Biology subjects

Benson, J. D.

Publications and source records attributed to Benson, J. D..

7 recordsLinked to original sources

bioRxiv2026

4D confocal microscopy of time dependent cell and nuclear osmotic response reveals differential osmoregulation and transport kinetics

A cell's ability to maintain its volume is a prerequisite for its essential physiological functions. Changes in extracellular osmolarity induce the flux of water and solutes across the cell and nuclear membrane and, therefore, alters cell and nuclear volumes. Here, we investigated the equilibrium and dynamic volumetric responses of cells and nuclei in adherent human hepatoma (HepG2) cell monolayers using live three-dimensional imaging. Through image segmentation, 3D reconstruction, and volume rendering, precise cell and nuclear volumes were quantified. We found that during hypoosmotic stress, cells swell and lose osmolytes through active volume regulation, and nuclei undergo greater relative expansion than cells. During hyperosmotic stress, we found that both cells and nuclei shrink rapidly, develop irregular morphologies and undergo passive volume regulation. Furthermore, we explored the osmotic responses in these cells by restoring isosmotic conditions following osmotic stress and found that nuclei returned to volumes greater than their initial isosmotic volume, whereas cytoplasmic volume returned to a value below its initial isosmotic volume following swelling. By fitting coupled transport models water and solute permeability estimates for both cells and nuclei were made from their dynamic responses to different anisosmotic media with and without the presence of permeating solutes dimethyl sulfoxide and ethylene glycol. Together, these quantitative measurements of cell, cytoplasm, and nuclear volumetric responses and associated transport parameters have important implications for understanding cell physiology and the mechanism of osmoregulation.

biophysics↗

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↗

bioRxiv2022

Machine learning and hypothesis driven optimization of bull semen cryopreservation media

Cryopreservation provides a critical tool for dairy herd genetics management. Due to widely varying inter- and within-bull post thaw fertility, recent research on cryoprotectant extender medium has not dramatically improved suboptimal post-thaw recovery in industry. This progress is stymied by the interactions between samples and the many components of extender media and is often compounded by industry irrelevant sample sizes. To address these challenges, here we demonstrate blank-slate optimization of bull sperm cryopreservation media by supervised machine learning. We considered two supervised learning models: artificial neural networks and Gaussian process regression (GPR). Eleven media components and initial concentrations were identified from publications in bull semen cryopreservation, and an initial 200 extender-post-thaw motility pairs were used to train and 32 extender-post-thaw motility pairs to test the machine learning algorithms. The median post-thaw motility after coupling differential evolution with GPR the increased from 52.6 {+/-} 6.9% to 68.3 {+/-} 6.0% at generations 7 and 17 respectively, with several media performing dramatically better than control media counterparts. This is the first study in which machine learning was used to determine the best combination of constituents to optimize bull sperm cryopreservation media, and provides a template for optimization in other cell types.

bioengineering↗

bioRxiv2022

Osmotic, temperature, and cytotoxic damage in sea urchin oocytes

Sea urchin (Paracentrotus lividus) oocytes are an important species for aquaculture and as a model species for multiple scientific fields. Despite their importance, methods of cryopreserved biobanking of oocytes are currently not possible. Optimized cryoprotectant loading may enable vitrification methods of cryopreservation and thus long-term storage of oocytes. Determining an optimized protocol requires membrane characteristics and models of damage associated with the vitrification loading protocol, namely osmotic, temperature, and cytotoxic damage. We present and experimentally evaluated state-of-the-art models alongside our novel models. We experimentally verify the damage models throughout time at difference treatment intensities. Osmotic damage experiments consisted of hypertonic solutions composed of seawater supplemented with NaCl or sucrose and hypotonic solutions composed of seawater diluted with deionized water. Treatment times ranged from 2 to 30 minutes. To test temperature damage (in particular chill injury), oocytes were exposed to 1.7 {degrees}C, 10 {degrees}C, and 20 {degrees}C (control) for exposure times ranging from 2 to 90 minutes. Cytotoxicity was investigated by exposing oocytes to solutions of Me2SO for exposure times ranging from 2 to 30 minutes. We identify appropriate models and use these to search for an optimal loading protocol, namely the time dependent osmotic damage model (for osmotic damage), the temperature dependent model (for temperature damage), and the external molality Arrhenius power model (for cytotoxicity). We combined these models to estimate total damage during a cryopreservation loading protocol and performed a exhaustive grid search for optimal loading for a given goal intracellular cryoprotectant concentration. Given our fitted models, we find sea urchin oocytes can only be loaded to 0.13 Me2SO v/v with a 50% survival, For reference, levels for vitrification are approximately 0.45 v/v. Our synthesis of damages is the first of its kind, and enables a fundamentally novel approach to modelling survival for cells in general.

