ANTICANCER ACTIVITY STUDIES: DIFFERENT MODELS

Jesil Mathew. A,MCOPS, Manipal University

US Mortality, 2005US Mortality, 2005

*Includes nephrotic syndrome and nephrosis.Source: US Mortality Data 2005, National Center for Health Statistics, Centers for Disease Control and Prevention, 2008.

1. Heart Diseases 652,091 26.6

2. Cancer 559,312 22.8

3. Cerebrovascular diseases 143,579 5.9

4. Chronic lower respiratory diseases 130,933 5.3

5. Accidents (unintentional injuries) 117,809 4.8

6. Diabetes mellitus 75,119 3.1

7. Alzheimer disease 71,599 2.9

8. Influenza & pneumonia 63,001 2.6

9. Nephritis* 43,901 1.8

10. Septicemia 34,136 1.4

Rank Cause of DeathNo. of deaths

% of all deaths

2008 Estimated US Cancer Deaths*2008 Estimated US Cancer Deaths*

ONS=Other nervous system.Source: American Cancer Society, 2008.

Men294,120

Women271,530

26% Lung & bronchus

15% Breast

9% Colon & rectum

6% Pancreas

6% Ovary

3% Non-Hodgkin lymphoma

3% Leukemia

3% Uterine corpus

2% Liver & intrahepaticbile duct

2% Brain/ONS

25% All other sites

Lung & bronchus 31%

Prostate 10%

Colon & rectum 8%

Pancreas 6%

Liver & intrahepatic 4%bile duct

Leukemia 4%

Esophagus 4%

Urinary bladder 3%

Non-Hodgkin 3% lymphoma

Kidney & renal pelvis 3%

All other sites 24%

Change in the US Death Rates* by Cause, 1950 & 2005

Change in the US Death Rates* by Cause, 1950 & 2005

* Age-adjusted to 2000 US standard population.Sources: 1950 Mortality Data - CDC/NCHS, NVSS, Mortality Revised.2005 Mortality Data: US Mortality Data 2005, NCHS, Centers for Disease Control and Prevention, 2008.

HeartDiseases

CerebrovascularDiseases

Influenza &Pneumonia

Cancer

1950

2005

Rate Per 100,000

What is cancer

• Neoplasia is any new or continued cell growth not needed for normal development or replacement of dead and damaged tissues.

• Tumor & Cancer : In medical language, a neoplasm is often referred as tumor.

• Malignant • Benign • Metastasis

Evolution of Cancer• Human body has ~1014 cells from a single egg cell.• Typical adult cell has gone through ~50 divisions.• Each division is accompanied by 1-10 errors typically - so total

around 50-500 mutations per cell. But 90% of mutations will do nothing.

• Additional mutations can occur from UV, radiation, virus, etc.• Mutations that favor growth of an individual cell over survival of

the organism lead to cancer.• Activation of pro-growth “oncogenes”• Suppression of anti-growth “tumor suppressor genes”• Occassionally can get cancer without mutation.• best example is teratoma - are the result of abnormal development

of pluripotent cells: germ cells and embryonal cells.

Normal cells Vs Cancer cellsNormal cells

• Have limited cell division• Undergo apoptosis• Show specific morphology• Have a small nuclear-cytoplasmic ratio• Perform specific differentiated functions• Adhere tightly together• Nonmigratory• Grow in an orderly and well-regulated manner• Contact inhibited

Cancer cells• Have rapid or continuous cell division• Do not respond to signals for apoptosis• Show anaplastic morphology• Have a large nuclear-cytoplasmic ratio• Lose some or all differentiated functions• Adhere loosely together• Able to migrate through embryonic cells• Grow by invasion

Normal cells Vs Cancer cellsNormal cells Vs Cancer cells

Characteristics of cancerCharacteristics of cancer• They divide - generally relatively fast.• Often

– Have chromosomes are fused/split, with some deleted and some duplicated.

– Have poor DNA repair mechanisms/high mutation rates.

– More fragile than normal cells.– Retain characteristics of normal cells they descended

from– Many breast cancers require estrogen and/or

progesterone to proliferate

Targets of anticancer drugs: conventional

• Cell division:– Pro - valid for all cancers. – Con - bad for bone marrow, gut, hair, etc.

• Recruitment of blood vessels:– Pro - prevents tumors from getting large. May make other

chemotherapy more effective. – Con - small tumors can still cause harm. Long term effects may reduce

overall circulation.• DNA repair:

– Pro - works on many cancers– Con - not all cancers have repair defects. Stresses other cells.

• Hormones:– Pro - relatively non-toxic. (Don’t *need* estrogen).– Con - doesn’t work on all cancers.

