Assoc. Prof. of Biotechnology Environmental conditions of animal cell culture Dr. Tarek Elbashiti Assoc. Prof. of Biotechnology

PHYSICOCHEMICAL PROPERTIES 1- pH Most of human cells grow well at pH 7.4, some normal fibroblast lines perform best at pH 7.4–7.7, and transformed cells may do better at pH 7.0–7.4 Phenol red is commonly used as pH indicator. It is red at pH 7.4 and becomes orange at pH 7.0, yellow at pH 6.5, lemon yellow below pH 6.0, more pink at pH 7.6, and purple at pH 7.8

2-CO2 and Bicarbonate Carbon dioxide in the gas phase dissolves in the medium, establishes equilibrium with HCO3 ions, and lowers the pH. Because dissolved CO2, HCO3 and pH are all interrelated, it is difficult to determine the major direct effect of CO2. The atmospheric CO2 tension will regulate the concentration of dissolved CO2, as a function of temperature.

This regulation in turn produces H2CO3, which dissociates according to the reaction H2O + CO2 ⇔ H2CO3 ⇔ H+ + HCO3 HCO3 has a low dissociation constant with most of the available cations so it tends to reassociate, leaving the medium acid.

The net result of increasing atmospheric CO2 is to depress the pH, so the effect of elevated CO2 tension is neutralized by increasing the bicarbonate concentration: NaHCO3 ⇔ Na+ + HCO3 The increased HCO3 concentration pushes equation (1) to the left until equilibrium is reached at pH 7.4. If another alkali (e.g., NaOH) is used instead, the net result is the same: NaOH + H2CO3 ⇔ NaHCO3 + H2O ⇔ Na+ +HCO3 + H2O (3)

Cultures in open vessels need to be incubated in an atmosphere of CO2, the concentration of which is in equilibrium with the sodium bicarbonate in the medium. Cells at moderately high concentrations (≥1 × 105 cells/mL) and grown in sealed flasks need not have CO2 added to the gas phase, provided that the bicarbonate concentration is kept low (∼4 mM), particularly if the cells are high acid producers.

At low cell concentrations, however (e. g At low cell concentrations, however (e.g., during cloning), and with some primary cultures, it is necessary to add CO2 to the gas phase of sealed flasks. When venting is required, to allow either the equilibration of CO2 or its escape in high acid producers, it is necessary to leave the cap slack or to use a CO2-permeable cap

3-Buffers Inorganic NaHCO3 ,CO2 Organic HEPES (N-2 hydroxyethyl peprazin N2 ethanosulfonic acid) Each has advantages and disadvantages

Culture media must be buffered under two sets of conditions: (1) open dishes, where in the evolution of CO2 causes the pH to rise and (2) overproduction of CO2 and lactic acid in transformed cell lines at high cell concentrations, when the pH will fall.

A buffer may be incorporated into the medium to stabilize the pH, but in (1) exogenous CO2 may still be required by some cell lines, particularly at low cell concentrations, to prevent the total loss of dissolved CO2 and bicarbonate from the medium.

Despite its poor buffering capacity at physiological pH bicarbonate buffer is still used more frequently than any other buffer, because of its low toxicity, low cost, and nutritional benefit to the culture. HEPES is a much stronger buffer in the pH 7.2–7.6 range and is used at 10–20 mM. It has been found that, when HEPES is used with exogenous CO2, the HEPES concentration must be more than double that of the bicarbonate for adequate buffering

4-Oxygen Most cells require oxygen for respiration in vivo, cultured cells often rely mainly on glycolysis Most cells use anaerobic ( O2 diffusion slow ) Large amount lead to free radical Scavengers ( glutathion ,2- mercaptoethanol) Most cell culture use ATMO O2 only

Because the depth of the culture medium can influence the rate of oxygen diffusion to the cells, it is advisable tokeep the depth of the medium within the range 2–5 mm (0.2–0.5 mL/cm2) in static culture. Selenium versus O2 WHY !!! as selenium is a cofactor in glutathione synthesis. Serum media don’t need

Most dispersed cell cultures prefer lower oxygen tensions, and some systems (e.g., human tumor cells in clonogenic assay and human embryonic lung fibroblasts do better in less than the normal level of atmospheric oxygen tension.

