Fabrication And Characterization Studies Of Human Umbilical Cord Decellularized Extracellular Matrix Hydrogel

BackgroundThe study of human organ development and diseased states is hampered by medical ethics and intrinsic differences between animal models and human biology.Advances in three-dimensional(3D)cell culture techniques facilitated by a deeper understanding of extracellular matrix(ECM)biology combined with greater knowledge about cellular microenvironment and stem cell differentiation programmes.The existing 3D cell culture strategies are dependent on animal-derived decellularized extracellular matrix(dECM)systems or chemically synthesized matrix systems,such as Matrigel,collagen and cross-linked polyethylene glycol hydrogels.The compositions of these heterologous animal-derived culture systems are fully different from those of human tissues,and their use often poses great risks when in downstream clinical applications,with immunogen and pathogen transfer being the substantial concerns.While,chemically synthesized culture matrix can only partially reproduce the native features and the corresponding microenvironment of real organs due to poor histocompatibility and simplex composition.As a result,it is still impossible to translate the basic research results of 3D cultured products(such as organoids)into clinical applications.This study proposed a new method for the preparation of human umbilical cord dECM hydrogel and explored its feasibility as a 3D cell culture matrix,that would be really utility in 3D cell culture and regenerative medicine in the future.Method1.UC-dECM hydrogel fabrication and standardized identification.2.Isolation,culture and identification of umbilical cord-derived mesenchymal stem cells(UCMSCs).3.3D culture of UC-MSCs based on UC-dECM hydrogel and identification of UC-MSCs growth and survival.4.induce differentiation UC-MSCs into hepatocyte like cells(HLCs)and hepatocyte function identification of HLCs.5.3D co-culture of UC-MSCs and HLCs based on UC-dECM hydrogel and related functional identification.Results1.The UC-derived dECM hydrogel maintained a natural extracellular matrix structure with characteristic biochemical compositions,3D network structure features,provide a favourable microenvironment,including structural adhesive networks and soluble signal storages,to support cell adhesion and growth.2.The UC-MSCs were successfully isolated from the umbilical cord.UC-MSCs cling closely to the hydrogels network via pseudopodia like microvilli that 3D structure creates a beneficial condition for cell proliferation.UC-MSCs proliferated gradually and evenly dispersed throughout the dECM hydrogel with the extension of time.Theresults confirm the proliferation rate in the UC-dECM hydrogel was significantly higher than that in matrigel and serum-free medium,and the UC-MSCs maintain the stem cell properties during the whole culture process.3.We efficiently induce differentiation MSCs into HLCs(hepatocyte-like cells)with cytokines and small-molecule compounds.UC-MSCs and HLCs were co-cultured in UC-dECM hydrogels at specific ratios.The rusult confirmed that the 3d co-culture system has the functions of MSCs and HLCs,indicating that the prepared human UC-dECM hydrogel can realize multi-cell co-culture.ConclusionThe human UC decellularized dECM hydrogel provides a soft adhesive network that facilitates cell expansion and provides a 3D structured microenvironment for cell co-culture.The human UC-derived dECM effectively retained the natural organ microenvironment and bioinformation,including 3D adhesive networks and dECM proteins,highly similar to those observed in human liver endoderm tissue.These homologous and compatible proteins are more suitable for the purpose than those found in animal-derived dECM but are difficult to reproduce via artificial synthesis.Thus,our human UC-derived dECM hydrogel is more suitable for UC-MSC growth and proliferation as well as 3D co-culture of multi-cells.In addition,the UC is human tissue with no ethical concerns or challenges.It is abundant,has low immunogenicity,and can be produced at a large-scale,providing a new choice for cell 3D culture and regenerative medicine.