Transcript 無投影片標題
Cell Culture Environment How to grow a plant ? Water Soil Air (CO2 and O2 ) Nutrition Water : Culture medium Soil : substrate for cell growth Air (CO2 and O2 ) : Air in incubation chamber Nutrition : growth supplement Culture Medium Functions of culture medium: 1. Maintain the pH 2. Maintain the osmolarity 3. Provide nutrient 4. Provide energy Basic components of cell culture environment The in vitro environment must meet the fundamental physiological requirements of the cell 1. Culture medium 2 . Physiological factors pH 7.27.5 osmolarity 280320 mOsmol/kg CO2 25% in air temperature 35oC-37oC 3. Stationary versus dynamic media supply enough replenish at proper time vol. 0.20.3ml/cm2 A complete culture media: 1. A basal medium specifies for all cellular requirements 2. A set of components that specify other cellular requirements and permit growth of cells in the basal medium Basic Energy source for cell growth Carbohydrate sugar starch Basal Medium Types of basal medium 1.Eagles medium and derivative e.g. BME, EMEM, AMEM, GMEM, JMEM 2. Media designed at Roswell Park Memorial Institute ( RPMI) e.g.RPMI 1629, RPMI 1630, RPMI1640 3. Basal medium designed for use after serum supplement e.g. Fischer’s Lieboutz, Trowell, Will 4.Basal medium designed for serum-free formulation e.g. CMRL 1060 Ham’s F10 and derivatives TC199 and derivatives NCTC Waymouth 5. For insects culture e.g. Grace’s medium Schneider’s medium Mitsuhashi and maramerosch medium IPL-41 medium Chiu and black medium D-22 medium What does cells need to grow better? Basic components of the culture medium 1.Buffer system ß - glycerophosphate organic components e.g. HEPES, Tes, Bes 2. Energy Source glucose, maltose, sucrose, fructose, galactose, mannose 3. Amino Acid essential amino acid 4. Vitamins precursor for the cofactors e.g. biotin, choline, folic acid, nicotinic acid… 5 . Hormones and growth factor e.g. insulin, hydrocortisone, NGF, EGF 6. Proteins and Polypeptide supplement with: fetuin, -globulin, fibronectin, albumin, transferrin…. 7. Fatty acid and Lipid 8. Accessory e.g. Zn,Cu,Se…. 10. Antibiotics factors to be considered: absence of cytoxisity broad anti-microbial spectrum accepted cost minimum tendency to induce formation of resistant micro- organism e.g. penicillin, streptomycin gentamycin. Buffer System Bicarbonate buffer Recommended CO2 concentration and gas phase to use with common basal media basal medium NaHCO3 concentration Eagle’s MEM(Hank's salt) grace’s (Hank salt ) IPL-41 (Hank salt ) TC100 (Hank salt ) Schneider’s (Hank salt ) IMDM TC199 DMEM/Ham’sF12 RPMI1640 Ham’sF12 DMEM 4 4 4 4 4 36 26 29 24 14 44 % of CO2 gas phase atmospheric atmospheric atmospheric atmospheric 5 5 5 5 5 10 Choice of basal medium from literature e.g. BME: for HeLa, L-cells, BHK, primary culture of human rodent and avian fibroblast e.g. RPMI: for human haemopoietic cells e.g. Iscove’s modified Dulbecco’s medium( IMDM): for haemopoietic origin, growth and differentiation of human and murine primary bone marrow culture e.g. sf9,sf21: for Drosophilla and insect culture Preparation of basal medium Factors to be considered: Avoid contamination Use pure water Use analytical chemicals Glassware used must be cleaned Equipment for preparation of media: High purity chemicals and biologicals Good analytical balance Hot plate with magnetic stirrer Volumetric flasks of various volume pH meter Osmometer Autoclave and membrane filtrate medium kept at 4oC storage: storage should not be over 3 months Serum Why use serum ? Advantages of using serum 1. Serum represent a cocktail of most of the factors 2. Required for cell proliferation and maintenance. 3. An universal growth supplement which is effective with most cells. 