Yuhao Wang1,2
Kexin Yang
Chao Yang- 1State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases and Engineering Research Center of Oral Translational Medicine, Ministry of Education and National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China
- 2Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China
- 3Chengdu Shiliankangjian Biotechnology Co., Ltd., Chengdu, Sichuan, China
- 4Sichuan Tianfu Cell Quality Detection and Evaluation Center Co., Ltd., Chengdu, Sichuan, China
Although mesenchymal stem cells (MSCs) are among the most promising cell types for regenerative medicine, the lack of mature “off-the-shelf” cryopreserved preparations limits their widespread clinical application. This represents a critical bottleneck and an often-underestimated complication of the cryopreservation process, which leads not only to significant reduction in viable cell yield but also to subtle yet consequential perturbations in therapeutic function. This review distinguishes itself by critically synthesizing recent advances through the lens of the integrated “vial-to-vein” pathway, emphasizing how cryopreservation-induced attrition of functional potency—particularly in immunomodulation and paracrine signaling—compromises clinical efficacy. We systematically analyze the evolution beyond conventional dimethyl sulfoxide (DMSO)-based media towards next-generation, bioinspired cryoprotectants and storage strategies designed to safeguard these critical biological attributes. We then review the cryopreservation effects on MSCs morphology, surface marker consistency, and multipotent differentiation as well as their fundamental immunomodulation. Subsequently, the review consider the efficiency of cryopreserved MSCs in different disease models like cardiovascular diseases — respiratory diseases and chronic kidney disease. Finally, we discuss the pivotal transition in quality control, arguing for a multi-pillar paradigm that integrates precise molecular identity testing with clinically relevant functional potency assays tailored to specific indications. Crucial in the pursuit of this integrated understanding is to ensure a set of consistent, reliable and coherent properties by which next-generation MSCs therapies can be evaluated. Yet correlating these in vitro metrics with clinical efficacy remains the single greatest hurdle.
1 Introduction
For a long time, MSCs have been central to cell therapy research (Hansen et al., 2022; Margiana et al., 2022). They are multipotent and can be gathered from tissues like bone marrow and fat. Furthermore, they are capable of forming bone, cartilage, and fat cells (Naji et al., 2019). Initially, it was believed that these cells repaired the body simply by replacing damaged tissue; however, this model is now outdated. Many studies have found that MSCs are effective even without long-term engraftment in the body (Spees et al., 2016), indicating that their mechanism of action is more complex.
Today, we see MSCs differently (Caplan, 2017). They are now considered “medicinal signaling cells” that act primarily through paracrine signals (Margiana et al., 2022). Instead of replacing tissue, MSCs release various factors such as cytokines and growth factors, which alter the local body environment (Spees et al., 2016). These secreted molecules have powerful effects. They can help cells survive, promote new blood vessel growth, reduce scarring, and, most importantly, regulate the immune system (Naji et al., 2019; Spees et al., 2016). For example, MSCs exert immunomodulatory effects through multiple mechanisms: they can inhibit the activation and proliferation of T-cells, induce macrophages to polarize toward the anti-inflammatory M2 phenotype, and modulate the function and differentiation of B-cells and dendritic cells. This makes them a promising treatment for many inflammatory diseases and for Graft-versus-Host Disease (GVHD) (Hansen et al., 2022; Naji et al., 2019).
Bringing MSCs from the lab to the clinic as a therapeutic product creates significant logistical challenges. Freshly cultured cells do not last long, are expensive, and hard to standardize for widespread use (Crow, 2019). This is why cryopreservation is so critical. It permits the creation of “off-the-shelf” allogeneic MSC therapies. Importantly, large and consistent batches can be produced. These products can then be stored for long periods and used as needed, offering a drug-like model of administration (Crow, 2019). This model could lower costs and make these therapies more accessible.
But there is a catch. The very process of freezing and thawing is harsh on the cells. It can damage them, affecting not just their viability but also their crucial biological functions. This leads to the central question of this review: are cryopreserved MSCs as effective as fresh ones? We are particularly concerned about their immunomodulatory capacity, which is key to their clinical success. Simple viability tests after thawing do not tell the whole story and often do not correlate with therapeutic potency (Hansen et al., 2022). Therefore, given the variability between donors and manufacturing processes, better functional assays are needed to ensure product quality (Hansen et al., 2022; Crow, 2019).
To address these issues, this review will systematically examine how cryopreservation truly impacts MSCs. We will look at the latest technologies, the effects on cell biology (like morphology and differentiation), and the performance in various disease models. We will also cover the essential manufacturing and regulatory steps needed to create standardized, cryopreserved MSCs products, as outlined in Figure 1.
Figure 1. Workflow for creating an “off-the-shelf” MSCs therapy. MSCs are isolated from tissues like bone marrow or umbilical cord and expanded under GMP. A key step is cryopreservation, shown here with two advanced methods: (A) using novel DMSO-free CPAs like trehalose-based polymers, and (B) preserving MSCs in their native tissue niche. After thawing, cells must pass several critical quality control (QC) tests. These confirm the cells’ identity (e.g., via transcriptomic signature), ensure their safety (free of contaminants), and measure their potency for the specific therapy, using assays like MLR for immunomodulation or MPS for vasculogenesis.
2 Fundamentals and development of MSCs cryopreservation technology
How can we create a successful “off-the-shelf” MSCs therapy? The answer starts with reliable cryopreservation methods that preserve cell viability and function. Here, we examine the fundamental science of freezing cells, tracing the evolution from traditional DMSO + fetal bovine serum (FBS)-based cryoprotectants to novel cryoprotective agents, native tissue niches, and vitrification cryopreservation, extending toward promising horizons such as automated manufacturing platform (Figure 2).
Figure 2. The evolving paradigm of MSC cryopreservation: from traditional DMSO + FBS-based cryoprotectants to novel cryoprotective agents, native tissue niches, and vitrification cryopreservation, extending toward promising horizons such as automated manufacturing platform.
2.1 Traditional cryopreservation media: composition and mechanisms
When cells are frozen, they are pushed to their physical limits. The formation of ice crystals and sudden changes in osmotic pressure are the two main culprits of irreversible damage (Figure 3). As the external medium cools, extracellular ice forms first. This causes solute concentration and creates a hypertonic environment. Consequently, water flows out of the cells, resulting in osmotic shock and dehydration. Conversely, rapid cooling can lead to lethal intracellular ice formation (IIF), physically rupturing membranes and organelles. This aligns with Mazur’s “two-factor hypothesis,” which states that both excessively slow and excessively fast cooling are lethal (Bojic et al., 2021). Moreover, during warming, ice recrystallization and devitrification impose additional mechanical damage. On a biochemical level, freeze–thaw processes may induce protein denaturation, mitochondrial dysfunction, reactive oxygen species generation, and premature activation of programmed cell death pathways (e.g., apoptosis and necrosis)—cumulatively determining post-thaw viability and function (Chang and Zhao, 2021; Li et al., 2022).
Figure 3. Mechanisms of cryoinjury in MSCs and a comparison of advanced protective strategies. This figure illustrates three key concepts in MSCs cryopreservation. On the left, the process of conventional cryopreservation is shown, where the use of agents like DMSO can still lead to significant cell damage from extracellular ice crystal formation (causing osmotic stress) and intracellular ice formation (causing mechanical rupture), in addition to direct cytotoxicity. In the center, the protective mechanism of novel, advanced CPAs like trehalose-based or zwitterionic polymers is depicted. These agents mitigate cryoinjury by inhibiting ice recrystallization (IRI), stabilizing the cell membrane, and balancing osmotic shifts without inherent toxicity. On the right, the concept of preserving MSCs within their native tissue niche is shown. The natural extracellular matrix (ECM) acts as a protective scaffold, physically buffering against stresses and maintaining the cellular microenvironment, which enhances post-thaw viability and recovery.
A comparative study on MSCs revealed that murine MSCs (mMSCs) showed a post-thaw viability of 91.5% ± 5.6%, whereas human MSCs (hMSCs) achieved only 82.9% ± 4.3%. This disparity is attributed to differences in cell volume and surface-area-to-volume ratios (SA/V): mMSCs exhibit an SA/V of approximately 0.65 μm-1, compared to ∼0.34 μm-1 for hMSCs. The higher SA/V facilitates more efficient dehydration, thereby reducing susceptibility to IIF—emphasizing the need for tailored cooling rates according to cell type, consistent with fundamental cryobiological principles (Jang et al., 2017; Capicciotti et al., 2015; Yi et al., 2014; Prickett et al., 2015). Importantly, such biophysical variations also exist between MSCs from different human tissue sources, which underlies their distinct cryopreservation vulnerabilities. For instance, adipose-derived MSCs (AD-MSCs) typically have a larger cell volume and lower SA/V ratio compared to the smaller, more spindle-shaped bone marrow MSCs (BM-MSCs). This fundamental difference in cellular geometry makes AD-MSCs theoretically more susceptible to intracellular ice formation during rapid cooling, necessitating tailored optimization of cooling rates for each cell type.
