Cryopreservation practices in clinical and preclinical iPSC‐based cell therapies: Current challenges and future directions

Abstract

Induced pluripotent stem cells (iPSCs) offer significant therapeutic potential, but cryopreservation challenges, particularly the reliance on cytotoxic Dimethyl Sulfoxide (Me₂SO), hinder their clinical application. This review examines current cryopreservation practices in clinical and preclinical iPSC‐based therapies, highlighting the consistent use of Me₂SO and the logistical challenges of post‐thaw processing. The findings underscore the urgent need for alternative cryopreservation techniques to ensure the safety and efficacy of off‐the‐shelf iPSC therapies.

1. INTRODUCTION

Induced pluripotent stem cells (iPSCs) represent a significant advancement in biotechnology, offering tremendous potential for cell therapy. iPSCs are created by reprogramming fully differentiated somatic cells into a pluripotent state, allowing them to differentiate into virtually any cell type. ¹ This technology opens doors for treating various conditions, including as heart failure, ² arthritis, ³ Parkinson's disease, ⁴ and spinal cord injury. ⁵ However, despite the promise of iPSC‐based therapies, cryopreservation remains a critical challenge that must be addressed to ensure the safety and efficacy of these treatments.

Cryopreservation is essential for storing and transporting iPSC‐based therapies. The gold standard involves using 5%–10% (v/v) Dimethyl Sulfoxide (Me₂SO) as a cryoprotective agent (CPA), ⁶ slow freezing, and storage in vapor‐phase liquid nitrogen. While effective, Me₂SO is cytotoxic at temperatures above 0°C, necessitating its removal post‐thaw through centrifugation before administration. This process, common in cell banking, poses significant risks when applied to cell therapies intended for patient use.

Intravenous administration of Me₂SO is routine for therapies like Chimeric Antigen Receptor (CAR)‐T cells and hematopoietic stem cell transfusions, but it carries potential risks. Most adverse events are minor, such as nausea and headaches, but rare fatalities have been reported. ^{7 , 8 , 9} The situation becomes even more complex with novel iPSC‐based therapies exploring alternative routes of administration, such as direct injections into the heart, ¹⁰ spine, ⁵ brain, ¹¹ and eye. ¹² Unfortunately, there is limited safety data for Me₂SO use in these contexts, and in vitro studies suggest that even low concentrations of Me₂SO can significantly reduce cell viability. ^{13 , 14}

Given these risks, clinical and preclinical sponsors often remove Me₂SO from the cell product post‐thaw through dilution, centrifugation, supernatant removal, and reconstitution in a saline buffer. This is typically a manual open process performed under aseptic conditions. While this procedure is standard in research settings, it introduces additional challenges, risks, and costs when performed at the point of care in cell therapy applications, which can be overcome by using Me₂SO‐free cryopreservation media.

In the US and EU, reconstitution of cell therapies falls outside the scope of GMP and occurs in hospital or treatment center pharmacies. ^{15 , 16} Open processing in pharmacy settings carries contamination risks. In the US, over 1000 contamination incidents occurred in compounding pharmacies from 2001 to 2013, some resulting in deaths. ¹⁷

Cell therapies are inherently complex and have high manufacturing failure rates, as high as 13%–25% for patients with hematologic malignancies receiving CAR‐T. ^{18 , 19} Simplifying the process by removing post‐thaw processing steps can potentially reduce overall risk of failure, improving product safety and availability.

Cost is another key factor. Manual labor accounts for nearly 50% of the total cost of cell therapies. ^{20 , 21 , 22} In the EU, cost barriers have already led manufacturers like bluebird bio to withdraw cell therapies due to reimbursement challenges. ²³

Automated cell washing instruments exist, like the CliniMACs Prodigy (Miltenyi), ²⁴ however they add significant cost such as the instrument itself, maintenance, qualification, validation, training, reagents and consumables which can add up to hundreds of thousands of dollars annually. ²⁵ Thus, Me₂SO‐free cryopreservation of cell therapies reduces risk of contamination due to open processing, reduces labor costs and simplifies the cell therapy administration, and reduces equipment expenditures on automated cell washing solutions.

