Fig. 2.

Soft substrate maintains cellular morphology, rate of expansion and proliferation during serial passage. Representative phase contrast images of (A) EP (passage number ≤6) and (B) LP (passage number >10) UC-hMSCs on collagen-coated TCP and (C) EP and (D) LP on soft gel. (E) Cell spread area increases with passage on TCP; however, spreading area of cells on gel showed no significant difference across passages. (N=3, n=150). (F) The number of protrusions, as shown by white arrows in B, are significantly increased in LP cells cultured on TCP (N=3, n=150). (G) LP UChMSCs on gel showed significantly lower traction than that on TCP. The experiment was performed using two biological samples (N=3, n=20). (H) DT of UC-hMSCs cultured on gel and TCP over the passage. While DT increases with passage when cultured on TCP, it remains unaffected when expanded on 5 kPa gel. The difference in DT for EP and MP are negligible while for LP the difference is significant (N=3, n=1). (I) CPD of UC-hMSCs cultured on gel and TCP over the passage. CPD increases linearly for UChMSCs on the gel but approaches a plateau for cells cultured on TCP (N=1, n=11). (J) Immunofluorescence images of nuclei co-stained with DAPI (blue) and BrdU (red) capturing a relative percentage of cycling cells. (K) Percentage BrdU positive cell for P14 (LP) from gels are significantly higher than the cells from TCP, though for EP (P6) cells there was no significant difference between gel and TCP. This data reconfirms that cells maintain their proliferative potential if cultured on the soft gel. Results are expressed as mean±s.e.m. *P<0.05, **P<0.001, ***P<0.0001. Scale bars: 100 µm.