Surviving suspended animation: a rapid ride across no man’s land

We developed a rapid (up to hundreds of millions °C/min) and scalable joule heating approach to improve the cryopreservation of biological systems. These systems range in size from cells (µm) to organisms (Drosophila embryos) to tissue slices (mm) and we demonstrate the ability to tailor the rewarming rates to achieve optimal outcomes in these biosystems.
Surviving suspended animation: a rapid ride across no man’s land

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Living biological systems can be put into suspended animation if successfully cooled to cryogenic temperatures such as in liquid nitrogen (-196 °C). Later, biological processes can resume at the desired time and location upon successful rewarming and recovery. We envision that, enabled by various cryopreservation techniques, off-the-shelf living products will be available on demand across the globe, therefore greatly advancing healthcare, biodiversity, food supply, and sustainability (See ATP-Bio).

During cooling of a biological system, the temperature zone between heterogeneous nucleation and glass transition is dubbed as “no man’s land”, a place where ice rapidly forms and grows. Vitrification happens when rapid cooling allows the system to pass over (not through) the no man’s land. In the ultra-cold, ice-free (or nearly ice-free) vitrified state, biological systems remain in suspended animation – viable, but with biological processes on indefinite hold.

To achieve this, it requires the use of cryoprotective agents (CPAs), which are comprised of biologically compatible anti-freeze molecules such as DMSO, ethylene glycol, and propylene glycol. CPAs are loaded into biosystems to make cooling and warming rates physically attainable for vitrification. With increasing CPA concentrations, vitrification is easier to achieve (i.e., lower cooling rates are required), but at the price of increased CPA toxicity. Recent studies show that lower CPA concentrations require ultra-rapid rewarming to avoid the “no man’s land” on the way from vitrification to physiological temperatures.

Since 2018, we have worked on a cryomesh approach for rapid and scalable vitrification and rewarming for small (µm to mm) biological systems. The CPA-loaded systems are transferred to a nylon mesh so that the excess CPA solution surrounding the biological system can be removed before cooling. This results in greatly reduced thermal mass and improved cooling and warming rates over what had been demonstrated prior. This approach has led to successful cryopreservation of a number of fruit fly (Drosophila melanogaster) embryo lines (see our paper) and pancreatic islets with high viability, function, recovery and clinical scalability for transplantation (see our paper).

The achievable warming rates of cryomesh using convective warming – i.e., plunging the cryomesh into warm water – is ~ 2×105 °C/min, which requires the use of high CPA concentration (i.e., > 4 M). Many biological samples are more susceptible to CPA toxicity, therefore requiring lower CPA concentration and necessitating even faster rewarming rates.

Figure 1. a. Schematics of biological systems cryopreservation using plunge cooling and joule heating. b. Joule heating can be applied to biological systems at different scales.

In the current study, we introduce a joule heating-based platform technology to achieve even faster warming rates. Briefly, vitrified (or partially vitrified) biological systems on a cryomesh are rapidly rewarmed by contact with an electrical conductor that is fed a voltage pulse specific to the size of the system (Figure 1). Pulse duration and magnitude are carefully designed to provide the fastest achievable warming rates, based on the heat transfer process. We demonstrate successful cryopreservation of three model biosystems with thicknesses across three orders of magnitude, including adherent cells (~4 µm), Drosophila melanogaster embryos (~50 µm after CPA dehydration) and rat kidney slices (~1.2 mm) using low CPA concentrations (2-4 M). Due to the intrinsic heating mechanism, the joule heating technique converts ~100% of input energy to heat. For instance, using a 10 µs pulse to rewarm adherent cells, numerical simulation projects a warming rate up to ~ 6×108 °C/min, the fastest warming rate reported in the field of cryobiology to the best of our knowledge.

Using a high-speed camera, we observed ice formation that lasted ~50 ms during rewarming inside Drosophila embryos treated with a lower CPA concentration (27% w/w ethylene glycol). Surprisingly, those embryos show a decent survival compared to embryos treated with a higher CPA concentration (33% w/w ethylene glycol) where no ice formation was observed during rewarming. Our direct videographic evidence demonstrates that certain small amounts of ice formation during rewarming are not lethal when rapid rewarming is applied. Combined with the precise control over the duration, magnitude, and shape of the voltage pulse, this platform rewarming technique can be readily scaled across biosystems of varying sizes and in throughput. This work comprises a significant breakthrough in cryopreservation technology by achieving new higher rewarming rates for biological systems in the cell to tissue size range.

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