The classic cryopreservation of MSCs has long relied on a simple formula utilizing two key agents: DMSO to prevent intracellular ice formation (Margiana et al., 2022; Naji et al., 2019) and FBS to provide protein stability and protect cell membranes (Svoradová et al., 2023; Gao et al., 2021; Duarte Rojas et al., 2024). Yet, this approach is far from ideal. DMSO is known to be toxic to cells (Spees et al., 2016; Crow, 2019). FBS presents a host of other challenges for clinical translation, including inconsistent performance between batches and the risk of immune reactions (Duarte Rojas et al., 2024). The search for better options was therefore inevitable. The adoption of human platelet lysate (HPL) as a safer alternative to FBS is a prime example of this shift (Svoradová et al., 2023; Duarte Rojas et al., 2024), driven by the fundamental weaknesses of the original method.
2.2 Optimization strategies for cryopreservation media
Researchers have taken two main approaches to address the limitations of the classic DMSO + FBS medium. The first approach is conservative: improving the existing formula by adding new ingredients, often referred to as “cryoprotective adjuvants.” Examples include polyethylene glycol (PEG), which physically protects the cell membrane (AlHindi and Philip, 2021), and non-penetrating sugars such as trehalose, which shield cells from extracellular ice damage (Gao et al., 2021; Andreoli et al., 2024). To solve the FBS problem, a defined protein like bovine serum albumin (BSA),which is derived from bovine sources, can be used instead for stability (Duarte Rojas et al., 2024).
However, the inclusion of animal serum may not be necessary for clinically applicable cells, mainly because of the risks of xenogenic viral transmission (e.g., prions) and variability in serum composition, sources, and production lots affecting cultured cell phenotypes (Cimino et al., 2017; Guiotto et al., 2020). A more radical approach, however, aims to completely remove the problematic ingredients. This has led to the design of fully serum-free and xeno-free systems. By replacing FBS and sometimes DMSO with synthetic, less toxic components, these formulations offer a much safer and more consistent product for clinical use. For instance, formulations using glycerol and specific amino acids like isoleucine have demonstrated good post-thaw survival and functional recovery of MSCs with reduced immunogenicity compared to DMSO-based protocols (de Camargo et al., 2025; Larsen et al., 2024). Studies indicate that glycerol provides cryoprotection by osmotically shielding cells from ice crystal damage, while amino acids such as proline act by preventing intracellular crowding and ectoine functions as an osmoprotectant against hypertonic stress. The clinical-scale feasibility of such xenofree strategies is further supported by advances in serum-free culture systems, exemplified by Lonza’s commercially available medium designed for expanding umbilical cord-derived MSCs while maintaining their critical biological properties (Wu et al., 2014). Using these cryoprotectants, either individually or in combination, a methylcellulose-based cryomedium was developed to replace DMSO/serum-containing solutions effectively. Results demonstrated 99% post-thaw viability with optimized formulations. Notably, a composite solution containing 1% proline and 10% ectoine achieved 90% viability (Freimark et al., 2011). The development of novel, polymer-based cryoprotective agents (CPAs)represents the next frontier, offering a move away from traditional small-molecule protectants altogether.
2.3 Cryopreservation via native tissue niche
The native tissue niche is an intrinsic three-dimensional microenvironment within organisms. It consists of tissue-specific ECM, signaling molecules, and adjacent cells. This architecture provides physical and biochemical cues essential for stem cell survival, self-renewal, and differentiation (Yang et al., 2016). Wharton’s jelly—the gelatinous matrix within umbilical cord tissue—features an ECM that forms a natural 3D scaffold. Research confirms this structure, enriched with collagens, hyaluronic acid, and other components, effectively mimics the in vivo stem cell niche by providing MSCs with adhesion sites and mechanical support. Remarkably, cryopreserved MSCs in processed umbilical cord tissue maintain expression of stemness markers (CD73/CD90/CD105) and multilineage differentiation potential. Experimental evidence demonstrates that post-thaw umbilical MSCs retain high efficiency in differentiating into osteoblasts, chondrocytes, and adipocytes. This validates the 3D niche’s role in preserving cellular functional stability (Ding et al., 2011; Seo et al., 2021). Within dental pulp niches, neurovascular networks deliver stem cell factor (SCF) and maintain hypoxic conditions that preserve dental MSCs quiescence (Sharpe, 2016). Therefore, native tissue niches serve as protective scaffolds that mechanically buffer external stresses and sustain cellular microenvironments, thereby enhancing post-thaw survival rates and functional recovery.
In practice, the cryopreservation of intact tissue niches generally requires the addition of CPAs to mitigate intracellular ice formation and osmotic stress during freezing and thawing. Both permeating CPAs (e.g., DMSO, ethylene glycol) and non-permeating CPAs (e.g., trehalose, sucrose, hydroxyethyl starch) have been employed to improve cell recovery within the preserved ECM. Importantly, the ECM itself does not usually require enzymatic removal or disruption prior to cryopreservation; rather, its intact structure functions synergistically with CPAs to protect embedded cells by reducing ice crystal propagation and maintaining local osmotic balance. Recent studies suggest that combining native ECM scaffolds with optimized CPAs cocktails achieves superior post-thaw viability and functionality compared with isolated cell suspensions, underscoring the complementary roles of chemical protection and structural microenvironments in tissue-level cryopreservation (Seo et al., 2021; Whaley et al., 2021). A comparative analysis of various cryopreservation media is listed in Table 1.
Table 1. Comparison of GMP-grade cryopreservation media for MSCs and future improvement opportunities.
2.4 Vitrification cryopreservation
Vitrification cryopreservation employs rapid cooling of highly concentrated cryoprotective agents (CPAs) in liquid nitrogen to achieve a glass-like amorphous solid state, effectively inhibiting ice crystal formation and thereby preserving cellular structural integrity and function (Fahy, 1986; Jomha et al., 2012; Rall and Fahy, 1985; Vajta et al., 1998; Dias et al., 2023). A typical protocol involves two sequential steps: initial equilibration with 20% (v/v) ethylene glycol at room temperature for 5 min, followed by direct immersion into a cryomedium containing 40% (v/v) ethylene glycol, 0.3 mol/L sucrose, 18% (w/v) ficoll and 20% fetal bovine serum in liquid nitrogen. Although this conventional formulation achieves high post-thaw viability, its clinical applicability is severely limited by several factors. The high concentrations of CPAs, particularly ethylene glycol, induce significant cytotoxicity and osmotic stress, compounded by risks associated with residual CPA exposure post-thaw (Han et al., 2023). Furthermore, the inclusion of FBS introduces xenogeneic components, posing immunogenicity risks, potential pathogen transmission, and substantial regulatory hurdles for clinical translation (Lawson et al., 2011). Consequently, research has shifted toward developing safer, xeno-free formulations. For instance, a DMSO-free cocktail comprising ethylene glycol, 1,2-propanediol, sucrose, and polyvinyl alcohol (PVA) as an additive has been validated for vitrification of umbilical cord blood-derived MSCs (UCB-MSCs). Comparative analyses demonstrate that this approach significantly outperforms programmed freezing; PVA supplementation elevated post-thaw viability from 71.2% to 95.4% (p < 0.01), whereas conventional programmed freezing yielded suboptimal survival (<45%) (Wang et al., 2011). Nevertheless, even improved formulations raise concerns regarding osmotic stress, potential cytotoxicity from high concentrations of alternative CPAs like EG or 1,2-propanediol, and the effects of residual additives. Therefore, while avoiding DMSO and serum marks a critical advance toward clinical translatability, further optimization of CPA composition and stepwise loading protocols remains essential to balance cell viability, functionality, and biosafety. Innovative approaches are further addressing these limitations. Techniques such as nanoparticle-enhanced rewarming and hydrogel encapsulation are enhancing vitrification efficiency, consistently achieving >95% cellular viability while mitigating toxicity risks and maintaining functional competence (Pu et al., 2025; Wang et al., 2016). These advances underscore the ongoing evolution of vitrification protocols toward clinically compliant, highly effective cryopreservation systems.
