Picoliter Droplets for Single Cell Cryopreservation

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DESCRIPTION (provided by applicant): Cryopreservation is a technique for preserving biological cells and tissues that relies on the fact that molecular diffusion, and thus cellular injury, are vastly reduced at cryogenic temperatures. In conventional cryopreservation, cooling rate is chosen to lie between rates that cause solution effects (slow rates) and those that cause intracellular ice formation (fast rates). For many cell types; however, these ranges overlap so that the addition of cryoprotectant chemicals (CPAs), tailored to each cell type, is necessary. One promising alternative is vitrification, or the formation of a non-crystalline glass-like solid on cooling. In theory, vitrification should avoid all damages from ice formation and could provide a simple and effective method for cell preservation independent of cell type. Unfortunately, it requires extreme cooling rates coupled with high and typically toxic CPA concentrations to prevent ice crystal formation at practically realizable cooling rates. One reason for this is that extracellular ice often initiates from particulate matter (heterogeneous nucleation) long before spontaneous (homogeneous) nucleation would occur. Both forms of ice nucleation are stochastic, which leads to the hypothesis of this work: cryopreservation of biological cells captured within microscopic aqueous droplets will enable the vitrified state to be achieved at low and nontoxic cryoprotectant concentrations. This follows from the realizations that dividing a solution into numerous droplets dramatically decreases not only the probability that a particular droplet contains an ice nucleator, but also the characteristic time in which homogeneous nucleation is likely to occur. To test this hypothesis, the first aim is to develop a microfluidic device for the systematic study of cryopreservation in an inverse (water-in-oil) emulsion. This will allow the creation of monodisperse aqueous-CPA droplets and their rapid cooling to cryogenic temperatures, either on-chip, within a plunge-cooled quartz micro-capillary, and/or inside a conventional cryostage. Next, the effects of droplet size and cooling rate on the critical CPA concentration for vitrification will be studied. This will entail the development of a physicochemical model of droplet vitrification which will guide the search for conditions enabling low-CPA vitrification. Vitrification will be assessed with methods such as X-ray diffraction, FTIR, and/or DSC. Finally, this information will be used to test a variety of indicated protocols on hepatocytes, with and without cocultured fibroblasts, where viability, proliferative ability and hepatospecific function will measure success. Relevance: The reduction in cryoprotectant levels required for vitrification gained by encapsulation of single cells in microscopic droplets of water could enable the preservation of heterogeneous cell suspensions.