[in Japanese]

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In vitro culture of bovine preimplantation embryos, in general, has been achieved in a serumsupplemented medium with a co- cultivation of somatic cells such as oviduct epithelial cells and cumulus/granulosa cells. This review describes an improved culture methodology of bovine preimplantation embryos that can be cultured in a serum- free medium without somatic cell support. Several improvements have been undertaken to establish a novel serum- free culture system in order to increase the efficiency of bovine embryo development. The optimal energy utilization of embryos was determined with a reduced concentration of glucose from a commercial nutrient TCM199 medium and added with pyruvate and lactate. Oxygen tension is one of critical factors in culture environment. Low oxygen concentration (5% O₂) was superior to the regular open air culture system (approximately 20% O₂) for bovine embryo development. Polypeptide factors such as FGF-2, TGF-β₁ and tissue inhibitor of metalloproteinase-1 (TIMP-1) were identified as embryotrophic activity. As a result, the newly developed serum- free culture system without somatic cell co-culture apparently increased the higher efficiency of bovine blastocyst formation than conventional serum- supplemented medium with somatic cells.

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We applied VNTR locus typing to the identification of cell lines. Using human leukemia/lymphoma cell lines, each locus (ApoB, D1S80 and DXS52) was amplified by PCR. Detection of theY-chromosome was also done. Sixteen cell lines derived from various individuals, (some cell lines from the same patient, others not) were selected for identification of their ApoB, D1S80and DXS52 VNTR loci and Y-chromosome. BALM-3, -4 and -5, three cell lines derived from one female patient, were identical for all VNTR loci tested. No specific bands for the Ychromosome were detected. BALM-13 and -14, derived from one male patient, were identical for all VNTR loci tested, and their Y-chromosome specific bands were clearly detected. BALM9 and its subclones (BALM-9K, -L, -KL and N), BALM-10, -11 and -12, all derived from one female patient, were as follows: the ApoB locus of BALM-9, B ALM-9KLa nd BALM-12w as detected as two bands of the same size, distinct from the other bands from clones from that patient. The DXS52 locus of BALM-11 was deleted, and BALM-12 showed two bands which were absent in the other clones from the same patient. However, the D1S80 locus was detected consistently in all clones. All results obtained by VNTR locus typing were confirmed by DNA profiling. In further studies, eight human leukemia/lymphoma cell lines, CCRF-HSB-2, MOLT-4, BALL-1, Namalwa, HAL-01, HL-60, KU-812 and EoL-1, were selected and their ApoB, D1S80 and DXS52 loci were investigated at two independent laboratories. The results obtained were compared by computer analysis. They showed identical patterns and bands sizes except for one cell line (Namalwa). This indicated that one cell line from one of the laboratories was mislabeled. We demonstrate here that VNTR typing amplified by PCR is a convenient (simple, speedy and accurate) and useful technique for cell line identification and for checking cross-culture contamination of cell lines.

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The present study was undertaken to confirm the presence of different types of microglia in the mixed brain cultures of neonatal mice and in tissue sections from adult mice, by immunohistochemical method with monoclonal antibodies, Mac-1 and F4/80 which recognize with surface antigens of monocyte-macrophage(MO) and ER-MP antibodies which recognize with surface antigens of bone marrow precursor cell. We identified ER-MP positive microglia in mixed brain cultures which was clearly distinguished from Mac-1 and F4/80 positive microglia. Furthermore, ER-MP positive microglia were observed in tissue sections from the brain. In contrast MO from lung did not express ER-MP antigens. In brain tissue from op/op mice which were deficient for monocyte-MO, the ER-MP positive cells were observed as a comparable level as normal mice, however, Mac-1 and F4/80 positive cells could be hardly observed. In conclusion, microglia in the brain had consisted with at least two different subtypes and ER-MP positive microglia might not be derived from blood monocytes.

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