Coculture assays allow the investigation of the role of endothelial cell and mural cell interactions in small vessel development and function. Different setups for coculture can be used to assay questions of interest. We include here methods for direct coculture, indirect coculture, and coculture in a three-dimensional extracellular matrix scaffold for studies of either a simple and direct association between the two cell types, the exchange of soluble molecules, or the interaction within a biomimetic tissue microenvironment.
Small intestinal submucosa grafts for vascular regeneration have produced variable patency (0-100%) that has been concurrent with variability in fabrication techniques. We hypothesized that 1) preservation (P) or removal (R) of the stratum compactum layer of the intestine and 2) a dehydrated (D) or hydrated (H) state of the graft, affect early patency and tissue regeneration. We combined both parameters through a 2(2) factorial experimental design into four groups (PD, RD, PH, RH), and compared them in an in vivo early response predictive model (swine, ID 4.5 mm, 7d, n = 4). Patency, thrombogenicity, vascularization, fibroblast infiltration, macrophage polarization profile, endothelialization, and biaxial mechanics were assessed. PD grafts remained patent (4/4) but had scarce vascularization and fibroblast infiltration. RD and RH had extensive vascularization and fibroblast infiltration, however, RD had sustained patency (4/4) and the highest number of regeneration-associated phenotype macrophages (M2), whereas RH had lower patency (3/4) and less M2 macrophages. PH had a modest cellular infiltration, but the lowest patency (2/4) and a dominant adverse macrophage phenotype. Elasticity of R grafts evolved toward that of native carotids (particularly RD), while P grafts kept their initial stiffness. We concluded that fabrication parameters drastically affected early patency and regeneration, with RD providing the best results.
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