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

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 (1)  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 (2)  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 CO 2, the concentration of which is in equilibrium with the sodium bicarbonate in the medium.  Cells at moderately high concentrations (≥1 × 10 5 cells/mL) and grown in sealed flasks need not have CO 2 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., 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 NaHCO 3, CO 2 -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 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 (O 2 diffusion slow )  Large amount lead to free radical  Scavengers (glutathion, 2- mercaptoethanol)  Most cell culture use atm O 2 only

 Because the depth of the culture medium can influence the rate of oxygen diffusion to the cells, it is advisable to keep the depth of the medium within the range 2–5 mm  (0.2–0.5 mL/cm2) in static culture.  Selenium versus O 2 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 plasma 290 mosmol/kg  Mice 310 mosmol/kg  ( ) mosmol/kg acceptable for most cells.  Should be kept consistent at ±10 mosmol/kg.  Slightly hypotonic medium may be better for Petri dish or open-plate culture to compensate for evaporation during incubation.

 1 osmole = 1 mole of osmotically active particles  It is particularly important to monitor osmolality if alterations are made in the constitution of the medium.  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.

 Optimal according to animal body or organ type (Skin and Testis)  Most human and warm-blooded animal cell lines is 37 ◦ C, close to body heat, but set a little lower for safety,  As overheating is a more serious problem than underheating.  Mammalian cells can survive several days at 4°c and can be frozen and cooled to -196°C.  But not 40°C (will die).  For avian cells: 38.5°C for maximum growth and more slowly at 37°C but satisfactory.

 For cold-blooded animals: tolerate a wide temperature range, between 15◦C and 26◦C.  If necessary, it can be maintained at room temperature, but the variability of the ambient temperature in laboratories makes this undesirable, and a cooled incubator is preferable.  A number of temperature-sensitive (ts) mutant cell lines have been developed that allow the expression of specific genes below a set temperature, but not above it.

 The two discriminating temperatures are usually only about 2–3◦C apart.  The use of ts mutants usually requires an incubator with cooling as well as heating, to compensate for a warm ambient temperature.  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.  In preparing a medium for the first time, it is best to make up the complete medium and incubate a sample overnight at 37◦C under the correct gas tension, in order to check the pH.

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.

Balanced Salt Solutions (BSS)  Inorganic  May has ( glucose, Bicarbonate, HEPES)  Function : -Dilution -Washing -Dissecting -Hours incubation ( passing media)

 The choice of BSS is dependent on both the CO2 tension and the intended use of the solution for tissue disaggregation or monolayer dispersal; in these cases Ca2+ and Mg2+ are usually omitted, as in Moscona’s calcium- and magnesium-free saline (CMF) or D-PBSA.  The choice of BSS also is dependent on whether the solution will be used for suspension culture of adherent cells.

 S-MEM is a variant of Eagle’s MEM that is deficient in Ca2+ in order to reduce cell aggregation and attachment.  HBSS, EBSS, and PBS rely on the relatively weak buffering of phosphate, which is not at its most effective at physiological pH.  HEPES (10–20 mM) is currently the most effective buffer in the pH 7.2– 7.8 range,  Tricine in the pH 7.4–8.0 range.

COMPLETE MEDIA  The term complete medium implies 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,  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.

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.  Antibiotics have a number of significant disadvantages: (1) They encourage the development of antibiotic resistant 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.

Antibiotics Used in Tissue Culture

SERUM  Contains growth factors, which promote cell proliferation, and adhesion factors and antitrypsin activity, which promote cell attachment.  source of minerals, lipids, and hormones, many of which may be bound to protein.  Most used are: bovine calf, fetal bovine, adult horse, and human serum.  Calf (CS) and fetal bovine (FBS) serum are the most widely used.  Human serum is sometimes used in conjunction with some human cell lines, but it needs to be screened for viruses, such as HIV and hepatitis B.

Constituents of Serum

SELECTION OF MEDIUM AND SERUM  All 12 media described were developed to support particular cell lines or conditions.  All now have more general applications and have become classic formulations.  Among them, data from suppliers would indicate that RPMI 1640, DMEM, and MEM are the most popular, making up about 75% of sales.  Other formulations seldom account for more than 5% of the total; most constitute 2–3%, although blended DMEM/F12 comes closer, with over 4%.

 Information regarding the selection of the appropriate medium for a given type of cell is usually available in the literature in articles on the origin of the cell line or the culture of similar cells.  Information may also be obtained from the source of the cells.  Cell banks, such as ATCC and ECACC, provide information on media used for currently available cell lines, and data sheets can be accessed from their websites.

