Characterization and Validation of Brain and Blood Vessel-derived Extracellular Matrix Bioinks for Recapitulating Human Blood-Brain Barrier in vitro

Abstract

Globally, neurodegenerative diseases, such as neuroinflammation-associated Alzheimer’s disease and Parkinson’s disease, have affected the health and quality of life of many people. The major regulator of neuroinflammation is the blood-brain barrier (BBB), which controls the molecular transport from brain vasculature to the central nervous system. Since the BBB is a complicated structure consisting of various cell types, animal models are difficult to use for in-depth mechanistic studies in neuroinflammation. Therefore, it is necessary to recapitulate the tissue-specific intrinsic function and pathophysiology of the human BBB in vitro. Previous researches demonstrated that decellularized extracellular matrix (dECM), derived from the target tissue, has advantage in providing tissue specific micro-environmental cues. In this study, we conducted characterization and functional validation of brain-derived dECM (BdECM) and blood vessel-derived dECM (VdECM) for developing human BBB model in vitro. BdECM and VdECM were obtained by decellularization process of porcine brain and aorta tissues, respectively. Briefly, the external blood of chopped tissues was removed with DW, followed by the treatment of sodium dodecyl sulfate (SDS), DNase, Triton-X 100, and peracetic acid. As a final step, the residual detergent was removed with PBS and DW. To validate the process, DNA and glycosaminoglycans (GAGs) quantification were conducted. To further investigate the preserved ECM components, proteomic analysis using LC-MS/MS was conducted. Finally, to validate the cell viability and proliferation and protein expression in BdECM and VdECM, human brain microvascular endothelial cells (HBMECs) and human brain vascular pericytes (HBVPs) were encapsulated in dECM. LIVD/DEAD assay and CCK test and immunofluorescence staining against PDGFR-β and VE-Cadherin were carried out. As a result, biochemical assay of BdECM and VdECM validated reduction of DNA content (25.90 ng/mg and 59.03 ng/mg) and preservation of GAGs (84.7% and 73.0% of native tissue). Proteomic analysis revealed that the main components preserved in BdECM are collagen and laminin, which is major constituent of basement membrane of BBB. On the other hand, the major components of VdECM are vimentin and tubulin, which regulate endothelial sprouting. Furthermore, over 95% cell viability and higher proliferation than collagen in BdECM and VdECM were verified. Finally, corresponding to the result of proteomic analysis, endothelial sprouting-like structure was observed in VdECM. Later, this study will be the basis for developing an in vitro platform for BBB to investigate the mechanism of neuroinflammation.