Essential nutrients for tissue culture

Provides all the essential nutrients • Amino acids, energy substrates, vitamins, minerals, salts, etc • Maintain constant pH • Hepes vs CO 2/HCO 3 buffering buffering • Phenol red indicator (yellow-orangemaroon) (acid>>>>>>>>alkaline)

Physicochemical Properties -Affecting Medium 1. pH - for normal cells – 7.4, for transformed cells 7.0 – 7.4 – phenol red 2. Buffering - – Commonly used systems: CO 2 , – Bicarbonate/HEPES, 3. Oxygen - dissolved oxygen – Correct O 2 tension so as to meet the requirement and avoid toxicity – Requirements depends upon type of culture – Selenium – guard against O 2 toxicity toxicity 4. Osmolality - 280-310 mOsm/kg – Measured using depression of the freezing point or elevation of the vapor pressure. – Helps guard against errors of weighing, dilution. – Addition of strong acids, bases like HEPES significantly affect. 5. Temperature • Keep at 4°C-Not use • Warm at 37C –before use to culture the cells

Animal Sera • Provides various hormones/ growth factors to stimulate cell proliferation and function • Less well defined than Serum-free culture systems • Often difficult to culture cells without serum, but may interfere with studies of specific factors (e.g. insulin, glucose) Serum Containing Medium • Traditional undefined medium • Major source of various nutrients such as growth factors, adhesion factors, minerals, lipids, trace elements etc. • Commonly used sera: Calf, Fetal bovine, adult horse & human sera • Horse sera: less metabolism of polyamines, more consistent batch to batch • Human sera: only used for few cell lines, screening for HIV, hepatitis B virus. • Promotes cell proliferation, adhesion factors, antitrypsin activity and cell attachment, source of various nutrients Contents of Serum • Protein Contents: Albumin – carrier of lipids & minerals. • Fetuin & Fibronectin: promote attachment • α2 macroglobulin: inhibits trypsin • Transferrin: makes Iron less toxic and bioavailable. • Growth Factors: main role is in growth stimulation & are mitogenic E.g. PDGF, FGF, EGF, VEGF, Angiogenin etc. • PDGF – major growth factor • Hormones: Insulin: promotes uptake of glucose & amino acids, mitogenic when bound to IGF – I receptor • IGF - 1/2: mitogenic and stimulate growth • Hydrocortisone: promote cell attachment • Minerals: Iron, Copper, Zinc, Selenium – essential trace elements required for cell growth. Selenium: important role in detoxifying free radicals by promoting glutathione synthesis. • Inhibitors: TGF – β, hydrocortisone – cytostatic. • Apart from these, Carbohydrates (1.0 2.0 mg/mL), various vitamins (10ng - 10µg/mL), amino acids are also present. Advantages of Serum • Provides various components • Modulates physiological properties of medium • Protease inhibitors • Provides nutrients not present in basal medium • Carrier proteins for low molecular weight substances (e.g. transferrin) • Help in solubilization of poorly dissolved substances (e.g. apolipoprotein) • Cell substrate attachment (fibronectin, vironectin) • Various enzymes • Proteins which prevent non specific adsorption (e.g. albumin) • Neutralization of detergents • Prevents essential nutrients e.g. fatty acids Disadvantages of Serum in culture medium i. Potential introduction of animal viruses ii. Antibodies against viruses, to which host cell is exposed. iii. Availability of high quality iv. Undesirable contaminants v. High running costs & capital requirements vi. Shelf Life & Storage – always purchased in bulk vii. Physiological variability & consistency viii. Downstream Processing ix. Characterization of final product laborious.