Biomedical subjects

Raimund Strehl

Publications and source records attributed to Raimund Strehl.

11 recordsLinked to original sources

PubMed2006

Derivation of a xeno-free human embryonic stem cell line.

Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [Stem Cells 2006; 24:221-229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611.

Cell Culture Techniques↗

PubMed2006

Pluripotent human stem cells as novel tools in drug discovery and toxicity testing.

Improved technologies are urgently needed to develop effective and safe new drugs in a cost-efficient manner. Cell-based assays have many advantages in drug research, particularly because these assays can be adapted in a high-throughput format. In addition, technological advances in the areas of instrumentation and automation are providing expanding opportunities for high-content analyses. However, in cell-based research, none of these systems is particularly useful unless the cells that are being evaluated are clinically relevant. Pluripotent human stem cells are expected to revolutionize the accessibility to a variety of human cell types. The possibility to propagate pluripotent human stem cells and to subsequently differentiate these cells into desired target cell types will provide a stable supply of cells for a range of applications in drug discovery and toxicity testing. This feature discusses some of the research opportunities for pluripotent human stem cells.

Cell Culture Techniques↗

PubMed2005

Long-term maintenance of human articular cartilage in culture for biomaterial testing.

Cartilage is a tissue that derives its unique mechanical and biological properties from the combination of relatively few cells and a large amount of a complex extracellular matrix. Furthermore, cartilage tissue is comparatively slow to respond to changes or harmful influences. To date, the optimal generation and long-term maintenance of cultured human articular cartilage for in vitro testing of biomaterials, poses an experimental difficulty. Experiments using cultured isolated chondrocytes in combination with scaffolds often fail to yield results comparable to the in-vivo situation. Consequently, our aim was to develop a culture method that allows in vitro maintenance of human hyaline cartilage explants in an optimal quality over an extended period of time. Such a culture could, for example, be used to determine the long-term effect of a new scaffold on intact cartilage, as an in vitro model for repair processes and to investigate biomaterial integration. In this study we compared conventional static cultures with and without serum supplementation to a serum-free perfusion culture for the ability to maintain human articular cartilage explants in a morphologically intact and differentiated state over an extended period of time of up to 56 days. Results were evaluated and compared by morphological, histochemical and immunohistochemical methods. The experiments showed that short-term maintenance of cartilage in a differentiated state for up to 14 days is possible under all culture conditions tested. However, best long-term culture results for up to 56 days were obtained with perfusion culture under serum-free conditions. Such a perfusion culture system can be used to perform biocompatabilty tests in vitro by long-term coculture of biomaterial and intact human articular cartilage.

Adult↗

PubMed2005

Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system.

We describe an improved and more robust protocol for transfer and subsequent propagation of human embryonic stem cells under feeder-free conditions. The results show that mechanical dissociation for transfer of the human embryonic stem cells to Matrigel resulted in highest survival rates. For passage of the cultures on the other hand, enzymatic dissociation was found to be most efficient. In addition, this method reduces the time, work, and skills needed for propagation of the human embryonic stem cells. With the present protocol, the human embryonic stem cells have been cultured under feeder-free conditions for up to 35 passages while maintaining a normal karyotype, stable proliferation rate, and high telomerase activity. Furthermore, the feeder-free human embryonic stem cell cultures express the transcription factor Oct-4, alkaline phosphatase, and cell surface markers SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, and formed teratomas in severe combined immunodeficient mice. This method provides distinct advantages compared with previous protocols and make propagation of human embryonic stem cells less laborious and more efficient.

Cell Culture Techniques↗

PubMed2005

Renal epithelia in long term gradient culture for biomaterial testing and tissue engineering.

