UNIT- 10 ANIMAL CELL CULTURE TECHNIQUES (3).pptx

1. History of Cell Culture
2. Introduction to cell culture
3. types of cell lines
4. culture media
5. serum in culture media
6. Applications of cell & tissue culture
7. Adherence
8. Cell line evolution
9. Passaging, revival and cryopreservation
10. cell culture laboratory layout

1. CELL CULTURE techniques

2. HISTORY OF CELL CULTURE Roux 1885: Embryonic chick cells were   maintained in saline Harrison 1907: Amphibian spinal cord cultivated in lymph produced axons in vitro Carrel 1913: Cells in a culture   can grow continuously under aseptic conditions Earle 1943: First continuous cell line from L-cell mouse fibroblast .  Gey et.al. , 1952 : Established continuous cell lines   derived from human cervical carcinoma – He La cells Levi Montaleini 1954: NGF stimulated the neuron growth. Eagle 1955: Developed by a systematic study a Basal medium of the nutrients and the serum for cell culture Puck et.al.,1956 Selected mutants with altered nutrient requirement Temin and Rubin 1958: Developed quantitative assay for infection of Rous sarcoma virus in cell culture

3. Hayflick and Moorehead 1961: Human fibroblasts in culture have finite life time Littlefield 1964: Introduced HAT medium for for selective growth of somatic cells Ham 1965: Introduced a defined serum free medium to support the clonal growth of certain mammalian cells Harris and Watkins 1965: produced first heterokaryon by virus indused fusion Augusti Tocco and Sato 1968: Neuroblastoma cultures Koehler and Milstein 1975: Monoclonal antibody secreting hybridoma cell line Sato et.al. 1976: Published the first series of papers defining the mixtures of hormones and growth factors for different cell lines in serum free media Wigler and Axel 1977: (Adopting method of Graham and Vander Eb ): Single copy mammalian gene in culture cells)

4. What is Tissue/Cell Culture? Refers to the removal of cells or tissues or organs from an animal or plant and their subsequent placement in a favorable artificial environment conducive to growth Environment usually consists of a suitable glass or plastic culture vessel containing a liquid or semisolid medium that supplies the nutrients essential for survival and growth. TYPES OF TISSUE CULTURE

5. How are cell cultures obtained Primary Culture Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). Explant culture Enzymatic dissociation

6. Cell Line After the first subculture, the primary culture becomes known as a cell line or subclone . Cell lines derived from primary cultures have a limited life span (i.e., they are finite) and as they are passaged , cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. Cell Strain If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain Buying and Borrowing Buy established cultures from organizations such as ATCC or Coriell institute of  Medical Research or RIKEN cell bank or National Centre for Cellular Sciences (NCCS) 

7. What are cultured cells like?

8. Types of Cells Cultured cells are usually described based on their morphology (shape and appearance) or their functional characteristics. There are three basic morphologies: Epithelial-like : cells that are attached to a substrate and appear flattened and polygonal in shape. 2. Lymphoblast-like : cells that do not attach normally to a substrate but remain in suspension with a spherical shape. 3. Fibroblast-like : cells that are attached to a substrate and      appear elongated and bipolar, frequently forming swirls in heavy cultures

9. Finite vs Continuous Cell Line Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence, these cell lines are known as finite. However, some cell lines become immortal through a process called transformation, which can occur spontaneously or can be chemically or virally induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line .

10. properties finite continuous Finite versus Continuous cell lines:

11. The artificial environment created in the laboratory is generally known as media. A media comprises an appropriate source of energy for the cells which they can easily utilize and compounds which regulate the cell cycle. The choice of media is cell type specific and often empirical and there is no “all purpose” medium. It should provide many nutrients, buffering capacity, isotonic, and should be sterile. Characteristics and compositions of the cell culture media vary depending on the particular cellular requirements as shown in Table 1 Culture media