• Targeting Signaling and associated molecules

Targets of anticancer drugs: novel approaches

There are many ways to die…..

Cell ViabilityCell Viability

• Functional assay

• DNA labeling assay

• Morphological assay

• Reproductive assay

• Membrane integrity assay

The major criteria employed in viability assayThe major criteria employed in viability assay

Cell countingCell counting

Membrane integrity assay Hemocytometer Trypan blue assay / Automatic trypan blue method LDH(lactate dehydrogenase) leakage

Fluorescent dyes

Functional Assay MTT / XTT assay Crystal violet / Acid phosphatase(AP) assay Alamar Blue oxidation-reduction assay / Neutral red assay [3H]-thymidin and BrdU incorporation

DNA Assay Enzymatic DNA labeling / DNA-binding dye labeling

Morphological assay Reproductive Assay Colony-forming efficiency

Laser scanning confocal microscopy Nuclear magnetic resonance methods

Membrane integrity assaysMembrane integrity assays

Dye exclusion methodsDye exclusion methodsDye exclusion methodsDye exclusion methods• Viability assays measure the percentage of a cell

suspension that is viable. • This is generally accomplished by a dye exclusion stain,

where cells with an intact membrane are able to exclude the dye while cells without an intact membrane take up the coloring agent.

• The dye used for exclusion stain is usually trypan blue but erythrosin and naphthalene black have also been used.

• A dye uptake stain can be used to measure viability as well. In this case, the dye is normally taken up by viable cells but not by the non-viable cells. Diacetyl fluorescein is an example of a dye used for dye uptake assays.

Trypan blue

A stain which will only enter across the membranes of dead/non-viable cells.

- Cause cancer in lab. animals - Appropriate precaution should be taken when handling trypan blue (use of

extraction hood and gloves)

Dilution by trypan blue

- Viable cells : small, round and refractive - Non-viable cells : swollen, larger, dark blue

HemocytometerThe most common routine method for cell counting which is efficient

and accurate is with the use of a hemocytometer.

Hemocytometer Cell Counts Hemocytometer Cell Counts

Volume : 0.1mm3

1 ml = 1 cm3 = 1000 mm3

1 mm

1 mm

0.1 mm

Dead cell

Materials and Equipment

Trypan blue (0.4 g trypan blue in 100 ml physiological saline) → pass through a 0.22 ㎛filter

Hemocytometer with coverslip

Hand-held counter

Microscope

Methods1) Clean hemocytometer & coverslip and wipe with 70% alcohol

before use

2) Place coverslip on hemocytometer

3) Mix the cell suspension gently

4) Aliquot 0.1 ml cell suspensions

5) Add 0.1 ml (2-fold dilution), 0.3 ml (4-fold dilution) or 0.9 ml (10-fold dilution) trypan blue : appropriate range of cells to be counted

6) Draw a sample into a Pasteur pipette after mixing

7) Draw the cell suspension in to fill the chamber

8) Using a light microscope at low power, count the number of cells

9) Count the viable & non-viable cells in both halves of the chamber

10) Calculations

A = Vol. Of cell solution (ml)

B = Dilution factor in trypan blue

C = Mean number of unstained cells

D = Mean number of dead/stained cells

104 = Conversion of 0.1 mm3 to ml

(1) Total number of viable cells

A B C 10Ⅹ Ⅹ Ⅹ 4

(2) Total dead cell count

A B D 10Ⅹ Ⅹ Ⅹ 4

(3) To give a total cell count

Viable cell count + dead cell count

(4) % viability

(Viable cell count/Total cell count) 100Ⅹ

Example

Dilution factor Vol.

of CSCell count Total viable cells

0.1 ml CS +

0.1 ml TB (2) 20 ml 23 20Ⅹ2 23 10Ⅹ Ⅹ 4 = 9.2 10Ⅹ 6 cells

0.1 ml CS +

0.3 ml TB (4) 15 ml // 15Ⅹ4 23 10Ⅹ Ⅹ 4 = 1.38 10Ⅹ 7 cells

0.1 ml CS +

0.9 ml TB (10)10 ml // 10Ⅹ10 23 10Ⅹ Ⅹ 4 = 2.3 10Ⅹ 7 cells

1) Vol. : Volume

2) CS : Cell Solution

3) TB : Trypan blue

Automated trypan blue method for optimal cell viability determination

www.innovatis.com

Introduction

The trypan blue dye exclusion assay is the most commonly used and accepted method for the measurement of cell viability

It relies on the alteration in membrane integrity as determined by the uptake of dye by dead cells, thereby giving a direct measure of cell viability

Based on optimal image analysis, the technology allow precise cell-viability and cell-density determination

