ABSTRACT
An increasing number of investigations is dealing with the repair of acute and chronic renal failure by the application of stem/progenitor cells. However, accurate data concerning the cell biological mechanisms controlling the process of regeneration are scarce. For that reason new implantation techniques, advanced biomaterials and morphogens supporting regeneration of renal parenchyma are under research. Special focus is directed to structural and functional features of the interface between generating tubules and the surrounding interstitial space. The aim of the present experiments was to investigate structural features of the interstitium during generation of tubules. Stem/progenitor cells were isolated from neonatal rabbit kidney and mounted between layers of a polyester fleece to create an artificial interstitium. Perfusion culture was performed for 13 days in chemically defined Iscove's Modified Dulbecco's Medium containing aldosterone (1 x 10(-7) M) as tubulogenic factor. Recordings of the artificial interstitium in comparison to the developing kidney were performed by morphometric analysis, scanning and transmission electron microscopy. The degree of differentiation was registered by immunohistochemistry. The data reveal that generated tubules are embedded in a complex network of fibers consisting of newly synthesized extracellular matrix proteins. Morphometric analysis further shows that the majority of tubules within the artificial interstitium develops in a surprisingly close distance between 5 and 25 mum to each other. The abundance of synthesized extracellular matrix acts obviously as a spacer keeping generated tubules in distance. For comparison, the same principle of construction is found in the developing parenchyma of the neonatal kidney. Most astonishingly, scanning electron microscopy reveals that the composition of interstitial matrix is not homogeneous but differs along a cortico-medullary axis of proceeding tubule development.
Subject(s)
Kidney Tubules/growth & development , Kidney Tubules/ultrastructure , Kidney/growth & development , Kidney/ultrastructure , Polyesters/chemistry , Animals , Cells, Cultured , Humans , Rabbits , Surface PropertiesABSTRACT
Tissue factory is a modular system designed to generate artificial tissues under optimal perfusion culture conditions. The microenvironment within the culture containers can be fine-tuned to meet the physiological needs of individual tissues, so that the generation of differentiated three-dimensional tissue constructs becomes possible. An optimal physiological environment is created by modulating a liquid phase as well as an artificial interstitium surrounding the growing construct. An innovative construction principle allows production of tissue culture containers, gas exchangers, and gas expanders at minimal material expenditure. Therefore it will be possible for the first time to produce sterile one-way perfusion culture modules for the generation of artificial tissues. The modules can be used separately as well as in a combined module. The system is designed to provide a possible platform for the standardized production of artificial tissues for future applications in biomedicine.
Subject(s)
Tissue Engineering/instrumentation , Cell Culture Techniques/methods , Temperature , Tissue Engineering/methodsABSTRACT
The development of renal histo-architecture substantially depends on the three-dimensional extension of the collecting duct (CD) ampulla, since under its influence, nephron induction takes place in the surrounding mesenchyme. Recently, micro-fibers were detected by soybean agglutinin (SBA), which line from the basal aspect of each CD ampulla through the mesenchyme towards the organ capsule in embryonic kidney. Their unique distribution suggests that they may play an important role in the control of CD ampulla growth and in forming the renal stem cell niche. A profound analysis of interstitial proteins between the CD ampulla and the nephrogenic mesenchyme is lacking. Consequently, the goal of the current investigation was to colocalize the micro-fibers detected by SBA with interstitial proteins. For this reason a detailed cell biological analysis of extracellular molecules at this site was carried out. Double labeling showed that the micro-fibers do not correspond to known collagens and other extracellular matrix molecules such as agrin, versican or MMP-9. In addition, it could be demonstrated that the micro-fibers do not contain epithelial or mesenchymal cell elements. Furthermore, two-dimensional electrophoresis with subsequent Western blotting yielded two different amino acid sequences (1: GHYADPTSPR; 2: NNGCCSSDYHA) obtained from SBA-labeled protein spots. Both amino acid sequences could not be assigned to known rodent proteins. The findings suggest that the SBA-labeled micro-fibers represent a new type of extracellular structure between the CD ampulla, the mesenchyme and the organ capsule.
