certain biomechanical properties, hence, this prolonged exposure may potentially decrease/inhibit cell-ECM interactions and jeopardize ECM integrity (33) (67). It is key to apply proper detergent exposure and peristaltic force to ensure the future applicability of the decellularized ECM (i.e. recellularization, characterization).   The bioreactor in our study was used for decellularization and we performed recellularization by hand. Previous studies utilize bioreactors for reseeding as well (68). For example, HUVECs can be reseeded through renal arteries of decellularized kidneys resulting in re-endothelialized kidney constructs across the entire kidney section including interlobar, arcuate arteries, glomeruli and peritubular capilaries (68). In our study, reseeded HUVECs were only found lining around a few circular structures without any involvement of renal glomeruli as described in section 3.3. The   81 differences in results may be due to different recellularization protocols. The perfusion approach provided long-lasted reseeding into the renal vasculature and the manual approach could only position cells on the surface of the renal vasculature. In this case, it is suggestive that cells reseeded by perfusion are more likely to reside in the kidney than that performed by hand. On the other hand, cell numbers being reseeded using bioreactors require very high concentration of cells that is much more than in our study (68). Therefore, we speculate that sufficient cell-number may be crucial for successful kidney ECM recellularization.   One of the novelties in this study is that to our knowledge, we are the first to successfully reseed renal cancer cells onto the decellularized kidney ECM. As described in section 3.3, ccRCC cell line was successfully reseeded under various conditions and formed aggregates in different locations of the decellularized ECM. It demonstrated that metastatic kidney cancer cells can be grown and proliferated on a 3D platform without strict conditions. It is speculated that these cancer cells may grow in an integrative and interactive manner with other cells in 3D conditions as well. Previous studies described the reseeding of colon (69) and breast cancer cells (70) to their corresponding organ specific decellularized ECMs. Although the reseeding principles are similar to that of our study, the integration of cancer cells to a matrix was not observed within the colon cancer cells reseeding model (69) which was distinct from the in vivo scenario. Meanwhile, integration of cells to the ECM was seen in a breast cancer model which demonstrated mimicry to the in vivo situation. Despite the methodology that injected cancer cells inside the ECM, which bypassed the   82 initiation of cell penetration, this study lacked evidence showing cell-ECM interactions (70). In our study, metastatic kidney cancer cells formed significantly larger interconnected multicellular aggregates (100 – 200 µm in size) inside the decellularized ECM with the methodology of loading cells onto the matrices. Obvious cell integration was observed. It is suggestive that our model achieved better mimicry of the in vivo condition with presentation of cell-ECM interactions during cell growth.    On the other hand, attempts to reseed the decellularized kidney ECM with HEK293 cells by hand was not successful. Currently, our bioreactor does not allow for infusion of cells. Modification of our bioreactor to allow for distribution of cells in addition to detergents may allow for integration of cells to the renal ECM. Therefore, future studies are planned to perform recellularization by perfusion after modification of our bioreactor. Also, a combination of cells (cancerous, endothelial and embryonic) can be reseeded together using perfusion approach to observe cellular distributions.  During the establishment of a bladder cancer model, a protocol with combined physical and chemical approaches for the immersion decellularization of porcine bladder squares was established, representing a preliminary step toward the building of a bioengineered scaffold for bladder tumor models. A recent study performed perfusion decellularization to rabbit bladders with non-ionic detergent passing through the urethra (71). Despite have different organ sources, we performed immersion decellularization with salt solutions and achieved satisfactory results with histological and SEM evaluations as described in section 3.2. Perfusion decellularization was not   83 performed on porcine bladder because it was not efficient to do so with such a sizable organ. Instead, we sectioned it into equal squares for easier manipulation. In addition, we tested non-ionic detergents as described in a related study (71) with immersion approach but did not achieve similar decellularization results. The main difference was organ thickness. Hence, these results suggest that non-ionic detergents may not pass through thicker tissues easily during immersion decellularization and alternative reagents may be required when using these tissues.   