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Improved Cryopreservation using Ice Binding Proteins

Final Report Summary - CRYOPRESERVATION (Improved Cryopreservation using Ice Binding Proteins)

Cryopreservation is the use of low temperatures to preserve biological samples. The ability to store biological materials without damaging them has taken on increasing importance in the face of a shortage of organs for transplantation. While preservation by freezing seems simple, freezing and thawing of cells and tissues typically compromise their integrity such that specialized protocols are needed for cryopreservation to be even partly successful. New and improved cryopreservation methods are therefore required. One direction to achieve this goal is to learn from organisms that live under low-temperature conditions how they avoid freeze damage.

Antifreeze proteins (AFPs) are a remarkable group of proteins that help cold-adapted organisms to survive at sub-zero temperatures. These proteins bind to ice crystals, thus inhibiting their growth. Highly active AFPs are termed hyperactive AFPs, while less active proteins are termed moderate AFPs. In frozen samples, damaging recrystallization processes, in which large ice crystals grow bigger at the expense of small crystals, are well inhibited by AFPs. The unique interactions of AFPs with ice crystals suggest their potential as additives for cryopreservation of foods, cells, tissues, and organs.

Our group investigates the interactions of AFPs with ice and promotes the use of AFPs to advance cryopreservation technologies. Several tools were developed in our laboratory to study these proteins: a custom-made computer-controlled cold microscope stage system for measuring AFP activity (published in the Journal of Visualized Experiments, Braslavsky and Drori, 2013); a novel microfluidic device allowing temperature control of small ice crystals in a microscopic environment (Celik et. al. PNAS, 2013, and in Drori et. al. Interface, 2014), and a miniature cold finger device (Haleva, Celik, Bar-Dolev, et al., Biophys J. 2016, and Bar-Dolev Interface 2016). Using these devices and fluorescently labeled AFPs, we explored the dynamic nature of the activity of these proteins. and the way they interact with ice. Ice crystals tend to be flat with hexagonal symmetry. The planes normal to the hexagonal planes are called basal planes. We have shown that both types of AFPs bind irreversibly to ice crystals (Celik et al. PNAS 2013); however, moderate AFPs are unable to bind to the basal plane whereas hyperactive AFPs do bind to the basal plane and have different ice binding kinetics (Langmuir, Drori, et al. 2014). Another way to distinguish moderate from hyperactive AFPs is by observing ice shaping, the evolution of the shapes of ice crystals grown in the presence of AFPs. We found that ice shapes in hyperactive AFP solutions are formed during melting (published in Bar-Dolev et al. Interface, 2012 and Liu et al. Proc, R. Soc A, 2012).

Ice recrystallization inhibition (IRI) of AFPs is also an important issue that we have investigated (PLoS One, Mizrahy et al. 2013, Mangiagalli et al. FEBES 2016). IRI and ice shaping are both involved in freeze damage to tissues and cells; hence, understanding and controlling these processes would have a great impact on the cryopreservation of biological samples and in the food industry. Further surprising rolls were found in nature for ice-binding proteins. We showed that an Antarctic bacterium uses its ice-binding protein to adhere to ice crystals as a platform to live on (Bar-Dolev et al., Interface 2016).
Large quantities of AFPs are required for cryopreservation investigations. We are currently in the course of developing methods for the large-scale production of AFPs. In our approach, the proteins are expressed in a bacterial system and purified by exploiting their affinity for ice.

To summarize, our study advanced the basic mechanisms underlying AFP activity, expand their possible use, and seek to develop approaches for using such proteins in cryopreservation applications.