2.5 Chemical strategies to improve cryopreservation
Recent advances in chemical biology have introduced novel strategies to mitigate cryoinjury and enhance cell recovery. First, cell encapsulation within hydrogels or biocompatible polymers has been shown to provide a protective matrix that buffers osmotic fluctuations and minimizes direct contact between cells and ice crystals (Ortiz Silva et al., 2024). Second, intracellular delivery of cryoprotectants has been explored to overcome the limitation of non-permeating CPAs. Approaches such as nanoparticle carriers, membrane transporters, or transient membrane permeabilization can introduce molecules like trehalose or glycerol into the cytosol. This process enhances intracellular protection (Murray et al., 2024; Stewart et al., 2018). Third, caspase inhibitors have demonstrated considerable efficacy in reducing apoptosis triggered by freeze–thaw stress. For example, in umbilical cord–derived MSCs, the broad-spectrum caspase inhibitor Z-VAD-FMK effectively suppressed activation of caspases-3 and -8, inhibited DNA degradation, and reduced cell death post-thaw. Selective inhibitors such as z-IETD-FMK, z-LEHD-FMK, and z-DEVD-FMK specifically attenuate both extrinsic and intrinsic apoptotic pathways, including cleavages such as Bid cleavage, thereby enhancing cell survival (Bissoyi and Pramanik, 2014). Together, these chemical strategies integrate extracellular protection with intracellular signaling modulation, offering a more comprehensive framework to maintain both viability and therapeutic potency of cryopreserved MSCs for clinical applications.
2.6 Advanced cryopreservation processes and equipment
Beyond the medium composition, the freezing process itself is a critical variable. Controlled-rate freezers allow for precise, programmed cooling protocols (e.g., 1 °C/min), which are considered the gold standard for optimizing cell survival by carefully managing the balance between dehydration and intracellular ice formation (Duarte Rojas et al., 2024; Abraham and Goel, 2025). Cells that were cryopreserved using programmed freezing consistently showed higher metabolic activity and better-preserved phenotypic characteristics upon thawing compared to simple passive freezing methods (Mohamed et al., 2024).
To meet the demands of clinical translation, the field has moved towards automated, closed-system manufacturing platforms. Systems like the Quantum® Cell Expansion System and the CliniMACS Prodigy® are designed to perform cell culture, expansion, and even formulation in a sterile, GMP-compliant environment, significantly reducing the risk of contamination and human error (Strecanska et al., 2025). In addition to cell processing, controlled-rate freezers and programmable vitrification devices—such as the Kryo 560-16, Planer KRYO 360, and VIA Freeze® systems—are increasingly integrated into these platforms to standardize the cryopreservation process. These automated platforms enhance the efficiency, scalability, and consistency of MSC production, ensuring that large, uniform batches of high-quality cells can be produced for clinical trials and commercial supply (Strecanska et al., 2025). The integration of cryopreservation steps into these automated workflows is a key enabler for the industrialization of cell therapy.
3 Effects of cryopreservation on the biological characteristics of MSCs
Having established the technological foundations of MSC cryopreservation, we now turn to a critical question: how does this process impact the fundamental biological characteristics that define their therapeutic identity? In 2006, the Mesenchymal Stem Cell Committee of the International Society for Cellular Therapy (ISCT) established and published minimal criteria for defining human MSCs: (1) Morphological adherence to plastic culture surfaces with characteristic spindle-shaped or fibroblast-like morphology; (2) Surface antigen expression profile demonstrating ≥95% positivity for CD105, CD73, and CD90, while exhibiting ≤2% positivity for CD45, CD14/CD11b, CD34, CD19/CD79α, and HLA-DR; (3) Multilineage differentiation potential confirmed through in vitro induction into osteoblasts, chondrocytes, and adipocytes under defined culture conditions (Dominici et al., 2006) (Figure 4). While modern cryopreservation protocols can achieve high post-thaw viability, it is crucial to understand the more subtle impacts on the biological attributes that define MSCs therapeutic potential (Table 2).
Figure 4. Minimal criteria for defining human MSCs.
Table 2. Summary of tissue-origin specific cryopreservation vulnerabilities and their functional implications in MSCs.
3.1 Changes in cell morphology and surface markers
A fundamental requirement is the maintenance of basic cellular characteristics. Most studies report that cryopreserved MSCs, upon thawing and re-culture, retain their characteristic spindle-shaped morphology and exhibit good attachment and proliferation capabilities, similar to their pre-cryopreservation state (Lin et al., 2021). The canonical MSC surface markers (CD73, CD90, CD105) largely maintain their expression levels post-thaw, which serves as a key identity criterion (Wang W. et al., 2022). However, some studies have noted more nuanced changes. For example, one study on rat adipose-derived MSCs (AD-MSCs) found that while the core markers were stable, the expression levels of some functional molecules, such as TGF-β1 and IL-6, were significantly decreased after cryopreservation (Farag et al., 2024). Notably, evidence indicates that cells require a recovery or adaptation phase after thawing, and several studies have shown that without this post-cryopreservation adaptation, the full therapeutic potential of stem cells cannot be restored. Therefore, it is possible that the impairment of functional indicators is alleviated during recovery; however, further investigation is required to confirm this (Linkova et al., 2022). This highlights that stability can vary depending on the specific marker and cell source, with stem cells from dental pulp (hDPSCs) showing high phenotypic stability (Wang W. et al., 2022), while AD-MSCs may exhibit some functional alterations (He et al., 2018).
3.2 Impact on multipotent differentiation potential
The hallmark of MSCs is their ability to differentiate into osteoblasts, chondrocytes, and adipocytes. The cryopreservation process can differentially affect these lineage potentials,and this effect is further complicated by the MSC tissue source. Several studies have reported a general decrease in the proliferation and differentiation capacity of cryopreserved MSCs compared to fresh cells, particularly for bone marrow-derived (BM-MSCs) and adipose-derived (AD-MSCs) sources (Pola-Silva et al., 2021; Cottle et al., 2022; Miroslava et al., 2020; Gao et al., 2020). The sensitivity appears to be lineage-specific. For instance, some cryopreservation protocols have been shown to preserve adipogenic potential while significantly reducing osteogenic potential in AD-MSCs (Ntege et al., 2020), whereas BM-MSCs might exhibit a different pattern of vulnerability. Chondrogenic differentiation also appears variably affected, with certain cryopreservation methods causing moderate decreases in cartilage matrix production, indicating that chondrogenic pathways may be partially susceptible to cryo-injury (Ali et al., 2024; Dicks et al., 2023). These observations suggest that cryopreservation can impair specific lineage signaling pathways, making post-thaw functional assessment essential to ensure therapeutic efficacy. The differentiation into more specialized lineages, such as cardiomyocytes, appears to be particularly sensitive. Cardiomyogenic differentiation is a complex process requiring precise signaling cues, which can be disrupted by the stresses of cryopreservation and thawing (Koung et al., 2023; Pilbauerova et al., 2022). The underlying mechanisms may involve cryo-induced dysfunction in key signaling pathways (e.g., Wnt, TGF-β), increased intracellular oxidative stress, or a decline in the cells’ overall self-renewal capacity, all of which can compromise their ability to respond to differentiation stimuli (Cottle et al., 2022; Kanazawa et al., 2022; El Assaad et al., 2024). This is a critical consideration for cardiac regeneration therapies, where ensuring the preservation of this specific potential is paramount.
3.3 Impact on immunomodulatory capacity
The paracrine-mediated immunomodulatory function is arguably the most important therapeutic mechanism of MSCs. Cryopreservation can impact this function by altering the secretome and the cells’ responsiveness to inflammatory signals (Table 3).
Table 3. Comparative analysis of the immunomodulatory function of fresh versus cryopreserved MSCs.
The impact of cryopreservation on the secretory profile of MSCs is both complex and pivotal. MSC expression of indoleamine 2,3-dioxygenase (IDO), a key molecule in suppressing inflammation, appears to vary with cryopreservation method. Some studies report an initial increase in IDO expression and activity, which might be an immediate stress response. This upregulation may decrease over longer culture periods (Tan et al., 2019). In contrast, other investigations demonstrate a significant reduction in IDO-mediated T-cell suppression, highlighting protocol-dependent variability (François et al., 2012). The synthesis of prostaglandin E2 (PGE2), another critical mediator, is highly dependent on continuous enzymatic activity. Cryoinjury can disrupt this biosynthetic pathway, frequently leading to a marked decrease in PGE2 secretion (Shi et al., 2018). Similarly, the production of key immunoregulatory molecules, such as transforming growth factor-beta (TGF-β) and human leukocyte antigen-G (HLA-G), can be compromised (Hoogduijn et al., 2016).
