• 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