Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells

Abstract

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.