Description:
OBJECTIVE: Develop methods and/or materials to allow long-term storage of vertebrate cells under ambient conditions for use in portable cell-based toxicity sensors. DESCRIPTION: As part of a research program to develop cell-based sensors for assessment of toxicity in environmental water samples the US Army Engineer Research and Development Center (ERDC) is seeking novel methods and materials for the long-term sustainment and storage of vertebrate cells. Unlike current biorecognition based sensors that utilize antibodies, aptamers, or other analyte-specific binding mechanisms, biosensors incorporating intact living cells can respond to emerging, unknown, or combinatorial chemical or biological threats and can act as broad spectrum screening tools(1). Many of the available rapid cell-based toxicity tests for water require significant control of environmental parameters, limiting their use under non-ideal field conditions(2). Current cell-based sensors are limited in utility and fieldability by the storage logistics and shelf life imposed by the use of vertebrate cell-based elements. The use of bacteria on portable, rapid toxicity sensors enables several long-term preservation methods such as freeze-drying or vacuum drying(3) that generally are not suitable for vertebrate cells. Importantly, the results of these bacterial cell-based biosensors cannot be directly extrapolated to vertebrate toxicity. Therefore, the use of vertebrate cells in portable, rapid toxicity sensors becomes ideal due to the direct translation of the toxicity information to a real time risk assessment. Significant advances in the development of biosensors utilizing mammalian cells have been made resulting in portable devices that improve the cell survivability(4) or storage conditions(5). To improve the fieldability and utility of cell-based sensing constructs we are seeking methods and/or materials to allow long-term storage of vertebrate cells under fieldable environmental conditions (see performance metrics below) that require minimal operating and preparatory steps prior to use. PHASE I: Provide proof of concept demonstration of methods and/or materials for long-term storage of vertebrate cells with minimal operating and preparatory steps and environmental requirements. The concept will be original or will represent significant advances or extensions of existing approaches. Design and performance metrics for the proof of concept are given below. Note that due to the performance requirements cryogenic storage is not an acceptable solution. 1) The method and/or material must sustain cells for a minimum of 3 months with greater than 80% viability. 2) The method and/or material must require minimal temperature or environmental control during storage. An ideal method/material will sustain cells under ambient conditions with storage temperatures up to 40 degrees C. Acceptable methods/materials may require controlled storage temperatures down to 4 degrees C. Methods/materials requiring lower storage temperatures are not acceptable. Preference will be given to methods/materials capable of cell storage under ambient conditions. 3) The method must not require substantial user interaction to maintain cell viability. For example, solutions requiring frequent media or reagent changes or additions are not acceptable. 4) The method and/or material must maintain the physiological functioning of the cells. 5) Cells must be ready to use after storage in 3 hours or less with minimal preparatory or operational steps; shorter times are favored. 6) The method and/or material must be applicable to and effective for a broad range of cell types, including fish, amphibian, avian, reptilian, and mammalian. No method/material must be developed that specifically precludes the potential use of human cell lines. 7) The method and/or material must be transferrable to a glass or fused silica microfluidic chip OR allow facile loading of reanimated cells onto a glass or fused silica microfluidic chip with minimal operational steps. Note that many of the criteria above apply only to the conditions required for storage/maintenance and not to initial preparation of the cells for storage/maintenance. PHASE II: Expand on the Phase I proof of concept work to demonstrate extended cell viability and functioning for 6 months with greater than 90% viability and be ready to use in 60 minutes or less with minimal operational steps under the performance parameters described in Phase I. Demonstrate that the method is applicable to a variety of cell types including fish, amphibian, avian, reptilian, and mammalian. Additionally, the material and/or method must either be contained on a glass or fused silica microfluidic chip OR allow facile loading of reanimated cells onto a glass or fused silica microfluidic chip with minimal operational steps. The total weight of components necessary for cell sustainment, including protective case, consumables, and any necessary hardware should be less than 2 lbs. Total volume, including protective case and any consumables should be less than 1.0 ft(3) note that this requirement applies only to cell storage/sustainment and not to initial cell preparation for storage. Smaller and lighter solutions are favored. Demonstrate that the method is viable under environmental conditions encountered in field testing. Phase II deliverables include equipment and materials for independent evaluation and testing. PHASE III: Evaluate the ability of the material/method to preserve cells used in cell-based sensor constructs during field testing. The material/method will be used with the SafePort water analysis system as well as other systems incorporating cell-based sensors(6). Methods to preserve cells for cell-based sensing have potential applications in a wide variety of areas including drug toxicity screening in pharmaceutical studies, toxicity screening of municipal and military water supplies, and studies of environmental toxicity. A well-formulated marketing strategy will be critical for success in these commercial applications.
