Biomedical Testing

Poseidon provides support services to the biopharmaceutical industries as a contract research organization. For pricing information on the following assays, please contact +1 646 341 7714 or email info@poseidonsciences.com

A. Angiogenesis Assays

1) Tube formation assay

Cell type Human umbilical vein endothelial cells(HUVEC)Primary cultured cells within passage 5
Control Vehicle-treated control
Method The measurement of total tube length on matrigel
Antagonist If active, minimal inhibitory concentration of compound is determined.

2) Basic FGF-induced cell proliferation assay

Cell type Human umbilical vein endothelial cells (HUVEC) Primary cultured cells within passage 5 or Bovine capillary endothelial cells (BCE)
Control 10 ng/ml bFGF
Method Colorimetric assay (XTT based)
Antagonist >50% Inhibition of bFGF-induced proliferation

3) VEGF-induced cell proliferation assay

Cell type Human umbilical vein endothelial cells (HUVEC) Primary cultured cells within passage 5
Control 10 ng/ml VEGF
Method Colorimetric assay (XTT based)
Antagonist >50% Inhibition of VEGF-induced proliferation

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4) Cell cytotoxicity assay

Cell type Human umbilical vein endothelial cells(HUVEC) or Ha-CaT cell
Control Vehicle-treated control
Method Colorimetric assay (XTT based)
Antagonist >50% Decrease in optical density relative to vehicle control

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5) MMP activity assay

MMP Source Purified human recombinant MMP-1, -2, -3, -7, -9, -13 Substrate : fluorogenic peptide substrate (e.g.Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 for MMP-2 and MMP-7)
Reaction MMP + fluorogenic substrate -> fluorescence increase
Method Spectrofluorometric quantitation of emission (Perkin Elmer LS50B)
Test concentration 10uM

6) Rat aortic ring angiogenesis assay

Species Spraque-Dawley rat (aorta)
Control Serum-treated control
Antagonist Increase in microvessel density without serum
Method Microvessel outgrowth from aortic ring on matrigel (microscopic picture data); The measurement of microvessel percent area
Test concentration One concentration per compound

Kruger E. A. and Figg W.D. Clinical Cancer Research 7:1867-1872,2001

7) Rat aortic ring angiogenesis assay

Species Spraque-Dawley rat (aorta)
Control Vehicle-treated control
Antagonist Decrease in microvessel density in the presence of serum
Method Microvessel outgrowth from aortic ring on matrigel (microscopic picture data); The measurement of microvessel percent area
Test concentration One concentration per compound

Kruger E. A. and Figg W.D. Clinical Cancer Research 7:1867-1872,2001

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8) Matrigel implant assay

Species C57BL6 mouse (n=8)
Control Vehicle-treated control
Dose Generally, 1 or 2 dose levels, as requested by client Dosing can be once or twice a day per mouse for 4 days
Criteria Hemoglobin content in matrigel
Route Subcutaneous injection of sample mixed with matrigel orintraperitoneal injection / or oral administration

Passaniti, A et al. Lab. Invest 67: 519-528, 1992

Anti-inflammatory Testing
Inflammation (ear edema) model

Species ICR mouse (n= 5 )
Inducer TPA
Dose One or Two concentrations of a sample as requested by client; Indomethacin as reference compound
Criteria Ear thickness before and after the challenge
Route Topical application

Goto Y et al. Euro J Pharmacol 438: 189-19,6 2002

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Inflammation Assay (Ear edema model)
Edema is induced in the ear by topical application of 10ul of TPA (Tetradecanoylphorbol acetate) in acetone (2.5 ug/ear) to both the inner and outer surface of one ear of each mouse. The experimental compound is diluted with acetone and is applied topically immediately after TPA. The reference drug, Indomethacin (0.5mg/ear), is administered as a positive control. The thickness of each ear is measured before treatment and 4 hours after induction of inflammation, using a micrometer (Mitutoyo Co.). Edema was expressed as the increase in ear thickness due to TPA application. Edema inhibition is expressed as the reduction in thickness with respect to the control group.


Effect of Test Compounds on HUVEC Proliferation
The effect of an experimental compound on endothelial cell proliferation is determined in vitro using HUVE (Human Umbilical Vein Endothelial) cells. HUVE cells are isolated from human umbilical cord veins by a method of Jaffe et al. (Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, C. R. Culture of Human endothelial cells derived from umbilical veins. J. Clin. Invest. 52: 2745- 2756,1973). HUVE cells are confirmed by immunostaining with antibody against factor VIII. The cells were grown in M199 medium(Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 mg /mL streptomycin, 50 mg /mL endothelial cell growth supplement, and 5 units /mL heparin at 37 oC in an atmosphere of 5% CO2-95% air.

1 x104 HUVE cells are plated in each well of 96 well plates. Five different concentrations ranging from 1 to 100mM of the test compound are tested in the presence of bFGF used as maximum proliferation control. Cells are cultured for additional 48h, and relative cell numbers in each well are determined by XTT Cell Proliferation Kit (Roche).


Cell cytotoxicity assay
Cell cytotoxicity assay is performed to evaluate the cytotoxicity of a test compound against normal cells. 5 x103 Ha-CaT cells are plated in each well of 96 well plates and HR008 is added at various concentrations. The plates are incubated for another 48 hours. The viable cells are measured using the XTT Cell Proliferation Kit (Roche).

100µM 50µM 10µM 5µM 1µM 0.1µM
Viability(%) 70.4 81.8 96.3 96.5 100.0 100.0


Rat aortic ring assay
The effect of a test compound on angiogenesis is studied by culturing aortic explants in three–dimensional matrix gels according to the procedure of Kruger and Figg (Kruger E. A. and Figg W.D. Protein Binding Alters the Activity of Suramin, Carboxyamidotriazole, and UCN-01 in an ex Vivo Rat Aortic Ring Angiogenesis Assay Clinical Cancer Research 7:1867-1872, 2001).

Thoracic aortas are excised from 8-week-old male Sprague Dawley rats and the fibroadipose tissue is removed. The aortas are sectioned into 1-mm-long cross-sections, rinsed with Human Endothelial-SFM(GIBCO), placed on the Matrigel-coated wells, covered with additional 50 ul Matrigel, and allowed to gel for more than 30min at 37 oC, 5% CO2. All the rings are cultured in Human Endothelial-SFM(GIBCO) supplemented with 200ug/ml of ECGS(Endothelial Cell Growth Supplement, Sigma) as an angiogenesis inducer. HR008 diluted with ethanol is added to the culture medium at final concentrations of 1, 10, and 100 uM. Ethanol alone (1%) is added to the culture medium as a vehicle control.

All assays are performed by using 5 aortic rings per sample. Aortic rings are photographed on day 10. The area of angiogenic sprouting is calculated using Image-Pro Plus software program (Media Cybernetics). Microvessel densities are reported in square pixels.

Below is an example of a typical assay result.

Figure above shows typical photographs of angiogenic sprouting and corresponding quantitation by image analysis at various concentrations of an experimental compound.

Table Image analysis quantitation of HR008 dose-response activity.

NO. control 1µM 10µM 100µM
1 18.3 18 15.1 17.3
2 17.8 15.7 13.5 10.1
3 17.2 14.6 13.2 8.8
4 16.8 11.5 10.4 8.5
5 8.7 7.3 8.9 8.2
mean 15.76 13.42 12.22 10.58
S.D 4.0 4.1 2.5 3.8
%inhibition 0 15 22 33

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