5-Osmolality Most cultured cells have a fairly wide tolerance for osmotic pressure Human 290 mosm/ kg Mice 310 mosm /kg Animal ( 260 -320) mosm /kg 1 osmole = 1 mole of osmotically active particles HEPES and drugs dissolved in strong acids and bases and their subsequent neutralization can all markedly affect osmolality.

6-Temperature The optimal temperature for cell culture is dependent on (1) the body temperature of the animal from which the cells were obtained, (2) any anatomic variation in temperature (e.g., the temperature of the skin and testis may be lower than that of the rest of the body) (3) the incorporation of a safety factor to allow for minor errors in regulating the incubator.

Apart from its direct effect on cell growth, the temperature will also influence pH due to the increased solubility of CO2 at lower temperatures and, possibly, because of changes in ionization and the pKa of the buffer. The pH should be adjusted to 0.2 units lower at room temperature than at 37◦C.

Optimal according to animal body Or organ type Most at 37 Overheating more serious than under heating 4c or 196- But not 40

7-Viscosity The viscosity of a culture medium is influenced mainly by the serum content and in most cases will have little effect on cell growth. Viscosity becomes important, however, whenever a cell suspension is agitated (e.g., when a suspension culture is stirred) or when cells are dissociated after trypsinization.

This becomes particularly important in low-serum concentrations, in the absence of serum, and in stirred bioreactor cultures Any cell damage that occurs under these conditions may be reduced by increasing the viscosity of the medium with carboxymethylcellulose (CMC) or polyvinylpyrrolidone (PVP)

8-Surface Tension and Foaming The effects of foaming have not been clearly defined, but the rate of protein denaturation may increase, as may the risk of contamination if the foam reaches the neck of the culture vessel. Foaming will also limit gaseous diffusion if a film from a foam or spillage gets into the capillary space between the cap and the bottle, or between the lid and the base of a Petri dish.

Foaming can arise in suspension cultures in stirrer vessels or bioreactors when 5% CO2 in air is bubbled through medium containing serum.

BSS Inorganic May has ( glucose, Bicarbonate, HEPES) Function : Dilution Washing Dissecting Hours incubation ( passing media)

Culture Vessels and Substrates Glass Plastic ( polystyrene ) Gama irradiation Chemically ( ionization) Protein coat ( poly-lysin, poly-ornithin) Matrix protein ( fibronectin, laminin, collagen)

Culture Vessels and Substrates

Fig. 8.2. microtitration) plates. Plates are availableMultiwell Plates. Six-well, 24-well, and 96-well (with a wide range in the number of wells, from 4 to 144 (see Table 8.1 for sizes and capacities).

Fig. 8.4. Plastic Flasks. Sizes illustrated are 10 and 25 cm2 (Falcon, B-D Biosciences), 75 cm2(Corning), and 185 cm2 (Nalge Nunc) (see Table 8.1 for representative sizes and capacities).

Multisurface Flask

Choice of vessel According to ; - cell mass -adherent or suspension Vented or sealed Frequency Analysis The cost

COMPLETE MEDIA The term complete medium involves a medium that has had all its constituents and supplements added and is sufficient for the use specified. It is usually made up of a defined medium component, some of the constituents of which, may be added just before use, as various supplements, such as serum, growth factors, or hormones.

Defined media range in complexity from the relatively simple Eagle’s MEM, which contains essential amino acids, vitamins, and salts, to complex media such as medium 199 (M199), CMRL 1066, MB 752/1, RPMI 1640, and F12 and a wide range of serum-free formulations.

The complex media contain a larger number of different amino acids, including nonessential amino acids and additional vitamins, and are often supplemented with extra metabolites (e.g., nucleosides, tricarboxylic acid cycle intermediates, and lipids) and minerals. Nutrient concentrations are, on the whole, low in F12 (which was optimized by cloning) and high in Dulbecco’s modification

Eagle’s MEM (DMEM), optimized at higher cell densities for viral propagation. Barnes and Sato [1980] used a 1:1 mixture of DMEM and F12 as the basis for their serum-free formulations to combine the richness of F12 and the higher nutrient concentration of DMEM. Although not always entirely rational, this combination has provided an empirical formula that is suitable as a basic medium for supplementation with special additives for many different cell types.