Beyond changes in TGF-β1 and IL-6, recent studies have directly assessed the effects of cryopreservation on MSCs’ immunomodulatory capacity. Functional assays measuring the suppression of T-cell proliferation, considered a gold-standard assessment, consistently indicate that cryopreserved MSCs generally retain their immunosuppressive capacity, albeit often with a quantifiable reduction in potency compared to their fresh counterparts (Vymetalova et al., 2020), showing impaired suppression of T-cell proliferation, reduced induction of regulatory T cells, and altered cytokine-mediated crosstalk with immune cells (François et al., 2012; Pollock et al., 2015). A primary immunomodulatory mechanism of MSCs is the induction of regulatory T-cells (Tregs). While thawed MSCs can still promote Treg expansion, this capability is frequently attenuated (Bárcia et al., 2017). Furthermore, the capacity of cryopreserved MSCs to modulate the balance between pro-inflammatory and anti-inflammatory T-helper cells is preserved but suboptimal. They typically maintain the ability to suppress the polarization of pro-inflammatory Th1 and Th17 cells; however, the efficiency of this suppression may be less pronounced than in fresh MSCs (Le Nail et al., 2018). Cryopreservation may compromise the immunomodulatory function of MSCs, arguably their most important therapeutic role. This is reflected by reduced production of key signaling molecules such as TGF-β1 and IL-6 after thawing (Farag et al., 2024), which could weaken their overall immunosuppressive effect (Farag et al., 2024). For example, diminished post-thaw MSCs activity has been linked to reduced inhibition of effector T-cell responses and altered modulation of macrophage polarization, highlighting a direct compromise of their immunoregulatory potential (Duffy et al., 2011; Li et al., 2023). Studies show that a reduction in TGF-β1 might impair their ability to control T-cell responses, while changes in IL-6 could disrupt their balancing act between pro- and anti-inflammatory signals (Martín-López et al., 2023; Gil-Chinchilla et al., 2024). On the other hand, some work has shown that engineering MSCs to produce more IL-6 actually boosts their therapeutic effect (Huang et al., 2022). This suggests that finding ways to maintain or even enhance key factor expression post-thaw is a promising path forward. Protecting this specific function during the freeze-thaw cycle remains a major research priority.
4 How cryopreserved MSCs perform in vivo
While in vitro assays are essential, the ultimate test of cryopreserved MSCs occurs in living organisms. The following section synthesizes their performance across diverse preclinical disease models and clinical trials, with a particular focus on the comparison between fresh and cryopreserved cells.
A pivotal consideration, often highlighted by comparative studies, is the functional differences between fresh and cryopreserved MSCs. Interspecies differences (e.g., between rodent and human MSCs) are important. However, directly comparing fresh and cryopreserved aliquots from the same donor source more accurately reveals the true impact of the freeze-thaw process on therapeutic potential. In terms of efficacy, numerous reports indicate that cryopreserved MSCs may exhibit impaired in vivo performance compared to their fresh counterparts, including reduced engraftment efficiency, shorter persistence at injury sites, and attenuated therapeutic effects in pre-clinical models of inflammatory diseases (Tan et al., 2019; Le Nail et al., 2018). This likely results from the combined effects of cryoinjury on cell viability, homing receptor integrity, and paracrine factor secretion, as discussed in Section 3.3. Regarding safety, the cryopreservation process itself introduces additional variables. The necessity for penetrating CPAs, most notably DMSO, raises concerns about infusion-related toxicity and potential adverse effects on patients, which are not a factor for freshly transplanted cells (Windrum et al., 2005). Furthermore, the risk of phenotypic drift or the selection of a specific subpopulation during freeze-thaw could, in theory, alter the long-term safety profile, though evidence for this is still emerging. Most critically, the issue of potency—a quantitative measure of a product’s biological activity—is central. Regulatory guidelines emphasize the need for potency assays, yet defining a universal assay for thawed MSCs remains challenging. A cryopreserved batch may meet release criteria based on viability but may possess significantly reduced immunosuppressive or pro-regenerative capacity compared to the pre-freeze culture.
4.1 Cardiovascular and respiratory diseases
In the context of cardiac repair using MSCs, clinical results have shown mixed outcomes, reflecting variable efficacy across studies. A randomized controlled trial enrolled 30 patients with chronic ischemic cardiomyopathy who received transendocardial injections of either autologous or allogeneic cryopreserved bone marrow-derived MSCs and reported their outcomes at 6-month follow-up. At 6-month follow-up, left ventricular ejection fraction (LVEF) increased by 4.9% (autologous) and 5.4% (allogeneic), with concomitant significant reduction in myocardial infarct size. No clinically significant alloimmune reactions were observed in either cohort (Ramireddy et al., 2017). A meta-analysis of clinical trials found that cryopreserved umbilical cord MSCs could produce a significant, though often short-lived, improvement in LVEF, especially if post-thaw viability was high (Safwan et al., 2025). The main challenge lies in sustaining the improvement in cardiac function over the long term. At a mechanistic level, these cells have been shown to encourage the expression of cardiac proteins like Troponin T, helping to improve heart muscle function (Clavellina et al., 2023; Ali et al., 2025). However, these outcomes cannot be unequivocally attributed to the act of cryopreservation or the resultant post-thaw cell state, as variability in therapeutic efficacy may also be influenced by factors such as MSCs source, patient selection criteria, and clinical protocol design. More conclusive evidence regarding the impact of cryopreservation on therapeutic efficacy would require direct comparative studies between fresh and cryopreserved MSCs, which would better clarify whether reduced long-term benefit arises specifically from cryoinjury or from other confounding variables (Fan et al., 2020).
4.2 Respiratory diseases
Cryopreserved MSCs retain significant therapeutic potential in pulmonary injury models, including pulmonary fibrosis and acute lung injury. Researchers established a cryobank containing 124 cryopreserved aliquots of lung-derived MSCs (L-MSCs) from Ovis aries fetuses. In vitro differentiation assays confirmed L-MSCs’ trilineage mesodermal differentiation capacity. Crucially, post-thaw L-MSCs maintained anti-fibrotic functionality after undergoing hepatic differentiation. This was evidenced by sustained secretion of urea and albumin, indicating their regenerative potential for pulmonary tissue repair and fibrotic mitigation (Dominguez-Pinilla et al., 2025). The cells have shown clearer promise in acute lung injury models like Ventilator-induced lung injury (VILI). Cryopreserved umbilical cord-derived mesenchymal stem cells (UC-MSCs) exert therapeutic effects through paracrine secretion of multifactorial cytokines and bioactive mediators, effectively mitigating inflammatory responses and facilitating pulmonary tissue regeneration (Dominguez-Pinilla et al., 2025; Mallis et al., 2022). After thawing, they still produce key immunomodulatory factors like IL-6 and IL-10 and can steer the lung’s immune environment toward an anti-inflammatory M2 macrophage phenotype (Mallis et al., 2022; Budgude et al., 2021). This confirms that their essential anti-inflammatory and paracrine functions were well preserved following cryopreservation, supporting their use in treating acute pulmonary conditions.
4.3 Kidney diseases
For chronic conditions like diabetic nephropathy (DN), cryopreserved MSCs are used to fight ongoing inflammation and fibrosis. In mouse models of DN, MSCs treatment lowered inflammatory cytokine levels in the kidneys and reduced scarring (Zhu et al., 2025; Wu et al., 2025). This effect is tied to their ability to home to the damaged kidney and shift the local immune cells toward an anti-inflammatory state (Chen et al., 2025; Bejugam et al., 2025). Researchers are also exploring ways to boost their efficacy, for example, by pre-treating the cells with GLP-1 receptor agonists before freezing, which enhances their therapeutic effects in DN models (Liu et al., 2024; Habib et al., 2021).
4.4 Graft-versus-host disease
Graft-versus-host disease (GVHD) is a serious complication following allogeneic hematopoietic stem cell transplantation, triggered by donor immune cells attacking recipient tissues. Patients with steroid-refractory acute GVHD (SR-aGVHD) have a particularly poor prognosis (Moreno and Cid, 2019). In 2020, the U.S. FDA approved the first mesenchymal stromal cell (MSC)-based therapy, Ryoncil® (remestemcel-L), for the treatment of pediatric SR-aGVHD, representing a major breakthrough in cellular therapy for this condition (ICHGCP, 2025). Critically, this “off-the-shelf” therapy is administered via intravenous infusion of MSCs immediately after thawing from cryopreserved stocks, underscoring the pivotal role of an effective cryopreservation protocol in enabling its practical clinical application. This therapy acts through multi-dimensional immunomodulatory mechanisms, including the secretion of anti-inflammatory factors, suppression of excessive T-cell activation, and promotion of regulatory T-cell generation, thereby reestablishing immune tolerance. Pivotal Phase III clinical trials demonstrated an overall response rate of 69% among treated patients, with a complete response rate of 47%, significantly improving survival rates. This approval not only provides the first “off-the-shelf” cellular therapeutic option for SR-aGVHD and advances the industrialization of cell therapies, but also lays the foundation for broader clinical applications of MSCs in other immune-related diseases (Kurtzberg et al., 2020). However, challenges such as heterogeneity in treatment response, standardization of manufacturing, and long-term safety require further investigation.
4.5 Other diseases
The applications extend beyond these common diseases. In veterinary medicine, they are used to treat systemic inflammation in horses (Uberti et al., 2022; MacDonald and Barrett, 2019). A particularly striking example comes from dentistry, where cryopreserved allogeneic bone marrow MSCs were used to regenerate pulp-like tissue in the necrotic teeth of children, restoring sensitivity and healing lesions (Gomez-Sosa et al., 2024). This case highlights the robust regenerative power these cells can retain even after cryopreservation.
