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AN ASSIGNMENT ON CELL CULTURE [pic] ASSIGNED BY | | | |Dr. Md. Bahanur Rahman |Dr. Sukumar Saha | |Professor |Associate Professor | |Dept. f Microbiology and Hygiene |Dept. of Microbiology and Hygiene | |Faculty of Veterinary Science |Faculty of Veterinary Science | |B. A. U. Mymensingh-2202 |B. A. U. Mymensingh-2202 |
SUBMITTED BY kohinur Aktar M. S. IN MICROBIOLOGY ROLL NO10VMicro JJ-04M REG. NO. : 31053 SESSION: 2004-2005 DEPARTMENT OF MICROBIOLOGY AND HYGIENE BANGLADESH AGRICULTURAL UNIVERSITY MYMENSINGH-2202 CONTENTS |SL. NO |Topics |Page no. | |01 Introduction |01 | |02 |Historical events in the development of cell culture |02 | |03 |Major development’s in cell culture technology |03 | |04 |Applications of cell culture |03 | |05 |Tissue culture and Types Of Tissue Culture |04 | |06 |Cell culture and Types of cell culture |05 | |07 |List of cell lines |07 | |08 |Cells Used For Cultivation Of Certain Viruses |08 | |09 |Contamination of cell culture |09 | |10 |Detection of contaminants |10 | |11 |Basic Procedure Of Cell Culture |10 | |12 |Techniques for culturing cells |14 | |13 |Chicken Embryo Fibroblast Cell Culture |15 | |14 |Rules for working with cell culture |19 | |15 |Basic aseptic conditions |20 | |16 |Safety aspect in cell culture |20 | |17 |References |21 | Introduction Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells.
Cell culture was first successfully undertaken by Ross Harrison in 1907. Roux in 1885 for the first time maintained embryonic chick cells in a cell culture. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. Animal cell culture became a common laboratory technique in the mid-1900s,[1] but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century. The 19th-century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body.
In 1885 Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture. Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907–1910, establishing the methodology of tissue culture. Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The Salk polio vaccine was one of the first products mass-produced using cell culture techniques.
This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures. Historical events in the development of cell culture • 1878: Claude Bernard proposed that physiological systems of an organism can be maintained in a living system after the death of an organism. • 1903: Jolly observed cell division of salamander leucocytes in vitro. • 1907: Harrison cultivated frog nerve cells in a lymph clot held by the ‘hanging drop’ method and observed the growth of nerve fibers in vitro for several weeks.
He was considered by some as the father of cell culture. • 1910: Burrows succeeded in long term cultivation of chicken embryo cell in plasma clots. He made detailed observation of mitosis. • 1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells. • 1927: Carrel and Rivera produced the first viral vaccine – Vaccinia. • 1933: Gey developed the roller tube technique. • 1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture. • 1948: Earle isolated mouse L fibroblasts which formed clones from single cells. Fischer developed a chemically defined medium, CMRL 1066. 1954: Abercrombie observed contact inhibition: motility of diploid cells in monolayer culture ceases when contact is made with adjacent cells. • 1961: Hayflick and Moorhead isolated human fibroblasts (WI-38) and showed that they have a finite lifespan in culture. • 1965: Ham introduced the first serum-free medium which was able to support the growth of some cells. • 1975: Kohler and Milstein produced the first hybridoma capable of secreting a monoclonal antibody. • 1985: Human growth hormone produced from recombinant bacteria was accepted for therapeutic use. • 1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially available. • 1989: Recombinant erythropoietin in trial. 990: Recombinant products in clinical trial (HBsAG, factor VIII, HIVgp120, CD4, GM-CSF, EGF, mAbs, IL-2). [pic]Applications of cell culture Areas where cell culture technology is currently playing a major role. • Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies • Toxicity testing Study the effects of new drugs • Cancer research Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells • Virology Cultivation of virus for vaccine production, also used to study there infectious cycle. • Genetic Engineering
Production of commercial proteins, large scale production of viruses for use in vaccine production e. g. polio, rabies, chicken pox, hepatitis B & measles. • Gene therapy Cells having a functional gene can be replaced to cells which are having non-functional gene. • Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biological products or the use of adjuvants. Tissue culture In vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture.
Different types of cell grown in culture includes connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor cells. Types Of Tissue Culture There are three main methods of initiating a culture ? Organ culture ? Primary explant culture and ? Cell culture Organ culture: Organ culture implies that the architecture, characteristics of the tissue in vivo, is retained at least in part, in the culture. Towards this end, the tissue is cultured at the liquid-gas interface (gel, grid, etc. ), which favors the retention of a spherical or three dimensional shape.