12. Most animal cell culture media are generally having following 10 basic components and they are as follows: 1. Energy sources: Glucose, Fructose, Amino acids 2. Nitrogen sources: Amino acids 3. Vitamins: Generally water soluble vitamines B & C 4. Inorganic salts: Na+, K+, Ca2+, Mg2+ 5. Fat and Fat soluble components: Fatty acids, cholesterols 6. Nucleic acid precursors 7. Antibiotics 8. Growth factors and hormones 9. pH and buffering systems 10. Oxygen and CO2 concentration. Basic components in the culture media

13. Media used for tissue culture may be grouped into two broad categories: 1. Natural media 2. Artificial media. The choice of medium depends mainly on the type of cells to be cultured (normal, immortalized or transformed), and the objective of culture (growth, survival, differentiation, production of desired proteins). Non-transformed or normal cells (finite life span) and primary cultures from healthy tissues require defined quantities of proteins, growth factors and hormones. But immortalized cells (spontaneously or by transfection with viral sequences) produce most of these factors, but may still need some of the growth factors present in the serum. In contrast, transformed cells (autonomous growth control and malignant properties) synthesize their own growth factors; in fact, addition of growth factors may even be detrimental in such cases. But even these cultures may require factors like insulin, transferrin, silenite, lipids, etc. Types of Media

14. Natural Media These media consist solely of naturally occurring biological fluids and are of the following three types: 1. Coagula or clots 2. Biological fluids 3. Tissue extracts. Artificial Media Different artificial media have been devised to serve one of the following purposes: 1. Immediate survival (a balanced salt solution, with specified pH and osmotic pressure is adequate), 2. Prolonged survival (a balanced salt solution supplemented with serum, or with suitable formulation of organic compounds), 3. Indefinite growth 4. Specialized functions.

15. The various artificial media developed for cell cultures may be grouped into the following four classes: ( i ) Serum containing media ( ii) Serum free media ( iii) Chemically defined media ( iv) Protein free media. SERUM Liquid yellowish, clear content left over after fibrin and cells are removed from the blood is known as serum. Calf (bovine), foetal bovine, or horse are used, in some cases human. Fetal bovine serum (FBS) (10-20% v/v) is the most commonly applied supplement in animal cell culture media.

16. Contents of Serum

17. Functions of Serum in the Culture Medium It provides the basic nutrients for cells ; the nutrients are present both in the solution as well as are bound to the proteins. It provides several hormones, e.g., insulin , which is essential for growth of nearly all cells in culture, cortisone, testosterone, prostaglandin, etc. It contains several growth factors, e.g., platelet derived growth factor (PDGF), transforming growth factor β (TGF-β), epidermal growth factor, etc.; these are present in concentrations of μ g/l. A major role of serum is to supply proteins, e.g., fibronectin, which promote attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they can begin to divide.

18. It provides several binding proteins, e.g., albumin, transferrin, etc., which carry other molecules into the cell. For example, albumin carries into cells lipids, vitamins, hormones, etc. Transferrin usually carries Fe in a non-basic form, but binding of transferrin to its receptor in cell membrane is believed to be mitogenic. It increases the viscosity of medium and thereby, protects cells from mechanical damages, e.g., shear forces during agitation of suspension cultures.  Protease inhibitors present in the serum protect cells, especially trypsinised cells, from proteolysis. The serum also provides minerals, like Na+, K+, Fe2+, Zn2+, etc. It also acts as a buffer.

19. Media should provide many nutrients, buffering capacity, isotonic and sterile. Main components are: Energy sources: Glucose, Fructose, Amino acids. Nitrogen sources: Amino acids. Vitamins: Water soluble vitamin B & C. Inorganic salts: Na + , K + , Ca 2+ , Mg 2+ . Fat and Fat soluble components: Fatty acids, Cholesterols. Nucleic acid precursors. Antibiotics. Growth factors and hormones. pH and buffering systems. Oxygen and CO 2 concentration. COMPONENTS OF MEDIUM MEDIA SUPPLEMENTS: 1.L-Glutamine 200mM, 2.Fetal Bovine serum 10%v/v, 3.HEPES- 25mM, 4.Antibiotics.