4. Serum buffers the cell culture system against a variety of perturbation Types of serum 1. FBS ( Fetal Bovine serum) from abattoir 2. Horse serum, calf serum from donor ) should pass virus test) Constituents of serum 1.Growth factors ; 5—30 kDa polypeptide 2.Albumin a. a carrier protein b. carry thyroxin and metal c. pH buffering 3. Transferrin-ion transport protein 4. Anti-protease: prevent cell from proteolytic damage 1-trypsin b2-macroglobulin Nutritional and protective factors which may be supplied by serum 1.Specific growth factors-EGF,PDGF,IGF,FGF, IL-1,IL-6 2.Trace elements-Iron, Zinc, Selenium ( Co,Cu,I,Mn,Mo,Cr,Ni,V,As,Si,F,Sn) 3. Lipids-- cholesterol, linoleic acid, steroids 4. Polyamines--putrescine, ornithin, spermidine 5. Attachment factors-- fibronectin, fetuin, Laminin 6. Mechanical protection– albumin 7. Buffering capacity– albumin 8. Neutralization of toxic factors– albumin 9. Transport of metals transferrin/Fe+3, ceruloplasmin/Cu+2 10.Protease inhibitors– antitrypsin,b1macroglobulin Potential problems with the use of serum 1. Lack of reproducibility Serum batches varies considerably The presence of specific antibodies may also affect the results Serum may vary during the process of collection Sterity 2. Risk of contamination To prevent viral infection: b-propiolactone -irradiation heat inactivation( 56oC, 1hr) 3. Availability and cost 4. Influence of downstream process difficulties in purifying proteins serum protein 4—8 mg/ml, recombinant protein---- 0.1ug/ml difficulties in purifying monoclone antibody Sourcing and selection of serum sourcing: dealing with reputable supplier selection: test the capability for growth on cheaper serum type test different batches of serum for growth performance test of cell growth from very low seeding test of cloning efficiency Serum storage and use kept frozen, -20oC thaw rapidly, mix gently kept at 4oC once thawed Replacement of serum in medium Replace by Controlled Process Serum Replacement ( CPSR) CPSR: derived from bovine plasma, lower endotoxin lower protein Replace by supplement or fortified serum fortified serum: supplement with mitogen, growth factor, hormones, proteins, protein stabilizer, trace elements Serum Free medium A properly designed serum free medium is reproducible is not reliant on economics of the world cattle market simplifies down stream purification has no unknown factors e.g. viruses, growth inhibitors The most common requirement of serum free medium e.g. polypeptide hormone, insulin, iron transport protein, transferrin Supplement of serum free medium e.g. steroid growth factors, trace elements reducing agents, diamines, vitamins albumin complexes with unsaturated fatty acid Design of serum free media Reduce serum gradually Adding other components Growth assay study in the presence of added supplements for basal medium Add components singly or in combination to a basal medium in a step wise manner Mechanical stabilizers and adhesion factors For suspension culture: To prevent shear damage: damage by air bubble, stirrer, shaker… improved by increase viscosity of the medium by adding: carboxy methyl cellulose ( viscosity modifier) and prolyvinylpyrrolidone( PVP), pluronic-F68 principles: formation of an interfacial structure of adsorbed molecule on cell surface) Considerations : Energy source of basal medium Carrier of lipids Proper environment for metabolism Growth function of cells ( good buffering capacity) Good maintenance of electrolyte balance Fe+2---Zn+2----Cu+2 trace elements: Na+,K+, Ca+2,Mg+2,Cl-,HPO4-2….. For anchorage dependent cells substrate treat with adhesive glycoprotein, fibronectin, laminin, chondrotin, serum spreading factors Difficulties that may encountered with serum free medium a. Design of a dedicated medium for each cell type b. Culture condition become more critical c. Serum free medium has a reduced capacity to inactivate or absorb toxic materials Selection of components e.g. transport protein,stabilizing proteins growth regulators, growth factors, attachment proteins, crude extracts, essential nutrient Practical hints on solubilizing specific components e.g. riboflavin, folic acid, tyrosine, cystein dissolve in NaOH e.g. fatty acid, lipids, fat soluble vitamins in alcohol solutions e.g. pluoronic F-68 soluble in cold water e.g. hypoxanthine heat to dissolve