Construction and testing of a rabbit cornea-equivalent for evaluation of chemical injury in vitro

ABSTRACTWe propose combination of the rabbit cornea endothelial and epithelial cells with bioengineered scaffolds in cell culture well insertsThis technology could significantly reduce the use of rabbits for the Draize rabbit eye irritation assayThe barrier to commercialization is the lack of an effective method for storingbankingof living bioengineered cornea equivalentsWe will combine cell expansion and scaffold technology developed by our collaboratorProfessor Soker at the Wake Forest Institute for Regenerative Medicinewith patented ice free cryopreservation strategies developed by scientists at our companyTissue Testing Technologies LLCCryopreservation using ice free vitrification has shown promise with several human cellderived construct modelsincluding EpiOcularThis work will be performed in three specific aimsIn the first aim a bioengineered gel scaffold originally formulated for human cells will be assessed for compatibility with rabbit endothelial and epithelial cells and optimized in culture well insertsThe scaffold and porousclear insert membrane represents the stroma of the corneaIn the second aim we will compare three experimental groupscorneal epithelial constructs without endothelial cellsconstructs with just an endotheliumandcornea constructs with an endothelium under and an epithelium on top of the scaffoldThey will be compared using established positive and negative control treatments to determine whether the less complicated constructs are more or less predictive of irritation than a construct with both endothelial and epithelial layersIn parallel with aim two the third aim will assess our lead ice free cryopreservation strategy for short termdaysstorageDuring these studies a panel of biological and physical assays will be performed to determine the feasibility of employing our constructs for irritation evaluation of new chemical formulationsThe Phase II plan will consist of further optimizationtesting against a larger panel of irritantslong term shelf life evaluation and head to head comparison with the in vivo Draize rabbit eye test that will be required for translationmarket acceptanceof cryopreserved bioengineered rabbit cornea equivalents to the test market!NARRATIVEDue to the hazardous nature of many materials and substances ocular toxicity testing is required to evaluate the dangers associated with new products ranging from householdindustrialagriculturalmilitarycosmeticstoiletriesand pharmaceutical productsThe Draize eye irritation test in rabbits is commonly usedIn vitro cell based assays are frequently used for cosmetics and other research but none of the models are derived from the rabbitIn an effort to further reduce animal use for ocular testingwe will develop a rabbit cornea cell based construct modeled upon the structure of the rabbit corneaSince most in vivo ocular studies are in the rabbit it is logical to anticipate that a rabbit cell based construct will be more likely be predictive of irritatingtoxic outcomes for compounds in the rabbit eye than alternative models based on other species and cell typesThe results will be compared with existing rabbit test dataIf the model turns out to provide predictive data relevant to humans it may help reduce the number of compounds that need to be tested in whole animal studies