Transcript Subculture and Cell linesx

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
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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
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