This mini‐review highlights the significant problem posed by current cryopreservation protocols for iPSC‐based cell therapy candidates, particularly the reliance on Me₂SO. While iPSCs hold promise as a scalable and ethical source for off‐the‐shelf therapies, the risks associated with traditional cryopreservation methods threaten to undermine their potential. The issues with Me₂SO, including cytotoxicity and the risks associated with its removal, emphasize the urgent need for alternative cryopreservation techniques. Exploring and developing these alternatives is crucial to ensuring the safety, efficacy, and widespread adoption of iPSC‐based therapies across various medical applications.

1.1. Cryopreservation practices in iPSC‐based cell therapy clinical trials

A comprehensive analysis of iPSC‐based clinical trials, informed by two systematic reviews ^{26 , 27} and additional literature, examined 57 clinical trials to understand current cryopreservation practices. Among these trials, 32% (18/57) disclosed the use of Dimethyl Sulfoxide (Me₂SO) as a cryoprotectant, and 9% (5/57) reported performing a post‐thaw wash step before administration (JPRN‐JMA‐IIA00384, ⁴ JPRN‐JMA‐IIA00385, ⁴ JPRN‐jRCTa031190228, ⁵ NCT06394232, ¹² NCT03763136 ² ). This post‐thaw washing and subsequent culture of the cell product are executed at the point of care, representing significant logistical challenges as they involve post‐processing, complicating the adoption of off‐the‐shelf cell therapies. Notably, 5% (3/57) of trials administered the iPSC‐based cell product fresh, following a culture period of up to 96 h post‐thaw and storage at 4–8°C before administration (JPRN‐JMA‐IIA00384, ¹¹ JPRN‐JMA‐IIA00385, ¹¹ JPRN‐jRCTa031190228 ⁵ ).

The reliance on post‐thaw processing at the point of care underscores the current limitations of cryopreservation techniques, as the need for washing indicates potential safety concerns with Me₂SO. A safe and effective Me₂SO‐free cryopreservation media could eliminate the need for such steps, simplifying the workflow and reducing risks associated with contamination and product damage. However, a significant limitation of this meta‐analysis is that only 22% (13/57) of the clinical trials disclosed their cryopreservation protocols. To gain additional insights, a review of cryopreservation practices in preclinical studies involving iPSC‐based cell therapies was conducted.

1.2. Cryopreservation practices in preclinical iPSC‐based cell therapies

An extensive review of preclinical studies focusing on iPSC‐based cell therapies was conducted to explore cryopreservation practices. Using the search terms “iPSC” and “cryopreservation” on PubMed, 74 articles published between 2017 and 2024 were identified, and 12 preclinical studies were selected for analysis based on their use of iPSC‐derived cell therapies with therapeutic intent. The selected studies span various species and disease models, with a significant focus on administering iPSC‐derived cell products directly into the affected organ or tissue, presented in Table 1.

TABLE 1.

Cryopreservation trends in preclinical iPSC‐based cell therapies.

Cell type	Disease area	Administration	Species	Reference
Hepatocytes	Inherited liver disease	Intrasplenic injection	Rat	28
Corneal endothelial cell	Corneal edema	Intra‐ocular injection	Monkey	29
Dopaminergic neurons	Parkinson's	Intraventricular injection	Rat	30
Dopaminergic neurons	Parkinsons	Intraventricular injection	Rat	31
Mid brain dopamine neurons	Parkinson	Intraventricular injection	Rat	32
Neural progenitor cell	Spinal cord injury	Spinal injection	Pig	33
mDA neurons	Parkinson	Intraventricular injection	Mouse	34
Retinal pigment epithelial cell	Vision restauration	Intra‐ocular injection	Rat	35
Megakaryocytes	Thrombocytopenia	IV transfusion	Mouse	36
CAR Macrophage	Pancreatic cancer	IV transfusion	Mouse	37
NK cells	Cancer	IV transfusion	Mouse	38
Neural stem/progenitor cell	Spinal cord injury	Spinal injection	Human	5

Note: Summary of preclinical studies on iPSC‐derived cell therapies across various species and disease models. The studies feature diverse administration routes, with the majority (75%, n = 9/12) delivering the cell product directly into the affected organ or tissue.