5 From lab to clinic: manufacturing and quality control
The translation of cryopreserved MSCs from a laboratory reagent to a reliable clinical product necessitates rigorous standardization and quality control. This final section addresses the manufacturing, regulatory, and logistical frameworks that underpin their successful clinical application.
5.1 GMP regulations and oversight
GMP regulations are non-negotiable; they are designed to ensure every batch of a cell therapy product is safe, pure, potent, and consistent (Bio-Techne, 2025). This means having tight control over the entire process, from screening donors to the final storage of the cryopreserved cells (Li et al., 2024). Key quality metrics for a cryopreserved MSC product are cell viability, purity, identity, and functional potency. Although a post-thaw viability above 70%–80% is a common release criterion, it alone does not guarantee product quality (Rusconi et al., 2024). Purity tests must confirm the absence of unwanted cells, and identity tests ensure the product actually contains MSCs. Importantly, studies have shown that MSCs produced under these strict GMP conditions can be just as functional as their research-grade counterparts, proving that high-quality cells can be made at scale (Mendt et al., 2021).
5.2 Quality assessment of cryopreserved MSCs in preclinical and clinical trials
A central challenge in the field is defining and measuring attributes that correlate with clinical efficacy. As discussed, post-thaw viability is a necessary but insufficient metric (Putra et al., 2021). This is underpinned by the complex relationship between pre-freeze and post-thaw cell status. A high pre-freeze viability (>90%) is a prerequisite for a successful outcome; however, the freeze-thaw process itself can induce a significant viability drop of 10%–30% due to inherent cryoinjury, meaning that even an optimal starting point does not guarantee a high-quality final product (El Kadi et al., 2025). The current focus is on potency assays that measure the product’s mechanism of action for specific indications, such as immunomodulatory capacity or pro-angiogenic activity (Dave et al., 2022). A systematic review found that while there were often no significant differences in vitro potency between cryopreserved and fresh MSCs, the in vivo efficacy could vary, highlighting a gap in our understanding (Dave et al., 2022). Furthermore, batch-to-batch consistency is paramount. Variations in cell source, culture conditions, and cryopreservation protocols can impact cell function and therapeutic outcomes (Wiese et al., 2022). For example, in cardiovascular trials, MSCs efficacy has been shown to correlate with patient-specific factors like baseline LVEF and inflammatory status, suggesting that a successful therapy will require both a consistent product and patient stratification (Patel et al., 2025). Establishing a clear correlation between in vitro quality attributes and clinical outcomes remains a key goal in the field.
5.3 Cryopreserved cell preparations: storage, transportation, thawing and recovery, and handling
Ultra-low temperature cryopreservation is used for storing cryopreserved preparations. Liquid nitrogen (−196 °C) or mechanical refrigeration systems (−150 °C to −80 °C) maintain cell viability for extended periods (Harris, 2014). The logistics involved in transporting frozen products, commonly referred to as the “vial-to-vein” process, is of crucial importance. This necessitates a robust cold chain management system. Prior to transportation, stem cells from different sources require customized formulations of cryoprotectants to prevent ice crystal damage caused by temperature fluctuations during transportation (Harris, 2014). During transportation, the products must remain at ultra-low temperatures (for example, in dry ice or liquid nitrogen vapor shippers) until they reach the clinical site (Weng, 2023; Wang et al., 2023; Heydarzadeh et al., 2022). Furthermore, real-time temperature monitoring and the utilization of shock-absorbing packaging can be employed (Harris, 2014).
At the bedside, a standardized operating procedure (SOP) for thawing is crucial. Moving beyond the conventional 37 °C water bath, which poses contamination risks and variability, standardized and qualified thawing systems are now considered best practice. Automated, closed-system dry-thaw devices are increasingly adopted in GMP settings. These devices precisely control the warming rate at ∼100 °C/min, ensuring a consistent and rapid thaw that maximizes cell recovery and minimizes the toxic effects of DMSO(15). This rapid thawing minimizes ice recrystallization damage (Weng, 2023). Post-thaw, the cell product must be administered within a short, validated timeframe to ensure viability and function are not compromised (Harris, 2014). Moreover, precise alignment between thawing operations and clinical treatment plans should be achieved, and the entire management process from storage to clinical application must be subject to strict control and documentation, so as to ensure product quality and patient safety (Harris, 2014; Kumar et al., 2024).
Finally, the latest frontier in ‘vial-to-vein’ logistics focuses on ensuring product quality at the moment of infusion. While post-thaw viability via trypan blue remains common, it is a poor predictor of function. Emerging technologies are enabling more sophisticated, rapid assessments at the point-of-care. Portable flow cytometers can now be deployed to provide a rapid immunophenotype confirmation within minutes of thawing (Gao et al., 2021). Furthermore, in-line viability sensors based on dielectric spectroscopy are under development for real-time, label-free monitoring of cell concentration and vitality during the thaw-and-wash process (Rosell-Valle et al., 2021).
6 Discussion and future perspectives
The significance of cryopreservation technology in the clinical translation of MSCs is increasingly prominent. Current research indicates that cryopreserved MSCs maintain basic biological characteristics and multi-directional differentiation potential; however, they still exhibit some functional decline, particularly in immunomodulation (Medrano-Trochez et al., 2021; Kamprom et al., 2024). Although clinical trials have demonstrated the safety and therapeutic potential of cryopreserved MSCs, inconsistent results and limited data have hindered their widespread application.
To develop MSCs as standardized, “off-the-shelf” therapeutics, researchers must overcome two main challenges: preserving post-thaw functionality and managing biological heterogeneity. The advancements synthesized in this review illuminate a clear, integrated path forward. A primary obstacle, the cytotoxicity of DMSO, is being addressed by two compelling strategies. Importantly, it should be noted that low concentrations of DMSO (≤5–10%) are generally considered clinically acceptable when carefully removed prior to administration, thereby mitigating acute toxicity risks (Santos et al., 2024). In parallel, another translational barrier arises from the xenogeneic components of FBS, which may introduce risks of immunogenicity and pathogen transmission (Martin et al., 2022; Dessels et al., 2016). To address this, serum-free and chemically defined cryomedia have been developed, while HPL has emerged as a clinically safer alternative to FBS, providing comparable growth factor support without xenogeneic concerns. These approaches collectively strengthen the clinical feasibility of MSCs cryopreservation by reducing both solvent-related cytotoxicity and serum-derived safety risks.
Three main approaches address these challenges: 1) rational design of synthetic, biocompatible CPAs, such as trehalose-based polyethers with IRI activity (El Kadi et al., 2025) and zwitterionic polymers that manage osmotic stress (Dave et al., 2022); 2) preserving MSCs within their native tissue “niche,” leveraging the natural extracellular matrix as a superior cryoprotective scaffold (Yang et al., 2016); and 3) upgrading equipment and technologies to enhance functional recovery by optimizing vitrification methods and developing advanced cryopreservation processes and devices. These innovations are paving the way for safer, more effective cryopreservation protocols (Strecanska et al., 2025; Wang et al., 2011).
To overcome the limitations of traditional, non-specific identity markers, the field is adopting robust core transcriptomic signatures. These signatures provide a definitive molecular fingerprint for MSCs and remain stable across manufacturing pressures (Wiese and Braid, 2020). Beyond identity, assessing therapeutic potency is shifting from simple viability counts to a matrix of validated, indication-specific functional assays. Rigorously validated methods like the mixed lymphocyte reaction (MLR) for quantifying immunomodulation (Nicotra et al., 2020) and advanced microphysiological systems (MPS) for measuring vasculogenic capacity exemplify this crucial trend (Lam et al., 2022). These assays provide the necessary tools to ensure batch-to-batch consistency and characterize products for their intended clinical use.
In synthesis, the path towards effective, off-the-shelf MSCs therapies requires an integrated quality control framework that incorporates: 1) advanced cryopreservation techniques using novel CPAs or niche-preserving storage; 2) robust molecular signatures for unambiguous cell identity; and 3) a panel of validated, indication-specific potency assays. However, despite clear progress, significant challenges remain. The foremost challenge is establishing a definitive correlation between in vitro potency measurements and in vivo clinical efficacy. This correlation remains the “holy grail” for the field. To bridge this gap, large-scale clinical trials linking detailed product characterization to patient outcomes are urgently needed. Furthermore, standardizing these advanced assays across different laboratories is essential. The development of cost-effective, rapid surrogate markers is also critical for practical implementation in a GMP manufacturing setting. Ultimately, the successful integration of these innovations in cryobiology and bio-analytics will be the key to delivering MSCs therapies with the predictable, consistent, and potent therapeutic benefits that patients require.
