Figure 6
Spheroid release and culture mode switching from floating to adhesion culture.
(A) Adding PEG/DEX-free fresh medium decreases the density of the DEX-in-PEG ATPS pattern; therefore floating spheroids settled. (B) EB formation and cardiac differentiation using DEX-in-PEG ATPS pattern. Ten EBs were formed with 5000 ES D3 cells using DEX-in-PEG ATPS pattern made of ES growth media in 96-well plates. (Day -2)Two days after pattern formation, formed EBs were released from the pattern apex and attached to the culture surface. (Day 0) 24 h later, EB attachment to the culture surface was confirmed using a microscope and media were replaced with cardiac differentiation media. (Day 1) On day 9, beating clusters were observed in some of the EB cultures; on day 12, the number of beating clusters was counted under microscope. Scale bar: 400 μm (C) Representative images of day 12 EBs. Black arrow heads: EB cores; white heads: beating clusters which have different tissue morphology. Scale bar: 400 μm (D) qPCR analysis of representative three germ layer lineage markers. Relative mRNA expression levels of one pluripotency (Pou5f1), two mesodermal lineage (Brachyury and Hand1), one endodermal lineage (Gata4) and two ectodermal lineage markers (Sox1 and Otx2) were measured. The expression levels of all the markers were normalized by actin expression and then further divided by the normalized expression levels of each marker from undifferentiated ES cells. Gene expression of spheroids formed using HD method were also analyzed to be compared with the results of ATPS method (n = 3).