The system performs automatic and reproducible measurements of human or animal suspension cell densities as well as standardized differentiation between viable and dead cells, based on the trypan blue dye exclusion method

The direct and automated optical analysis by means of modern pattern recognition methods allows cell identification and a standardized differentiation between viable and dead cells and also cell debris

The system consist of three functional part: the liquid handling unit, image capture hard ware, and a data processing system, including the user interface

Fig. Cedex workbench The IP Result viewer enables the user to control whether the Cedex system recognizes the cells correctly, and whether it reliably differentiates and between viable and dead cells

Result view of the image processing

Marked viable and dead cells

Viable cell Dead cell

Fig. a) Distribution histogram of the viable cell diameter for a human leukemia cell sample. b) Histogram of compactness for a human leukemia cell sample. The abscissa represents the ratio of cell circumstance to cell area normalized to the value of 1 for an ideal sphere

(a) (b)

Vi-CELL Vi-CELL TMTM CELL Viability Analyzer CELL Viability Analyzer

Principle

Trypan Blue dye Exclusion Methods

Run results

LDH Release

• One parameter of cell death is the integrity of the cell membrane.

• It can be measured by cytoplasmic enzyme activity released by damaged cells.

• Lactate dehydrogenase is a stable cytoplasmic enzyme present in all the cells.

• The LDH is rapidly released to the culture supernatant upon damage of the plasma membrane.

Quantitative value for the loss of cell viability

The activity of LDH can be measured as the reduction of pyruvate to lactate.

The reduction is coupled to the oxidation of NADH to NAD+, which is followed spectrophotometrically at 340nm

LDH Pyruvate + NADH + H+ NAD⇌ + + lactate

As NADH has a high absorbance at 340nm compared to NAD+, the reaction is measured as the rate of decrease in absorbance at 340nm.

LDH (lactate dehydrogenase) Leakage

Requirement of LDH assay

Greater process productivity (e.g. High-cell-density entrapped-culture systems) : difficulty of cell isolation

→ Metabolic parameters(glucose uptake) : compromised because uptake/production rates can alter as a result of the cell switching carbon source.

→ Analysis of the release of intracellular enzymes can be used enzyme in cell culture studies is LDH.

Assumptions of LDH assay : Intracellular enzymes are only released after damage to the cell

membrane : Rapidly released from damaged cells.

Pitfalls of LDH assay

The release of LDH activity can be related to the total No. of dead & lysed cells.

The stability of LDH can vary considerably, ranging from the loss of a few percent per day to a half-life of 12h depending upon the cell type.

Assumed that the release of LDH occurs rapidly after damage to the cell membrane. This assumption is not necessarily correct.

The release of LDH can be complete in cells which are considered dead by dye exclusion methods.

Complete release may only occur upon cell lysis.

This point is further complicated because dye exclusion methods do not measure lysed cells.

Reagents and Solutions

Buffer (Tris 81.3 mmol/L ; NaCl 203.3 mmol/L ; pH 7.2)

: Dissolve 4.92 g Tris and 5.95 g NaCl in 400 ml water and adjust to pH 7.2 at 30 with HCl. Make up to a final volume of 500 ℃ml with water

NADH solution(-NADH 0.17 mg/ml)

: Dissolve 3.4 mg NADH in 20 ml buffer

Pyruvate solution(9.76 mmol/L)

: Dissolve 0.107 g monosodium pyruvate in 90 ml buffer. Make up to a final volume of 100 ml with buffer.

Buffer is stable at 0-4 .℃

The NADH solution is kept at 0-4℃ and must be prepared fresh daily

The pyruvate solution should be dispensed into 1.5ml aliquots and stored at -20 .℃ After thawing, each aliquot should be discarded.

The pyruvate solution is stable for 2 months.

Stability of solutions

Materials and Equipment

NADH solution

Pyruvate solution

Narrow-bandwidth spectrophotometer, fitted with a thermostatted cuvette holder capable of temperature control within 0.1 and a chart-recorder.℃

Assay Conditions

Incubation temperature : 30.0 ℃

Wavelength : 340 nm

Final reaction volume : 1.07 ml

Light path : 1.0 cm

• The LDH activity is determined by an enzymatic test.

• The first step is the reduction of NAD+ to NADH/H+ by the LDH catalysed conversion of lactate to pyruvate.

• In the second step, the catalyst diaphorose trasfers H/H+ from NADH/H+ to the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phnyltetrazolium chloride (INT), which is reduced to a red formazan.

