introduction to animal cell culture
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• Fibroblast-like
• Epithelial-like
• Lymphoblast-like
Morphology of Cells in Culture
Based on shape and appearance: 3 types
Fibroblast-like cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate
Morphology of Cells in Culture
•Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.
Morphology of Cells in Culture
•Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface.
Morphology of Cells in Culture
Anchorage-dependent
Anchorage-independent
Morphology of Cells in Culture
Depending on the adherence property : 2 types
Anchorage-dependent (adherent or monolayer culture):
Must be cultured while attached to a solid or semi-solid substrateExample: MCF7, PC-3
Anchorage-Independent (suspension culture):
Can be grown floating in the culture mediumExample: K562
Morphology of Cells in Culture
Adherent Cell Culture Suspension Cell Culture Attached to a solid or semi-solid substrate Grown floating in the culture medium
Easy visual inspection under inverted microscope
Requires daily cell counts and viability determination to follow growth patterns
Cells are dissociated enzymatically (e.g. trypsin) or mechanically
Does not require enzymatic or mechanical dissociation
Growth is limited by surface area Growth is limited by concentration of cells
No agitation required Requires agitation (i.e., shaking or stirring) for adequate gas exchange
Used for cytology, harvesting products continuously, and many research
applications
Used for bulk protein production, batch harvesting, and many research applications
Adherent Cell vs Suspension Cell
Cell Culture Environment
Physico-chemical environment
• Growth media (pH, osmotic pressure, O2 and CO2 tension)
• Temperature
Physiological environment
• Hormone and nutrient concentrations
The culture medium is the most important component of the culture environment, because it provides:
Necessary nutrients
Growth factors
Hormones for cell growth
Regulating the pH of the culture
Osmotic pressure of the culture
Cell Culture Media
The three basic classes of media are:
a. Basal media
b. Reduced-serum media
c. Serum-free media
Cell Culture Media
Basal Media
• Contains amino acids, vitamins, inorganic salts, and a
carbon source such as glucose.
• Basal media formulations must be further supplemented
with serum
Cell Culture Media
Reduced-Serum Media
• Basal media formulations enriched with nutrients and
animal-derived factors with reduced amount of serum
Cell Culture Media
Serum-Free Media• Appropriate nutritional and hormonal formulations replaces serum completely
• Serum-free medium in combination with growth factors has the ability to make the selective medium for primary cell culture.
Cell Culture Media
• Commonly used Medium: GMEM, EMEM,DMEM, RPMI etc.
• Media is supplemented with Antibiotics (penicillin, streptomycin)BSANa-bicarbonate L-glutamateNa-pyruvateHEPESGrowth factors etc.
Cell Culture Media
a) Serum:
• Vitally important source of growth factors, adhesion factors, hormones, lipids and minerals
• Regulates cell membrane permeability:
• Serves as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell.
Factors affecting culture environment
b) pH Level:
Cell line Optimal pH
Mammalian cell lines 7.4
Transformed cell lines 7.0 – 7.4
Normal fibroblast cell lines 7.4 – 7.7
Insect cell lines 6.2
Factors affecting culture environment
• The growth medium controls the pH of the culture and buffers the cells in culture against changes in the pH.
• Buffering is achieved by an organic (e.g., HEPES) or CO2-bicarbonate based buffer
• 4 – 10% CO2 is common for most cell culture experiments
c) CO2 Level
Factors affecting culture environment
• The optimal temperature for cell culture largely depends on the body temperature of the host from which the cells were isolated.
d) Temperature
Cell line Optimal Temperature
Human and mammals 36°C - 37°Insect cells 27°C Avian cell lines 38.5°C Cold-blooded animals (e.g., amphibians, cold-water fish)
15°C - 26°C
Factors affecting culture environment