SBIR-STTR Award

The search for alternative methodologies to reduce the number of animals used in biomedical research and toxicology testing has focused on in vitro methods of cultured cells. Our concept is to develop an off-the-shelf frozen microtiter plate ("Cryoplate") which will provide a readily available cell delivery system for in vitro assaying of cytotoxicity. We propose to examine the general hypothesis that cells dispensed in a tissue culture plate and attached to either a natural or synthetic substrate can withstand the rigors of freezing and thawing to provide a cell delivery system for in vitro cytotoxicity assays. Initial studies will utilize cultured bovine corneal cells which permit convenient examination of tolerance to the stresses of cryopreservation, either as individual cells attached directly to the synthetic substrate of the tissue culture plate or as a layer of cells attached to the natural extracellular matrix (ECM) laid down by these cells in culture. It is envisaged that optimization of cryobiological variables will enable development of the Cryoplate concept for use in a wide variety of toxicology and diagnostic laboratories. This product will not only eliminate the need for plating and expanding cells for in vitro testing purposes, but will possibly require handling fewer cells than conventional methods. The basic technology developed in this research may eventually lead to the cryopreservation of cells on other substrates such as miniaturized synthetic chips carrying sensors or other diagnostic tools. PROPOSED COMMERCIAL APPLICATION: It is anticipated that cryopreserved cell delivery systems will be commercialized for chemical testing and diagnostic assays. The methods may be applicable to the development of in vitro cell assay kits for a variety of cell types and purposes. Licensing opportunities will be sought with industry partners for alternative applications.

Thesaurus Terms:
cryopreservation, cytotoxicity, evaluation /testing, technology /technique development, tissue /cell culture corneal endothelium, extracellular matrix

Advancements in high throughput screening, and mandates all over the world to reduce animal testing, have driven the development of a variety of assays to evaluate potentially new drugs and their side effects. Environmental monitoring of potentially harmful chemicals and by-products has also driven the development of new assays. Convenient cell based systems for testing a wide variety of products with a variety of cell types has become a great need. In Phase I, we proposed the development of a system providing ready to use cells on microtiter plates for any desirable assay. We demonstrated that bovine corneal endothelial cells (BCE) attached to tissue culture plastic or an extracellular matrix can be cryopreserved and be viable after thawing. We have also shown that we are able to uniformly cool and warm microtiter plates and improve overall retention of cells to their substrate. The goal of the Phase II proposal, is to further define and optimize the parameters that will provide successful cryopreservation of BCE cells attached to microtiter plates. It was shown that cells attached to an extracellular matrix demonstrated better viability after cryopreservation than cells attached to tissue culture plastic. Further examination of extracellular matrix components will be performed to determine if adjustments to the composition of the matrix can improve viability and attachment. In addition other cryobiological variables will be evaluated including optimization of cooling, the choice of cryoprotectant vehicle solution and its influence on cell survival as well as long term storage of cells after cryopreservation on microtiter plates. All these experiments are designed to further optimize the system and provide cells on plates that are ready to use for a variety of applications. Validation of cryopreserved BCE cells will be provided by a battery of functional assays including, but not limited to, responses to toxicants, extracellular matrix formation, barrier function and nitric oxide production. It is anticipated that optimization of procedures for corneal endothelial cells will provide a technology platform that can be applied, with further development, to other cell types