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Biomedical subjects

W W Minuth

Publications and source records attributed to W W Minuth.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

Biomedical Engineering↗
PubMed1995

Development of renal podocytes cultured under medium perifusion.

BACKGROUND: In the past, podocytes have been described as highly susceptible to dedifferentiation under cell culture. Whether this process resulted from insufficient culture conditions or whether it was a consequence of missing cellular interactions remained unclear. A further reason could be that podocytes within the maturing kidney are irreversibly growth-arrested at a very early point of development because proliferating cells have been detected at the S-shaped body stage but not at the capillary loop stage or in the maturing glomeruli. These were important reasons that hindered the establishment of podocyte cell culture systems. EXPERIMENTAL DESIGN: The aim of our present study was to culture podocytes under the most organotypic conditions possible to maintain typical cellular characteristics. Cortex explants of neonatal rabbit kidneys consisting of nephrogenic tissue were used as a source for podocytes. No serum additives were given for the whole culture period of 13 days. An organ-specific environment was obtained by keeping the podocytes within the surrounding renal tissue and by ensuring a permanent exchange of medium. RESULTS: mAb were used to characterize podocytes and the other glomerular cell types. Cultured podocytes and parietal cells of Bowman's capsule were identified by EnPo 1. Ks 19.2.105, a marker for cytokeratin 19, was used to discriminate among these epithelial cells because cytokeratin 19 is expressed by the parietal cells of Bowman's capsule but not by podocytes. The Ab EC1 specifically detected endothelial cells. Glomerular endothelium cultured under medium perifusion expressed these typical Ag and thus could be unequivocally discriminated. Furthermore, by means of the proliferation marker Ki-67, it could be demonstrated that glomerulus-like structures developed under culture by proliferation of visceral and parietal cells of Bowman's capsule. CONCLUSIONS: A culture model is presented that allows the maintenance of developing podocytes within the organ-specific tissue environment and under permanent medium perifusion.

Animals↗
PubMed1995

Transitional differentiation patterns of principal and intercalated cells during renal collecting duct development.

The developing renal collecting duct epithelium of neonatal rabbits exhibits 3 different zones. The ampullary tip epithelium acts as an embryonic inducer and is responsible for the generation of all of the nephron anlagen. It pilots the whole microarchitecture of the kidney. In the ampullary neck epithelium multiple cell divisions cause the elongation of the embryonic collecting duct so that the organ can grow. Finally, the cells in the ampullar shaft transdifferentiate into the functional collecting duct epithelium (CD) consisting of Principal (P) and various kinds of Intercalated (IC) cells. It is unknown by which morphogenic mechanisms the ampullar cells develop into the heterogeneously composed collecting duct epithelium. Using both morphological and immunohistochemical methods, we investigated the transdifferentiation patterns leading from the ampullar epithelium to the P and IC cells in the neonatal kidney. An electron microscope analysis of the cortico-medullary course of the developing collecting duct revealed that conspicuous morphological alterations start in the neck of the ampulla. The lumen of the neck region is narrowed to a slit. While most of the cells in the ampullar tip exhibit few, short microvilli, the neck cells bear numerous, extremely long microvilli at their apical cell poles. All of the neck cells exhibit the same cytoplasmic staining pattern and the same number of mitochondria. Farther down in the shaft, clearly recognizable P and IC cells are found. Thus, differentiation into P and IC cells starts with a transitional precursor cell type in the ampullar neck. Perfusion culture experiments with the embryonic collecting duct epithelium made it possible to generate transitional and differentiated cell types for the first time under in vitro conditions. The cultured epithelial cells showed characteristics common to both P and IC cells. Immunohistochemical findings revealed that morphological differentiation starts before the functional properties of P and IC cells can be detected.

Animals↗
PubMed1994

Interrelationship of renal vascular development and nephrogenesis.

Within the cortex region of the neonatal rabbit kidney the developing microvasculature was investigated by means of two endothelium-detecting antibodies (EnPo 1 and EC1). Rows of antibody-labelled cells were found within tissue regions that had previously been described as avascular. We conclude that these vessel-like structures detected by EnPo 1 and EC1 are capillary precursors without lumina. Furthermore, beneath the fibrous capsule within the morphologically homogeneous mesenchyme two cell populations can be discriminated by use of differential antigen expression. The EnPo 1 antigen, which is abundant on endothelial cells and podocytes at different developmental stages, was detected on a subpopulation of mesenchymal cells. These cells were exclusively detected surrounding the tip of the collecting duct ampulla. Due to the unique specificity of EC1 and EnPo 1 the process of microvascular development can be readily followed on serial optical sections gained by laser scan microscopy. (1) Adjacent to EnPo 1-positive mesenchymal cell islets vessel-like structures are found that are in contact with the differentiated vasculature. (2) The renal vesicle is enclosed by a network of vessel-like structures establishing contact with differentiated vessels. (3) No guidance of invading capillary sprouts toward the developing glomerulus and nephron is required, since vascular elements already accompany the earliest detectable nephron stage.

