Description: This is a derviative of the CHO/dhFr- cell line, ECACC catalogue no. 94060607 which has been adapted to grow in the animal component free medium CHO DHFR (Sigma C8862). The CHO/dhFr- cell line lacks the enzyme dihydrofolate reductase (DHFR) which is necessary for purine synthesis. In the absence of exogenous purines, this enzyme is required for growth. Hence the CHO/dhFr- cell line has growth requirements for hypoxanthine (or adenine), glycine and thymidine. However, the cells will continue to grow without the addition of hypoxanthine and thymidine because the cells can obtain sufficient from the medium. Alpha MEM minus does not contain the ribonucleosides or the deoxyribonucleosides, and will not support the growth of CHO/dhFr- or its serum free adaptation as neither contains the gene to produce DHFR. (See attached data sheet).

Also Known As:

Species: Hamster

Tissue: Ovary

Growth Properties: Aggregates in suspension

Morphology: Suspension

Growth Medium: CHO DHR medium (Sigma C8862) + 2mM Glutamine + 100?M Hypoxanthine + 16?M Thymidine (e.g. use HT supplement Sigma H0137). Addition of Aminopterin (Sigma A5159) 0.4?M is also advised. When freezing cells use freeze medium composed of 50:50 fresh:conditioned medium + 10% DMSO.

Subculturing Procedure: Maintain cultures between approximately 3-9×100,000 cells/ml; 5% CO2; 37°C. Cells grow as aggregates in suspension. Viability may be poor on resuscitation and may initially decrease further. Full recovery may take up to 2 weeks. A centrifugation step to remove the cryoprotectant is essential. Rapidly thaw the frozen ampoule in a water bath at 37°C for 1-2 minutes. Transfer the contents to a centrifuge tube and slowly add 5-10ml of pre-warmed growth media. Remove a sample for counting. Centrifuge at 100g for 2-3 minutes to pellet cells and seed at a relatively high density of 5-7 x 100,000 cells/ml. Leave culture flask upright and observe regularly until viable proliferating cells are seen. Once established use a split ratio of 1:2 approximately every 4 to 5 days.

Release Conditions: No

Depositor: Dr. E. Penn, European Collection of Cell Cultures, Health Protection Agency, Porton Down, Salisbury, SP4 0JG, UK

Originator: Yes

References: None Specified By Depositor

Hyperlink to ECACC Cell Line Data Sheet: 05011002