Biomedical Testing

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

2) Basic FGF-induced cell proliferation assay

3) VEGF-induced cell proliferation assay

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4) Cell cytotoxicity assay

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5) MMP activity assay

6) Rat aortic ring angiogenesis assay

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

7) Rat aortic ring angiogenesis assay

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

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8) Matrigel implant assay

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

Anti-inflammatory Testing
Inflammation (ear edema) model

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).

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.

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