Line name: [K]iPS4F-8
Origin: Biopsy of foreskin keratinocytes

Data related to the biological sample:
Donated: 26.04.2008
Received at the Banco de Líneas Celulares of CMR[B]: 27.04.2008

General description of the process:

The biopsy is collected in keratynocyte serum-free low calcium medium (Epilife, Invitrogene) containing three types of antibiotics (100U/mL penicillin-streptomycin and 10 mg/mL amphotericin). The biopsy is then placed in a 10 mm dish, cut in several small pieces, and incubated for 12-18 hours at 4°C in dispase diluted in keratinocyte medium to separate the dermis from epidermis. The epidermis is separated from dermis using a pair of forceps, is incubated for 10 minutes at 37°C in trypsin (0.25%) in a 15 mL tube. Fresh keratynocite medium is added and centrifugate for 5 min at 1200 RPM. The pellet of cells is resuspended and cultured in Epilife medium on dish coated with type I collagen.

Under these conditions, only keratin-positive keratinocytes grow. After adding calcium physiological levels they differentiate, and form a completely differentiated stratified epidermis.
Factors OCT4, SOX2, KLF4, c-Myc and GFP are cloned in a retrovirus that favours transcriptional activation. After two rounds of infection, cells are tripsinized on Day 4 and are planted on an irradiated monolayer of mouse embryonic fibroblasts (MEF) on an hES cell culture medium. On Days 2–3 growth of small fast-growing colonies is detected, and 10 days after infection they present a similar morphology to embryonic stem cells.
The next mechanical passagings were carried out on human foreskin fibroblasts (HFF-1).

Cell and culture medium used for derivation:
Support: human foreskin fibroblasts (ATCC, American Type Culture Collection, CCD1112Sk).
Culture medium: Knockout Dulbecco’s modified Eagle’s medium supplemented with 2 mmol/l
GlutaMAX (Gibco, InVitrogen corporation), 0,05mmol/l 2-mercaptoethanol (Gibco, InVitrogen corporation), 8 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen), 1% non-essential amino acids (Cambrex), 20% Knockout Serum Replacement (InVitrogen) y 0,5% Penicillin-Streptomycin (Gibco, InVitrogen corporation).

Characterization [K]iPS4F-8

Karyotype [K]iPS4F-8

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[K]iPS4F-8