1. 1. by Dr Norshuhaila Mohamed Sunar CHAPTER 1 CELL CULTURE TECHNOLOGY
2. 2. Outline 1.1 Definition and Introduction 1.2 Culture Environment 1.3 Culture Media 1.4 Fields Of Applications, Techniques And Products of Culture Technology
3. 3. 1.1 Definition and Introduction
4. 4. Cell Culture in vitro - A brief history 1885: Roux maintained embryonic chick cells alive in saline solution for short lengths of time 1912: Alexis Carrel cultured connective tissue and showed heart muscle tissue contractility over 2-3 months 1943: Earle et al. produced continuous rat cell line 1962: Buonassisi et al. Published methods for maintaining differentiated cells (of tumour origin) 1970s: 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 date: Tissue culture becomes less of an experimental research field, and more of a widely accepted research tool
5. 5. Isolation of cell lines for in vitro culture Resected Tissue Cell or tissue culture in vitro Primary culture Secondary culture Sub-culture Cell Line Sub-culture Immortalization Successive sub-cultureSingle cell isolation Clonal cell line Senescence Transformed cell line Immortalised cell line Loss of control of cell growth
6. 6. Primary cultures Derived directly from animal tissue embryo or adult? Normal or neoplastic? Cultured either as tissue explants or single cells Initially heterogeneous become overpopulated with fibroblasts(a cell in connective tissue) Finite life span in vitro Types of cell cultured in vitro
7. 7. Making a Primary Culture
8. 8. Types of cell cultured in vitro Secondary cultures Derived from a primary cell culture Isolated by selection or cloning Becoming a more homogeneous cell population Finite life span in vitro Cells taken from a primary culture and passed or divided in vitro. These cells have a limited number of divisions or passages. After the limit, they will undergo apoptosis. (Apoptosis is programmed cell death)
9. 9. Types of cell cultured in vitro Continuous cultures Derived from a primary or secondary culture Immortalised: Spontaneously (e.g.: spontaneous genetic mutation) By transformation vectors (e.g.: viruses &/or plasmids) Serially propagated in culture showing an increased growth rate Homogeneous cell population Infinite life span in vitro Genetically unstable
10. 10. Cell morphologies vary depending on cell type Fibroblastic Endothelial Epithelial Neuronal
11. 11. 1.2 Culture Environment
12. 12. Cell culture environment (in vitro) What do cells need to grow? Substrate or liquid (cell culture flask or scaffold material) chemically modified plastic or coated with proteins suspension culture Nutrients (culture media) Environment (CO2, temperature 37o C, humidity) Oxygen tension maintained at atmospheric but can be varied Sterility (aseptic technique, antibiotics and antimycotics) Mycoplasma tested
13. 13. 1.3 CULTURE MEDIA PLANT AND ANIMAL CELL CULTURE MEDIUM
14. 14. Animal Culture Medium Basics Medium components Buffer(s) to maintain pH Salts for osmosis and cell needs Amino acids essential and other Growth stimulants (hormones and agonists) Serum (fetal calf/bovine) Lipids including cholesterol Vitamins Food (typically glucose) Trace Minerals for metabolism/enzyme function Nucleic Acids
15. 15. Animal Culture Medium Basics Base medium Just components without additives. no serum or antibiotics Complete medium 5 or 10% serum Basic medium Antibiotics or antimycotics low serum or starving medium Same as complete but with no or low levels of serum (0.5%).
16. 16. Selecting Media (animal cells) The choice of cell culture medium is extremely important, and significantly affects the success of cell culture experiments. Different cell types have highly specific growth requirements, and the most suitable medium for each cell type must be determined experimentally.
17. 17. RPMI Media
18. 18. Eagles Minimum Essential Media
19. 19. Culture Media These contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and are available either as a powder or as a liquid from various commercial suppliers.