Previous studies demonstrated sophisticated procedures and set-ups to rebuild a functional bladder involving the use of stem cells and various scaffolds (72) (73). Since our focus is to create a tumor mimicry of in vivo conditions instead of reforming the organ functionality, both normal/mortal and cancerous cells were reseeded to the decellularized bladder ECM. One of the novelties of this study is that, to our knowledge, we are the first group successfully reseed urothelial cancer onto the decellularized bladder ECM. As described in section 3.4, UM-UC3 cell line was successfully reseeded on the urothelium of the bladder and showed obvious invasiveness compare to that reseeded with urothelial cells. Multi-cell layers formed on the urothelium as bladder cancer during cell reseeded. Also, the ECM underneath was partially degraded by cancer cell invasion. Thus, it demonstrated the aggressiveness of ccRCC, which cannot be observed in a 2D system. These results reveal that this model better translates the cancer biology and serves the purpose of bridging the gap between 2D models and “true” scenarios.    84 In this study, benign urothelial cells (SV-HUC) were also reseeded by hand and majority of cells were observed lining on the urothelium without penetrating the basement membrane. Similar study with by-hand recellularization approach showed almost identical results (74)  show that the bladder decellularization protocol described in section2.2.3 preserves the integrity of the basement membrane.   Cell migration is a key process during the development of tissue (75). Most cell migration assays were performed in 2D models and very few studies of cell movements were generated in 3D scenarios. In our study, we observed fibroblasts’ penetration during bladder recellularization and since fibroblast plays a vital role in cancer initiation, invasion and metastasis (76), I explored their penetration pattern quantitatively. A related study performed fibroblast recellularization by-hand onto a decellularized bladder ECM and showed little cell penetration to the stroma (77). In our study, we successfully reseeded fibroblasts onto stroma of the bladder and incubated two times longer than that of the related study. Significant cell penetration was observed as described in section 3.5, which suggests that our model maintained the appropriate environment for cell growth.   This study demonstrates the success of multi-type cell reseeding (HUVECs + Fibroblasts + SV-HUCs) as described in section 3.4. Different cells were proved to be positioned at the correct locations: HUVECs and fibroblasts are in stroma when SV-HUCs are in the urothelium. This has not been shown previously and it suggests the potential to optimize it into a bladder’s replica to mimic clinical conditions.    85 Tumor progression is highly associated with biochemical and mechanical cues provided by the ECM (78) (79). To accurately produce a urogenital tumor model from decellularized ECMs, the ECMs’ composition is pivotal since it provides those cues for cell growth. On the other hand, ECM integrity is also important since cell proliferation is higher on more rigid surface (80), which explained the fact that cells attach more easily in 2D petri-dish than in 3D model. It further illustrates the reason why 3D tumor models mimic the in vivo condition better than that of 2D system. In the present study, decellularized kidney and bladder ECM were characterized/profiled as described in sections 3.1.3 and 3.2.3. Notably, the comprehensive full-protein profile of decellularized ECMs (kidney and bladder) has not been documented before and it demonstrates high compositional similarities (structural, functional proteins and certain growth factors) compare to that of native organs based on the measured coverage. This finding validated the effectiveness of our established decellularization protocols and set the foundation of successful cell proliferation after recellularization.   Above all, our in vitro urogenital tumor models have several advantages. 1) The scaffolds were derived from biological tissues that maintain native biocompatibility, biodegradability and certain mechanical properties for cell growth. 2) The scaffolds were cost-effective since they were retrieved from animal organs supposed to be discarded. 3) High-throughput production can be achieved. 4) Our models are potentially suitable for multi-cell type reseeding that provide biomimetic environment for cell growth. 5) The models have potential to be used to culture patient-derived cells in vitro to predict therapeutic outcomes in a personalized manner.    86 Chapter 5: Conclusions and Future Directions  5.1 Summary  In summary, this thesis is a description of preliminary model-making methodologies with its related precautions and possible experimental obstacles. Within this study, tissue dissociation protocols of each organ were established and decellularization protocols for both kidneys and bladders were successfully optimized and established. The evaluation and characterization of the decellularized ECMs were accomplished with evidence of the preservation of structural proteins and growth factors. Afterward, the bladder ECM recellularization protocol was successfully optimized and established while the kidney ECM recellularization protocol was considered partially established due to inconsistent experimental reproducibility. In conclusion, this work establishes the model as proof-of-principle and sets the foundation for future patients’ studies.  