Cell lines Basics


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This work is done in IIT-M (Indian Institute of Technology- Madras) with help of Indian Academy of Science during June 2011-Oct 2011 under Dr Karunagaran Devarajan sir

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Cell lines Basics

  1. 1. Basics of Cell Culture K. Manohar Babu Research Scholar Under Guidance of Dr. M. Hema Prasad Dept. Of Toxicology Institute Of Genetics and Hospital for Genetic DiseasesWork is carried in IIT-MAs a part of Summer Research ProgramBy - Indian Academy of SciencesDuring ---Aug to Oct -2011
  2. 2. Introduction• Cell culture is the process by which animal cells or plant cells are grown under controlled conditions.• 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
  3. 3. Types of celllines• On the basis of morphology or on their functional characteristics. They are divided into three.• Epithelial like-attached to a substrate and appears flattened and polygonal in shape Ex: Adherent cell lines • Cervical cancer celllines: HeLa • Breast cancer celllines: MCF-7 • Lymphoblast like- cells do not attach remain in suspension with a spherical shape Ex: Suspension cell lines • Leukemia celllines: K562 • Fibroblast like- cells attached to an substrate appears elongated and bipolar • Carotid celllines: HacCat
  4. 4. HeLa MCF-7 K562
  5. 5. Topics to be covered………• General maintenance of cell lines • Media preparation • Cell count and seeding • Trypsinization of cell lines • Cryopreservation• Drug sensitivity assay • Drug calculation • MTT assay/Alamar blue assay • Cell cycle analysis by flow cytometry • Soft Ager assay • Wound healing assay • DNA damage by Hoechst nuclear staining
  6. 6. Why is cell culture used for? 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• 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.• Genetic Engineering : Production of commercial proteins e.g. polio, rabies, hepatitis B & measles• Gene therapy
  7. 7. 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• Primary cells have limited life span• Cells such as macrophages and neurons do not divide in vitro so can be used as primary cultures Continous celllinesCell lines which either occur spontaneously or induced virally or chemically transformed into Continous cell lines -Fast growth and have aneuploid chromosome number -ability to grow upto higher cell density -stop expressing tissue specific genes
  8. 8. Culture media• Choice of media depends on the type of cell being cultured• Commonly used Medium are EMEM,DMEM etc.• Media is supplemented with antibiotics viz. penicillin, streptomycin etc.• Prepared media is filtered and incubated at 4 C
  9. 9. Trypsinization of cell lines/ Passazing:• Culture flasks were observed under 10X microscope, if cell lines are confluent proceed for trypsinization.• Nutrient media (DMEM) is removed and washed with 1 ml of PBS buffer discard the buffer.• 500 ul of tripsin is added to the culture flask and incubate at 37oC for 5 min in CO2 incubator.• Observe the cell under the microscope. Cells will be round and detaching from the surface. Mix well by using pipet.• Take the detached cells by pipette to 1 ml ependroff and centrifuge at1500 RMP for 4 min.• Add fresh nutrient medium and resuspend
  10. 10. Cell count and seeding• Celllines after confluent, trypsinized and collected in 1 ml eppendroff.• Cells were centrifuged, decanted the supernent and resuspended with 1 ml fresh medium• 10 ul of Suspended culture is diluted with 90ul of fresh medium.• 10 ul of diluted culture is loaded in hemocytometer.• Count the number of cells in four different chambers
  11. 11. Number of cells will be seeded by
