Development of an in vitro platform for epithelial-stromal interactions: A basement membrane-containing scaffold from decellularized porcine bladders

Abstract

The first organized extracellular matrix that appears during mammalian embryogenesis is a basement membrane (BM), BM is present in all adult epithelia, endothelia, muscle, nerve and fat tissues. BM is a sub-micrometer thick compact lattice of macromolecules that is maintained by the adhered cells. Systems such as collagen gels, Matrigel® or synthetic polymeric scaffolds have been proposed to mimic the BM and to study the interactions between different cell types, but all lack a structured BM. Here we aimed to obtain and characterize a natural, thin basement membrane-containing scaffold from pig urinary bladders that are subjected to blunt dissection of layers and decellularization steps, preserving the near native BM with a few layers of underlying connective tissue to maintain its structural integrity. The scanning electron microscopy, confocal multiphoton microscopy and immunohistochemistry helped confirm the presence of the BM. A veil-like network composed of thin fibers was present on top of a course network, and glycosaminoglycans, collagen and basement membrane proteins were present. The scaffold’s ability to repopulation and basement membrane barrier function were further confirmed when HaCaT and MRC5 cells attached and remained respectively on the epithelial and mesenchymal side without any crossover. Cells remained viable till 2 weeks. This BM-containing scaffold allows to create in vitro models of epithelial-mesenchymal tissues through a structured basement membrane and investigate basement membrane dynamics. The basement membrane-containing scaffold was found to be isotropic under uniaxial tension with a failure strain of 0.25 allowing its use to investigate strain induced basement membrane dynamics.