Subculture and Cell lines Chapter 13 Propagation of Subculture • Subculture – important transition from culture • Need to SC – primary culture has occupied all substrate • Biological significance – culture can be propagated, characterized and stored Cell line and Strain • Primary culture is subcultured – Cell line • Cell line transforms in vitro – Continuous Cell line • Specific properties – Cell Strain • Selected or cloned and characterized Continuous Cell Strain – Table 13.3 SC • Passage number – number of times the culture has been subcultured • Generation number – number of doublings the culture has undergone • No account of cell loss through necrosis, apoptosis, premature aging and withdrawal from cycle Culture Age • Cell lines with limited culture life spans – finite cell lines • Reproduce - limited cell generations • Cell lines escaped from senescence Continuous Cell line - Generation no is less imp., and no of passages is imp., - Split ratios, cell conc., at SC is imp., Cell line designations • Given code or designation – accession number by cell bank • NHB – Normal Human Brain • NHB1, NHB2 – cell strain and cell line number • If cloned – NHB2-1, NHB2-2 • Split ratios – NHB2/2 Choosing a cell line • Finite vs. continuous cell line - prefer continuous lines • Normal or transformed – prefer nontumorgenic • Species – nonhuman cell line • Growth characteristics • Availability – stocks or prepare own line Choosing a cell line • • • • Validation – characterized or not Phenotypic expression Control cell line Stability – cloned or not, length of cloning and can you make frozen stocks? Routine maintenance • Periodic medium change or feeding • Nonproliferating cultures - medium change is needed – exhausted • Proliferating cultures – trypsinization + medium change are needed • Intervals vary – cell lines Significance of cell morphology • Check for contamination – granularity around nucleus, cytoplasmic vacuolation and rounding of detached cells from substrate • Indicates inadequate toxic medium or serum, microbial contamination or senescence of cell line • Indicates medium change or SC Replacement of medium • Four factors indicate replacement • A drop in pH – Cells stop growing – pH 7.0 to 6.5, lose viability btwn 6.5 and 6.0 - Medium changes from red through orange to yellow - Change 0.1 pH units/day - can be left longer - Change 0.4 pH units/day – change 24-48 hrs Replacement of medium • Cell concentration – High cell conc. Exhaust faster – indicated by pH change • Cell Type – Normal cells deteriorate less than transformed cells • Morphological deterioration – allowed longer can lead to apoptosis Replacement of medium • Holding medium – used when mitosis is undesirable at high cell densities • Will hold finite life span lines without using limited cell generations • Serum conc. Is less or absent • Not suitable to transformed cell lines Replacement of medium • Volume, depth and surface area - Medium volume to surface area = 0.20.5ml/cm2 - Cells with high o2 do better in shallow medium – 2mm - Cells with low o2 do better in deep medium – 5mm - > 5mm – gaseous diffusion limitation When to Subculture? • Seeding - Lag period • Log/expo. Phase – cell density or no more growth • Removal of medium + dissociation of cells with trypsin + diluting in new bottles • Sensitivity of cells to proteolysis Criteria for subculture • Density of Culture – Normal and transformed cells – confluency – reseeded • Exhaustion of medium – fall in pH • Time since last SC – routine SC - Less density – more seeding and viceversa • Requirements – No SC in lag period - Taken btwn middle of log phase and time before plateau phase of previous SC Propagation in suspension • Nonadhesive or mechanical suspension • No trypsin treatment and media replacement • Culture diluted and expanded or diluted and excess discarded • 2-5 mm medium for gaseous exchange + agitation by pendulum Subculture of suspension cells • Cell concentration – should not exceed 1 x 10 6 cells/ml • pH – declines as cell conc. Goes up • Time since last SC – regular schedule • Contamination increases with any buildup of minor spillage on neck of flask during dilution SC • Check the use of antibiotics • Maintain provenance • • • This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity. 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