  12. 12. Cell viability• 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 this dye• Stain the cells with trypan dye and load to haemocytometer and calculate % of viable cells - % of viable cells= Nu. of unstained cells x 100 total nu. of cells
  13. 13. Cryopreservation• After trypsinization some cells are stored at low temperature i.e Liquid nitrogen• Cells were washed with PBS• In a two ml eppendroff, 10% DMSO is taken and 90% of FBS. To the mixture of solution pellet is added and kept at -80 oC and then liquid nitrogen.• 4. The cell stored at -80 oC is viable up to 6 months• Cell stored at -160 oC (i.e liquid nitrogen) viable for 2 years
  14. 14. Detection of contaminants• In general indicators of contamination are turbid culture media, change in growth rates, abnormally high pH, poor attachment, multi-nucleated cells, inclusion bodies and cell lysis• Yeast, bacteria & fungi usually shows visible effect on the culture• Mycoplasma detected by direct DNA staining with intercalating fluorescent substances e.g. Hoechst 33258• The best and the oldest way to eliminate contamination is to discard the infected cell lines directly
  15. 15. 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
  16. 16. Topics to be covered………• General maintenance of cell lines • Media preparation • Cell count and seeding • Trypsinization of cell lines • Cryopreservation• Drug sensitivity assay • Drug calculation • MTT assay/Alamar blue assay • Cell cycle analysis by flow cytometry • Soft Ager assay • Wound healing assay • DNA damage by Hoechst nuclear staining
  17. 17. Drug calculations• Find out the solubility of given extract/ product in water, DMSO or alcohol.• If the extract is crude (mixture of components or plant derived substance) prepare 1 mg/ml solution.• If it is known compound take directly molar concentrations i.e 10 nm, 100 nm, 1000 nm, 10 um, 50 um etc.• Day 1: Seed 5000 cells/well in 96 well plate, let the cells grow for 24 hours at 370C in CO2 incubator.• Day 2: Change the media aseptically to all wells. One row untreated, one row vehicle control or solvent treated and rest of the wells are treated with increasing concentrations in triplicate.• Day3: Let the exposure of drug and cell lines for 24 hours. After exposure time proceed for MTT or Alamar blue assay.
  18. 18. S.No Size Seeding density Cells at Trypsin-0.05% Growth Media confluence EDTA –0.53 mMCulture Dishes 1 35 mm 0.3 X 106 1.2 X 106 1 ml 2 ml 2 60 mm 0.8 X 106 3.2 X 106 2 ml 3 ml 3 100 mm 2.2 X 106 8.8 X 106 3 ml 10 ml 4 150 mm 5.0 X 106 20 X 106 8 ml 20 mlCulture plates 1 6 well 0.3 X 106 1.2 X 106 2 ml 3-5 ml 2 12 well 0.1 X 106 0.4 X 106 1 ml 1-2 ml 3 24 well 0.05 X 106 0.2 X 106 0.5 ml 0.5-1 mlCulture Flasks 1 T-25 0.7 X 106 2.8 X 106 3 ml 3-5 ml 2 T-75 2.1 X 106 8.4 X 106 5 ml 8-10 ml 3 T-160 4.6 X 106 18.4 X 106 10 ml 30. l
  19. 19. Alamar blue assay• Alamar Blue works as a cell viability and proliferation indicator through the conversion of resazurin to resorufin. Resazurin, a non- fluorescent indicator dye, is converted to highly red fluorescent resorufin via reduction reactions of metabolically active cells. The amount of fluorescence produced is proportional to the number of living cells. Resazurin (Purple) Resorufin (Red)
  20. 20. • Principle of Flowcytometry:• A beam of laser light of a single wavelength is directed onto a hydrodynamically-focused stream of liquid.• A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) and one or more fluorescencedetectors..• Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the ray, and fluorescent chemicals excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and, by analysing fluctuations in brightness at each detector .• FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).
