Cancer is a corporate term used for a group of diseases that are characterized by the loss of control of the growing, division, and spread of a group of cells, taking to a primary tumour that invades and destroys next tissues. It may besides distribute to other parts of the organic structure through a procedure known as metastasis, which is the cause of 90 % of malignant neoplastic disease decease.
The footings malignant neoplastic disease, malignant tumor ( new growing ) and malignant tumour are synonymous ; they are distinguished from benign tumours by the belongingss ofdedifferentiation,
invasiveness and the ability to run into stasis ( spread to other party of the organic structure ) . ( Both benign and malignant tumours manifest uncontrolled proliferation. )
Carcinoma
Carcinoma is a malignant tumor of epithelial beginning. It is a tumour that arises in the tissues that line the body`s variety meats like the olfactory organ, the colon, prostrate, urinary vesica, and the urethra. Most of all malignant neoplastic disease instances are carcinomas. The common symptoms include is dysphagia and weight loss.Growth of the tumour, be related to get downing, or be related to metastases into the environing esophageal lymph nodes may consequences in hurting.
Sarcoma
These are the tumours originate in fat, musculus, bone or linking tissue. The tumour may originate in the thighbone ( thigh bone ) or the pelvic girdle. It may besides develop in the skull or the level castanetss of the bole. Clinical symptoms are few. The most common is pain and on occasion swelling at the site of the tumour. Fever may besides be present.The tumour spreads easy, frequently to the lungs and other castanetss.
Leukemia
Leukemia ‘s are malignant neoplastic diseases of the blood or blood-forming variety meats. When leukaemia develops, the organic structure produces a big figure of unnatural blood cells. In most types of leukaemia, the unnatural cells are white blood cells. The leukaemia cells normally look different from normal blood cells, and they do non work decently. Leukemia can either be acute or chronic. In acute leukaemia the unnatural blood cells are blasts that remain really immature and can non transport out their normal maps. The figure of blasts additions quickly, therefore making a greater and earlier impact on the victim. In chronic leukaemia, some blast cells are present which are relatively more mature, and therefore can transport out some of their normal maps.
Lymphoma
Lymphomas affect the lymphatic system, a web of vass and nodes that acts as the body`s filter. The lymphatic system distributes foods to blood and tissue, and prevents bacteriums and other foreign “ encroachers ” from come ining the blood stream. There are over 20 types of lymphoma. Hodgkin`s disease is one type of lymphoma. All other lymphomas are grouped together and are called non-Hodgkin`s lymphoma. Non-Hodgkin`s lymphoma may happen in a individual lymph node, a group of lymph nodes, or in another organ. This type of malignant neoplastic disease can distribute to about any portion of the organic structure, including the liver, bone marrow, and lien.
Cell rhythm suppression
The cell rhythm refers to the procedure of cell division. In normal tissues this procedure is tightly controlled by the activity of a figure of cardinal cellular proteins. A loss of cell rhythm control may take to an inappropriate proliferation of cells and finally leads to tumor formation. The cell rhythm consists of:
G1 = growing and readying of the chromosomes for reproduction
S = synthesis of DNA ( and central bodies )
G2 = readying for chromosome duplicate
M = mitosis
Chemotherapy
Chemotherapy is the intervention of malignant neoplastic disease with drugs ( anticancer drugs ) that can destruct malignant neoplastic disease cells. In current use, the term “ chemotherapy ” normally refers to cytotoxic drugs which affect quickly spliting cells in general.
Chemotherapy refers to the usage of medicine and drugs for intervention of malignant neoplastic disease. Most chemotherapeutic drugs are cytotoxic and they work by killing cells. They do this by forestalling the formation of new DNA or by barricading some other indispensable map in the cell. There is a bound on how much of these drugs could be safely used. Since all the chemotherapy drugs interfere with growing of the cells, they all have some signifier of unsought side effects.
ASSAY FOR PROLIFERATION STUDIES
Tryphan blue assay
Principle
Trypan Blue is a bluish acid dye that has two azo chromophores group. Trypan blue will non come in into the cell wall of works cells grown in civilization. Trypan Blue is an indispensable dye, usage in gauging the figure of feasible cells present in a population. It is based on the rule that live cells possess integral cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells do non. In this trial, a cell suspension is merely assorted with dye and so visually examined to find whether cells take up or except dye. In the protocol presented here, a feasible cell will hold a clear cytol whereas a nonviable cell will hold a bluish cytol. An advantage of the trypan bluish exclusion trial is that is a simple and rapid method able to supply approximative consequences.
METHOLDOLOGY
Material REQUIRED IN MEM:
Monolayer civilization bottle of Hep 2 cells
5ml, 10ml serological pipette
Minimal indispensable media ( MEM ) with 10 % , 2 % fetal calf serum
TPVG ( Trypsin, PBS, versene, glucose )
Discarding jar, inverted microscope, dessicator
Baseball gloves, spirit, cotton, label tablet, marker pen
Material REQUIRED IN CYTOTOXICITY ASSAY:
Monolayer civilization in log stage
Drug infusion ( different concentrations )
MEM without FCS
0.4Aµ filter
5ml unfertile storage phial
Tissue paper, spirit, cotton, marker pen and baseball mitts
Micropipette and tips
Discarding jar
MINIMAL ESSENTIAL MEDIA PREPARATION:
Media is defined as a complex beginning of nutritionary supplementation vital for the growing proliferation and care of cells in vitro.
