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MacDonald, Katherine Nicole

University of British Columbia

Regulatory T cell therapy has shown promise in treating autoimmune disorders, transplant rejection and graft-versus-host disease in early clinical trials. However, efficient manufacturing of clinical grade cells is still a significant hurdle that must be overcome before these therapies can see widespread use. Previous work showed that large numbers of pure, naïve Tregs can be isolated from pediatric thymus. This research aims to investigate the variables governing Treg expansion with serum-free media and non-cell-based activation reagents to develop manufacturing protocols to produce therapeutic doses of thymus-derived Tregs. First, we tested activation reagents, cell culture media, restimulation timing, and cryopreservation to develop good manufacturing practice compatible protocols to expand and cryopreserve Tregs. Cryopreservation tests revealed a critical effect of timing: only cells cryopreserved 1-3 days, but not > 3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. We next investigated how changing cell density and feed frequency influenced Treg expansion, viability, and phenotype in a 3-week expansion protocol and found that Treg viability and expansion were correlated with the cell density at restimulation. Tregs restimulated at low cell densities (1x10⁵ cells/cm²) initially had high growth rates, viability, and FOXP3 expression, but at later culture times these parameters were reduced compared to slower growing Tregs restimulated at higher cell densities (5x10⁵ cells/cm²). High density expansion was associated with lower nutrient concentrations and higher accumulations of lactate, but this could be alleviated by decreasing the interval between feeds. We tested platforms to scale up Treg manufacturing and observed that Tregs expanded in gas permeable cell expansion bags were of higher quality than those expanded in agitated suspension culture or the G-Rex. Finally, we tested labelling expanded Tregs with a ¹⁹F-perfluorocarbon (PFC) nanoemulsion to enable in vivo tracking using MRI. While Tregs could be labelled with the ¹⁹F-PFC and detected in vivo in immunocompromised mice, labelling during expansion reduced cell viability, particularly after cryopreservation. Together, this research developed protocols and process understanding necessary to efficiently produce clinical grade Tregs, laying the groundwork for the first clinical trial of thymic Treg cell therapy in Canada.

Text

2022-03-28

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/81038

Biomedical Engineering

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/