In the organism epithelia perform perfect barrier functions. Strong rheological and mechanical influences constitute the normal environment of this tissue throughout life. Most epithelia are exposed to different fluids at the luminal and basal sides. To obtain realistic information about tissue development in modern biomaterial testing and tissue engineering it is necessary to mimick the natural environment of epithelia. Cultured cells are brought in contact with an artificial extracellular matrix to determine whether proper development into a functional epithelium occurs. As under natural conditions the cultures have to withstand mechanical and fluid stress over a prolonged period of time in close contact to a selected biomaterial. However, development of tissue-specific features such as polarization, tightness and transport under in vitro conditions will only occur, if the biomaterial and the culture conditions support tissue development. Leakage, edge damage and pressure differences during culture have to be avoided so that the natural functions of the growing epithelium can develop. Our aim is to generate functional epithelia derived from renal explants containing stem cells, which are microsurgically isolated and placed into specific O-ring carriers for optimal handling. The cells develop in combination with a collagenous matrix from an embryonic into a functional collecting duct (rCD) epithelium. To achieve optimal culture conditions the tissue is placed in a gradient culture container. A typical environment can be simulated by superfusing different culture media at the luminal and basal sides. Within days epithelia growing inside the gradient container build up a physiological barrier, which is maintained during the whole culture period. The described method allows to investigate the influence of new biomaterials over prolonged periods of time.

Animals↗

PubMed2003

Urea restrains aldosterone-induced development of peanut agglutinin-binding on embryonic renal collecting duct epithelia.

Peanut agglutinin (PNA) represents a commonly used marker for beta-type intercalated (IC) cells and their distribution in the corticomedullary course of the collecting duct (CD) in the mature rabbit kidney. It has been shown that aldosterone is able to generate >90% of PNA-binding cells in an embryonic CD epithelium in vitro. In adult kidney, a maximum of only 25% PNA-positive cells is found in the cortical segment of the CD, and PNA-binding completely disappears in the inner-medullary CD. Molecules that regulate the gradual development of CD-specific cells during organ growth are unknown. In the present experiments, it was found that addition of physiologic concentrations of urea to the culture medium is able to restrain the action of aldosterone in embryonic CD epithelia. Urea antagonizes in a concentration-dependent manner the action of aldosterone finally leading to only 10% of PNA-binding cells. The data point to a urea-specific effect, because osmolytes such as NaCl and mannitol did not affect PNA binding. In addition, urea did not influence expression of principal-cell typical markers such as AQP2 and 3. The findings may explain that a higher number of PNA-positive cells is found in the cortical region of the kidney correlated with a low concentration of urea as compared with only few PNA-binding cells in the medullary CD, where a high concentration of urea occurs. Thus, an increasing concentration of urea may trigger the number of PNA-positive cells in the cortical-medullary course of the CD during organ development.

Aldosterone↗

PubMed2002

Detection of glycosylated sites in embryonic rabbit kidney by lectin chemistry.

The reciprocal cell biological interaction between mesenchymal and epithelial tissue plays a critical role during nephrogenesis. It is unknown to date whether the tissues interact during nephron induction by pure diffusion of substances or whether cellular contacts via gap junctions or focal adhesion molecules are involved. In neonatal rabbit kidney the interface between both tissues shows unique features. It consists of a distinct space, which is filled with specific extracellular matrix consisting of glycosylated proteins such as fibronectin, laminin, collagen, and proteoglycans. In the present experiments we tested by histochemistry whether it is possible to detect additional glycosylated proteins using Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Ulex europaeus I agglutinin (UEA I), and Peanut agglutinin (PNA) as molecular markers. All tested lectins showed distinct labeling patterns in embryonic renal tissue. Within the collecting duct ampulla, DBA and UEA I revealed intensive cellular reaction. In contrast, PNA and SBA reacted at the basal aspect of the collecting duct ampulla tip in addition to a cellular reaction. To identify the individual molecules labeled by the lectins, embryonic tissue was fractionated and separated by electrophoretic methods. For the first time, we were able to show by two-dimensional electrophoresis and subsequent western blot experiments that lectins bind to a series of individual protein spots, which have not been identified to date.

Animals↗

PubMed2002

Advanced technique for long term culture of epithelia in a continuous luminal-basal medium gradient.

