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Cell culture

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Effective information about culturing cell in outside the cell which is now a days big issue on medical research...
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Cell culture

  1. 1. CELL CULTURE AND CELL LINES AND ITS IMPLICATION IN FISHERY A SEMINAR ON BY: ASIK IKBAL 3RD YR B.F.Sc , WBUAFS
  2. 2.  Cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment.  Cell culture refers to a culture derived from dispersed cells taken from original tissue, from a primary culture, or from a cell line or cell strain by enzymatic, mechanical, or chemical disaggregation.  They require------- ongoing care, ------- adequate nutrition, ------- a proper environment and ------- regular checkups. Introduction Cell culture
  3. 3.  Fish cells removed from tissues, will continue to grow if supplied with the appropriate nutrients and conditions.  When it is carried out in a laboratory, the process is called Cell Culture.  cell culture became a common laboratory technique in the mid-1900s, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.  However, there are also cultures of plants, fungi and microbes, including viruses, bacteria and protists. Introduction
  4. 4. History • 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. • Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. • The research of fish cell culture has developed rapidly since Wolf and Quimby established the first fish cell line RTG-2 in 1960s for the first time. • After then, fish cell culture has become an essential research technology which has been used extensively.
  5. 5. 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.
  6. 6. 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  Cancer research  Virology  Genetic Engineering  Gene therapy Why is cell culture used for?
  7. 7. 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 tissues grown in culture include connective tissue elements such as fibroblasts, skeletal tissue, cardiac tissue, epithelial tissue (liver, skin, kidney) and many different types of tumor cells.
  8. 8. Primary culture  Cells, when surgically or enzymatically removed from an organism and placed in suitable culture environment, will attach and grow and it is 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.  Lineage of cells originating from the primary culture is called a cell strain. Cell culture
  9. 9. Continuous cell lines  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 continuous cell lines.  Characteristics of continuous 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 donor tissue. -stop expressing tissue specific genes. Established human cell lines Fish cell lines
  10. 10. 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.
  11. 11. Types of cell Cultures  Cell cultures may contain the following three types of cells:  1. Stem cells,  2. Precursor cells and  3. Differentiated cells  Stem cells- they are undifferentiated cells which can differentiate under correct inducing conditions into one of several kinds of cells; different kinds of stem cells differ markedly in terms of the kinds of cells they will differentiate into.  Precursor cells- these cells are derived from stem cells, are committed to differentiation, but are not yet differentiated; these cells retain the capacity for proliferation.  Differentiated cells- they usually do not have the capacity to divide.  Some cell cultures, e.g., epidermal keratinocyte cultures, contain all the three types of cells.
  12. 12.  Cell cultures can be grown as:  Cells need a surface or substrate to adhere for proliferation.  Cells that are unable to adhere to a substrate are unable to divide, i.e., their growth is anchorage dependent.  The various kinds of substrates used in cell cultures are(1) glass, (2) plastics, (3) metals. Cell culture
  13. 13. Suspension culture • At this stage, cells are usually heterogeneous but still closely represent the parent cell types as well as in the expression of tissue specific properties. • After several sub-cultures onto fresh media, the cell line will either die out or 'transform' to become a continuous cell line. • Such cell lines show many alterations from the primary cultures including change in morphology, chromosomal variation. • Fish cells can be grown either in an unattached suspension culture or attached to a solid surface. • It has been successfully developed to quite large bioreactor volumes, with successful production of viruses and therapeutic proteins.
  14. 14. Types of Suspension culture  Suspension cultures are of following types:  1. Batch Culture (fixed medium volume; as the cell grow, medium is gradually depleted; eventually cells cease to divide),  2. Fed Batch Culture (gradual addition of fresh medium leading to an increase in culture volume),  3. Semi-continuous Batch Culture (at regular intervals, a constant fraction of the culture, including cells, is withdrawn and an equal volume of fresh medium is added to the culture),  4. Perfmion Culture (at regular intervals, a constant volume of spent medium, without cells, is withdrawn and an equal volume of fresh medium is added); and  5. Continuous-flow Culture (continuous withdrawl of culture along with cells and addition of equal volume of fresh medium so that the culture is maintained in a steady state).
  15. 15. Media and supplements used in fish cell culture  The nutrient media used for culture of animal cells and tissues must be able to support their survival as well as growth, i.e., must provide nutritional, hormonal and stromal factors. The various types of media used for tissue culture may be grouped into two broad categories: 1. Natural Media and 2. Artificial Media. Culture media
  16. 16. Natural media  These media consist solely of naturally occurring biological fluids and are of the following three types:  (1) cagula or clots,  (2) biological fluids and  (3) tissue extracts.  Biological Fluids----various biological fluids used as culture medium (e.g., amniotic fluid, ascitic and pleural fluid, aqueous humour from eye, insect haemolymph, serum etc.). Serum is the most widely used biological fluids.