Primary explant culture: In primary exaplant culture, a fragment of tissue is placed at a glass-liquid interface. Where following attachment migration is promoted in the plane of the solid substrate. Cell culture: Cell culture implies that the tissue, or outgrowth from the primary explant, is dispersed (mechanically or enzymatically) into a cell suspension, which may then be cultured as an adherent manslayer on a solid substrate or as a suspension in the culture medium. [pic] Fig. Epithelial cells in culture, stained for keratin (red) and DNA (green) Cell culture: Culture prepared with dispersed cells are designated as cell cultures. Types of cell culture: Three types of monslayer cell cultures – ? Primary cell culture Diploid cell culture / cell line and ? Continuous / established / heteroptoid cell culture or cell lines Primary cell culture: Tissues/cells which are used one time for the preparation of cell culture after collecting them from the animals. • Primary cells are the first generation cell preparations that grow from the tissues of origin. • Predominantly epithelial cells • Normal diploid chromosomes of the same number as the parent tissue. • Cells when surgically or enzymatically removed from an organism and placed in suitable culture environment will attach and grow are called as primary culture • Primary cells have a finite life span Primary culture contains a very heterogeneous population of cells • Sub culturing of primary cells leads to the generation of cell lines • Cell lines have limited life span, they passage several times before they become senescent • Cells such as macrophages and neurons do not divide in vitro so can be used as primary cultures • Lineage of cells originating from the primary culture is called a cell strain • eg. Primary rhesus monkey kidney (pRMK), primary human embryonic kidney (pHEK) Diploid cell culture: Diploid cell lines as their name suggests are those which continuously maintain their diploid chromosome number throughout serial passages, these cells usually die out after 50th passage. eg. Human Lung fibroblast (MRC-5 or WI-38). Continuous Cell Culture: CCC demonstrates heteroploid chromosome numbers during repeated subculturing. • Originate from malignancies. • Replicate vigorously. • Usually not difficult to culture. • Being capable of indefinite passaging. eg. Human cervical carcinoma (HeLa) `Most cell lines grow for a limited number of generations after which they ceases • Cell lines which either occur spontaneously or induced virally or chemically transformed into Continous cell lines • Characteristics of continous cell lines -smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio -Fast growth and have aneuploid chromosome number -reduced serum and anchorage dependence and grow more in suspension conditions -ability to grow upto higher cell density -different in phenotypes from donar tissue -stop expressing tissue specific genes Types of cells On the basis of morphology (shape & appearance) or on their functional characteristics.
They are divided into three. • Epithelial like-attached to a substrate and appears flattened and polygonal in shape • Lymphoblast like- cells do not attach remain in suspension with a spherical shape • Fibroblast like- cells attached to an substrate appears elongated and bipolar List of cell lines Human cell lines [pic] Fig. cultured HeLa cells • DU145 (Prostate cancer) • Lncap (Prostate cancer) • MCF-7 (breast cancer) • MDA-MB-438 (breast cancer) • PC3 (Prostate cancer) • T47D (breast cancer) • THP-1 (acute myeloid leukemia) • U87 (glioblastoma) • SHSY5Y Human neuroblastoma cells, cloned from a myeloma • Saos-2 cells (bone cancer) Primate cell lines Vero (African green monkey Chlorocebus kidney epithelial cell line initiated 1962) Rat tumor cell lines • GH3 (pituitary tumor) • PC12 (pheochromocytoma) Mouse cell lines • MC3T3 (embryonic calvarial) Plant cell lines • Tobacco BY-2 cells (kept as cell suspension culture, they are model system of plant cell) Other species cell lines • zebrafish ZF4 and AB9 cells. • Madin-Darby Canine Kidney (MDCK) epithelial cell line • Xenopus A6 kidney epithelial cells. Cells Used For Cultivation Of Certain Viruses |Cells |Viruses | |i) Hela |Polio, equine abortion, NDV, measles, herpes simplex. |ii) Human embryonic intestine |Some arboviruses | |iii) Human amnion |Polio, adenovirus, measles | |iv) human embryonic skin |Varicella, Measles | |v) Mouse fibroblast |Herpes simplex | |vi) rabbit kidney |Herpes simplex and admo | |vii) Canine kidney |Canine distemper, canine hepatitis | |viii) Bovine fetus kidney |FMD, Infectious-bovine rhinotrachetis, bovine enterovirus. | |ix) Whole chicken embryo |Herpes simplex, NDV, influenza | |x) Chicken embryo CAM |NDV, Influenza, Infections bronchitis of chicken | |xi) Chicken embryo lung |NDV, Influenza | |xii) chicken embryo kidney |Infections bronchitis of chicken. | Contamination of cell culture
All cell cultures, whether commercially purchased or originated and maintained in house, may be subject to contamination by bacteria, fungi or mycoplasma. Sources for Contamination: The sources for contamination are many and include- • the usual throat flora of laboratory personnel • clinical specimens • contaminated cell culture reagents and additives such as bovine sera. Bacterial/Fungal Contaminants:Usually produce gross changes in the appearance of the culture. Culture media become turbid, the pH of the media becomes acid, cells appear granular and may detach from the culture vessel. Detection: Bacterial and fungal contamination are not always obvious.
