Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA370

Poster Communications

Organization of vascular smooth muscle cells as multilayered organoids triggers their resting state

M. Jäger1, A. Feldner1, M. Hecker1, C. Arnold1, T. Korff1

1. Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany.

In vitro propagation of cells is usually based on culture media supplemented with growth factors and/or fetal calf serum (FCS) to promote cellular growth and activation. However, for vascular smooth muscle cells (VSMCs) these conditions attenuate the quiescent/contractile phenotype that predominates in vivo and is characterized by low proliferation as well as increased abundance of proteins of the contractile apparatus. Thus, delineation of environmental parameters which support the resting/contractile state of cultured VSMCs as in vivo remains an important challenge. In this context, our study intended to define culture conditions promoting the contractile phenotype of easy accessible human umbilical artery smooth muscle cells (HUASMCs) by i) exposing them to serum-free medium, ii) organizing them as 3D multilayered organoids and iii) enabling communication with human umbilical vein endothelial cells (HUVECs) in 3D co-culture organoids. While HUASMCs are relative undemanding cells and were grown in DMEM supplemented with 15% FCS (standard conditions), they nevertheless showed robust expression of VSMC markers such as smooth muscle myosin heavy chain or α-smooth muscle actin (αSMA). Serum-free medium did not affect the marker expression but decreased proliferation (Ki67 positive cells (mean ±S.D.); standard vs. serum-free: 33.48±12.60 % vs. 10.08±6.60 %, p<0.05, n=3). However, organization of HUASMCs as 3D organoids promoted a contractile phenotype as evidenced by increased VSMC marker abundance (αSMA; fold change vs. control: 1.84±0.59, p<0.01, n=8) and decreased proliferation (Ki67 positive cells; control vs. organoids: 13.65±0.82 % vs. 0.01±0.01 %, p<0.001, n=3). This finding was further supported by microarray-based gene set enrichment analysis (log2-fold change vs. control: e.g. cell cycle: -2.51, p<0.01, n=3). In addition, co-culture of HUASMCs with HUVECs further enhanced the acquisition of a contractile phenotype shown by an increase in the abundance of the VSMC marker calponin (fold change vs. control: 2.12±0.48, p<0.01, n=5). To investigate whether VSMCs cultured as organoids can reenter an activated state, they were seeded in 3D collagen gels supplemented with 15% FCS. Under these conditions, HUASMCs increase their proteolytic capacity to invade the extracellular matrix which was inhibited by treatment with the MMP-Inhibitor GM6001 (cumulative sprout length after 24h [µm]; control vs. inhibitor: 1523±121 vs. 479±94, p<0.0001, n=4). In conclusion, culture of VSMCs in 3D organoids rather than exposure to serum-free medium promoted a resting phenotype thus displaying a superior culture technique for enabling organotypic in vivo-like differentiation of VSMCs. Since organoid VSMCs can reenter an activated state, this method could serve as an excellent basis for tissue engineering purposes or functional analyses of compounds targeting VSMCs.

Where applicable, experiments conform with Society ethical requirements