Timilsina Suraj, McCandliss Kaitlyn Faith, Trivedi Evan, Villa-Diaz Luis G
Department of Biomarkers and Investigative Pathology Unit (BIPU), Charles River Laboratories, Mattawan, MI 49071, USA.
Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA.
Bioengineering (Basel). 2023 Aug 24;10(9):999. doi: 10.3390/bioengineering10090999.
Human embryonic stem cells and induced pluripotent stem cells (hPSC) have an unprecedented opportunity to revolutionize the fields of developmental biology as well as tissue engineering and regenerative medicine. However, their applications have been significantly limited by the lack of chemically defined and xeno-free culture conditions. The demand for the high-quality and scaled-up production of cells for use in both research and clinical studies underscores the need to develop tools that will simplify the in vitro culture process while reducing the variables. Here, we describe a systematic study to identify the optimal conditions for the initial cell attachment of hPSC to tissue culture dishes grafted with polymers of N-(3-Sulfopropyl)-N-Methacryloxyethyl-N, N-Dimethylammoniun Betaine (PMEDSAH) in combination with chemically defined and xeno-free culture media. After testing multiple supplements and chemicals, we identified that pre-conditioning of PMEDSAH grafted plates with 10% human serum (HS) supported the initial cell attachment, which allowed for the long-term culture and maintenance of hPSC compared to cells cultured on Matrigel-coated plates. Using this culture condition, a 2.1-fold increase in the expansion of hPSC was observed without chromosomal abnormalities. Furthermore, this culture condition supported a higher reprogramming efficiency (0.37% vs. 0.22%; < 0.0068) of somatic cells into induced pluripotent stem cells compared to the non-defined culture conditions. This defined and xeno-free hPSC culture condition may be used in obtaining the large populations of hPSC and patient-derived iPSC required for many applications in regenerative and translational medicine.
人类胚胎干细胞和诱导多能干细胞（hPSC）为发育生物学以及组织工程和再生医学领域带来了前所未有的变革机遇。然而，由于缺乏化学成分明确且无动物源的培养条件，它们的应用受到了显著限制。对于用于研究和临床研究的高质量、规模化细胞生产的需求凸显了开发工具的必要性，这些工具既能简化体外培养过程，又能减少变量。在此，我们描述了一项系统性研究，以确定hPSC在接种了N-（3-磺丙基）-N-甲基丙烯酰氧乙基-N，N-二甲基铵甜菜碱（PMEDSAH）聚合物的组织培养皿上，结合化学成分明确且无动物源的培养基进行初始细胞附着的最佳条件。在测试了多种补充剂和化学物质后，我们发现用10%人血清（HS）预处理PMEDSAH接种的培养板能支持初始细胞附着，与在基质胶包被培养板上培养的细胞相比，这使得hPSC能够长期培养和维持。使用这种培养条件，观察到hPSC的扩增增加了2.1倍，且无染色体异常。此外，与未明确的培养条件相比，这种培养条件支持体细胞重编程为诱导多能干细胞的效率更高（0.37%对0.22%；<0.0068）。这种化学成分明确且无动物源的hPSC培养条件可用于获得再生医学和转化医学许多应用所需的大量hPSC和患者来源的诱导多能干细胞。
