Cell culture media
- 2. INTRODUCTION • The influence of cell culture technology on human society has been immeasurable. • Progress in biology in recent years has depended heavily on cell culture technology. • In addition, cell culture based practical technologies have been developed in various areas, including the assessment of the efficacy and toxicity of new drugs, manufacture of vaccines, and biopharmaceuticals and assisted reproductive technology. • Hence, cell culture technology is regarded as a foundation for further development and popularization. • In this context, the cell culture medium is the most important factor in cell culture technology. 2
- 3. • A medium supports cell survival and proliferation, as well as cellular functions, hence the quality of the medium directly affects the research results, the biopharmaceutical production rate and treatment outcomes of assisted reproductive technology. • It is essential therefore, for investigators who are working with cell culture to select an appropriate medium that is suitable for their aims. • In some cases researchers should modify a medium themselves. • In addition, when facing problems, researchers have to know the properties of the medium in order to identify the cause of any problem with their experiments. Conti… 3
- 4. 4 HISTORY • In 1882, Sydney Ringer developed Ringer’s solution, a balanced salt solution of a composition that is close to that of bodily fluids, and successfully kept frog hearts beating after dissection and removal from the body. • This is said to be the first instance of in vitro cultivation of animal tissue. Balanced salt solutions were developed one after another in the wake of Ringer’s report, including Locke’s solution, Tyrode’s solution, the Krebs–Ringer bicarbonate solution, Gey’s solution, Earle’s solution and Hanks’ solution. • The composition of these balanced salt solutions is simple and includes only inorganic salts, sometimes with glucose added as a nutrient. • Nonetheless, their pH, osmotic pressure, and inorganic salt concentrations were calibrated to physiological conditions -
- 5. 5 and these solutions can be used successfully to keep tissues and cells outside the body alive for short periods, generally up to a few days. • In 1907, Ross G. Harrison successfully monitored an apparent outgrowth of nerve fibers of a frog for several weeks in lymph fluid that had been freshly drawn from the lymph sacs of an adult frog. • This experiment is considered to be the beginning of animal cell cultivation. Conti…
- 6. 6 TYPES OF CELL CULTURE MEDIA Mainly 2 types of culture media are used in animal tissue culture : 1) NATURAL MEDIA 2) ARTIFICIAL / SYNTHETIC MEDIA The type of medium depends on the type of cells to be cultured and its objectives.
- 7. 7 NATURAL MEDIA DEFINITION : • Consisting of natural biological substances such as plasma, serum and embryo extract. • Natural media consists solely of naturally occurring biological fluids. • Natural media are very useful and convenient for a wide range of animal cell culture.
- 8. 8 NATURAL MEDIA MEDIA TYPE EXAMPLES COAGULANT OR CLOTS TISSUE EXTRACTS BIOLOGICAL FLUIDS Plasma separated from heparinized blood, serum and fibrinogen Extracts of chicken embryos, liver and spleen and bone marrow extract. Plasma, serum, lymph, amniotic fluid and pleural fluids Conti…
- 9. 9 SYNTHETIC MEDIA / ARTIFICIAL MEDIADEFINITION : • Composed of a basal medium and supplements, such as serum, growth factor and hormones. • It is prepared by adding nutrients (both organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, cofactors etc. • Different artificial media have been devised to serve one or more of the following purposes: • 1) immediate survival (a balanced salt solution, with specific pH and osmotic pressure) • 2) prolonged survival (a balanced salt solution supplemented with various formulation of organic compounds and/or serum) • 3) indefinite growth • 4) specialized functions.
