TA2_001. Deep supercooling of cells and tissue structures for long term storage

Berk Usta, MGH
Martin Yarmush, MGH
Korkut Uygun, MGH
Aslihan Gokaltun, MGH

Engineering systems to eliminate the water-air interface in cells and tissues in solution to limit ice nucleation

Due to the thermodynamically unstable nature of supercooled liquids, preservation of large biological samples in media at subzero temperatures for extended periods of time without the nucleation of ice has been impossible. To fill this technical gap, we recently developed a method called “Deep Supercooling (DSC) via Surface Sealing” where the aqueous sample is sealed with an immiscible liquid to eliminate the water-air interface, a large source of ice nucleation. We anticipate that DSC of red blood cells (RBC) can provide practical, high-quality, and long-term storage as an alternative to the current clinical standard (42 days) via refrigeration. Our approach is to develop an improved RBC storage solution that also includes novel ice-(re)crystallization inhibitors and additives to minimize cellular injury and reduce metabolism.

We also aim to use DSC to extend the preservation of hepatic (and other) cells and tissue constructs. Data showed that hepatocytes stored in improved solutions showed similar viability and functionality after 48h storage compared to fresh controls. Further work is being performed on 2D well-based hepatocyte cultures and 3D spheroids, and also targets oxidative stress injuries and diverse injury mechanisms that are possibly unique to supercooled storage.

TA2_002. Characterization of naturally occurring deep eutectic systems (NADES)

Allison Hubel, UMN

Characterize the changes in the freezing behavior of water at different concentrations of molecules; use this to lower cryoprotectant concentrations

Sugars and sugar alcohols can control the behavior of water during freezing, specifically the freezing temperature and amount of ice present. The purpose of this study is to (i) characterize the changes that take place in the freezing behavior of water at different concentrations of molecules; and (ii) use our understanding of this behavior to streamline cryopreservation. A wide range of sugar:sugar-alcohol molar ratios were synthesized and characterized. Some of these compositions result in Natural Deep Eutectic Systems (NADES) and studying their interactions with water across dilution and temperature is inherently important for maximizing their utility. NADES were found to have greater viscosity, reduced heat of fusion, greater absolute molar excess volume, lower water activity, and greater hydrogen bonding using low temperature Raman than non-NADES solutions. The results contradict the conventional wisdom in cryopreservation that more cryoprotectant is better. Specifically, we found a distinct decrease in molar excess volume observed for certain compositions, suggesting that interactions between sugars, sugar alcohols and water are more complex and there is a narrow range of concentrations/mole fraction that result in the strongest interactions with water. This knowledge should enable us to reduce the experimentation required to determine a composition for conventional slow freezing that results in higher post thaw recovery. In addition, understanding interactions amongst molecules and water should enable design of vitrification solutions that are lower in total cryoprotectant concentration.

TA2_003. Isochoric supercooled preservation and revival of human cardiac microtissues

Boris Rubinsky, UCB
Kevin Healy, UCB
Matthew Powell-Palm, UMN

Isochoric supercooling, to achieve high-stability and predictable supercooling of the University of Wisconsin (UW) organ preservation solution at −3 °C

Low-temperature biopreservation and 3D tissue engineering present two unique routes towards eventual on-demand access to transplantable biologics, where recent advances in both fields present critical new opportunities for crossover between them. In this project, we employ a recently developed technique, isochoric supercooling, to achieve high-stability and predictable supercooling of the University of Wisconsin (UW) organ preservation solution at −3 °C, in which we successfully preserve a functional 3D hiPSC-derived cardiac microphysiological system (MPS) for multiple days without the addition of non-physiological cryoprotectants such as dimethyl sulfoxide (DMSO) or glycerol. The cardiac MPS combines human cells with microfluidics to promote 3D self-assembly into a microtissue that faithfully recapitulates complex human heart muscle structure and function. Our work suggests that functional 3D engineered tissues may provide an excellent high-content, low-risk testbed to study organ preservation in a genetically human context. It also suggests that isochoric supercooling provides a thermodynamically simple and procedurally streamlined method of ice-free preservation at sub-0 °C temperatures. Given its non-reliance on specific cryoprotective agents, we suggest that it may be applied for the preservation of arbitrary biologics in the high-subzero temperature range.

