Vulgar Name: Human
|Age/Ethinicy:||49 YEARS OLD|
|Additional info:||SV40-Adeno vector transformed cornea cells. 64kD keratin producing. Choleratoxin-free medium. This cell line must only be distributed with depositor's authorization.|
|Culture Medium:||1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate, 5μg/ml Insulin, 10ng/ml hEGF and 5%fetal bovine serum.|
Remove medium, and rinse with PBS without calcium and magnesium.
Remove the solution and add an additional 1 to 2 mL of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 37°C) until the cells detach. Add fresh culture medium, aspirate and dispense into new culture flasks.
NOTE: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 12 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 6th edition, published by Alan R. Liss, N.Y., 2010.
Medium Renewal: twice/week
Subcultivation ratio: 1:8 split
|Culture Conditions:||Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37°C|
|Cryopreservation:||95% FBS + 5% DMSO (Dimethyl sulfoxide)|
|Thawing Frozen Cells:||SAFETY PRECAUTION: Is highly recommend that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris. 1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the Oring and cap out of the water. Thawing should be rapid (approximately 2 minutes). 2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions. 3. For cells that are sensitive to DMSO is recommended that the cryoprotective agent be removed immediately. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 7 minutes. 4.Discard the supernatant and Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). 5. Incubate the culture in a appropriate atmosphere and temperature (see "Culture Conditions" for this cell line). NOTE: It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).|
|References:||Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, Handa H Invest Ophthalmol Vis Sci 1995; 36:614-21. PubMed ID: 7534282 Zhang Y, Araki-Sasaki K, Handa H, Akhtar, R A Curr Eye Res 1995; 14:563-71. PubMed ID: 7587302 Araki K, Ohashi Y, Sasabe T, Kinoshita S, Hayashi K, Yang, X Z, Hosaka Y, Aizawa S, Handa H Invest Ophthalmol Vis Sci 1993; 34:2665-71. PubMed ID: 7688358|
|Depositors:||Marize Brittes Rott - Universidade Federal do Rio Grande do Sul|