In-Vitro

In vitro studies are used to aid in the prioritization of active compounds, the evaluation of lead compounds, and the assessment of drug to drug interactions. Our scientists provide qualitative and quantitative data to complement preclinical testing and to allow proper design of clinical studies.


Ocular Cell Lines

Bovine Retinal Capillary Endothelial Cells & Pericytes

Currently EyeCRO has developed the techniques to isolate primary bovine retinal capillary endothelial cells (BRCECs) and pericytes. The Purity of BRCECs and pericytes were confirmed by binding of Dil-Ac-LDL to LDL receptor on the surface of BRCECs and immunolabeling with anti-smooth muscle antibody. These cells have been used in the discovery and development of new ocular drugs. BRCECs and pericytes are isolated from bovine retina. The Purity of BRCECs and pericytes were confirmed by binding of Dil-Ac-LDL (red) to LDL receptor on the surface of BRCECs and immunolabeling with specific antibody against α-smooth muscle actin (green). Nuclei of both cells were counterstained with DAPI (blue).

Cytokine Production

ICAM-1 & VEGF

EyeCro provides cell-based assays to determine the effect of new drugs on the expression of angiogenic stimulators and inflammatory cytokines, e.g. vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1). VEGF expression in human retinal capillary endothelial cells (HRCECs) and human retinal pigment epithelial cells (ARPE-19) is elevated by cobalt chloride (CoCl2). ICAM-1expression in ARPE-19 is stimulated by tumor necrosis factor-alpha (TNF-α). The VEGF and soluble ICAM-1 levels in cell-free conditioned medium of each treatment were measured by Enzyme-linked immunosorbent assay (ELISA). In chart 1 below the red bar represents a normal cell, and the blue bar represents a cell that is induced with angiogenic(left) and inflammatory(right) cytokines. By administering a compound that inhibits these cytokines you can see the results.

Flow Cytometry Analysis of Cell Cycle

Cell cycle and apoptosis

EyeCRO provides the analysis of cell cycle and apoptotic cells to determine whether the effect of new drugs is caused by slowing cell growth or inducing apoptosis. Endothelial and cancer cells were treated with new drugs for 24 h. After treatment, cells were fixed and stained with propidium iodide (PI). DNA content of cells was analyzed by flow cytometry. The percent of cells in each phase and apoptosis was analyzed by ModFit LT (Verity Software). The chart below shows the different stages of cellular growth. Chart A shows a flow cytometric analysis of untreated BRCEC cells. Chart B shows a flow cytometric analysis of BRCECs treated with 4 µM of a compound. Chart C data represents the percentage of cell distribution.

Cell Viability Assays

MTT Assay

MTT assay is used to evaluate whether new drugs have potent activity in inhibiting the growth of a variety of cells, including endothelial cells, pericytes, cancer cells, fibroblast, etc. Below, you can see that the compounds inhibited the growth of primary bovine retinal capillary endothelial cells (BRCECs). BRCECs were treated with 4 compounds at the concentrations of 0.5, 1, 2, 4, 8 µM for 3 days. After the treatment, viable cells were quantified using MTT assay.

Immunocytochemistry

EyeCRO has established in vitro models of the nuclear translocation of hypoxia induced factor-1alpha (HIF-1α) and nuclear factor kappa B (NFқB) to explore whether new drugs affect the activation of key proteins in the pathogenesis of ocular diseases and cancer in ocular cells or cancer cells. The nuclear translocation of these proteins is detected by Immunocytochemistry method and Western blotting. In the chart 4 you can see that the compound effectively inhibits the protein HIF-1α. The immunflourescence makes the protein much easier to see.

Immunocytochemistry - figure 1Immunocytochemistry - figure 2

Cell-based Assays

Cell-based Assays
 

 

Preclinical Ophthalmic Contract Research