Inoculate samples from suspicious cultures on to blood and/or Sabourauds agar and into thioghycollate or similar broth medium to test for growth of contaminating bacteria or fungi are usually discarded because contamination is difficult to eliminate. Mycoplasmal Contaminants: Mycoplasma contamination may go undetected through many generations of cells. Detection of contaminants Mycoplasma isolation requires the use of broth or plate media that are enriched with yeast extract and hones serum. An incubation period of 48 hours to 7 days may be required for growth of the organisms on agar and subculturing of broth media to agar plates at 10 ~ 14 days is recommended for detection of small quantities of the organisms. • In general indicators of contamination are turbid culture media, change in growth rates, abnormally high pH, poor attachment, multi-nucleated cells, graining ellular appearance, vacuolization, inclusion bodies and cell lysis • Yeast, bacteria & fungi usually shows visible effect on the culture (changes in medium turbidity or pH) • Mycoplasma detected by direct DNA staining with intercalating fluorescent substances e. g. Hoechst 33258 • Mycoplasma also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycoplasmal RNA • The best and the oldest way to eliminate contamination is to discard the infected cell lines directly. Basic Procedure Of Cell Culture 1) Equipments and its preparation 2) Solutions and constituents of media 3) Culture media 4) Techniques for culturing cells 5) Control of contamination 6) Infection of cell cultures ? Subculturing ? Freezing (Preservation) ? Reactivation(thawing) Basic equipments used in cell culture Laminar cabinet-Vertical are preferable • Incubation facilities- Temperature of 25-30 C for insect & 37 C for mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approx. 38. 5 C • Refrigerators- Liquid media kept at 4 C, enzymes (e. g. trypsin) & media components (e. g. glutamine & serum) at -20 C • Microscope- An inverted microscope with 10x to 100x magnification • Tissue culture ware- Culture plastic ware treated by polystyrene Cell Culture media and stock solution Many different cell-culture media can be used and all have the following basic composition: 1) A balanced salt solution ) A protein supplements such as serum or amniotic fluid, or protein derivatives such as lactalbumin hydrolysate or tryptoe-phosphate broth, or amino acid. 3) An antibiotic mixture to control accidental microbial contamination. Balanced Salt Solutions (BSS): + PBS Hanks BSS (HBSS) and Earles Bss are frequently used as bases for growth media. Their function is to maintain a physiologic pH (7. 2-7. 6) and osmotic pressure and to provide water, glucose and inorganic ions needed for a normal cell metabolism. BSS and PBS are also frequently used to wash inocula and dead cells from cultures to remove serum-containing media before trypsinization and to dilute trypsin solutions. BSS consists of essential inorganic chemicals dissolved in glass-distilled or deionized water to form isotonic solutions.
Table: Composition of some BSS and PBS |Solution |Component |HBSS |EBSS |Dulbecco’s BSS |PBS | |A |Water |800 |800 |800 |1000 | | |NaCl |8. 0 |6. 8 |8. 0 |8. 0 | | |KCl |0. 4 |0. 4 |0. 2 |0. 2 | | |MgSO4. 7H2O |0. 2 |0. |- |- | | |KH2PO4 |0. 06 |- |0. 2 |0. 26 | | |Na2HPO4. 2H2O |0. 06 |- |0. 14 |0. 57 | | |NaH2PO4. H2O |- |0. 14 |- |- | | |Glucose |1. 0 |1. 0 |- |- | | |Phenolred, Na |0. 017 |0. 017 |0. 017 |0. 17 | | | | | | | | |B |Water |100 |100 |100 |- | | |CaCl2 |0. 14 |0. 2 |0. 1 |- | | | | | | | | |C |Water |100 |100 |100 |1. 8 | | |MgCl2. H2O |- |- |1. 0 |- | | |NaHCO3 |0. 35 |2. 2 |- | | ? Body Fluid: Sera, plasma and other body fluids were the earliest nutrient media employed for cell culture. They are still widely employed but usually in combination with other natural substances or as supplements to chemically defined media. • Sera: Horse, cattle, and lambs calves about 6 months of age is satisfactory. Function: are not clearly understood. However certain serum proteins promote attachment and spreading of cells on glass surfaces.