• The resulting formazan absorbs maximally at 492nm and can be measured quantitatively at 490nmReferences 1.Decker, T. and M. L. Lohmann-Matthes (1988). "A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity." J Immunol Methods 115(1): 61-9.2.Korzeniewski, C. and D. M. Callewaert (1983). "An enzyme-release assay for natural cytotoxicity." J Immunol Methods 64(3): 313-20.3.Lappalainen, K., I. Jaaskelainen, et al. (1994). "Comparison of cell proliferation and toxicity assays using two cationic liposomes." Pharm Res 11(8): 1127-31.4.Nachlas, M. M., S. I. Margulies, et al. (1960). "The determination of lactic dehydrogenase with a tetrazolium salt." Anal Biochem 1: 317-26.

Ethidium bromide (EtBr) and propidium iodide (PI)PI is impermeable to intact plasma membrane.

PI binds to nucleic acids upon membrane damage : flow cytometric techniques depend on fluorescence, PI is ideally suitable for the rapid evaluation of the permeability properties of large numbers of cells while maintaining good statistical accuracy.

Intercalates with DNA or RNA red

Fluorescent dyesFluorescent dyes

Fluorescein diacetate (FDA) is a nonpolar ester

which passes through plasma membranes and is hydrolyzed by intracellular esterases to produce free fluorescein, the polar fluorescein is confined within cells which have an intact plasma membrane and can be observed under appropriate excitation conditions.

Undamaged cell : highly fluorescent fluorescein dye

Damaged cell : fluoresce only weakly

greenish-yellow at 450-480 nm

Intact cell –PI and FDA is added

Fluorescein in intact cells

Schematic illustration of the principle of PI/FDA cell viability assay

● FDA (Fluorescein diacetate)● PI (Propidium iodide)

Plasma membrane is damaged ; fluorescein leaks out

PI enters and strains nucleic acids

A group of hepatoma cells exposed to a diffusing wave of digitonin. Intact cells (green) are damaged by digitonin, loose the green fluorescence and acquire

red fluorescence of PI.

Example 1 ; Observation of cell death

Functional assaysFunctional assays

Evaluate viability by examining the metabolic components that are necessary for cell growth, on the premise that cellular damage will inevitably result in the loss of ability to maintain and provide energy for metabolic function and growth.

Colorimetric assay

Rapid and accurate assessment of viable cell number

Miniaturized into 96-well plates

Measure using an Microplate reader

Permit many sample to be analyzed rapidly

Reduce medium and plastics costs

These assay are read at 570 nm (except for the Acid Phosphatase (A.P) assay-wavelength is 405 nm) on a ELISA plate reader, using a 620 nm filter as reference wavelength.

It is important to remove any bubbles from the well before absorbance readings.

Examples are

MTT / XTT assay

Crystal violet dye elution (CVDE)

Acid phosphatase (AP) assay

Alamar blue oxidation-reduction assay

Neutral Red (NR) assay

[3H]-thymidine and BrdU incorporation

MTT Assay

Introduction

This assay is a sensitive, quantitative and reliable colorimetric assay that measures viability, proliferation and activation of cells.

The assay is based on the capacity of mitochondrial dehydrogenase enzymes in living cells to convert the yellow water-soluble substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan product which is insoluble in water.

The amount of formazan produced is directly proportional to the cell number in range of cell lines.

metabolically active Cell

MTT Formazan

Insoluble

Materials and equipment MTT solution (5 ㎎ / ㎖ in phosphate buffered saline (PBS) pH 7.5),

HCl, Propan-2-ol

96-well microtiter plate, ELISA reader

Procedure (suspension and monolayer cells)1. Prepare MTT stock solution and fiter through a 0.2 ㎛ filter to

sterilize and remove the small amount of insoluble residue

2. To 100 ㎕ cell suspension or cell monolayer in each microtiter well add 10 ㎕ MTT

3. Incubate in a humidified incubator at 37 for 3 h℃4. Add 100 ㎕ 0.04 M HCl in propan-2-ol to each well and mix

thoroughly to dissolve insoluble dark blue formazan crystals

5. Read plate on a ELISA reader using a test wavelength of 570 nm and reference wavelength of 630 nm

Advantages of MTT assay

• Considered a major advance; used the most prevalent in vitro assay

• Rapid, versatile, quantitative and highly reproducible

• Adaptable to large-scale screening; relevant for most

cells

• MTT reduction correlates to indices of cellular protein

and earlier cell number

• More sensitive and earlier predictor of toxicity than

classical LDH or neutral red measurements

Disadvantage of MTT assay

1. Production of the MTT product is dependent on the MTT concentration in the medium. The kinetics and degree of saturation are dependent on cell type.

