High throughput method for simultaneous screening of membrane permeability and toxicity for discovery of new cryoprotective agents

Abstract

Vitrification is the most promising method for cryopreservation of complex structures such as organs and tissue constructs. However, this method requires multimolar concentrations of cell-permeant cryoprotective agents (CPAs), which can be toxic at such elevated levels. The selection of CPAs for organ vitrification has been limited to a few chemicals; however, there are numerous chemicals with properties similar to commonly used CPAs. In this study, we developed a high-throughput method that significantly increases the speed of cell membrane permeability measurement, enabling ~100 times faster permeability measurement than previous methods. The method also allows assessment of CPA toxicity using the same 96-well plate. We tested five commonly used CPAs and 22 less common ones at both 4 {degrees}C and room temperature, with 23 of them passing the screening process based on their favorable toxicity and permeability properties. Considering its advantages such as high throughput measurement of membrane permeability along with simultaneous toxicity assessment, the presented method holds promise as an effective initial screening tool to identify new CPAs for cryopreservation. SignificanceCryoprotective agent (CPA) toxicity is the most limiting factor impeding cryopreservation of critically needed tissues and organs for transplantation and medical research. This limitation is in part due to the challenge of rapidly screening compounds to identify candidate molecules that are highly membrane permeable and non-toxic at high concentrations. Such a combination would facilitate rapid CPA permeation throughout the sample, enabling ice-free cryopreservation with minimal toxicity. This study presents a method for rapidly assessing the cell membrane permeability and toxicity of candidate CPAs, identifies several novel high-permeability low-toxicity CPAs for further testing, and lays the groundwork for additional high throughput screening to discover novel CPAs with the potential to improve cryopreservation of complex tissues and organs.