Cell and Tissue Culture Lecture 1 PDF

CELL AND TISSUE CULTURE Prepared by: Christian Joseph N. Ong, MSc, LPT Department of Biology College of Science Bulacan State University 1 Outline Introduction : Historical Perspective What is cell culture?...

CELL AND TISSUE CULTURE Prepared by: Christian Joseph N. Ong, MSc, LPT Department of Biology College of Science Bulacan State University 1 Outline Introduction : Historical Perspective What is cell culture? Basic Requirements of Cell Culture Types of Tissue Culture Advantages of cell culture Problems and Limitations Applications of Cell Culture 2 3 Cell culture Removal of cells from an organism (plant or animal) and their subsequent growth in a favorable (controlled) artificial environment Cells may be removed directly and disaggregated (if in one piece is an aggregate of cells) by mechanical and/or enzymatic means before culturing May be derived from a cell line that has already been immortalized 4 5  Sydney Ringer Ross Granville Harrison Wilhelm Roux (1835-1910) (1879-1959) (1850-1924) Developed salt Extracted neural tube 1885, excised the solution to support and lymph fragments medullary plate of an and maintain the from frog embryos and embryonic chicken beating of the observed the develop- and maintained it in a animal heart ment of the nerve fibres warm saline solution. separated from its on a glass slide body Schiff, Judith Ann. (2002) 6 Cell Culture in Vitro: A Brief History 1912 Alexis Carrel cultured connective tissue and showed heart muscle tissue contractility for 2-3 months 1943 Earl et al., produced a rat cell line 1962 Buonassisi et. al published methods for maintaining differentiated cells of tumor origin 1970 Gordon Sato et al., published the specific growth factor and media requirements for many cell types 1979 Bottenstein and Sato defined a serum free medium for neural cells 1980 to now Tissue has become less of an experimental research field but more of widely accepted research tool 7 Historical background ▪ 1951 Salk cultured monkey kidney cells and used these cells to grow viruses. ▪ Cultured three types of polioviruses. ▪ 1953 announced that he developed an inactivated polio vaccine ▪ The vaccine is now on the WHO list of Jonas Salk and his polio essential medicines. vaccine 8 Cell Culture in Vitro: A Brief History 1943 W.R. Earle established L cell mouse fibroblast cell line, the first continuous cell line 1951 George Otto Gey developed the HeLa cells, the first cancer cell line, from cervical cancer (Helen Lacks, is the patient of surgeon Howard Jones, from whom the malignant cervical cancer tissue came from) 1952 Dulbecco first discovered the use of trypsin for generation of replicate subcultures 1955 Eagle developed a defined media (Eagle’s medium) 1961 Sorieul and Ephrussi discovered cell dusion for somatic cell hybridization 9 A drop of liquid (lymph) with specimen is hanging upside down from the underside of a cover slip https://microbiologynote.com/hanging-drop-method 10 Significant landmarks 1885 Present Materials to be cultured Culture media and supplements Culture wares and equipment Culture methods Methods for analysis 11 History of Cell Culture: The Corning Story 12 Major developments in Cell Culture Technology 1. Use of anti-biotics and fungicides in in vitro cell cultures 2. Use of trypsin to remove adherent cells to further sub-culturing. The formulation of chemically defined media 13 14 Basics of cell and tissue culture (1) Culture and media ingredients (2) Culture Wares (3) Proper Work Environment (4) Tissue to be cultured 15 (1) Culture Media and Media Ingredients Nutrients of the cell: amino acids, vitamins etc... Cell should be grown/maintained in aqueous medium Different media are used to suit the need of the sample Regular changes of culture medium to sustain growth of cells 16 Factors to be considered in choosing a culture media Solubility of materials Tyrosine is soluble only in acidic solution Lipids in alcoholic solution Compatibility of components Purity of materials Chemical instability Storage condition (e.g. culture media with glutamine has a shorter life span) 17 Natural media for culturing cells 1. Plasma 2. Collagen 3. Biological Fluids Serum (allow blood to coagulate) Amniotic fluid Aqueous humor Coconut water (supplement in cultures of plant tissues); autoclaved and filtered 18 Blood Plasma accounts for 55% of the blood’s total volume Made up of : 91.5% water, 7% proteins and 1.5% other components (electrolytes, vitamins, hormones) * Use of anti-coagulants in cell culture (blood) 19 Collagen An integral part of the framework that holds cells and tissues together A ubiquitous protein found mostly in every tissue Most abundant in dermis, tendon and bone A useful