Biology subjects

Ahmadkhani, N.

Publications and source records attributed to Ahmadkhani, N..

4 recordsLinked to original sources

bioRxiv2026

A high throughput platform for measuring and predicting vitrification behavior in multicomponent aqueous solutions

Cryopreservation depends critically on suppression of ice formation by cryoprotective agents (CPAs), but limited data is available on the CPA concentration required for vitrification (Cv). Here, we introduce a high-throughput 384-well platform that integrates automated liquid handling, randomized plate layouts, and a binary-search strategy to rapidly determine Cv across hundreds of formulations. Relative to conventional methods, this approach increases throughput by [~]50-fold, compressing a year of measurements into one week, while markedly reducing manual labor. Across [~]200 CPA compositions, we demonstrate that environmental boundary conditions strongly influence vitrification behavior: plates sealed with silicone mats exhibited lower Cv than open plates, indicating that sealed configurations promote vitrification. Further, the data reveal a decrease in Cv with increasing CPA molecular weight, consistent with enhanced ice suppression by larger molecules. We also present a simple mixture model that accurately predicts Cv for a broad range of CPA formulations, including mixtures containing up to seven CPAs (R{superscript 2} [≥] 0.93), and use this model to evaluate published CPA toxicity data to identify formulations that operate near their vitrification threshold while maintaining relatively low toxicity. Together, these results establish a framework for rapid Cv determination, predictive modeling of vitrification behavior, and rational design of CPA formulations.

bioengineering↗

bioRxiv2025

Screening for cryoprotective agent toxicity and toxicity reduction in mixtures at subambient temperatures

Organ transplantation faces major challenges in preserving and transporting organs due to the limitations of existing cold storage methods. Cryopreservation offers a promising alternative for extending preservation time, but it remains a challenge to avoid toxicity from the high concentrations of cryoprotective agents (CPAs) required to prevent ice formation. In this study, we expanded a previously reported high-throughput CPA toxicity screening platform by retrofitting an automated liquid handling system with subambient cooling capabilities. This enabled systematic assessment of CPA toxicity at 4 {degrees}C, a temperature commonly used for CPA equilibration in tissue and organ cryopreservation. Overall, we screened 22 individual CPAs and a wide range of binary mixtures at concentrations up to 12 mol/kg, allowing us to identify CPA combinations that reduce toxicity. Our findings revealed that at 4 {degrees}C, CPA toxicity was significantly reduced compared to room temperature. Several CPA combinations resulted in significantly lower toxicity than their constituent CPAs at the same concentration, including 12 CPA mixtures at 6 mol/kg and 8 CPA mixtures at 12 mol/kg. Toxicity neutralization was also observed in 9 cases, especially in combinations involving formamide, acetamide, dimethyl sulfoxide, and glycerol. For example, exposure to 6 mol/kg formamide alone resulted in 20% viability, but the addition of 6 mol/kg glycerol to create a mixture with a total concentration of 12 mol/kg eliminated this toxicity, resulting in a viability of 97%. These findings support the rationale for using multi-CPA cocktails and underscore the potential of rational mixture design to reduce toxicity.

pharmacology and toxicology↗

bioRxiv2025

High-Throughput Evaluation of Cryoprotective Agents for Mixture Effects That Reduce Toxicity

Vitrification is a promising approach for cryopreserving complex biological structures such as organs. However, to prevent ice formation, high concentrations of cell-permeable cryoprotective agents (CPAs) are required, which can be highly toxic. The current reliance on a small number of CPAs limits optimization of low-toxicity compositions for vitrification. To address this, there is growing interest in uncovering novel chemicals with analogous protective qualities. This may not only enhance vitrification efficacy but also mitigate toxic effects. In the current study, we employed a high throughput method to assess the toxicity of 21 compounds at room temperature, both individually and in binary combinations. Our analysis revealed that toxicity increases with both exposure duration and concentration, and that several CPA combinations result in reduced overall toxicity. Notably, among all tested mixtures, four binary combinations--formamide/glycerol, dimethyl sulfoxide/1,3-propanediol, 1,2-propanediol/diethylene glycol, and 1,3-propanediol/diethylene glycol--produced a statistically significant decrease in toxicity, resulting in significantly higher viability for the 6 mol/kg mixture than both corresponding 6 mol/kg single CPA solutions. The high-throughput approach presented here will aid in building a comprehensive CPA toxicity database, which will improve our understanding of toxicity mechanisms and support the development of predictive models for identifying novel CPA mixtures with low toxicity.

pharmacology and toxicology↗

bioRxiv2024

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

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.

bioengineering↗