20. 20. Culture Media Requires Ions; Na+ , K+ , Ca++ , Mg++ , Cl- , PO4 - , HCO3 - Trace elements; iron, zinc, selenium Sugars such as glucose Amino Acids
21. 21. Media Selection Cell Line Cell Type Species Tissue Medium 293 Fibroblast Human Embryonic Kidney MEM 10% HI Horse Serum 3T6 Fibroblast Mouse Embryo DMEM 10% FBS A549 Epithelial Human Lung Carcinoma F-12K 10% FBS H9 Lyphoblast Human T-Cell Lymphoma RPMI-1640 10% FBS HeLa Epithelial Human Cervix Carcinoma MEM 10% FBS
22. 22. Serum Serum is a partially undefined material that contains growth and attachment factors, and may show considerable variation in the ability to support growth of particular cells.
23. 23. Serum Fetal calf serum (FCS) is the most frequently used serum, but for some applications less expensive sera such as horse or calf serum can be used. Different serum batches should be tested to find the best one for each cell type.
24. 24. FCS vs FBS Fetal Calve / Bovine Serum typically used interchangeably but are different. Fetal Calf Serum taken from newborn Calves Fetal Bovine Serum from fetus Some variation in content of growth factors Variability in lot to lot and location to location often times tested for several viruses that my impact cells. Either can be different if mother or calve has been nursing or grazing .
25. 25. L-glutamine L-glutamine is an unstable amino acid that, with time, converts to a form that cannot be used by cells, and should be added to medium just before use. Provides nitrogen for nucleotides Serves (like pyruvate) as secondary energy source for metabolism Breaks down (40%) within 3 to 4 weeks, faster if in cultured cells. In culture, glutamine breakdown generates ammonium Some supplements (glutamax) are more stable and can replace glutamine for long term culturing of slow cells
26. 26. Antibiotic & Antimycotic Antibiotics and fungicides can be used as a supplement to aseptic technique to prevent microbial contamination.
27. 27. Antibiotic & Antimycotic Antibiotic / Antimycotic Working Concentration Stability at 37C Amphotericin B 2.5 g / ml 3 days Ampicillin 100 g / ml 3 days Penicillin 100 U / ml 3 days Streptomycin 100 g / ml 3days Puromycin 20 g / ml Unknown Kanamycin 100 g / ml 5 days
28. 28. Media & Components Media, serum, and supplements should be tested for sterility before use by incubation of a small aliquot at 37C for 48 hours. If microbial growth has occurred after this incubation, the medium or supplement should be discarded.
29. 29. Plant Culture Medium Plant Culture Medium Requirements Varies: Plant cell type (woody, fern, orchid) Maintenance of callus or shoot formation (stage II) Stimulation of Root and de-differentiation Protoplast, suspension or batch cultures General Components: Macronutrients, micronutrients, vitamins, amino acids, nitrogen, phosphorous, sugar, organic supplements and solidifying agents/support systems AND growth regulators (hormones)
30. 30. Plant Culture Medium Macronutrients (macroelements) - Needed in media in large amounts and make up ~0.1% of dry weight of plant: Nitrogen supplied in form of ammonium ion (H4NO3 + ) and nitrate (KNO3) best if both are present and together act to buffer pH. Some amino acids can supplement N requirements or take place as the N is removed via TCA and transglutaminases High ammonium causes a pale, glassy culture (vitrification ) Potassium come as counter ion with NO3 - and PO4 -2 Phosphorus K2HPO4, H4NO3(HPO4)2, High concentrations of phosphate will lead to ppt with Ca+2 and other cations
31. 31. Plant Culture Medium Micronutrients (Microelements): Trace amount elements and salts necessary for growth: Fe (FeSO4) The complex allows for a slow continuous release and avoids free metal generation of radical oxides after reaction with water. Others include: Zn, Cu, B, and Mo. Carbon and Energy Source cultures do little if any photosynthesis (heterotrophs). Must supply carbon to metabolize ATP and other energy molecules. Sucrose is usually used Galactose, sorbitol and maltose also are used.