extracellular matrix for improving cell culture Exert effects on the adhesion, morphology, growth, migration and differentiation of a variety of cell types 20 Blood Serum Plasma without the fibrinogen Serum includes all proteins not used in blood clotting (coagulation) all the electrolytes, antibodies, hormones, and any exogenous substances Serum is an essential factor for the self-renewal of embryonic stem cells in combination with the cytokine leukemia inhibitory factor (LIF) 21 Serum Blood allowed to clot (removal of clot) Important in growth of cells > 1,000 different components in serum Proteins, electrolytes, lipids, carbohydrates, hormones, enzymes. Growth factors, nutrients needed for proliferation and differentiation etc... 22 Serum components Albumin, primary protein in serum Functions as carrier of small molecules Carries negative charge: binds readily to salts (Na, K etc.. ) Hormones , vitamins and free fatty acids Contributes to the overall mitogenic property of serum by binding growth promoting components and presenting them to the cell at active and mitogenic levels Alpha1, alpha2: contain major protease inhibitors Growth factors: EGF, FGF, IGF, PDGR 23 Difference between serum and plasma micriobiologyinfo.com 24 Fetal Bovine Serum (FBS) Produced by ISO9001 facility; Origin should be is EDQM certified Derived from clotted whole blood, collected from the foetus via cardiac puncture under refrigerated condition. Blood is immediately centrifuged; serum is frozen and transferred to processing facilities 25 FBS On receipt at the processing plant, the serum is thawed, tested for acceptability, pooled and passed through three 100nm (0.1um) sterilizing filters Serum is bottled through an aseptic filling process Quality Control procedure to ensure the serum meets raw material specifications before it is further processed FBS must be free of bovine spongiform encephalopathy (BSE), food and mouth disease and other highly infectious disease 26 Production of serum Each fetus will yield between 0.2 to 0.5 liter of blood, half of which will be serum 3 months fetus yields about 150 ml of raw FBS, at 6 months 350 ml and at 9 months (near- term) 550 ml An expensive component of the culture requirement for cell culture 27 High levels of serum proteins is not good for cell culture Serum proteins can negatively influence cell culture environment Fibroblasts or other unwanted cells may overgrow in the culture 10% serum may have 5 mg/ml serum proteins Disadvantages of high serum proteins in cell culture (e.g. unwanted effects of stimulation or inhibition of growth and cellular function in some cell cultures) 28 Fetal Bovine serum contains growth factors which are necessary for the growth of cells in vitro 29 Classification of Culture Media Essential for immediate survival Essential for prolonged survival Essential for indefinite growth Essential for specialized functions 30 Types of culture media Natural culture media: those that come from from tissue extraction or animal body fluids, such as plasma, lymph, and serum Synthetic culture media 31 Natural Medium: Amniotic Fluid (AF) Structural matrix protein fibronectin Use in pre-natal diagnosis for many decades Enzymatic proteins (inborn errors of metabolism) Subset of cells in AF capable of maintaining prolonged and undifferentiated proliferation as well as capable of differentiating into multiple tissue type. 32 Natural Medium: Coconut water Ability to promote growth Mostly used in plant tissue cultures Contains sugars, sugar alcohols, lipids, nitrogenous compounds, organic acids, various enzymes Auxins and cytokinins (in plants) 33 Synthetic Culture Media Commercially available Liquid or powder Prepared under Sterile conditions Commonly used media: Roswell Park Memorial Institute (RPMI 1640) Minimum Essential Medium (MEM) Dulbecco Modified Essential Medium (DMEM) 34 RPMI 1640 Media Components 23 Amino acids : L – Alanine, L-Arginine, L-Glutamine L- Asparagine, L-Aspartic Acid, L-Cystine 14 Vitamins: Ascorbic acid i-Inositol Folic acid Biotin Niacin Thiamine Vitamin B12 Riboflavin Nicotinamide Bactopeptone is an enzyme digest of an animal protein 35 Culture Media RPMI Media 1640 has a wide range of applications for mammalian cells, including the culture of fresh human lymphocytes, fusion protocols, and growth of hybrid cells. DMEM delivers superior quality, consistency and control for your mammalian cell culture. Iscove's Modified Dulbecco’s media are a highly enriched synthetic media well suited for rapidly proliferating, high-density cell cultures 36 Dulbeccos Modified Eagle Medium (DMEM) Choice of: ▪ High/Low/No Glucose ▪ GlutaMax/With glutamine/No glutamine ▪ Phenol Red/No phenol red ▪ Powder/Liquid 37 Liquid Media stable for