PorFocal - Porcine Cornea Confocal Assay

Slight vs. Non-Irritating Ocular Irritation Screening

Novel Ultra low level ocular irritation screening. Confocal Imaging with Fluorescent staining.

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Background

Many eye use products require multiple applications, such as eye drops. Historically, this was done using a rabbit eye test. The need for effective toxicity screening methods that are able to detect the ocular irritation potential of pharmaceutical formulations earlier in their developmental is very important and ultimately reduces costs and helps maintain an adequate level of human eye safety and health. Overall product development cost and human test subject pain and distress can be reduced while promoting robust product safety for public health.

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). Draize rabbit eye testing is insensitive to lower levels of irritation; rendering discrimination between sub-mild irritant product formulations difficult. The only reliable choice for sub-mild irritant testing is human clinical testing. Pre-screening with a in vitro/alternative assay would significantly reduce expensive human testing for products that need to be tested for sub-mild ocular irritation are products that are used daily in the eye area (such as products used in the eye, such as eye drops or cosmetic items). These eye products are often prescribed for daily use for several weeks—for example, a course of ocular antibiotics for conjunctivitis. Human testing using these parameters can become cost prohibitive for manufacturers.

PorFocal - Assay Summary

The PorFocal Assay uses excised porcine corneas (a normal waster product in food production) in a specialized tissue culture method that allows for maintaining viable tissues for up to 21 days. These corneas are then treatable with topically applied test materials (liquids & solids). Corneas can be dosed over multiple days. Each cornea is then 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.