20. CONSTITUENTS OF SERUM Proteins and Polypeptides Albumin Fetuinb Fibronectin Globulins Protease inhibitors: α1- antitrypsin, α2- macroglobulin. Transferrin . Growth factors: EGF, PDGF, IGF-I and -II, FGF, IL-1, IL-6. Amino acids Lipids Cholesterol Fatty acids Linoleic acid Phospholipids. Hormones: Hydrocortisone Insulin Triiodothyronine Thyroxine . Vitamins: Vitamin A Folate . Polyamines: Putrescine , spermidine . Urea Inorganics : Calcium Chlorides Iron Potassium Phosphate Selenium Sodium Zinc. Carbohydrates Glucose Hexosamine Lactic acid Pyruvic acid

21. The chemical composition of these supplements may vary between lots, even from a single manufacturer. The supplements of animal or human origin may also be contaminated with infectious agents (e.g., mycoplasma and viruses) which can seriously undermine the health of the cultured cells when these contaminated supplements are used in cell culture media formulations and may pose a health risk in cell therapy and other clinical applications.  A major fear is the presence of prions causing spongiform encephalopathy in humans or animals.  Cell surface chemistry, which is a critical portion of the in vitro microenvironment for many cell types, can be adversely modified via  adsorption or incorporation of serum or extract proteins. Limitation of Media with Serum

22. The use of undefined components such as serum or animal extracts also prevents the true definition and elucidation of the nutritional and hormonal requirements of the cultured cells, thus eliminating the ability to study, in a controlled way, the effect of specific growth factors or nutrients on cell growth and differentiation in culture. Undefined supplements prevent the researcher from studying aberrant growth and differentiation and the disease related changes in cultured cells. Using cell culture media in the industrial production of biological substances, serum and animal extract supplementation of culture media can also complicate and increase the costs of the purification of the desired substances from the culture media due to nonspecific copurification of serum or extract proteins.

23. DISADVANTAGES OF SERUM 1.Physiological Variability: Reason : components include nutrients (amino acids, nucleosides, sugars, etc.), peptide growth factors, hormones, minerals and lipids, the concentrations and actions of which have not been fully determined. 2.Shelf Life and Consistency:   Serum deteriorating during the storage time. So It must be replaced with another batch that may be selected as similar, but will never be identical, to the first batch.. 3.Quality Control: Extensive testing to ensure that the replacement is as close as possible to the previous batch. 4.Specificity: If more than one cell type is used, each type may require a different batch of serum, so that several batches must be held on reserve simultaneously. 5.Availability: the supply of serum is restricted now-a-days because of drought in the cattle-rearing areas, the spread of disease among the cattle, or economic or political reasons.

24. 6.Downstream Processing:  The presence of serum creates a major obstacle to product purification 7.Contamination: Serum is frequently contaminated with viruses, 8.Cost: serum constitutes the major part of the cost of a bottle of medium (more than 10 times the cost of the chemical constituents) 9.Growth Inhibitors: As well as its growth-promoting activity, serum contains growth-inhibiting activity, and although stimulation usually predominates, the net effect of the serum is an unpredictable combination of both inhibition and stimulation of growth. 10.Standardization: Standardization of experimental and production protocols is difficult, both at different times and among different laboratories, because of batch-to batch variations in serum. All of the above problems can be eliminated by removal of serum and specific media with required factors can be used, thus there will be control over the proliferation and differentiation.

25. SERUM-FREE MEDIA Media, which often are specifically formulated to support the culture of a single cell type, incorporate defined quantities of purified growth factors, lipoproteins and other proteins usually provided by the serum or extract supplement. Since the components (and concentrations thereof) in such culture media are precisely known, these media are generally referred to as “defined culture media” and often as “serum-free media” or “SFM.” Use of SFM facilitates the investigation of the effects of a specific growth factor or other medium component on cellular physiology, which may be masked when the cells are cultivated in serum- or extract-containing Media SFM typically contain much lower quantities of protein (indeed, SFM are often termed “low protein media”) than those containing serum or extracts, rendering purification of biological substances produced by cells cultured in SFM far simpler and more cost-effective. 