up to 2 years (1 year if they contain L-Glutamine). must be stored at 2-8°C in the dark Media concentrates are stored at 8-10°C and are stable for 1 year at the original pH. Each product label indicates required storage conditions and expiry date 38 Complete Media for cell culture Should contain Sera Amino acids Essential amino acids (those which are not synthesized in the body) Nonessential amino acids Vitamins Salts (contribute to the osmolality of the medium) Glucose Hormones and Growth Factors Organic supplements Antibiotics 39 Amino acids 20 amino acids as building blocks. Organisms considerably vary in their ability to synthesise essential amino acids. Man and the Albino rat can make only 10 of the 20 common amino acids. The rest must be supplemented by the media. Non-essential amino acids can be synthesised directly by the organism but they are also added to cell culture media to save energy for the cells 40 Vitamins Cannot be synthesized by mammalian cells Act as co-factors for many enzymes and are essential for their function. Absence of vitamins in culture may lead to decrease in cell growth, cell death or loss of productivity Vitamin B12, Ascorbic acid, Folic acid, D- biotin, (vitamin H) 41 L-Glutamine L-Glutamine is an Amino acid essential for cell growth Serves as an auxiliary energy source when cells are rapidly dividing Unstable and breaks down easily to D-glutamine, a form which cannot be used by cells Must be stored frozen to retain its stability Best to add it to the culture medium just before its use. 42 Inorganic Ions Na, Ca, Mg, K, Fe, CO3, PO4, SO4 Na, K: maintenance of osmotic pressure within the medium Ca, Mg ions function of some intracellular enzymes; spreading of cells over glass surface Ca involve in the sol-gel alternation of the cytoplasm HCO3 fundamental biochemical cell processes PO4 energy metabolism; buffer substance SO4 actively incorporated into cellular proteins 43  For applications where Ca2+ and Mg 2+ ions interfere with enzyme activity (e.g. Trypsin); Use the modified HBSS (Hanks buffered salt solutions products. 44 Buffers Sterile To provide the right osmotic pressure and appropriate ionic strength Provide an environment that will maintain the structural and physiological integrity of cells in vitro. 45 Buffers Aqueous solution consisting of a weak acid and its conjugate base Simple to complex types Used in culture medium to keep the pH more or less constant pH of the solution changes very little when a small amount of strong acid or base is added 46 47 Things to be noted when buying culture media Batch size (lot number) Storage and Stability “Shelf life” Volume of use over time Ease of use, budget, space and time 48 Batch Size Batch size is the number of units manufactured in a production run Batch sizes for powdered media range from 1 to 10,000 Litres; For liquid media from 1 to 2,000 Litres. 49 Storage and Stability Powdered media stable for 3 years when stored at 2-8 °C in their original containers and in dry, dark conditions. Proper storage is required if not entirely used. Performance is greatly affected by heat, light and humidity. 50 51 PHENOL RED Commonly used as a pH indicator pH 7.8 = Purple pH 7.4 = Red 7.0 = Orange 6.5 = Yellow < 6.5 = lemon Yellow 52 53 Theory HA + H2O ------- H3O+ + A− When hydrogen ions are into the solution, equilibrium shift is to the LEFT When hydroxide (OH) ions are added to the solution, the equilibrium shift is to the RIGHT as hydrogen ions are removed in the reaction H + OH = H2O 54 Antibiotics/Fungicides suppress the growth of microorganisms Anti-bacterial or anti-fungal May be different in various laboratories Anti-biotics: Penicillin, streptomycin, gentamycin Anti-fungal: Fungizone 55 Mitotic inducing agents (Mitogens) Substance Source Cells activated PHA Phaseolus T- cells Phytohemagglu vulgaris tinin (red bean) Concanavalin A Canavalia T-cells ensiformis (Jack bean) Pokeweed Phytolacca T and B-cells america 56 Colcemid (Colchicine) An arresting agent Inhibits the assembly of microtubules Arrests cells at metaphase Ideal for cytogenetic studies 57 (2) Culture Wares Plastic/glass pipets, micropipets Flasks, petri dishes, tubes Graduated cylinder Mini tubes Measuring glass wares 58 Culture flasks in various volumes and forms 59 60 Multi- channel pippetor Dispensing very small amount of liquids 61 3. Proper Work Environment (1) Aseptic Techniques Swabbing, capping, flaming, handling of bottles and flasks Pipeting, pouring (2) Sterilization Autoclave Media Sterile Filtration (microporous filters: 0.20-0.45 µm) 62 3.1 Laminar Flow A kind of cabinet or tissue culture hood A carefully enclosed bench designed to prevent contamination of biological samples Gives protection from dust and