| Freezing material is at the heart of maintaining robust tissue archives. But what are the consequences of some of these freezing processes in terms of tissue quality over time? Before deciding how to store samples, both the sensitivity of the downstream analysis (e.g., RNA stability) and how long that process needs to be viable should be considered. The cryopreservation field is rapidly evolving, especially for human samples. For example, standard practice for understanding tumor physiology was to fix in formalin, embed in paraffin, and store at room temperature. However, delicate RNA can degrade over time under these conditions but remains robust if stored at −80 °C (Baena-Del Valle et al., 2017). Thus, coral RNA and enzyme specimens may best be stored at −80 °C, potentially remaining stable for up to 10 years at these low temperatures and making them suitable for additional downstream analyses. For corals, storing at −80 °C allows for the highest number of downstream analyses (Fig. 2). However, longer-term stable storage (>tens of years) at liquid nitrogen temperatures (−196 °C) is preferable (Ortega-Pinazo et al., 2019; Kelly et al., 2019), though highly impractical for many researchers due to the cost and equipment needs associated with ultra-cold storage. In contrast, many laboratory analyses can be reliably performed on specimens stored at −20 °C (Fig. 2) for 2 to 5 years. |
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Frozen But Alive: Cryopreservation Holds Material Safely for Many Years
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| Cryobiology is the study of cells and tissues at cold temperatures. The central principle in cryopreservation is to avoid the formation of lethal intracellular ice. Generally, cryopreservation uses permeating cryoprotectants or solutes, such as dimethyl sulfoxide (DMSO), methanol or propylene glycol, and non-permeating solutes, such as sugars (e.g., glycerol), to allow the permeating cryoprotectants to enter cells and block ice crystal formation, and to permit the non-permeating solutes to dehydrate and remove intracellular water to reduce and avoid ice formation. Once cells and tissues are safely cryopreserved and held at liquid nitrogen temperatures, most biological processes are reduced. Theoretically, if cells are maintained at liquid nitrogen temperatures, they can survive for thousands of years with minimal damage. Thus, cryopreservation of living coral tissue and maintenance in liquid nitrogen (e.g., cryobanks) provides access to a multitude of scientific and restoration uses because the tissues are frozen, but also alive. Once the cryoprotectants are warmed and the cells are rehydrated, they are alive, and any number of analyses can be done post-thawing. However, cryopreserved cultured cells are equally robust at either −196 °C or −80 °C using a number of metrics over 8 years (Miyamoto et al., 2018). Even in properly cryopreserved samples, tissue degradation can occur if samples are removed from a freezer to subsample and then refrozen or exposed to heat transients by opening and closing of a freezer door. Thus, avoiding any changes in freezer temperatures is ideal. |
| To date, cryopreservation processes have been used to preserve coral sperm from over 48 species worldwide (Hagedorn et al., 2012). This international collaboration has used frozen sperm to subsequently fertilize coral eggs and create new coral larvae (Hagedorn et al., 2017). Moreover, frozen sperm has also been used to demonstrate the feasibility of assisted gene flow in the critically threatened coral Acropora palmata (Hagedorn et al., 2021). Frozen coral material is now archived in biorepositories around the world, and some of the material for the assisted gene flow experiments was stored for up to 10 years before successful use in fertilization experiments. |
