Introduction to animal cell culture

Introduction to animal cell culture

1. Fibroblast-like Epithelial-like Lymphoblast-like Morphology of Cells in Culture Based on shape and appearance : 3 types

2. Fibroblast-like cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate Morphology of Cells in Culture

3. 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

4. Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface. Morphology of Cells in Culture

5. Anchorage-dependent Anchorage-independent Morphology of Cells in Culture Depending on the adherence property : 2 types

6. Anchorage-dependent ( adherent or monolayer culture ): Must be cultured while attached to a solid or semi-solid substrate Example: MCF7 , PC-3 Anchorage-Independent ( suspension culture ): Can be grown floating in the culture medium Example: K562 Morphology of Cells in Culture

7. 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 a gitation 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

8. Cell Culture Environment Physico-chemical environment   Growth media (pH, osmotic pressure, O 2  and CO 2  tension) Temperature Physiological environment   Hormone and nutrient concentrations

9. The culture medium is the most important component of the culture environment, because it provides: N ecessary nutrients G rowth factors H ormones for cell growth R egulating the pH of the culture O smotic pressure of the culture Cell Culture Media

10. The three basic classes of media are:   Basal media   Reduced-serum media   Serum-free media Cell Culture Media

11. 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

12. Reduced-Serum Media Basal media formulations enriched with nutrients and animal-derived factors with reduced amount of serum Cell Culture Media

13. 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

14. Commonly used Medium: GMEM, EMEM,DMEM, RPMI etc. Media is supplemented with Antibiotics (penicillin, streptomycin) BSA Na-bicarbonate L-glutamate Na- pyruvate HEPES Growth factors etc. Cell Culture Media

15. 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

16. b) pH Level: Cell line Optimal p H 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

17. 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 CO 2 -bicarbonate based buffer 4 – 10% CO 2  is common for most cell culture experiments c) CO 2  Level Factors affecting culture environment

18. 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