contamination by a constant, stable flow of filtered air passing over the work surface Protects specimens but not the user 63 Laminar flow Hood (1) horizontal air flow= air blows from the side facing you, parallel to the work surface and is not recirculated (2) Vertical = air blows down from the top of the hood onto the work surface and is drawn through the work surface and either recirculated or vented 64 3.1b. Biological Safety Cabinet (BSC) enclosed protection compartments used in laboratory environments Different from Laminar flow Provides protection to the sample, the researcher (personnel) and the environment 65 Classes of (Biological Safety cabinets) Class I- offer significant levels of protection to lab personnel and environment when good microbial techniques are in place. Class II- designed for work involving BSL 1,2,3 materials. Provide aseptic environment necessary for cell culture expt. , for potentially hazardous materials Class III- highest attainable level of protection to personnel and the environment 66 A biological Safety cabinet A laminar flow hood 67 Schematic diagram of a biological safety cabinet 68 Ultra Low Freezer, Incubator and centrifuge 69 70 3.2 Temperature Controls the rate of many cellular processes and cell viability Cells cooled below freezing point are destroyed due to the formation of ice crystals within the cytoplasm Optimal temperature depends on the body tem of the host cells: Mammals and humans 36-37 C Insects: 27-30 C; Avian: 38.5C Cold blooded animals: 15-26 C 71 3.2 Osmotic pressure (Os) OS of medium is critical For mammalian cells normal OS at 38C is about 7.6 atmospheres Cells are affected of change +/- 10% OS in most biological fluids is mainly due to dissolved crystalloids ( glucose, NaCl etc..) “Larger molecules in the medium contribute little to the osmotic pressure” 72 3.3 Gases: CO2 and O2 Animal cells will unlikely survive indefinitely in the absence of oxygen. CO2 is a by-product of metabolism and is immediately fixed by cations present in the medium. HCO3 most important buffering ion in most culture media 73 3.4 Hormones Surprisingly in many animal tissue cultures cells in vitro seem to be able to metabolize and grow perfectly well without hormones (insulin). Effect of hormones is much more clearer in plants 74 Types of tissue culture Cell culture Primary Explant culture: A small piece of tissue or pieces of the tissue are attached to a glass or treated plastic culture vessel and immerse in culture medium Organ culture: Explantation and growth in vitro of organs or part of organs in which the various tissue components, such as parenchyma and stroma, and their anatomical relationship and function: explanted tissue closely resembles its parent tissue in vivo. 75 4. Tissues/Cells to be cultured Blood (WBC) Amniotic Fluid Body fluids (e.g.pleural effusion) Abortus Material Solid Tissues Normal vs Malignant tissues 76 Blood Easy to obtain Easy to handle/culture Shorter growth curve (1) whole blood (microculture) (2) lymphocytes (macroculture) 77 Primary Cell Culture: Duration: (1) short term: 24, 48, 72, 96h (2) long term (weeks, months) 78 Primary Cell Culture Cells that are isolated and cultured directly from a subject (e.g human) Cells proliferate under appropriate conditions until they occupy all the available substrate CONFLUENCE: When cells reach confluency these are ready for harvest or will be subcultured (i.e passaged) Transfer to a new vessel, and provided with fresh supply of medium 79 Primary Cell Culture Maintenance and growth of cells directly removed from the tissue or original source. Material (tissue) needs to be reduced to smaller pieces/ single cells by: mechanical disaggregation or by using enzymes (e.g. Collagen, trypsin, DNase etc.....) Limited growth potential Finite life span 80 Types of Cell Culture Technique Microculture : Use of whole blood Macroculture: Entails separation of lymphocytes from whole blood Subjected to density gradient centrifugation 81 Macroculture technique Lymphocyte separation Lymphoprep, a separation medium (sterile and endotoxin tested) Lymphocyte-rich region 82 Cells/ml = average cell count x 11x10,000 106 Hemocytometer 83 Cell Viability Total cells no. of cells – No. of dead cells % of viable cells Total cells no. of cells 84 Cell Line Cloning selection: One cell lineage is selected, 🞄 Specific properties identified in the bulk of cells Cells with highest growth capacity predominate Cell population with uniformity in genotype and phenotype Commercially available: ATTC Stem Cell Line : is a family of constantly dividing cells, the product of a single group of cells 85 The ATCC Cell Biology Collection American Type Culture Collection A private, non profit organization dedicated to