| BackgroundEnd stage renal disease(ESRD)is the terminal stage of a variety of chronic kidney diseases characterized by complete and permanent loss of kidney function,which has become one of the most highly fatal diseases in the world.The number of ESRD patients is very large and the dilemma of donor shortage is getting worse since the number of patients increasing every year with the number of donor kidneys for transplantation does not increase significantly.In order to alleviate this contradiction,scientists tried to regenerate the kidney through cell-scaffold technology in order to partially or completely replace the normal kidney function.One of the most important parts of cell-scaffold technology is kidney scaffold.Scientists have already tried kidneys of monkey,rat,sheep,pig and human,pig kidney is currently considered to be a suitable substitute for human kidney regardless of size and structure.The pig kidney scaffolds were identified further as a good source for cell-scaffold techniques.However,the kidney regeneration needs a huge number of cells for scaffold,which is not suitable for many researches.In this study,rat kidney scaffold is still used for study.There are two commonly used methods for decellularization of kidney:whole organ perfusion method and tissue section immersing and agitation method.In this study,we will compare scaffolds of two kinds of decellularization methods and choose a better way to get more satisfying scaffold.Another important part of this technology is the need for suitable seed cells.Scientists had already tried embryos stem cells(ESCs),neonatal rat kidney cells,fetal kidney cells,human umbilical vein endothelial cells(HUVECs)and achieved good results.However,these cells are not suitable for clinical application.There are ethical problems,tumorigenicity problems,cell regulation problems and etc.Adipose tissue-derived stem cells(ADSCs)as emerging pluripotent stem cells recent years.ADSCs have been used for regeneration of bone tissue,adipose tissue,muscle tissue and nerve tissue.Studies have shown that ADSCs as mesoderm-derived stem cells can differentiate into mesoderm-derived cells such as osteoblasts,adipocytes,tubular cells,muscle cells,nerve cells and other adult cells.This study intends to use ADSCs as seeding cells and proper rat kidney scaffold for renal regeneration.The growth status and differentiation potential of ADSCs will be assessed.Part 1 Preparation and characterization of decellularized kidney scaffold PurposesTwo kinds of commonly used rat kidney scaffolds will be compared and the better one will be used for further renal regeneration studies.Methods(1)Rat kidney retrieval250g adult male Wistar rats were used in this study.The animals were anesthetized well and both kidneys were removed completely with renal artery,vein and renal ureter after thoroughly perfused with PBS.(2)Decellularization processThe artery and ureter were catheterized in whole organ perfusion group,and kidneys were cut into about 4 sections with thickness around 2-3mm.0.5%SDS was selected as decellularized liquid.Record the time taken for decellularization to visible translucent condition in both groups.Then the scaffolds were washed using PBS for 24 hours.(3)Compare two kinds of kidney scaffolds Various of methods were used to compare the kidney scaffolds obtained using both methods to select the proper one used for next step.Including:1)Time used for decellularization process from normal colour to white transparent appearance in both groups.2)Morphological comparison using HE staining to observe the morphological differences between the two groups.3)Movat staining was used to observe whether there is significant difference in tissue staining between the two groups.4)Scanning electron microscopy(SEM)was performed to observe the appearance,thickness,and shape of the fibrous strands of both groups.5)Immunohistochemistry and immunofluorescence were used to detect the distribution of structure proteins such as fibronectin,elastin,and collagenin IV in the two groups of scaffolds.6)Tissue dry weight,DNA content,protein content,sGAG content and cytokines content were detected,and the differences between the two groups were compared.Results(I)The two groups of kidney were decellularized to the similar white transparent appearance,and the time used were significantly different.Decellularization efficiency of whole scaffold group(6.6 ± 0.27h)was significantly higher than the scaffold section group(25.21 ± 0.62h).(2)HE staining shows that the structure of two kinds of acellular scaffolds are similar without structure damage and no typical cellular compartments could be observed.Movat staining showed that whole scaffold dyed visible deeper in collagen and elastic fiber than the scaffold section.SEM showed a similar fiber structure of two kinds acellular scaffold.Immunofluorescence and immunohistochemistry staining showed similar distribution of structural proteins.