The review reveals that all preclinical iPSC‐based cell therapy candidates (12/12) used Me₂SO as a cryoprotectant. Additionally, 67% (8/12) of the studies employed a uniform freeze rate of 1°C/min, with the remainder not disclosing their freeze rate. Importantly, all studies (12/12) included a post‐thaw wash step to remove Me₂SO, which introduces post‐processing at the point of care and represents a barrier to developing off‐the‐shelf cell therapies.

These findings, summarized in Figure 1, highlight the consistent use of Me₂SO across both clinical and preclinical studies, despite its associated risks and the logistical challenges of post‐thaw processing. The review underscores the need for innovation in cryopreservation techniques, particularly those that can support the development of off‐the‐shelf iPSC‐based therapies without the drawbacks of current methods. The limited disclosure of cryopreservation protocols in clinical studies further complicates efforts to establish product comparability, especially if preclinical processes are modified during clinical development. The ICH Q5 guideline stresses that any manufacturing changes potentially impacting product quality may require additional preclinical studies, ³⁹ reinforcing the importance of early exploration of alternative, Me₂SO‐free cryopreservation solutions.

FIGURE 1.

Cryopreservation trends in iPSC‐based preclinical studies. Trends in cryopreservation protocols across 12 preclinical studies involving iPSC‐derived cell products. The figure highlights the consistent use of Me₂SO (Dimethyl Sulfoxide) as a cryoprotectant and the prevalence of uniform freeze rates and post‐thaw washing procedures.

1.3. Future directions in Me₂SO‐free cryopreservation of cell therapies

Research on Me2SO‐free cryopreservation media for cell therapies is ongoing, with no clinical tests yet. Scientists are exploring combinations of FDA‐approved CPAs, including sugars, alcohols, and proteins, showing promising results and, in some cases, outperforming Me₂SO. Machine learning has optimized a five‐component Me2SO‐free formulation, improving post‐thaw viability and reducing intracellular ice in iPSCs. ^{40 , 41}

Ultrasonication with microbubbles has transported trehalose inside MSCs, overcoming its inability to penetrate cell membranes. This approach, using trehalose as a potent ice inhibitor, achieved post‐thaw viability comparable to DMSO. ⁴²

Commercial Me2SO‐free media, like PrimeXV FreezIS DMSO‐free (FujiFilm), have shown safety in preclinical mouse studies. ⁴³ Further improvements may come from refining freeze rates and multistep protocols. ^{41 , 44} Advancements in this field will require collaboration across disciplines, including formulation science, machine learning, and innovative cryoprotectant delivery techniques.

2. CONCLUSION

In conclusion, while iPSC‐based therapies represent a groundbreaking advancement in biotechnology with the potential to revolutionize treatment for numerous diseases, the challenges associated with cryopreservation cannot be overlooked. This review has highlighted the pervasive reliance on Me₂SO as a cryoprotectant in both clinical and preclinical studies, alongside the significant risks and logistical challenges it presents, particularly regarding post‐thaw processing. The consistent use of Me₂SO, despite its cytotoxicity and the complications of its removal, underscores the critical need for innovative cryopreservation strategies that can eliminate these risks.

The findings emphasize the urgency of developing alternative cryopreservation techniques that can safely and effectively preserve iPSC‐based therapies, enabling their widespread adoption as off‐the‐shelf treatments.

AUTHOR CONTRIBUTIONS

Michael Dobruskin: Conceptualization; investigation; methodology; visualization; formal analysis; data curation; writing – original draft; writing – review and editing. Geoffrey Toner: Funding acquisition; supervision; resources. Ronald Kander: Funding acquisition; supervision; resources.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Dobruskin M, Toner G, Kander R. Cryopreservation practices in clinical and preclinical iPSC‐based cell therapies: Current challenges and future directions. Biotechnol. Prog. 2025;41(4):e70031. doi: 10.1002/btpr.70031

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.