The Primary Study Of Building Bioartificial Renal Tubule Assist Device

BackgroudAcute and chronic renal failure are familiar diseases in clinic. Mostly, the treatment is the renal substitution therapy with hemodialysis or hemofiltration treament. Although current renal substitution therapy has had a dramatic life-sustaining effect in patients suffering from both acute or chronic renal failure, these clinical disorders are still serious medical conditions. This persistently poor prognosis of these disorders may be due to the fact that hemodialysis and hemofiltrations are not complete renal therapies. These techniques provide only a clearence or filtration function for small solute but not replace the lost transport ,metabolic, and endocrine functions of the kidney, which are predominantly found in the tubular elements of the organ.The renal tubule cell's role in gulutathione reclamation, glutathione peroxidase synthesis, other middle molecule metabolism, and activatin of vitamin D3,are well recongnized pathways to maintain import tissue integrity and host defense under stress conditions. A less recongnized role of the kidney and the renal tubule cells is its potential immunoregulatory functionl.Loss of renal function is an independent predictor of mortality in hospitalized patients weiht ARE At the same time , Loss of renal function not only is the sign of seriousness of disease, but also is anindependent predictor of mortality in hospitalized patients with ARF.The propensity of patients with ARF to develop SIRS and sepsis suggests that renal function, specifically renal tubule cell function secondary to ATN, plays a critical immunomodunlatory role in individuals under stress states. Additional,renal tubule cells are injured early in septic shock. So, we hypothesis that replacement of their activity with cell therapy will improve immunoregulation of the SIRS of sepsis and thereby provide additional physiologic activities that improve the current natural history of this disease process. Cell therapy is dependent on cell and tissue culture methodologies to expand specific cells to replace important differentiated processes deranged or lost in various disease states. Bioartificial renal tubule is just the applacations in renal disease based on cell therapy.In order to replace the renal tubule funtion, people has developed an extracorporeal device using a standard hemofiltratin cartridge containing many renal tubule cells ,which has the function of renal tubule. According to the studies of Humes HD et al, In vitro studies of the renal tubule assist device(RAD)have shown that the cells retain differntiated active transport properties, differntiated metabolic activities,and important endocrine proceses. Further studies have shown that the RAD, when incorporated in series with a hemofiltration cartridge in an extra-corporeal blood perfusin circuit,replaces filtrations, transport, metabolic, and endocrine functions of the kidney in acutely uremic dogs. There is in the growing appreciation that most disease processes are not due to the lack of a single protein but develop due to alterations in complex interactions of a variety of cell products. Bioartificial renal tubule based on cell therapy is an exciting approach to the treament of acute and chronic renal disease. Based on these abundance backgrounds, this study focus on the building of bioartifical renal tubule assist device, in order to creating a working flat for the further investigation correlated animal and clinical studies.ObjectiveTo study the buildup of the bioartificial renal tubule asisit device, simulate the proliferation condition of the implantation cells, and investigate the method of building bioartificial renal tubule asisit device.Methods—. Renal Tubule Asisit Device. Design and Cell SeedingF60 high-fix hemofiltration cartridge containing polysulfone hollow fibers was choosen as bioreactor. Then connected with tubings for the next work.Firstly,50ml serum free RPMI 1640 medium was perfused into the intracapillary. To prepare the intraluminal surface of the hollow fiber for epithelial attachment and confluent growth, serum free RPMI 1640 medium containing laminin (2ng/m 2 )was perfused into the intracapillary and allowed to remain for 60min.secondly,renal tubule cells were dislodged from the culture plates with hosphate buffered saline containing 0.25%trypsin. the cells were suspended in 80ml culture medium with 1% fetal calf serum at a total number of 6.2 X 107.Thirdly, 150ml culutre medium with 10% fetal calf serum was perfused into the extracapillary. Fourthly, the cell suspension was introduced into the intracapillary form two ports of the filtration cartridge, and allowed to sit for 90 min. After this time, the bioreactor was rotated 90°,and another seeding of cells was performed. This process was repeated two more times to complete a 360° seeding procedure. The final cell suspension was flushed from the intracapillary, and the bioreactor was placed in a cell incubator .After 24 hr, the bioreactor was perfused with culture media through the extracapillary space(ECS) at 150nl/min.Four days after cell seeding, the bioreactor was perfused intraluminally with culture media at a rate of 10^/min,lasting for about 10 days.—-. Histologic ProcessingDrew out the polysulfone hollow fibers from the hemofiltration cartridge onthe lth,4th, and 14th day after seeding cells, respectively. Samples were fixed with 95 alcohol or 4% fonnaldehyd. For light microscopy, samples were embedded in paraffin, and sectioned and processed with HE staining methods.ResultsIn the sections of fibers drew out from the hemofiltration cartridge on the lth day after seeding LLC-PKl,we found some attached cells in the light microscopy. While on the 4th and 14th day, there was no cells to be observed. Counting the cells in the outflewed medium from intracapillary on the 5th, 10th, 14th day, respectively, the outcome of the cell counting was: 25.2X 10\ 28.4X 10\ 7.6X 106, amount to about 6.1 X 107, which was not obvious difference with the total of seeding cells at first.ConclusionIn the construction of RAD, the initial steps in the construction of a RAD had already finished to optimize renal substitution therapy . The method of building RAD had explored initially. Summarized some problems in the construction, pointed out the reasons which influenced the success of model to build up, also had accumulated some experiences for the next work.