Lect. 4
The following definition should be considered before studying cell culture :Cell culture : establishment and maintenance of cultures derived from dispersed cells taken from original tissues , primary culture or from a cell line or cell strain.
Cell line : immortalized cell, which have undergone transformation and can be passed indefinitely in culture.
Cell strain : cells which can be passed repeatedly but only for a limited number of passages.
Cell clones : individual cells separated from the population and allowed to grow.
Primary culture : cells resulting from the seeding of dissociated tissues. Primary cultures often lose their phenotype and genotypes within several passages.
Cell passage : the splitting (dilution) and subsequent redistribution of a monolayer or cell suspension into culture vessels containing fresh media.
Monolayer : a layer of cells one cell thick , grown in a culture.
Cryopreservation
If a surplus of cells are available from subculturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below –130°C (cryopreservation) until they are needed.
Suspension culture : cells which do not require attachment to substratum to grow , i.e. anchorage independent . cell culture derived from blood are typically grown in suspension . cells can grow as single cells or clumps. To subculture the cultures which grow as single cells they can be diluted . however , the cultures containing clumps need to have the clumps disasociated prior to subculturing of the culture.
Hatch : to bring cells out of the freezer , to start a culture from a freezer stock .
Thaw : same as hatch .
Defreeze : same as hatch .
Carry : to maintain a cell line by subculturing in tissue culture medium containing nutrients that will maintain the phenotype and genotype of the cell line.
Equipment
A . laminar flow hoods
B . Co2 incubators
C . Microscopes
D . Vessels
Medium formulations :
Basic cell culture medium
1 L Dulbecco’s modified eagle medium (DMEM)
-4.5 g/L D-glucose
-L-glutamine
-110 mg/L sodium pyruvate
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110 ml Defined fetal bovine serum
11 ml 100x antibiotic-antimycotic (penicillin, streptomycin , amphotericin B ).
Reduced serum medium for virus preparation :
1 L DMEM
20 ml Defined Fetal Bovine Serum
10 ml 100x antibiotic –antimycotic
Culture Conditions
Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins,minerals), growth factors, hormones, and gases (O2, CO), and regulates the physicochemical environment (pH, osmotic pressure, temperature).
Most cells are anchoragedependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture).
Morphology of Cells in Culture :
Cells in culture can be divided in to three basic categories based on their shape and appearance (i.e., morphology).• Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate.
• Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.
• Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface.
