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Figure 3 | BMC Developmental Biology

Figure 3

From: Developmental cues and persistent neurogenic potential within an in vitro neural niche

Figure 3

Light microscopy of 2- and 3-D cultures grown in the presence of various ECM components. Ai-Gi, Following 4-/4+ retinoic acid neural induction of mouse embryonic stem cells, the cells are plated in 96-well plates at 250,000 cells/cm2 on the substrate as indicated. Images are from Day 14 cultures. Aii-Gii, Cells are plated in 3-D using 0.15% Puramatrix hydrogel with the addition of the ECM substrate as indicated. In all panels, arrows indicate process development and arrowheads indicate compact aggregate formation. Images are from Day 14 cultures. H-K, Higher magnification of Day 13 cultures grown in PuraMatrix hydrogel at 60 μg/cm2 ECL reveals the best formation of in vitro NSC niche in 3-D of any conditions tested. All conditions tested produced in vitro neural niche in 2-D, but only 60 μg/cm2 ECL is able to consistently support its formation in 3-D. Scale bar in Ai is 100 μm and applies to panels Ai-H. Scale bar in J is 50 μm and applies to J and K. * Concentrations of ECM components are expressed per surface area of the culture well. ECM components for 3-D cultures are added at the same concentration as 2-D cultures. The volume of PuraMatrix assembled from 50 μl of 0.15% PuraMatrix in each well increases the surface area for ECM attachment, thus diluting the effective concentration of the ECM components. Note the 10-fold increase in effective concentration of ECM components in 3-D cultures as compared to 2-D cultures.

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