26. DISADVANTAGES OF SERUM-FREE MEDIA: Multiplicity of Media   Each cell type appears to require a different recipe tumors may vary in requirements from tumor to tumor, even within one class of tumors. It presents a problem for laboratories maintaining cell lines of several different origins. Selectivity   Some media may select a sub-lineage and even in continuous cell lines, some degree of selection is required.    Cells at different stages of development (e.g. stem cells vs. committed precursor cells) may require different formulations, particularly in the growth factor and cytokine components. Expensive:   As the removal of serum also removes the protective, detoxifying action so, high degree of aseptic conditions must be maintained. Purity of the reagents and water must be high. Cell Proliferation : Growth is often slower in serum-free media. Availability: Although improving steadily, the availability of properly quality-controlled serum-free media is limited.

27. PHYSICOCHEMICAL PROPERTIES OF CULTURE MEDIUM 1. pH : Most cell lines grow well at pH 7.4. Transformed cells may do better at ph 7.0–7.4. Phenol red is commonly used as an indicator. It shows- More pink at pH 7.6, Purple at pH 7.8 Red at pH 7.4, Orange at pH 7.0, Yellow at pH 6.5, Lemon yellow below pH 6.5. 2. CARBON DIOXIDE : To maintain buffering capacity of the medium and to compensate the lost dissolved gas from medium. Generally 0-10% CO2 is supplied. 5% gas supply from cylinders is widely used. CO2 levels are measured by FYRITE UNIT. DISADVANTAGE Phenol red has weak estrogenic activity.

28. 3. BUFFERING : It is required under two conditions. (1) open dishes, wherein the evolution of co2 causes the pH to rise and (2) overproduction of CO2 and lactic acid in transformed cell lines at high cell concentrations, when the pH will fall. Widely used buffers: 1.Bicarbonate buffer. 2.Tris buffer. 3.Zwitter ionic buffer (HEPES). (N-2-Hydroxy ethyl piperazine-N-2-ethane sulfonicacid ) Low Toxicity, Low Cost, and Nutritionally Benefit. 4. OSMOLALITY Osmolalities between 260 mosmol /kg and 320 mosmol /kg are quite acceptable for most cells but, once selected, should be kept consistent at ±10 mosmol /kg. Slightly hypotonic medium may be better for petri dish or open-plate culture to compensate for evaporation during incubation. Modifiers : NaCl , Dextrose

29. 5. OXYGEN : As there is no oxygen carrier (Hb)cells rely chiefly on dissolved O2, which can be toxic due to the elevation in the level of free radicals. Providing the correct O2 tension is therefore, always a compromise between fulfilling the respiratory requirement and avoiding toxicity.  So free radical scavengers, such as Glutathione,                                    2-Mercaptoethanol ( β- mercaptoethanol),                                                              or Dithiothreitol , are added into the medium. SELENIUM is incorporated into the medium which is cofactor for GLUTATHIONE thus avoid toxicity. 6. TEMPERATURE The temperature recommended for most human and warm-blooded animal cell lines is 37◦C, close to body heat. Cultured mammalian cells will tolerate considerable drops in temperature, can survive several days at 4◦c, and can be frozen and cooled to −196◦c.

30. MEDIUM APPLICATION AUTHORS Basal medium Growing cells with serum Eagle 1965 Minimal essential medium (MEM) Growing cells with dialyzed serum Eagle 1959 Dulbecco’s modified Eagle’s medium Virus transfected cells, high density growth with serum Dulbecco and Freeman 1959 Ham’s F10 medium Chick embryo cells, serum Ham 1963 RPMI 1640 Human leukemic cells and Hybridomas Moore and Kitamura 1968 Mc Coy’s5A medium Human lymphocytes Mc Coy et.al 1959 Neuroblast medium CNS Neurons Brewer et al 1994 MCDB 131 Human endothelium Knedler and Ham 1987 Medium199 Chick embryo fibroblasts Morgal et.al 1950 SOME COMMON COMMERCIALLY AVAILABLE MEDIA DMEM (Dulbecco's Modified Eagle Medium) Adherent culture. RPMI (Roswell Park Memorial Institute medium ) Suspension culture.

