Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions.

The value of cultured cells in cell biological, pharmaceutical or biotechnological research depends on the degree of terminal cell differentiation. In conventional Petri dishes or tissue culture plates it is often difficult to achieve culture conditions which resemble the in situ situation of intact tissue, as regards optimal cell adhesion, exchange of nutrients and metabolic products. These limitations prompted us to develop simple laboratory tools which optimize the environment of cultured cells. A perfusion apparatus with various culture containers and compatible cell holder sets was constructed which allows the simulation of organotypic conditions. (i) The cells can be kept on individual and interchangeable support materials for an optimal cell attachment. (ii) Culture medium can be perfused during the whole culture period. (iii) One type of the new culture container can be perfused with different media at the apical and basal side of the cells, thus mimicking the organotypic environment that applies for epithelial monolayers. Cell culture experiments with renal collecting duct epithelia exhibited an excellent morphological appearance showing typical features of principal and intercalated cells.

Developing renal microvasculature can be maintained under perfusion culture conditions.

The cortex corticis of the neonatal rabbit kidney consists of developing nephrons, vessels, collecting duct ampullae and the nephrogenic mesenchyme. Inductive interactions between embryonic mesenchyme and collecting duct ampullae lead to the coordinated development of the nephrons and the collecting duct system. The factors regulating nephrogenesis and vascular development within this tissue region are unknown. In order to analyze the hormonal regulation of vascular development an organotypic culture system was established. Cortex explants from neonatal rabbit kidneys were prepared, mounted in a set of holding rings and cultured under serum-free conditions for 14 days in conventional culture plates or under permanent medium perfusion in a newly developed culture container. The detection of endothelial cells was carried out by means of two monoclonal antibodies. Within the renal cortex corticis EnPo 1 detected developing vasculature as well as podocytes and a subset of mesenchymal cells. EC1 displayed exclusive specificity for endothelial cells. The antibody did not discriminate between arteries and veins. Endothelial cells of different developmental stages were labeled with the same intensity. A combination of both antibodies allowed the discrimination between developing endothelial cells and podocytes. Following 14 days of culture under permanent medium exchange, excellent tissue preservation as well as endothelial cell proliferation was observed in cortex explants. In contrast, tissue kept in stationary culture revealed a high degree of disintegration. Endothelial antigen expression was also severely disturbed. Tissue maintenance under stationary conditions was improved by the application of a hormone mixture consisting of aldosterone and 1,25-hydroxyvitamin D3. However, the high degree of spatial organization shown by developing endothelial cells in vivo was maintained exclusively in explants cultured in the presence of hormone under permanent perfusion.

Approach to an organo-typical environment for cultured cells and tissues.

If cells or tissues are taken out of an organ and put in culture, normally they lose morphological, physiological and biochemical features. This dedifferentiation process starts during the isolation procedure and continues during the whole culture period. It is caused by the stagnant liquid condition and the inadequate anchorage of cells at the bottom of tissue culture plasticware. The use of filters as basement membrane substitutes and the coating of cultureware with extracellular matrix proteins improve the environmental factors for cultured cells but do not consider the paracrine influence of cytokines or the nutritional needs of individual cell types. To limit cellular dedifferentiation in culture, we constructed a new system, which adapts, as far as possible, cell and tissue cultures to an organo-typical environment. The system is based on a compatible cell carrier arrangement, which allows individual selection of supports for optimal cell anchorage and differentiation. The cell carriers are placed in a newly constructed container, which is permanently perfused with fresh culture medium. The system runs outside an incubator with simple laboratory tools; only a peristaltic pump, a warming table and pH-stabilized media are necessary. Without any subculturing, acute and chronic influences of drugs or the quality of medical implantation grafts can be studied over months.

Production of monoclonal antibodies against principal cells of the renal collecting duct by in vitro immunization with unsolubilized antigens.

We report the production of monoclonal antibodies (MAb) by an in vitro technique which react with principal cells of the renal collecting duct. Spleen cells were directly simulated in vitro with unsolubilized antigens, i.e., by direct contact with the apical site of cultivated principal cells or by contact with cell fragments. Out of several others two antibodies, IV1 and IV2, were selected, which specifically reacted with the principal cells of the collecting duct. MAbIV1 also reacted with Type A intercalated cells, indicating the existence of a common antigen in the apical membrane of both cell types. Type B intercalated cells were consistently unreactive. All other parts of the uriniferous tubule were also unreactive. In Western blot analysis MAb IV1 showed immunoreactivity with a 40 KD and a 43 KD antigen. Our experiments demonstrate the possibility of producing antibodies against unsolubilized antigens by a simple in vitro technique. The activity of particular lymphocyte in this in vitro system is shown by the specificity of the antibodies.

