Cells Culture medium composition

Transcription

1 Cells Culture medium composition

2 Cell Culture Media When a cell is removed from its original tissue and placed in culture, the medium must provide all the environmental conditions (i.e., nutritional, hormonal, stromal and physiological conditions) that the cell has been exposed to in vivo.

3 Media Development Initial Media Were Body Fluids, Lymph, Serum, Chick Embryo Extract Need For Larger Amounts Gave Rise to Chemically Defined Media Eagle s Basal Medium (1955) Followed by MEM (Minimum Essential Medium) (1959) Horse Serum, Calf Serum or Human Serum Supplementation Common Media: MEM, DMEM, RPMI 1640 (hematopoietic cells) Serum-free is Preferred (minimize infectious agents)

4 What do cells need to grow? What do cells need to grow? Substrate or liquid (cell culture flask or scaffold material) chemically modified plastic or coated with ECM proteins suspension culture Nutrients (culture media) Environment (CO2, temperature 37 o C, humidity) Oxygen tension maintained at atmospheric but can be varied Sterility (aseptic technique, antibiotics and antimycotics) Mycoplasma tested

5 Physiological Properties of Culture medium 1) ph 2) CO2 and Bicarbonate 3) Osmolality 4) Oxygen 5) Buffering 6) Temperature

6 ph Control ph can affect Cell metabolism Growth rate Protein synthesis Availability of nutrients ph - For Most Cells ph= (while transformed cells may be better at ph , epidermal cells could grown at ph Phenol Red is Used as ph Indicator - Purple at ph>7.8, ph<6.5 yellow (see Fig. 1) Figure 1. Phenol red is commonly used for visible detection off ph in the medium - ph of the medium is dependent on the delicate balance of dissolved carbon dioxide (CO2) and bicarbonate (HCO3), changes in the atmospheric CO2 can alter ph of the medium - necessary to use exogenous CO2 when using medium buffered with CO2-bicarbonate basal buffer

7 Buffering There are two buffering systems: 1) a natural buffering system where gases CO2 balanced with the CO2/HCO3 - content of the culture medium. 2) Chemical buffering using HEPES Ad1. needs to maintain in an atmosphere of 5-10% CO2. Bicarbonate is low cost, nontoxic and also provide other benefits for cells. Ad2. has superior capacity at ph , expensive, and can be toxic at higher concentrations. Do not required gasses atmosphere.

8 Buffering Bicarbonate Buffer Most Common Cheap Nutritional benefit Non-toxic HEPES Buffer Stronger Expensive Toxic At High Concentrations Why Use CO2? Why Not Use HEPES? Turns out HCO3 - and CO2 Help Growth

9 CO2 and Bicarbonate CO2 (carbon dioxide) in the gas phase dissolved in the medium, establishes equilibrium with HCO3 - ions, and lowers the ph. Concentration of carbon dioxide (CO2), bicarbonate and ph are interrelated. To Compensate Na HCO3 is Added (4mM). Lack of Dissolved CO2 and HCO3 - Limits Cell Growth NaHCO3 needs to be in Equilibrium With Dissolved CO2 The Higher the CO2, The More HCO3 - Needed Sodium Pyruvate Yields Endogenous CO2

10 Oxygen Cultured Cells consist Mainly on Glycolysis (culture cell is more anaerobic when compare to in vivo cells and the depth of culture medium influence the rate of O2 diffusion) Atmospheric O2 Is More Than Adequate Organ Cultures Require High (95%) Dispersed Cultures Require Low Oxygen Tension Cultured cells mostly rely on the dissolved O2 in the medium which may be toxic at high concentration due to the generation of free radicals. Mercaptoethanol Used To Neutralise Oxygen Radicals, Adequate quantities of O2 must be supplied so that the cellular requirements are met avoiding toxic affects. Addition of selenium to the medium is also advocated to reduce O2 toxicity (selenium is a cofactor for synthesis of glutathione)

11 Osmolality Measured by Depression of Freezing Point or elevation of the vapour pressure of the medium Put Cells in Distilled Water, They explode Put Cells in Salty Water, They Shrink Measuring of osmolality is necessary when you prepare medium from powder. Human plasma=290 mosmol/kg, osmolality of culture medium mosmol/kg Acceptable Once an osmolality is selected for a medium it must be maintained at the level (+/- 10 mosm/kg) Osmolality Is Primarily From Inorganic Salts, Amino Acids and Vitamins When added an additional acids, bases, drugs etc. to the medium osmolality should be re-controlled

