Composition of Cell Culture Medium

1. Basal medium

• Inorganic salts: NaCl, KCl, CaCl₂, MgSO₄, phosphates, and bicarbonate. They maintain osmotic pressure, ionic balance, membrane potential, and cell adhesion.
• Buffering system: Sodium bicarbonate with CO₂ incubation, or HEPES buffer, maintains pH usually around 7.2-7.4. Phenol red may be added as a pH indicator.
• Carbohydrate source: Usually glucose, supplying energy for cellular metabolism.
• Amino acids: Essential and non-essential amino acids are needed for protein synthesis. L-glutamine is an important nitrogen and energy source but is unstable, so it is often added freshly.
• Vitamins: Such as B-complex vitamins, folic acid, and choline, which act as coenzymes in metabolism.
• Other metabolic precursors: Sodium pyruvate, nucleosides, and trace elements may support specific cell types.

2. Supplements

• Serum, usually fetal bovine serum (FBS), commonly 5-20%, supplies proteins, hormones, attachment factors, lipids, trace elements, and some growth factors. It also protects cells from mechanical damage.
• Serum-free supplements may be used instead of serum for better experimental control. These include insulin, transferrin, selenium, albumin, lipids, and defined trace elements.
• Hormones and signalling molecules: Insulin, hydrocortisone, thyroxine, and epinephrine may be added depending on the cells cultured.
• Attachment factors: Collagen, fibronectin, laminin, or poly-L-lysine may be supplied, especially for adherent primary cells.
• Antibiotics and antifungals: Penicillin-streptomycin, gentamicin, or amphotericin B may be added temporarily to reduce contamination. However, they should not replace aseptic technique and prolonged use can mask contamination or harm cells.

3. Growth factors

• EGF: Promotes epithelial and keratinocyte growth.
• FGF or bFGF: Supports fibroblasts, endothelial cells, and stem cells.
• PDGF: Stimulates fibroblast and mesenchymal cell proliferation.
• VEGF: Supports endothelial cell growth.
• NGF: Promotes neuronal survival and differentiation.
• IGF-1: Supports growth and metabolism in many cell types.
• TGF-β: Regulates proliferation and differentiation, depending on cell type.
• Cytokines, such as IL-2, IL-3, GM-CSF, and EPO, are used for selected immune and hematopoietic cells.

Classification of Cell Culture Media

1. Natural media
   • Derived from biological fluids or tissue extracts.
   • Examples: plasma clot, serum, embryo extract, tissue extracts, and conditioned medium.
   • They contain nutrients and growth-promoting substances but their composition is not precisely known.
2. Synthetic or artificial media
   • Prepared from chemically defined constituents such as salts, glucose, amino acids, vitamins, and buffers.
   • Examples: MEM, DMEM, RPMI-1640, Ham's F-12, and M199.
   • They may be further classified into:
     • Basal media: Basic nutrient medium, usually requiring serum supplementation.
     • Serum-containing media
     • Serum-free media
     • Chemically defined media: Every constituent and its concentration are known.
     • Specialized/selective media: Designed for particular cells, for example neuronal, epithelial, stem-cell, or hybridoma media.

Serum-containing versus Serum-free Media

A. Serum-containing media

Advantages

1. Easy and convenient for routine culture of many cell lines.
2. Serum supplies multiple nutrients, hormones, attachment proteins, and growth factors simultaneously.
3. It enhances cell survival, proliferation, and attachment, especially after subculture.
4. It protects cells against mechanical damage and toxic substances.
5. Less optimization is needed for many established cells.

Disadvantages

1. Undefined composition: exact amounts of growth factors and proteins are unknown.
2. Batch-to-batch variation in serum causes poor reproducibility of experiments.
3. Serum can contain contaminants, including mycoplasma, viruses, endotoxins, or unwanted proteins.
4. It may stimulate or inhibit cell growth and alter cell differentiation unpredictably.
5. Serum is expensive and creates ethical concerns related to animal use.
6. It complicates purification of secreted products, such as monoclonal antibodies or recombinant proteins.

B. Serum-free media

Advantages

1. Known and controlled composition, giving better reproducibility.
2. Reduces variability between experiments and batches.
3. Lowers the risk of contamination from animal-derived serum.
4. Allows selective growth of a desired cell type by adding specific growth factors.
5. Facilitates study of the effects of individual hormones, cytokines, or drugs.
6. Better for downstream purification of cell products and for large-scale biopharmaceutical manufacturing.
7. Reduces animal-derived components and associated ethical concerns.

Disadvantages

1. Requires careful optimization for each cell type.
2. Some primary cells and fastidious cells grow poorly unless the correct growth factors are supplied.
3. Cells may require gradual adaptation from serum-containing to serum-free conditions.
4. Media are often more expensive than conventional basal medium plus serum.
5. Cells may show reduced survival after thawing, passaging, or transfection unless suitable supplements are added.