  21. 21. Cell cycle analysis by flow cytometry• Seed cells in a 12 well plate with 4X105 cells/well cell densities. Label the wells accordingly the treatment.• Incubate for 24 hours at 37oC in 5% CO2 incubator. Remove the spent medium and add 500-750 ul of medium containing the drug at required concentration.• Allow the drug, medium to stand for required exposure time.• Harvest the cells, i.e collect the cells in spent medium, The cells in the wells are washed with PBS-EDTA and remove by tripsinization, which is then collected in the same tube used for cells collected in spent , Add 300 ul of PBS-EDTA, resuspend cells by tapping• Count the cells using tryptophan blue inclusion assay. Count all cells (live + dead), estimate the cell density.• Add 700 ul of 70% ethanol (ice cold) to the suspended cell solution with mild vertexing during drop wise addition of 70% ethanol.• Keep these fixed cells in 4oC until analysis in flow cytometery
  22. 22. • Centrifuge cells at 3000 rpm for 5 min., Decant supernent.• Wash with ice cold PBS containing 1%FBS and re-centrifuge.• Decant supernent and resuspend cell pellet in 300 ul PBS-EDTA• Add 5 ul of 10 mg/ml RNase and incubate for 1 hour at 37 oC• 1mg/ml propidium Iodide is added to final concentration of10 ug/ml (3ul).• Keep in dark at 4oC (If stored)• Analyze in flow cytometer at 488nm
  23. 23. Soft Ager assay • Melt 1% Agar (DNA grade) in microwave, cool to 40°C in a waterbath. Warm 2X RPMI + 20% FCS to 40°C in waterbath. Allow at least 30 minutes for temperature to equilibrate. • Mix equal volumes of the two solutions to give 0.5% Agar + 1X RPMI + 10% FCS. • Add 1.5mL/ 35 mm dish (2.5mL), allow to set. The plates can be stored at 4°C for up to 1 week.• Top Agar • Melt 0.7% Agar (DNA grade agarose) in microwave, cool to 40°C in a waterbath. Also warm 2X RPMI + 20% FCS to the same temperature. • Require 5,000 cells/35mm plate. Add 0.1ml of cell suspension to centrifuge tubes. • For plating add 3mL (5mL) 2X RPMI + 10% or 20% FCS and 3mL (5mL) 0.7% Agar to tube with cells, • Incubate assay at 37°C in humidified incubator for 10 - 14 days. • Stain plates with 0.5mL of 0.005% Crystal Violet for >1 hour, count colonies using a dissecting microscope.
  24. 24. Wound healing assay• The assay is a “wound gap” in a cell monolayer is created by scratch, followed by monitoring the “healing” of this gap by cell migrating and growth towards the center of the gap, hereby filling up the “gap”. Factors that alter the motility and growth of the cell can lead to increased or decreased rate of “healing” of the gap.
  25. 25. • Cells were grown in DMEM supplemented with 10% FBS.• Cells were seeded into 24-well tissue culture plate, they should reach ~70-80% confluence as a monolayer.• Gently scratch the monolayer with a new 1 ml pipette tip across the center of the well. Scratch a straight line in one direction.• After scratching, gently wash the well twice with medium to remove the detached cells. Replenish the well with fresh medium.• Grow cells for additional 48 hours (or the time required).• Wash the cells twice with 1x PBS then fix the cells with 3.7% paraformaldehye for 30 minutes.• Fixed cells are stained with 1% Crystal Violet in 2% ethanol for 30 minutes.
  26. 26. DNA damage by Hoechst nuclear staining• Count cells.• To confluent cell lines, aspirate the media and replace with fresh media• Mix gently. Add 5 μl of Hoechst 33342 stock solution and mix again. Incubate at 37oC for 45 min.• Preparation of Hoechst 33342 stock solution:• Dissolve 1 mg of Hoechst 33342 powder in 1 ml of distilled water. Store at 2-8oC protected from light for up to 1 month.
  27. 27. Effect of IL-18 cytokine on different Cancer cell lines and combinational effect with CurcuminUnder supervision ofDr. Karunagaran DevarajanDept. of Biotechnology, IIT-M
  28. 28. IL-18 is also called as Interferon inducing factor-g IL-18 is a pro inflammatory cytokine with anti cancer activitySchismatic representation of IL-18 gene and promoter polymorphism
