BIOREACTOR SYSTEM (ERT 314)

1. BIOREACTOR SYSTEM(ERT 314) Huzairy Hassan School of Bioprocess Engineering UniMAP

2. ANIMAL CELL Cultures

3. Biochemistry of Animal Cells - Animal cells vary in size (10 to 30 µm) and shape (spherical, ellipsoidal). - Typically eukaryotes - Do not have a cell wall, but are surrounded by a thin and fragile plasma membrane that is composed of protein, lipid and carbohydrate. - This structure results in significant shear sensitivity. - Microvilli – increase surface area of the cell and provide more effective passage of materials across the plasma membrane. - The surface of the cell is negatively charged, and cells tend to grow on positively charged surfaces, such as Sephadex or collagen  anchorage-dependent cells. - Some animal cells such as hybridomas are non-anchorage-dependent and grow in suspension culture.

4. Figure 1: Animal cell

5. - Animal cells have a cytoskeleton: a system of protein filaments that provide the cells mechanical strength, control cell shape, and guide cell movement  critical components in controlling cell response to mechanical forces such as from fluid flow or from attachment to surfaces. - A typical growth medium: contains glucose, glutamine, nonessential and essential amino acids, serum (horse or calf) and mineral salts, vitamins, trace elements, growth factors, and buffers in water.

6. Cultivation of Animal Cells - Animal (mammalian) cells grow at 37 ºC and pH ≈ 7.3. - Typical doubling times are 12 to 20 hours. - Usually, 5% CO2-enriched air is used to buffer the medium pH around pH = 7.3. - Kinetics of growth similar to microbial growth. - Usually, the stationary phase is relatively short, and the concentration of viable cells drops sharply thereafter due to accumulation of toxic metabolic products such as lactate and ammonium. - Product formation such as monoclonal antibody (MAb) by hybridoma cells, can continue under non-growth conditions. - Most of the products are mixed-growth associated, and product formation takes place both during the growth phase and after growth ceases.

7. - Kinetics of product formation (e.g., MAb); where α is YPX, yield of product from biomass and β is specific rate of product formation due to maintenance (kg biomass -1 s-1). The term in the left is for growth-associated production, and in the right is for non-growth associated.

8. PHYSICOCHEMICAL PROPERTIES pH - the optimum pH for cell growth varies relatively among different cell strains - most cell lines grow wells at pH 7.3 - 7.4 - to determine the optimum pH, do a brief growth experimentor a special function analysis Buffering - culture media must be buffered under two sets of conditions: • Open dishes, wherein the evolution of CO2 causes the pH to rise • Overproduction of CO2 and lactic acid in transformed cell lines at high concentration, when the pH will fall - buffer is incorporated into medium to stabilize the pH Buffering-HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) Medium buffering with 4mM NaHCO3 and 10mM of HEPEScan be used to support culture in the absence or breakdown of 5% CO2 incubator system.

9. PHYSICOCHEMICAL PROPERTIES-cont’d • CO2 and Bicarbonate - in medium, the carbon dioxide appears as dissolved CO2 in equilibrium with HCO3- and lowers the pH (CO2, HCO3- and pH are interrelated) - the atmospheric CO2 will regulate the concentration of dissolved CO2 directly and in turn produces H2CO3 H2O + CO2 ↔H2CO3 ↔ H+ + HCO3- (1) - HCO3- has low dissociation constant with most cations and tends to re-associate, leaving the medium acid. The net result of increasing atmospheric CO2.is to depress the pH. - this effect of elevated CO2 is neutralized by increasing the bicarbonate concentration: NaHCO3 ↔ Na+ + HCO3- - the increased HCO3- concentration pushes the eq.(1) to the left until the equilibrium is reached at pH 7.4.

10. PHYSICOCHEMICAL PROPERTIES-cont’d • Oxygen • cultures vary in their oxygen requirement. • lower oxygen tensions are preferable for most cell cultures • but some organ cultures require up to 95% O2 in the gas phase. • the depth of the culture medium can influence the rate of oxygen diffusion to the cells (it is advisable to keep within the range 2 – 5 mm in static culture.) .

