2 Propagation of Subculture Subculture – important transition from cultureNeed to SC – primary culture has occupied all substrateBiological significance – culture can be propagated, characterized and storedHomogeneity inc. and hetero dec.
3 Cell line and Strain Primary culture is subcultured – Cell line Cell line transforms in vitro – Continuous Cell lineSpecific properties – Cell StrainSelected or cloned and characterized - Continuous Cell Strain – Table 13.3Finite cell lines - die
4 SC Passage number – number of times the culture has been subcultured Generation number – number of doublings the culture has undergoneNo account of cell loss through necrosis, apoptosis, premature aging and withdrawal from cycleCell loss occurs btwn sc
5 Culture AgeCell lines with limited culture life spans – finite cell linesReproduce - limited cell generationsCell lines escaped from senescence - Continuous Cell lineGeneration 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
6 Cell line designations Given code or designation – accession number by cell bankNHB – Normal Human BrainNHB1, NHB2 – cell strain and cell line numberIf cloned – NHB2-1, NHB2-2Split ratios – NHB2/2Cell bank – should follow exactly the no
7 Choosing a cell lineFinite vs. continuous cell line - prefer continuous linesNormal or transformed – prefer non-tumorgenicSpecies – nonhuman cell lineGrowth characteristicsAvailability – stocks or prepare own linecontinuous 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
8 Choosing a cell line Validation – characterized or not Phenotypic expressionControl cell lineStability – cloned or not, length of cloning and can you make frozen stocks?
9 Routine maintenance Periodic medium change or feeding Nonproliferating cultures - medium change is needed – exhaustedProliferating cultures – trypsinization + medium change are neededIntervals vary – cell lines
10 Significance of cell morphology Check for contamination – granularity around nucleus, cytoplasmic vacuolation and rounding of detached cells from substrateIndicates inadequate toxic medium or serum, microbial contamination or senescence of cell lineIndicates medium change or SC
11 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 6.0Medium changes from red through orange to yellowChange 0.1 pH units/day - can be left longerChange 0.4 pH units/day – change hrs
12 Replacement of mediumCell concentration – High cell conc. Exhaust faster – indicated by pH changeCell Type – Normal cells deteriorate less than transformed cellsMorphological deterioration – allowed longer can lead to apoptosis
13 Replacement of mediumHolding medium – used when mitosis is undesirable at high cell densitiesWill hold finite life span lines without using limited cell generationsSerum conc. Is less or absentNot suitable to transformed cell lines
14 Replacement of medium Volume, depth and surface area Medium volume to surface area = ml/cm2Cells with high o2 do better in shallow medium – 2mmCells with low o2 do better in deep medium – 5mm> 5mm – gaseous diffusion limitation
15 When to Subculture? Seeding - Lag period Log/expo. Phase – cell density or no more growthRemoval of medium + dissociation of cells with trypsin + diluting in new bottlesSensitivity of cells to proteolysisMedium must be changed or cells divided
17 Criteria for subculture Density of Culture – Normal and transformed cells – confluency – reseededExhaustion of medium – fall in pHTime since last SC – routine SCLess density – more seeding and viceversaRequirements – No SC in lag period- Taken btwn middle of log phase and time before plateau phase of previous SCContamination or exhaustion
18 Propagation in suspension Nonadhesive or mechanical suspensionNo trypsin treatment and media replacementCulture diluted and expanded or diluted and excess discarded2-5 mm medium for gaseous exchange + agitation by pendulum
19 Subculture of suspension cells Cell concentration – should not exceed 1 x 10 6 cells/mlpH – declines as cell conc. Goes upTime since last SC – regular scheduleContamination increases with any buildup of minor spillage on neck of flask during dilutionStandardize amount and type of media with serum conc.
20 SCCheck the use of antibioticsMaintain provenance
21 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 ). 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; andagainst 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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