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 non-tumorgenic

• 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 =

0.2-0.5ml/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

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

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