Mammalian cell culture Download

Transcript
Isolated Tissue
Cell or tissue culture in vitro
Primary culture
Sub-culture
Secondary culture
Sub-culture
Cell Line
Single cell isolation
Successive sub-culture
Immortalization
Cloned cell line
Loss of control
of cell growth
Transformed cell line
Cell Strain
Senescence
Revive frozen cell population
Isolated from tissue
Maintain in culture (aseptic cinditions)
Typical
cell culture flask
Sub-culture (passaging)
‘Mr Frosty’
Used to freeze cells
Cryopreservation
DOUBLING TIME :
Time taken by cells to
double the population.
CELL LINE :
When the primary culture is
first sub-cultured or passaged.
CELL STRAIN :
When a particular type of cell
lineage is selected, characterized and cloned it is called
a cell strain.
PASSAGE NUMBER :
The number of
times a culture has been sub-cultured.
Types of cell cultured
Primary cultures
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Derived directly from animal tissue,
embryo or adult.
Cultured either as tissue explants or single cells.
Initially heterogeneous – containing various types of
cells.
Finite life span in vitro.
Retain differentiated phenotype.
Mainly anchorage dependant.
Exhibit contact inhibition.
Types of cell cultured
Secondary cultures
• Derived from a primary cell culture.
• Isolated by selection or cloning.
• Becoming a more homogeneous cell population that
is contains a specific cell type.
• Finite life span in vitro.
• Retain differentiated phenotype.
• Mainly anchorage dependant.
• Exhibit contact inhibition.
Types of cell cultured
Continuous cultures
• Derived from a primary or secondary culture
• Immortalised:
• Spontaneously
• By transformation
• Serially propagated in culture showing an increased
growth rate
• Homogeneous cell population
• Loss of anchorage dependency and contact inhibition
• Infinite life span in vitro
• Differentiated phenotype:
• Retained to some degree in cancer derived cell lines
• Very little retained with transformed cell lines
• Genetically unstable
Check confluency of cells
Remove spent medium
NEED TO PASSAGE CELLS:
To maintain cells in culture.
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To increase cell number for experiments/storage
Checking confluency
of cells
Wash with PBS
Incubate with
trypsin/EDTA
Resuspend in serum
containing media
Transfer to culture flask
70-80% confluence
100% confluence