Stem Cell Cryopreservation | Corning

Cryopreservation, or the storage of cells and tissues at very low temperatures, is a commonly used technique. As described in Integrative Medicine Research, the cryopreservation process maintains the structural integrity of living cells so that when they're thawed from storage, they are capable of functioning once again. Tom Bongiorno, PhD, a Field Application Scientist with Corning, says that cryopreservation is suitable for stem cell work, including induced pluripotent (iPS) lines. However, he also notes that these cells are extremely sensitive. Without careful treatment, cryopreservation can induce differentiation or decrease their viability.

Although low temperatures around 4⁰C can suspend many biological processes, Frontiers in Medicine; Gene and Cell Therapy describes how very low temperatures, such as those used in cryopreservation, are better for long-term storage. Storage temperatures below -130⁰C completely inhibit cellular activity, and there is a range of labware suitable for safe storage under these conditions. Prolonged, and possibly indefinite, storage of cryopreserved cells and tissues can offer benefits for many cell-based therapies, including therapies involving transplantation, fertility, and stem cells.

Cryopreservation Basics

There are four major steps to the cryopreservation process:

  1. The starting tissue or cells are selected, they are usually mixed with a cryoprotective agent (CPA) that helps prevent cryoinjury during the freezing and thawing process. CPAs, which include dimethyl sulfoxide (DMSO), ethylene glycol (EG), and glycerol, among others, are usually liquid and can penetrate cells relatively easily. Ideally, they should be non-toxic or possess low toxicity. CPAs protect the cells against cryoinjury by minimizing ice crystal formation during the freezing phase.

"It's important to fully characterize your stem cell type so you know what works for them," says Bongiorno, noting that DMSO may not be the best CPA for all cells. "Refer to the existing literature to find out what CPA works best with your cells."

It's also important to consider the end use for the stem cell line. Genetic Engineering News notes that storing T-cells requires less toxic CPAs that are appropriate for onward treatments.

2. Once the sample is mixed with CPA, it is frozen for storage. BMC Biology describes two common freezing processes: Freezing is either conducted slowly in a controlled environment, such as the Corning CoolCell, to avoid intracellular ice formation, or by faster vitrification, where samples flash freeze from an aqueous to glass state by immersion in liquid nitrogen. Bongiorno again advises learning as much as possible about your cells and suggests that vitrification with specialized CPAs is better for embryonic stem cells.

3. As the samples thaw for use, it is critical to control the rate at which the sample warms to avoid cell damage.

4. The final step is to remove all traces of the CPA for optimal cell recovery. Equipment such as the Corning X-WASH™ system, a functionally closed system that safely washes and harvests cells, helps to scale up for high-throughput processing. When thawing mesenchymal stem cells, the X-WASH reduced the DMSO concentration from 10,000 ppm in the cryovial to about 200 ppm after washing.

Cryopreservation for Long-term Storage in Cell-based Therapies

Being able to extend storage is considered extremely important for delivering successful cell-based therapies. Not only does cryopreservation extend shelf life, but it also helps with scaling up to increase volume.

Bongiorno suggests using cryopreservation to "pause" cell culture. Using peripheral blood mononuclear cells (PBMCs) as an example, scientists can use cryopreservation at each stage of preparation — after obtaining the blood sample, in isolating the cell type, and even following reprogramming to iPS prior to expansion.

The benefits of cryopreservation in the stem cell workflow can help mitigate some of the common problems encountered in developing cell-based therapies, namely characterization and scaling up. Cryopreservation can provide timelines with flexibility in these areas.

Scheduling flexibility is helpful, especially for clinical processing. Being able to start and stop the process not only decreases waste but also optimizes use of specialized facilities. Being able to pause cell production to fit in with the availability of a manufacturing suite, for example, is a huge benefit.

Testing flexibility means that processes can be optimized and validated before they are launched into full-scale manufacturing. Applications often require testing ahead of time. Being able to verify the quality of cell lines you are considering before committing to expansion not only saves precious resources but also allows scientists to build up a bank of qualified cell lines suitable for treatment.

Scaling Up and Expansion is essential to building sufficient volume for cell-based therapies, but it's tricky to make sure everything is ready all at once. For example, during research and development it might not be possible to predict exactly how many cells are required. In these cases, cryopreservation allows stocks to be saved for later amplification, scaling up from stock as required. Timeline flexibility also helps with supply chain challenges; with cryopreservation, you can freeze stocks and then get everything else set up and ready to go ahead of time before committing the cells.

Cryopreservation technology is evolving. Research is refining processes to support cell viability in various applications, including tissue transplantation, cell-based regenerative medicine, and organoids for drug screening. Keeping up-to-date with research and technology innovations is essential in stem cell therapies. Although there is still work to be done, Bongiorno notes that in the future, researchers may be able to cryopreserve cells following differentiation.

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