(3)Two groups of scaffolds were compared,showing that the dry weight of whole scaffold group(0.023 ± 0.005g)was significantly higher than scaffold section group(0.016 ± 0.002g);the DNA content of whole scaffold group(2.07 ± 0.11ng/mg)was slightly higher scaffold section group(1.06±0.13 ng/mg);the protein content of whole scaffold group(0.228 ± 0.01 μg/mg)is much more than scaffold section group(0.039 ± 0.004 μg/mg);sGAG of whole scaffold group(9.62 ± 0.08 pg/mg)is higher than the scaffold section group(1.19 ± 0.02 pg/mg)and content of cytokines were significantly higher in whole scaffold group than the scaffold section group.Conclusions(1)Whole organ perfusion method and kidney sections immersing and agitation method can both abtain scaffolds efficiently using 0.5%SDS.However,the two kinds of scaffolds are not identical and the two methods can not be simply interchanged.than.(2)Whole organ perfusion method is more efficient,and can retain more cytoskeleton structure protein sGAG and cytokines in scaffolds compared with kidney section immersing and agitation method.With more structure component remained,whole organ perfusion maybe more suitable for kidney regeneration research.Part 2 ADSCs isolation,scaffold recellization and related identification PurposeMethods to isolate ADSCs from tissues were studied,and isolated ADSCs were identified.The cultured ADSCs were used as seed cells to regenerate the kidney.After culturing for different days,the growth of ADSCs in the scaffolds was observed and identified.Methods(1)Fatty pads were taken from the inguinal area of rats and cells were isolated using type I collagenase and modified isolation methods for primary culture.It takes about 5 to 7 days to 80%cell fusion density.(2)After passage,the osteogenic,adipogenic differentiation and flow cytometry of ADSCs were performed for indentification.(3)After cell expansion cultivation,the cells were digested with 0.25%trypsin.The cells were centrifuged and resuspended to adjust the cell concentration to 1～5 × 107.Each time about 8～10ml cell suspension were needed.(4)Recellularizationa.The cell suspension was injected into the rat artery at a rate of 1 ml/min,and about 2 ml were injected into the renal artery of each rat kidney scaffold.Then connect the ureter and the interface channel,the channel is connected to the cell suspension.Then the renal artery channel was and waste outflow channel were closed.The channel to balance pressure was connected to the negative pressure pump.Turn on the negative pressure pump,adjust the negative pressure to-40cmH2O,control the rate of cell injection at about 1ml/min,about 2ml cell suspension were injected into ureter.Recellularized scaffold was placed overnight for cells to adhere.b.Using the same method for SDF-1 addition group.The prepared whole scaffold was soaked in 0.25 ng/mL SDF-1 solution.The cells were similarly perfused into the scaffold and was allowed to adhere overnight.(5)Cultivation of regenerated kidney Before starting the perfusion process,connect the balance gas containing 5%CO2 and 95%air to the air bottle,then connect the gas to sterile filter and sterile gas went through the fresh DMEM/F12 medium.Regenerated kidney was perfusion using this medium and cultured at 37 0 C for different days.(6)Identification of regeneration kidney HE staining was used to observe ADSCs status in regenerated kidneys cultured for different days and group with addition of SDF-1.Immunofluorescence and immunohistochemistry staining were used to find cells differentiation status.Results(1)ADSCs harvested using our modified method shows satisfying cell morphology and growth rate.Osteogenenic,adipogenic differentiation showed positive result.Flow cytometry identification CD90,CD44 was positive and CD45 is negative,indicating that the status of ADSCs is good.(2)After recellularization,cells attached to the scaffold evenly in glomerular and tubular structures,forming glomerular-like and tubular-like strucutres.The number of cells reached its peak in day 5.After day 5,cells reduced gradually.(3)With addition of SDF-1 and cultivation for 5 days,the number of cells were much more than control group.But ADSCs formed cell clusters without clear structures.(4)Immunohistochemistry and immunofluorescence analyses showed that cells in regenerated kidney underwent differentiation toward endothelial cells and tubular cells in both groups.Expression of CD31,Vwf,aquaporin I and α1 Na+-K+ATPase were positive in both groups.Conclusion(1)Cells obtained using our method were in good cell state and could be used for further study.(2)ADSCs attached to scaffold reached the peak in about five days in regenerated kidney.(3)The cells attached to scaffold can express endothelial and tubular markers,indicating that ADSCs differentiated toward glomerular and tubular direction.ADSCs can attach to the decellularized scaffold,grow and differentiate toward vascular endothelial cells and tubular cells.It is feasible to use ADSCs as seeding cells.(4)SDF-1 could attract more cells toward the scaffold lumen and cavity and form cell clusters without clear glomerular and tubular structures.The use of SDF-1 still need further study. |
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