| Microenvimoment plays an important role in maintaining cell viability and functions.As the basic structure and functional unit of the kidney,nephron has the functions of glomerular filtration,renal tubular reabsorption and secretion.Due to the special structure and functions of the nephron,the cellular microenvironments with different types and in different regions are signally different,.In vitro experiments with the advantages of few influencing factors and controllable conditions play an important role in the study of renal injury mechanism and drug screening.However,the existing two-dimensional culture and three-dimensional in vitro culture platforms have low degree of biofidelity,so it is difficult to simulate the complex microenvironment of kidney.Microfluidic technology with the advantages of fluid controllable,flexible design and integration,supplied by cells with physiological functions and biomimic arrangement could build kidney-on-a-chip with similar structure and core functions.At present,there have been some reports about kidney-on-a-chip.But these works have low biofidelity in structure and functions,because the types of the cells used in the works are few and only a part of nephron are mimiced.The physiological process that substrances are filtered through glomeruli and secreted or reabsorted by renal tubule can not realize in these works.To explore and verify the key factors to improve kidney-on-a-chip biofidelity,from simple to complex,the design and fabrication of single renal tubular epithelial cell chip,renal peritubule chip and nephron-on-a-chip with glomerular model and tubular model are described in this paper.The first two chips that can be used to preliminary simulate renal tubular functions,while nephron-on-a-chip can be used to preliminary simulate the substrances conversion between free and binding states,glomerular filteration,renal tubular secretion,renal tubular reabsorption,which are the four main large dynamic processes influencing on renal toxicity.Subsequently,the necessity of relatively complete structure and functions of nephron-on-a-chip is verified,and the functions and potential of nephron-on-a-chip are discussed.In the first chapter,a novel method of isolation and culture of several primary cells for construction of the nephron-on-a-chip is described.Podocytes,renal tubular peritubular endothelial cells and renal tubular epithelial cells can be obtained simultaneously from the same rat in one test by this approach.The optimal methods of isolation and culture were established by investigating the effects of obtained methods on tissue acitivity,the effects of colleagenase and digesting time on purity and acitivity of tissues,the effects of coating conditions on proliferation and function maintained,and the effects of medium ingredient on cell viability and functions maintained.In the second chapter,two different designs for renal tubule chip construction are described.Based on the "sandwich structure",a porous membrane covered with renal tubular epithelial cells is used to build the single layer renal tubular epithelial cell chip.Two porous membranes covered with renal tubular epithelial cells and peritubular endothelial cells respectively,composes the renal tubule peritubular chip.The compactness and functions of the renal tubular epithelial cells and peritubular endothelial cells layers are evaluated.Then,the effects of renal tubule chip on cellular function reconstruction are investigated.Furthermore,cisplatin is used to assess the cellular polarization and different renal tubule chip designs for cisplatin-induced renal damage test.Compared with the result of single layer renal tubular epithelial cell chip,result of chip with two different cell layers is closer to the situation in vivo.In the third chapter,a design for nephron-on-a-chip is described.Based on the "sandwich structure",the chip contains two modules:the glomerular module consisted of podocytes and endothelial cells respectively,and the renal tubular module consisted of rat primary tubular epithelial cells and endothelial cells.The glomerular module has functions of molecule-selective and charge-selective filtration,the renal tubular module had the function of p-aminohippuric acid secretion and glucose reabsorption.Bovine serum albumin(BSA)extra added in "artificial blood" promotes cellular function reconstruction.The nephron-on-a-chip is also compared with two types of renal tubule chips by cisplatin-induced renal damage test.Both architectural design and drug test results of the nephron chip are much closer to the situation in vivo.In the fourth chapter,set the drug-induced injury as an example,the functions and application potentials of nephron-on-a-chip are discussed.The chip can be used to investigate cell impairments in glomerular module and renal tubular module caused by cisplatin concentration gradients and compare cell damage orders in the affection of cisplatin and doxorubicin.Except that,the effects of extra added BSA in "artificial blood" on cisplatin and doxorubicin-induced renal damage.Some of the data are similar to the reported in vivo results,indicating that the nephron-on-a-chip has great potential in the study of drug-induced renal injury.In conclusion,this dissertation introduces a novel design of nephron-on-a-chip,preliminary simulates the substrances conversion between free and binding states,glomerular filteration,renal tubular secretion,renal tubular reabsorption,which are the four main large dynamic processes influence on renal toxicity.Compared with the traditional platforms,the chip has high biofidelity in structure and functions.The chips can be used for nephrotoxity screening in vitro,studing the functions of kidney,and especially invesitigating the pathogenesis of renal injury These make the nephron-on-a-chip become an applicable and valuable model for drug toxicity screening and renal damage research.The chip is expected to fill the gap in the lack of high fidelity models in vitro studies and become an effective approach for renal disease research and new target and drug development. |
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