Cell Culture All PPT PDF

BIOL3402 Cell Biology and Cell Technology Dr. W. Y. Lui (6 lectures) Office: Rm4N09 KBSB Email: [email protected] (use BIOL3402 as subject when email me) BIOL3402 Cell Biology and Cell Technology (WYL lectures) Learning outcomes: Through the studies, you should be a...

BIOL3402 Cell Biology and Cell Technology Dr. W. Y. Lui (6 lectures) Office: Rm4N09 KBSB Email: [email protected] (use BIOL3402 as subject when email me) BIOL3402 Cell Biology and Cell Technology (WYL lectures) Learning outcomes: Through the studies, you should be able to Describe and explain the media formulation Explain various cell dissociation and separation techniques Distinguish different types of cell culture (primary cell culture vs culture using cell line) Describe the characteristics of transformed cells Explain various methods for modification of cells in culture and the corresponding selection methods Explain how modification of cells in culture can help answering some scientific questions Describe the essential laboratory facilities for cell culture Describe the method of cryopreservation Why do we perform tissue/cell culture? 1. To study cell physiology Without HGF With HGF 2. To synthesize valuable cell product Gene cloning + ! Recombinant protein Cell culture  Metabolism Energy Food Cellular molecules (Polymeric molecules) (Small) Cellular macromolecules Cellular Cellular structure functions Energy (i) Enables organism to carry out cellular physiology (ii) Required for biosynthesis (iii) Activation of biomolecules (iv) Intermediate compounds of energy pathway are cellular molecules How to obtain energy? Intermediate products in cellular respiration serve as precursors for the synthesis of essential molecules Culture medium (1) Eagle’s Minimal Essential Medium (EMEM, MEM) Used for a wide variety of cell lines (e.g. HeLa cell) L-Amino Acid (mM) Vitamins (mM) Salts (mM) (i) Glucose Arginine 0.1 Biotin 10 -3 NaCl 100 -3 Cysteine 0.05 Choline 10 KCl 5 -3 Glutamine 2 Folic acid 10 NaH2PO4·H20 1 (ii) 13 Essential amino acids Histidine 0.05 Nicotinamide 10 -3 -3 NaHCO3 20 Isoleucine 0.2 Pantothenic acid 10 CaCl2 1 -3 Leucine 0.2 Pyridoxal 10 MgCl2 0.5 -3 (iii) Vitamins Lysine Methionine 0.2 0.05 Thiamine Riboflavin 10 10 -4 Phenylalanine 0.1 Threonine 0.2 (iv) Salts Tryptophan 0.02 Tyrosine 0.1 Valine 0.2 Add if needed Miscellaneous (v) Antibiotics Glucose 5 mM Penicillin* 0.005% Streptomycin* 0.005% (vi) Serum (e.g. FBS) Dialyzed horse serum 5% *To prevent microbial contamination Source: Science 1955 122:501 Culture medium (2) From MEM to Ham’s F12 (R.G. Ham, 1965) Satisfy the fastidious requirements of some cell lines Basic components of MEM plus the following: Glucose (i) Pyruvate Glycolysis (ii) 7 non-essential amino acids Pyruvate (iii) Trace elements (mM or nM) Krebs cycle (e.g. Fe++, Cu++) Oxidation (iv) Organic compounds phosphorylation (e.g. adenine, thymidine, hypoxanthine) Add if needed (v) Antibiotics (vi) Serum Culture medium Disadvantages of using serum-supplemented media: 1. Serum is chemically undefined and variation between batches, inconsistent results may occurs. 2. Serum is expensive and accounts for 70-80% of the cost of media. 3. Frequently contaminated by virus. (3) From serum-supplemented media to serum-free media Develop supplements of growth factors to replace serum Not all cell lines are able to grow in serum-free condition Culture medium Why use growth factors-supplemented serum-free media ? 1. To study endocrine regulation 2. To simply product purification 3. Consistency (Reproducible) Growth factors Polypeptides stimulate the growth of particular cell types Growth-promoting quality rather than its nutritional value Not take part in metabolic pathway Effective at very low concentration Common supplements for most cell types: (i) Insulin – glucose uptake and metabolism amino acid uptake (ii) Transferrin – iron-carrying protein (iii) Epidermal growth factor – mitogen for epithelial and fibroblastic cells (iv) Other growth factors (e.g. NGF, TGF) High conc of Fe2+ Steroid Low conc of Fe2+ No steroid Attachment factors/extracellular matrix Required by some cells to sandwiched between cell surface and the substratum (surface of the plastic culture dish) in serum-free condition Mimic in vivo situation Common attachment factors/extracellular matrix Source (i) Collagen