- 10. 10 SYNTHETIC MEDIA MEDIA TYPE EXAMPLES SERUM CONTAINING MEDIA SERUM FREE MEDIA XENO FREE MEDIA Human, bovine, equine or other serum is used as a supplement Crude protein fractions such as bovine serum albumin or α or β globulin are supplements Human source components such as human serum albumin are used as supplements but animal components are not allowed as supplements PROTEIN FREE MEDIA CHEMICALLY DEFINED MEDIA Undefined components such as peptide fractions are used as supplements Highly purified components such as recombinant proteins are appropriate supplements Conti…
- 11. 11 USE OF NATURAL MEDIA • Alexis Carrel was a French surgeon and biologist who received the Nobel Prize in Physiology and Medicine in 1912 for his research on the vascular suture and transplantation of blood vessels and organs. • He contributed greatly to tissue culture technology by devising a prototype of the cell culture flask that is used widely today and by establishing the aseptic manipulation technique. • The first success of animal cell culture by Harrison inspired Carrel to send Montrose T. Burrows to work under Harrison’s supervision in 1909. • There, Burrows found that lymph is unsuitable for the cultivation of cells from warm-blooded animals and used plasma instead. • Thereafter, blood plasma had become a major culture medium for a variety of animal cells.
- 12. 12 • He successfully cultivated chicken embryonic cells by using chicken blood plasma, which is readily available, and later successfully cultivated mammalian cells as well. • In 1912, Carrel demonstrated that the long-term cultivation of the cells that have been obtained from the connective tissues of chick fetuses is possible (for several months) with a periodic exchange of the medium. • In 1913, he discovered that adding embryonic extract to blood plasma can dramatically increase cellular proliferation and extend the culture period of fibroblasts from the chick embryo heart. Conti…
- 13. 13 • Meanwhile, because the composition of the lymph, plasma, and embryonic extract was unknown, it became a new scientific inquiry regarding which of their components affected the survival and growth of animal tissues and cells. • This situation led to a period when researchers attempted to identify the growth-promoting substances within these ingredients of natural origin and to replace them with ingredients of definite composition. Conti…
- 14. 14 ARTIFICIAL MEDIA (mainly composed of a BASAL media) • Each basal medium has been designed in each case on the basis of the cell type, the origin (animal species), and the purpose of the culturing. • In fact, the medium composition can differ greatly depending on such background factors. Whether supplementation with natural products is allowed is another important presupposition for the choice of a basal medium. • For example, MEM (developed by Eagle) was designed under the assumption of serum supplementation and accordingly includes only the minimum necessary components (inorganic salts, sugar, essential amino acids, and water-soluble vitamins). • In contrast, Medium 199 and Ham’s F-12, intended for serum-free culture, contain various other components.
- 15. 15 TYPES AND CHARACTERISTICS OF BASAL MEDIUM
- 16. 16 Conti…
- 17. 17 1) SERUM CONTATINING MEDIUM • Serum contains growth factors, which promote cell proliferation, and adhesion factors and antitrypsin activity, which promote cell attachment. Serum is also a source of minerals, lipids, and hormones, many of which may be bound to protein. • The sera used most in tissue culture are bovine calf, fetal bovine, adult horse, and human serum. Calf (CS) and fetal bovine (FBS) serum are the most widely used, the latter particularly for more demanding cell lines and for cloning. • 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. • Serum also adds buffering capacity to the medium and binds or neutralizes toxic components.
- 18. 18 • Horse serum is preferred to calf serum by some workers, as it can be obtained from a closed donor herd and is often more consistent from batch to batch. • Horse serum may also be less likely to metabolize polyamines, due to lower levels of polyamine oxidase; polyamines are mitogenic for some cells. Conti… FETAL BOVINE SERUM (FBS)
- 19. 19 COMPONENTS OF SERUM 1. Protein: • proteins are a major component of serum, the functions of many proteins in vitro remain obscure; it may be that relatively few proteins are required other than as carriers for minerals, fatty acids, and hormones. • Those proteins for which requirements have been found are albumin, which may be important as a carrier of lipids, minerals, and globulins. • fibronectin (cold-insoluble globulin), which promotes cell attachment, although probably not as effectively as cell-derived fibronectin; and α2-macroglobulin, which inhibits trypsin. • Fetuin in fetal serum enhances cell attachment and transferrin binds iron, making it less toxic and bioavailable. Other proteins, as yet uncharacterized, may be essential for cell attachment and growth.
