The Construction And Biocompatibility Of Decellularized Rat Kidney

Background: The incidence of chronic kidney disease has been increasing over theworld, and the number of patients suffered from end stage renal disease is alsoincreasing. Dialysis and kidney transplantation are the major replacement therapycurrently, and kidney transplantation is the most effective and ideal therapy, but thedonors are limited. Moreover, kidneys allograft will be attacked by immune system ofhosts, so the immunosuppressive drugs are needed for patients received kidneytransplantation. In recent years, the development of tissue engineering has maderegenerative organs as transplanting organs possible. Tissue engineering material ofhigh quality is very important to organ regeneration, and the kidney structure ofkidney is so complex that the artificial scaffold cannot achieve this kind of complexity.The invention of decellularized organ scaffold has made acellular kidney scaffold astissue engineering material scaffold possible. The complex structure of kidney ispreserved in decellularized kidney scaffold, and there are signals which can promotethe localization and differentiation of cells in the scaffold, all of which are beneficialto kidney regeneration. At present, there have been some research on kidneydecellularization abroad, but there are few in our country.Objective： Construct acellular kidney scaffold by decellularizing cells；Determinethe microstructure and ingradients of extracellular matrix.； Determine thecytocompatibility of the scaffold；Observe the survival condition of seeded iPSCs indecellularized kidney scaffold.Methods:(1) The kidney was harvested with artery and vein, and perfused by1%SDS solution and1%Triton-X100solution continually. Observe the morphology andcolor of kidney scaffold, determine whether there is cell residue and by HE staining,observe the microstructure of scaffold by transmission electron microscope. Immunohistochemistry staining is used to determine the preservation of ingredients ofextracellular matrix.(2) Treat cells with medium incubated with kidney scaffold, anddetermine the cytocompatibility by MTT assay and flow cytometry.(3) Harvest iPSCswith a number of1×107, and inject them into kidney scaffold, then incubate for24hours and observe the survival of iPSCs.Results:(1)1%SDS was perfused continually for12h, then1%Triton-X100wasperfused for30min. After the perfusion, the kidney became white and translucent. HEstaining results indicated that there was no cell residue in the scaffold, and thestructure of glomerulus and tubule was preserved intact; The Periodic Acid-Schiff(PAS) staining and immunohistochemistry staining results indicated that theingredients of collagen Ⅳ，Elastin, Laminin and glycoprotein were preserved. Thetransmission electron microscope results showed that there was no cell residue, andthe basement membrane of decellularized kidney scaffold was continuous and intact.The mesangial matrix was also preserved.(2) The results of MTT assay showed that the medium incubated with kidneyscaffold had no effect on the proliferation ability of rat mesangial cells,49F cells andHK-2cells. The flow cytometry results indicated that there was no significantdifference in apoptotic rate and necrotic rate between cells treated by mediumincubated with kidney scaffold and cells incubated under normal condition.(3) There are survival iPSCs in decrllularized kidney scaffold.Conclusions:(1) Kidney cells were decellularized successfully by1%SDS and1%Triton-X100, there was no cell residue in the kidney scaffold and the structure ofglomerulus and tubule was preserved intact. The important ingradients of extracellularmatrix were also preserved.(2) The proliferation ability and apoptosis rate of ratmesangial cells,49F cells, HK-2cells and MPC5cells treated by medium incubatedwith kidney scaffold have no significant difference with those cells incubated innormal medium.(3) The seeded iPSCs could partly survive in the kidney scaffold.