Subject(s)
Kidney Tubules, Collecting/chemistry , Animals , Animals, Newborn , Collagen/chemistry , Collagen/metabolism , Collagen/ultrastructure , Extracellular Matrix Proteins/metabolism , Kidney/chemistry , Kidney Cortex/chemistry , Kidney Cortex/embryology , Kidney Tubules, Collecting/ultrastructure , Mesoderm/chemistry , Mesoderm/ultrastructure , Microscopy, Electron, Scanning , Plant Lectins/metabolism , Rabbits , Soybean Proteins/metabolism , Staining and Labeling , Surface PropertiesABSTRACT
The nature of the primary functional events of nephron induction is still unknown, making it impossible to completely understand the mechanism of tissue interaction between collecting duct ampulla and the surrounding nephrogenic mesenchyme. Soluble morphogenic substances are known to be exchanged in the process and it is assumed that nephron induction requires close contact between both tissues involved. Contrasting with that assumption our previous investigation revealed a thick fibrous meshwork separating nephron inducer and mesenchyme. Our present investigation focused on the molecular characterization of the mab (CD)Amp1 antigen, which is found only in this meshwork. The protein was shown immunohistochemically to be located exclusively at the embryonic collecting duct ampulla and could be clearly distinguished from other extracellular matrix proteins such as collagen type IV, laminin, reticulin, and fibronectin. Two-dimensional electrophoresis of the soluble form of P(CD)Amp1 showed a molecular weight of 87,000 and an isoelectric point of 4.3-4.4. Results from N-terminal sequencing indicated a partial sequence homology of P(CD)Amp1 to collagen type IV alpha 2-chain precursor but additionally yielded unknown sequences. Thus P(CD)Amp1 is a novel, collagen-related protein, restricted to the fibrous meshwork at the mesenchymal-epithelial interphase, which is the site of primary epithelial-mesenchymal interaction.
Subject(s)
Antibodies, Monoclonal/analysis , Antigens, Surface/immunology , Epithelial Cells/chemistry , Kidney Tubules, Collecting/chemistry , Amino Acid Sequence , Animals , Animals, Newborn , Antigens, Surface/chemistry , Blotting, Western , Cells, Cultured , Collagen Type IV/chemistry , Collagen Type IV/immunology , Electrophoresis, Gel, Two-Dimensional , Epithelial Cells/cytology , Extracellular Matrix/chemistry , Extracellular Matrix Proteins/analysis , Fluorescent Antibody Technique, Indirect , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/growth & development , Microscopy, Confocal , Molecular Sequence Data , Molecular Weight , Rabbits , Sequence Homology, Amino AcidABSTRACT
Epithelia perform barrier functions being exposed to different fluids on the luminal and basal side. For long-term testing of new biomaterials as artificial basement membrane substitutes, it is important to simulate this fluid gradient. Individually-selected biomaterials can be placed in tissue carriers and in gradient containers, where different media are superfused. Epithelia growing on the tissue carriers form a physiological barrier during the whole culture period. Frequently however, pressure differences between the luminal and basal compartments occur. This is caused by a unilateral accumulation of gas bubbles in the container compartments resulting in tissue damage. Consequently, the occurence of gas bubbles has to be minimized. Air bubbles in the perfusion culture medium preferentially accumulate at sites where different materials come into contact. The first development is new screw caps for media bottles, specifically designed to allow fluid contact with only the tube and not the cap material. The second development is the separation of remaining gas bubbles from the liquid phase in the medium using newly-developed gas expander modules. By the application of these new tools, the yield of embryonic renal collecting duct epithelia with intact barrier function on a fragile natural support material can be significantly increased compared to earlier experiments.
Subject(s)
Epithelial Cells/cytology , Organ Culture Techniques/instrumentation , Organ Culture Techniques/methods , Tissue Engineering/instrumentation , Tissue Engineering/methods , Air , Animals , Carbon Dioxide/chemistry , Cell Differentiation , Culture Media , Electrolytes , Kidney/cytology , Rabbits , Temperature , Time FactorsABSTRACT
BACKGROUND: During kidney development, the embryonic collecting duct (CD) epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. It is unknown by which molecular mechanism the different cell types arise. We have experimental evidence that the electrolyte environment is involved in the process of terminal cell differentiation. METHODS: Embryonic CD epithelia from neonatal rabbit kidneys were microsurgically isolated and maintained in gradient perfusion culture for 13 days under serum-free conditions. Controls were maintained in the same medium (Iscove's modified Dulbecco's medium; IMDM) on basal and luminal sides. Experimental series were performed with IMDM only on the basal side, while on the luminal side IMDM with increasing concentrations of NaCl was used. Finally, the development of principal and intercalated cell features was registered by immunohistochemical labeling with markers specific for adult CD cells. RESULTS: Immunohistochemical markers show that the differentiation pattern is quite different when the embryonic CD epithelia are cultured in IMDM only as compared with specimens kept in IMDM supplemented with 3-24 mmol/l NaCl on the luminal cell side. First signs of changes in development were seen when low doses of 3-6 mmol/l NaCl were added. CONCLUSIONS: We conclude that facultative protein expression in embryonic CD epithelium is influenced by the electrolyte environment and starts to be upregulated after administration of unexpectedly low doses of 3-6 mmol/l NaCl added to the luminal perfusion culture medium and increases in a concentration-dependent manner.