Fetal bovine serum is particularly rich in protein. • Plasma: Chicken plasma is preferable specially from cockerels (< 1 year of age). Use: i) For physical support ii) Nutrient supplement for a variety of cells. • Bovine Amniotic Fluid: incorporated in media for supporting cell growth. • Embryo extract: Saline extract of chick or bovine embryo is used to provide growth factor. ? Bacteriological Media: Sources of amino acids e. g. I. Lactalbumin hydrolysate, II. Yeast extract, III. Trypotase phosphate and IV. Peptones. ? Cell Dispersing Agents: Proteolytic enzyme Trypsin 0. 5% solution, PBS without Ca and Mg ions. The solution is sterilized by filtration. Others are: Collagenase Profeinase Antibiotic solution: Antibiotic-antimycotic® solutions are commercially available (e. g. from GIBCO-BRL). These solutions contain Penicillin-G sodium ( 10,000 IU/ml Streptomycin sulfate ( 10,000 µg/ml Amphotericin-B (Fungizone®) ( 25 µg/ml Dose: 1 ml. of this solution to 100 ml. of medium ? Buffering system: Incorporation of a buffering system in the medium is mandatory to aid in maintaining proper pH. Media with HBSS contain a low concentration of sodium bicarbonate and function effectively in a closed system (the culture flasks are incubated with caps tightly closed in an ambient air incubation).
Media with EBSS contain a higher concentration of sodium bicarbonate and function well in an open system (incubated with caps loosely in place in an incubator with a 5% CO2 environment). ? pH Indicator: All media, both outgrowth and maintenance, contain a pH indicator, usually phenol red. ? A salmon-pink colour indicates a pH of 6. 9-7. 2, optimal for cell culture maintenance. ? Colour changes to bright pink, indicating increased pH, when the buffering system is disturbed (as when caps are not tightly closed) or when cell are not metabolizing well. Colour change to bright yellow indicates excess acid production and usually is seen in cultures infected by bacteria or fungi or in cutters that proliferated excessively and are need of refeeding or subculturing. Culture Media: ) Media containing biological substances b) Synthetic media e. g. M-199 and Earle’s Minimum essential media (EMEM). I. Outgrowth media/growth promoting media: Employed to aid the attachment of cells to glass and to initiate growth. Basic media enriched with 10% serum serve as outgrowth media for freshly subcultured or newly established cell cultures when rapid cell proliferation is desired. The amino acid L-glutamate is also added in a concentration of 3% outgrowth media to stimulate cell growth. II. Maintenance Media: In this case, the serum content is reduced to 2% and used for cultures that have reached confluency and are to be maintained in a steady state.
