Development of a High Throughput Method for Vitrification

ABSTRACT: In an effort to reduce the use of animals for toxicity testing, companies have developed bioengineered in vitro skin models and other human tissue constructs for predictive toxicity testing to replace traditional assays such as the Draize skin irritation test and also for use as preclinical animal models. Presently, tissues or skin constructs are made-to-order and require a lead time of several weeks before they can be used. Quality control checks are conducted post shipment creating serious recall liabilities for the manufacturer. The ability to cryopreserve tissue models would solve key logistical issues creating “on-demand” banks of tissues which can be prescreened to ensure quality control. This would create economies of manufacturing scale and reduce costs to manufacturers and end users. We have developed a patented ice-free vitrification method for cryopreservation of bioengineered skin constructs. Using this patented process, we have consistently demonstrated andgt;80% viability of several types of human bioengineered epithelial constructs. These viability results persisted for several days post-rewarming and both viability and functional assessments meet the manufacturerandapos;s acceptance criteria for use in toxicology tests. However, a limiting factor for preservation at larger scale is the ability to vitrify multiple constructs at one time. Our current method allows for processing of up to 6-12 constructs at one time. Processing of more than 12 results in reduce viability due to cytotoxicity from exposure to cryoprotectants for too long. In this Phase I, proposal we plan to test the feasibility of using a specially designed cassette that can hold up to 24 constructs at once. The cassette would allow preservation of multiple constructs streamlining the vitrification process while removing variation between constructs due to operator error. The outcome will be measured by assessment of tissue viability and functional assays required by manufacturers for quality control. The success of this proposal will be inline with our company goal of commercializing our vitrification process by storing on-demand human bioengineered tissues for distribution to customers for drug or chemical screening and research applications. After demonstration of feasibility in Phase I, we will submit a Phase II proposal with the primary objective of custom development of a fully automated robotic sample handling system for construct cryopreservation and removal of cryoprotectants prior to use for predictive toxicity testing.PROJECT NARRATIVE Bioengineered human tissue constructs are being developed to replace the use of more traditional animal toxicity tests, such as the Draize irritation and corrosion tests, and preclinical animal models. An effective preservation method for validated skin constructs and other tissues in development will create the opportunity for tissue banking, providing an “on-demand” supply of tissues for research purposes. Vitreous preservation will provide a less expensive, consistent, better quality controlled product. This project will test the feasibility of a cassette for preservation of multiple constructs at once. Streamlining the vitrification process would make it amenable to automation development in Phase II. Reducing manufacturing costs of bioengineered tissue equivalent constructs and increasing the availability of human test models that will be more predictive of outcome for humans than currently employed live animal models.