| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Since our final goal if to establush a cellular model for a congenital skin disease, it is crucial to utilize a proper culture system. Therefore, in this project, we attempted to establish the multicellular spheroid method which has been widely studied for cancer research and developmental biology. Normal human epidermal keratinocytes were co-cultured with fibroblasts on the dishes coated with a thermo-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm) as described in detail elsewhere. Later, the keratinocyte-attached fibroblast monolayrs were thoroughly detached from the PNIPAAm collagen substratum as a self-supporting sheet. By the end of fourth day, 5-7 layrs were seen. In spheroid culture systems.salient markers of keratinization such as keratohyalin granules and a compact stratum corneum were not seen. As dayspassed, some pyknotic changes and deletion of nuclei at the core was seen. Although our model does not show the same morphology as shown in epidermis in vivo, the characteristics of cells in the spheroid support the notion that spheroid is a good in vitro experimental model reflecting the in vivo status of cells in living organs. Moreover, we attempted to transfect genes into keratinocytes by the electroporation method. As a preliminary study, we transfected a hair Keratin gene into COS-1 cells. Using a monoclonal antibody to hair keratins as a marker, we could successfully detected the expression of hair keratins in the COS-1 cells. However, this conditions were not suitable for keratinocytes, since the viability of keratinocytes decreased remarkably after electroporation. We keep investigating to find a suitable condition for keratinocytes, therefore the transfection efficiency will be improved in near future.
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