Author contributions
YW: Conceptualization, Writing – review and editing, Writing – original draft. KY: Writing – original draft, Writing – review and editing, Data curation, Investigation, Visualization. SY: Data curation, Investigation, Visualization, Writing – original draft, Writing – review and editing. FH: Data curation, Investigation, Visualization, Writing – original draft, Writing – review and editing. CY: Writing – review and editing, Conceptualization, Supervision. WT: Conceptualization, Funding acquisition, Supervision, Writing – review and editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the National Key Research and Development Program of China (2022YFA1104400) and the National Natural Science Foundation of China (U21A20369).
Conflict of interest
Author CY was employed by Sichuan Tianfu Cell Quality Detection and Evaluation Center Co., Ltd.
Author CY was employed by Chengdu Shiliankangjian Biotechnology Co., Ltd.
The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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References
Abraham, M., and Goel, S. (2024). Comprehensive characterisation and cryopreservation optimisation of buffalo (Bubalus bubalis) adipose tissue-derived mesenchymal stem cells. Cryobiology 115, 104896. doi:10.1016/j.cryobiol.2024.104896
Abraham, M., and Goel, S. (2025). Species-specific optimisation of cryopreservation media for goat and buffalo adipose-derived mesenchymal stem cells. Cryobiology 118, 105211. doi:10.1016/j.cryobiol.2025.105211
AlHindi, M., and Philip, M. R. (2021). Osteogenic differentiation potential and quantification of fresh and cryopreserved dental follicular stem cells-an in vitro analysis. J. Stem Cells Regen. Med. 17 (1), 28–34. doi:10.46582/jsrm.1701004
Ali, E. A. M., Smaida, R., Meyer, M., Ou, W., Li, Z., Han, Z., et al. (2024). iPSCs chondrogenic differentiation for personalized regenerative medicine: a literature review. Stem Cell Res. Ther. 15 (1), 185. doi:10.1186/s13287-024-03794-1
Ali, S. A., Mahmood, Z., Mubarak, Z., Asad, M., Sarfraz Chaudhri, M. T., Bilal, L., et al. (2025). Assessing the potential benefits of stem cell therapy in cardiac regeneration for patients with ischemic heart disease. Cureus 17 (1), e76770. doi:10.7759/cureus.76770
Andreoli, V., Berni, P., Conti, V., Ramoni, R., Basini, G., and Grolli, S. (2024). Mesenchymal stromal cells derived from canine adipose tissue: evaluation of the effect of different shipping vehicles used for clinical administration. Int. J. Mol. Sci. 25 (6), 3426. doi:10.3390/ijms25063426
Bárcia, R. N., Santos, J. M., Teixeira, M., Filipe, M., Pereira, A. R. S., Ministro, A., et al. (2017). Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing. Cytotherapy 19 (3), 360–370. doi:10.1016/j.jcyt.2016.11.008
Bejugam, D., Bu, S., Nguyen, A. N., Yaltaghian, M., and Smolen, K. K. (2025). New frontiers in type I diabetes treatment: the impact of mesenchymal stromal cells on long-term complications. Front. Clin. Diabetes Healthc. 6, 1586061. doi:10.3389/fcdhc.2025.1586061
Bio-Techne (2025). GMP raw materials: meeting the demand for cell therapy. Bio-Techne. Available online at: https://www.bio-techne.com/resources/blogs/gmp-raw-materials-meeting-the-cell-therapy-demand. (Accessed September 7, 2025).
Bissoyi, A., and Pramanik, K. (2014). Role of the apoptosis pathway in cryopreservation-induced cell death in mesenchymal stem cells derived from umbilical cord blood. Biopreservation Biobanking 12 (4), 246–254. doi:10.1089/bio.2014.0005
Bojic, S., Murray, A., Bentley, B. L., Spindler, R., Pawlik, P., Cordeiro, J. L., et al. (2021). Winter is coming: the future of cryopreservation. BMC Biol. 19 (1), 56. doi:10.1186/s12915-021-00976-8
Budgude, P., Kale, V., and Vaidya, A. (2021). Cryopreservation of mesenchymal stromal cell-derived extracellular vesicles using trehalose maintains their ability to expand hematopoietic stem cells in vitro. Cryobiology 98, 152–163. doi:10.1016/j.cryobiol.2020.11.009
Bueno, C., Montes, R., and Menendez, P. (2010). The ROCK inhibitor Y-27632 negatively affects the expansion/survival of both fresh and cryopreserved cord blood-derived CD34+ hematopoietic progenitor cells: Y-27632 negatively affects the expansion/survival of CD34+HSPCs. Stem Cell Rev. Rep. 6 (2), 215–223. doi:10.1007/s12015-010-9118-5
Capicciotti, C. J., Kurach, J. D. R., Turner, T. R., Mancini, R. S., Acker, J. P., and Ben, R. N. (2015). Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations. Sci. Rep. 5, 9692. doi:10.1038/srep09692
Caplan, A. I. (2017). Mesenchymal stem cells: time to change the name. Stem Cells Transl. Med. 6 (6), 1445–1451. doi:10.1002/sctm.17-0051
Chang, T., and Zhao, G. (2021). Ice inhibition for cryopreservation: materials, strategies, and challenges. Adv. Sci. Weinh Baden-Wurtt Ger. 8 (6), 2002425. doi:10.1002/advs.202002425
Chen, C., Xu, B., Li, W., Chen, J., Yang, M., Gao, L., et al. (2025). New perspectives on the treatment of diabetic nephropathy: challenges and prospects of mesenchymal stem cell therapy. Eur. J. Pharmacol. 998, 177543. doi:10.1016/j.ejphar.2025.177543
Chinnadurai, R., Copland, I. B., Garcia, M. A., Petersen, C. T., Lewis, C. N., Waller, E. K., et al. (2016). Cryopreserved mesenchymal stromal cells are susceptible to T-Cell mediated apoptosis which is partly rescued by IFNγ licensing. Stem Cells Dayt Ohio. 34 (9), 2429–2442. doi:10.1002/stem.2415
Cimino, M., Gonçalves, R. M., Barrias, C. C., and Martins, M. C. L. (2017). Xeno-free strategies for safe human mesenchymal stem/Stromal cell expansion: supplements and coatings. Stem Cells Int. 2017, 6597815. doi:10.1155/2017/6597815
Clavellina, D., Balkan, W., and Hare, J. M. (2023). Stem cell therapy for acute myocardial infarction: Mesenchymal stem Cells and induced pluripotent stem cells. Expert Opin. Biol. Ther. 23 (10), 951–967. doi:10.1080/14712598.2023.2245329
Cottle, C., Porter, A. P., Lipat, A., Turner-Lyles, C., Nguyen, J., Moll, G., et al. (2022). Impact of cryopreservation and freeze-thawing on therapeutic properties of mesenchymal stromal/stem cells and other common cellular therapeutics. Curr. Stem Cell Rep. 8 (2), 72–92. doi:10.1007/s40778-022-00212-1
Crow, D. (2019). Could iPSCs enable “Off-the-Shelf” cell therapy? Cell 177 (7), 1667–1669. doi:10.1016/j.cell.2019.05.043
Dave, C., Mei, S. H. J., McRae, A., Hum, C., Sullivan, K. J., Champagne, J., et al. (2022). Comparison of freshly cultured versus cryopreserved mesenchymal stem cells in animal models of inflammation: a pre-clinical systematic review. eLife 11, e75053. doi:10.7554/eLife.75053
de Camargo, G. C., da Cruz Landim-Alvarenga, F., Maciel, A. P., Dos Santos, D. B., de Paula Freitas Dell’Aqua, C., E Alvarenga, M. L., et al. (2025). Polyvinyl alcohol can replace the fetal bovine serum during cryopreservation of canine adipose mesenchymal stromal cells. Vitro Cell Dev. Biol. Anim. 61 (4), 369–373. doi:10.1007/s11626-025-01046-x
de Witte, S. F. H., Lambert, E. E., Merino, A., Strini, T., Douben, HJCW, O’Flynn, L., et al. (2017). Aging of bone marrow- and umbilical cord-derived mesenchymal stromal cells during expansion. Cytotherapy 19 (7), 798–807. doi:10.1016/j.jcyt.2017.03.071
Dessels, C., Potgieter, M., and Pepper, M. S. (2016). Making the switch: alternatives to fetal bovine serum for adipose-derived stromal cell expansion. Front. Cell Dev. Biol. 4, 115. doi:10.3389/fcell.2016.00115
Dias, C., Commin, L., Bonnefont-Rebeix, C., Buff, S., Bruyère, P., and Trombotto, S. (2023). Comparative evaluation of the in vitro cytotoxicity of a series of chitosans and chitooligosaccharides water-soluble at physiological pH. Polymers 15 (18), 3679. doi:10.3390/polym15183679
Dicks, A. R., Steward, N., Guilak, F., and Wu, C. L. (2023). Chondrogenic differentiation of human-induced pluripotent stem cells. Methods Mol. Biol. Clifton NJ. 2598, 87–114. doi:10.1007/978-1-0716-2839-3_8
Ding, D. C., Shyu, W. C., and Lin, S. Z. (2011). Mesenchymal stem cells. Cell Transpl. 20 (1), 5–14. doi:10.3727/096368910X
Dominguez-Pinilla, N., González-Granado, L. I., Gonzaga, A., López Diaz, M., Castellano Yáñez, C., Aymerich, C., et al. (2025). Consecutive intrabronchial administration of Wharton’s jelly-derived mesenchymal stromal cells in ECMO-supported pediatric patients with end-stage interstitial lung disease: a safety and feasibility study (CIBA method). Stem Cell Res. Ther. 16 (1), 164. doi:10.1186/s13287-025-04289-3
Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., et al. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 8 (4), 315–317. doi:10.1080/14653240600855905
Duarte Rojas, J. M., Restrepo Múnera, L. M., and Estrada, M. S. (2024). Comparison between platelet lysate, platelet lysate serum, and fetal bovine serum as supplements for cell culture, expansion, and cryopreservation. Biomedicines 12 (1), 140. doi:10.3390/biomedicines12010140
Duffy, M. M., Ritter, T., Ceredig, R., and Griffin, M. D. (2011). Mesenchymal stem cell effects on T-cell effector pathways. Stem Cell Res. Ther. 2 (4), 34. doi:10.1186/scrt75
El Assaad, N., Chebly, A., Salame, R., Achkar, R., Bou Atme, N., Akouch, K., et al. (2024). Anti-aging based on stem cell therapy: a scoping review. World J. Exp. Med. 14 (3), 97233. doi:10.5493/wjem.v14.i3.97233
El Kadi, K., Murad, S., and Janajreh, I. (2025). Ice crystallization kinetics in supercooled droplets from a molecular perspective. J. Colloid Interface Sci. 703 (Pt 2), 139192. doi:10.1016/j.jcis.2025.139192
Fahy, G. M. (1986). Vitrification: a new approach to organ cryopreservation. Prog. Clin. Biol. Res. 224, 305–335.