2. Assay is less effective in the absence of cell proliferation.

3. MTT cannot distingulish between cytostatic and cytocidal effect.

4. Individual cell numbers are not quantitated and results are expressed as a percentage of control absorbance.

5. Test is less effective if cells have been cultured in the same media that has supported growth for a few day, which leads to underestimation of control and untreated samples.

Example; NIH J2 3T3 cell

in medium

MTT solution

elution

Absorbance at 540 nm

XTT assay

Introduction

The assay is based on the cleavage of the yellow tetrazolium salt XTT

to form an orange formazan dye metabolic active cells.

This conversion only occurs in viable cells.

The formazan dye formed is soluble in aqueus solution and is directly

quantified using Microplate reader.

Both MTT and XTT work by being to a formazan dye only by

metabolic active cells.

metabolically active cell

Water soluble

Materials and equipments XTT labeling regent, electron-coupling reagent 96-well microtiter plate, Microplate reader → XTT labeling mixture : mixed 5 ㎖ XTT labeling reagent

with 0.1 ㎖ electron coupling reagent

Procedure1. Cell are grown in microtiter plates in a final volume of 100

㎕ culture medium per well. The incubation period of the cell cultures depends on the particular experimental approach and on the cell line.

2. After incubation period, add to each well 50 ㎕ of the XTT labeling mixture

3. Incubate the microtiter plate for 4 to 24 h in incubator4. Read plate on a Microplate reader using a wavelength

between 450 and 500 nm (reference wavelength of 650 nm)

Compare with MTT assay and XTT assay

Culture cells in a MTP for a certain period of time (37 )℃

MTT assay XTT assay

Prepare labeling mixture

Incubate cells (0.5-4 h, 37 )℃

Add solubilizing solution(Isopropanol) and incubate

Measure absorbance using an ELISA reader

Add XTT labeling mixtureAdd MTT labeling reagent

Insoluble formazan Soluble formazan

Example: MTT and XTT

MTT XTT

Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002. Evaluation of redox indicators and the use of digital scanners and spectrophotormeter for

quantification of microbial growth in microplates. J. Micro. Methods. 50:63-73

Principle

Dye elution: Cell up-taken dye was measured colorimetric method after acetic acid

dye elution.

Nuclei counting

Incubation of cell samples in a mixture of citric acid and crystal violet causes cells to lyse and the released nuclei to stain purple.

Crystal violet

Procedure

Dye elution ① After removal of medium, rinse 96

well plates with 100 ㎕ /well of PBS and stain with 100 ㎕0.25% (g/10ml) aqueous crystal violet for 10 min.

② Rinse plats four times in tap water. ③ Dry the outsides of the plates with

paper to help avoid water stains, and then dry the plates at 37 . ℃When dry, add 100 ㎕ per well of 33% glacial acetic acid (33 ml/100ml) and mix the contents of each well before reading at 570 nm.

Nuclei counting

① Allow microcarriers from a culture sample (1ml) to settle to the bottom of a centrifuge tube.

② Removed clear supernatant by aspiration.

③ Add 1ml of crystal violet reagent.

④ Incubate at 37 at least 1 h.℃ ⑤ Introduce a sample into the

hemocytometer chamber and count the purple-stained nuclei as for whole cells.

Example; Monolayer culture

Ref.:Journal of Orthopaedic Resarch19 (2001)

※ OP : Osteoporosis

Crystal Violet - 60minutes

Non-OP OP

Example; Microcarrier culture

Rabbit oral mucosal cell cultured by microcarrier Cell counting

Acid phosphatase (AP) assay

Introduction

The action of this enzyme in many of tissue is to cleave a waste product called pyrophosphate and effectively convert it to a useable phosphate.

P-nitrophenyl phosphate will be the substrate and nitrophenol is the product of this reaction.

Nitrophenol is colorless but when the pH of the reaction solution is alkaline, it is appears yellow. The pH of the reaction solution will be changed by the addition of NaOH.

P-nitrophenyl phosphate + Acid phosphatase Nitrophenol + HPO4

-2

Materials and equipment Substrate-containing buffer : 10 mM P-nitrophenyl phosphate in

0.1 M sodium acetate pH 5.5, 1 M NaOH

96-well micro titer plate, Microplate reader

Procedure1. At end of cell growth period, remove medium and rinse wells in

100 ㎕ PBS

2. Add 100 ㎕ substrate-containing buffer to each well

3. Incubate for 2 h in incubator. Read plates at 405 nm, and either reincubate for a further time if increased sensitivity is required, or ‘stop’ with addition of 50 ㎕ /well of 1 M NaOH to cause an electrophilic shift in the p-nitrophenol chromophore and thus develop the yellow color, giving greatly increased sensitivity

Principle In the presence of cellular metabolism the color of Alamar Blue (ALB)

changes from a fully oxidized, non fluorescent blue to a fully reduced, fluorescent red. ALB will be reduced by a variety of enzymes and small molecules, including the cytochrome system, FMN, FAD, NAD, and NADP.