11. PHYSICOCHEMICAL PROPERTIES-cont’d • Osmolality • Osmolality: the concentration of a solution expressed as the total number of solute particles per kilogram • in practice, osmolalities between 260 mOsm/kg and 320 mOsm/kg are quite acceptable for most cells, but once selected, should be kept consistent at ± 10 mOsm/kg. • osmolality is usually measured by depression of the freezing point, or elevation of the vapor pressure, of the medium. • The measurement of osmolality is to ensure that there are no errors in weighing, dilution etc in preparing your media.

12. PHYSICOCHEMICAL PROPERTIES-cont’d • Temperature • the optimal temperature for cell culture is dependent on: • The body temperature of the animal from which the cells were obtained • Any anatomical variation in temperature (e.g. the temperature of the skin) • The incorporation of a safety factor to allow for minor errors in regulating the incubator. • thus, the temperature recommended for most human and warm-blood animal cell lines is 37°C, close to body heat. • cultured cells can survive several days at 4°C and can be frozen and cooled to -196°C but cannot tolerate temp > 2oC for more than few hours and die rapidly at 40oC and over. • the consistency of the temperature is essential to ensure reproducible results. • temperature not just influences the effect on cell growth, but also the pH due to the increased solubility of CO2 at lower temperature.

13. PHYSICOCHEMICAL PROPERTIES-cont’d • Viscosity - viscosity of a culture medium is influenced mainly by the serum content and have little effect on cell growth. - however it becomes important whenever a cell suspension is agitated or cells are dissociated after trypsinization. - any cell damage that occurs under these conditions may be reduced by increasing the viscosity of the medium (add CMC, PVP). • Surface Tension and Foaming - the rate of protein denaturation may increase, as may the risk of contamination if the foam reaches the neck of the culture vessel. - foaming limits gaseous diffusion - the addition of silicone antifoam helps prevent foaming by reducing the surface tension and may also protect cells against shear stress from bubbles.

14. BALANCED SALT SOLUTIONS • A balanced salt solution (BSS) is composed of inorganic salts, sodium bicarbonate and in some cases, glucose. • BSS forms the basis of many complete media (e.g. Eagle’s MEM with Hanks’s salts) • BSS is also used as diluents for concentrates of amino acids and vitamins to make a complete media, as washing or dissection medium, and for short incubations up to 4 h. • The choice of BSS is dependent on: • The CO2 tension • The intended use of the solution for tissue disaggregation or monolayer dispersal (Ca2+ and Mg2+ is omitted) • Whether the solution will be used for suspension culture of adherent cells (Ca2+ is omitted)

15. COMPLETE MEDIA Complete media contains: • Amino acids (essential aa) • Vitamins • Salts • Glucose • Organic Supplements (protein, nucleosides, CAC intermediates) • Hormones and Growth Factors • Antibiotics Example : DMEM, Eagle’s MEM, RPMI-1640, Iscove’s, CMRL-1066, L-15 medium (Leibovitz), McCoy’s 5A F12

16. Antibiotics for animal cell culture

17. Serum - is a cell-free liquid recovered from blood. - Examples: Fetal Bovine serum (FBS), Calf serum (CS), Horse serum (HS). - Functions: 1) to stimulate cell growth and other cell activities by hormones and growth factors 2) to enhance cell attachment by certain proteins such as collagen and fibronectin 3) to provide transport proteins carrying hormones, minerals, and lipids. - Disadvantages: expensive, cause further complication in cultivation and downstream processing, potential contamination with virus, mycoplasma or prions, foams formation, rapid deterioration (1 year).

18. SERUM Serum contains: • Protein - carriers for minerals, fatty acids, and hormones. e.galbuminas carrier of lipid or mineral, fibronectin which promote cell growth. Fetuin in fetal serum enhances cell attachment. Protein increases the viscosity of the medium, reduce shearing during pipetting and add buffering capacity. • Growth factors – promote cell proliferation, and adhesion factors and antitrypsin activity which promote cell attachment. E.g PDGF from platelet during clotting, stimulate cell proliferation. • Hormones – insulin promotes the uptake of glucose and amino acids. Hydrocortisone promote cell attachment and proliferation.

