Basement membrane (BM) is a compact and continuous sheet of 50–350 nm thickness and located underneath epithelium, endothelium, etc. It helps tissue repair and morphogenesis, and stem cells' differentiation to mature phenotypes. We have devised several culture models for cellular assembly of BM in vitro and developed a thorough removal of the covering BM-forming cells in order to utilize the bared BM as a culture substratum. Thus de novo synthesized BM (sBM) substrata of LN-511 isoform normally guided tissue progenitors or embryonic stem cells to terminal differentiation and morphologically and functionally mature phenotypes, for example, airway ciliated cells, hepatocytes and pancreatic β cells. Therefore sBM has been proven to be a matrix, that is, solid-phase cradle. BM formation, which we introduce in this chapter, has the advantages of it not being necessary to prepare each BM component, but also being free from clearing irregular aggregates that come from malassembly among them. We simply culture epithelial and endothelial cells etc. on the fibrillar collagen substratum that has been coated beforehand with N-acetylglucosamine ligands-branching or -sprouting hydrophobic polymer, and wait for automatic assembly of BM structure by these cells. rLN-10 cells, genetically modified cells, are advantageous for forming an optimized sBM.