Engineering of cartilage tissue using bioresorbable polymer carriers in perfusion culture.

Bioresorbable polymer fleeces with a high internal surface area were used as temporary matrices to establish three-dimensional cultures of isolated human articular chondrocytes. The polymer surface was coated with poly-L-lysine to support cell attachment. The resulting cell-polymer tissues were cultured in perfusion culture chambers to achieve a constant supply of nutrients by diffusion. Retention and accumulation of extracellular matrix components synthesized by the chondrocytes were improved by encapsulation of the cell-polymer integrate in agarose gel. The cell-polymer tissues formed abundant collagen fibrils in vitro with a typical cross-triation clearly visible in electron microscopy analysis. Chondrocytes and intercellular matrix stained positively with monoclonal antibodies specific for differentiated chondrocytes and type II collagen. Synthesis of proteoglycans and collagen was also evident by further analysis with alcian blue and azan staining of cell-polymer tissue sections. The presented experimental tissue culture technique offers a novel concept for the in vitro formation of vital cartilage implants for reconstructive surgery or treatment of destructive joint diseases and possibly for the in vitro engineering of human tissues in general, with applications in drug testing and replacement of animal experiments.

Tissue engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new cell culture techniques.

The engineering of living tissues in vivo requires new concepts in cell culture technology. In contrast to conventional cell cultures, the development of tissues depends on a three-dimensional arrangement of cells and the formation or synthesis of an appropriate extracellular matrix. Special emphasis is given to the major role of the extracellular matrix and cell differentiation in an artificial tissue. New technical approaches of in vitro tissue engineering are compared to the natural development of tissues in vivo. Current methods using resorbable biomaterials, tissue encapsulation and perfusion culture are discussed. Major consideration is given to scaffold structures of biomaterials that define a three-dimensional shape of a tissue or guide matrix formation. The different goals of tissue engineering such as in vitro models and transplant production are taken into account in the described techniques. Practical concepts comprising cell multiplication and differentiation in subsequent steps for future clinical applications are outlined.

Tissue factory: conceptual design of a modular system for the in vitro generation of functional tissues.

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.

Characterization of micro-fibers at the interface between the renal collecting duct ampulla and the cap condensate.

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.

The influence of culture media on embryonic renal collecting duct cell differentiation.

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.

Artificial tissues in perfusion culture.

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.

Engineering of cartilage tissue using bioresorbable polymer fleeces and perfusion culture.

Replacement of injured or diseased skeletal tissues by either autograft or allograft cartilage has increased steadily during recent decades. The ideal method is to use autologous cartilage; however, this is extremely limited due to the scarcity of donor sites. We present a new approach to the in vitro formation of cartilage grafts for autologous grafting in reconstructive surgery. Bioresorbable polymer fleeces of polylactic acid were used as temporary cell carrier matrices to establish three-dimensional cultures of human chondrocytes. The polymer surface was coated with poly-L-lysine before cell integration. These cell-polymer tissue constructs were encapsulated with low melting point agarose and then placed in perfusion culture chambers to provide a constant supply of nutrients into the cultures. The culture medium consisted of Ham's F12 supplemented with 2% fetal calf serum and 50 micrograms/ml ascorbic acid. The cell-polymer tissues were harvested and frozen for toloudine and alcian blue staining as well as electron microscopic examination after different periods of time in culture. A monoclonal antibody specific for collagen type II was used to characterize the cell phenotype. With this culture procedure chondrocytes maintained a differentiated phenotype with synthesis of collagen and proteoglycan. Collagen fibrils with clear cross-striation were evident in electron microscopic images. The results show that our organotypic cell culture method allows the in vitro production of bioartificial cartilage for transplantation.

The interface between generating renal tubules and a polyester fleece in comparison to the interstitium of the developing kidney.

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.

Neonatal rabbit kidney cortex in culture as tool for the study of collecting duct formation and nephron differentiation.

By stripping off the capsula fibrosa of neonatal rabbit kidneys a consistently thin tissue layer consisting of collecting duct anlagen, S-shaped bodies and nephrogenic blastema is obtained. This thin layer seems to be an excellent object for investigation of epithelium formation and nephron differentiation. Three different tissue culture protocols are described: 1. A polarly differentiated collecting duct epithelium with 'tight' characteristics consisting only of principal cells, grown on specific renal support 2. A morphologically dedifferentiated collecting duct principal cell monolayer grown on the unspecific bottom of a plastic culture dish 3. An embryonic tissue layer with numerous S-shaped bodies which might be a suitable model for investigation of the development of maturing nephron structures in serum-free culture medium.

Cell associated glycoproteins synthesized by cultured renal tubular cells.