Rat tendons (ii) Fibronectin Human blood plasma (iii) Laminin Mouse sarcoma (iv) Poly-lysine Synthetic Cell dissociation and separation techniques (1) Tissue culture Tissue Dissociation Cell culture Perfusion ‐ Prevent blood contamination ‐ e.g. Liver and kidney Dissociation Methods: (i) Mechanical ‐ e.g. Spleen and thymus (ii) Enzymatic ‐ Trypsin, collagenase or hyaluronidase Dissociation Removal of blood from tissue by perfusion Dissect and chop into small pieces A series of enzymatic digestion (depend on cell types) (i) Trypsin – non‐specific protease cleaves peptide bonds on the carboxyl side of lysine and arginine residues NH K A C K G R G COOH (i) Collagenase and hyaluronidase – digest extracellular matrix containing glycoprotein such as collagen and hyaluronic acid (iii) DNase – disperse DNA released from lysed cells Isolate a particular pool of cells Primary culture Isolation of testicular cells from the testis Cross‐section of testis Preparation of Sertoli cells for culture by enzymatic digestion (i) Testes isolated from rats (ii) Decapsulate testes (iii) Chop tubules into small pieces by scissors Wash and discard supernatant (x3) (iv) Incubate with 0.1% trypsin/0.002% DNase solution (30 min, 37 oC) Cent’ and discard supernatant (x1) (v) Incubate with 0.01% soy bean trypsin inhibitor/0.002% DNase in glycine/EDTA solution (10 min, Rm T) Wash and discard supernatant (x2) (vi) Incubate with 0.05% collagenase/0.0005% DNase solution (10 min, 37 oC) Cent’ and discard supernatant Preparation of Sertoli cells for culture by enzymatic digestion (vii) Incubate with 0.1% collagenase/0.0005% DNase solution (30 min, 37 oC) Wash and discard supernatant (x3) (viii) Incubate with 0.1% hyaluronidase/0.0005% DNase solution (30 min, 37 oC) (ix) Wash by centrifugation at least five times (x) Resuspend Sertoli cells in serum‐free F12/DMEM containing insulin, transferrin, EGF (xi) Check the purify of Sertoli cell culture Preparation of Germ cells for culture by Mechanical method (i) Testes isolated from rats (ii) Decapsulate testes (iii) Mince tubules into small pieces by scissors (iv) Centrifuge at 300 rpm (2 min) and collect supernatant (v) Repeat steps (iii) and (iv) to collect more supernatant nylon mesh (vi) Pool all supernatants and filter using Mira cloth (vii) Pool all supernatants and filter through 100 μm nylon mesh Preparation of Germ cells for culture by Mechanical Method (viii) Filter the resulting suspension through glass wool (Observe under the microscope) (ix) Collect filtrate and centrifuge at 1000 rpm (10 min) and discard supernatant (x) Resuspend the pellet with media and wash the pellet three times (xi) Filter the resulting suspension through 20 μm nylon mesh and centrifuge at 1000 rpm (10 min) (xii) Resuspend germ cells in in serum‐free F12/DMEM supplemented with insulin, transferrin, EGF, sodium lactate and sodium pyruvate Purify a pool of germ cells Spermatogonia Primary spermatocytes Secondary spermatocytes No spermatid  Tissue/organ (e.g. testis) Different separation methods (e.g. Enzymatic or Mechanical) Enzymatic Mechanical Sertoli cells Germ cells Cell Counting (i) Coulter Counter (ii) Hemocytometer Five cell types can be isolated from animal (1) Epithelial cells A A layer of cells cover the organs Polygonal and rectangular B (2) Fibroblasts Cells are spindle‐like shape on attachment to a solid surface (3) Muscle cells Myoblasts are capable of differentiation to form the myotubes Myotubes further differentiate to form muscle fiber (4) Nerve cells Highly differentiated and not divide in culture (5) Blood cells Culture in suspension e.g. lymphocytes From primary cell culture to cell lines (1) Primary cell culture Very few Established when the cells taken (2) Continuous cell line directly from animal tissue Freshly isolated cells in culture – primary cell culture Majority of cells die within several days or weeks (depend on cell types) A very very small cell population continues growing through many sub‐ culture Unlimited proliferation – immortalized – continuous cell line  Normal cells Transformation Continuous cell line (finite growth) (infinite growth) Transformation Cells lose their sensitivity to the stimuli associated with growth control How can cells be transformed or “immortalized”? 