31. The three basic classes of media are: Basal media, Reduced-serum media, Serum-free media. Differ in their requirement for supplementation with serum. BASAL MEDIA The majority of cell lines grow well in basal media, which contain amino acids, vitamins, inorganic salts, and a carbon source such as glucose, but these basal media formulations must be further supplemented with serum. REDUCED-SERUM MEDIA Another strategy to reduce the undesired effects of serum in cell culture experiments is to use reduced-serum media. Reduced-serum media are basal media formulations enriched with nutrients and animal-derived factors, which reduce the amount of serum that is needed. SERUM-FREE MEDIA: Replacing the serum with appropriate nutritional and hormonal formulations. TYPES OF MEDIUM

32. ROLE OF MEDIUM Nutrient support Maintenance of pH and osmolality Buffering system Growth factors Metabolic support Antioxidants and reducing agents Most media (MEM and DMEM) were developed with serum supplementation Some are tailored without the serum support to study the specific effects of defined growth factors “Special Use Media” are now commercially available for specific cell lines and contain undisclosed components

33. Applications of tissue CULTURE

34. CATEGORY ADVANTAGES ADVANTAGES OF TISSUE CULTURE

35. DISADVANTAGES OF TISSUE CULTURE CATEGORY EXAMPLES

36. TYPES OF TISSUE CULTURE ORGAN CULTURE: Three dimensional culture of un-dis-aggregated tissue retaining some or all of the histological features of tissue in vivo. CELL CULTURE: Culture derived from dispersed cells taken from the original tissue, dispersed by enzymatic, mechanical or chemical dis aggregation. The source of cells can also be from primary cultures already developed else where. HISTOTYPIC CULTURE: Reaggregation of cells into a three dimensional tissue like structure by cultivation of high density cells in artificial media or matrix (e.g. Collagen gel). ORGANOTYPIC CULTURE: Recombining cells of different lineages into organ like structures.

37. BIOLOGY OF CULTURED CELLS Good environment of culturing cells is based on: 1.Nature of substrate. 2.Degree of contact with other cells. 3.Constitution of medium. 4.Constitution of gas. 5.Incubation temperature. Cadherins Integrins Lminin , Fibronectin , Hyaluronan Transmembrane proteoglycans .

38. Cell–cell adhesion molecules Cell–substrate interactions Cell Adhesion Molecules ( CAM s) are proteins located on the cell surface involved with the binding with other cells or with the extracellular matrix(ECM) in the process called cell adhesion. INTEGRINS: Are the receptors for matrix molecules such as fibronectin , entactin , laminin and collagen which bind to them via a specific motif usually containing the arginine – glycine –aspartic acid (RGD) Sequence. Each integrin comprises one α and one β subunit, the extracellular domains of which are highly polymorphic, thus generating considerable diversity among the integrin . These proteins are self-interactive; that is, homologous molecules in opposing cells interact with each other E- cadherin ------ Epithelia cell N- cadherin ------ Nerve cell P- cadherin -------Placenta Selectins : which binds to the fucosylated receptors CD34, GlyCAM-1( glycosylation dependent cell adhesion molecule-1), PSGL-1(P- selectin glycoprotein ligand-1). E- Selectin L- Selectin

39. CADHERINS INTEGRINS

40. Cell Adhesion

41. Evolution of a Cell Line Lag Phase : The time the cell population takes to recover from sub culture, attach to the culture vessel Log Phase: cell number begins to increase exponentially. Plateau Phase : growth rate slows or stops due to exhaustion of growth medium or confluency

42. Preparation of primary culture

43. Adherent Suspension Culture There are two basic systems for growing cells in culture, as monolayers on an artificial substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture).

44. REPLACEMENT OF MEDIUM OR FEEDING A Drop in pH The rate of fall and absolute level should be considered. Most cells stop growing as the pH falls from pH 7.0 to pH 6.5 and start to lose viability between pH 6.5 and pH 6.0, so if the medium goes from red through orange to yellow, the medium should be changed. Cell Concentration Cell Type Morphological Deterioration.

