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

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 Diseases
Work is carried in IIT-M
As a part of Summer Research Program
By - Indian Academy of Sciences
During ---Aug to Oct -2011

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. 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. HeLa MCF-7
K562

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. 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. 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 celllines
Cell 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. 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. 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. 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. Number of cells will be seeded by

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. 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. 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. 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. 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. 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. S.No Size Seeding density Cells at Trypsin-0.05% Growth Media
confluence EDTA –0.53 mM
Culture 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 ml
Culture 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 ml
Culture 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. 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. • 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. 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. • 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. 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. 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. • 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. 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. Effect of IL-18 cytokine on different
Cancer cell lines and combinational
effect with Curcumin
Under supervision of
Dr. Karunagaran Devarajan
Dept. of Biotechnology, IIT-M

28. IL-18 is also called as Interferon inducing factor-g
IL-18 is a pro inflammatory cytokine with anti
cancer activity
Schismatic representation of IL-18 gene and promoter polymorphism

29. S. Polym Res Type of Patient Associated/N Cancer out Author & year
N orphi ults Cancer s Vs come
o sm Control
-607
C/A
IL-18
polycystic ovary 118 Vs 79
1 C Not Associated Protective role Yang et al., 2010
syndrome Chinese
73 Vs 97 Farjadfar
2 A IL-18 lung cancer Associated cancer risk
Iranian Et al., 2009
232 Vs
3 A IL-18 GI cancers 312 Associated Protective role Haghshenas et al., 2009
Iranian
Nasopharyngeal 250 Vs 270
4 A IL-18 Associated cancer risk Nong et al., 2009
carcinoma Chinese
85 Vs 158
5 A IL-18 ovarian cancer No t Associated No role Samsami et al., 2009
Iranian
6 A IL-18 breast cancer 250 Vs 206 Associated Protective role Khalili et al., 2009
head and neck
7 A IL-18 squamous cell 111 Vs 212 Not Associated No role Asefi et al., 2009
carcinoma
Indian Increased Cancer
8 C IL-18 Cervical cancer Associated Sobti et al., 2008
opulation risk
nasopharyngeal 163 Vs 164 Increased cancer
9 A IL-18 Not Associated Farhat et al., 2008
carcinoma Tunisian risk
hepatocellular Increased risk of
10 C IL-18 Associated Bouzgarrou et al., 2008
carcinoma Cancer
11 A IL-18 oral cancer 149 Vs 89 Not associated No role Vairaktaris et al., 2007
Esophageal
235 Vs 250
12 C IL-18 squamous cell Associate Increased risk Wei et al., 2007
Chinese
carcinoma
265 Vs 280
13 A IL-18 prostate cancer Associate Increased risk Liu et al., 2007
Chinese

30. Hypothesis: IL-18 cytokine therapeutical
response 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. • 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. K562-IL-18
IL-18 enhances the cell proliferation 3.5 folds in K562 celllines

33. K562-Curcumin
IC 50 value Curcumin is 5 uM in K562 celllines

34. K562- Curcumin +3ng IL-18
IC 50 value of curcumin in the presence of 3 ng/ul IL-18 enhances
to 15uM in K562 cell lines

35. K562- IL-18 + 25uM Curcumin
IL-18 enhances the cell proliferation in the presence of 25 uM
curcumin in K562 cell lines

36. MCF7- Curcumin
IC 50 value Curcumin is 20 uM in MCF-7 cell lines

37. MCF-7; IL-18
IL-18 did not effect prolifiration in MCF-7 cell lines

38. MCF-7;IL-18 +25uM Curcumin
IL-18 did not effect the inhibitory activity of Curcumin in MAF-7

39. MCF-7; Curcumin + 3ng IL-18
IC 50 value of curcumin did not effected by 3 ng/ul IL-18

40. HeLa - Curcumin
IC 50 value Curcumin is15 uM in HeLa cell lines

41. HeLa IL-18
IL-18 enhances the cell proliferation dose dependent manner up to
2 ng/ml

42. HeLa- Curcumin +3ng IL-18
IL-18 proliferation is inhibited by Curcumin

43. HeLa IL-18 + 25um Curcumin
IL-18 proliferation is inhibited by Curcumin

44. HacCat celllines
IL-18
IL-18 enhances the cell proliferation dose dependent manner in
HacCat cell lines

45. HacCat celllines
Curcumin
IC 50 value Curcumin is 30 uM in HacCat cell lines

46. Hela cell lines

47. Hela cell lines

48. MCF-7 cell lines

49. MCF-7 cell lines

50. HeLa cell lines treated with Curcumin

51. HeLa cell lines treated with 3 ng/ml IL-18

52. K-562 cell lines

53. K-562 cell lines

54. Curcumin treated K-562 cell lines

55. Control
IL-18 --3ng/ml
0h 24 h

56. Conclusion
IL-18 enhances cell proliferation in HeLa, K-562
and HacCat cell lines but not in MCF-7
IL-18 increase IC50 value of curcumin in K562
cell lines and HeLa cell lines, infers may provide
drug resentence.
Recombinant IL-18 may not best choice of drug
for Breast cancers

57. Thank you