Techniques for culturing cells: Cells are used in two main forms- ? as sheet about one cell thick ( Monolayer ? as suspension of either tissue fragments or dispersed cells. Tissue and organs from which cell cultures are to be derived should ideally be taken from fetuses or from gnotobiotic animals to minimize the risk of contamination with latent viruses. Chicken Embryo Fibroblast Cell Culture Preparation of 10% fcs containing media: Mix MEM (500 ml) + L-glutamate (5 ml) + antibiotic (1+ 1 cryovial) ( Adding 50 ml serum and well mixing (When 10 ml serum is used, it is maintenance media (2%) ( Adjustment of pH (7. 0-7. ) by adding Sodi-bi-carb up to light red colour Preparation Of Chicken Embryo Fibroblast Cell: 8-10 days embryonated egg ( Soaking with Tr. of iodine ( Shell rupture ( Membrane removed ( Embryo on a petridish containing PBS ( Separate of head, extremites and intestine from the body ( Washing with PBS ( Body is cut into small pieces ( Taking in a conical flask ( Adding to Trypsin 0. 3 ml (Warm trypsinzation 0. 25%) ( Stirring on hot plate stirrer with magnetic bar for 10-15 minutes ( Sieving by delipidized gauze in funnel ( Neutralization of trypsin by MEM ( Centrifugation 3-5 minutes to remove MEM and trypsin (1000-1200 rpm) ( Discard the supernatant (
Washing the cell with PBS for 3 times to remove RBC and trypsin ( clear cells are found Method of cell culture: Taking of 0. 3 ml cell in each cell culture tube ( Adding of MEM in culture tube around 3 ml ( Observation of the cell under microscope ( Placing in the incubator ( After 24 hour observe in the inverted microscope. Viability test: Viability test of cells can be made at this stage by trypan blue stain 0. 25% Trypan blue stock. Preparation of inoculum: • Swab samples (150 µl) and Antibiotic (75 µl) in test tube ( Seated by parafilm and remain 20-30 min. at room temperature ( Keep at 40C until use • Take 10 ? PBS (100 µl) and 10 ? PBS (300 µl) in a series of test tube (
Inoculate feces of pigeons to make 10-20% suspension (eye estimation) ( Seated the test tube with parafilm and centrifuge at 2500 rpm for 5 minutes ( Collection of supernatant (200 µl) in another test tube ( Add antibiotic (100 µl) and PBS (50 µl) seated with parafilm Infection of cell by virus: • 10% MEM (growth promoting) and 2% (maintenance) MEM is made. • Cell culture flask (test tube) is taken from incubator ( Slight burning of cap by flame and remove fluid from the cell culture flask (2. 5 ml) ( Add 0. 2 ml inoculums and remain 40-60 minutes in room temperature ( Remove all fluid from cell culture flask by syringe ( Slight burning of the cap by flame (
Add 2% MEM of 2-2. 5 ml in each flask ( Again slight burning of the cap by flame Sub culture: Removal of media ( Washing with PBS ( 0. 25% trypsin (break intercellular junction) + 0. 02% EDTA (detach the cell from wall of bottle) ( Incubation at 370C/room temperature for 5-10 minutes ( Shaking bottle for detachment ( Reduction of trypsin activity by using 10% fetal calf serum ( Centrifugation ( Wash with PBS ( Tabbing of cell suspension ( Counting ( 2nd passage 8 ? 106 cell/ml Preservation: 1. Check the culture for the followings: a) healthy b) freedom from contamination c) Specific characteristics 2. Grow the culture up to the late log phase. 3.
Dilute the preservatives in growth medium to make freezing medium a) Add dimethyl sulphoxide (DMSO) 5-10% or b) Add glycerol (10-15%) 4. Resuspend the cells freezing medium at approximate 1 ? 106 – 1 ? 107 cells/ml 5. Dispense the cell suspensions into cryovials/ampoules. 6. Place the cryovial/ampoules on canes for the canister storage. 7. Lay the ampoules on cotton wool in a polystyrene foam box with a wall thickness of 15 mm. 8. The box is placed at -200C for overnight, then at -700C for overnight and finally transfer them to a liquid nitrogen (N2) freezer. Thawing: 1) Preparation of medium. 2) Retrieve the ampoule from the freezer, and place it in 10 cm water bath at 370C in a bucket. ) When the ampoule is thawed, then soak the ampoule thoroughly with 70% alcohol and open it. 4) Transfer the contents of the ampoule to a culture flask. 5) Add medium slowly to the cell suspension. 6) The cells in the ampoule may be stained with naphthalene black or trypan blue to determine their viability. Rules for working with cell culture Never use contaminated material within a sterile area Use the correct sequence when working with more than one cell lines. • Diploid cells (Primary cultures, lines for the production of vaccines etc. ) • Diploid cells (Laboratory lines) • Continous, slow growing line • Continous, rapidly growing lines • Lines which may be contaminated • Virus producing lines Basic aseptic conditions If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen • Swab all bottle tops & necks with 70% ethanol • Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame • Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger • Work either left to right or vice versa, so that all material goes to one side, once finished • Clean up spills immediately & always leave the work place neat & tidy Safety aspect in cell culture • Possibly keep cultures free of antibiotics in order to be able to recognize the contamination • Never use the same media bottle for different cell lines.
