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 Homogeneity inc. and hetero dec.

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 Finite cell lines - die

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 Cell loss occurs btwn sc

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., One cell line grown under same conditions – should show same doublings. CCl show inc. cell proliferation and satura. Density

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 Cell bank – should follow exactly the no

Choosing a cell line Finite vs. continuous cell line - prefer continuous lines Normal or transformed – prefer non-tumorgenic Species – nonhuman cell line Growth characteristics Availability – stocks or prepare own line continuous lines are easy to maintain, grow faster, more clones, can adapt to serum – free media. With non-human cl there are less biohazard restrictions and more tissue is accessible

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 = 0.2-0.5ml/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 Medium must be changed or cells divided

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 Contamination or exhaustion

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 Standardize amount and type of media with serum conc.

SC Check the use of antibiotics Maintain provenance

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