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    • In summary we have shown that various Matrigel coated microc

    2018-10-29

    In summary we have shown that various Matrigel coated microcarriers can support long term propagation of undifferentiated hESC. HES-2 and HES-3 were propagated for over 17 passages on Matrigel coated DE53 and Cytodex 1 microcarriers (over 11 passages). Similar results were reported by Nie et al. (2009), the authors demonstrate that by coating Cytodex 3 microcarriers with Matrigel, they were able to propagate H9 cells for at least 10 passages while expressing pluripotent markers at levels comparable to those from 2D cultures on Matrigel coated plates (Nie et al., 2009). The size and shape of the microcarriers has an effect on the mode of propagation and cell yield. hESC grew as compact cell-microcarrier opioid receptors on the cylindrical shaped (L 130μm×D 35μm) positive charged cellulose microcarriers (DE52, DE53 and QA52) and as a less compact cell-microcarrier aggregate on the beaded 190μm diameter Cytodex1 microcarrier (Figs. 2 and 3). These different modes of propagation did not affect cell yield and pluripotency. Reduction of the bead diameter from 190μm (Cytodex1) to 65μm (Tosoh65 PR) and 10μm (Tosoh10 PR) resulted in generation of more dense cell-microcarrier aggregates (Fig. 2). In fact, the 10μm spherical microcarriers which are smaller than the cells serve only as a linker between the cells for the generation of condensed cell-microcarrier aggregates. These tight structures led to a decrease in cell yield (Fig. 1E) probably as a result of limited access of nutrient and growth factors to the cells. Cell yields from the macroporous microcarrier cultures (Cytopore 2 and Cultispher G) were also relatively low (Fig. 1E). We assume that the macroporous beads might provide a non-uniform exposure of cells to nutrients and growth factors, whereby cells inside the pores have less access to growth factors (Nie et al., 2009; Lock and Tzanakakis, 2009). Moreover, Cultispher G cultures resulted in a decrease in pluripotency after the second passage (52–64% cells expressing Tra-1-60) probably due to the low Matrigel adsorption onto Cultispher G. The use of laminin as an alternative substrate for Matrigel in 2D plate cultures has been reported by several groups (Xu et al., 2001; Beattie et al., 2005). In this study, we have shown that mouse laminin can also replace Matrigel in 3D microcarrier cultures. Two cell lines (HES-2 and HES-3) were propagated for long periods (10 passages) on two different laminin coated, positively charged microcarriers (Cytodex 1 and DE53). The cultured cells showed stable karyotype and retained pluripotency. hESCs were capable of differentiating into cells of the three germ layers by in vitro spontaneous differentiation via embryoid bodies, and teratoma formation in SCID mice. In general, similar cell yields were obtained in cultures of laminin coated DE53 microcarrier compared to Matrigel coated ones. Recently, Rodin et al. (2010) identified laminin-511 within the human laminin family as the important substrate supporting long term cultivation of undifferentiated hESC. Moreover, they showed that laminin-511 has better adhesion property than laminin-111, which is found in purified natural mouse laminin. Thus, it is possible that coating of microcarriers with human laminin-511 could improve cell yields. We have demonstrated recently that the effect of agitation on cell differentiation is cell line specific. HES-2 cells propagated on Matrigel coated DE53 in agitated spinner flasks maintained pluripotency, while HES-3 cells tend to differentiate during propagation (Leung et al., 2010). This phenomenon was accentuated when using laminin coated microcarriers. HES-2 cells on laminin coated microcarriers showed similar expression of pluripotent markers to Matrigel coated controls. But HES-3 cultured on laminin coated microcarriers completely lost their expression of pluripotent markers (Fig. 7C). Moreover the viability of HES-3 cells propagated on laminin coated microcarriers was considerably lower than on Matrigel coated ones. These results show that the Matrigel coating may offer a shear protective element, which we assume, arises from the gelatinous polymeric nature of the thick coating. hESC propagated on a laminin-111 coated surface has lower cell contact area when compared to Matrigel, as reported by Rodin et al. (2010). Hence we postulate that under agitated conditions, the lower cell contact of HES-3 on laminin coated microcarriers might have resulted in cell detachment resulting in lower viability, differentiation and consequently a reduced final cell yield (Fig. 7C). It will be of interest to test laminin-511 which was shown to have greater cell contact area, in spinner flask cultures (Rodin et al., 2010).