Research On Use Of Decellularized Pig Kidney Scaffold For Kidney Regeneration

Purpose of studyCurrently there are two major methods for treatment of end-stage renal disease: hemodialysis and kidney transplant. Although hemodialysis can prolong the life of end-stage renal disease patients, but it has poor excretory function, the current efficiency is only 10%, and with no endocrine function. The quality of life and survival time is not ideal. Kidney transplant 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.Regenerative medicine brings hope to solve this problem. Broadly speaking kidney regeneration is available in four main ways:(1) simple cell-based regeneration; (2) embryonic kidney transplant; (3) biosynthesis of artificial materials; (4) scaffold-based whole organ regeneration. Kidney regeneration based on cells currently focuses on the repair mechanism of stem cells in kidney injury. For stem cells to repair kidney function, they usually enter the body through the blood system and are not specific, and by now there has been no test confirming their repair mechanism. For 3D cell culture, such as induced pluripotent stem cells and embryonic stem cells, they in vitro form glomerular and renal tubule-like structures, but cannot form a complete kidney. The metanephros is the precursor of a grown kidney. With transplant of embryo metanephros to the receptor on the greater omentum, the metanephros will have new blood vessels growing into the receptor and it can grow to a normal kidney size. In theory the metanephros is indeed a good source for regeneration of kidney, but its application still faces many problems, such as ethical arguments when applied to the human embryo, and long-term survival has not been reported. The xenograft using a non-human metanephros by means of blastocyst complementation may solve the problem of immune rejection, but a new kidney has poorly structured vessels and is not ready for transplant. And being on the peritoneum affects the form, structure and functionality. The kidney has a complex structure and contains a variety of cells, with urinary tract and endocrine functions. Man-made synthetic materials as scaffold are not proper for use in the kidney.In recent years, using decellularized organs as scaffolds for generation of the whole organ has become a hot topic of study. A decellularized scaffold consists of the extracellular matrix (ECM) and is a support network with a 3D structure and functions. The ECM can provide a 3D scaffold for cells, secrete and store growth factors and cytokines, and regulate signal transduction. The ECM of one’s own provides a good support for organ regeneration due to good biocompatibility, essential substances for cell survival (proteins and polysaccharides), stromal growth factors and cytokines, complete blood vessels, and the ability to induce differentiation of progenitor cells into organ-specific cells. Progress of cutting-edge technologies in recent years allows researchers to start exploring the use of extracellular matrix of one’s own organ for kidney regeneration. In short, decellularized scaffolds provide a good platform for regeneration by retaining a micro-environment essential for cell survival. Research success has been reported in use of decellularized scaffolds for organ regeneration in heart, lung, liver, aortic valve, and tendon. But whether the decellularization reagent destroys some key parts of the ECM is not clear. Full decellularization is very important for organ regeneration, because residual cell components may cause apoptosis of newly injected cells and inflammation. Therefore, decellularization is an essential process. Studies have shown use of rat kidney cells for re-cellularization of decellularized scaffold of rat kidney, obtaining functionally transplantable kidneys. However, the kidneys are too small for clinical use. The pig kidney has a similar anatomy to the human being, and it features low incidence of branched and aberrant renal arteries. From the anatomy point of view, the pig kidney meets the requirements of xenotransplantation.In response to these issues, we intend to use pigs as experimental animals to prepare decellularized kidney scaffold. Through relevant identifications, we will use embryonic stem cells to re-cellularize decellularized scaffold in order to obtain functional transplantable large animal kidneys and provide the scientific basis for human kidney regeneration.Methodology of study1. Make a pig kidney decellularized scaffold. Using a domestic pig as the experimental subject, anesthetize it and take out its kidney. Use a self-made kidney perfusion device to connect the decellularization agent and pig kidney artery, immediately use sodium phosphate buffer solution (PBS) to flush the kidney blood to prevent clotting. Then perfuse 0.5% 12 alkyl sulfonate (SDS) and 1% Triton-x100 to the pig kidney at 8ml/min to