Subject(s)
Cell Differentiation/physiology , Kidney Tubules, Collecting/embryology , Water-Electrolyte Balance/physiology , Animals , Cells, Cultured , Culture Media , Electrophoresis, Polyacrylamide Gel , Epithelial Cells/cytology , Immunoblotting , Immunohistochemistry , RabbitsABSTRACT
While more and more humoral factors are being implicated in nephrogenesis, there is no detailed knowledge of the morphological structures at the interface of the nephron inducer and the surrounding mesenchyme. Hence we examined this area in the cortex of neonatal rabbit kidneys by scanning and transmission electron microscopy. Our interest was focused on the basal aspect of the collecting duct ampulla and the surrounding competent mesenchyme where morphogenic signals are exchanged during nephron induction. Close contact between these two tissues is assumed during nephrogenesis to allow direct cellular contact or diffusion of soluble factors across a short distance. However, our data show the presence a wide cleft around the collecting duct ampulla spatially separating the inducer and the competent mesenchyme during nephron induction. This cleft is filled with a characteristic fibrillar mesh-work.
Subject(s)
Animals, Newborn/anatomy & histology , Nephrons/embryology , Nephrons/ultrastructure , Animals , Embryo, Mammalian/physiology , Embryonic and Fetal Development , Immunohistochemistry , Mesoderm/ultrastructure , Microscopy, Electron , Microscopy, Electron, Scanning , RabbitsABSTRACT
Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture.
Subject(s)
Biomedical Engineering/methods , Cell Culture Techniques/methods , Cell Differentiation/physiology , Materials Testing/methods , Animals , Culture Media , Diffusion Chambers, Culture/methods , HumansABSTRACT
An in vitro model was used to investigate the terminal differentiation mechanisms leading from embryonic to adult renal tissue. For these experiments the capsula fibrosa with adherent embryonic tissue was isolated from neonatal rabbit kidneys. These explants were mounted onto special tissue carriers and cultured in medium containing serum for 24 h. During that time collecting duct (CD) cells grew out and formed a monolayered epithelium covering the whole surface of the explant. The carriers were then transferred to perfusion culture containers to obtain an optimal degree of differentiation. A special type of container allowed us to continuously superfuse the epithelia with individual media on the luminal and basal sides. Using this method it became possible to culture embryonic CD epithelia in a fluid gradient for weeks. The epithelia were superfused with standard Iscove's modified Dulbecco's medium (IMDM) on the basal side, while IMDM containing additional NaCl was used on the luminal side. In controls IMDM was superfused on both the luminal and basal sides. It was found that the degree of differentiation in the CD epithelia is dependent on the influence of fluid gradient exposure. Perfusion culture under isotonic conditions revealed that less than 5% of cells were immunopositive for principal and intercalated cell features, while epithelia cultured in a luminal-basal gradient showed more than 80% positive cells. Immunoreactivity for characteristic markers started to develop after an unexpectedly long latent period of 3-6 days, then increased continuously during the following 5 days and reached a maximum on day 14. After switching back from the gradient to isotonic culture conditions the immunoreactivity for some markers decreased within 5 days, while other characteristic features remained stable. Thus, differentiation was not only under the control of growth factors but was also regulated by the electrolyte environment.
Subject(s)
Cell Culture Techniques , Cell Differentiation , Kidney/cytology , Animals , Cell Differentiation/drug effects , Culture Media , Electrolytes/pharmacology , Embryo, Mammalian , Epithelial Cells/cytology , Kidney Tubules, Collecting/cytology , Perfusion , RabbitsABSTRACT
During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove's modified Dulbecco's medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams' medium E, McCoys 5A medium, and Dulbecco's modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.
