10.014 pRSV-T細胞

器官來源： 眼

ATCC Number： CRL-11515?

數量： 大量

細胞類型： 其他細胞類型

是否是腫瘤細胞： 0

物種來源： 人

運輸方式： 凍存運輸

組織來源： cornea

生長狀態： 貼壁生長

細胞形態： 上皮樣

Designations: 10.014 pRSV-T

10.014 pRSV-T細胞Depositors: CR Kahn, Gillette Medical Evaluation Laboratories

Biosafety Level: 2 [Cells contain SV-40 and Rous Sarcoma viral sequences ]

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Homo sapiens

Morphology: epithelial

Source: Organ: eye

Tissue: cornea

Cell Type: epithelial transfected with a plasmid containing the SV40 early region

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DNA Profile (STR): Amelogenin: X,Y

CSF1PO: 10

D13S317: 12,13

D16S539: 8,13

D5S818: 12,13

D7S820: 8,10

10.014 pRSV-T細胞THO1: 6,8

TPOX: 8,10

vWA: 15

Comments: A primary culture of normal corneal epithelium was immortalized by transfection with plasmid pRSV-T using lipofectamine overnight at 37C. pRSV-T contains the SV40 early region genes and the Rous Sarcoma virus long terminal repeat.

Propagation: ATCC complete growth medium: Keratinocyte-Serum Free medium (GIBCO-BRL 17005-042) with 5 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract, 0.005 mg/ml insulin and 500 ng/ml hydrocortisone.

Temperature: 37.0°C

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Subculturing: 10.014 pRSV-T細胞Protocol:Subculture when 80% confluent

remove medium

briefly rinse the cell layer with 0.05% (w/v) Trypsin-53mM EDTA solution

add Trypsin-EDTA solution to flask and incubate at 37?C for up to three minutes to facilitate dispersal. Observe cells under an inverted microscope. Hit or shake the flask if some cells remain attached

neutralize the trypsin with growth medium, centrifuge at 300 x g for 8 minutes

resuspend the pellet in fresh growth medium and dispense into new COATED flasks. Flasks used to propagate these cells must be coated (for at least 2 hours at 37C) with 0.01 mg/ml bovine serum albumin, 0.01 mg/ml fibronectin and 0.03 mg/ml bovine collagen type I.

Incubate at 37C with 5% CO2. Cells will not be well attached for the first 24 hours after subculture.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended when 80% confluent

Medium Renewal: Every 2 to 3 days

Preservation: Freeze medium: complete growth medium supplemented with 7.5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase

References: 34094: Walker TL, Kahn CR. 10.014 pRSV-T細胞Human corneal epithelial cell lines with extended lifespan. US Patent 5,672,498 dated Sep 30 1997

46895: Kruszewski FH, et al. Evaluation of a human corneal epithelial cell line as an in vitro model for assessing ocular irritation. Fundam. Appl. Toxicol. 36: 130-140, 1997. PubMed: 9143482