(B-71) High-Throughput Methods for Simultaneously Analyzing Cell Membrane Permeability and Toxicity for Discovery of Novel Cryoprotective Agents.

Introduction:: Millions of lives could be saved if enough organs were available to meet the demand for transplantation. The supply-demand imbalance is exacerbated by challenges with maintaining viability during ex vivo storage. For example, about 70–80% of all donated hearts and lungs are discarded in part because of concerns about loss of viability during storage. By extending the storage time, cryopreservation can decrease the number of discarded organs. Despite some successes with small samples like embryos, it remains a challenge to cryopreserve larger specimens like tissues and organs. A major challenge is identifying the cryoprotective agents (CPAs) that permeate cell membranes and promote formation of a glass, without causing cytotoxicity. To address this challenge, we developed high throughput methods to detect cell permeability and toxicity of CPAs, essential for identifying less toxic CPAs and designing cryopreservation procedures without causing osmotic stress.


Materials and Methods:: The experiments were carried out using endothelial cells cultured in 96 well plates and stained with calcein-AM. A FlexStation plate reader was used to automate CPA addition and subsequent fluorescence measurements at 1.5 s intervals at 4 °C and 25 °C for 27 different chemicals. The resulting fluorescence data was used to estimate the cell membrane permeability to CPA and water. The same well plate was then used to assess CPA toxicity. Fluorescence was measured after the cells had been exposed to CPA for ~10 min, then again after removing the liquid from the well. Toxicity was estimated from the ratio of these fluorescence values.




Results, Conclusions, and Discussions:: Figure 1A illustrates high throughput measurement of cell membrane permeability using a FlexStation plate reader. Using the automated plate reader, we observed similar changes in relative fluorescence intensity and cell volume as we have determined in our previous study [1]. Hypertonic solutions cause cell shrinkage, while hypotonic solutions cause cell swelling. Likewise, cellular shrinkage results in a decrease in fluorescence intensity, whereas cellular swelling results in an increase. Figure 1B shows the toxicity and permeability of cells at 4 °C for 27 different chemicals. For chemicals with extremely high permeability or significant toxicity, the precise permeability value could not be determined. However, for cryopreservation procedure design purposes, knowledge of the high permeability of CPA is sufficient because the main objective of measuring permeability is to develop a multistep addition and removal process for CPAs to prevent osmotic damage to cells. Osmotic damage is not a concern for chemicals with very high CPA permeability as they do not cause osmotic damage. Also, high-toxicity CPAs should be excluded as cryopreservation candidates. Of the CPAs tested, with the exception of glycerol (which has a relatively low permeability), most commonly used CPAs have high cell membrane permeability and low toxicity. Our experiments have revealed some other less commonly used chemicals that meet these criteria, including dimethylacetamide, 2-methoxyethanol and diethylene glycol. An entire well plate can be measured in less than an hour, which is 100x faster than our previous microscope-based method for measuring CPA permeability, while also offering a simultaneous estimate of CPA toxicity.

We have established high throughput measurements of CPA toxicity and membrane permeability, laying the groundwork for identifying novel CPAs and designing improved cryopreservation methods.


Acknowledgements (Optional): : This work was supported by funding from the National Institutes of Health (R01 EB027203), as well as a generous donation from the Cryonics Institute.


References (Optional): : [1] Fry, AK, Higgins AZ, Cell Mol Bioeng (2012) 5(3): 287-298.