Fan, X. L., Zhang, Y., Li, X., and Fu, Q. L. (2020). Mechanisms underlying the protective effects of mesenchymal stem cell-based therapy. Cell Mol. Life Sci. CMLS 77 (14), 2771–2794. doi:10.1007/s00018-020-03454-6
Farag, A., Ngeun, S. K., Kaneda, M., Aboubakr, M., Elhaieg, A., Hendawy, H., et al. (2024). Exploring the potential effects of cryopreservation on the biological characteristics and cardiomyogenic differentiation of rat adipose-derived mesenchymal stem cells. Int. J. Mol. Sci. 25 (18), 9908. doi:10.3390/ijms25189908
François, M., Copland, I. B., Yuan, S., Romieu-Mourez, R., Waller, E. K., and Galipeau, J. (2012). Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing. Cytotherapy 14 (2), 147–152. doi:10.3109/14653249.2011.623691
Freimark, D., Sehl, C., Weber, C., Hudel, K., Czermak, P., Hofmann, N., et al. (2011). Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells. Cryobiology 63 (2), 67–75. doi:10.1016/j.cryobiol.2011.05.002
Galipeau, J. (2013). The mesenchymal stromal cells dilemma--does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy 15 (1), 2–8. doi:10.1016/j.jcyt.2012.10.002
Gao, S., Ogawa, M., Takami, A., Takeshita, K., and Kato, H. (2020). Practical and safe method of long-term cryopreservation for clinical application of human adipose-derived mesenchymal stem cells without a programmable freezer or serum. Cryo Lett. 41 (6), 337–343.
Gao, L., Zhou, Q., Zhang, Y., Sun, S., Lv, L., Ma, P., et al. (2021). Dimethyl sulfoxide-free cryopreservation of human umbilical cord mesenchymal stem cells based on zwitterionic betaine and electroporation. Int. J. Mol. Sci. 22 (14), 7445. doi:10.3390/ijms22147445
Gil-Chinchilla, J. I., Bueno, C., Martínez, C. M., Ferrández-Múrtula, A., García-Hernández, A. M., Blanquer, M., et al. (2024). Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics. Front. Immunol. 15, 1385691. doi:10.3389/fimmu.2024.1385691
Gomez-Sosa, J. F., Cardier, J. E., Wittig, O., Díaz-Solano, D., Lara, E., Duque, K., et al. (2024). Allogeneic bone marrow mesenchymal stromal cell transplantation induces dentin pulp complex-like formation in immature teeth with pulp necrosis and apical periodontitis. J. Endod. 50 (4), 483–492. doi:10.1016/j.joen.2024.01.002
Guiotto, M., Raffoul, W., Hart, A. M., Riehle, M. O., and di Summa, P. G. (2020). Human platelet lysate to substitute fetal bovine serum in hMSC expansion for translational applications: a systematic review. J. Transl. Med. 18 (1), 351. doi:10.1186/s12967-020-02489-4
Habib, H. A., Heeba, G. H., and Khalifa, M. M. A. (2021). Effect of combined therapy of mesenchymal stem cells with GLP-1 receptor agonist, exenatide, on early-onset nephropathy induced in diabetic rats. Eur. J. Pharmacol. 892, 173721. doi:10.1016/j.ejphar.2020.173721
Han, Z., Rao, J. S., Ramesh, S., Hergesell, J., Namsrai, B. E., Etheridge, M. L., et al. (2023). Model-guided design and optimization of CPA perfusion protocols for whole organ cryopreservation. Ann. Biomed. Eng. 51 (10), 2216–2228. doi:10.1007/s10439-023-03255-5
Hansen, S. B., Højgaard, L. D., Kastrup, J., Ekblond, A., Follin, B., and Juhl, M. (2022). Optimizing an immunomodulatory potency assay for mesenchymal stromal cell. Front. Immunol. 13, 1085312. doi:10.3389/fimmu.2022.1085312
Harris, D. T. (2014). Stem cell banking for regenerative and personalized medicine. Biomedicines 2 (1), 50–79. doi:10.3390/biomedicines2010050
He, Q., Ye, Z., Zhou, Y., and Tan, W. S. (2018). Comparative study of mesenchymal stem cells from rat bone marrow and adipose tissue. Turk J. Biol. Turk Biyol. Derg. 42, 477–489. doi:10.3906/biy-1802-52
Heng, B. C. (2009). Effect of rho-associated kinase (ROCK) inhibitor Y-27632 on the post-thaw viability of cryopreserved human bone marrow-derived mesenchymal stem cells. Tissue Cell 41 (5), 376–380. doi:10.1016/j.tice.2009.01.004
Heydarzadeh, S., Kheradmand Kia, S., Boroomand, S., and Hedayati, M. (2022). Recent developments in cell shipping methods. Biotechnol. Bioeng. 119 (11), 2985–3006. doi:10.1002/bit.28197
Hoogduijn, M. J., de Witte, S. F. H., Luk, F., van den Hout-van Vroonhoven, MCGN, Ignatowicz, L., Catar, R., et al. (2016). Effects of freeze-thawing and intravenous infusion on mesenchymal stromal cell gene expression. Stem Cells Dev. 25 (8), 586–597. doi:10.1089/scd.2015.0329
Huang, P., Zhang, C., Delawary, M., Korchak, J. A., Suda, K., and Zubair, A. C. (2022). Development and evaluation of IL-6 overexpressing mesenchymal stem cells (MSCs). J. Tissue Eng. Regen. Med. 16 (3), 244–253. doi:10.1002/term.3274
ICHGCP (2025). First cell-based gene therapy for adult patients with relased or refractory MCL. Available online at: https://ichgcp.net/zh/news/fda-approves-first-cell-based-gene-therapy-for-adult-patients-with-relapsed-or-refractory-mcl. (Accessed December 16, 2025).
