Pilot Study Of A Kind Of Bioartificial Glomerular Filtration Membrane

PARTIFabrication of artificial glomerular filtration membraneBackground:The kidneys perform a wide array of functions, Including regulation of water and electrolyte balance, excretion of metabolic waste and so on. Firstly, the kidneys receive an enormous blood flow. Some plasma filters selectively into bowman's space, and then through the tubular reabsorption and secretion to complete this function. Now hemodialysis is the commonest substitute method in ESRD patients. Its core component is the dialysis membrane. Dialysis membrane is a polymer material, processed through the spinning process. Such membrane is very heterogeneous in size and thickness. It is very difficult to further add other processing elements, such as sensor, micro-switch. To this end, we hope to choose new materials and processes, to fabricate a new filtration membrane. In the future, maybe we can add some processing components, such as the capacity of receptors, micro motors, switches, and so on.Methods:We use MEMS technology, silicon is the main frame and metal film is a porous film. In the bottom of silicon, there are many rectangular array windows, in the other surface, there is a metal membrane with a lot of slit pores. Detailed steps including:1,we select double-sided polishing and oxidation silicon as the substrate, and remove the surface adsorption of organic pollutants and particles by bombardment of high energy ion beam.2,coating photoresist in the both faces of silicon. The photoresist thickness is 5 microns.3, baking photoresist in a special procedure.4, the silicon dioxide was then patterned by photolithography, The wavelength of UV light is 4000A and Exposure power is 275 watts.5, selective dissolution of exposured photoresist. Examine the image by the microscopy.6, washing the silicon and baking photoresist.7, buffered hydrofluoric acid is used to remove the silicon dioxide.8, on the other side, chromium and copper were sputtered, respectively.9, patterned silicon through coating photoresist, exposuring, developing. A filtration membrane was formed by electroplating nickel.10, Remaining photoresist was removed.11, hydrofluoric acid is used to remove the silicon and silicon dioxide.12, electroplating aurum.Result:Through standards-based MEMS technology, including etching, sputtering, electroplating and a series of processing, we fabricated a porous thin film. The various processing parameters were carefully optimized for this study. We have cutted it into 1cm×1cm or 2cm×2cm silicon chip. Finally, we examined and observed all samples carefully under the microscopy. We found that thin film has regular slit pores and fully meet design requirements.Conclusion:The filtration membrane pores are completely of the same size. Pore distribution is regular. The film thickness is about 2-3 microns. PART IIBiocompatibility and cytotoxic evaluation of artificial glomerular filtration membraneIntroduction:All materials contacted with body fluids or implanted in body, should have a good biocompatibility. Moreover, in order to better simulate the glomerular function, we plan to culture podocytes or endothelial cells on the artificial membrane filtration. Firstly, we need to detect biocompatibility and cytotoxicity of the artificial glomerular filtration membrane. Secondly, we plan to observe the morphology of cultured cells in the artificial filtration membraneMaterials and methods:After passing through test, the artificial filtration membrane were soaked with diluted hydrochloric acid to remove residual various impurities. After sterilization, they were coated by collage type I, type IV, and laminin. We observe the impact on the pore size of coating material by SEM. MDCK cell was cultured on the surface of artificial membrane. The live/dead cell was counted after trypan blue stain. The proliferation of cells was observed by MTT test, the extracellular matrix (FN and type IV collagen) and inflammatory cytokines (IL-1 BETA, and TNF alpha) in the cell culture medium were test by ELISA. In the end, we observed MDCK cell morpholopy by scanning electron microscopy.Results:(1) The size of artificial filtration membrane pores has not changed after coated with extracellular matrix. (2) Our research showed that when the pore size is 15μm or more, the cells is difficult of adhesion and proliferation. When we narrowed the pore size, the cell was sparse, however, the cell was easier to adhesion, proliferation and formation of monolayer on the surface of thin membrane of pre-coated extracellular matrix. (3) We found that cells adhesion and growth on different substrates coated membranes were not the same. Cells were easily to proliferation on the type IV collagen-coated thin membranes in the initial phase (1-2 days). There was significant difference between the groups. However, the differences between groups disappeared in the later time. Cells count reached a peak on days 4. The cells count showed a downward trend on extend time (see Figure 2-3). (4) When cells were cultured on the membrane, the number of dead cells was about 5-13%in the total number of cells. It was very similar to the results of cell cultured on glass. The results indicated that membrane had no apparent cytotoxic effects. The result was further confirmed by MTT test. (5) The release of inflammatory factor IL-1 beta and TNF-ALPHA in the cell culture supernatant in porous film were similar in the cell culture glass plate. There was no significant difference between groups. (6) in the cell culture supernatant, We have detected free collagen type IV and fibronectin. artificial Filtration membrane didn't stimulate or weaken the synthesis of extracellular matrix. (7) MDCK cells formed a flat single-cell fusion in day 3 or 4. Cell morphology on the surface of silicon filtration membrane was similar to conventional MDCK cells. If we continue to culture cell, they will show a cubic shape, and they are linked closely together. There is a large number of microvillus on the top of cells.Conclusion:Our research shows that porous membrane based on MEMS technology has not significant cytotoxicity. It does not stimulate cell release of inflammatory factors and it does not promote cell death. We explored the impact of extracellular matrix adsorption on to conventional MEMS materials, on cell growth, attachment and morphology. The film allowed highly differentiated growth of renal epithelial cell. Cell growth and differentiation on the thin film replicated the growth and differentiation observed in standard tissue culture material systems. Part IIIDetermination of filtration propertiesIntroduction:Current researches show the major determinants of glomerular permeability are endothelial fenestration, glomerular basement membrane and podocyte slit diaphragms. Slit diaphragms is the most important portion. Therefore, we cultured podocytes on the surface of bioartificial glomerular membrane and detected properties of that composite filter membrane.Methods:we cultured immortalized podocytes on the surface of poly silicon film. When cells covered the entire film, it would be put into a cell made by ourselves. There are some solutes with different molecular weight in the cell culture media. The cell culture medium flowed through the film, we examined the concentration of various solutes in the inflow and outflow fluid and the amount of filtration fluid and calculated the filtration fraction and the apparent clearance of the solutes.Results:(1) In this study, transmembrane pressure by adjusting the air pump was set to 10 mmHg. the cell culture medium flowed through film with a velocity of 2 ml per minute, in a certain period of time, the velocity of filtration fluid was 0.52±0.07 ml/min. The result indicated that the membrane filtration fraction is about 26%. The apparent clearance rate of vitamin B 12 was 1, the apparent clearance rate of albumin was about 0.136, and the apparent clearance rate of human immunoglobulin G was 0.019. (2) We changed transmembrane pressure by adjusting the air pump and we will found red blood cells in the filtration solution when the transmembrane pressure was greater than 40 mmHg, it indicated that membrane was prone to rupture at this pressure. If the transmembrane pressure is lower than 20 mmHg, we can't find red blood cells in the filtration fluid. (3) if the liquid flow is 2ml/min and transmembrane pressure was 10 mmHg, we were failed to find podocytes in the filtration fluid and cell culture medium within two hours. However, if we continue to extend the test time. We may be found podocytes in the cell culture media.Conclusion:the filtration fraction of this composite filter membrane was 26%. Small molecules can be completely filtered, but albumin and immune globulin is filtered Partially. These results suggested that we need to further improve that filter membrane.