  29. 29. S. Polym Res Type of Patient Associated/N Cancer out Author & yearN orphi ults Cancer s Vs comeo sm Control -607 C/A IL-18 polycystic ovary 118 Vs 791 C Not Associated Protective role Yang et al., 2010 syndrome Chinese 73 Vs 97 Farjadfar2 A IL-18 lung cancer Associated cancer risk Iranian Et al., 2009 232 Vs3 A IL-18 GI cancers 312 Associated Protective role Haghshenas et al., 2009 Iranian Nasopharyngeal 250 Vs 2704 A IL-18 Associated cancer risk Nong et al., 2009 carcinoma Chinese 85 Vs 1585 A IL-18 ovarian cancer No t Associated No role Samsami et al., 2009 Iranian6 A IL-18 breast cancer 250 Vs 206 Associated Protective role Khalili et al., 2009 head and neck7 A IL-18 squamous cell 111 Vs 212 Not Associated No role Asefi et al., 2009 carcinoma Indian Increased Cancer8 C IL-18 Cervical cancer Associated Sobti et al., 2008 opulation risk nasopharyngeal 163 Vs 164 Increased cancer9 A IL-18 Not Associated Farhat et al., 2008 carcinoma Tunisian risk hepatocellular Increased risk of10 C IL-18 Associated Bouzgarrou et al., 2008 carcinoma Cancer11 A IL-18 oral cancer 149 Vs 89 Not associated No role Vairaktaris et al., 2007 Esophageal 235 Vs 25012 C IL-18 squamous cell Associate Increased risk Wei et al., 2007 Chinese carcinoma 265 Vs 28013 A IL-18 prostate cancer Associate Increased risk Liu et al., 2007 Chinese
  30. 30. Hypothesis: IL-18 cytokine therapeuticalresponse depends on type of cancer.• Some cancers are very sensitive and give good response to IL-18 and inhibit tumor development and some types may resistant and not influenced by IL-18.• Aim: To evaluate effect of IL-18 in different cell lines and to find out IC 50 value of IL-18 and Curcumin on different cell lines.• Methodology: Different cell lines are tested against IL-18 and finding sensitivity of IL-18 in different cell lines. Cell cycle analyses, MTT assay, RT-PCR are performed to see the sensitivity.• Possible outcome: Different cell line might have different IC50 values; this data may be useful for those conducting clinical studies with recombinant IL-18 therapy.
  31. 31. • Curcumin is a anti inflammatory agent and IL-18 is pro inflammatory cytokine.• Combination of these two agents might effective in anti cancer therapy. Treatment with IL-18 alone Treatment with Curcumin alone Treatment with IL-18 along with IC50 value of curcumin Treatment with Curcumin along with standard value of IL-18
  32. 32. K562-IL-18IL-18 enhances the cell proliferation 3.5 folds in K562 celllines
  33. 33. K562-CurcuminIC 50 value Curcumin is 5 uM in K562 celllines
  34. 34. K562- Curcumin +3ng IL-18IC 50 value of curcumin in the presence of 3 ng/ul IL-18 enhancesto 15uM in K562 cell lines
  35. 35. K562- IL-18 + 25uM CurcuminIL-18 enhances the cell proliferation in the presence of 25 uMcurcumin in K562 cell lines
  36. 36. MCF7- CurcuminIC 50 value Curcumin is 20 uM in MCF-7 cell lines
  37. 37. MCF-7; IL-18IL-18 did not effect prolifiration in MCF-7 cell lines
  38. 38. MCF-7;IL-18 +25uM CurcuminIL-18 did not effect the inhibitory activity of Curcumin in MAF-7
  39. 39. MCF-7; Curcumin + 3ng IL-18IC 50 value of curcumin did not effected by 3 ng/ul IL-18
  40. 40. HeLa - CurcuminIC 50 value Curcumin is15 uM in HeLa cell lines
  41. 41. HeLa IL-18IL-18 enhances the cell proliferation dose dependent manner up to2 ng/ml
  42. 42. HeLa- Curcumin +3ng IL-18IL-18 proliferation is inhibited by Curcumin
  43. 43. HeLa IL-18 + 25um CurcuminIL-18 proliferation is inhibited by Curcumin
  44. 44. HacCat celllines IL-18IL-18 enhances the cell proliferation dose dependent manner inHacCat cell lines
  45. 45. HacCat celllines CurcuminIC 50 value Curcumin is 30 uM in HacCat cell lines
  46. 46. Hela cell lines
  47. 47. Hela cell lines
  48. 48. MCF-7 cell lines
  49. 49. MCF-7 cell lines
  50. 50. HeLa cell lines treated with Curcumin
  51. 51. HeLa cell lines treated with 3 ng/ml IL-18
  52. 52. K-562 cell lines
  53. 53. K-562 cell lines
  54. 54. Curcumin treated K-562 cell lines
  55. 55. Control IL-18 --3ng/ml0h 24 h
  56. 56. ConclusionIL-18 enhances cell proliferation in HeLa, K-562and HacCat cell lines but not in MCF-7IL-18 increase IC50 value of curcumin in K562cell lines and HeLa cell lines, infers may providedrug resentence.Recombinant IL-18 may not best choice of drugfor Breast cancers
  57. 57. Thank you