- 20. 20 2. Growth factor: • PDGF stimulates growth in fibroblasts and glia, but other platelet-derived factors, such as TGF-β, may inhibit growth or promote differentiation in epithelial cells. • Fibroblast growth factors (FGFs), epidermal growth factor (EGF), endothelial cell growth factors such as vascular endothelial growth factor (VEGF) and angiogenin and insulin- like growth factors IGF-I and IGF-II which have been isolated from whole tissue or released into the medium by cells in culture, have varying degrees of specificity and are probably present in serum in small amounts. 3. Nutrients and Metabolites: • Serum may also contain amino acids, glucose, oxo(keto) acids, nucleosides, and a number of other nutrients and intermediary metabolites. Conti…
- 21. 21 • These may be important in simple media but less so in complex media, particularly those with higher amino acid concentrations and other defined supplements 4. Lipids, Minerals and Inhibitors: • Linoleic acid, oleic acid, ethanolamine, and phosphoethanolamine are present in serum in small amounts, usually bound to proteins such as albumin. Conti…
- 22. 22 ADVANTAGES DISADVANTAGES • Serum contains various growth factors and hormones which stimulates cell growth and functions. • Helps in attachment of the cells • Acts as a spreading factor • Acts as a buffering agent which helps in maintaining the pH of the culture media. • Functions as a binding protein • Lack of uniformity in the composition of serum. • Testing needs to be done to maintain the quality of each batch before using. • May contain some of the growth inhibiting factors • Increase the risk of contamination. • Presence of serum in media may interfere with the purification and isolation of cell culture products
- 23. 23 BASIC COMPONENTS OF A COMPLETE MEDIA • Components vary among cell lines, and these differences are partly responsible for the extensive number of medium formulations • Each component performs a specific function: Buffering systems: Regulating pH is critical for optimum culture conditions and is generally achieved by one of the two buffering systems:
- 24. 24 Natural buffering system : • In a natural buffering system, gaseous CO2 balances with the CO3/HCO3 content of the culture medium. • Cultures with a natural buffering system need to be maintained in an air atmosphere with 5-10% CO2, usually maintained by an CO2 incubator • Natural buffering system is low cost and non-toxic.
- 25. 25 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid • Chemical buffering using a zwitterion, HEPES, has a superior buffering capacity in the pH range 7.2-7.4 and does not require a controlled gaseous atmosphere. • HEPES is relatively expensive and toxic at a higher concentration for some cell types. • HEPES has also been shown to greatly increase the sensitivity of media to phototoxic effects induced by exposure to fluorescent light.
- 26. 26 The essential amino acids are required by cultured cells, • Cysteine • Arginine • Glutamine and tyrosine • Other non essential amino acids are often added as well. • The concentration of amino acids usually limits the maximum cell concentration attainable, and the balance may influence cell survival and growth rate. Amino acids
- 27. 27 • Glutamine is required by most cells, although some cell lines will utilize glutamate. • Glutamine is also used by cultured cells as a source of energy and carbon. • Glutamine is unstable in culture medium with a half-life of between 3 and 5 days, and it generates ammonia, which can be toxic as a by-product. • Glutamax (Invitrogen) is an alanyl-glutamine dipeptide that is stable an bioavailable due to the action of dipeptidase Glutamine
- 28. 28 • Eagle’s MEM contains only the water-soluble vitamins excluding biotin. • Biotin is present in most of the more complex media, including the serum free recipes. • p aminobenzoic acid (PABA) is present in M199, CMRL 1066 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. • Amino acids, vitamin requirements have been derived empirically and often relate to the cell line originally used in their development. Vitamins
- 29. 29 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. • 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. • Calcium is reduced in suspension cultures in order to minimize cell aggregation and attachment.
- 30. 30 • Na+, K+, and Cl− regulate membrane potential. • SO4 2−, PO4 3−, and HCO3− have roles as anions required by the matrix and nutritional precursors for macromolecules, as well as regulators of intracellular charge. • The sodium bicarbonate concentration is determined by the concentration of CO2 in the gas phase and has a significant role in buffering capability.