Animals↗
PubMed1994

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.

Adult↗
PubMed1994

Maturation of renal collecting duct cells in vivo and under perifusion culture.

The embryonic collecting duct epithelium of neonatal kidney undergoes profound functional changes during maturation. In its initial state as inductor epithelium it appears homogeneous, but differentiates into a heterogeneously composed collecting duct epithelium consisting of principal and intercalated cells. The mechanism of this terminal differentiation process is unknown. We used morphological and immunohistochemical methods to investigate the maturation of the collecting duct system in neonatal rabbit kidney and under organotypic culture conditions. The new perifusion culturing method allowed us to follow the differentiation of the ampullary collecting duct epithelium under conditions as close as possible to the situation within the organ. With this technique we were able to induce a differentiation process similar to that in the in situ situation. This process led to the appearance of a mixed cell population consisting of principal and intercalated-like cells, respectively. A continuous perifusion of the medium made it possible to stabilize the microenvironment under culture conditions and thus to maintain the heterogeneous composed collecting duct epithelium in a differentiated status over long periods of time.

Animals↗
PubMed1994

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.

Aldosterone↗
PubMed1993

Histochemical markers reveal an unexpected heterogeneous composition of the renal embryonic collecting duct epithelium.

The ampullary collecting duct epithelium acts as an inductor in the embryonic and neonatal kidney. It induces the formation of all nephron generations and thus determines the whole architecture of the kidney. As the organ matures, the collecting duct epithelium itself transdifferentiates. The ampullary inductor epithelium, which appears homogeneous as revealed by light microscopy, develops into the well-known heterogeneous epithelium of the mature collecting duct consisting of light principal and dark intercalated cells. Up to now the mechanisms initiating and regulating this transdifferentiation step are unknown. Only very few data are available concerning functional characteristics of the ampullary epithelial cells of neonatal rabbit kidney. Therefore, a characterization of the collecting duct ampullary cells was carried out by means of immunohistological techniques using a set of different monoclonal antibodies and the lectin peanut agglutinin. All epithelial cells within the ampullary tip and neck were positive for cytokeratin 19, an intermediate filament protein. On the other hand, the monoclonal antibody CD 7 revealed a clear cut boundary between the ampullary neck and the ampullary tip region. Furthermore, after incubation with the monoclonal antibody BO-7 specifically reacting with intercalated cells of the mature collecting duct, both labeled and unlabeled cells were observed within the whole ampullary epithelium. These results were confirmed by scanning electron microscopical investigations which revealed two distinct epithelial cell populations. Thus, an unexpected heterogeneity of the ampullary epithelium could be demonstrated.

Amino Acid Sequence↗
PubMed1993

Aldosterone modulates PNA binding cell isoforms within renal collecting duct epithelium.

To investigate the differentiation of the ampullary collecting duct cells into adult principal and intercalated cells, the embryonic cortex of newborn New Zealand rabbit kidney was isolated and brought in culture. With this culture technique the ampullary cells formed a polarized collecting duct epithelium which was kept under permanent exchange of medium and in the presence of aldosterone, arginine vasopressin and/or insulin. After 14 days of perfusion culture the epithelia showed light and dark cells resembling the principal and intercalated cells of the adult collecting duct. The differentiation from embryonic into adult collecting duct cells was controlled by applying the monoclonal antibody CD 7. Independent of the hormonal treatment all of the epithelial cells matured in culture and expressed the CD 7 antigen. This corresponded with the situation found within the adult kidney, where the CD 7 antigen was localized in all principal and intercalated (IC) cells, whereas the embryonic ampullary epithelium in the neonatal kidney remained negative. A differentiation feature of the beta-type intercalated cell was investigated by labeling the cultured epithelia with peanut agglutinin (PNA). In contrast to the CD 7 antigen the development of PNA binding was highly dependent of time and individual hormone administration. While in control epithelia only 8% of PNA positive cells were found, aldosterone induced epithelia revealed 72% PNA labeled cells. The combination of aldosterone and insulin increased the number of PNA-positive cells to 90%. By scanning electron microscopy it could further be shown that several isoforms of cells were reactive with PNA. Thus, in culture the PNA label is not restricted to the typical beta-type IC cells.

Aldosterone↗
PubMed1993

Silver-enhanced colloidal-gold labelling of rabbit kidney collecting-duct cell surfaces imaged by scanning electron microscopy.

The luminal cell surfaces of rabbit kidney cortical collecting-duct cells were labelled with peanut lectin (PNA) and investigated by scanning electron microscopy. Labelling was performed either on 20-microns-thick cryostat sections from prefixed and cryoprotected rabbit kidney tissue or on cultured collecting-duct epithelium using biotinylated PNA and a 6-nm colloidal-gold-coupled antibody against biotin. Colloidal-gold labels were detected at low magnification (2000-4000x) using silver enhancement. Coating with chromium allowed simultaneous imaging of both cell-surface morphology and labelling topography in the backscattered electron imaging mode. Our results show that PNA binding is specific for a subtype of intercalated cells equipped with microvilli on the luminal surface. The presented method promises to be useful for the identification of specific cell types in heterogeneous tissues.

