Concentration of Cryoprotectant in water-in-oil microdroplets for single cell vitrificaton

Abstract

(cont.) Droplets with an initial concentration of IM were found to be concentrated to about 3-4M in 90s while droplets starting at 2M were concentrated to 6M in about the same time. The entire process takes place over a time scale of about one minute, fast enough to minimize exposure times but slow enough to be precisely controllable. This phenomenon is demonstrated to dynamically concentrate cryoprotectants within single cell-containing droplets. These droplets of sizes of about 30 micron diameter were concentrated to 3-4M from a starting concentration of IM in about 300s. The cells are tolerant to this concentration process and do not die when subjected to it. The process may be used in practice to innocuously concentrate cell encapsulating droplets which may then be vitrified before they are exposed to high temperatures for fatally long time scales. With appropriate characterization, the controllability of the process will allow for choosing exact cryoprotectant concentration levels used for vitrification. The demonstrated phenomenon has several other applications in cryobiology. Its controllability and speed may be used to dynamically modulate cryoprotectant concentrations in preservation protocols that require stepwise concentration or dilution. In addition, the process was found to be reversible and may thus be used for unloading cryoprotectants by controlled cooling as opposed to heating.

Among the several challenges associated with vitrification of cells, a major roadblock is the requirement of high concentrations of cryoprotectant chemicals and the damages caused by exposure of cells to these high concentrations at physiological temperatures. It is thus desirable to minimize the time of exposure of cells with high concentrations of cryoprotectants to physiological temperatures. In addition, vitrification requires very rapid cooling rates. As cooling rates of a sample are limited by its size, it becomes ideal to use the minimum sizes of the sample to be preserved. Certain organic oils, such as soybean oil, are made of triacylglycerols and are capable of dissolving small amounts of water due to the presence of ester groups, a property which enhances significantly with increasing temperature. This phenomenon was exploited to accomplish temperature controlled concentration of cryoprotectants in single water droplets with and without cells dispersed in the organic phase. The organic phase used in the present work is soybean oil while glycerol is used as the cryoprotectant. Glycerol was found to be comparatively insoluble in soybean oil at 35 'C for up to 10 minutes. The present work employed heating on a temperature controlling stage and temperature increases of about 10K. Solutions of glycerol in DI Water were mixed with soybean oil and emulsions made by vigorous agitation. The water to oil concentration was kept at 0.1% v/v to simulate an infinite dissolution medium and to prevent different droplets from affecting each other. To prevent premature dissolution, the oil is saturated with water at room temperature by incubating for 48 hours. Micro-liter-sized droplets of the emulsion are placed on a heating/ cooling stage and droplets of 15-20 micron diameter are visually selected from polydisperse emulsion for observation under a microscope. Upon increasing temperature, water dissolves into the oil rendering the droplet highly concentrated with the oil-insoluble cryoprotectant. The experiment involved heating to 35 °C from room temperature, so that all water eventually dissolved into the oil.