| Publication number |
CLPUB00528 |
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| Authors |
Zabrenetzky V., Vilner L., Thomson A., Hay R.J. |
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| Title |
The isolation, immortalization and characterization of human fetal keratinocytes. |
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| Citation |
In Vitro Cell. Dev. Biol. Anim. 33 Suppl. 1:34A.4-34A.4(1997) |
|---|
| Abstract |
Cultured keratinocytes are useful research models for the study of wound
repair and skin substitutes for the treatment of severe burns and ulcers.
Migrating wound epidermal cells do not terminally differentiate e.g.
produce keratin, as do keratinocytes of normal epidermis. Immortalized
human keratinocytes have research applications in the study of drug
toxicity and pharmacology. We report the immortalization of human fetal
keratinocytes which retain differentiated characteristics. Fetal skin
samples (112 and 126 days gestation) were digested by collagenase: trypsin
treatment. The digested products were plated on collagen, fibronectin, BSA
coated flasks in Gibco KSFM media supplemented with epidermal growth
factor (EGF) and bovine pituitary extract. Cells were passaged by trypsin-
versene treatment. At passage 2, cells were frozen. Thawed cells, which
had been passaged at least once were transfected with the E6/E7, gene of
human papillonia virus 16 (HPV16) in the presence of polybrene. Five
days later the cells were exposed to G418. Untransformed cells died. After
50 population doublings, the cells continued dividing and retained
epithelial, cuboidal appearance. The cells were characterized for keratin
expression by immunohistochemistry. The cells were positive for epithelial
specific antigen using a monoclonal antibody produced from the hybridoma
Ep16 (ATCC HB-155). Further studies will determine the expression and
attachment of these cells to various matrix molecules and migration to
various chemotactic factors.
|
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| Cell lines |
CVCL_3297; CCD-1102 KERTr CVCL_3300; CCD-1106 KERTr |
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