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1.
Generation of renal tubules at the interface of an artificial interstitium.   总被引:2,自引:0,他引:2  
During kidney development a multitude of tubular portions is formed. Little knowledge is available by which cellbiological mechanism a cluster of embryonic cells is able to generate the three-dimensional structure of a tubule. However, this know-how is most important in tissue engineering approaches such as the generation of an artificial kidney module or for the therapy of renal diseases using stem cells. To obtain cellbiological insights in parenchyme development we elaborate a new technique to generate under in vitro conditions renal tubules derived from the embryonic cortex of neonatal rabbits. The aim of the experiments is to establish a specific extracellular environment allowing optimal three-dimensional development of renal tubules under serum-free culture conditions. In the present paper we demonstrate features of the renal stem cell niche and show their isolation as intact microcompartments for advanced tissue culture. Perfusion culture in containers exhibiting a big dead fluid volume results in the development of a flat collecting duct (CD) epithelium at the surface of the tissue explant. In contrast, by fine-tuning the dead fluid volume within a perfusion culture container by an artificial interstitium made of a polyester fleece shows the generation of tubules. It is an up to date unknown morphogenetic information which tells the cells to form tubular structures.  相似文献   

2.
The generation of tissue‐like structures in vitro is of major interest for various fields of research including in vitro toxicology, regenerative therapies and tissue engineering. Usually 3D matrices are used to engineer tissue‐like structures in vitro, and for the generation of kidney tubules, 3D gels are employed. Kidney tubules embedded within 3D gels are difficult to access for manipulations and imaging. Here we show how large and functional human kidney tubules can be generated in vitro on 2D surfaces, without the use of 3D matrices. The mechanism used by human primary renal proximal tubule cells for tubulogenesis on 2D surfaces appears to be distinct from the mechanism employed in 3D gels, and tubulogenesis on 2D surfaces involves interactions between epithelial and mesenchymal cells. The process is induced by transforming growth factor‐β1, and enhanced by a 3D substrate architecture. However, after triggering the process, the formation of renal tubules occurs with remarkable independence from the substrate architecture. Human proximal tubules generated on 2D surfaces typically have a length of several millimetres, and are easily accessible for manipulations and imaging, which makes them attractive for basic research and in vitro nephrotoxicology. The experimental system described also allows for in vitro studies on how primary human kidney cells regenerate renal structures after organ disruption. The finding that human kidney cells organize tissue‐like structures independently from the substrate architecture has important consequences for kidney tissue engineering, and it will be important, for instance, to inhibit the process of tubulogenesis on 2D surfaces in bioartificial kidneys.  相似文献   

3.
Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.  相似文献   

4.
While a variety of genetic mutations have been shown to be associated with renal cyst formation, mechanisms of renal cyst formation are largely unknown. In prior communications we described alterations in E-cadherin assembly in cultured cystic epithelial cells (Charron AJ, Nakamura S, Bacallao R, Wandinger-Ness A. J Cell Biol 149: 111-124, 2000). Using the same cell line we assayed cadherin expression by RT-PCR using primer pairs that anneal to highly conserved sequences of cadherin genes but flank informative regions of cadherins. Using this approach we found that autosomal dominant polycystic kidney disease (ADPKD) cells express cadherin 8, a neuronal cadherin with limited expression in the kidney. Immunohistochemistry confirmed cadherin 8 expression in cystic epithelia. To test the functional significance of cadherin 8 expression in renal epithelial cells, we adapted a three-dimensional collagen culture method in which HK-2 cells form tubule structures and microinjected adenovirus into the matrix space surrounding tubule structures. Adenovirus expressing cadherin 8 under the control of a tet promoter caused cyst structures to grow out of the tubules when coinjected with adenovirus expressing a tet transactivator. Microinjection of single adenovirus expressing either tet transactivator or cadherin 8 failed to cause cyst formation. When doxycycline was added to the culture, following coinjection of adenovirus, there was a dose-response reduction in cadherin 8 expression and cyst formation. Similarly, HK-2 cells transfected with Flag-tagged cadherin 8 form cysts in addition to tubular structures. HK-2 cells transfected with Flag-tagged N-cadherin do not form cysts. These data suggest that ectopic expression of cadherin 8 in renal epithelial cells is sufficient to cause the morphogenic pattern of cyst formation.  相似文献   

5.
Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin(+) and CD133/1(+) cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1(+) cells. Isolated CD133/1(+) papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1(+) progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.  相似文献   

6.
Human proximal tubular (PT) epithelial cells were isolated from urine and monoclonally cultured as monolayers for 1 wk, after which they were subcultured between two layers of collagen gel, designated a "collagen gel sandwich." Under these culture conditions, PT cells formed three-dimensional tubular structures exhibiting distinct polarized cell morphology. Scanning and transmission electron microscopic studies showed that they bore numerous microvilli at the apical surface and that they closely contacted the collagen gel at the basal surface. These studies indicate that PT cells exfoliated in urine still exhibit the potential to proliferate and form organized structures mimicking in vivo tubules. Because of the current lack of useful culture systems for human tubular epithelial cells originating from kidney tissue, we suggest that this unique culture system using voided PT cells in urine could open up new avenues to study not only the mechanisms of morphogenesis but also the physiology of human PT cells.  相似文献   