Subject(s)
Culture Media, Serum-Free , Kidney Tubules, Collecting/cytology , Animals , Cell Culture Techniques/methods , Cell Differentiation , Cells, Cultured , Kidney Tubules, Collecting/embryology , RabbitsABSTRACT
While more and more humoral factors involved in nephrogenesis are being discovered, there is no detailed knowledge of the morphological structures at the interface of the nephron inducer and the surrounding mesenchyme. For that reason we examined this area in the cortex of neonatal rabbit kidneys by scanning electron-microscopical and transmission electron-microscopical techniques. Our interest was focused on the basal aspect of the collecting duct ampulla and the surrounding competent mesenchyme, where morphogenic signals are to be exchanged during nephron induction. Close contact between these two tissues involved in nephrogenesis is assumed to allow direct cellular contact or diffusion of soluble factors across a short distance. Our data, however, show the presence of a dense fibrillar meshwork around the collecting duct ampulla, spatially separating the inducer and the competent mesenchyme during nephron induction.
Subject(s)
Nephrons/growth & development , Nephrons/ultrastructure , Animals , Animals, Newborn , Antibodies, Monoclonal , Basement Membrane/ultrastructure , Cell Differentiation/physiology , Kidney Tubules, Collecting/growth & development , Kidney Tubules, Collecting/ultrastructure , Mesoderm/cytology , Microscopy, Confocal , Microscopy, Electron , Microscopy, Electron, Scanning , Nephrons/immunology , RabbitsABSTRACT
The collecting duct (CD) epithelium of the mammalian kidney is an extraordinary structure with respect to its functional changes during development and its heterogeneous composition when matured. All of the different nephron epithelia of the mammalian kidney consist of one single cell type. In contrast, the differentiated CD is composed of at least three distinct cell types [principal, alpha intercalated-, and beta intercalated cells] that are responsible for the multiple physiological functions of this kidney compartment. During development the function of the CD changes: initially, the CD ampulla serves as an embryonic inducer, while the matured epithelium plays a key role in maintaining the homeostasis of body fluids. At present the process of CD maturation is not well understood. Neither the time course of development nor the morphogenic factors leading to the heterogeneously composed epithelium are known. In the present study the differentiation of the CD epithelium was investigated using newly developed monoclonal antibodies and well-characterized antisera. The morphological changes induced during differentiation were monitored by immunohistochemistry and scanning electron microscopy. The experiments were performed on neonatal and adult rabbit kidneys. Results obtained by light microscopical techniques and scanning electron microscopy revealed that the ampullary tip can be distinguished from the ampullary neck, as well as from the maturing CD. A number of proteins that were not detectable in the ampulla were detected in the neonatal CD and were found at even higher concentrations in the adult CD (PCD8, chloride/bicarbonate exchanger). Other proteins (PCD9) were downregulated during differentiation. For the first time the transient character of the differentiation stage of the neonatal CD could be demonstrated unequivocally. Furthermore, considerable heterogeneity in protein expression patterns (PCD6 and PCD9) was demonstrated within the beta IC cell population of the mature CD.
Subject(s)
Antigens, Differentiation/immunology , Kidney Tubules, Collecting/embryology , Aging , Animals , Antibodies, Monoclonal/metabolism , Antigens, Differentiation/analysis , Antiporters/metabolism , Chloride-Bicarbonate Antiporters , Immunohistochemistry , Kidney Tubules, Collecting/chemistry , Kidney Tubules, Collecting/immunology , Kidney Tubules, Collecting/ultrastructure , Microscopy, Confocal , Microscopy, Electron, Scanning , RabbitsABSTRACT
During kidney organogenesis the development of renal vessels must be synchronized with the maturation of nephrons and the collecting duct system. Several reports showed that hormones and mitogenic peptides as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) are involved in this regulatory process. It is a known fact that bFGF receptors are expressed by differentiating tubular epithelium and mesenchyme, but little information is available about the function of bFGF in kidney organogenesis. The role of bFGF during kidney development was investigated using an organotypic culture system and immunohistological techniques. Renal cortex explants were prepared from the kidneys of neonatal rabbits with a microsurgical method, retaining the natural tissue composition. The explants were cultured serum free under continuous medium perfusion. Our results indicate a new and unexpected role of bFGF during the differentiation process. When bFGF alone was applied, vessels could no longer be detected. The inhibitory influence of bFGF could be overcome by addition of VEGF or hormones such as retinoic acid and aldosterone/vitamin D3. The combination of these factors with bFGF resulted in the expression of small vessel-like structures. We conclude that bFGF has a morphogenic rather than a mitogenic function during kidney vessel development.