The MEM phial is dissolved in the pre sterilized Millipore distilled H2O and assorted good, closed and sterilized at 15lbs 121°c for 15mins. Let ingredients in the measure, depending on the concentration of fetal calf serum ( 2 % or 10 % ) mix good by agitating. Take attention avoid spills base on balls CO2 utilizing unfertile pipette, Shake the bottle, look into Ph and adjust to 7.2 to 7.4. The MEM bottles are kept for 2 yearss at 37°c and checked for asepsis, PH bead and drifting atoms they are so transferred to the icebox.
Preparation OF INGREDIENT:
Penicillin and Streptomycin: ( Concentration 100IU of Penicillin and 100 Aµg 0f Streptomycin )
Dissolve both antibiotics in unfertile Millipore distilled H2O, so as to give a concluding concentration 100 IU of penicillin and 100Aµg of streptomycin/ml. Mix good and administer in 1ml aliquots. Shop at -20° C Check asepsis.
Fungizone ( amphotericin B ) : ( conc: 20Aµg/ml )
Dissolve in unfertile Millipore distilled H2O so as to give a concluding concentration of 20Aµg/ml and administer in 1ml aliquots in phials. Shop at -20°c. Check asepsis.
L.glutamine: 3 %
Weigh 3g of l-glutamine accurately and fade out in 100ml unfertile Millipore distilled H2O and mix good. Filter through Millipore membrane filter 0.22Aµ and administer in 5ml aliquots in phials. Shop at -20°c. Check asepsis.
4. 7.5 % sodium-bi-carbonate
Weigh needed measure of sodium-bi-carbonate ( to give 7.5 % solution ) accurately and fade out in 100ml of unfertile Millipore distilled H2O. Filter through what adult male filter paper No.4, distribute into bottles and at 121°c, 15lbs, 15mins. Cool and shop at +4°c.
5. Fetal calf serum
Bring FCS at room temperature. Inactivated at 56°c in H2O bath forA? hr and cool at room temperature. If floating atoms are seen filter through Seitz filter. Distribute in 100ml,50ml, 20ml measures in unfertile bottles. Shop at -20°c.
6. Trypsin, PBS, versene, glucose solution: ( TPVG )
2 % Trypsin: 100ml
Weigh 2g of trypsin accurately ; fade out in 100 milliliter sterile Millipore distilled H2O with magnetic scaremonger for A? hr. Filter through membrane filter. Shop at -20°c.
0.2 % EDTA ( versene )
Weigh 200mg of EDTA accurately. Dissolve in 100 milliliter of unfertile Millipore distilled H2O. Autoclave at 15lbs/15mins.
10 % Glucose -100ml
Weigh 1g of glucose accurately. Dissolve in 100 milliliter of unfertile Millipore distilled H2O and filter through whatman filter paper and autoclave at 15lbs/15mins.
TPVG-100ml
PBS – 840ml
2 % trypsin -50ml
0.2 % EDTA -100ml
10 % glucose -5ml
Penicillin & A ; streptomycin -5ml
Mix all ingredients and adjust the pH to 7.4 with 0.1 N HCl or 0.1 N NaOH. Distribute in 100 ml aliquots. Shop at -20°c.
MAINTENANCE OF CELL LINE:
Hep-2 cell line obtained from National Centre for cell scientific discipline, Pune.
Care of cells involves the undermentioned operations:
Dispersion and Sub culturing ( seeding ) of cells.
Preservation of cells in depository.
Revival of cells from depository.
SUBCULTURING AND MAINTENANCE OF CELL LINE:
1. Bring the medium and TPVG to room temperature for dissolving.
2. Detect the tissue civilization bottles for growing, cell devolution, pH and turbidness by seeing in upside-down microscope.
3. If the cells become 80 % feeder it goes for bomber culturing procedure
4. Wipe the oral cavity of the bottle with cotton soaked in spirit to take the adhering atoms.
5. Discard the growing medium in a discarding jar maintain distance between the jar and the flask.
6. Then add 4 – 5 milliliter of MEM without FCS and gently rinsed with tilting. The dead cells and extra FCS are washed out and so fling the medium.
7. TPVG was added over the cells. And incubate at 37° C for 5 proceedingss for disaggregation. The cells become single and it ‘s present as suspension.
8. Add 5ml of 10 % MEM with FCS by utilizing serological pipette.
9. Gently give passaging by utilizing serological pipette. If any clumbs is present so repeat the procedure.
10. After passaging split the cells into 1:2, 1:3 ratio for cytotoxicity surveies for plating method
“ Seeding of cells ” :
After homogenize take one milliliter of suspension and pour in to 24 good home bases. In each well add 1ml of the suspension and maintain in a desiccators in 5 % CO2 atmosphere. After 2 yearss incubation observe the cells in upside-down microscope. If the cells became 80 % feeder.