acquire, preserve, authenticate and distribute (APAD) biological materials Most comprehensive and diverse of its kind in the world, consisting of over 3,600 cell lines from over 150 different species Over 950 cancer cell lines, 1,000 hybridomas, Stem cells Low passage Authenticated cell line identity Free of contamination 86 Cell lines commonly used HT29 Colorectal adenocarcinoma; human epithelial Adherent, McCoy’s medium Oncogene: myc +; ras +; myb +; fos +; sis +; p53 +; abl -; ros -; src – First Propagated: 1964 The Memorial Sloan-Kettering Cancer Center (IP) For research purposes only 87 pt.slideshare.net 88  STEPS IN PRIMARY CELL CULTURE Surgical Tissue (colon) Mechanical disaggregation Enzymmatic disaggregation Single cell suspension 89 Culturing cells from a solid tumor 90 Cell Line A primary culture that is subcultured (passaged or transferred) A transformed cell population with the ability to divide indefinitely because of random mutation or deliberate modification (artificial expression of telomerase gene) Numerous cell lines are well established as representative of particular cell types 91 Cell line Immortalized Cell lines : Cancer Cell lines Normal Cell lines (e.g. derived from stem cells); usually these are transformed cell lines 92 Examples of Cell Lines 1. Human Cancer cell lines 2.1 Breast Cancer cell lines (MCF-7) 2.2 Colorectal Cancer cell lines (HT-29) 2.3 Leukemia Cell Lines (K562) 2 Animal Cell lines: 2.1 Frog cell lines 2.2 Mouse cell lines 2.3 Rat cell lines 93 Cell passaging or splitting A technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Subculturing cells into a new vessel Cells should be passaged or split when they are 90%-100% confluent. 94 Passaging Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Adherent or suspension cultures For adherent cultures, cells first need to be detached using Trypsin-EDTA soln. Cell scraper can also be used to scrape the cells that adhere to the surface of the flask 95 Passaging of cells (suspension type of cells) in cell culture 96 Passage number Number of times a cell culture has been sub- cultured More passages increase the risk of contamination Passage number should be recorded Should not get too high 97 Passaging This is to prevent use of cells undergoing genetic drift and other variations Generally, the ATCC recommends that cell culture should be limited to five passages (for medical and biopharmaceutical applications). 98 99 Where to buy/get Cell Lines American Type Culture Collection (ATCC) European Culture Collection German Collection of Microorganisms and Cell Cultures (DSMZ) 100  Some Cell Lines that can be ordered from ATTC Designation ATTC Organ Disease Catalog # /Organism /Morphology HCT 116 CCL 247 Colon/ Homo Colorectal sapiens Cancer/ Epithelial HT 29 HTB 38 Colon/ Homo Colorectal sapiens Adenocarcinom a/ Epithelial MDA-MB-231 HTB 26 Mammary gland Adenocarcinom breast/ a/ Homo sapiens Epithelial HeLa CCL2 Cervix/Homo Adenocarcinom sapiens a/ Epithelial 101 Cell Kinetics A science that has as its objective on understanding how cells mature with time and how they respond to outside influences (e.g., exposure to radiation and drugs). Phases: Lag, Log, and Plateau 102 Kinetics of Cell Growth: Established Cell Lines (1) Lag phase (2) Logarithmic phase increase in cell population (3) Stationary phase follows after cells reached their maximum population is reached 103 Lag Phase Time following subculture and reseeding Very little evidence of increase in cell number Period of adaptation Cell replaces elements of the cell surface and extra cellular matrix lost during trypsinization; Attaches to the substrate and spreads out. Cytoskeleton reappears which is needed in the spreading out process. Increases synthesis of DNA polymerase, synthesis of DNA 104 Log Phase Exponential increase in cell number Terminates in one or two population doublings after confluence is reached Length depends on seeding density, growth rate of cells, density that inhibits cell proliferation Cells randomly distributed in the Cell cycle; Need for cell synchronization 105 Plateau Phase Toward the end of Log phase Culture becomes confluent All of the available growth surface is occupied. Reduced Growth rate Cell proliferation ceases almost completely Cells become less motile, ruffling of membrane 106 Confluency Refers to the proportion of the surface which is covered by cells. 