Aging↗
PubMed1992

Characterization of an endothelial protein in the developing rabbit kidney.

A new protein (EnPo 1 antigen) abundant on endothelial cells and glomerular podocytes has been characterized by means of the mouse monoclonal antibody EnPo 1. Following electrophoretical separation of rabbit kidney homogenates EnPo 1 recognized a protein with a molecular weight of 110 kDa and an isoelectric point of 5.9 in Western blots. Using immunohistological techniques, the EnPo 1 antigen has been localized in high concentrations on glomerular podocytes of different developmental stages. Furthermore, the EnPo 1 antigen was expressed on endothelial cells of all adult rabbit organs tested so far. Detailed analysis of neonatal rabbit kidney revealed the abundance of EnPo 1 antigen on both differentiated vessels as well as on immature endothelial cells and endothelium of the microvasculature. Thus, for the first time a marker for in situ investigations of angiogenic processes within the mammalian kidney is available. Analysis of kidney cryosections by confocal laser scan microscopy revealed a direct connection between mature and differentiating vessels in the outer kidney cortex. Furthermore, two differentially organized cell populations discriminated by their EnPo 1 binding pattern were localized in the embryonic renal cortex. Morphologically, these cells were not distinguishable from other mesenchymal cells.

Animals↗
PubMed1992

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.

Animals↗
PubMed1990

A compatible support system for cell culture in biomedical research.

The lack of a suitable system to culture epithelial cells for a long period under a luminal-antiluminal medium gradient, was the reason to develop a new system. It consists of an interchangeable sheet of permeable support material, which is set in place by two tight fitting holding rings. For special demands the supports can be coated with extracellular matrix proteins improving cellular attachment and terminal differentiation. The handling of the sheets by forceps proceeds easily and quickly, thus fastening the transfer of cultured cells without additional manipulations. The sheets can be transferred to a newly developed microperfusion chamber on which an apical and a basal perfusion over a long culture period parallel to a transepithelial electrophysiological registration becomes possible. The chamber has an extremely low amount of fluid dead space. The separate perfusion of cultured cells under isotonic, hypotonic or hypertonic conditions opens new possibilities. Thus, culture can be performed under most natural conditions e.g., that found within the kidney.

Animals↗
PubMed1990

Successive lectin-binding changes within the collecting duct during post-natal development of the rabbit kidney.

Histological sections of neonatal rabbit kidney of various ages were analysed to study the ontogeny of the collecting duct system. In addition, the sections were incubated with fluorescent wheat germ (WGA) and peanut agglutinin (PNA) to investigate the terminal differentiation of collecting duct cells. With both lectins we found different binding behaviours in the three segments of the collecting duct anlagen, the ampullary bud, the ampullary neck and the collecting duct. During the observed steps in development the lectin-binding pattern in the collecting duct and the ampullary neck appeared unchanged. In the collecting duct most cells reacted with WGA and only a few with PNA, while in the ampullary neck all cells bound both WGA and PNA. In the ampullary bud, however, the lectin-binding pattern changed between unlabelled and completely labelled stages with both lectins. The results indicate that both intercalated and principal cells originate from a common precursor cell.

Animals↗
PubMed1990

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.

Animals↗
PubMed1989

Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney.

Immunohistochemical experiments with monoclonal antibodies (mabs) on the kidney of neonatal rabbits revealed that the primary expression of collecting duct typic structures does not occur in a continuous and parallel, but in a subsequent developmental process. Only mabs RCT-30 A, and CD 4-V revealed immunoreactivity at the ontogenetically oldest parts of the collecting duct, the ampullae, while the other used markers (CD 1-3, CD 5-V, RCT-30 and RMCX) did not. In contrast, all of the tested antibodies showed positive reactions at the medullary and cortical collecting duct of the neonatal kidney as well as of the adult kidney. Additional incubations with wheat-germ agglutinin (WGA) a marker of terminal-differentiated collecting duct cells demonstrated weak-labelled ampulla cells beside intensively labelled ampullary neck and medullary collecting duct cells. With peanut agglutinin (PNA) labelling a 3 step transition could be illuminated: weak-labelled ampulla cells were found beside continuously bright labelled ampullary neck cells and finally a punctuate pattern downwards to the papilla. If the ampullary neck is the zone of proliferation, our findings of WGA- and PNA-co-labelling in this zone indicate, that in early developmental stages characteristic structures of different mature cells, probably principal and intercalated cells, are co-expressed within one single cell type. Thus intercalated cells might derive from principal cells.

Animals↗