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Analysis of stem and progenitor cell responses to variations in bioprocess culture variables Chaudhry, Muhammad Arshad S.

Abstract

The realization of many potential cell therapies will depend on the development of culture protocols that consistently expand stem and progenitor cell numbers while retaining their developmental properties. Hematopoietic cell lines were used to develop predictive mathematical models of cytokine-dependent proliferation. First, Hill-type empirical models provided a quantitative understanding of how cytokines (IL-3 and GM-CSF) individually influenced the growth rate. Then a competitive model of cytokine interactions was developed that effectively predicted the growth rates for TF-1 and MO7e cells exposed to combinations of IL-3 and GM-CSF, over wide concentration ranges. Mouse embryonic stem cells (mESC) were used to study the influence of various culture variables on their proliferation and embryoid body (EB) formation potential. The medium pH, osmolality, basal medium composition, as well as serum and serum replacement (SR) concentrations were found to modulate mESC responses. For most of these variables, conventional cultivation methods did not expose the cells to levels that significantly decreased culture output. However, for feeder-containing cultures, the osmolality increased to over 400 mOsm/kg and, when ESC were cultured in such a medium, the EB yield was decreased by more than 50% compared to media at 300 and 335 mOsm/kg. Likewise, pH dose response experiments revealed that, within 48 h, compared to a medium initially at pH 7.3, the EB yield decreased by approximately 50% when R1 cells were cultured at either pH 7.15 or 7.45, with even lower yields at more extreme pH conditions. The mESC responses were also found to be serum concentration-dependent with maximal results at 5% serum in DMEM compared to the commonly used 15%. In DMEM:F12, a reduced dependency on serum was observed. When SR was used, both the proliferation and EB formation potential were maximal with 5% SR in DMEM. In DMEM:F12, 1.7% SR was sufficient to maintain the EB yield of mESC lines with a similar growth rate. Taken together, these results demonstrate that multiple critical culture variables can profoundly influence the responses of mESC and that these variables need to be kept within defined ranges to maintain the mESC in culture without compromising their developmental potential.

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