5.2 Future Directions  Moving forward, testing the validity and reliability of our model and recellularization is essential. Confirmation of the recapitulation of various genitourinary organs, with or without malignant cells, will be important to further develop our model for clinical use. Eventually, I envision our model being developed into a personalized medicine   87 platform in which patients’ tumors are seeded in our model and then tested with various agents to determine optimal treatments. Optimization of our model may involve incorporation of the patient’s own “normal” cells including immune cells. Genetic validation of the model can be performed and comparison between that and patients; tumor specimen can be generated. Eventually, with patients’ cells being reseeded, different drug-testing assays can be established to identify the optimal treatment.                          88 Bibliography  1. Canadian Cancer Statistics. Canadian Cancer Society's Advisory Committee on Cancer Statistics Toronto, ON: Canadian Cancer Society; 2017 [Available from: http://www.cancer.ca/en/cancer-information/cancer-type/bladder/statistics/?region=on. 2. Kindey Cancer Canada. 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Trial #3 dH2O 1000 mL 200 min. 1% Triton X-100 2000 mL 400 min. 1% Triton X-100 1000 mL 1000 min. 0.1% SDS 1000 mL 200 min. Trial #4 dH2O 1000 mL 200 min. 0.6 M KCl  1000 mL 200 min. 1 M KI 1000 mL 200 min. dH2O 1000 mL 200 min. Trial #5 dH2O 1000 mL 200 min. 0.6 M KCl  2000 mL 400 min. 1 M KI 2000 mL 400 min. dH2O 1000 mL 200 min.  DNase I (2,000 unit) were perfused into the kidney at the end of decellularization cycles to eliminate remnant DNA fragments.         94 Appendix B  Trial protocols: Decellularization by immersion – urinary bladder Trial #1 dH2O   30 min. 1% Triton X-100   120 min. 0.1% SDS  120 min. dH2O   overnight Trial #2 dH2O   30 min. 1% Triton X-100   180 min. 0.1% SDS  180 min. dH2O   overnight Trial #3 dH2O   30 min. 1% Triton X-100   180 min. 0.5% SDS  180 min. dH2O   overnight Trial #4 dH2O   30 min. 0.6 M KCl  120 min. 1 M KI  120 min. dH2O  overnight Trial #5 dH2O   30 min. 0.6 M KCl  180 min. 1 M KI  180 min. dH2O  overnight   All bladder immersion trials were tested in 50 mL centrifuge tube (Sigma-Aldrich, CLS40829, Damstadt, Germany), 150 mm x 25 mm tissue-culture treated culture dishes (Sigma-Aldrich, CLS430599, Damstadt, Germany) and 100 mm x 20 mm tissue-culture treated culture dishes (Sigma-Aldrich, CLS430167, Damstadt, Germany) respectively. DNase I (1kU/ml) were used at the end of immersion cycles to eliminate remnant DNA fragments.             95 Appendix C Trial protocols: Decellularization by immersion – kidney Trial #1 dH2O   30 min. 1% Triton X-100   120 min. 0.1% SDS  120 min. dH2O   overnight Trial #2 dH2O   30 min. 1% Triton X-100   180 min. 0.1% SDS  180 min. dH2O   overnight Trial #3 dH2O   30 min. 1% Triton X-100   180 min. 0.5% SDS  180 min. dH2O   overnight Trial #4 dH2O   30 min. 2% Triton X-100   180 min. 0.5% SDS  180 min. dH2O   overnight Trial #5 dH2O   30 min. 0.6 M KCl  120 min. 1 M KI  120 min. dH2O  overnight Trial #6 dH2O   30 min. 0.6 M KCl  180 min. 1 M KI  180 min. dH2O  overnight                96 Appendix D Trial protocols: Recellularization – kidney  Number of cells Tissue Pre-immersed  Media used for recellularization Timespan Trial #1 4,000  In FBS Corresponding cell growth media 6 days Trial #2 10,000  In FBS Corresponding cell growth media 6 days Trial #3 4,000  In corresponding growth media Matrigel 21 days Trial #4 10,000  In corresponding growth media Matrigel 21 days Trial #5 4,000 In corresponding growth media Corresponding cell growth media 6 days               97 Appendix E  Trial protocols: Recellularization – Urinary bladder (single cell-type)  Number of cells Tissue Pre-immersed  Media used for recellularization Timespan Trial #1 1,000,000  In FBS Corresponding cell growth media 6 days Trial #2 500,000  In FBS Corresponding cell growth media 6 days Trial #3 1,000,000 In corresponding growth media Matrigel 21 days Trial #4 500,000 In corresponding growth media Matrigel 21 days Trial #5 1,000,000 In corresponding growth media Corresponding cell growth media 6 days              98 Appendix F  Trial protocols: Recellularization – Urinary bladder (multi cell-type) Total cell # = 5,000,000 Ratio (HUVEC: Fibroblast: SV-HUC) Tissue Pre-immersed  Media used for recellularization Timespan Trial #1 1 :1 :1 In FBS Corresponding cell growth media 6 days Trial #2 2 :1 :2 In FBS Corresponding cell growth media 6 days Trial #3 1 :1 :1 In corresponding growth media Matrigel 21 days Trial #4 2 :1 :2 In corresponding growth media Matrigel 21 days Trial #5 2 :1 :2 In corresponding growth media Corresponding cell growth media 6 days       

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