The majority of epithelia in our organism perform barrier functions on being exposed to different fluids at the luminal and basal sides. To simulate this natural situation under in vitro conditions for biomaterial testing and tissue engineering the epithelia have to withstand mechanical and fluid stress over a prolonged period of time. Leakage, edge damage and pressure differences in the culture system have to be avoided so that the epithelial barrier function is maintained. Besides, the environmental influences on important cell biological features such as, sealing or transport functions, have to remain upregulated and a loss of characteristics by dedifferentiation is prevented. Our aim is to expose embryonic renal collecting duct (CD) epithelia as model tissue for 14 days to fluid gradients and to monitor the development of tissue-specific features. For these experiments, cultured embryonic epithelia are placed in tissue carriers and in gradient containers, where different media are superfused at the luminal and basal sides. Epithelia growing on the tissue carriers act as a physiological barrier during the whole culture period. To avoid mechanical damage of the tissue and to suppress fluid pressure differences between the luminal and basal compartments improved transport of the medium and an elimination of unilaterally accumulated gas bubbles in the gradient container compartments by newly developed gas expander modules is introduced. By the application of these tools the yield of embryonic renal collecting duct epithelia with intact barrier function on a fragile natural support material could be increased significantly as compared to earlier experiments. Epithelia treated with a luminal NaCl load ranging from 3 to 24 mmol l were analyzed by immunohistochemical methods to determine the degree of differentiation. The tissue showed an upregulation of individual CD cell features as compared to embryonic epithelia in the neonatal kidney.

Animals↗

PubMed2002

Proliferating cells versus differentiated cells in tissue engineering.

The efficiency of cell or tissue cultures is usually judged by how quickly confluence is reached within a Petri dish or on a scaffold. Growth factors and fetal bovine serum are employed to drive cultured cells from one mitosis to the next as quickly as possible. The tissue specific interphase is extremely short under these conditions, so that the degree of differentiation desired in tissue engineering cannot be achieved. To reach the goal of functional differentiation in vitro mitosis and interphase must be separated experimentally and tailored to the specific requirements of the cell-type used. This could be achieved by a three step concept for tissue-engineering in vitro as we present here. The expansion phase is followed by a phase in which tissue differentiation is initiated. The final phase serves to express and maintain histotypical differentiation of the generated tissue.

Animals↗

PubMed2002

SBA-positive fibers between the CD ampulla, mesenchyme, and renal capsule.

During kidney development, the CD shows two peculiarities. First, the tip of the CD ampulla is always found at a specific distance from the organ capsule. Second, the CD growth occurs as a perfectly straight elongation. It is unknown whether the CD-specific growth is dependent on hormonal action or on structural elements. Histochemical experiments on neonatal rabbit kidney yielded new insight into the interface of the CD ampulla and the surrounding nephrogenic mesenchyme. Incubation of tissue sections with soybean lectin (SBA) showed the existence of fibers extending in a radial course from the ampullar tip through the mesenchyme toward the organ capsule. SBA labeling did not colocalize with collagen type I, III, IV, V, and VI, laminin, fibronectin, and tenascin. It is assumed that while the kidney increases in volume the structural fixation of the ampullar tip by the SBA-positive fibers causes CD ampullae to maintain a constant distance from the organ capsule. The connection would explain the linear extension of the CD in relation to the organ capsule. In addition, the presented data suggest that the SBA-positive fibers between ampullar tip and organ capsule create a structural microcompartmentation of the nephrogenic zone.

Animals↗

PubMed2002

Cyclooxygenases in the collecting duct of neonatal rabbit kidney.

COX 1 and 2 expression during the terminal phase of kidney development is poorly understood. To obtain information about this process we followed the primary appearance of cyclooxygenases in collecting duct (CD) epithelia of the neonatal rabbit kidney with immunohistochemical and two dimensional electrophoretical methods. In the fully embryonic cortical zone immunohistochemical expression of COX 1 is seen in all cells of the CD ampulla, while COX 2 is lacking within the nephron inducer. Within the matured cortical collecting duct (CCD) COX 1 and 2 immunoreactivity could not be detected. In contrast, a heterogeneous expression profile for COX 1 and 2 is found in the outer medullary CD (OMCD), since not all cells showed immunohistochemical labeling. Within the inner medullary CD (IMCD) nearly all cells express both cyclooxygenases. As revealed by western blot experiments generated embryonic CD epithelia in perfusion culture demonstrate high COX 1 presence at the begin of culture, while COX 2 is found to a minor degree. From day 3 until day 14 continuous levels of COX 1 and 2 expression are detected. Administration of 1 x 10(-7) mmol/l aldosterone does not influence COX expression, while application of 100 mmol/l NaCl increases COX 2 fourfold. The upregulation of COX 2 by a chronic NaCl load in embryonic epithelia suggests in part a constitutive and in part a facultative expression during CD cell differentiation.

Animals↗