It is clear that mycoplasmal infection of mammalian cell cultures continue to introduce serious artifacts in in vitro investigations. All cell culture laboratories must institute a practical and efficient system to prevent, detect and control mycoplasmal infection. Simple procedures are available and prudent and will increase the reliability and reproducibility of in vitro experimentation.

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Serum-free media developed for general use were examined for the capability of shortterm or long-term growth of various cell lines. The examined media were ASF 103, ASF 104, and GIT. Most established cell lines grew fairly or very well in these media, although slower growth was obvious in some cell lines at a lower cell inoculum. Primary cultured cells from Syrian hamster embryos also grew fairly well when EGF was added to these media. ASF 103 and GIT media supplemented with EGF supported the extended growth of Syrian hamster embryo cells, even though the control cells cultured in a serumcontaining medium showed in vitro senescence. These media had little effect on the production of IgG in hybridoma cell lines and of CA 19 - 9 in a stomach cancer cell line. Growth capacity of HeLa cell line in ASF 103 and GIT media having been kept at 4°Cor -20°C for as long as 9 months proved to be fairly good.

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During the process of X-ray-induced neoplastic transformation of Syrian / golden hamster embryo (SHE) cells, cell morphology altered progressively. While normal SHE cells showed flat and well extended morphology, anchorage-independent clones were slightly refractive and tumorigenic cells became spindle-shaped and highly refractive.
By immunostaining method, we observed that actin microfilaments were well organized in normal SHE cells. Although same pattern still existed in anchorage-independent clones, actin microfilaments completely disappeared in tumorigenic cells. In the case of tubulin fibers, we detected an increment of the number of fibers also in tumorigenic cells. Fibronectin meshwork gradually decreased during the malignant progression of Xirradiated cells. Only vimentin filaments did not altered during the progression of neoplastic transformation.
From western blot analysis, we could not find differences in the amount of actin and tubulin among SHE cells, anchorage-independent clones and tumorigenic cells.
These results suggest that multistep changes of cytoskeletal organization and extracellular matrix cause the stepwise change in cell morphology, and that disappearance of these organization may play a role in the acquisition of tumorigenicity.

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DNA synthesis of pancreatic islet cells of 6-8 wk-old rats was examined during a 14day-culture period by autoradiography using ³H-thymidine. Pancreatic islets were isolated from rats by collagenase and dispersed into single cells with EDTA and Dispase. The dispersed islet cells or isolated islets were cultured in a Microplate and the DNA synthesizing capacity (labeling index) of islet beta cells was determined in the presence of 5.5 or 27.5 mM glucose. The labeling index in 5.5 mM glucose was 0.2 % on the first culture day and this level was the same as that determined in the in vivo experiment. The labeling indices increased and reached a peak of 2-3 % on day 3-5, declined to 1-2 % on day 7 and further to 0.2-0.4 % on day 14. The labeling indices in 27.5 mM glucose attained to the higher levels of 3-4 % on day 3-7, and decreased slowly thereafter. The inability of beta cells to maintain their proliferating capacity under sustained stimulation of high glucose might be one of the causes to induce diabetes in adult age.

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The present study informs the spinner culture of cells with poor anchorage dependency by embedding in collagen droplets. The collagen gel droplets were formed by pouring an ice-cold cell suspension in collagen solution into prewarmed liquid paraffin while stirring. The cells in droplets grew in three dimensional forming clusters. Insulin-secreting In-R1-I10 cells proliferated from 10⁷ on inoculation to 3.6 x 10⁹ per flask during 21 culture days and the amounts of insulin secreted for 24 hr were 0.8, 2.1and 3.9 ug/flask on the 10th,15th and 21st day, respectively. The extent of cell growth could also be monitored by the measurement of consumption of glucose in the medium. This culture system makes it possible to obtain a large number of cells and to maintain functional integrity of cells in culture, and thus enables us to collect cellular products repeatedly during a long term culture period.