32. 32. Plant Culture Medium Organic Supplements Wide range of various needs Amino Acids can provide nitrogen and support for metabolism as well as biosynthesis for new proteins, lipids and nucleotides Casine (milk protein) hydrolysates typically are the source of amino acids Vitamins: Vitamin B1 (thiamin) and Vitamin B6 (nicotinic acid pyridoxine), and myo-inositol. The latter is not a vitamin but used as one for plant culture media. Activated Charcoal (AC) Used for its ability to bind hydrophobic compounds which inhibit growth. The actual role isnt always clear nor is it always included in medium. Gelling Agents (support systems) Solidified surface typically from the complex carbohydrates (non-digestible) extracted from seaweed (agar). Lots of variation between batches and suppliers Gums from plants, agarose can also be used
33. 33. Plant Culture Medium Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. Auxins- Promote cell division and growth most auxins are synthetic and not found in plants. Naturally produced 1H-indol-3-acetic acid, is unstable to both heat and light. Naturally produced in apical and root meristems seeds and developing fruit Alters proton pump and ATP production in target cells Induces cell elongation Suppresses lateral bud growth and stimulates adventitious roots Synthetic form(s) include 2-4 dichorophenoxyacetic acid (2-4D) Acts as a herbicide by inducing unsustainable growth in broad leaf (dicot) weeds corn, rice and wheat all have one leaf (monocot). Can be used for trees to hold fruit for development 2-4-D
34. 34. Plant Culture Medium Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. Cytokinins Promote cell division and are produce in young leaves fruits and seeds. Used to stimulate cell division, induce shoot formation and auxiliary shoot proliferation while inhibiting root formation. Not good for stage III. Delays cell aging and increases as some fruits bloom and grow Used to induce bud growth in orchids and daylilies Prevents browning in salads When mixed with gibberellins can increase the size of a fruit (30-50% in pears and mangos) Zeatin first isolated from corn Kinetin first isolated herring spirm
35. 35. Plant Culture Medium Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. Ratio of Auxin and cytokinin control root formation Root initiation occurs when more auxin than cytokinin is in media and adventitious and shoot growth takes place when more cytokinin than auxin ratio
36. 36. Plant Culture Medium Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. Gibberellins & Abciscic acid- Regulate cell elongation and determine plant height. Gibberellins increase growth of low-density cultures, enhance callus growth and elongate dwarf plants Abscisic acid alters callus growth, enhance bud and shoot formation, and inhibit cell division. Commonly used in somatic embryogenesis
37. 37. Plant Culture Medium Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. Ethylene- volatile gas produced during ripening, stress, mechanical damage or infection. Produced from methyl group of methionine Nearly all plant tissues can produce Natural role is to encourage fruit ripening and flower blooming Used commercially to initiate flowering and ripen tomatoes, citrus and bananas why brown bags? Specific protein receptors for ethylene have been found which act as transcription factors Can be a problem in culture without proper air circulation H2C=CH2
38. 38. SOLUTIONS USED IN CELL CULTURE
39. 39. Phosphate Buffered Saline - Ca2+ Mg2+ Free (PBS) Used to wash/remove excess serum that inhibits the function of Trypsin-EDTA. trypsin is inhibited by serum Must be warmed in the water bath before use so cells are not shocked by cold liquid. PBS without Ca2+/Mg2+ wash adherent cell cultures before detaching them from the growing surface with trypsin.
40. 40. Trypsin EDTA An enzyme used to detach the cells from a culture dish. Trypsin cleaves peptide bonds (LYS or ARG) of the extracellular matrix. EDTA chelates calcium ions in the media that would normally inhibit the function of trypsin. Trypsin will self digest and become ineffective if left in water bath more than 20 minutes. Trypsinizing cells too long will reduce cell viability
41. 41. Trypan Blue An exclusion dye(dye exclusion method. ) Living cells cannot take up the dye and will appear bright and refractile. Live cells or tissues with intact cell membranes are not coloured. Dead cells with broken membranes will absorb the dye and appear blue. Usually add 200 l of trypan blue to 200 l of cell suspension in eppendorf tube
42. 42. Bleach Used to destroy any remaining cells in dishes and tubes before they are tossed in the trash can. Add enough to change media to clear, wait 5 minutes, rinse solution down sink throw away the dish/flask/plate in the trash can.
43. 43. 1.3 FIELDS OF APPLICATION, TECHNIQUES AND PRODUCTS
44. 44. CO2 incubator maintains CO2 level (5- 10%), humidity and temperature (37o C) to simulate in vivo conditions.
