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MTT assay for cell proliferation
induced tumors models
Transplantable
Sulphorhodamine B Assay
tumors models
Evaluation:
Evaluation:
Evaluation
• percentage reduction of tumor incidence compared
with carcinogen control.
N, N-Diethylnitrosamine (DEN)-induced Lung
Adenocarcinoma in Hamster
Procedure
• 17.8 mg DEN/kg body weight twice weekly by
subcutaneous injection for 20 weeks starting at age
7 to 8 weeks usually produces tracheal tumors in
90-100% and lung tumors in 40-50% of male Syrian
hamsters.
Evaluation:
• The percentage reduction in tumor incidence in
treatment group is compared control group
DMBA-induced Oral Cancer in Hamster
Procedure:
Evaluation:
• Tumor size,
• Tumor number and
• Tumor burden of drug treated animals can be
compared with control animals
Benzopyrene-induced For Stomach Tumors in
Mouse
Procedure:
• 1 mg of benzopyrene in 0.1 ml peanut oil twice
weekly for 4 weeks given to mice by gavage.
Evaluation:
• Tumor incidence and
• Tumor burden in drug treated animals can be
observed and compared with carcinogen control
animals at the end of the experiment.
• Transplantable tumor
models
• These models are based on the use of cancer
cell lines or tissues that can be grown in mice or
rats.
• Methods of transplantation
A. Heterotopic transplantation
• To the site other than that of origin
• e.g. pancreatic cancer implanted
subcutaneously
B. Orthotopic transplantation
• To the site of origin
• e.g. lung cancer implanted in lung
• Transplantable tumor
models
B. Heterotopic transplantation
• transplantation of tumor cells or tissue at the site
other than its site of origin.
• Subcutaneous transplantation 1. Intraperitoneal transplatation
• A tumor cell suspension is • Tumor cell suspension is
injected into the flank of the injected in peritoneal cavity.
animal.
• Require between a few days to • Grows within few days.
a few months to grow.
• Develop as solid tumor. • Develops as ascites.
Evaluation parameters:
Volume of peritoneal fluid
Viability of tumor cells in peritoneal fluid (% viability)
% Increase in survival time as compared to control
A.Xenograft models
• For tumor models that more closely resemble the
clinical disease, transplantable tumors of human
origin should be used.
• But transplantation of such human tumors in mice
may result in severe immune rejection.
• For this purpose athymic (nude) mice or severe
combined immunodeficiency (scid) mice are used.
• These animals lack immune response to such foreign
transplanted material.
• Transplantation of tumor cell lines into nude mice can
be accomplished via multiple routes: s.c., i.p., i.v.,
intracranial, intrasplenic, renal subcapsular.
1. Intraperitoneal Microencapsulated
Tumor Assay
Procedure:
Evaluation
• Counting viable tumor cells by Heamocytometer in treated
versus control animals.
2. Hollow fiber Assay
Procedure:
• Polyvinylidene fluoride (PVDF) hollow fibers (500k Da M.wt.
exclusion, 1mm i.d.) containing target cells are heat, sealed and
cut at 2 cm intervals and implanted into rodents.
• 3 or more tumor cell lines can be grown concurrently, in 2
physiologic sites, i.p. and s.c. within each mouse.
• The mice are treated with experimental compounds once daily
for four days. Fibers are collected 24 hr following the last dose
of compound.
Evaluation:
• After collection the viable cell mass is determined using an MTT
dye conversion assay.
• The cytostatic/cytocidal effect of a compound is determined
from differences in the viable cell mass in fibers from
compound treated Vs diluent treated mice.
• Methods for evaluation of drug
effect
1. Tumor size
2. Tumor weight
3. Excision clonogenic assay
4. End point dilution assay
5. Increase in Survival time
Excision clonogenic assay
• Determines the fraction of cells in a tumor population retaining
proliferative capability after drug exposure.
• Tumor bearing animals are treated with drug.
• At 24 hours, the tumors are excised from treated and
untreated animals.
Tumor
Cell suspension
Procedure:
• The HUV-ECs are cultured at 37 °C and 5% CO2 in 90% Ham’s F12K,
10% fetal bovine serum, 30 μg/ml endothelial cell growth factor, 100
μg/ml heparin, and 4 mM L-glutamine.at a density of 3 × 103 cells/well in
24-well plates.
• After 24 hrs, the test compound in various concentrations of the vehicle
are added, and plates are incubated for 72 h.
• Cells are then harvested with trypsin/EDTA and counted by a
hemocytometer.
Evaluation:
• Inhibition of cell proliferation is compared in vehicle treated and
compound treated cultures.
Chorioallantoic membrane assay
Rationale:
• Angiogenesis on chorioallantoic membrane of chicken eggs is used as a
model system.
Procedure:
• Fertilized White Leghorn chicken eggs incubated at 37 °C
• On day 3, a square window is opened in the shell and 2 to 3 ml of albumen
is removed to allow detachment of the developing CAM.
• The window is sealed with a glass and the eggs are returned to the
incubator.
• On day 8, 1 mm 3 gelatin sponges loaded with 3 μl phosphate buffered
saline (negative control) or containing 3 μg of the basic fibroblast growth
factor (positive control), or with various doses of test compound, are
implanted on top of the CAM.
• CAM are examined daily until day 12, when the angiogenic response peaks.
Evaluation:
• On day 12, blood vessels entering the sponge within the focal plane of the
CAM are recognized, counted and photographed. Only transversely
sectioned microvessels i.e. capillaries and venules with or without a 3 to 10
μm lumen are counted
Mesenteric window angiogenesis model
Rationale:
• The mesenteric window assay in rats for quantitative
measurement of induction and inhibition of angiogenesis
Procedure:
• i.p. injection of the mast cell secretagogue compound 48/80 twice
daily for 4-5 days to male S D rats ( 225 g.)
• Test compounds or saline are injected s.c. 1 h before each
injection of compound 48/80.
• Specimens are fixed on slides and stained with toluidine blue to
measure the relative vascularized area.
• Three randomly selected vascular view fields per mesenteric
window spread are analyzed for microvascular length per unit
area of vascularized tissue.
• The total microvascular length is computed from the vascularized
area of each animal multiplied by the mean microvascular length
for the corresponding treatment group.
Evaluation:
• The total microvascular length in control and drug treated groups
are compared by statistical analysis of the observations.
The Rat Aortic Ring Assay
Procedure: It involves three steps:
Preparation of the aorta
• Thoracic and abdominal aorta is isolated transferred to a
culture dish containing MCDB 131 culture medium remove
the surrounding fibro-adipose tissues cut into 1mm ring
sections and rinsed in culture medium to remove blood
residues.
Making agarose culture wells
• Concentric rings from solidified agarose are prepared in a Petri
dish and 4 wells are transferred in each culture dish.
Culturing of the aortic rings
• Bottom of each well is coated with 150μl of clotting fibrinogen
solution.
• Aortic rings are embedded into the well by filling up all the
agarose wells completely with fibrinogen solution.
• Then 10ml of MCDB 131 culture medium is added into each
Petri dish and kept at 37 °C with 5% CO2.
Evaluation:
• Quantification of microvessel growth is done manually or by
computer assisted image analysis (i.e. pixel integrated density
is calculated from the image).
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