Cytotoxicity check:
In order to analyze the antitumor activity of a new drug, it is of import to find the cytotoxicity concentration of the drug. Cytotoxicity trials define the upper bound of the extract concentration, which is non-toxic to the cell line. The concentration nontoxic to the cells is chosen for antiviral check.
After the add-on of the drug, cell decease and cell viability was estimated. The consequence is confirmed by extra metabolic intercession experiment such as MTT assay.
PERFORMANCE OF DRUG CYTOTOXICITY ASSAY
Cytotoxicity is the toxicity or harm caused to the cells on add-on of drug. After the add-on of the drug, cell viability is estimated by staining techniques, where by cells are treated with Trypan blue. Trypan blue is excluded by unrecorded cells, but stains dead cells bluish. The consequences are confirmed by extra metabolic intercession experiments such as MTT checks.
Materials Required
Monolayer civilizations in log stage.
Drug infusion ( different concentrations )
MEM without FCS
0.45 i? filter
5mL unfertile storage phial
Tissue paper, Marker pen, Spirit, Cotton and Baseball gloves
1 milliliter, 2 milliliter pipettes, Micropipette and tips
Discarding jar with 1 % Hypochlorite solutio
Drug Dilutions
Stock drug concentration
100 milligram of drug is dissolved in 10 milliliter of serum free MEM giving a concentration of 10mg / 1 milliliter. The stock is prepared fresh and filtered through 0.45 i? filter before each check. Working concentrations of drug runing from 10mg/ml to 0.3125mg/mlare prepared as follow
Preparation of working stock of 1 milligrams /mL
To 4.5 milliliters MEM add 0.5 milliliter of stock to give a on the job concentration of 1mg/mL
Drug concentration can be prepared from the working stock in MEM without FCS. Prepare needed volume of trial sample for each concentration.
1.48hr monolayer civilization of Hep2 cells at a concentration of one hundred thousand /ml /well ( 10 cells / milliliter / good ) seeded in 24 good titer home base.
2. The home bases were microscopically examined for feeder monolayer, turbidness and toxicity if the cells become feeder.
3. The growing medium ( MEM ) was removed utilizing micropipette. Care was taken so that the tip of the pipette did non touch the cell sheet.
4. The monolayer of cells was washed twice with MEM without FCS to take the dead cells and extra FCS.
5. To the washed cell sheet, add 1ml of medium ( without FCS ) incorporating defined concentration of the drug in several Wellss.
6. Each dilution of the drug ranges from 1:1 to 1:64 and they were added to the several Wellss of the 24 good titer home bases.
7. To the cell control wells add 1ml MEM ( w/o ) FCS.
8. The home bases were incubated at 37°c in 5 % CO2 environment and observed for cytotoxicity utilizing inverted microscope.
MTT ASSAY:
MTT check is called as ( 3- ( 4, 5-dimethyl thiazol-2yl ) -2, 5-diphenyl tetrazolium bromide.MTT check was foremost proposed by Mossman in 1982.
Procedure:
After incubation, take the medium from the Wellss carefully for MTT check.
In each well wash with MEM ( w/o ) FCS for 2 – 3 times. And add 200Aµl of MTT conc of ( 5mg/ml ) .
And incubate for 6-7hrs in 5 % CO2 brooder for Cytotoxicity.
After incubation add 1ml of DMSO in each well and mix by pipette and go forth for 45sec
If any feasible cells present formazan crystals after adding solublizing reagent ( DMSO ) it shows the violet colour formation.
The suspension is transferred in to the cuvette of spectrophotometer and an O.D values is read at 595nm by taking DMSO as a space.
Cell viability ( % ) = Mean OD/Control OD x 100
Sample: Cadmium – 8
S.no
Concentration ( Aµg/ml )
Dilutions
Optical density
Cell viability
1
1000
Neat
0.13
22.03
2
500
1:1
0.19
32.20
3
250
1:2
0.23
38.98
4
125
1:4
0.27
45.76
5
62.5
1:8
0.34
57.62
6
31.25
1:16
0.41
69.49
7
15.625
1:32
0.48
81.35
8
7.8125
1:64
0.55
93.22
9
Cell control
–
0.59
100
TOXICITY-1 TOXICITY-2
TOXICITY-3 NORMAL Hep 2 CELLLINE
Sample: CD-6
S.no
Concentration ( Aµg/ml )
Dilutions
Optical density
Cell viability
1
1000
Neat
0.05
8.47
2
500
1:1
0.16
27.11
3
250
1:2
0.24
40.67
4
125
1:4
0.29
49.15
5
62.5
1:8
0.33
55.93
6
31.25
1:16
0.37
62.71
7
15.625
1:32
0.48
81.35
8
7.8125
1:64
0.57
96.61
9
Cell control
–
0.59
100
TOXICITY-1 TOXICITY-2
TOXICITY-3 Normal Hep 2 Cell line