TA2_004. Transient phase identification for vitreous biological systems

Chris Dames, UCB

Develop a non-invasive, volumetric method of identifying phases in a sample during transient cooling and heating to enable better preservation protocols

Quantitative detection of ice remains a prominent barrier to further integration of vitrification as a biopreservation method. Rapid heating of inorganic samples may leave no indication of transient ice nucleation after a sample is fully thawed. The aim of this project is to develop a non-invasive, volumetric method of identifying phases in a sample during transient cooling and heating.

Our approach is to build a cryogenic stage to easily heat and cool the samples by contact methods and to incorporate switchable substrates to allow for different measuring methods of crystallinity. One such measurement is the 3-omega method, sampling for thermal conductivity which differs considerably between vitreous and crystalline states and thereby allows us to determine the extent of crystallization in the sample. Our measurements have focused so far on CPA-loaded water solutions of various concentrations and we have optimized the substrate to maximize the sensitivity of this method. We have also tested in situ transmissive optical measurements to distinguish between vitreous and crystalline state of the CPA-loaded water samples as the crystalline state exhibits birefringence unlike the vitreous state. By adapting this to an acoustic measurement scheme, we aim to provide a high-speed alternative which can sample large tissue volumes. Here, we explore thermal and acoustic means of phase identification to enable better clinical and research implementation of preservation protocols.

TA2_005. Development of 3D Cryoprinter for Printing soft biomaterials

Boris Rubinsky, UCB
Kevin Healy, UCB

3D Cryoprinter for the printing of soft biomaterials by freezing or vitrifying them as they are extruded onto the print plate

We have developed a 3D Cryoprinter for the printing of soft biomaterials. The cell-laden bioinks used in 3D bioprinting are often soft and low viscosity, making it difficult to print complex structures. Cryogenic temperatures can stabilize soft materials during 3D printing by freezing or vitrifying them as they are extruded onto the print plate, expanding the possible designs. The 3D printed objects can be cryopreserved in liquid nitrogen until they are needed for tissue culture.

We have also developed a method of thawing frozen 3D printed objects and crosslinking them before they lose their printed shape. We have named this method “Freezing-modulated-crosslinking,” as the speed of crosslinking is modulated by the speed of thawing. Using this method, we have successfully 3D cryoprinted various a-cellular scaffolds for use in tissue engineering.

TA2_006. Permeabilization of Cryptosporidium oocysts to CPAs for vitrification

Rebecca Sandlin, MGH
Mehmet Toner, MGH

Developing methods to volumetrically scale-up methods for cryopreservation of Cryptosporidium parasites by vitrification

In collaboration with the Cummings School of Veterinary Medicine at Tufts University, we are developing methods to volumetrically scale-up methods for cryopreservation of Cryptosporidium parasites by vitrification. The existing method to cryopreserve Cryptosporidium parvum oocysts by ultra-rapid cooling previously developed by our lab is restricted to 2 µL. The primary objectives of the project are i) to scale-up the volume of vitrified C. parvum samples to ~100µl ii) translate the protocol for compatibility with C. hominis oocysts. The main difficulty of scaled-up vitrification is the inherent reduction of cooling rate, thus requiring increased CPA concentration to maintain formation of stable glass. This introduces challenges related to permeability to CPA, its toxicity, as well as apparent between- and within-species variability such as the age of the oocysts. To overcome these challenges, we translated protocols for insect embryo permeabilization as well as strategies to mitigate CPA toxicity by implementation of stepwise CPA exposure.

TA2_007. Rapid Vitrification of Insects

Rebecca Sandlin, MGH
John Bischof, UMN
Mehmet Toner, MGH

Develop a method to cryopreserve whole insects through vitrification solutions, CPA damage mitigation, heat transfer maximization and CPA distribution

The overall goal of this project is to develop a method to cryopreserve insects using cutting-edge techniques to achieve ice-free cryopreservation. Towards this long-term goal, specific milestones and aims include (i) development of vitrification solutions which minimize toxicity, (ii) identification of recovery solutions which mitigate long term CPA damage (iii) maximize heat transfer during cooling and rewarming, and (iv) spatiotemporal analysis of CPA distribution. The main challenges to achieve successful vitrification are (i) high CPA toxicity, (ii) requirement of CPA loading at room temperature, and (iii) organism size and cellular heterogeneity.