Production Of Decellularized Porcine,Leporine And Murine Renal Biological Scaffold And Co-culture Exploration Of Renal Scaffolds Slices With HEK Cells In Vitro

[Objective] Currently kidney transplantation is one of the most optimal replacement therapies to end-stage renal disease(ESRD).After recepting the living donor or cadaver donor kidney transplantation,the receptor patient need long term administration of immunosuppressive drug.However,the transplantation of bio-artificial non-rejection tissue engineered kidney maybe solve this problem.And meanwhile porcine kidney were expected to much for its similarity with human kidney in physiological structure,but the species difference of co-culture with heterogeneous cell has not been reported as yet.So the purpose of this study is to produce the decellularized biological renal scaffold of pig,New Zealand white rabbit and SD rat and to explore the influence of porcine,leporine and murine renal biological scaffolds to the co-cultured seed cells HEK.[Methods] Adult healthy kidneys from 10 pigs,28 New Zealand white rabbits,and 28 SD rats were fetched and divided into normal groups and scaffold groups randomly and equally.The scaffold group kidneys were perfused for decellularization with heparinized PBS,1% Triton X-100 and 1% SDS through the renal artery successively.All the kidneys of each group were characterized in histomorphology and detected for mechanical properties.And we conducted the co-culture experiments with the slices of each group and HEK cells,then analyzing the expression of PCNA in HEK cells by immunofluorescence assay and the value of gray level.[Results] In each scaffold group after perfusion,the HE staining of cell nucleus in the slices is negative,Masson's trichrome staining of collagen is positive,and immunofluorescence staining of the Collagen I and Collagen IV is also positive.The DNA content in the normal group of the three species was much higher than that in the scaffold group,and the difference was statistically significant(P<0.05).And with the scanning electron microscopy,faveolate orifice structure and typical glomerulus-like structure are observed.Characterizing the mechanical properities,the Young's modulus of each scaffold group are similar with that of the corresponding normal group.Analyzing the gray level value of the HEK's cellular immunofluorescence images after the co-culture,it is revealed that the value of PCNA/DAPI in animal group was higher than that in the blank control group,and the difference was statistically significant(P<0.05).Furthermore,there is no significant difference on the PCNA/DAPI of HEK between the pig,New Zealand white rabbit and SD rat renal scaffolds.[Conclusions] The decellularization method of our study can wipe off the cells in porcine,leporine and murine kidney,retain the extracellular matrices and reserve the spatial structure and similar mechanical strength.And it is an effective way to produce the decellularized pig,New Zealand white rabbit and SD rat renal biological scaffolds.Importantly,the produced decellularized porcine,leporine and murine kidney scaffold slices can improve the proliferation activity of heterogeneous HEK cells when co-cultured in vitro,and there is no species difference on the improvement between pig,New Zealand white rabbit and SD rat.