1. Spontaneous 2. Mutagens 3. Virus ‐ Infection by retrovirus express activated oncogenes (e.g. ras)  Transformation Normal cells Continuous cell line (finite growth) (infinite growth) Characteristics of transformed cells: 1. May lose anchorage dependence shaking shaking Anchorage dependence Anchorage independence 2. May lose contact inhibition features 3. Show chromosome fragmentation 4. Alteration from the normal diploid state (e.g. increase in chromosome number) 5. High capacity for growth Where to obtain cell lines? (i) The American Type Culture Collection (ATCC) (ii) The European Collection of Animal Cell Culture (ECACC) What kinds of services they provided? (i) Stock of commonly used cell lines such as CHO cells, MDCK cells (ii) Provide safe storage service of private cell lines (iii) Offer tests for contamination of cell lines (iv) Characterize cell lines using isoenzyme analysis and DNA fingerprinting Growth Parameter Several days later...................... Inoculate cells in growth Cells stop growing Cell line obtained (i) limited nutrient medium at a density of (ii) toxic metabolite accumulated from the culture (e.g. 104‐105 cell/cm2) collection (iii) lacking growth surface Sub‐culturing/passaging ‐ Trypsinize monolayer ‐ Dilute cells (1:2 to 1:10) in fresh media............. Passage number ‐ indicates the number of sub‐cultures performed since the cells were obtained Four phases of a culture (i) Lag Phase No apparent increase in cell concentration Synthesis of growth factors Duration varies Dependent on medium formulation and the initial concentration (ii) Growth/Log Phase An exponential increase in cell number N=N0∙2x N= final cell concentration N0= initial cell concentration X = generation numbers of cell growth Used to determine the doubling time of cell line Doubling time = Total time x The proportion of cells in cycle is high A cell inoculum of 105 cells/ml which increases to a density of 106 cells/ml in 3 days, how many generations have been passed through and what is the doubling time? Log10N=log10N0+ xlog102 Log10106=log10105 + xlog102 x = 3.32 Doubling time = Total time X Doubling time = 3 x 24 3.32 = 21.69 hour (iii) Stationary/Plateau Phase No further increase in cell concentration Cell growth=cell death Cell growth is limited (i) Depletion of nutrient Unable to support further cell growth (ii) Accumulation of toxic metabolic by‐product (iii) No free growth surface Cells stop growing when a single monolayer covers the available substratum (confluence) Cells are still metabolically active (iv) Decline Phase Cell death exceeds cell growth Viable cell concentration decreases Death is associated with the build‐up of toxic products Via two pathways: Apoptosis and necrosis Modification of cells in culture (i) Cell fusion (ii)Expression of cloned gene in cultured cells (transfection or viral transduction) (i)Cell Fusion Production of somatic cell hybrid by fusion of cells to form heterokaryons Combine their characteristics and form a hybrid cell with one fused nucleus Fusion techniques: (A) Fusogen such as polyethylene glycol (PEG) (B) Electrofusion Application: Fusion of antibody-producing B lymphocyte with myeloma cell line to produce antibody and chromosome mapping (i) Cell Fusion (A) Fusogen myeloma B cell Two cells (diploid each) Fusogens Membrane fusion Heterokaryon Heterokaryon Nuclear fusion with elimination of chromosome Nuclear fusion Hybrid cell (one fused nucleus) Mitosis Cell line Hybrid cell (B) Electrofusion Non-specific Apply current in low voltage to generate an electric field myeloma Two populations B cell of cells -ve +ve Followed by higher voltage with longer duration -ve +ve Hybrid cell Proliferate (B) Electrofusion Specific (biotin-avidin system) Crosslink myeloma cells and antigen with biotin and avidin respectively Biotin attached Avidin linked B cell to myeloma to antigen Antigen binds specifically to B cells Biotin binds to avidin with high affinity Two cells electrically fuse together to form hybrid cells Application of cell fusion techniques (a) Antibody production Normal antibody-producing lymphocytes have limited life span Myeloma have infinite growth capacity Desire property of Infinite growth antibody synthesis + capacity of of B-lymphocyte myeloma Fusion A mixed population of cells (lymphocytes, myelomas and hybridomas) HAT Selection Desire hybridomas Unlimited antibody production (b) Chromosome mapping Fusion of human and