Advantages Simple, rapid, inexpensive, required no lysis, extraction or washing of sample

Disadvantages Unstable during storage (absorbance of oxidized ALB-3day, reduced ALB-

increase from one day to the next)

Characteristics - Sensitivity : Propidium iodide (PI), Sulforhodamine B (SRB) > ALB=MTT - The ALB assay is faster, simpler, and less artefact prone than the MTT assay.

Alamar Blue oxidation-reduction assay

Procedure

Neutral Red assay(3-amino-7dimethyl-2-methyphenazine hydrochloride)

Principle

- The incorporaton of NR into the lysosomes of viable cells after their incubation with test agents.

Use

- Industrial, pharmaceutical, environmental and other testing laboratories concerned with acute toxicity testing.

Advantages

- Simplicity, speed, economy, and sensitivity

Materials and Equipments

Solution ① Neutral red

4mg/ml stock solution Dilute 1:100 into medium , incubate

overnight at 37 and centrifuge for ℃10 min at 1500 g before use.

② 1% CaCl2/0.5% formaldehyde Mix 6.5 ml 37% formaldehyde with

50 ml 10% CaCl2 and 445 ml distilled water.

③ 1% acetic aicd/50% ethanol Mix 4.75 ml acetic acid with 250 ml

95% ethanol and 245 ml distilled water.

Equipment ① Complete media suitable for chosen cell type.

② Culture petri dish

③ 96well tissue culture plate

④ Inverted microscope

⑤ ELISA-type spectrophotometer

⑥ Microplate shaker

⑦ Eight-channel pipette

Procedure

① Resuspend cells of actively growing culture and count cells and accurately allocate appropriate number suspended in medium.

② Seed 0.2 ml containing desired number of cells to each well of 96 well plate and incubate at 37 for 24 h or longer.℃

③ Removed the medium and add fresh medium containing graded dilutions of test agent. Incubate for desired length of time. Examine at least 4-8 wells per concentration of test agent.

Keep serum concentration as low as possible during this step.

(prevent or to reduce adsorption of xenobiotic to serum components)

④ After incubation for desired time interval, remove medium with test agent and incubate cells with fresh medium containing 40 ㎍ /ml NR dye.

⑤ Continue incubation for 3h to allow for incorporation of vital dye into survival cells.

⑥ Remove medium by inverting the plate and rapid rinse with a mixture of 1% CaCl2 / 0.5%formaldehyde.

⑦ Extract dye into suprernate with 0.2 ml of solution of 1% acetic acid/50% ethanol.

After10 min at room temperature and rapid agitation for a few seconds on a micrometer plate shaker, scan the plate with an ELISA-type spectrophotometer equipped 540 nm filter.

Principle

The rate of DNA synthesis is a reflection of proliferation under many condition.To measure the proliferative rates by [3H]-thymidine uptake, cells are cultured in microtitre wells, thymidine is added, and the uptake by DNA is measured , after lysing and washing on, by scintillation counting. Bromodeoxyuridine(BrdU) can be incorporated instead of [3H]-thymidine and the incorporation can be assayed with antibodies to BrdU in a non-radioactive assay.

[3H]-thymidine and BrdU incorporation(DNA synthesis measurement)

Schematic diagram of [3H]-TdR and BrdU

Labeling index with [3H]-thymidine

① Set up the culture at 2x104 cells/ml~ 5x104 cells/ml in 24 well plates containing cover-slips. Grow to the desired cell density.

② Add [3H]-thymidine to the medium . 100KBq/ml(~5μCi/ml)and incubate for the cultures 30 min.

③ Remove the labeled medium, and discard it into a designed container for radioactive waste.

④ Wash the cover-slips three times with PBSA. ⑤ Add 1:1 PBSA: acetic methanol, 1ml per well, and remove it immediately ⑥ Add 1ml of acetic methanol at 4 to each well, and leave the cultures for

10min. ⑦ remove the cover-slips, and dry them with a fan ⑧ mount the cover-slip on a microscope slide with the cells uppermost. ⑨ Leave the mountant to dry overnight.

DNA synthesis by [3H]-thymidine

① Grow the culture to the desired density.

② [3H]-TdR, 40 KBq /ml(~1.0μCi), 2 MBq/mol(~50 μCi/mol) in HBSS.

※1Ci =3.7*1010 회 / s = 3.7*1010 Bq , 1Bq =1 회 /s

③ Incubate the cell for 1-24 h.

④ Remove the radioactive medium carefully.