19. SERUM – cont’d • Nutrients and metabolites – glucose, ketoacids, nucleosides and a number of nutrients and intermediary metabolites • Lipids – linoleic acids, oleic acid, ethanolamine and phosphoethanolamine are present in serum in small amounts usually bound to proteins such as albumin f. Minerals – trace elements and iron, copper, and zinc may be bound to serum protein. - e.g. selenium helps to detoxify free radicals as a cofactor for GSH synthetase • Inhibitors – serum may contain substances that inhibit cell proliferation such as negative growth regulators (TGF-β). examples: fetal bovine serum (FBS), horse serum, human serum

20. Serum are quite expensive. Fetal bovine serum is used for more demanding cell lines. • Before use, serum is heat inactivated by incubating at 56° C for 30 mins (may remove mycoplasma) • Selection of Medium and Serum • Type of media to use for a particular cell lines-available in the literature • Most common-DMEM, MEM, RPMI-1640 • For most cell lines and primary cultures-they can be maintained in media like MEM with calf serum. • More complex media-stem cell culture, specialized function (to grow virus)

21. DISADVANTAGES OF SERUM • Physiological Variability - serum also contains a wide range of minor components that effect on cell growth and their concentrations and actions of which have not been fully determined. • Shelf Life and Consistency - serum varies from batch to batch, and will last one year and perhaps deteriorating during that time. It must be replaced but the new one will never be identical to the first batch. • Quality Control – changing serum batches requires extensive testing to ensure that the replacement is as close as possible to previous batch.

22. DISADVANTAGES OF SERUM • Specificity - if more than one cell type is used, each type may require a different batch of serum • Availability - the supply of serum is restricted due to drought in the cattle-rearing areas, the spread of disease among the cattle, or economic or political reasons. • Downstream Processing - the presence of serum creates a major obstacle to purification

23. DISADVANTAGES OF SERUM • Contamination - serum is frequently contaminated with viruses and mycoplasma • Cost: it is more expensive (> 10x) • Growth inhibitors - serum also contains growth-inhibiting activity • Standardization - standardization of experimental and production protocols is difficult, both at different times and among different laboratories.

24. ADVANTAGES OF SERUM-FREE MEDIA Selective Media One of the major advantages of the control over growth-promoting activity afforded by serum-free media is the ability to make a medium selective for a particular cell type. Regulation of Proliferation and Differentiation Add to the ability to select for a specific cell type the possibility of switching from a growth-enhancing medium for propagation to a differentiation-inducing medium by altering the conc. and types of growth factors and other inducers.

25. DISADVANTAGES OF SERUM-FREE MEDIA • Multiplicity of Media – each cell type appears to require a different recipe. While the degree of specificity may be an advantage to those isolating specific cell types, it presents a problem for laboratories maintaining cell lines of several different origins. • Selectivity– cells at different stages of development may require different formulations, particularly in the growth factor and cytokine components. • Reagent Purity – the removal of serum also requires that the degree of purity of reagents, water and the degree of cleanliness of all apparatus as removal of serum also removes the protective, detoxifying action that some serum may have.

26. DISADVANTAGES OF SERUM-FREE MEDIA – cont’d • Cell Proliferation – growth is often slower in serum-free media, and fewer generations are achieved with finite cell lines. • Availability – the availability of properly controlled serum-free media is quite limited, and the products are often more expensive than conventional media.

27. REPLACEMENT OF SERUM Serum-free Subculture Adhesion Factors. When serum is removed, it may be necessary to treat the plastic growth surface with fibronectine or laminin by adding directly to the medium. Protease Inhibitors. Protease inhibitors such as soya bean trypsin inhibitor must be added to serum-free media after subculture. Trypsin Temperature. Special care is required when trypsinizing cells from serum-free media, as the cells are fragile and need to be chilled to reduce damage.

28. REPLACEMENT OF SERUM – cont.d Hormones Hormones that have been used to replace serum include growth hormone (somatotropin), insulin which enhances plating efficiency, and hydrocortisone, which improves the cloning efficiency of glial and fibroblasts. Nutrients in Serum Iron, copper, and a number of minerals have been included in serum-free recipes

29. REPLACEMENT OF SERUM-cont’d Growth Factors Growth factors and cytokines which have wide range of specificity is added. E.g. Keratinocyte growth factor (KGF), besides showing activity with epidermal keratinocytes, will also include proliferation and differentiation in prostatic epithelium. Proteins and Polyamines The inclusion in medium of proteins such as bovine serum albumin or tissue extracts often increases cell growth and survival, but adds undefined constituents to the medium.

30. REPLACEMENT OF SERUM-cont’d Matrix One of the properties of serum is to provide a number of proteins (e.g. fibronectin) that coat the plastic and make it more adhesive. In the absence of serum, the plastic substrate may need be coated with fibronectin or polylysine.

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