| Publication number |
CLPUB00347 |
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| Authors |
Zabrenetzky V., Thomson A., Vilner L., Hay R.J. |
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| Title |
The isolation, characterization and immortalization of human fetal kidney cells. |
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| Citation |
In Vitro Cell. Dev. Biol. Anim. 33 Suppl. 1:34A.3-34A.3(1997) |
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| Abstract |
Immortalized kidney cells have diverse research applications in the study
of renal physiology, biochemistry, toxicology and pathology. The human
fetal kidney, resembles histologically, Wilms tumor, a complex childhood
neoplasm that recapitulates the differentiation of the normal nephron_
Immortalized kidney cells are being shown to have the same sensitivity to
human viruses as the primary cells used for human virus detection and
isolation. This is the first report of the successful immortalization of a
human fetal line by human papilloma virus 16 (HPV16). Human fetal kidney
at 115 and 133 days were enzymatically dissociated by treatment with a
mixture collagenase:trypsin (1,1). After inoculation and neutralization,
the cells were plated on collagen, fibronectin and RSA coaled flasks in
Clonetics REGM (low serum media). The cells were grown and passaged twice
before freezing. Immortalization was performed on cells that had been
passaged at least once after thawing. Transfection of the fetal cells with
the E6/E7 genes of HPV16 occurred in the presence of polybrene. Control
cells were in polybrene without viral genes. To test the efficacy of the
transfection, control and treated cells were exposed to G418 three days
after transfcction. The cells not transfected stopped dividing and came
off the surface of the flask. The immortalized cells continued to divide.
Immortalized cells underwent at least 50 population doublings (PDL) and
retained epithelial morphology. Primary cells underwent 6 PDL before
ceasing division. Immortalized cells were characterized by
immunohistochemistry of keratin and acid phosphatase expression. These
cells were positive for anti-keratin AE1/AE3 which recognizes the acidic
subfamily characteristic of keratinocytes. The cells were also positive
for leukocyte acid phosphatase which is 'characteristic' of proximal
tubule brush border membrane (PTC BBM). These cells demonstrate possible
research significance as an immortalized fetal line with differentiated
characteristics.
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| Cell lines |
CVCL_3298; CCD-1103 KIDTr CVCL_3299; CCD-1105 KIDTr |
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