Three-dimensional scaffold-free fusion culture: the way to enhance chondrogenesis of in vitro propagated human articular chondrocytes.

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-b2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-b2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.

Radioresistance of rat glioma cell lines cultured as multicellular spheroids. Correlation with electrical cell-to-cell-coupling.

Two chemically induced rat glioblastomas, RG2 and F98, were cultured as monolayers and as multicellular spheroids and subjected to Co-gamma-irradiation. In parallel, intercellular communication between cells was determined as electrical coupling between neighbouring cells using micro-electrode techniques. A third glioblastoma with known radiobiological response (9L) was assayed with respect to intercellular communication and included into this analysis. Electrical coupling was low for RG2, intermediate for F98, and high for 9L. Radioresistance of spheroids, as expressed in terms of the mean inactivation dose computed from the survival curves increased in the same direction (RG2: 2.4 Gy; F98: 5.1 Gy; 9L: 6.5 Gy). A comparison of these parameters demonstrates a correlation between solid tumor radioresistance and gap-junctional cell-to-cell communication, at least for the class of glioblastomas analysed in this study.

[Organization of the Pediatric Tumor Cell Bank of the Society of Pediatric Oncology and Hematology (GPOH)]. / Aufbau der Kindertumorzellbank der Gesellschaft für Pädiatrische Onkologie und Hämatologie (GPOH).

Characterized cell lines are absolutely necessary in applied research of cell biology and medicine. For the completion of diagnosis and therapy especially in pediatric oncology we are establishing a Cell Bank for Pediatric Tumors. The Cell Bank for Pediatric Tumors collects tissue samples of different types of solid malignant tumors from children and young adults. The specimens are transferred to in vitro culture (guidelines of the American Type Culture Collection-ATCC), the resulting cells are characterized to assure accordance with the histogenesis of the original tumor and stored in liquid nitrogen. The cell cultures are characterized morphologically (phase contrast microscopy) and immunocytochemically (ABC-method). To prove the malignancy of cells in primary culture the amount of hypertetraploid cells was determined (DNA-Scanning-Cytophotometry). Cell lines are checked to find out whether they develop tumors in nude mice followed by an analysis of the karyotype. Additional investigations (e.g. in vitro test of cytostatic drug resistance) are carried out on request by the sender. Part of the tumor tissue which is used to start the cell culture is in parallel diagnosed histopathologically at the Children's Tumor Register, Kiel and/or at the Charité. By the end of the year 1995 the Cell Bank for Pediatric Tumors had received 183 different specimens including 123 solid tumors (e.g. 24 neuroblastomas, 18 osteosarcomas, 12 Wilms' tumors, 13 rhabdomyosarcomas), 44 tissue specimens without any malignant cells, 8 probes without vital cells and 8 leukemias and lymphomas. We were able to establish primary cell cultures of 50% of the sterile tumor tissue probes, to cultivate them for a minimum of 5-10 passages, to characterize and freeze them. Six out of these tumor cell lines were already cultivated for one year and are available to the scientific community.

Cadherin-11 is highly expressed in rhabdomyosarcomas and during differentiation of myoblasts in vitro.

Rhabdomyosarcomas bear a morphological and genetic resemblance to developing skeletal muscle. Apart from myogenic marker genes (bHLH factors, myosin, actin), cell adhesion molecules such as N-cadherin and N-CAM have been reported to be expressed both in rhabdomyosarcomas and during myogenesis. The present study demonstrates the expression of another cadherin, cadherin-11, in rhabdomyosarcomas and during differentiation of myoblasts in vitro: cadherin-11, a predominantly mesenchymal cell adhesion molecule, is highly expressed in embryonal rhabdomyosarcomas and alveolar rhabdomyosarcomas, which do not bear the Pax-3-FKHR fusion previously described. Cadherin-11 is down-regulated in normal skeletal muscle and after myotube formation in vitro. The results of this study suggest that cadherin-11 might be involved in myogenesis and that rhabdomyosarcomas may re-express or fail to down-regulate cadherin-11. Since alveolar rhabdomyosarcomas bearing the t(2;13) translocation do not express cadherin-11, it is postulated that Pax-3 and cadherin-11 might be linked and involved in the same myogenic pathway.