Thin cortical kidney explants from newborn New Zealand rabbits were cultured in Dulbecco's MEM containing 10% fetal bovine serum. Within 24 h the explants formed globular bodies which were completely covered by a monolayered epithelium. The cells show polar differentiation and resemble the renal collecting duct epithelium. By culturing the globular bodies in Dulbecco's MEM with D-valine instead of L-valine additionally a monolayer of renal collecting duct cells was obtained. For the study of glycoprotein synthesis the globular bodies and the collecting duct monolayers were incubated with various labelled carbohydrates, protein and collagen precursors and then fractionated into coarse membrane pellets. The synthesized glycoproteins were regained in 600 x g and 12,000 x g coarse membrane fractions and extracted with Triton X 100 buffer for column chromatography and SDS-polyacrylamide electrophoresis in 6 M urea. In addition to a 85,000 d glycoprotein, a carbohydrate rich collagen like protein (apparent molecular weight in column chromatography 200,000 d, in the SDS-polyacrylamide electrophoresis 150,000 d) was found. The 150,000 d glycoprotein incorporates favorably radioactive proline, sulfate, and smaller amounts of lysine, and leucine. Compared to the 85,000 d glycoprotein a double amount of glucosamine and galactose and four fold amount of fucose was detected. The 85,000 d protein has to be ascribed as a usual glycoprotein, in contrast the 150,000 d protein shows an unusual combination of characteristics and has to be considered as a new type of renal glycoprotein.

Glycoprotein synthesis of renal collecting duct epithelium cultured as globular bodies.

Cortical kidney explants from newborn New Zealand rabbits were cultured in Dulbecco's minimum essential medium (DMEM) containing 10% fetal calf serum. Within 24 h the explants formed 'globular bodies' which were completely covered by a monolayered epithelium. The cells were differentiated and resembled collecting duct epithelium. By culturing the globular bodies in Dulbecco's MEM with D-valine instead of L-valine, a monolayer of collecting duct cells was obtained and used for control experiments. For analysis and identification of synthesized glycoproteins, the globular bodies were incubated with various labelled carbohydrates and amino acids, and then fractionated. Glycoproteins secreted into the culture medium were not detected. Cell-associated glycoproteins were found in crude membrane fractions and then extracted with Triton X-100 for column chromatography, SDS-polyacrylamide electrophoresis in 6 M urea, isoelectrofocusing, and two-dimensional electrophoresis. Two prominent glycoproteins containing galactose and glucosamine were synthesized during the spreading of the epithelium, with an apparent molecular weight of 150,000 and 85,000 (SDS-PAGE). The synthesized glycoproteins differ in their content of radioactive glycoprotein precursor and leucine. The 85,000 dalton monomer glycoprotein has an isoelectric point of 3.5 and was identified by two-dimensional electrophoresis.

Induction and inhibition of outgrowth and development of renal collecting duct epithelium.

Renal cortical explants consisting of capsula fibrosa with an adherent thin layer of collecting duct anlagen, S-shaped bodies, and nephrogenic blastema were isolated from newborn New Zealand rabbits and cultured in Dulbecco's Modified Eagle's medium for 24 hours. Within this period of time, the explants formed globular bodies surrounded by an epithelium of differentiated collecting duct cells. The outgrowth of the collecting duct cells and the formation of the epithelium occurred only when serum was added to the cultivation medium. Different types and different concentrations of serum were tested; fetal bovine serum and newborn and adult rabbit sera at concentrations of at least 5% induced the outgrowth and spreading of the cells. The surrounding epithelium did not develop in the absence of serum. The outgrowth of the collecting duct cells in serum-supplemented cultivation media was arrested by inhibitors of protein and glycoprotein synthesis and by cytoskeletonal-blocking agents such as cycloheximide (1 x 10(-6) M), actinomycin C1 (2 micrograms/ml), tunica-mycin (1 microgram/ml), 6-diazo-5-oxo-norleucine (2 x 10(-5) M), vinblastine (5 x 10(-6) M), colchicine (1 x 10(-3) M), and cytochalasin B (2 micrograms/ml). In contrast, inhibitors of DNA synthesis, e.g., cytosine arabinoside (2.5 x 10(-5) M), mitomycin (1 x 10(-6) M), and hydroxyurea (2.5 x 10(-3) M), had no influence on the outgrowth.

Mesodermalization of amphibian gastrula ectoderm in transfilter experiments.

Transfilter experiments were carried out with competent gastrula ectoderm of Triturus vulgaris and Ambystoma mexicanum as the reacting materials and blastoporal lip, alcohol-treated bone marrow and archenteron roof as the inducing materials, It was shown that a mesodermal inducing substance is able to penetrate Nuclepore filters of different pore sizes (3.0, 1.0, 0.4, 0.2 micrometer). The degree of mesodermal differentiations depends on the reacting materials, the inductors and the filters which were used in the transfilter experiments.