7.
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from The Polycystic Kidney Foundation. We gratefully acknowledge the support of the Children’s Medical Research Institute and Children’s Miracle Network Foundation.  相似文献   

8.
Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin+ and CD133/1+ cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1+ cells. Isolated CD133/1+ papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1+ progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.  相似文献   

9.
New and effective treatment for acute kidney injury remains a challenge. Here, we induced mouse hematopoietic stem and progenitor cells (HSPC) to differentiate into cells that partially resemble a renal cell phenotype and tested their therapeutic potential. We sequentially treated HSPC with a combination of protein factors for 1 wk to generate a large number of cells that expressed renal developmentally regulated genes and protein. Cell fate conversion was associated with increased histone acetylation on promoters of renal-related genes. Further treatment of the cells with a histone deacetylase inhibitor improved the efficiency of cell conversion by sixfold. Treated cells formed tubular structures in three-dimensional cultures and were integrated into tubules of embryonic kidney organ cultures. When injected under the renal capsule, they integrated into renal tubules of postischemic kidneys and expressed the epithelial marker E-cadherin. No teratoma formation was detected 2 and 6 mo after cell injection, supporting the safety of using these cells. Furthermore, intravenous injection of the cells into mice with renal ischemic injury improved kidney function and morphology by increasing endogenous renal repair and decreasing tubular cell death. The cells produced biologically effective concentrations of renotrophic factors including VEGF, IGF-1, and HGF to stimulate epithelial proliferation and tubular repair. Our study indicates that hematopoietic stem and progenitor cells can be converted to a large number of renal-like cells within a short period for potential treatment of acute kidney injury.  相似文献   

10.
Summary Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured, to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occured during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occured only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.  相似文献   

11.
Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occurred during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occurred only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.  相似文献   

12.
Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.  相似文献   

13.
Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.  相似文献   

14.
Antithrombin is a serine protease inhibitor that is critical in maintaining a thromboresistant vasculature. The association between low serum antithrombin concentration and renal disease suggests that the kidney plays a role in the conservation of plasma antithrombin. We used immunohistochemical techniques to determine the spatial distribution, heparin binding characteristics, and intracellular and intercellular localization of antithrombin in biopsy specimens (n = 53) of human donor kidneys obtained at the time of transplantation. In the renal cortex, double antibody techniques demonstrated the presence of intracellular antithrombin in proximal tubule epithelial cells. The reactivity was granular and was co-localized with vesicle-like structures. Distal and collecting tubules did not demonstrate intraepithelial antithrombin reactivity. No tubule structures in the medullary region demonstrated intracellular antithrombin, but all these structures showed intense basement membrane antithrombin reactivity. Double antibody techniques also demonstrated that the heparin binding domain of intraepithelial antithrombin was occupied. Semiquantitative scores for intraepithelial antithrombin were significantly decreased in renal biopsy specimens obtained 30 min after anastomosis compared with biopsies from the same organ obtained before anastomosis. These findings suggest that antithrombin, probably in association with heparin or heparan sulfate, is internalized by renal proximal epithelial cells. Although the ultimate fate of intraepithelial antithrombin is not known, this may represent a mechanism by which the kidney helps to maintain plasma antithrombin concentrations.  相似文献   

15.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease. ADPKD is characterized by cyst development that leads to abnormal kidney structure. Renal tubules are a fundamental unit of architecture, so controls of tubular growth and formation are important for proper kidney function. The molecular mechanisms of tubulogenesis are being actively studied as the basis of diagnosis and treatment of ADPKD. Mxi1 is a member of the MAD family of proteins that functions in terminal differentiation, inhibition of cell cycle progression and tumor suppression, while the Myc protein, which is antagonized by Mxi1, causes renal cystogenesis. Based on these molecular relationships, the present study implicated Mxi1 with ADPKD be demonstrating that curtailed Mxi1 gene expression caused cyst formation in Mxi1-deficient mice. To ascertain whether Mxi1 affects renal epithelial cell tubulogenesis, three-dimensional cultures (3D culture) of mIMCD-3 cells and stably Mxi1 over-expressed mIMCD-3 cells were established. The results indicated that over-expression of the Mxi1 gene plays a role in the regulation of tubulogenesis by regulating some genes participating in renal epithelial branching tubulogenesis such as matrix metalloproteinase 9 (MMP9), integrins, fibronectin, and E-cadherin. The results support the suggestion that over-expression of Mxi1 can suppress renal epithelial tubulogenesis. In particular, MMP9 is greatly affected by the expression level of Mxi1. It can be concluded that mIMCD-3 cells that stably over-express Mxi1 fail to form renal epithelial tubules because of abnormally reduced expression of MMP9.  相似文献   