Subject(s)
Fibroblast Growth Factor 2/pharmacology , Kidney Cortex/blood supply , Kidney Cortex/cytology , Neovascularization, Physiologic/drug effects , Animals , Antibody Specificity , Antineoplastic Agents/pharmacology , Capillaries/cytology , Capillaries/drug effects , Cell Division/drug effects , Cells, Cultured , Endothelial Growth Factors/pharmacology , Hormones/pharmacology , Ki-67 Antigen/analysis , Ki-67 Antigen/immunology , Lymphokines/pharmacology , Rabbits , Renal Circulation/drug effects , Tretinoin/pharmacology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
A new field in biomedical science has been established. Cell biologists, engineers, and surgeons now work within a team. Artificial connective, epithelial, or neuronal tissues are being constructed using living cells and different kinds of biomaterials. Numerous companies and laboratories are presenting dynamic developments in this field. Prognoses predict that, at the beginning of the coming century, the industry of tissue engineering will reach the importance of the present genetic technology. An enormous demand for organ and tissue transplants motivates research activities and drives the acquisition of innovative techniques and creative solutions. At the front of this development is the creation of artificial skin for severely burned patients and the generation of artificial cartilage for implantation in articular joint diseases. Future challenges are the construction of liver organoids and the development of an artificial kidney on the basis of cultured cells. In this paper we show strategies, needs, tools, and equipment for tissue engineering. The presupposition for all projects is the induction, development, and maintenance of differentiation within the tissue under in vitro conditions. As experiments in conventional culture dishes continued to fail, new cell and tissue culture methods had to be developed. Tissues are cultured under conditions as close as possible to their natural environment. To optimize adherence or embedding, cells are grown on novel tissue carriers and on individually selected biomatrices or scaffolds. The tissues are subsequently transferred into different types of containers for permanent perfusion with fresh culture medium. This guarantees constant nutrition of the developing tissue and prevents the accumulation of harmful metabolites. An organo-typical environment for epithelial cells, for example, is obtained in gradient containers, which are permanently superfused at the apical and basal sides with different media. Long term experiments result in cultured tissues in a quality thus far unreached.
Subject(s)
Biomedical Engineering/trends , Culture Techniques/trends , Cell Differentiation , Cells/cytology , Culture Techniques/methods , HumansABSTRACT
The influence of electrolytes on the development of renal principal and intercalated collecting duct cells is unknown. Consequently embryonic collecting duct epithelia were exposed to different electrolyte concentrations, and their degree of differentiation was registered by immunohistochemical methods. Embryonic collecting duct epithelia were isolated from neonatal rabbit kidneys and placed on tissue carriers. The apical urine and the basal serum compartments were simulated in a gradient culture container. The two sides of the epithelium were each constantly superfused with medium for 13 days. In controls the medium on both apical and basal side was standard Iscove's modified Dulbecco's Medium (IMDM) with 112 mmol/l Na+ and 85 mmol/l Cl-. In experimental series the NaCl concentration at the basal side of the epithelium was increased up to 137 mmol/l Na+ and 99 mmol/l Cl- as found in the serum of neonatal rabbits. Light microscopy revealed morphologically faultless epithelia following gradient perfusion culture in standard and NaCl-adapted IMDM. The development of principal and intercalated cell features was monitored with the monoclonal antibodies 703, 503, PCD9, and peanut lectin. Cells immunopositive for monoclonal antibody 703, for example, increased from less than 10% in controls to more than 80% in NaCl-adapted IMDM. It is a new finding that the development of collecting duct cell features is influenced by the extracellular electrolyte environment.