45. Subculturing or passaging Subculturing is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain. When to Subculture? Density of Culture Time Since Last Subculture Exhaustion of Medium. Requirements for Other Procedures

46. Subculturing of monolayer

47. Subculturing of suspension culture

48. (a) Bacteria. (b) Yeast . (c) Mold. (d) Mycoplasma Types of Contamination

49. Characteristic features of microbial contamination are as follows: (1) A sudden change in pH, usually a decrease with most bacterial infections, very little change with yeast  until the contamination is heavy, and sometimes an increase in pH with fungal contamination. (2) Cloudiness in the medium , sometimes with a slight film or scum on the surface or spots on the growth surface that dissipate when the flask is moved. (3) Under a low-power microscope (∼×100), spaces between cells will appear granular and may shimmer with bacterial contamination. (4) Under high-power microscopy (∼×400), it may be possible to resolve individual bacteria and distinguish between rods and cocci.

50. ROUTES TO CONTAMINATION Technique manipulations, pipetting , dispensing, etc. Work Operator hair, hands, breath, clothing surface. Materials and reagents Solutions. Glassware and screw caps. Culture flasks and media bottles in use. Equipment and Facilities Room air. Laminar-Flow Hoods. CO2, humidified incubators. Importation of Biological Materials Tissue samples. Incoming cell lines.

51. Cell lines in continuous culture are prone to variation due to selection in early-passage culture senescence in finite cell lines and genetic and phenotypic instability in continuous cell lines. why cryopreservation has to be done? Reasons are as follows: (1) Genotypic drift due to genetic instability. (2) Senescence and the resultant extinction of the cell line. (3) Transformation of growth characteristics and acquisition. of malignancy-associated properties. (4) Phenotypic instability due to selection and dedifferentiation. (5) Contamination by microorganisms . (6) Cross-contamination by other cell lines. (7) Misidentification due to careless handling. (8) Incubator failure. (9) Saving time and materials maintaining lines not in immediate use. (10) Need for distribution to other users. Cryopreservation

52. PRINCIPLES OF CRYOPRESERVATION Optimal freezing of cells for maximum viable recovery on thawing. This is achieved (a) by freezing slowly to allow water to leave the cell but not so slowly that ice crystal growth is encouraged, (b) by using a hydrophilic cryoprotectant to sequester water, (c) by storing the cells at the lowest possible temperature to minimize the effects of high salt concentrations on protein denaturation in micelles within the ice, and (d) by thawing rapidly to minimize ice crystal growth and generation of solute gradients formed as the residual intracellular ice melts. Cell Concentration Normally at 1 × 10 5 /mL, 1 × 10 7 should be frozen in 1 mL of medium, and, after thawing the cells, the whole 1 mL should be diluted to 20 mL of medium, giving 5 × 10 5 cells/mL .

53. Freezing Medium Dimethyl sulphoxide (DMSO)- widely used. Glycerol, Polyvinyl pyrrolidine (PVP), Polyethylene glycol (PEG), Hydroxy Ethyl Starch (HES), Trehalose . DMSO : drawbacks Should be colorless. Stored in glass containers if not it will leach impurities from plastic or rubber. Should be diluted if not toxic to cells. Glycerol : On long storage toxic to cell lines. Widely used freezing mixture 40%v/v medium with 10%serum, 40%v/v FCS, 20%V/V DMSO/Glycerol.

54. Procedure for freezing cells

55. Thawing Cells

56. LABORATORY LAYOUT, EQUIPMENT AND MATERIALS

57. Medium-Sized Tissue Culture Laboratory layout

58. Medium-Sized Tissue Culture Laboratory layout

59. Layout for open work bench in laminar airflow hood

60. EQUIPMENT AND MATERIALS REQUIRED ASEPTIC AREA: Laminar flow hoods, Inverted microscope, Centrifuge, Waterbath , Refrigerator, Freezer (-20 C, -80 C), Liquid nitrogen freezers, Trolleys and carts, INCUBATORS: Normal incubator, Humid CO 2 Incubator, Hemocytometer slides, Cell counter. Mr. Frosty WASH UP AREA: Washing sink, Pipette washer, Pipette drier, Glassware washer, MEDIA PREPARATION: Millipore water purifier, Suction pump, Conductivity meter, Magnetic stirrers, Digital balance, P H meter, Osmometer . STERILIZATION EQUIPMENT: Autoclave, Hot air oven, Membrane filters. CONSUMABLES: Pipettes, Culture flasks, Petri dishes, Multiwell plates, Tip and tipboxes , Centrifuge tubes, Glass bottles and measuring cylinders.