  17. 17. 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, and  (4) specialized functions.
  18. 18. Artificial Media  The various artificial media developed for cell cultures may be grouped as:  (i) serum containing media  (ii) serum-free media,
  19. 19. Serum Containing Media  The various defined media, (e.g., Eagle's minimum essential medium etc.) when supplemented with 5- 20% serum are good nutrient media for culture of most types of cells.  It provides the basic nutrients for cells; the nutrients are present both in the solution as well as are bound to the proteins.  A major role of serum is to supply proteins (e.g,fibrobnectin), 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.
  20. 20. Serum-free media Various defined serum free media is used due to  1. Improved reproducibility of results  2.variation due to batch change of serum is avoided.  3. Easier downstream processing of products from cultured cells.  4. Toxic effects of serum are avoided.  5. Biassays are free from interference due to serum proteins.
  21. 21. Serum-free media  Specific media like Leibovitz L15 is used to eliminate the need of adding CO2 and NaHCO3.  After the initiation of primary culture, fish serum is added at 1% final concentration.
  22. 22. Basic Constituents of media Inorganic salts Carbohydrates Amino Acids Vitamins Fatty acids and lipids Proteins and peptides
  23. 23. Basic equipments used in cell culture Laminar cabinet Incubation facilities Refrigerators Microscope Tissue culture ware
  24. 24. Buffering Systems •Most cells require pH conditions in the range 7.2 - 7.4 and close control of pH is essential for optimum culture conditions (however there are major variations to this optimum). •Fibroblasts prefer a higher pH (7.4 - 7.7) whereas, continuous transformed cell lines require more acid conditions pH (7.0 - 7.4).
  25. 25. Buffering Systems •Regulation of pH is particularly important immediately following cell seeding when a new culture is establishing and is usually achieved by one of two buffering systems; •A "natural" buffering system where gaseous CO2 balances with the CO3 / HCO3 content of the culture medium and •Chemical buffering using a zwitterions.
  26. 26. Explant culture • It is a technique used for the isolation of cells from a piece or pieces of tissue. Tissue harvested in this manner is called an explant. • It can be a portion of the shoot, leaves, or some cells from a plant, and can be any part of the tissue from an animal. • Primary cultures are derived directly from normal animal tissue and cultured either as an explant culture or dissociation into a single cell suspension by enzyme digestion.
  27. 27. Explant Preparation  Preparation of donor fish:  The donor fish is usually starved for a day or two to reduce the possibility of gross contamination from feces and unconsumed feed.  During this period, fish is allowed to swim in well aerated autoclaved water for reducing the microbial load adhered on to the skin and gills, then sacrificed by plunging in ice for 10-15 min.  Adequate care should be taken to minimize the possible routes of contamination.
  28. 28. Explant Preparation  Decontamination:  The decontaminating solutions includes chlorine solution (500 ppm), 70% ethanol, iodophore solution (0.5 w/v iodine) etc. are used.  Strong disinfectants are avoided because they damage the tissue.  The commonly used antibiotics are penicillin (400 IU/ml) and streptomycin (400 μg / ml) with an anti- fungal amphotericin B (10 μg/ml).  The tissue of interest is aseptically picked up and washed three to five times with the antibiotic solution.
  29. 29. Explant Preparation  Dissection and / or Disagregation: Two major methods for initiating a primary culture followed are given below.  (i) Tissues are disaggregated into its component cells. This is achieved by enzymatic digestion using trypsin or collagenase supplemented with EDTA.  These enzymes digest the extra celluar matrix and EDTA chelates divalent cations like Ca2+ and Mg2+ required for the integrity of the matrix.  The tissue was also dispersed by mechanical means like slicing, sieving, forcing through a needle and repeated pipetting.  The collected cells are then seeded in the vessel at 5 x 105 cells per ml of medium.
  30. 30. Explant Preparation  (ii) The required tissue is picked up and cut in small pieces to prepare explants of 1 mm3 size and planted in culture dishes.  1 or 2 fragments of tissue are seeded in 1 cm2 area.  The caudal fin, heart and gills are aseptically excised from fingerlings are rinsed individually with phosphate-buffered saline (PBS), 70% ethanol and Iodine antiseptic (0.5% w/v iodine).  The head and gut of the fry should be carefully discarded while the remaining tissue mass is washed as above.  Explants of 1mm3 sizes are prepared and washed thrice with PBS containing antibiotics for 5-10min.
  31. 31. Culture of cells following seeding  The explants are seeded in 25 cm2 tissue culture flask and kept semi dry for a few minutes.  The adherence of explants is accomplished by incubation with 0.5 mL of PBS at 28° C. After 8-10 hrs, L-15 (Leibovitz) is added gently. 50% of the media is recommended to be exchanged once in every 3 days. Daily observations are made using an inverted microscope. The dispersed cells adhere on the culture substrate and start to proliferate.