12 Temperature and Humidity Typically temperature is 37 C for warm-blooded animal cells Cell cultures from birds C, cold blooded animals C Cells Tolerate Lower Temp (35 C) Cells Can Survive For -196 C and can survive several days at 4 C Do Not Tolerate High Temp (39.5 C), Few Hours and They Die Temperature changes directly influencing growth of cells and affect the ph due to the increased solubility of CO2. Humidity must be maintained at saturating level as evaporation can lead to changes in - Osmolarity - Volume of media and additives

13 Balance Salt Solution (BSS) The basis for the cell culture media was the balanced salt solution (BSS) - used to create the physiological ph and osmolality required to maintain the cell in vitro Solution Composed of Inorganic Salts, ex. KCl, CaCl2, NaCl Earle s vs Hanks Earle s good for CO2 Hanks good for sealed flasks and atmospheric air as gas phase HEPES Buffer Can Be Added, Expensive (!) If HEPES Added, NaCl Omitted to Maintain Osmolality BSS Used For Tissue Dissaggregation BSS Rely on Phosphate for Buffering, Relatively Weak Buffering Capacity

14 Balanced salt Solutions (BSS) Biological Buffers Known as physiological or balanced salt solution (BSS). BSS forms the basis of many complete media, and commercial suppliers will provide major culture mediums. BSS are used for washing tissue and cells and usually combined with other agents to tread the tissue and cells The function of this salt solution in the media are to - maintain proper ph ( ) - May contain Sodium bicarbonate - required atmospheric CO2 - HEPES - maintain ideal osmotic pressure ( mosm) - provide a source of energy, e.g., glucose

15 Cell culture environment (in vitro) Basal Media Maintain ph and osmolarity ( mosm/l). Provide nutrients and energy source. Components of Basal Media Inorganic Salts Major components to maintain osmolarity The most common ions are: Ca 2+,Na +, K +,Cl, HCO 3 - Ions for cell attachment and enzyme cofactors Most media deliver most of the salts form BBS Ca 2+ required by some cells for adhesion (cadherin), also plays role in signal transduction Na +, K +,Cl regulate membrane potential Calcium is reduced in suspension culture to minimise cell aggregation and attachment HCO 3 - plays crucial role in medium buffering

16 Cell culture environment (in vitro) Other Components of Basal Media Amino Acids (L-glutamine) Essential amino acid (not synthesised by the cell) Energy source (citric acid cycle), used in protein synthesis Unstable in liquid media - added as a supplement All used an a source of energy and carbon Other arginine, tyrosine - individual for different cell types Vitamins Precursors for numerous co-factors B group vitamins necessary for cell growth and proliferation Common vitamins found in basal media is riboflavin, thiamine and biotin MEM medium contain only the water-soluble vitamins (B group, plus choline, folic acid, exclude biotin ) Other derived from serum

17 Cell culture environment (in vitro) Other Components of Basal Media Non-essential amino acids (NEAA) Usually added to basic media compositions Energy source, used in protein synthesis May reduce metabolic burden on cells Growth Factors and Hormones (e.g.: insulin) Stimulate glucose transport and utilisation Uptake of amino acids Maintenance of differentiation Glucose Included In Most Media Energy Source Metabolized into pyruvate May! Enter CAC (citric acid cycle) This Explains Need For Glutamine/glutamate For Carbon and Energy Needs

18 Cell culture environment (in vitro) Components of Basal Media Keto acids (oxalacetate and pyruvate) Intermediate in Glycolysis/Krebs cycle Keto acids added to the media as additional energy source Maintain maximum cell metabolism Carbohydrates Energy source Glucose and galactose Low (1 g/l) and high (4.5 g/l) concentrations of sugars in basal media Some media contain also maltose and fructose

19 Antibiotics Reduce the risk of bacterial and fungal contamination Antibiotics Should Be Avoided Mask cryptic contaminations (mycoplasma) Can generate antibiotic resistant bacteria Good aseptic technique should be sufficient Cells can become antibiotic resistant changing phenotype Several Antibiotics/Anti-mycotics Ampicilin, Gentamycin, Amphotericin B, Tetracyclin Primary Cultures-Use Antibiotics Preferably avoided in long term culture

20 Serum Several Types of Serum FCS - foetal calf serum Horse Serum Human Serum (has to be tested for viruses like HIV or hepatitis B) with some human cell line FBS - fatal bovine serum Anti-trypsin Activity Serum Contain: - minerals - Lipids - Hormone - Proteins (Albumins Major Component Of Serum) - Growth Factors Promote Cell Proliferation and growth ex. IGF-I, Insulin, FGF, EGF, VEGF, IL-1, IL-6