mouse cells to form hybrid cell Human chromosome are lost from the hybrid cell lines, usually only few chromosomes are retained A complement of hybrid cell lines retaining different human chromosomes are developed A DNA marker specific to a gene is used to perform PCR or hybridization, the chromosome carrying that particular gene can be identified (ii) Expression of cloned gene in cultured cells (A) Transfection (transfer of “naked” DNA) (A) Calcium phosphate method - DNA co-precipitate with Ca3(PO4)2 (B) DEAE-dextran - DNA complexed with the DEAE-dextran (C) Liposome - Liposome complex are formed positively charged lipid and DNA - DNA are encapsulated in liposome vesicle (D) Microinjection - Insert DNA by a microneedle into the nucleus of individual cell (E) Electroporation - Promote the uptake of DNA by electrical impulse (B) DNA transfer by viral transduction - Transfer DNA into mammalian cells : Extremely efficient Reasons: (i) They replicate to high copy number, mean that several copies of the required gene within each cell (ii) Some viruses integrate efficiently into the animal cell genome - Widely used viruses for infection : Adenoviruses Retroviruses Selection System Select the successful hybridomas or clones from the population Selectable markers - for hybridomas selection and stable transfection Marker genes (i) Thymidine kinase (TK) (ii) Hypoxanthine guanine phosphoribosyltransferase (HGPRT) (iii) Neomycin resistance gene (NeoR) Two pathways in the biosynthesis of nucleotides Folic acid dihydrofolate X Aminopterin de novo biosynthesis Glycine Tetrahydrofolate Aspartate dGTP DNA dTTP dATP IMP TMP Salvage pathway HGPRT TK Hypoxanthine Thymidine  Folic acid Selection System for hybridoma dihydrofolate X Aminopterin de novo biosynthesis Glycine Tetrahydrofolate B-lymphyocyte Aspartate Myeloma preselected preselected by by 8-azaguanine dGTP dATP DNA dTTP bromodeoxyuridine IMP TMP Salvage pathway HGPRT TK Hypoxanthine TK- B-lymphocytes HGPRT- myeloma Thymidine survive survive Fusion (A) TK marker gene: Bromodeoxyuridine A mixed population of cells TK+ Die (lymphocytes, myeloma and hybridoma) TK- Survive HAT Selection (B) HGPRT marker gene: 8-Azaguanine Hybridoma (TK+ and HGPRT+) survive HGPRT+ Die HGPRT- Survive Selection System Neomycin resistance gene (NeoR) NeoR gene codes for aminoglycoside phosphotransferase Presence of this enzyme confers resistance to aminoglycoside group of antibiotics such as G418 G418 prevents replication of mammalian cells Cells carrying NeoR gene after transfection are able to replicate in medium containing G418 and form cell clones Geneticin NeoR gene Phosphorylated G418 (G418) (Unharmful compound to cells) (Harmful to cells) X Transfection X X Expression vector containing desire protein and NeoR gene Selection medium Replicate containing G418 Cells do not contain NeoR gene X Death Expression vector containing desire protein and NeoR gene Stable transfection Clone the gene of interest into a vector containing NeoR Generate the “Kill curve” for a particular cell line to determine the dosage of G418 for selection Transfection Selection Subculture those clones that survive in medium containing G418 Check whether the gene is overexpressed in those clones by PCR and western blot Laboratory facilities (i) Water Source (ii) Glassware/Plastic ware (iii) Laminar Flow Hood (iv) CO2 incubator (v) Microscope (vi) Sterilization and Disinfection (a) Autoclaving (b) Filtration (c) Irradiation (d) Chemicals (vii) Culture contamination (a) from culturing techniques (b) Other sources: medium serum tissue or organ equipment (i) Water Source Water is the most critical constituent of all media and reagent Requires three‐ or four‐stage process Recycling Soft water First Stage: Second Stage: Third Stage: Fourth stage: Distillation Carbon High Grade Micropore or filtration deionization filtration reverse osmosis Softener Ultrapure water Hard water Media preparation RO system (stage 1) Milli‐Q H2O system (stages 2‐4) (ii) Glassware/Plastic ware In the past, glassware were used and recycled Washing and autoclaving are required Pre‐sterilized plastic ware (flasks, dishes and pipettes) suitable for cell culture – convenience and reduce contamination The plastic is sulphonated, the surface suitable for cell attachment (iii) Laminar Flow Hood (a) Horizontal Flow Hood Air filtered