Chen Allen Kuan-Liang, Chen Xiaoli, Choo Andre Boon Hwa, Reuveny Shaul, Oh Steve Kah Weng
Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore.
Stem Cell Res. 2011 Sep;7(2):97-111. doi: 10.1016/j.scr.2011.04.007. Epub 2011 May 11.
A variety of microcarriers may be used for the expansion of human embryonic stem cells (hESC) for cell therapy applications. This study investigated the effects of 10 types of microcarriers on hESC attachment efficiency, growth and pluripotency. High attachment efficiency was observed on uncoated microcarriers, however poor cell growth and/or gradual loss of pluripotency occurred during continuous passaging. Coating of the microcarriers with Matrigel resulted in higher cell yields and stable pluripotent states for at least three passages. Positively charged cylindrical cellulose microcarriers (DE52, DE53 and QA52) and large (190 μm) positively charged spherical microcarriers (Cytodex 1) exhibited high cell expansion potential and levels of pluripotency. Lower cell yields were obtained using smaller diameter spherical (65 μm and 10 μm) or macroporous beads. Instead of Matrigel, laminin coated microcarriers (DE53 and Cytodex 1) are capable of supporting the long term propagation and pluripotency of HES-2 and HES-3 cell lines. HES-2 cell line which was shown earlier to be shear resistant achieved similar cell growth and expression of pluripotent markers when cultured on both Matrigel (84% Tra-1-60, 1.43×10(6) cells/ml) and laminin (74% Tra-1-60, 1.37×10(6) cells/ml) coated microcarriers in spinner flasks. In contrast, HES-3 exhibited a decrease in cell yield, viability and pluripotent markers on laminin as compared with Matrigel coated microcarriers possibly due to shear sensitivity. Conventional microcarriers intended for propagation of mammalian cells are not suitable for long term propagation of hESC. Matrigel or laminin coating is essential for stable long term propagation of hESC on a variety of microcarriers.
多种微载体可用于人类胚胎干细胞（hESC）的扩增，以用于细胞治疗。本研究调查了10种微载体对hESC附着效率、生长和多能性的影响。在未包被的微载体上观察到高附着效率，但在连续传代过程中细胞生长不佳和/或多能性逐渐丧失。用基质胶包被微载体可产生更高的细胞产量，并使多能状态稳定至少三代。带正电荷的圆柱形纤维素微载体（DE52、DE53和QA52）以及大尺寸（190μm）带正电荷的球形微载体（Cytodex 1）表现出高细胞扩增潜力和多能性水平。使用较小直径的球形（65μm和10μm）或大孔珠可获得较低的细胞产量。与基质胶不同，层粘连蛋白包被的微载体（DE53和Cytodex 1）能够支持HES - 2和HES - 3细胞系的长期增殖和多能性。先前显示具有抗剪切能力的HES - 2细胞系，当在转瓶中基质胶（84% Tra - 1 - 60，1.43×10(6) 个细胞/ml）和层粘连蛋白（74% Tra - 1 - 60，1.37×10(6) 个细胞/ml）包被的微载体上培养时，实现了相似的细胞生长和多能性标志物表达。相比之下，与基质胶包被的微载体相比，HES - 3在层粘连蛋白包被的微载体上细胞产量、活力和多能性标志物有所下降，这可能是由于其对剪切敏感。用于哺乳动物细胞增殖的传统微载体不适用于hESC的长期增殖。基质胶或层粘连蛋白包被对于hESC在多种微载体上的稳定长期增殖至关重要。