⑤ Wash the cell carefully with 2 ml of HBSS, PBSA, and 2 ml ice-cold 0.6 M TCA for 10 min.

⑥ Wash the cell with TCA twice 5 min each time.

⑦ 0.5 ml of 2 M perchloric acid, a hot plate at 60 for 30min and allow the solution to ℃cool.

⑧ Add 0.5ml SLS in NaOH incubate the solution at 37 for 30 min or overnight at room ℃temperature.

⑨ Collect the solubilized pellet and determine the radioactivity.

DNA labeling assayDNA labeling assay (using fluorescent probes assay)(using fluorescent probes assay)

Two types of nonviable cells

Two methods: Enzymatic DNA labeling and DNA-binding dye labeling

dNTP dUTP

Direct Indirect

X Fluoresein, FITC, PE etc Biotin DIG

Avidin conjugated with

fluoresein, AP, POD

Anti-DIG antibody conjugated with fluoresein,

AP, POD

Ⅰ. Enzymatic DNA labeling

Fig. Immunostaining of apoptotic cells (dark brown) by TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and

peroxidase staining in rabbit endometrium

DNA-binding dyes: fluorochrome

DyePermeability via intact membrane

Staining

DNA RNA

Acridine orange Yes Green Red-orange1

Hoechst 33342 Yes Blue No

Hoechst 33258 Yes Blue No

DAPI(4,6-diamidino-2-

phenylindole)

Yes Bright blue No

EtBr(Ethidium bromide)

No Orange Slightly red1

PI(Propidium iodide)

No Red No2

1 RNase treatment is required2 RNase treatment is required because PI could stain double strand RNA

Ⅱ. DNA-binding dye labeling

Dye

Apoptosis

Necrosis

Early apoptosis Late apoptosis*

Acridine orangeGreen

Condensed

Green

Fragmented

Green

Diffuse, Intact

Hoechst 33342Blue

Condensed

Blue

Fragmented

Blue

Diffuse, Intact

Hoechst 33258Blue

Condensed

Blue

Fragmented

Blue

Diffuse, Intact

DAPIBlue

Condensed

Blue

Fragmented

Blue

Diffuse, Intact

Ethidium bromide

No

(Orange, Condensed

if permeabilized)

Orange

Fragmented

Orange

Diffuse, Intact

Propidium iodide

No

(Red, Condensed

if permeabilized)

Red

Fragmented

Red

Diffuse, Intact

Pattern of dye staining according to color and chromatin morphology

* late apoptosis is regarded as the stage of membrane fragmentation and secondary necrosis

Fig. In Hoechst 33258 / PI double staining, cells with blue intact nuclei were viable cells, whereas those with blue fragmented nuclei were early apoptotic cells. Cells with pink intact nuclei were necrotic cells, whereas cells with pink fragmented nuclei were late apoptotic cells. (blue against Hoechst33258, red against PI)

Apoptotic(0%)

Necrotic(10.5%)

Apoptotic(85.2%)

Necrotic(11.2%)

Apoptotic(1.2%)

Necrotic(92.5%)

Fig. DAPI staining of condensed nuclei of apoptotic cells

Acridine orange

Fluorescent protein biosensors measuring the molecular dynamics of macromolecules, metabolites, and ions in single cells have emerged from the integrative use of contemporary synthetic organic chemistry, biochemistry, and molecular biology.

Vascular endothelial cells (ECs) play and important role in physiologic hemostasis and blood vessel permeability, express immune related functions in monocytes and macrophages, and the viability of ECs is important in predicting the post-operative function and durability of cryopreserved vessels for implantation.

Tetrameric Griffonia simplicifolia agglutins (GS1) shows prominent binding only to the a-D-galactosyl residue of blood vessel ECs.

Staining with fluorescein isothiocyanate(FITC) conjugated with GS1 differentiates ECs from the other cells in flow cytometry.

PI intercalates DNA double strands in dead cells without regard to cell types, as their membrane lose integrity.

GS1-FITC and PI double staining: presumed to immediately determine the differential viability of ECs from whole cells

The use of fluorescent probes enables the rapid determination of the viability of ECs and whole cells from the same tissue without separating ECs from whole cells, and of the viability of each step in the cryopreservation process

Fig. Each part of quadrant statistics was observed under fluorescence microscopy. Live ECs preferentially expressed the green color of GS1. Dead ECs are double stained by the green color of GS1-FITC and the red color of PI, which results in yellow. Dead cells except dead ECs are identified by only the red color of PI.

Morphological assayMorphological assay

Large-scale, morphological changes that occur at the cell surface, or in the cytoskeleton, can be followed and related to cell viability.

