14 February 2023 Elizabeth Tan

What does thawing look like in cryopreservation?

The freezing and thawing of cells is a technique that has been used for the preserving and storing of biological materials for a long time. The process, known as cryopreservation, has made it possible to halt the cellular clock, which results in significant reproducibility because it allows for the cells to be used when required, saving time, costs, and resources. Thawing is critical for all the applications of cryopreservation, spanning a broad range including the preservation of cell and gene therapies.

The goal of the entire freezing-storage-thawing process is to ensure high cell survival. And thawing in cryopreservation is the process of warming cryopreserved biological samples to a temperature so that they can be viable and functional for use.

Different cryopreserved cells have varying methods of thawing, and the process can be stressful to the cells. Using the right technique can increase the chances of cells surviving the procedure, and more importantly, achieve predictable and reproducible cell behaviour after cryopreservation. To attain this, various guidelines and protocols have been created over time to ensure that there is standardization when it comes to thawing frozen cells. Standardization of the thawing protocol brings about great benefits to results since having a consistent cell survival rate reduces the need to thaw more cells to secure sufficient stock. Variations in the freezing or thawing protocol leads to differences in the cells’ phenotype and hence variations in outcomes.

The common ways of thawing cells include water baths, bead baths, and dedicated thawing devices. Water baths consist of immersing the samples into 37°C water while attempting to keep the top of the vial dry. Water is an excellent conductor of heat energy, and hence a water bath for the samples would mean even heating. It is unlikely to have any hot and cold zones so the entire bath can be used and expected to have the same temperature. Water, however is also a source of contamination. A water bath needs to be constantly monitored and refilled to prevent any element burn-out from evaporation.

Bead baths, like water baths, consist of putting the sample into beads which have a temperature of 37°C. These beads are usually made of aluminum and transfer heat energy less efficiently and can potentially heat less uniformly. This means that bead baths could be warmer in some areas and colder in others. The absence of liquid in these baths has the advantage of reducing the chances for contamination. Bead baths heat up quicker than water baths and can be used with minimal supervision, they are also more efficient since they require less electricity and do not need to be refilled.

Dedicated devices include dry automated thawers and vial thawing systems. The main difference between dry automated thawers and vial thawing systems is that dry automated thawers allow for cryobags to be thawed whereas the vial thawing systems are for vials only. Dry automated thawers are instruments that use a dry heat technology and multiple sensors to determine when thawing is complete, so there is an exact endpoint that maximizes cell viability and minimizes uncertainties associated with manual operations. The thawing temperature can be set using the dry automated thawers, ranging from 37°C to 50°C. Vial thawing systems accommodates a cryogenic vial with controlled profile thawing. The adaptive sensing technology detects and monitors the vial temperature and thawing profile, and the vial is gently ejected once the endpoint is determined. Homogenous temperature around the vial is ensured by using conducive heating blocks that are coupled to the vial to be thawed through an inert, malleable conducive material that conforms to any irregularities in the vial wall, providing a uniform heat transfer surface.

There are also lab-specific considerations that play a role when choosing the method to thaw cells. For example, if cryopreservation is required for a product with clinical application, the thawing process would likely be conduced in a cleanroom and since water baths are a source of contamination, other methods need to be considered.

At PanTHERA, we are passionate about the process of cryopreservation, our novel Ice Recrystallization Inhibitors (IRIs) increase overall post-thaw cell recovery and quality by reducing damage throughout the cryopreservation process. In addition to their positive impact in stabilizing cell and gene therapies for medical applications, PanTHERA’s IRI technology can be used across several areas that includes assisted reproductive technologies, animal and aquaculture husbandry, cell-based diagnostics, and biopharmaceutical production. More information on the IRIs and their benefits can be found here.

About PanTHERA CryoSolutions

PanTHERA CryoSolutions is a Canadian corporation that designs and manufactures cryopreservation solutions for cells, tissues and organs for research and clinical markets. Our patented ice recrystallization inhibitor (IRI) technology exceeds other products by providing superior cryopreservation and increasing post-thaw cell recovery and function for our customers. The technology enables the use of significantly less costly storage and transportation systems limiting the need for liquid nitrogen use for some cell therapy applications.


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