Figure 4
From: Bioengineering functional smooth muscle with spontaneous rhythmic contraction in vitro
Maintenance of intestinal smooth muscle cell mixture (ISMC Mix) and vascular aortic smooth muscle cell line (MOVAS cells) cultured on STO cells in vitro. Non-sorted cells from enzymatically digested intestinal muscle strips were used as ISMC Mix. All ISMC Mix and MOVAS cells were cultured separately in FBS medium for the first 4 days followed by FBS or F12 medium. (a–c) 100 k ISMC Mix were cultured on STO cells or Ge with FBS or F12 medium for up to 3 weeks. (a) Immunofluorescence of ISMC Mix with ICC markers showing co-localization (yellow) of Kit (red) and Ano1 (green), SMCs marker MHC (red), and neuronal marker β-tubulin (green) at day 7. Scale bar, 100 µm. (b) GFP + ISMC Mix were analyzed for mRNA expression (Kit, Myh11, Tubb3, Kitl, and Acta2) and gfp DNA (n = 3). (c) Spontaneous contraction demonstrated the functionality of ISMC Mix cultured on STO cells with FBS or F12 medium (Supplementary Video 2 and 3) and frequency was measured (n ≥ 5). (d) MOVAS cells were analyzed for mRNA expression (Myh11) at day 7 (n = 3; triplicate samples). FBS = 15% FBS in DMEM. F12 = advanced DMEM/F12. *Samples were normalized to de-epithelialized intestine. **Samples were normalized to 100 k cells from day 0. Error bars, s.d. ***P < 0.0001, *P < 0.05.