21 Serum Among the biological fluids that have proved successful for culturing cells, serum is the most popular. Generally, 5-20% serum is usually needed for optimal cell growth in culture. Some of the major functions of serum are to provide: - Basic nutrients (both in solution and bound to proteins) - Hormones and growth factors, e.g., insulin - Attachment and spreading factors, e.g., fibronectin - Binding proteins (albumin, transferrin) for carrying hormones, vitamins, lipids, etc. - Non-specific protection factors against mechanical damage provide viscosity to reduce shear forces during agitation of cell suspension - Protease inhibitors - ph buffering

22 Hormones, minerals and other Hormones Insulin - promote uptake of glucose and amino acids Hydrocortisone (particularly foetal bovine serum) - promote cell attachment and cell proliferation some case can induce cell differentiation Minerals Iron, Zinc, copper, selenium and tricarboxylic acid intermediates Inhibitors Bacterial toxin or antibodies Physiological negative growth regulators - TGF-beta Heat Inactivation (56 o C for 30 mins) why? - Destruction of complement and immunoglobulins - Destruction of some viruses (also gamma irradiated serum)

23 Proteins in the serum The more presence is albumin which may be important as a carrier of lipids, minerals and globulins Fibronectin - promote cells attachments alfa-macroglobulin - inhibit trypsin Fetuin (fetal serum) enhances cell attachment Transferrin - bind iron, making less toxic and bioavailable Moreover, proteins in serum may reduce shear stress during the pipetting and stirring, and medium buffering capacity.

24 Disadvantages of using serum - For most of the cells, serum is not the physiological fluid that they come in contact with in the original tissue except during wound healing and blood clotting. - Serum may contain many unknown components that we do not how effect on our culture (amino acids, sugars, growth factors, peptides ) - Some growth inhibitors (like PDGF- platelet derived growth factor) may inhibit or promote growth/proliferation - Serum can sometimes be cytotoxic. It can contain bacterial toxins and other inhibitors. - Batch-to-batch variations in serum can necessitate time consuming and costly serum screening. - Quality control of new batch (time and money consuming) - Availability - economical, political, low restriction - Serum provides a risk of contamination (viruses) - Sterilisation problems associated with filtration of colloids and particulate content - Cost

25 Advantages of Serum-Free medium - Improved reproducibility between cultures and avoidance of batch-to-batch variations - Standardization of media formulations among different laboratories - Improved economy - Easier purification of culture products - Less protein interference in bioassays - Avoidance of serum cytotoxity - No serum proteases to degrade sensitive proteins - Selective culture of differentiated and functional cell types from heterogeneous populations of primary cultures

26 Replacement of serum Adhesion factors : necessary to treat surface with fibronectin, poly-l-lysin or added laminin directly to the medium Protease inhibitor: soya bean trypsin inhibitor or aprotinin to block trypsin activity or wash cells by centrifugation Replace trypsin with with recombinant trypsin which is less toxic and aggressive to the cells cultured without serum Hormones Growth factors Minerals Proteins

27 Culture medium cannot be autoclaved It is filtered through 0.2u membrane filters

28 How to used correct medium Check Literature 75% of Media Sales is RPMI 1640, DMED and MEM DMEM is Basically MEM With Higher Amino Acid (2x) Vitamins (4x) HCO3 - and CO2 (10%) To Improve Buffering If No Info Available, Do Simple Growth Expteriments

29 Fibroblast Culture Medium: - Dulbecco s Modified Eagle Medium (DMEM) - supplemented with 10% Fetal Bovine Serum (FBS; Invitrogen,), - 2mM L-glutamine - and 0.1mM (0.7µl/100ml final media volume) 2-mercaptoethanol - Antibiotics can also be added at final concentrations of 50 units/ml penicillin and 50 g/ml streptomycin. Fibroblast Culture Medium is filter sterilized, stored at 4 C, and used for up to 2 weeks.

30 Mouse embryo fibroblast (MEF) medium - Dulbecco s modified Eagle medium (DMEM) with high glucose (4500 mg/liter), - 10% (v/v) FBS, heat inactivated for 30 min at 56 C - nonessential amino acids (from 100 stock; Life Technologies) - 2 mm L-glutamine (from 100 stock; Life Technologies) mm 2-mercaptoethanol - 20 mm HEPES, ph penicillin/streptomycin when indicated (from 100 stock; Life Technologies) - Store up to 2 weeks at 4 C

31 How to prepare culture medium??

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