45. 45. Water bath To warm media PBS before placing on cells Can harbor fungi and bacteria, spray all items with 70% ethanol before placing in the hood. Usually takes 10 -15 minutes for media to warm, 5-10 min to thaw
46. 46. Vacuum pump For permanent aspiration of liquids (media, PBS). Use unplugged glass pasteur pipets, throw into sharps box when done.
47. 47. Inverted Phase Microscope A phase contrast microscope with objectives below the specimen. A phase plate with an annulus will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast
48. 48. Mechanics of phase microscopyMechanics of phase microscopy Shifting of phase by a wavelength Add and subtract amplitudes to create more contrast
49. 49. A comparison Phase contrast microscopy Light microscopy Can be used on living cells requires stain, thus killing cells
50. 50. Technique for cultivating animal cell
51. 51. How do people culture cells in the laboratory? Revive frozen cell population Isolate from tissue Maintain in culture (aseptic technique) Sub-culture (passaging) Cryopreservation Count cells Containment level 2 cell culture laboratory Typical cell culture flask Used to freeze cells
52. 52. Passaging Cells Why passage cells? To maintain cells in culture (i.e. dont overgrow) To increase cell number for experiments/storage How? 70-80% confluency Wash in PBS to remove dead cells and serum Trypsin digests protein-surface interaction to release cells (collagenase also useful) EDTA enhances trypsin activity Resuspend in serum (inactivates trypsin) Transfer dilute cell suspension to new flask (fresh media) Most cell lines will adhere in approx. 3-4 hours Check confluency of cells Remove spent medium Wash with PBS Resuspend in serum containing media Incubate with trypsin/EDTA Transfer to culture flask 70-80% confluence 100% confluence
53. 53. Passage cells Resuspend cells in serum containing media Centrifuge & Aspirate supernatant Transfer to cryovial Freeze at -80o C Resuspend cells in 10% DMSO in FCS Why cryopreserve cells? Reduced risk of microbial contamination. Reduced risk of cross contamination with other cell lines. Reduced risk of genetic drift and morphological changes. Research conducted using cells at consistent low passage. How? Log phase of growth and >90% viability Passage cells & pellet for media exchange Cryopreservant (DMSO) precise mechanism unknown but prevents ice crystal formation Freeze at -80o C rapid yet slow freezing Liquid nitrogen -196o C Transfer to liquid nitrogen storage tank Cryopreservation of Cells
54. 54. Contamination A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use. 1-Chemical Contamination Media Incubator Serum water 2-Biological Contamination Bacteria and yeast Viruses Mycoplasmas Cross-contamination by other cell culture How Can Cell Culture Contamination Be Controlled?
55. 55. Technique of cultivating animal cells Excise tissues from specific organ of animals (lung, kidney) under aseptic conditions. Transfer tissues into a growth medium containing serum and antibiotics in small T-flasks. These cells form a primary culture that usually attach onto the glass surface of flask in monolayer form. The cells growing on support surfaces are known as anchorage-dependent cells.
56. 56. Some cells grown in suspension culture and are known to be nonanchorage-dependent cells. Then a cell line appear from the primary culture and known as secondary culture. Remove cell from the surface of flasks using trypsin and add serum to the culture bottle. The serum containing suspension is then use to inoculate secondary cultures.
57. 57. Condition for animal cell culture
58. 58. Culture condition It is critical to find a HAPPY environment for cell cultures. Happy environment = allows cells to increase in number by undergoing cell division (mitosis). Provide the cells with appropriate temp, good substrate for attachment and proper culture medium.
59. 59. Temperature Usually set at the same point as the body temp of the host from which the cell obtained Cold-blooded vertebrates 18-25C Mammalian cells 36-37C Temp maintained by use of carefully calibrated and frequently checked incubators
60. 60. pH Most cells in culture grow best at pH 7.4 Common used buffer bicarbonate-CO2 or HEPES Keep the pH medium in a range 7-7.4 When using bicarbonate-CO2 buffer, need to regulate the amount of CO2 dissolved in the medium Done by using an incubator with CO2 control set to provide an atmosphere with between 2% and 10% CO2.
61. 61. Thank you