Presentation on cell line by Shakira 2


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Presentation on cell line by Shakira 2

  1. 1. Cell Culture and handling of the cell lines By Shakira Ghazanfar PhD Scholar PIASA 20-04-2011
  2. 2. Contents • Cell Culture • Cell Lines • Continues Cell Lines • Subculture • Handling of cell lines • Cryopreservation of cell lines
  3. 3. 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 R. Harrison in 1907
  4. 4. Cell Culture Definition: In vitro cultivation of cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is known as Cell Culture.
  5. 5. Cell Culture • Tissues Individual cells trypsin & mechanical shaking • Cells are washed, counted & suspended in a growth medium. • Growth medium – Minimum Essential Medium (MEM): essential aminoacids, vitamins, salts, glucose & bicarbonate in 5% CO2 with 5% fetal calf or calf serum, antibiotics & phenol red indicator
  6. 6. MAIN TYPES OF CELL CULTURE according to the cultivation period (and survival in culture) • Routinely used for growing viruses • Classified into 3 types: – Primary cell culture – normal cells taken from body & cultured, limited growth 1. Chick embryo fibroblast 2. Human amnion cell culture – Diploid cell strains – cells of single type (fibroblast cells) that can be sub cultivated for limited number of times, mostly 50 1. WI-38: human embryonic lung cell – Continuous cell lines – indefinite subcultivtion 1. HeLa: Human Ca of cervix cell line 2. HEP-2: Human epithelioma of larynx 3. Vero: Vervet monkey kidney
  7. 7. Classification 1. Primary Cultures – Derived directly from tissue and cultured either as • Outgrowth of tissue in culture • Cut into single cells (by enzymatic digestion or mechanical dispersion) – Advantages: • usually retain many different characteristics of the cell in vivo – Disadvantages: • initially heterogeneous but later become dominated by fibroblasts. • the preparation of primary cultures is labor intensive • can be maintained in vitro only for a limited period of time.
  8. 8. Classification 2. Continuous Cultures – derived from subculture (or passage, or transfer) of primary culture • Subculture = the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate in the culture – can be serially propagated in culture for several passages Types: – There are two types of continuous cultures • Cell lines • Continuous cell lines
  9. 9. Types of continuous cultures 1) Cell lines • short time life, die after approximately thirty cycles of division • usually diploid and maintain some degree of differentiation.
  10. 10. Types of continuous cultures • 2. 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 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 Reduced serum and anchorage dependence and grow more in suspension conditions
  11. 11. Common cell lines • Human cell lines • -MCF-7 breast cancer • HL 60 Leukemia • HEK-293 Human embryonic kidney • HeLa Henrietta lacks • Primate cell lines • Vero African green monkey kidney epithelial cells • Cos-7 African green monkey kidney cells • And others such as CHO from hamster, sf9 & sf21 from insect cells
  12. 12. American Type Culture Collection.
  13. 13. Practical
  14. 14. Design and Equipment for the Cell Culture Laboratory • 1. Laboratory design- in a safe and efficient manner • 2. safety cabinets
  15. 15. Cell Culture Room Close Small AC/Heater ROOM FOR ANIMAL CELL CULTURE sterile conditions (disinfection of the work surfaces, microbiological safety cabinets) Hood
  16. 16. Basic equipments used in cell culture • Laminar cabinet- • Incubation- Temperature is 37 C for mammalian cells, Co2 2- 5% & 95% air at 99% relative humidity. • Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutamine & serum) at -20 C • Microscope- An microscope with 10x to 100x magnification • Cell culture tubes- • Autoclave-
  17. 17. Laminar- flow hood
  18. 18. Laminar- flow hood • The working environment is protected from dust and contamination by a • constant, stable flow of filtered air • Two types:  Horizontal, airflow blow from the side facing you, parallel to the work surface, and is not circulating;  Vertical, air blows down from the top of the cabinet onto the work surface and is drawn through the work surface and recalculated
  19. 19. Laminar- flow hood
  20. 20. Laminar- flow hood • Routine maintenance checks of the primary filters are required (every 3-6 months). • They might be removed and discarded or washed in soap and water. • Every 6 months the main high efficiency particulate air (HEPA) filter above the work surface should be checked for airflow and hole
  21. 21. Precaution Measure Inside The Hood Incubator Gloves are always worn The pipettes are disposable Lab coat
  22. 22. Cell Culture Incubator
  23. 23. Cell Culture Incubator • It requires a controlled atmosphere with high humidity and super controlled of CO2. • The incubator should be large enough, like 50- 200 l have forced air circulation • Temperature should be + 0.5oC • It should be stainless steel, and easily cleaned
  24. 24. Autoclave A simple autoclave may be sufficient.