If caps are dropped or bottles touched unconditionally touched, replace them with new ones • Necks of glass bottles prefer heat at least for 60 sec at a temperature of 200 C • Switch on the laminar flow cabinet 20 mts prior to start working • Cell cultures which are frequently used should be subcultered & stored as duplicate strains References 1. ^””CellCulture””. http://www. bioteach. ubc. ca/Bioengineering/CellCulture/index. htm. Retrieved 2006-04-19. 2. ^ “”Some landmarks in the development of tissue and cell culture. “”. http://www. ncbi. nlm. nih. gov/books/bv. fcgi? db=Books=mboc4. table. 1516. Retrieved 2006-04-19. 3. ^””Animals and alternatives in testing. “”. http://caat. jhsph. du/pubs/animal_alts/appendix_c. htm. Retrieved 2006-04-19. 4. ^ Schiff, Judith Ann. “”An unsung hero of medical research. “”. http://www. yalealumnimagazine. com/issues/02_02/old_yale. html. Retrieved 2006-04-19. Yale Alumni Magazine, February 2002. 5. ^ “LipiMAX purified lipoprotein solution from bovine serum”. Selborne Biological Services. 2006. http://www. selbornebiological. com/products/lipimax. htm. Retrieved 2010-02-02. 6. ^ Portela VM, Zamberlam G, Price CA (April 2010). “Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells”. Fertil. Steril. 93 (6): 2050–5. doi:10. 1016/j. ertnstert. 2009. 01. 151. PMID 19324349. 7. ^ Drexler, HG; Dirks; Macleod (Oct 1999). “False human hematopoietic cell lines: cross-contaminations and misinterpretations”. Leukemia 13 (10): 1601–7. doi:10. 1038/sj/leu/2401510. ISSN 0887-6924. PMID 10516762. 8. ^ Drexler, HG; Macleod; Dirks (Dec 2001). “Cross-contamination: HS-Sultan is not a myeloma but a Burkitt lymphoma cell line” (Free full text). Blood 98 (12): 3495–6. doi:10. 1182/blood. V98. 12. 3495. ISSN 0006-4971. PMID 11732505. http://www. bloodjournal. org/cgi/pmidlookup? view=long=11732505. 9. ^ Cabrera, CM; Cobo, F; Nieto, A; Cortes, JL; Montes, RM; Catalina, P; Concha, A (Jun 2006). Identity tests: determination of cell line cross-contamination”. Cytotechnology 51 (2): 45–50. doi:10. 1007/s10616-006-9013-8. ISSN 0920-9069. PMID 19002894. 10. ^ a b Chatterjee, R (Feb 2007). “Cell biology. Cases of mistaken identity. “. Science (New York, N. Y. ) 315 (5814): 928–31. doi:10. 1126/science. 315. 5814. 928. ISSN 0036-8075. PMID 17303729. 11. ^ Liscovitch, M; Ravid (Jan 2007). “A case study in misidentification of cancer cell lines: MCF-7/AdrR cells (re-designated NCI/ADR-RES) are derived from OVCAR-8 human ovarian carcinoma cells. “. Cancer letters 245 (1-2): 350–2. doi:10. 1016/j. canlet. 2006. 01. 013. ISSN 0304-3835. PMID 16504380. 12. Macleod, RA; Dirks; Matsuo; Kaufmann; Milch; Drexler (Nov 1999). “Widespread intraspecies cross-contamination of human tumor cell lines arising at source. “. International journal of cancer. Journal international du cancer 83 (4): 555–63. ISSN 0020-7136. PMID 10508494. 13. ^ Masters, JR (Apr 2002). “HeLa cells 50 years on: the good, the bad and the ugly. “. Nature reviews. Cancer 2 (4): 315–9. doi:10. 1038/nrc775. ISSN 1474-175X. PMID 12001993. 14. ^ a b Dunham, J. H. and Guthmiller, P. (2008) Doing good science: Authenticating cell line identity. Cell Notes 22, 15–17. 15. ^ Ceb. com 16. http://jvi. asm. org/cgi/content/abstract/80/4/1959 | accessdate = 2010-01-31 }} 17. “NIAID Taps Chiron to Develop Vaccine Against H9N2 Avian Influenza”. National Institute of Allergy and Infectious Diseases (NIAID). 2004-08-17. http://www3. niaid. nih. gov/news/newsreleases/2004/h9n2. htm. Retrieved 2010-01-31. ———————– Major development’s in cell culture technology • First development was the use of antibiotics which inhibits the growth of contaminants. • Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel • Third was the use of chemically defined culture medium. In sterile saline -1960C Night Over -800C Night Over -200C Night Over O0C 2 ml + 2 ml cell suspension