16.
Spermatogonial stem cells (SSCs) are responsible for life-long, daily production of male gametes and for the transmission of genetic information to the next generation. Unequivocal detection of SSCs has relied on spermatogonial transplantation, in which functional SSCs are analyzed qualitatively and quantitatively based on their regenerative capacity. However, this technique has some significant limitations. For example, it is a time-consuming procedure, as data acquisition requires at least 8 weeks after transplantation. It is also laborious, requiring microinjection of target cells into the seminiferous tubules of individual testes. Donor-recipient immunocompatibility for successful transplantation and large variations in data obtained represent further limitations of this technique. In the present study, we provide evidence that a recently developed SSC culture system can be employed as a reliable, short-term in vitro assay for SSCs. In this system, donor cells generate three-dimensional structures of aggregated germ cells (clusters) in vitro within 6 days. We show that each cluster originates from a single cell. Thus, by counting the clusters, cluster-forming cells can be quantified. We observed a strong linear correlation between the numbers of clusters and SSCs over extended culture periods. Therefore, cluster numbers faithfully reflect SSC numbers. These results indicate that by simply counting the number of clusters, functional SSCs can be readily detected within 1 week in a semi-quantitative manner. The faithfulness of this in vitro assay to the transplantation assay was further confirmed under two experimental situations. This in vitro cluster formation assay provides a reliable short-term technique to detect SSCs.  相似文献   

17.
Early innervation of the metanephric kidney   总被引:2,自引:0,他引:2  
During kidney differentiation, the nephrogenic mesenchyme converts into renal tubules and the ureter bud branches to form the collecting system. Here we show that in the early undifferentiated kidney rudiment there is a third cell type present. In whole-mount preparations of cultured undifferentiated metanephric kidneys, neurones can be detected by immunohistochemical means with antibodies against the neurofilament triplet, 13AA8, and against neuronal cell surface gangliosides, Q211. Clusters of neuronal cell bodies can be seen in the mesenchyme close to the ureter bud. The terminal endings of neurites are found around the mesenchymal condensates that later become kidney tubules. A similar distribution of neurites can be revealed in tissue sections of kidney grafts growing in the chicken chorioallantoic membranes. In primary cultures of the ureter bud cells, neurones are constantly present. In another report, we have shown that, in experimental conditions, neurones are involved in regulation of kidney morphogenesis. The present results raise the possibility that neurones of the metanephric kidney may have this function in vivo as well.  相似文献   

18.
Recent evidence suggests that a local reninangiotensin system is operational in the kidney and that it mediates some of the actions of angiotensin II on renal tubules. In this study the ontogeny and renal distribution of the unique precursor to angiotensin II formation, angiotensinogen, was investigated in rats by use of immunohistochemistry, immuno-electron microscopy and non-isotopic hybridization histochemistry. At the light-microscopic level, intense staining for angiotensinogen was found in the proximal convoluted tubules of the cortex, with lighter staining in the straight proximal tubules of the outer stripe. The strongest immunostaining was found in the kidneys of neonatal rats, where glomerular mesangial cells and medullary vascular bundles were also immunopositive. The angiotensinogen content of the kidneys in late gestation embryos and neonates showed the presence of angiotensinogen by day E18 and a peak content in the neonate. Non-isotopic hybridization histochemistry with biotinylated oligodeoxynucleotide probes confirmed the presence of angiotensinogen mRNA expression in the proximal convoluted tubules of the renal cortex. Electron-microscopic immunohisto-chemistry showed staining of relatively few electron-dense structures close to the apical membrane of proximal convoluted tubule cells in the adult kidney. In the neonatal rat kidney, angiotensinogen immunostaining at the electron-microscopic level was found throughout the proximal tubule cells and was markedly stronger than that seen in adult kidney. The presence of angiotensinogen, from embryonic day 18, in the proximal tubules, mesangial cells and vasculature of the kidney suggests multiple potential sites of intrarenal angiotensin II generation with an ontogeny in late gestation.  相似文献   

19.
We examined the distribution of gamma-aminobutyric acid-like immunoreactivity (GABA-LI) in the rat kidney by light and electron microscopy. In vibratome sections, GABA-LI was present in both the renal medulla and cortex. The inner stripe of the outer medulla was most heavily and almost homogeneously labeled, whereas GABA-LI in the cortex was mainly confined only to some tubules. GABA-positive structures involved the epithelial cells of the thin and the thick ascending limbs of the loop of Henle, the connecting tubules, and the collecting ducts. In GABA-positive connecting tubules and collecting ducts the immunoreactivity was present in the cytoplasm of about half of the epithelial cells. As revealed by electron microscopy, the labeled cells in the collecting tubules were the light (principal) cells. No GABA-LI occurred in neuronal structures. These findings are consistent with the presence of a non-neuronal GABA system in the rat kidney. Furthermore, the specific distribution of GABA in the tubular epithelium suggests a functional significance of this amino acid in tubular transport processes.  相似文献   

20.
This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 °C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 µM resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.  相似文献   

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