Culture Information For SMB-Type Cells Culture Information For SMB-Type Cells SMB.s15 cell line SMB-PS cell line SMB[22F].F4 cell line MEDIUM: Medium 199 with Earle's Salts [containing 2.2 g/l sodium bicarbonate and glutamine as per formula], supplemented with newborn calf serum 10% and foetal calf serum 5%. A 5% CO2 gas atmosphere is needed. For subculture use trypsin/EDTA for subculture containing 0.05 % porcine trypsin and 0.02% EDTA [e.g. Sigma T-4174 10x solution diluted in Hank’s balanced salt solution (HBSS, w/o Ca, Mg, phenol red)]. ROUTINE CULTURE: Subculture approx. every 7 to 10 days at 1:4 to 1:5 split ratio (1:6 for SMB-PS). Change medium every 3 to 4 days. These cells grow slowly and do not like to be at low density, a density of not less than 1.2 × 104 / cm2 is recommended for good growth and maintenance of the scrapie agent. If cloning is necessary use conditioned medium from SMB.PS cells at ratio of 1:3. All SMB-type cells have a very poor cloning efficiency b ut have been successfully selected as clones either by limiting dilution cloning or by selection in cloning-rings from low-density plate cultures. 22F.F4 cells do not grow well in vessels above 80 cm2 size – this has been consistently found when using flasks but holds true with Petri dishes to some extent. For a number of reasons the Compton group has used 33°C for routine culture BUT all the SMB cell types generated to date will also grow (and SMB.s15 & SMB[22F] also propagate scrapie) at 37°C. At Compton the growth rate has not bee n significantly higher at 37°C than at 33°C. SMB-PS grow slightly faster than the scrapie-infected lines so the subculture period will be shorter (5 to 8 days) but for optimal growth do not split much lower than the recommended 1:4 to 1:6. ANTIBIOTICS: All the SMB-types tolerate penicillin and streptomycin sulphate (in combination) or gentamycin at standard concentrations. Routine culture in the Compton laboratories is done in the absence of antibiotics. FREEZING AND RECOVERY: Use 10 % dimethyl sulphoxide (DMSO) v/v in 20% foetal calf serum 70 % Medium 199 (or 10 % DMSO in 90 % foetal calf serum). As a routine approx. 75% confluent cultures (in a 75 cm2 vessel) have been fro zen in 2 vials, 1 ml/vial. Changing medium 24 hr prior to freezing is beneficial. Store in liquid nitrogen vapour phase. Thaw one frozen vial at 37°C, add 12 ml pre-warmed medium, centrifuge at 100 × g for 5 min, then re-suspend the cell pellet in 10 ml fresh medium pre-warmed to 37°C. Seed the 10ml suspension into a single 75 cm2 vessel (i.e. a 9 cm Petri or T75 flask) or 2xT25 flasks. TRANSFECTION: All the SMB-type cells respond badly to shock and therefore it is recommended that the shock stage of transfection procedures e.g. glycerol shock, is omitted. Electroporation might be possible but under the conditions that have been tested SMB do not survive. It might be that shocked cells can be induced to survive by the addition of SMB-PS cells to the outgrowing culture (they would then be negatively selected using antibiotic etc.) or that SMB-PS-conditioned medium would be beneficial to the shocked cultures. Calcium-transfection has proved to be universally reliable. BIOHAZARD: In the UK scrapie-infected cells are handled under Class 2 containment. See local SOP or CoP for routine handling methods. The surest means of disinfection is sodium hypochlorite used at 20,000 ppm available chlorine. ADVENTITIOUS AGENTS: All the SMB-type cells have occasionally been screened for adventitious agents; routinely they are free of bacteria, fungal agents, mycoplasma and BVDV. Certain lines have been tested (by inoculation into mice) for 12 common mouse viruses, and were determined to be negative for all 12. Routine inoculations into mice under SPF-barrier conditions have not resulted in adventitious infections. All end-users should verify the absence of adventitious agents to their own standards. Reference for SMB.s15 and derivative cells: Birkett CR, Hennion RM, Bembridge DA, Clarke MC, Chree A, Bruce ME, Bostock CJ "Scrapie strains maintain biological phenotypes on propagation in a cell line in culture" The EMBO Journal, Vol. 20, No. 13 pp. 3351-3358, 2001. This article was published on 2024-09-02