  25. 25. Refrigerators and Freezers • 4C •-20C •-80C • Liquid N2 tank
  26. 26. Microscope Large stage so plates and flasks can be used. Magnification; 5X, 10X, 20X, 40X
  27. 27. Culture vessels and medium for animal cell culture Culture vessel Culture Media removes a tray of stem cell cultures from an incubator
  28. 28. Anaerobic Jar
  29. 29. Multiwell plates
  30. 30. Cell Culture Bottles / Tubes
  31. 31. Heating of media on heater
  32. 32. Centrifuge
  33. 33. Culture media • Choice of media depends on the type of cell being cultured • Commonly used Medium are GMEM, EMEM,DMEM etc. • Media is supplemented with antibiotics viz. penicillin, streptomycin etc. • Prepared media is filtered and incubated at 4 C • Culture Medium – Basic Composition of Media • Inorganic salts • Trace elements • Buffering systems – pH range should be 7,2 – 7,4 • Carbohydrates • Aminoacids • Vitamins • Proteins and peptides • Fatty acids and lipids • Serum – fetal bovine serum • Antibiotics
  34. 34. Before use • Ultraviolet lights are used to sterilize the air and exposed work surfaces in laminar flow cabinets between use. • Detergent • 70% alcohol
  35. 35. Culture Morphology • Suspension (as single cells or small free-floating clumps) • or as a monolayer that is attached to the cell culture flask. • The form taken by a cell line reflects the tissue from which it was derived ... • From blood tend to grow in suspension • From solid tissue (lungs, kidney) tend to grow as monolayer's. • Attached cell lines can be classified as endothelial, epithelial, or fibroblasts and their morphology tell the area within the tissue of origin
  36. 36. Anchorage dependent or independent • Cell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate. • Suspension cells are anchorage- independent e.g. blood cells
  37. 37. Adherent cells • Cells which are anchorage dependent • Add enough trypsin/EDTA to cover the monolayer • Incubate the plate at 37 C for 5 mts • Tap the vessel from the sides to dislodge the cells • Add complete medium to dissociate and dislodge the cells • with the help of pipette which are remained to be adherent • Add complete medium depends on the subculture • requirement either to 75 cm or 175 cm flask
  38. 38. Suspension cells • Easier to passage as no need to detach them • As the suspension cells reach to confluency • Aseptically remove 1/3rd of medium • Replaced with the same amount of pre- warmed medium
  39. 39. confluency • Once the available substrate surface is covered by cells (a confluent culture) growth slows & ceases.
  40. 40. hep 3B - 70% confluency Gaps
  41. 41. 100% confluency
  42. 42. After 24 h
  43. 43. confluency • Cells to be kept in healthy & in growing state have to be sub-cultured or passaged , It’s the passage of cells when they reach to 80-90% confluency in flask/dishes/plates • Enzyme such as trypsin, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surface
  44. 44. Cell culture contaminants • Two Types: • Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible • Biological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines
  45. 45. Safety
  46. 46. Why sub culturing.? • Once the available substrate surface is covered by cells growth slows & ceases. • Cells to be kept in healthy & in growing state have to be sub-cultured or passaged • It’s the passage of cells when they reach to 80-90% confluency in flask/dishes/plates • Enzyme such as trypsin, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surface
  47. 47. 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
  48. 48. Cell Culture Used 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
  49. 49. • 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 Cell Culture Used
  50. 50. Advantage Viral Vaccines: Continuous Cell Lines Cell Substrate Live Vaccines Inactivated Vaccines African green monkey kidney: Vero Poliovirus Poliovirus (Europe) (U.S.)
  51. 51. Disadvantages • Cell characteristics can change • Cell can adapt to different nutrients • If mixed cells cultivated some types will disappear. • Activity of enzymes may altered by environment.
  52. 52. Cryopreservation Definition Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as, −196 °C (the boiling point of liquid nitrogen). Liquid Nitrogen
  53. 53. Principles of cryopreservation • Water in cell: Around 90% of water is free (water) while the remaining 10 % bounds to other molecular components of the cell (proteins, lipids, nucleic acids and other solutes). This water does not freeze and called hydrated water – Removal of water is necessary during freezing to avoid ice crystal formation, dehydration is limited to the free water – Removal of hydrated water could have adverse effect on the cell viability and the molecular function (freezing injuries)
  54. 54. Cryopreservation of Cell Lines • The aim of cryopreservation is to enable stocks of cells to be stored to prevent the need to have all cell lines in culture at all times. It is invaluable when dealing with cells of limited life span. The other main advantages of cryopreservation are: • Reduced risk of microbial contamination • Reduced risk of cross contamination with other cell lines • Reduced risk of genetic drift and morphological changes • Work conducted using cells at a consistent passage number (refer to cell banking section below) • Reduced costs (consumables and staff time)
  55. 55. Successful Cryopreservation of cell lines • There has been a large amount of developmental work undertaken for successful cryopreservation of a wide variety of cell lines of different cell types. • The basic principle of successful cryopreservation is a • Slow freeze • Quick thaw. • Cell lines should be cooled at a rate of –1ºC to –3ºC per minute and thawed quickly by incubation in a 37ºC water bath for 3-5 minutes..
  56. 56. • A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%. • Use a cryoprotectant such as dimethyl sulphoxide (DMSO) or glycerol to help protect the cells from rupture by the formation of ice crystals. • The most commonly used cryoprotectant is DMSO at a final concentration of 10%. Successful Cryopreservation of cell lines
  57. 57. Vocabulary  Cell-Culture-In vitro cultivation of cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is known as Cell Culture.  Culture – growth of microorganisms in a special medium; the process of growing microorganisms in the laboratory.  Anchorage-dependent- Cell lines derived from normal tissues are considered as anchorage- dependent grows only on a suitable substrate  Primary Culture :Cells when surgically or enzymatically removed from an organism and placed in suitable culture environment will attach and grow are called as primary culture  Cell Lines: Sub culturing of primary cells leads to the generation of cell lines  Cell Strain :Lineage of cells originating from the primary culture is called a cell strain  Anchorage-independent- Suspension cells are anchorage- independent e.g. blood cells
  58. 58. Next practical • Transformation • See you next week!
  59. 59. Thanks