50%, 60% or 80% 50% confluent means roughly half of the surface is covered and there is still room for cells to grow 100% percent confluent means the surface is completely covered by the cells; no more space left for the cells to grow as a monolayer 107 Kinetics of Cell Culture 108 Buffering of culture Media Open cultures: evolution of carbon dioxide will cause pH to rise and bicarbonate Overproduction of CO2 and lactic acid in transformed cell lines at high cell concentration, will cause pH to fall Can be incorporated in the media or exogenous application of CO2 to prevent total loss of CO2 and bicarbonate from the medium 109 pH pH of biological fluids has to be very near neutral 7.0 to permit survival of the whole animal pH 6.6 to 7.8 = average cell survival Ph 7.2 to 7.4 optimal growth (general rule) Above 7.8 and below 6.8 many cell types will die within 24 hours 110 Characterization Cytology Structure and function of cells Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. Immmunohistochemistry (fluorescent dyes and enzymes) Flow cytometry 111 ADVANTAGES OF TISSUE CULTURE 1. Control of the environment 2. Characterization and Homogeneity of Sample 3. Economy, Scale and Mechanization 4. In Vivo Modeling 112 Control of the Environment Physiological Conditions Physio-chemical Environment Hormone Nutrient concentrations Control of pH Temperature Humidity Osmotic pressure Carbon dioxide osmolarity 113 Economy, Scale and Mechanization Use of lower volumes/concentration of reagents Screening with many variables is cheaper (e.g. 96 well tray) Quantitation is easy Does away with legal, moral and ethical issues/questions on animal experimentation Robotics and microtitration (small volumes of liquid) 114 Homogeneity of sample/cell line Initially heterogenous Subculturing with replicates Series of passages result to homogeneous or at least uniform constitution Maybe brought about by: Selective pressure of culture conditions Selective Media Cloning Replicates (identical) reduces the need for statistical analysis of variance Preservation in liquid nitrogen or ultralow freezer (-78 to 80 C) 115 Importance of Cell and Tissue culture An important tool for the study of the biology of cells from multicellular organisms Provides an in vitro model of the tissue in a well defined environment which can be easily manipulated and analysed 116 Significance of cell Culture Monoclonal antibody technique Provides insights into the mechanism of action of antibodies Control of gene expression Cell interactions and intracellular control mechanisms in cell differentiation and development 117 LIMITATIONS OF CELL CULTURE 1. Expertise 2. Quantity 3. Dedifferentiation and Selection 4. Origin of Cells 5. Instability 118 (1) Expertise Skill in handling Strict adherence to aseptic requirements Microbial contamination Chemical contamination Able to diagnose problems as they arise Cross contamination Mislabeling, switching of samples Ability to address these problems - Patience and understanding 119 (2) Quantity and Cost Equipment Outlay Consumables: culture media, dyes, buffers, serum, glass slides Wares Personnel Electricity, water etc... 120 (3) Dedifferentiation and Selection Dedifferentiation – process by which a cell loses its differentiated phenotype (typical of the tissue from which it was derived); reverse of differentiation Caused by overgrowth of undifferentiated cells of the same or a different cell lineage Use of selective media to isolate specific cell lineage 121 (4) Origin of Cells Loss of differentiated properties will be make it very difficult to relate cultured cells to functional cells in the tissue from which they are derived. Need for cell surface markers to establish cell origin 122 Instability Cell lines are prone to genetic instability Continuous cell lines from tumors are very unstable; this instability is the major reason for their undergoing the necessary mutations to become continuous Due to unstable aneuploid chromosome condition (monosomy, trisomy) 123 Genetic Variations Causes: (1) Spontaneous mutation rate appears to be higher in vitro (high rate of cell proliferation) (2) Mutant cells are not eliminated unless growth capacity is impaired 124 MAJOR DIFFERENCES IN VITRO From 3D Geometry to 2D substrate Specific cell interactions characteristics of the histology of the tissue are lost as cells spread out and become mobile Growth faction of the cell population increases Absence of some systemic components involved in homeostatic regulation in vivo (nervous and endocrine systems). Energy metabolism largely by Glycolysis; citric acid cycle plays a minor role 125 Applications of Cell Culture Cytogenetic studies Diagnostics Environmental toxicology Drug Resistance/Sensitivity Assays Many more ….. 126 Study of Cell Behavior/kinetics in Real Time Confocal Microscopy Applications Live Cell Imaging 127 References Websites of: ATCC Culture Guide Life Technologies Biosera en.wikipedia.org/wiki/Tissue_culture Perspectives in Cell Culture, GIBCO Cell culture Animal Culture 128 Reference Books 129 Thank You for listening!!!! 130