- 31. 31 • 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 be oxidized to form CO2 and water. Glucose
- 32. 32 The most commonly used proteins are : 1) Albumin • Albumin binds to water, salts, free fatty acids, hormones, and vitamins, and transport them between tissues and cells. • Also remover of toxic substance from media. 2) Aprotinin • Aprotinin is a protective agent in cell culture systems, stable at neutral and acidic pH and resistant to high temperatures and degradation by proteolytic enzymes. • Inhibit proteases such as trypsin Proteins 3) Fibronectin • Fibronectin in cell attachment. 4) Transferrin
- 33. 33 Trace elements are often supplemented to serum-free media to replace those normally found in serum. • Trace elements like copper, zinc, selenium are needed in minute amounts for proper cell growth. • These micronutrients are essential for many biological processes, e.g. the maintenance of the functionality of enzymes. Trace elements
- 34. 34 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 Media supplements Antibiotics: • Antibiotics were originally introduced into culture media to reduce the frequency of contamination. • Antibiotics have a number of significant disadvantages: (1) They encourage the development of antibiotic-resistant organisms.
- 35. 35 (2) They hide the presence of low-level, cryptic contaminants that can become fully operative if the antibiotics are removed, the culture conditions change. (3) They may hide mycoplasma infections. (4) They have anti metabolic effects that can cross-react with mammalian cells.
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- 37. 37 • Most of the commercially available culture media include phenol red as a pH indicator, which allows constant monitoring of pH. • During the cell growth, the medium changes colour as pH is changed due to the metabolites released by the cells. • At low pH levels, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple. • Medium is bright red for pH 7.4, the optimum pH value for cell culture. Reagents – Phenol Red
- 38. 38 Disadvantages of using Phenol red : 1) Phenol red mimics the action of some steroid hormones, particularly estrogen .Thus it is advisable to use media without phenol red for studies using estrogen-sensitive cells like mammary tissue. 2) Presence of phenol red in some serum-free formulations interferes with the sodium potassium homeostasis. 3) Phenol red interferes with detection in flow cytometric studies.
- 39. 39 • The choice of cell culture media is extremely important, and significantly affects the success of cell culture experiments. • The selection of the media depends on the : Type of cells to be cultured The purpose of the culture Resources available in the laboratory • Different cell types have highly specific growth requirements, therefore, the most suitable media for each cell type must be determined experimentally. Eg : MEM for adherent cells and RPMI-1640 for suspension cells. Criteria for selecting Media
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- 41. 41 • Balanced salt solution (BSS) is composed of inorganic salts and may include sodium bicarbonate and, in some cases glucose. • HEPES buffer (5–20 mM) may be added to these solutions if necessary and the equivalent amount of NaCl omitted to maintain the correct osmolality. • BSS forms the basis of many complete media. • commercial suppliers will provide Eagle’s MEM with Hanks’s salts or Eagle’s MEM with Earle’s salts, indicating which BSS formulation was used. Balanced Salt Solution (BSS)
- 42. 42 • BSS is also used as a diluent for concentrates of amino acids and vitamins to make complete media. • As an isotonic wash or dissection medium, and for short incubations up to about 4 h (usually with glucose present). • Used to maintain pH and osmatic pressure of the medium. BSS Uses
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- 44. 44 • Eagle’s Minimum Essential Medium (EMEM) • Dulbecco’s Modified Eagle’s Medium (DMEM) • RPMI-1640 • Ham’s Nutrient Mixtures • DMEM/F12 • Iscove’s Modified Dulbecco’s Medium (IMDM) Common cell culture media
- 45. 45 1. Switch on the laminar flow cabinet 20 min prior to start working. 2. Swab all bottle tops & necks with 70% ethanol. 3. If working on the bench use a Bunsen flame. 4. Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame. 5. Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger. 6. Work either left to right or vice versa, so that all material goes to one side, once finished. Aseptic Conditions
- 46. 46 7. Clean up spills immediately & always leave the work place neat & tidy. 8. Never use the same media bottle for different cell lines. 9. If caps are dropped or bottles touched unconditionally touched, replace them with new ones. 10. Necks of glass bottles prefer heat at least for 60 sec. 11. Never use stock of materials during handling of cells.
- 47. 47 THANK YOU