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Transformation was carried out by infecting retro virus carrying cytomegaro virus promoter and c-myc oncogene to the growth cartilage (GC) cells from rat ribs. After the screening and cloning, cell clones with different characteristics were obtained and these characteristics were examined by histochemical staining. Two clones showed a spindle shape morphologically, a negative alkaline phosphatase activity (ALP), and an active fiber formation. The clones with these characteristics were assigned as fibroblast type. Three clones showed a polygonal shape morphologically, a negative ALP, and an active fiber formation. The clones with these characteristics were assigned as resting cartilage cell type. Other clones showed characteristics of each GC cells. Depending on their characteristics, they were assigned as the proliferative GC cells, early hypertrophic GC cells and late hypertrophic GC cells. Some of the nodule forming clones reacted with a GC specific antibody. Some clones changed their characteristics before and after their confluency.

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As an animal model with cartilagenous disease, some characteristic features of the osteochondrodysplasia rat (ocd/ocd) are presented. The affected state is lethal in nature, and inherited by an autosomal recessive gene ocd. The affected neonates show a typical dwarfing syndrome with systemic subcutaneous edema. The bone is one of the most severly affected organs. Histologically, there is an unique necrotic area of the chondrocytes spread in the mid portion of the cartilage plate of the affected neonate. Decrease in amount of the ECM substances, especially glycosaminoglycans and hyaluronic acid, are revealed by histochemistry, electronmicroscopy, and biochemical analysis of the affected cartilage. Preliminary findings on the pathogenesis and cultured chondrycytes are also presented.

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Osteoclasts are multinucleated cells responsible for bone resorption. Several lines of evidence have indicated that osteoclasts are derived from hemopoietic stem cells. However, the nature of osteoclast progenitors and their differentiation process into osteoclasts are still not known. We previously reported that osteoclast-like multinucleated cells were formed in mouse marrow cultures and in co-cultures of mouse spleen cells and osteoblastic cells. Using these culture systems, we studied the nature and differentiation process of osteoclast progenitors. Following results were obtained: 1) Osteoclasts are erived from not only immature cells but also mature cells of the monocyte-macrophage lineage.2 ) Osteclast progenitors differentiate into osteoclasts through the interaction of cell-to-cell contact with osteoblastic cells.3) M-CSF produced by osteoblastic cells is an important factor for maintaining growth and differentiation of osteoclast progenitors. These findings have significant implications for the study ofosteoclast biology.

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We examined the spatio-temporal pattern of collagen types I, II, IX and X collagen mRNAs in embryonic chick vertebrae undergoing endochondral ossification by in situ hybridization. Before stage 35, non-hypertrophic chondrocytes expressed both type II and IX collagen genes in the vertebrae. At stage 36. hypertrophic chondrocytes having a moderate amount of type X collagen mRNA were first detected in a few vertebral body segments as an island of cells in the dorsal part of midvertebrae.. Hypertrophic chondrocytes were increased in number at stage 37 and 38, and spread to larger areas.. These cells contained high levels of type X mRNA and decreased levels of type II and IX mRNAs.. From stage 38, blood vessels invaded deeply into the midvertebral hypertrophic chondrocytes area. As. As the vertebrae grew further at stage 40, the leading front of active hypertrophic chondrocytes with high levels of type X mRNA shifted from the midvertebral perivascular area towards intervertebral borders. Later at stage 45, comparison of the localization of collagen types II, IX and X mRNAs in the vertebral segment revealed a clearly reversed pattern of distribution between type X collagen mRNA and types II and X mRNAs. Type I collagen started to deposit in the cartilage matrix adjacent to invading capillaries and in the lacunae enclosing terminal hypertrophic chondrocytes at stages 38 and 39. From stage 40, perivascular mesenchymal cells and/or osteoblasts, bordering between the endochondral bone and marrow cavities, expressed high levels of type I collagen mRNA.

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[in Japanese]

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[in Japanese]

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