In the present review article, the notable and important development and contributions by Japanese scientists in the field of tissue culture are described. The Japanese Tissue Culture Association (JTCA) was founded in 1956 and the 65 meetings of the JTCA have held in various area in Japan. The JTCA has played an important role in the progress of tissue culture studies and in exchanging new informations on research works and practical techniques of tissue culture in Japan. Several committees concerning the cell bank, the biotechnology, the education system, and the edition of the Journal, “Tissue Culture Research Communications”, are effectually assisting the activity of the JTCA. The creative and unique contributions done by Japanese scientists during past 3 decades were described in eight fields of tissue culture, molecular biology, cytogenetics, cell biology, developmental biology, in vitro carcinogenesis, invertebrate cell culture, hereditary diseases and cellular aging. The idea and enthusiasm displayed by these Japanese scientists are still new and fresh. These may stimulate young scientists to proceed their research works in future.

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Recently, considerable success has been achieved in in vitro culture of embryos from livestock species (pigs, cattle, sheep). In all cases, advances with methods for livestock embryos was made possible by research with laboratory mammals, especially the mouse and hamster. One of the most significant of these contributions was the discovery of methods for overcoming 'in vitro developmental blocks' common to most species. Successful techniques such as oviduct organ culture and oviduct cell cultures were pioneered with laboratory mammals. Modifications of culture media, based on research with mice and hamsters, have resulted in high rates of pig embryo development in vitro. Experiments with bovine and ovine embryos are now beginning to show similar promise. Expectations are high for repeatable, successful culture in vitro of early stage embryos from livestock species using any of a number of currently available methods.

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The growth and development of human conjunctival epithelial cells on threedimensional collagen gels were influenced by the type of fibroblasts dispersed in the collagen gels and by the exposure of confluent epithelial cells to the air-liquid interface. By plating primary human conjunctival epithelial cells on Swiss 3T3-dispersed collagen gels and their subsequent exposure to air-liquid interface upon confluent, a conjunctival equivalent with characteristics of a real conjunctival epithelium was developed.

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Nuclear transplantation in enucleated oocytes facilitates the development of embryonic and differentiated nuclei in vitro. After nuclear transplantation, reconstituted mouse eggs receiving foreign nuclei even from somatic cells, could develop into blastocysts normally. In mammals, during in vitro culture, this is important since the formation of blastocysts is considered to be the first differentiation event. Though the trigger for blastulation is not very clear, factors affecting the timing of blastulation have been suggested. The nucleo/cytoplasmic ratio is a possible factor and was clearly shown to determine the timing of blastocyst formation in the mouse.
In this paper, the in vitro development of mouse eggs after nuclear transplantation and factors affecting the timing of blastulation are discussed.

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Hybridoma Data Bank develops a database on clones and their immuno -reactive products. Categories of data items are reactance, methods and availability. There are two data collecting centers in USA and Japan. Data are disseminated by regional centers in USA, Canada, India and Japan. Media of data dissemination are printed catalogues, letters and facsimile, and an on-line database.

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In order to investigate the molecular mechanisms involved in tissue formation, we tried to form a three-dimensional tissue from isolated cells in culture and succeeded by adding ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative to the culture medium of human skin fibroblasts. In this review we describe mechanisms of action of Asc 2-P on the growth of the cells and metabolism of the extracellular matrix (ECM), applications of the culture method for the construction of three-dimensional liver tissue and the effects of defective expression of collagen genes on the formation of tissue.

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In order to establish an in vitro screening assay for cleft palateinducing teratogens, we devised two methods using human embryonic palatal mesenchyme (HEPM cell). One is preferential cytotoxic assay which identifies the in vivo cleft palate-inducing ability by the differention of cell growth inhibition for human embryonic HEPM and MRC-5 cells. The other is cell spreading assay which identifies the cleft palte-inducing ability by the delay of cell spreading in HEPM cells. The sensitivity and specificity of the tests were higher than other in vitro assays. The index of preferential growth inhibition and cell spreading of HEPM cells will be useful for human risk studies, especially cleft palate-inducibility.

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Developmental retardation in vitro of mammalian embryos has been known for 30 years over species. The cause (s) of this phenomenon has not been clarified, but recently, evidences were accumulated suggesting that block to development in embryos may be due to oxygen toxicity.
In this article, culture condition-developmental efficiency correlation is described and discussed in terms of oxidative stress.

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Organ culture of a mature and aged nervous system was established by introduction of collagen gel culture. The effects of nerve growth factor (NGF) and hepatocyte secreted factor on neuronal regeneration was demonstrated in the organ culture of dorsal root ganglion (DRG) and retina from an adult mouse or rat. Neurite growth from dissociated sensory neurons was srongly enhanced by NGF in an embryonic stage, but the enhance ment decreased rapidly after birth. However, NGF strongly promoted neurite regeneration from adult DRG explants embedded in collagen gel after treatment with collagenase and trypsin in a similar manner as that from embryonic one. This activation of neurite regeneration by NGF was perfomed by the three dimesional cell aggregation of dissociated purified adult DRG neurons. Though adult neurons could not express strong response to NGF in a single form, the three dimensional aggregation evoked it. On the other hand, the hepatocyte secreted factor promoted neurite regeneration from transected nerve terminals of adult DRG and supported its survival. This effect could not be seen by the treatment with other known trophic factors (NGF, EGF, aFGF, bFGF, IGF- I, IGF-II). The factor secreted from hepatocytes is thought to be a new protein like neurotrophic factor. The factor could not enhance neurite regeneration from a single cell This factor might activate Schwann cells around neurons to release neurotrophic factors. Retina from an adult rat 7 days after transection of optic nerves was cultured in collagen gel and regenerated neurites. The hepatocyte secreted factor enhanced neurite regeneration from the explant and supported its suvival.
To analyze the effect of drugs on neuronal regeneration in vivo requires three different levels of culture system, single cell culture, cell aggnegation culture, and explant culture. Results from the application of drugs to the three culture systems are expected to elucidate mechanism of drugs in neuranal regeneration in vivo.

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