  32. 32. Culture of cells following seeding  Suspended dead cells can be removed by subsequent medium exchange. The optimum pH should be maintained nearly 7.4 and incubation temperature should be maintained 22-28° C for culture of fish cells.  Suspension cells are anchorage-independent e.g. blood cells.  Transformed cell lines either grows as monolayer or as suspension.
  33. 33. Sub-culturing • Once the available substrate surface is covered by cells (a confluent culture) growth slows & ceases. • Cells to be kept in healthy & in growing state sub-cultured or passaged has been done. • Enzyme such as trypsin, dipase, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surface. • Cells intolerant to trypsin can be scraped using a cell scraper or dispersed by rocking followed by gentle pipetting. • Detached cells can then be distributed to 2 to 4 flasks containing fresh medium depending on the split ratio required.
  34. 34. Growth pattern  Cells initially goes through a quiescent or lag phase that depends on the cell type, the seeding density, the media components, and previous handling.  The cells will then go into exponential growth where they have the highest metabolic activity.  The cells will then enter into stationary phase where the number of cells is constant, this is characteristic of a confluent population. Cell Culture of fin of L. rohita
  35. 35. Maintenance Cultures should be examined daily, observing the morphology, the color of the medium and the density of the cells. Epithelial cell culture
  36. 36. Harvesting  Cells are harvested when they reach a population density which suppresses growth.  Ideally, cells are harvested when they are in a semi-confluent state and are still in log phase. Cultured cells
  37. 37.  Cell viability is determined by staining the cells with trypan blue.  As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to viable cells.  The cells are stained with trypan dye and loaded to haemocytometer and calculation of % of viable cells is done. Number of unstained cells x 100 % of viable cells= total number of cells Cell viability
  38. 38. Cell culture contaminants of two types- Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible. Biological-causes visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines.
  39. 39.  Cytotoxicity causes inhibition of cell growth.  It effects on the morphological alteration in the cell layer or cell shape.  Cytotoxicity is determined by substituting materials such as medium, serum, supplements flasks etc. at a time.
  40. 40. BASIC ASEPTIC CONDITIONS TO BE MAINTAINED IN THE CELL CULTURE LABS  If working on the bench uses 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.
  41. 41. Applications for Fish cell cultures  To investigate the normal physiology or biochemistry of cells. For instance, studies of cell metabolism.  To test the effect of various chemical compounds or drugs on specific cell types (normal or cancerous cells, for example).  To study the sequential or parallel combination of various cell types to generate artificial tissues.  Therapeutic proteins can be synthesized in large quantities by growing genetically engineered cells in large-scale cultures.  Creation of viral vaccines from large scale cell cultures.  Cytotoxicity and genotoxicity studies.
  42. 42. Advantages of cell culture  The major advantage of using cell culture is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.  The materials are cheap and easy to obtain.  The experimental condition could be controlled accurately.  Can control all external factors.  Can easily test what the cells are doing.  Cells are easy to manipulate and propagate.  All of the cells are the same hence results of experiments will be consistent.  Cheaper to maintain.
  43. 43. Safety aspect in cell culture •Possibly cultures should be kept free of antibiotics. •The same media bottle for different cell lines never be used. If caps are dropped they are replaced with new ones. •Necks of glass bottles prefer heat at least for 60 secs at a temperature of 20°C. •Switch on the laminar flow cabinet 20 minutes prior to start working. •Cell cultures which are frequently used should be subcultured & stored as duplicate strains.
  44. 44.  After a period of continuous growth, cell characteristics can change. Cells can also adapt to different culture environments by varying the activities of their enzymes.  Expertise handling is needed to check chemical, microbial and cross contamination.  Require a control environment in the workplace for incubation, pH control containment and disposal of biohazards.
  45. 45.  Quantity and cost involvement is more.  Genetic instability like heterogeneity and variability may appear.  Sometimes the phenotypic characteristics of the tissue may get lost due to dedifferentiation and adaptation.  If the differentiated properties are lost, it is difficult to relate the cultured cell with the functional cell in the tissue.
  46. 46.  As the anatomy and physiology of fish is complex, there are many different cells and proteins which are interacting continuously for normal body functioning.  Being complex in nature, these events are difficult to watch individually in vivo.  Moreover, fish being delicate are usually harmed and get stressed while observing biological events.  Hence it is necessary to develop a parallel cell observation system particularly in vitro in nature to which a cell culture system strongly supplements. Conclusion
  47. 47. References  Basics of Cell Culture--- Paras Yadav1, Annu Yadav1, P. Kumar1, J.S. Arora1, T.K.Datta1, S. De1, S.L. Goswami1, Mukesh Yadav2, Shalini Jain3, Ravinder Nagpal4 and Hariom Yadav3 An Overview Of Cell Culture In Fish---Mohan R. Badhe, Priyanka C. Nandanpawar (CIFE, Mumbai). Cell culture --- From Wikipedia. www.google.com

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