Selecting a Suitable Medium

 Many continuous cell lines (e.g., HeLa, L929, BHK21), primary cultures of human, rodent, and avian fibroblasts, and cell lines derived from them can be maintained on a relatively simple medium such as Eagle’s MEM, supplemented with calf serum.  If information is not available, a simple cell growth experiment with commercially available media and multiwell plates can be carried out in about two weeks.

 It is to be hoped that as serum requirements are reduced and the purity of reagents increases, the standardization of media will improve.  Finally, you may have to compromise in your choice of medium or serum because of cost.  Thus, if a culture grows to 1×10 6 /mL in serum A and 2×10 6 /mL in serum B, serum B becomes the less expensive by a factor of two, given that product formation or some other specialized function is the same.

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 +, Mg 2+, Ca 2+, Cl −, SO4 2−, PO4 3−, and HCO 3 − 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 Ca 2+, are required by some cell adhesion molecules, such as the cadherins.  Ca 2+ also acts as an intermediary in signal transduction and the concentration of Ca 2+ in the medium can influence whether cells will proliferate or differentiate.  Na +, K +, and Cl − regulate membrane potential, whereas SO 4 2−, PO 4 3−, and HCO 3 1- 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,

 Which 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.

Testing Serum  The quality of a given serum is assured by the supplier, but the firm’s quality control is usually performed with one of a number of continuous cell lines.  If your requirements are more demanding, then you will need to do your own testing.  There are four main parameters for testing serum.

1. Plating efficiency:  Plate the cells out at 10 to 100 cells/mL, and look for colonies after 10 days to two weeks.  Stain and count the colonies, and look for differences in plating efficiency (survival) and colony size (cell proliferation).  Each serum should be tested at a range of concentrations from 2% to 20%.  This approach will reveal whether one serum is equally effective at a lower concentration, thereby saving money and prolonging the life of the batch, and will show up any toxicity at a high serum concentration.

2. Growth curve:  A growth curve should be plotted for cell growth in each serum, so that the lag period, doubling time, and saturation density (cell density at ‘‘plateau’’) can be determined.  A long lag implies that the culture has to adapt to the serum; short doubling times are preferable if you want a lot of cells quickly; and a high saturation density will provide more cells for a given amount of serum and will be more economical.

3. Preservation of cell culture characteristics:  Clearly, the cells must do what you require of them in the new serum, whether they are acting as host to a given virus, producing a certain cell product, differentiating, or expressing a characteristic sensitivity to a given drug.

4. Sterility:  Serum from a reputable supplier will have been tested and shown to be free of microorganisms.  However, in the unlikely event that a sample of serum is contaminated but has escaped quality control, the fact that it is contaminated should show up in mycoplasma screening.

Serum Heat Inactivation  Serum is heat inactivated by incubating it for 30 min at 56◦C.  It may then be dispensed into aliquots and stored at −20◦C.  Claims that heat inactivation removes mycoplasma are probably unfounded, although heat treatment may reduce the titer for some mycoplasma.

OTHER SUPPLEMENTS 1. Amino Acid Hydrolysates:  Bactopeptone, tryptose, and lactalbumin hydrolysate are proteolytic digests of beef heart or lactalbumin and contain mainly amino acids and small peptides.  Bactopeptone and tryptose may also contain nucleosides and other heat- stable tissue constituents, such as fatty acids and carbohydrates.

2. Embryo Extract:  Embryo extract is a crude homogenate of 10-day-old chick embryo that is clarified by centrifugation.  The crude extract was fractionated to give fractions of either high or low molecular weight.  The low-molecular-weight fraction (contains peptide growth factors) promoted cell proliferation, whereas the high-molecular- weight fraction (proteoglycans and other matrix constituents) promoted pigment and cartilage cell differentiation.

3. Conditioned Medium  It is found that the survival of low density cultures could be improved by growing the cells in the presence of feeder layers.  This effect is probably due to a combination of effects including conditioning of the substrate and conditioning of the medium by the release into it of small molecular metabolites and growth factors.  Conditioning of culture medium that was necessary for the growth and differentiation of myoblasts was due to collagen released by the feeder cells.

 Using feeder layers and conditioning the medium with embryonic fibroblasts or other cell lines remains a valuable method of culturing difficult cells.  Conditioned medium contains both substrate-modifying matrix constituents, like collagen, fibronectin, and proteoglycans, and growth factors, such as those of the heparin-binding group (FGF, etc.), insulin-like growth factors (IGF-I and -II), PDGF, and several others.

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 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 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