Optimization of cryoprotectant loading into murine and human oocytes

Abstract

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethylsulfoxide (Me2SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me2SO exposure time, revealing that neither shrinkage nor Me2SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me2SO addition appears to result from interactions between the effects of Me2SO toxicity and osmotic stress. We also investigated Me2SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me2SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me2SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.

Insertion of Vertically Aligned Nanowires into Living Cells by Inkjet Printing of Cells

“…Therefore, a surfactant or chemical additive that is capable of reducing the surface tension without cytotoxicity is required for stable evolution of spherical droplets as well as for long‐term cell viability. We selected dimethyl sulfoxide (DMSO) as a biocompatible surface tension modifier as this reagent is commonly used as a cryoprotectant additive (CPA) and as an agent to induce differentiation of cultured cells . Various concentrations of DMSO (0, 5, and 10 vol%) were added to the bioink that was then jetted using the same jetting conditions to determine the optimal DMSO concentration.…”

Foundations of modeling in cryobiology—I: Concentration, Gibbs energy, and chemical potential relationships

“…While not complete in their description of the processes, the predictions of these models have been extremely useful in guiding experimental design, demonstrating feasibility of approaches, etc. [11,12,20,36,44,[48][49][50]69,78,84,122]. Many advances in computational complexity, transport and solution theories [10,28,32,57,97], as well as experimental parameter identification techniques [24][25][26]58,116,117] have been made along the way, facilitating more accurate models and better predictions of intracellular ice formation.…”

Synergistic Development of Biochips and Cell Preservation Methodologies: a Tale of Converging Technologies

“…Each step can induce damage to cells. Osmotic shock occurring during loading and unloading of CPA is one of the major causes of cell damage during the cryopreservation process [63, 64]. A microfluidic approach to minimize osmotic shock to cells during cryopreservation has been introduced by Song and colleagues [11].…”