Ultra low level ocular irritation screening. Confocal Imaging with Fluorescent staining.
Many eye use products require multiple applications, such as eye drops. Historically, this was done using a rabbit eye test.
MB Research has developed a novel non-animal method (PorFocal), which utilizes corneas excised from porcine eyes (a normal waste by-product in food production). Utilizing a special long term culture system, porcine corneas can maintained in cultured for several weeks. This allows for multiple dosing (ex. corneas can be dosed 3-4 times per day).
After multiple doses, each cornea was stained with a dead cell marker stain (ethidium homodimer) and then imaged using a confocal microscope. Ethidium Homodimer is non-membrane permeable, and only enters dead or dying cells with compromised plasma membranes. Upon binding to DNA, this dye fluoresces brightly, allowing for quantification of dead cells per tissue volume. Dead cells are counted per volume of tissue using a maximum projection of a z-stack image series. Statistical analysis is performed to determine the extent of injury of treated corneas.
This high degree of sensitivity allows for detection of low-level irritants.
PorFocal has many applications, such as testing eye drops, drugs to treat ocular disease, or substances repeatedly applied on the face or near the eyes.