61. Cell Culture Hoods unidirectional flow of HEPA-filtered air over the work area. HORIZONTAL Blowing parallel to the work surface. Provides protection to the culture (if the air flowing towards the user) or to the user (if the air is drawn in through the front of the cabinet by negative air pressure inside). VERTICAL Blowing from the top of the cabinet onto the work surface. Vertical flow hoods also provide significant protection to the user and the cell culture.

62. HORIZONTAL VERTICAL

63. Dry incubators are more economical but require the cell cultures to be incubated in sealed flasks to prevent evaporation. Placing a water dish in a dry incubator can provide some humidity, but they do not allow precise control of atmospheric conditions in the incubator. Humid CO2 incubators are more expensive, but allow superior control of culture conditions. They can be used to incubate cells cultured in Petri dishes or multi-well plates, which require a controlled atmosphere of high humidity and increased CO2 tension. DRY INCUBATOR HUMID CO2 INCUBATOR

64. STERILIZATION EQUIPMENT: FREESTANDING AUTOCLAVE HOT AIR OVEN

65. SPECIALIZED EQUIPMENT Flow cytometer RT PCR Colony counter

66. Culture vessels and their characteristics

67. SUBSTRATES ATTACHMENT FOR THE GROWTH AND SPREADING Cells shown to require attachment for growth are said to be ‘ anchorage dependent’. cells that have undergone transformation and can grow in suspension are said to be ‘ anchorage independent’. SUBSTRATE MATERIALS Disposable plastic: 1.Single-use sterile polystyrene flasks : They are usually of good optical quality. The growth surface is flat, Provides uniformly distributed and reproducible monolayer cultures. As manufactured, polystyrene is hydrophobic and does not provide a suitable surface for cell attachment, so tissue culture plastics are treated by corona discharge, gas plasma, or γ -irradiation, or chemically, to produce a charged, wettable surface.

68. 2.Polytetrafluorethylene (PTFE; Teflon): Two forms charged (hydrophilic) uncharged (hydrophobic) monolayer cells, organotypic culture. Suspension cultures, macrophages. Others: polyvinylchloride (PVC), polycarbonate, Melinex , Thermanox (TPX), Polymethyl methacrylate . Fibres : Rayon, Nylon, Poly-l-lactic acid (PLA), Polyglycolic acid. Derivatized substrates with: RGD tripeptide , N- Hydroxy sulphosuccimide etc.

69. TREATED SURFACES 1.Extracellular matrix (ECM) Some cell lines like 3T3 or MRC-5 cells (mouse & human fibroblasts respectively) produce ECM. When flask is washed with triton x-100 the ecm will be remained. These flasks can be used for culturing cell lines of our interest. 2.Substrate coating: Treat the surface with collagen, fibronectin , laminin . 3.Commercially available matices : Matrigel , Natrix , Laminin . Suspension cultures: Attatchment is not required but uniform distribution of cell lines must be there i.e , no sedimentation should occur. So suspending agents are used. Ex : Agar, Agarose , Methocel

70. GOOD ASEPTIC TECHNIQUES:

71. GOOD ASEPTIC TECHNIQUES:

72. METHODS OF STERILIZATION

73. STERILE FILTRATION Filtration through 0.1- to 0.2-μm microporous filters is the method of choice for sterilizing heat-labile solutions

74. REFERENCES Ian Freshney Culture of Animal Cells A Manual of Basic Technique, 5th Edition. Handbook for cell culture basics ( Gibco ).