Amino Acids The essential amino acids (i.e., those that are not synthesized in the body) are required by cultured cells, plus cystine and/or cysteine, arginine, glutamine, and tyrosine, although individual requirements for amino acids will vary from one cell type to another. Other nonessential amino acids are often added as well, to compensate either for a particular cell type’s incapacity to make them or because they are made but lost by leakage into the medium.

The concentration of amino acids usually limits the maximum cell concentration attainable, and the balance may influence cell survival and growth rate. Glutamine is required by most cells, although some cell lines will utilize glutamate; evidence suggests that glutamine is also used by cultured cells as a source of energy and carbon . Glutamax is a alanyl-glutamine dipeptide which is more stable than glutamine.

Vitamins Eagle’s MEM contains only the water-soluble vitamins (the B group, plus choline, folic acid, inositol, and nicotinamide, but excluding biotin); other requirements presumably are derived from the serum. Biotin is present in most of the more complex media, including the serum free recipes, and p-aminobenzoic acid (PABA) is present in M199, CMRL 1066 (which was derived from M199), and RPMI 1640.

All the fat-soluble vitamins (A, D, E, and K) are present only in M199, whereas vitamin A is present in LHC-9 and vitamin E in MCDB 110 . Some vitamins (e.g., choline and nicotinamide) have increased concentrations in serum-free media. Vitamin limitation—for example, by precipitation of folate from concentrated stock solutions—is usually expressed in terms of reduced cell survival and growth rates rather than maximum cell density.

Like those of the amino acids, vitamin requirements have been derived empirically and often relate to the cell line originally used in their development;

Salts The salts are chiefly those of Na+, K+, Mg2+, Ca2+, Cl−, SO4 2−, PO4 3−, and HCO3− and are the major components contributing to the osmolality of the medium. Most media derived their salt concentrations originally from Earle’s (high bicarbonate; gas phase, 5% CO2) or Hanks’s (low bicarbonate; gas phase, air) BSS. Divalent cations, particularly Ca2+, are required by some cell adhesion molecules, such as the cadherins. Ca2+ also acts as an intermediary in signal transduction and the concentration of Ca2+ in the medium can influence whether cells will proliferate or differentiate

Na+, K+, and Cl− regulate membrane potential, whereas SO4 2−, PO43−, and HCO3 have roles as anions required by the matrix and nutritional precursors for macromolecules, as well as regulators of intracellular charge. Calcium is reduced in suspension cultures in order to minimize cell aggregation and attachment . The sodium bicarbonate concentration is determined by the concentration of CO2 in the gas phase and has a significant nutritional role in addition to its buffering capability.

Glucose Glucose is included in most media as a source of energy. It is metabolized principally by glycolysis to form pyruvate, which may be converted to lactate or acetoacetate and may enter the citric acid cycle and is oxidized to form CO2 and water. The accumulation of lactic acid in the medium, particularly evident in embryonic and transformed cells, implies that the citric acid cycle may not function entirely as it does in vivo, and recent data have shown that much of its carbon is derived from glutamine rather than glucose. This finding may explain the exceptionally high requirement of some cultured cells for glutamine or glutamate.

Hormones and Growth Factors Hormones and growth factors are not specified in the formulas of most regular media, although they are frequently added to serum-free media Antibiotics Antibiotics were originally introduced into culture media to reduce the frequency of contamination. However, the use of laminar-flow hoods, coupled with strict aseptic technique, makes antibiotics unnecessary. Indeed, antibiotics have a number of significant disadvantages: (1) They encourage the development of antibioticresistant organisms.

(2) They hide the presence of low-level, cryptic contaminants that can become fully operative if the antibiotics are removed, the culture conditions change, or resistant strains develop. (3) They may hide mycoplasma infections. (4) They have antimetabolic effects that can cross-react with mammalian cells. (5) They encourage poor aseptic technique.

Hence it is strongly recommended that routine culture be performed in the absence of antibiotics and that their use be restricted to primary culture or large-scale labor-intensive experiments with a high cost of consumables. If conditions demand the use of antibiotics, then they should be removed as soon as possible, or, if they are used over the long term, parallel cultures should be maintained free of antibiotics

A number of antibiotics used in tissue culture are moderately effective in controlling bacterial infections However, a significant number of bacterial strains are resistant to antibiotics, either naturally or by selection, so the control that they provide is never absolute. Fungal and yeast contaminations are particularly hard to control with antibiotics; they may be held in check, but are seldom eliminated