The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating
Pajęcka K., Nygaard Nielsen M., Krarup Hansen T., Williams J.M.
Experimental Cell Research, 2017
Primary glomerular endothelial cells (GEnCs) is an important tool for studying glomerulosclerotic mechanisms and in drug development. Primary GEnCs are commonly cultured either on gelatin or plasma-derived fibronectin-coated surfaces, yet neither of the substrates is a major component of the healthy mature GBM. Here, the authors set out to establish a simple, reproducible model of quiescent hGEnC monolayer in vitro by determining the best commercially available ECM substrate- recombinant human laminin (rhLN)- 521, -511, -111, fibronectin (FN), collagen type IV and collagen I – based Attachment Factor. All ECM matrices except recombinant human laminin 111 (rhLN111) supported comparable cell proliferation. Laminin-521, laminin-511, and FN were best at supporting hGEnC attachment and spreading. Culturing hGEnCs on rhLN521, rhLN511 or fibronectin resulted in a physiologically relevant barrier to 70 kDa dextrans which was 82 % tighter than that formed on collagen type IV. Furthermore, only hGEnCs cultured on rhLN521 or rhLN511 showed plasma-membrane localized zonula occludens-1 and vascular endothelial cadherin indicative of proper tight and adherens junctions. The authors’ recommendations based on the results are that hGEnCs is best cultured on the mature glomerular basement membrane laminin – rhLN521 – which, as the only commercially available ECM, promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined adherens junctions, and physiological perm-selectivity.
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