decellularize it. When the kidney turns white and slightly transparent, stop perfusion A and cleanse with PBS for 4 days until the SDS is clear. Thus the decellularized scaffold is obtained.2. Identification of pig kidney decellularized scaffold. Use immunohistochemistry, immunofluorescence quantitative DNA analysis, polymerase chain reaction (PCR), and determination of collagen content to identify that there are no cell residues. Masson staining is used to find out whether the cell components are completely removed and the retention of collagen; immunofiuorescence is used for the identification of type Ⅳ collagen and laminin; angiography is used to identify the integrity of the vascular system; DNA and collagen detection is used to detect the content of DNA and collagen before and after the decellularization; Elisa detection is intended to detect the change of some cytokines; the PCR method is for the determination PERV virus; the molecular biology method is used for determining that there is no liquid residue in decellularization. With these identification methods we finalize the method for preparation of kidney decellularized scaffold.3. Culture and identification of embryonic stem cells. After culturing mouse embryonic stem cells, we use the immunofluorescence method to identify three stem cell markers:OCT4, SOX2 and NANOG, to determine their stem cell properties. We use massive proliferation for embryonic stem cells to prepare for cell seeding in the decellularized scaffold.4. Cell seeding. The massively proliferated embryonic stem cells are used to prepare cell suspension of 10ml at a concentration of about 5×107/ml. Use a 28G trocar to puncture and inject the suspension into the kidney through multiple points. Inject once every 24h for three times. After the last injection, leave the kidney undisturbed for 24h. After each injection, place the kidney in a cell culture environment of 37℃ and 5% CO2 and 95% air mixture.5. In vitro organ culture. After injection of kidney cells, use related culture medium for continuous perfusion culture. First add a mixture of 5% CO2 and 95% air, which should be cleaned by filtering. The entire perfusion culture system is placed in a clean environment of 37℃ for continuous culture often days. Then after tissue fixation use HE staining and immunofluorescence for identification.Results5. In gross morphology, after 30h of 5% SDS and 1% Triton-X100 continuous infusion rinse, the kidney gradually becomes slightly transparent and white, producing a decellularized scaffold.6. Masson identification shows that the cellular components are completely removed, leaving only blue collagen; DNA quantitative analysis shows that only a small amount of DNA content is left, below 5% of the normal content; no difference is found in content of collagen and some cytokines before and after decellularization; immunofluorescence indicates that IV collagen and laminin remain intact after decellularization. Angiography shows that the contrast agent branches into the various levels of blood vessels after entering the renal artery, and finally seeping from peripheral vessels. Throughout the process there is no leakage of contrast agent on from the main vessels, demonstrating a good integrity of the blood vessels of the scaffold. PCR detection shows that the porcine endogenous retrovirus (PERV) is negative, meaning that the seeding cells are not infected.7. The embryonic stem cells grow well. After continuous subculture, they still maintain a good clone status with smooth edges. By immunofluorescence assay, the stem cell-related markers OCT4, SOX2 and NANOG are well expressed, which proves that after massive proliferation the embryonic stem cells still maintain good stem cell properties.8. The cell cultivation process goes smoothly and cell attachment is fine. After continuous culture using the self-made large animal organ culture system, HE staining and DAPI immunofluorescence assay show cell growth in the kidney scaffold, proving that continuous organ perfusion culture can maintain proper nutrition supply to the adhered cells in the scaffold.Conclusion4. We set up a fast and effective large animal decellularization device and a scaffold preparation process. By combined perfusion of 0.5% SDS and 1% Triton, a properly structured and functional decellularized kidney scaffold can be prepared.5. The prepared scaffold can retain a normal kidney’s complete 3D structure, vascular system and collection system. The extracellular matrix proteins and cytokines are well preserved to promote stem cell attachment and proliferation.6. Through multi-point puncture for cell seeding, a simple and effective large animal decellularized scaffold seeding process is established. The stem cells can achieve proper attachment and proliferation, providing a good basis for cell seeding in large animal decellularized kidney scaffold in future.