||78 Year /
||Established from undifferentiated thyroid carcinomas of a 78 year old female patient. Pathologically this primary carcinoma tissue contained residual well differentiated components suggesting well differentiated to undifferentiated carcinoma progression. Tumour suppresser genes p53, Rb, APC and MCC were analysed and sequence analysis confirmed a C:G to G:C transversion at the first base of p53 gene codon 248. Polymerase chain reaction-loss of heterozygosity assays confirmed allelic deletion of p53 gene. Loss of heterozygosity of tumour suppresser genes was not observed
||Dulbecco's Modified Eagle's Medium (DMEM) with + 2 mM Glutamine + 1% Non Essential Amino Acids (NEAA) + 10% of Fetal Bovine Serum (FBS).
||Split sub-confluent cultures (70-80%) 1:3 to 1:6 i.e. seeding at 2-4 x 10,000 cells/cm² using 0.25% trypsin/EDTA; 5% CO2; 37°C. Saturation density at confluency is 1x100,000 cells/cm².
Doubling time is 36 hours.
|Subculturing Subcultivation Ratio
||1:3 to 1:6 i.e. seeding at 2-4 x 10,000 cells/cm²
||Atmosphere: air, 95%; carbon dioxide (CO2), 5%
||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).
||Cancer Res 1992;52:1369; Int J Oncology 1994;4:583
||Etel Rodrigues Pereira Gimba - Instituto Nacional de Câncer