cell biology↗

bioRxiv2022

An agent based model of intracellular ice formation and propagation in small tissues

Successful cryopreservation of tissues and organs would be a critical tool to accelerate drug discovery and facilitate myriad life saving and quality of life improving medical interventions. Unfortunately success in tissue cryopreservation is quite limited, and there have been no reports of successful long term organ cryopreservation. One principal challenge of tissue and organ cryopreservation is the propagation of damaging intracellular ice. Understanding the probability that cells in tissues form ice under a given cryopreservation protocol would greatly accelerate protocol design, enabling rational model-based decisions of all aspects of the cryopreservation procedure. Established models of intracellular ice formation (IIF) in individual cells have previously been extended to small linear (one-cell-wide) arrays to establish the theory of intercellular ice propagation in tissues. However these small-scale lattice-based tissue ice propagation models have not been extended to more realistic tissue structures, and do not account for intercellular forces that arise from the expansion water into ice that may cause mechanical disruption of tissue structures during freezing. To address these shortcomings, here we present the development and validation of a lattice-free agent-based stochastic model of ice formation and propagation in small tissues. We validate our Monte Carlo model against Markov chain models in the linear two-cell and four-cell arrays presented in the literature, as well as against new Markov chain results for 2 x 2 arrays. Moreover we expand the existing model to account for the solidification of water into ice in cells. We then use literature data to inform a model of ice propagation in hepatocyte disks, spheroids, and tissue slabs. Our model aligns well with previously reported experiments, and demonstrates that the mechanical effects of individual cells freezing can be captured. Author summaryThe widespread ability to successfully store, or cryopreserve, tissues and organs in liquid nitrogen temperatures would be game changing for human and animal medicine and drug discovery. However, success is limited to a select number of small tissues, and no organs can currently be stored in a frozen or solid state and survive thawing. One major contributor to damage during this process is the formation of intracellular ice, and its associated cell level damage. This ice formation is complicated in tissues by the number of intercellular connections facilitating intercellular ice propagation. Previous researchers have developed and experimentally validated simple one dimensional models of ice propagation in tissues, but these fail to capture complex tissue geometries, and have many fewer intercellular connections compared to three dimensional tissues. In this paper, we adopt previous models of ice formation and propagation to a model capable of capturing arbitrary cell orientations in three dimensions, allowing for realistic tissue structures to be modelled. We validated this tool on simple models and with experimental data, and then test it on three structures made of digital liver cells: disks, spheroids, and slabs. We show that we can capture new information about the interaction of cooling the tissue, the formation of intracellular ice, the movement of ice from one cell to another, and the mechanical disruption that occurs during this process. This allows for novel insights into a mechanism of damage during cryopreservation that is cooling rate and tissue structure dependent.

biophysics↗

bioRxiv2022

Meta analysis and experimental re-evaluation of the Boyle van 't Hoff relation with osmoregulation modelled by linear elastic principles and ion osmolyte leakage

In this study we challenge the paradigm of using the Boyle van t Hoff (BvH) relation to relate cell size as a linear function of inverse extracellular osmotic pressure for short time periods (~5 to 30 mins). We present alternative models that account for mechanical resistance (turgor model) and ion-osmolyte leakage (leak model), which is not accounted for by the BvH relation. To test the BvH relation and the alternative models, we conducted a meta-analysis of published BvH datasets, as well as new experiments using a HepG2 cell line. Our meta-analysis showed that the BvH relation may be assumed of the hypertonic region but cannot be assumed a priori over the hyper- and hypotonic region. Both alternative models perform better than the BvH relation but are nearly indistinguishable when plotted. The return to isotonic conditions plot indicated neither alternative model accurate predicts return volumes for HepG2 cells. However, a combined turgor-leak model accurately predicts both the BvH plot and the return to isotonic conditions plot. Moreover, this turgor-leak model provides a facile method to estimate the membrane-cortex Youngs modulus and the cell membrane permeability to intracellular ions/osmolytes during periods of osmotic challenge, and predicts a novel passive method of volume regulation without the need for ion pumps.

biophysics↗