Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells

Abstract

45Induced pluripotent stem cells (iPSCs) are an attractive cell source for regenerative medicine and 46 the development of therapies, as they can proliferate indefinitely under defined conditions and 47 differentiate into any cell type in the body. Large scale expansion of cells is limited in adherent 48 culture, making it difficult to obtain adequate cell numbers for research. It has been previously 49 shown that stirred suspension bioreactors (SSBs) can be used to culture mouse and human stem 50 cells. Pigs are important pre-clinical models for stem cell research. Therefore, this study 51 investigated the use of SSBs as an alternative culture method for the expansion of iPSCs. Using 52 an established porcine iPSC line as well as a new cell line derived and characterized in the current 53 study, we report that porcine iPSCs (piPSCs) can grow in SSB while maintaining characteristics 54 of pluripotency and karyotypic stability similar to cells grown in traditional two-dimensional static 55 culture. This culture method provides a suitable platform for scale up of cell culture to provide 56 adequate cell numbers for future research applications involving porcine induced pluripotent stem 57 cells. 58 59 60 61 62 63 64 65 66 68 Pluripotent stem cells (PSCs) are defined by two main features: the ability to proliferate 69 indefinitely in an undifferentiated state under defined conditions, and the potential to become any 70 cell type arising from the three germ layers, endoderm, ectoderm and mesoderm, in the body. 71 Human and mouse embryonic stem cell (ESC) lines have significantly contributed to the progress 72 in biomedical research and cell therapy development (1; 2), however, the ethical and safety 73 concerns surrounding the use of human embryonic stem cells (hESCs) have limited their potential 74 applications (3). The emergence of induced pluripotent stem cells (iPSCs) as an alternative source 75 of cells that recapitulate the phenotype and function of ESCs, helped overcome these concerns and 76 energized the stem cell research field (4; 5). 77Outbred, large animal models that are closer in size, longevity and physiology to humans are 78 essential to expand our knowledge gained from rodents and facilitate translation of stem cell-based 79 approaches to human applications. iPSCs have been derived from large ungulate species including 80 pigs that share many physiological characteristics with humans, making the pig is a powerful 81 model for biomedical research and drug testing (6). 82Large numbers of cells are required for many research and therapeutic applications, which can be 83 challenging to obtain through conventional two-dimensional static culture systems in tissue culture 84 plates and flasks. Two-dimensional culture systems have limited surface area, often require a large 85 number of culture vessels and can be labor intensive. This additional handling can lead to cell loss 86 and increased risk for contamination. Moreover, the use of numerous plates and flasks may 87 introduce culture heterogeneity ...