| In recent years, chronic kidney disease (CKD) has an increasing incidence and end stage renal failure(ESRD) has been a public problem. Currently there are two therapies for the disease:hemodialysis and kidney transplantation. Although hemodialysis can prolong the life of end-stage renal disease patients, but it has poor excretory function and could bring multiple complications for the patients. The quality of life and survival time is not ideal. Kidney transplantation is currently the best treatment, but donor kidneys are short, which poses a serious impediment to the widespread application of kidney transplant, resulting in the death of many patients waiting for organs. In addition, the rejection after transplantation and use of immunosuppressive drug bring side effects which affect the long-term survival of transplant and recipient. In recent years a promising approach named cell-scaffold technique for functional organ replacement has emerged.The cell-scaffold technique is based on the decellularization of native organs and recellularization of the acellular organ scaffold. The purpose of "decellularization" of native organs is to remove cellular components which cause an immunogenic responses upon implantation. The acellular matrix is then recellularized with organ-specific cell types that repopulate the organ and restore organ function. Embryonic stem cells are good cell sources for kidney regeneration, which has pluripotent cell capability and could be induced to renal linage differentiation.Towards this end, the current study aims to establish an effective recellularization technique using embryonic stem cells that are capable of creating fully functional renal tubular structures within acellular kidney scaffolds.Part I preparation and characteristics detection of acellular kidney scaffoldsPurposeThe goal of the present study was to develop an effective rat kidney decellularization method for whole organ engineering using either the ionic (SDS) or non-ionic (distilled water) detergents.Methods1. kidney retrievaladult male Wista rats weighing 200-300 g were used. After anesthesia with 10% choral hydrate; a longitudinal abdominal incision was made and the both kidneys, aorta, vena cava, and ureter were identified. Then, the kidney was perfused using PBS to remove blood.2. Kidney decellularizationAn ad hoc device has been designed and constructed to perfuse kidney under controlled conditions with different solutions for the decellularization process. The kidney was transferred at room temperature into the chamber containing the solution used for the organ decellularization. The cannula previously inserted in the renal artery was connected to a peristaltic pump. The kidney was perfused with detergent solutions (0.5% SDS, distilled water, PBS) at a flow rate of 0.5 mL/min until the kidney was semi-transparent.3. Characterization of the decellularized scaffold(1)To assess cell and nuclear clearance as well as preservation of collagen, hematoxylin & eosin and Masson Trichrome staining were performed. Immunohistochemistry was performed to decetect the expression of collagen IV, laminin and fibronectin. Transmission electron microscope was used to examine the extracellular matrix in the DC kidney scaffolds.(2) The DNA and collagen content was tested using the kit. The cytokines in DC kidney scaffolds, such as HGF, TGF and VEGF, was extracted using an ELISA kit(3) To confirm the integrity of the vascular tree and demonstrate that fluid injected into the vasculature flowed throughout rather than extravasated throughout the organ. And orthotopic transplantation of the acellular scaffolds was also performed.Results1. At the end of the perfusion, a white acellular scaffold was obtained, which retained the overall shape and the macroscopic structure of kidney.2. Histologic evaluation with H&E and masson staining showed no typical of cellular nuclear material was observed as compared to normal rat kidney staining.3. Immunochemistry showed collagen IV and fibronectin, which were similar to that in native rat kidney tissue4. TEM analysis documented the complete removal of cellular components and uniform preservation of basement membrane integrity.5. We removed approximately 95% of DNA in comparison to the native organ, while still retaining total collagen levels similar to those in cadaveric kidney tissue. ELISA assays showed levels of the cytokines HGF, TGF-b and VEGF in kidney scaffolds that were similar to that of the intact kidney.6. We were able to retain an intact vascular network that retained hierarchical branching structures through the renal artery as depicted with contrast media.ConclusionWe have simplifed the decellularization process and evaluate the decellularized scaffold from as many aspects as possible and provided sufficient evidence for kidney regeneration through the decellularization-recellularization technology.Part II recellularization of the kidney scaffoldsPurposeRecellularization of the kidney scaffolds with murine embryonic stem cells to get a functional and transplantable kidney.Methods1. We choose the murine embryonic stem cells as the cell source. Immunochemistry was perform to detect the stem cell markers, such as OCT4, NONOG, SOX2.2. In vitro biocompatibility was tested with mouse ES cells. After decellularization and sterilization, the scaffolds were seeded with 5 × 107/ml diluted in 2 ml medium from the renal artery and ureter. Cells were allowed to attach overnight, after which perfusion culture resumed.3. Perfusion media was infused through sterile access ports (Millipore) to minimize the risk of contamination. Media was allowed to equilibrate with 5% CO2 and 95% room air before reaching the cannulated renal artery at 1 ml/min for 7 days.4. H&E staining was performed to test the histology of the regenerated kidney, we performed orthotopic transplantation of regenerated kidneys just as the protocol above for transplantation of acellular scaffold for functional test.Results1. Oct4, Nanog and SOX-2 in cultured mouse embryonic stem cells was well expressed.2. These cells were able to attach and grow in the ECM after cell seeding and perfusion. Using hematoxylin and eosin staining, we showed that the cells were localized in vessels, gromeruli and renal tubules.3. Regenerated kidneys produced less urine than decellularized kidneys (1.8 ± 0.7μl min-1 (mean ± s.d.) compared to 4.9 ±1.4 (0,1 min-1 in decellularized kidneys) with higher creatinine (1.4 ± 1.4 mg dl-1) and urea (26.5±1.6 mg dl-1) than decellularized controls.ConclusionsWe confirmed that this is feasible with stem cells in a decellularized kidney scaffold. Overall, this study indicates that our ultimate goal of delivering a transplantable, immune-tissue free, engineered kidney to patients is possible. |
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