The Cellular Biocompatibility Of Whole-kidney Acellular Matrix In Rats By Perfusion

Since kidney is one of the vital organs of human body,renal diseases have severe effect on patients'health and quality of life.Researchers explored many methods for the treatment of renal diseases.In them,dialysis and renal allotransplantation are most effective to replace kidney function of end-stage renal disease as well as the acute renal failure.However,dialysis replaces only a small fraction of normal kidney function. Whereas,renal allotransplantation is restrained by donor shortage,allograft failure,and long-term immunosuppression.Therefore,it is urgent to explore new methods to give a supplement to current methods.The emergence of tissue engineering brings new hopes for the functional and biological replacement of end-stage renal disease as well as acute renal failure.There are many tissues having been engineered until now.Tissue engineering is a rising subject in recent years, which applies cells, engineering material methods, and creates suitable biochemical and physio-chemical factors to improve or replace biological functions of tissues. It is the study direction of treating function failure and decline of human tissues and organs. The basic theory and method is culturing cells to a certain amount in vitro, after which the cells are inoculated to a scaffold with certain space structure, to construct a cell-scaffold complex that is later transplanted in vivo to repair or replace injured tissue. In recent years, rapid development can be seen in tissue engineering, especially in the field of the urologic system. There are reports of the emergence of tissue engineered organ corresponding to various tissues, such as bones, cartilages, blood vessels and bladders. Oversea scholars even have put tissue engineered bladder into clinical experiments already, which become the first artificial tissue engineered organ implanted in human body. This not only established an example for numerous researchers, but also unsealed a wider unknown domain:Can all human tissues have artificially-created organs through approaches of tissue engineering? Up to the end of this research, no success in construction of tissue engineered kidney in vitro is found domestically or overseas. Emphasis of researches has been put on how to grow renal cells in vitro. There is little mention of suitable cellular scaffold. Our department produced a whole-kidney acellular matrix (ACM) scaffolds in rats by perfusion.At present,there is little related report about it. The cellular biocompatibility of the ACM is poorly understood.Objective1. To explore and discuss the conditions and approaches of preparation of whole-kidney acellular matrix (ACM) scaffold in rats by perfusion.After comparison of various cellular scaffolds, ACM is found a favorable supporting scaffold. It possesses the macro and micro structure of original tissues, and it is rich in collagen, elastic fibers. Moreover, it contains fibrillin, fibronectin, laminin, protein polysaccharides, hyaluronic acid and chondroitin sulfate, etc. After decellularization, the remaining extracellular matrix components have good bio-compatibility. Respectively, every component carries or receives different signals and attracts different cells, and therefore has significant influences on the adherence, migration and transfer of cells. At present, the method of preparing ACM is basically limited to soaking and eluting cells with enzyme and eradicator, which normally can not achieve decellularization for parenchymal organs. Using eradicator as the dissolvent for elution, the research introduces perfusion as the method to elute the internal renal cells via natural platforms including blood vessels, renal glomerulus and renal tubules. This study also involves exploring and discussing the approaches of preparation, and finally the establishment of a systematic method.2. To observe and prove the prepared ACM has a regular three-dimensional space structure and good porosity, and cells are completely eluted.As cell, the most important component to engender antigenicity during transplants, is eluted, no MHC antigen exists in the remaining ACM. The immunoreaction ACM triggers is Th-2 reaction. Different from the typical Th-1 reaction triggered by heterotransplantation, this reaction has no rejection to the implanted ACM, which shows that ACM contains merits including little otherness between species and excellent bio-compatibility. The ACM prepared with the methods established by the research is evenly white and semitransparent under macroscopic observation. It is examed by scanning electron microscope,,to prove ACM has favorable micro hole structure that facilitates cell growth and complete decellularization.3. Evaluate the cytocompatibility of ACMEvaluate the cytocompatibility of ACM with the L929 cells invitro to assess the possibility of ACM as the cytoskeleton and tissue-engineered urinary organ construction.Methods1. Preparation of whole-kidney ACM in ratsBoth kidneys from twenty 12-week old whistar rats were removed under anesthesia at abdomens with 10% chloral hydrate, and the ureters, venae renales and renal arteries were kept. For every couple of kidneys, one is randomly put in the control group while the other in the experimental group. Intravenous catheters were inserted through renal arteries for all kidneys to construct channels for perfusion. After a slight push of heparinized PBS solution to test the obturation of the channels, the experimental group was perfused in order with heparinized PBS solution,1% sodium dodecylsulphate (SDS) solution, deionized water,1% TritonX-100 solution and PBS solution that contains mycillin. The best approach was achieved after adjustments of the pressure and time of perfusion.2. The micro observation of whold-kidney ACM in ratsThe whold-kidney ACM was observed under scanning electron microscope after being fixed with 2.5% glutaraldehyde, dehydrated with acetone using the gradient method, replaced by isoamyl acetate, dried with critical point method, and finally sprayed in vacuum.3. Cytocompatibility of whole-kidney ACM in rats with L929 cells in vitor(1)Samples were randomly divided into blank group(without any cells),negative control group(culture media), experimental group(rat kidney ACM leaching liquor),and positive control group(culture media containing 0.64%phenol).L929 cells in the logarithmic phase were seeded in 96-well plates at the density of 4×103/well,with 5 wells in each group.At 24,72,and120 hours after incubation,cells were stained with MTT method to detect absorbance at 490 nm and calculate relative growth rate.(2)Control group(culture medium),experimental group(rat kidney ACM leaching liquor),and positive control group(culture media containing 0.64% phenol)were set up to detect cell apoptosis at 48 hours after culture using flow cytometry.Results1. The process of perfusion for preparing whole-kidney ACM in rats Twenty rats were used in the experiment. During the process, the speed of perfusion was slow at the beginning, about 10 to 40 drops per minute, and it increased gradually afterwards. After the solution was changed to 1% SDS, the color of kidneys gradually changed to white and semitransparent from external to internal, and it was segmented and lobate. After 120 to 150 minutes, the change of color was obvious, and the branching and canal structure was seen clearly with unaided eyes. When it reached 180 to 240 minutes, the kidney was evenly white and semitransparent. The speed of perfusion was kept at 100 to 120 drops per minute. The use of solution was measured to be 400 to 600 ml at this time.2. Results of micro observation of whole-kidney ACM in ratsUnder the scanning electron microscope, renal glomerulus and proximal convoluted tubule was found to have complete structure in the control group. As for the experimental group, a reticulate structure was found that was formed by basilar membranes around renal glomerulus and tubules and extra cellular matrix including collagen protein. All other spaces were regular-shaped, cell-free holes.3. Results of the cellular biocompatibility of whole-kidney ACM in rats(1)The actual absorption coefficient of experimental groups was0.620± 0.032,0.957±0.007 and 1.336±0.011,after24,72 and 120 hours.The actual absorption coefficient of negative control group was 0.602±0.017,0.965±0.005 and 1.347±0.006.There was no significant difference between the experimental group and negative control((P=0.902>0.05)). The grade of the cytotoxicity of the whole-kidney ACM in rats was 0-1 at every time intervals(24,72,and 120 hours).(2) At 48 hours after incubation.There was no significant difference in cell apoptotic rate between experimental group and negative control group(P>0.05).ConclusionsPerfusion is a simple and reliable approach for preparation of whole-kidney ACM scaffold. This scaffold is ideal as it has no cell residue, possesses complete physical structure, provides nice three-dimensional environment in favor of the growing of cells. Moreover, it is bio-compatible, not easy to induce rejection, and it has little otherness between species.