Subject(s)
Cell Differentiation/drug effects , Kidney Tubules, Collecting/embryology , Sodium Chloride/pharmacology , Animals , Cell Division/drug effects , Culture Media , Culture Techniques , Epithelial Cells/cytology , Epithelium/embryology , Gluconates/pharmacology , Immunohistochemistry , Kidney Tubules, Collecting/cytology , Lithium Chloride/pharmacology , Potassium Chloride/pharmacology , RabbitsABSTRACT
During organogenesis the ampullar epithelium of the renal collecting duct acts as an inducer which generates all of the nephron anlagen. As development proceeds, one part of the collecting duct cells in the ampullar tip retain their inducer capability, while others develop into the functional epithelium consisting of principal and intercalated (IC) cells. The events leading from the embryonic inducer to the mature tissue are unknown. We investigated the maturation of embryonic collecting duct epithelium derived from neonatal rabbit kidney under in vitro conditions. To prevent dedifferentiation the epithelia were cultured on kidney-specific support material within a tissue carrier. Apical and basal compartments of the epithelia were simulated in a gradient culture container. The two sides of the epithelium were each constantly perfused with a different medium. During the 14-day incubation the tissue was not subcultured. The development of collecting duct cell features was investigated with morphological and immunohistochemical methods. Both light and electron microscopy revealed morphologically intact epithelia following gradient culture. The polarized cells rested on a uniformly developed basement membrane. The continuous application of aldosterone during the culture modulated the development of collecting duct cell characteristics. Both basal and luminal administration of aldosterone initiated differentiation in the embryonic epithelia. Using the sodium (Na) channel blocker amiloride, it was demonstrated that Na channels are involved in the differentiation of the IC cell phenotype.
Subject(s)
Kidney Tubules, Collecting/cytology , Aldosterone/pharmacology , Amiloride , Animals , Antibodies, Monoclonal , Cell Differentiation/drug effects , Cells, Cultured , Diffusion Chambers, Culture , Diuretics , Epithelial Cells , Histocytochemistry , Kidney Tubules, Collecting/ultrastructure , Microscopy, Electron , Rabbits , Sodium Channels/drug effects , Sodium Channels/metabolismABSTRACT
In the stagnant environment of traditional culture dishes it is difficult to generate long term experiments or artificial tissues from human cells. For this reason a perfusion culture system with a stable supply of nutrients was developed. Human chondrocytes were seeded three-dimensionally in resorbable polymer fleeces. The cell-polymer tissues were then mounted in newly developed containers (W.W. Minuth et al, Biotechniques, 1996) and continuously perfused by fresh medium for 40 days. Samples from the effluate were analyzed daily, and the pH of the medium and glucose concentration remained stable during this period. The lactid acid concentration increased from 0.17 mg/ml to 0.35 mg/ml, which was influenced by the degradation of the resorbable polymer fibers used as three dimensional support material for the cells. This perfusion system proved to be reliable especially in long term cultures. Any components in the culture medium of the cells could be monitored without disturbances as caused by manual medium replacement. These results suggest the described perfusion culture system to be a valuable and convenient tool for many applications in tissue engineering, especially in the generation of artificial connective tissue.
Subject(s)
Cartilage/cytology , Culture Techniques/trends , Hyalin/metabolism , Polymers/chemistry , Adult , Aged , Biocompatible Materials/metabolism , Cell Differentiation/physiology , Cell Separation , Cells, Cultured , Culture Media , Humans , Hydrogen-Ion Concentration , Lactic Acid/metabolism , Middle AgedABSTRACT
Kidney function depends on a well-developed vascular system. Any impairment of the blood supply disturbs the integrity and function of the organ. The differentiation of renal vessels has been investigation for many years, but little is known about the relationship between nephrogenesis and vessel development. In the present work the spatial organization of the differentiating vessels was analyzed in precisely oriented tissue sections and in optical sections acquired by laser scan microscopy. Developing vessels as well as small capillaries were visualized with two endothelium-detecting antibodies. Small vessels running in parallel towards the organ capsule were detected in numerous cortico-medullary-oriented tissue sections. Cross-sections of the nephrogenic zone showed a regularly arranged network, which was composed of cells detected by both monoclonal antibodies. Parts of this network were localized in regions of the nephrogenic zone which have been assumed to be free of vessels or vessel-like structures for a long time. These results were confirmed by the laser-scan-microscopic analysis of complete cortex explants. The extraordinarily regular arrangement of the endothelial network in the nephrogenic zone allowed us to reconstruct the developing vascular system. The results presented here underline the close relationship between nephrogenesis and vessel development.