Strategies for Stem Cell Cryopreservation

Cryopreservation is an important process in not just routine stem cell culture but also the clinical viability of cell-based therapies. Cell freezing is critical to the storage and timely delivery of consistent, functional cell products to patients. The underlying principle is straightforward—freeze cells to arrest metabolic activity while preventing intracellular or extracellular ice crystal formation. The effective and reproducible execution of this can be more complex, especially with varying cell types. In cell therapy workflows, cryopreservation may occur at multiple points, from initial isolation through final product formulation. Deviations from optimal protocols at any stage can compromise cell recovery and disturb downstream processing, possibly even impeding therapeutic use. This article examines useful considerations for stem cell cryopreservation, with benefits across clinical applications, including biobanking, regenerative medicine, and stem cell-based therapies. 

Across distinct cell types, cellular properties such as osmotolerance, permeability, and surface-to-volume ratio will vary, resulting in varying responses to the cryopreservation process. Accordingly, stem cells should be approached individually, along with unique considerations in methodologies and protective agents.

“As with any cell cryopreservation process, optimization is key. Different cells will prefer different freeze densities, freeze media formulation and thawing conditions,” notes Hilary Sherman, Senior Scientist at Corning Life Sciences. “Stem cells can be very sensitive to the already stressful cryopreservation process so care must be taken to maintain as high of viability as possible. The preferred cryoprotectant formulation may vary depending on the cell type being preserved.”

Tailoring cryopreservation to the stem cell type

Going further, cryopreservation should be tailored to specific stem cell types, including hematopoietic (HSC), mesenchymal (MSC), embryonic (ESC), and induced pluripotent stem cells (iPSC). “Challenges do differ across stem cell types because cells vary in osmotic tolerance and membrane permeability. That means the safe CPA loading/unloading steps and cooling rates are not identical for all cells,” says Mariya Hrynchak, Ph.D., Product Portfolio Manager at NIPPON Genetics EUROPE. “The practical takeaway is that cryopreservation is rarely one-size-fits-all: protocols need to be tuned to the cell type to protect both survival and downstream function.”

After thawing, tests should be conducted to verify that stem cells have retained their defining characteristics and have not undergone unwanted differentiation or phenotypic drift. Some important considerations include confirmation of cell identity, maintenance of lineage-specific markers, negative markers for differentiation, and preservation of functional potency relative to the pre-freeze population.

“I always look at my cells after 24 hours and ensure the morphology looks as it should. Additionally, as soon as I have enough cells, I will perform flow cytometry to ensure cells express the appropriate markers,” shares Sherman.

“In general, HSCs are relatively robust and reproducible with standard controlled-rate DMSO-based protocols, while MSCs can be more sensitive to handling conditions that influence consistent post-thaw performance, and ESCs/iPSCs often have the narrowest handling window and are particularly sensitive to stress during dissociation, freezing, and early recovery,” says Hrynchak.

More stem cell type-specific considerations are listed below.   

Hematopoietic stem cells (HSCs)

Before freezing, HSCs characterization methods can include volume measurement, nucleated cell counting, and CD34+ quantification by flow cytometry. Cells should be frozen within no later than 72 hours after collection, with 48 hours being most ideal. HSCs tolerate standard controlled-rate freezing protocols well, with optimal results achieved at 1–2 °C per minute cooling rates followed by cryogenic nitrogen storage below −140°C.

As a cryoprotectant, DMSO is generally used at recommended concentrations between 5–10%. It should be noted that DMSO is hyperosmotic, and immediate exposure of cryopreserved cells to the isosmotic blood system may lead to osmotic injury and excessive cell expansion. Reduced-DMSO formulations supplemented with alternative cryoprotectants such as human serum albumin or trehalose have been reported to maintain post-thaw viability and engraftment potential while minimizing infusion-related toxicity. Post-thaw, it is recommended to perform clonogenic assays to confirm functional potency and hematopoietic capacity.

Mesenchymal stem cells (MSCs)

MSC cryopreservation generally follows slow controlled-rate freezing or vitrification. Slow freezing, typically at cooling rates up to −3°C/min until reaching −80°C, followed by transfer to liquid nitrogen. This method is often used due to its operational simplicity, lower cryoprotectant requirements, and reduced risks of toxicity and contamination. Vitrification offers an alternative that eliminates the need for programmable freezers by rapidly plunging cells directly into liquid nitrogen, though this requires substantially higher CPA concentrations.