Damage can be identified by large decreases in volume secondary to losses in protein and intracellular ions of due to altered permeability to sodium or potassium.

Necrotic cells: nuclear swelling, chromatin flocculation, loss of nuclear basophilia

Apoptotic cells: cell shrinkage, nuclear condansation, nuclear fragmentation

Example; Morphological feature (Human skin keratinocyte)

Fig. Morphological feature of (A) normal human skin keratinocyte, and differentiated human skin keratinocyte(B).

(A) (B)

Example; Morphological feature (Human skin fibroblasts)

Fig. Morphological feature of (A) normal human skin fibroblasts, and aging human skin fibroblasts(B).

(A) (B)

Reproductive AssayReproductive Assay

Clonogenic Cell:

Defined as a cell with the capacity for sustained proliferation

Have undergone a minimum of 5-6 doublings to give rise to colonies containing at least 50 cells

Colony-forming Efficiency

Colony-forming Efficiency (CFE) The ability to form colonies is used as a measure of reproductive integrity

It is often referred to as plating efficiency (PE) Number of colonies formed CFE = 100% Number of cell plates

Clonogenic assays Be used to reflect stem cell content

The basis of assays for determining the lethal effects of cytotoxic agents

Determining the PE of an established

adherent cell line

Materials and Equipment

Cell growth medium : Eagle’s basal medium (BME)

100 iu/ml penicillin,

0.1 mg/ml streptomycin

Trypsin-EDTA

Gentain violet stain

Procedure

1. Trypsinize monolayer cultures or use cell suspension

cultures and determine the viable cell count

2. Dilute cells in growth medium to 1000 , 2000 and

5000 cells/10ml

3. Inoculate nine replicate Petri dishes with 4 ml growth medium plus 1ml cell suspension

4. Place plates in a humidified 5% CO2 plus air incubator are normal growth temperature and rock shelf or tray gently to and fro three times. The plates must not be moved now until colonies are stained

5. Stain and count three replicate per cell density at 1,2 and 3 weeks (murine lines) or 2 , 3 and 4 weeks (human lined)

6. Calculate the optimum cell densities for seeding and duration of incubation

Example; Rat keratinocytes

(A) (B)

(C) (D)

Colony forming Non-colony forming

48 hr after

subculture

6 days after

subculture : colony , : Single cells

Reference1. Cell quantification, module 4B:1, Hemocytometer cell counts and viability

studies, 1.1 – 1.5

2. http://www.embl-heidelberg.de/ ExternalInfo/karsenti/countingcells. html

3. http://www.bd.com/clinical/POL/products/equipment/hemacytm.asp

4. Park JC, Hwang YS, and Suh H., Viability Evaluation of engineered

Tissue., Yonsei Medical Journal 2000 41(6): 836-844

5. Ashish A., Sonia SY, Mark AH, and Minas TC., Pressure Related

apoptosis in Neuronal Cel Lines., Journal of Neuroscience Research

2000 60: 495-503

6. Islam TC, Skarin T, Sumitran S, and Toftgard R., Retinoids induce

apoptosis in cultured keratinocytes., British Journal of Dermatology

2000 143:1709-719

7. Maria L. Anthony, Shane N. O. Williams, and Kevin M. Brindle, Nuclear magnetic resonance methods of monitoring cell metabilism., Methods in Biotechnology, Vol. 8: Animal Cell Biotechnology, 165-175, Edited by: N.

Jenkins, Humana Press Inc. Totowa, NJ. 1999. 8. Macdonald JM, Grillo M, Schmidlin O, Tajiri DT, James TL., NMR

spectroscopy and MRI investigation of a potential bioartificial liver., NMR Biomed. 1998 Apr; 11(2):55-66.

9. Flendrig LM, la Soe JW, Jorning GG, Steenbeek A, Karlsen OT, Bovee WM, Ladiges NC, te Velde AA, Chamuleau RA., In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates., J Hepatol. 1997 Jun;26(6):1379-92.

10. Fluorescence and confocal microscopy (http://helios.mol.uj.edu.pl/conf_c/main.htm)11. Breuls RG, Mol A, Petterson R, Oomens CW, Baaijens FP, Bouten CV.,

Monitoring local cell viability in engineered tissues: a fast, quantitative, and nondestructive approach., Tissue Eng. 2003 Apr;9(2):269-81.

12. Griffiths JB, Newell DG, and Doyle A., Cell & Tissue Culture: Laboratory procedures., Cell Quantification, 4B, JOHN WILEY & SONS Ltd, UK.1993

In-vivo models

Models of transplantable tumour

• Murine solid tumour models with Daltons lymphomatic cells

Ascites tumor model

Normal mouse Tumor bearing mouse