through High‐Efficiency Particulate Air (HEPA) filter Flows out from the hood Provides an isolated area Use if the culture is safe and not infectious Not suitable to handle hazardous materials such as primate cell lines This design cannot protect the operator (iii) Laminar Flow Hood (b) Vertical Flow Hood A vertical flow of air is drawn into the working area through a HEPA filter 70‐80% of the air is re‐circulated to form an air curtain to maintain sterile operation and protect the operator The most common used cabinet: Class II cabinet Suitable for work with moderate toxic and infectious agents Class III cabinet: design to handle high‐risk pathogens (iv) Water jacketed CO2 Incubator Maintains the optimal temperature and pH for cell growth Water jacket provides a constant temperature environment Carbon dioxide supplied from a gas cylinder Bicarbonate‐CO2 buffering system ‐ + H2O + CO2 HCO3 + H The level of CO2 in gas phase of the incubator is 5‐10% (v) Microscope Routine checking to determine if there are any significant morphological changes (A) Standard binocular microscope Requires the specimen placed on a glass slide and covered by a cover slip (B) Inverted microscope Having the light source on the top, enable large object to be placed on the stage for viewing Binocular microscope Inverted microscope (vi) Sterilization and Disinfection Kill and remove micro‐organisms (a) Autoclaving Steam heat and dry heat Steam heat: 121oC, at 1 kg/cm2 (15 psi) for 20‐30 min Dry heat: 180oC for 20 min (b) Filtration Cellular ester filters (0.2 μm is considered‐the standard for removing bacteria and fungi)  4 5 3 (c) Irradiation 6 2 Induce covalent bonds (i) Ultraviolet light (UV) 1 ‐ Inactivate micro‐organisms and virus P P P ‐ Acts on nucleic acid UV ‐ Poor penetrating power P P (ii) Gamma rays P Break phosphodiester bonds ‐ Not actually performed in the laboratory ‐ Important method of sterilization XP XP XP ‐ Good penetrating power ‐ Acts on nucleic acids and produce free radicals that kill bacteria ‐ Commonly used with items that cannot be heat‐sterilized ‐ Pre‐sterilized disposable plastic wares are exposed to γ‐rays 4 5 Induce covalent bond 3 4 2 1 6 Gamma radiation 1 P P P P P (d) Chemicals (i) Gases ‐ Ethylene oxide and formaldehyde gas ‐ Effective against all types of micro‐organisms including virus ‐ Acts on nucleic acid and protein components of the micro‐organism ‐ Ethylene oxide is used for sterilizing items, particularly in hospital ‐ Formaldehyde gas is used to decontaminate or sterilize laminar flow cabinet ‐ Both ethylene oxide and formaldehyde gas are very toxic (ii) Solutions (Disinfection) ‐ Alcohol ‐ Optimal at a concentration of 70% (vii) Culture contamination (a) From culturing techniques (b) Other sources: medium serum tissue or organ equipment (i) Bacterial (ii) Mycoplasma (iii) Fungi (iv) Virus Cryopreservation and freezing and thawing of cells (i) Storage at low temperature (ii) Physical changes during freezing (iii) Cryo‐injuries (iv) Procedure to freeze and thaw cells (i) Storage at low temperature ‐196 oC Liquid nitrogen ‐120‐130 oC Liquid nitrogen vapor ‐70 oC Dry‐ice, deep freezer (Unstable) How to achieve ‐196 oC and recover to 37oC? (Freezing and thawing) Moderate cooling 1‐2 oC/min Rapid warming Critical temperature range in cooling ‐5 oC to ‐40oC (ii) Physical changes during freezing < ‐10 oC Dehydration Slow Rapid ‐2 oC Formation of ‐5 oC intracellular ice crystal  (iii) Cryoprotectants ‐ Small molecules are permeable to cells ‐ Glycerol and DMSO ‐ Delay intracellular freezing ‐ Increase extracellular fluid volume during extracellular ice formation (iv) Cryo‐injuries due to low thawing rate (a) Slow warming – recrystallization of intracellular ice (b) Some cryoprotectants are toxic to cells at warm temperature (iv) Procedure to freeze and thaw cells Resuspend cells Freezing: in freezing medium (e.g.medium with DMSO)....... 400 x g, 5‐10 min Aliquot into freezing vials Lid ‐70 oC freezer or Liquid Nitrogen overnight Tank (~ ‐1 oC/min) Freezing container containing isopropanol  (iv) Procedure to freeze and thaw cells Thawing: on i gat ifu ntr Prewarm Prewarmed.... Ce medium to 37oC medium.......... Discard medium containing DMSO and resuspend cells in fresh medium Pick a frozen Thaw the vial at vial from liquid 37 oC water bath........ N2 Tank Seed cells in culture dish