As a CPA, formulations with up to 10% DMSO generally provide high post-thaw viabilities. Formulation optimization has focused on supplementing DMSO with high-molecular-weight macromolecules such as FBS, polyethylene glycol, or polyvinylpyrrolidone to enhance cryoprotection. However, animal component-free media will be more ideal to meet regulatory and safety requirements for clinical applications.

Post-thaw quality controls should confirm that MSCs retain their defining characteristics. This includes verification of plastic adherence under standard culture conditions, positive expression of phenotypic markers (such as CD29, CD44, CD73, CD90, and CD105), and absence of negative markers (CD45, CD34, CD14, CD11b, CD79a, CD19, and HLA-DR). To ensure the MSC proliferative capacity, the population doubling time (PDT) of cells should also be measured.

Induced pluripotent stem cells (iPSCs)

iPSCs can be cryopreserved either as aggregates or single-cell suspensions, each with distinct advantages. Aggregate freezing benefits from cell-cell contacts that support survival and enables faster post-thaw recovery since cells do not need to re-form colonies. However, size heterogeneity can lead to variable CPA penetration, potentially compromising viability in aggregate cores. Single-cell cryopreservation offers good quality control through accurate cell counting and consistent vial-to-vial recovery. However, cells are subject to dissociation-induced apoptosis (anoikis) and typically require the addition of a rho kinase (ROCK) inhibitor.

Leading up to freezing, iPSCs should be harvested during the logarithmic or exponential growth phase, ideally determined empirically through growth curve analysis. Cultures with substantially differentiated cell populations should also be discarded, as these cells not only represent undesirable contaminants but may also induce differentiation of remaining pluripotent cells within colonies.

Standard slow-freezing protocols at −1°C/min using 10% DMSO are commonly used, particularly when cells are dissociated with EDTA. Alternative formulations have also been explored and show promise. Ethylene glycol demonstrates reduced toxicity and improved pluripotency maintenance, while knockout serum replacement-based media and optimized CPA combinations achieve comparable or better results. When thawing, the survivability of the cells can be improved by maintaining a high cell seeding density.

Post-thaw characterization

Diligent testing post-thaw is a worthwhile investment for stem cells, as this can ensure appropriate viability, pluripotency, and function for downstream work. Hrynchak advises assessing the recovery over the first 24 to 48 hours after conducting standard viability tests. This includes attachment efficiency for adherent cells, morphology, time to resume normal growth, and proliferation rate.

“This is important because some freeze-thaw injury shows up later as delayed apoptosis or slow recovery, not as immediate cell death,” says Hrynchak. “For pluripotent cells, ALP staining can be used as a quick screen. ALP-positive, well-formed colonies are an early indicator of an undifferentiated culture, while immunostaining, flow cytometry, or a short qPCR panel provides a more specific confirmation that pluripotency markers remain high. Overall, the goal is to obtain a read on identity, early stress, and recovery before committing to any downstream studies.” 

References

Whaley D, Damyar K, Witek RP, Mendoza A, Alexander M, Lakey JR. Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transplant. 2021;30:963689721999617. doi:10.1177/0963689721999617

Hunt CJ. Technical Considerations in the Freezing, Low-Temperature Storage and Thawing of Stem Cells for Cellular Therapies. Transfus Med Hemother. 2019;46(3):134-150. doi:10.1159/000497289

Erol OD, Pervin B, Seker ME, Aerts-Kaya F. Effects of storage media, supplements and cryopreservation methods on quality of stem cells. World J Stem Cells. 2021;13(9):1197-1214. doi:10.4252/wjsc.v13.i9.1197

Wuchter P. Processing, Cryopreserving, and Controlling the Quality of HSC. 2024 Apr 11. In: Sureda A, Corbacioglu S, Greco R, et al., editors. The EBMT Handbook: Hematopoietic Cell Transplantation and Cellular Therapies [Internet]. 8th edition. Cham (CH): Springer; 2024. Chapter 20.

Wang, J., Li, R. Effects, methods and limits of the cryopreservation on mesenchymal stem cells. Stem Cell Res Ther 15, 337 (2024). doi:10.1186/s13287-024-03954-3

Uhrig M, Ezquer F, Ezquer M. Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells. Cells. 2022;11(5):799. Published 2022 Feb 24. doi:10.3390/cells11050799