Jang, T. H., Park, S. C., Yang, J. H., Kim, J. Y., Seok, J. H., Park, U. S., et al. (2017). Cryopreservation and its clinical applications. Integr. Med. Res. 6 (1), 12–18. doi:10.1016/j.imr.2016.12.001
Jomha, N. M., Elliott, J. A. W., Law, G. K., Maghdoori, B., Forbes, J. F., Abazari, A., et al. (2012). Vitrification of intact human articular cartilage. Biomaterials 33 (26), 6061–6068. doi:10.1016/j.biomaterials.2012.05.007
Kamprom, W., Tangporncharoen, R., Vongthaiwan, N., Tragoonlugkana, P., Phetfong, J., Pruksapong, C., et al. (2024). Enhanced potent immunosuppression of intracellular adipose tissue-derived stem cell extract by priming with three-dimensional spheroid formation. Sci. Rep. 14 (1), 9084. doi:10.1038/s41598-024-59910-x
Kanazawa, S., Okada, H., Riu, D., Mabuchi, Y., Akazawa, C., Iwata, J., et al. (2022). Hematopoietic-mesenchymal signals regulate the properties of mesenchymal stem cells. Int. J. Mol. Sci. 23 (15), 8238. doi:10.3390/ijms23158238
Kaushal, R., Jahan, S., McGregor, C., and Pineault, N. (2022). Dimethyl sulfoxide-free cryopreservation solutions for hematopoietic stem cell grafts. Cytotherapy 24 (3), 272–281. doi:10.1016/j.jcyt.2021.09.002
Koung, N. S., Shimizu, M., and Kaneda, M. (2023). Characterization of rabbit mesenchymal stem/Stromal cells after cryopreservation. Biology 12 (10), 1312. doi:10.3390/biology12101312
Kumar, A., Ramesh, S., Walther-Jallow, L., Goos, A., Kumar, V., Ekblad, Å., et al. (2024). Successful transport across continents of GMP-manufactured and cryopreserved culture-expanded human fetal liver-derived mesenchymal stem cells for use in a clinical trial. Regen. Ther. 26, 324–333. doi:10.1016/j.reth.2024.06.012
Kurtzberg, J., Abdel-Azim, H., Carpenter, P., Chaudhury, S., Horn, B., Mahadeo, K., et al. (2020). A phase 3, Single-arm, prospective study of Remestemcel-L, Ex Vivo culture-expanded adult human mesenchymal stromal cells for the treatment of pediatric patients who failed to respond to steroid treatment for acute Graft-versus-Host disease. Biol. Blood Marrow Transpl. J. Am. Soc. Blood Marrow Transpl. 26 (5), 845–854. doi:10.1016/j.bbmt.2020.01.018
Lam, J., Lee, B., Yu, J., Kwee, B. J., Kim, Y., Kim, J., et al. (2022). A microphysiological system-based potency bioassay for the functional quality assessment of mesenchymal stromal cells targeting vasculogenesis. Biomaterials 290, 121826. doi:10.1016/j.biomaterials.2022.121826
Larsen, K., Petrovski, G., and Boix-Lemonche, G. (2024). Alternative cryoprotective agent for corneal stroma-derived mesenchymal stromal cells for clinical applications. Sci. Rep. 14 (1), 15788. doi:10.1038/s41598-024-65469-4
Lawson, A., Ahmad, H., and Sambanis, A. (2011). Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions. Cryobiology 62 (2), 115–122. doi:10.1016/j.cryobiol.2011.01.012
Le Nail, L. R., Brennan, M., Rosset, P., Deschaseaux, F., Piloquet, P., Pichon, O., et al. (2018). Comparison of Tumor- and bone marrow-derived mesenchymal stromal/stem cells from patients with high-grade osteosarcoma. Int. J. Mol. Sci. 19 (3), 707. doi:10.3390/ijms19030707
Li, X., Qian, S., Song, Y., Guo, Y., Huang, F., Han, D., et al. (2022). New insights into the mechanism of freeze-induced damage based on ice crystal morphology and exudate proteomics. Food Res. Int. Ott Ont. 161, 111757. doi:10.1016/j.foodres.2022.111757
Li, H., Dai, H., and Li, J. (2023). Immunomodulatory properties of mesenchymal stromal/stem cells: the link with metabolism. J. Adv. Res. 45, 15–29. doi:10.1016/j.jare.2022.05.012
Li, G., Zhao, Y., Liu, R., Zhang, Y., Zhang, Y., Du, W., et al. (2024). Highly effective strategy for isolation of mononuclear cells from frozen cord blood. J. Immunol. Methods 534, 113762. doi:10.1016/j.jim.2024.113762
Li, X., Wei, Y., Li, W., Xu, R., Xu, C., Luo, B., et al. (2025). Acidic pH-Triggered membrane fusion enables lyophilized lipid nanoparticles as potent cryoprotectants for human erythrocytes. Adv. Healthc. Mater 3, e02455. doi:10.1002/adhm.202502455
Lin, A. D. Y., Tung, M. C., and Lu, C. H. (2021). The hernia sac-A suitable source for obtaining mesenchymal stem cells. Surg. Open Sci. 6, 40–44. doi:10.1016/j.sopen.2021.08.002
Linkova, D. D., Rubtsova, Y. P., and Egorikhina, M. N. (2022). Cryostorage of mesenchymal stem cells and biomedical cell-based products. Cells 11 (17), 2691. doi:10.3390/cells11172691
Liu, L., Chen, Y., Li, X., Wang, J., and Yang, L. (2024). Therapeutic potential: the role of mesenchymal stem cells from diverse sources and their derived exosomes in diabetic nephropathy. Biomed. Pharmacother. Biomedecine Pharmacother. 175, 116672. doi:10.1016/j.biopha.2024.116672
Luz-Crawford, P., Kurte, M., Bravo-Alegría, J., Contreras, R., Nova-Lamperti, E., Tejedor, G., et al. (2013). Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells. Stem Cell Res. Ther. 4 (3), 65. doi:10.1186/scrt216
MacDonald, E. S., and Barrett, J. G. (2019). The potential of mesenchymal stem cells to treat systemic inflammation in horses. Front. Vet. Sci. 6, 507. doi:10.3389/fvets.2019.00507
Mallis, P., Chatzistamatiou, T., Dimou, Z., Sarri, E. F., Georgiou, E., Salagianni, M., et al. (2022). Mesenchymal stromal cell delivery as a potential therapeutic strategy against COVID-19: promising evidence from in vitro results. World J. Biol. Chem. 13 (2), 47–65. doi:10.4331/wjbc.v13.i2.47
Margiana, R., Markov, A., Zekiy, A. O., Hamza, M. U., Al-Dabbagh, K. A., Al-Zubaidi, S. H., et al. (2022). Clinical application of mesenchymal stem cell in regenerative medicine: a narrative review. Stem Cell Res. Ther. 13 (1), 366. doi:10.1186/s13287-022-03054-0
Martin, K. E., Kalelkar, P. P., Coronel, M. M., Theriault, H. S., Schneider, R. S., and García, A. J. (2022). Host type 2 immune response to xenogeneic serum components impairs biomaterial-directed osteo-regenerative therapies. Biomaterials 286, 121601. doi:10.1016/j.biomaterials.2022.121601
Martín-López, M., Rosell-Valle, C., Arribas-Arribas, B., Fernández-Muñoz, B., Jiménez, R., Nogueras, S., et al. (2023). Bioengineered tissue and cell therapy products are efficiently cryopreserved with pathogen-inactivated human platelet lysate-based solutions. Stem Cell Res. Ther. 14 (1), 69. doi:10.1186/s13287-023-03300-z
Medrano-Trochez, C., Chatterjee, P., Pradhan, P., Stevens, H. Y., Ogle, M. E., Botchwey, E. A., et al. (2021). Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations. Stem Cell Res. Ther. 12 (1), 565. doi:10.1186/s13287-021-02627-9
Mendt, M., Daher, M., Basar, R., Shanley, M., Kumar, B., Wei Inng, F. L., et al. (2021). Metabolic reprogramming of GMP grade cord tissue derived mesenchymal stem cells enhances their suppressive potential in GVHD. Front. Immunol. 12, 631353. doi:10.3389/fimmu.2021.631353
Miroslava, J., Pavel, S., Doris, V., Peter, B., Alzbeta, F., Stanislav, F., et al. (2020). New cryopreservation technology of hMSCs: first preclinical results using DMSO-containing medium. Cryo Lett. 41 (1), 50–56.
Mohamed, H. M., Sundar, P., Ridwan, N. A. A., Cheong, A. J., Mohamad Salleh, N. A., Sulaiman, N., et al. (2024). Optimisation of cryopreservation conditions, including storage duration and revival methods, for the viability of human primary cells. BMC Mol. Cell Biol. 25 (1), 20. doi:10.1186/s12860-024-00516-6
Moll, G., Geißler, S., Catar, R., Ignatowicz, L., Hoogduijn, M. J., Strunk, D., et al. (2016). Cryopreserved or fresh mesenchymal stromal cells: only a matter of taste or key to unleash the full clinical potential of MSC therapy? Adv. Exp. Med. Biol. 951, 77–98. doi:10.1007/978-3-319-45457-3_7
Moreno, D. F., and Cid, J. (2019). Graft-versus-host disease. Med. Clin. Barc. 152 (1), 22–28. doi:10.1016/j.medcli.2018.07.012
Murray, A., Kilbride, P., and Gibson, M. I. (2024). Trehalose in cryopreservation. Applications, mechanisms and intracellular delivery opportunities. RSC Med. Chem. 15 (9), 2980–2995. doi:10.1039/d4md00174e
Naji, A., Eitoku, M., Favier, B., Deschaseaux, F., Rouas-Freiss, N., and Suganuma, N. (2019). Biological functions of mesenchymal stem cells and clinical implications. Cell Mol. Life Sci. CMLS 76 (17), 3323–3348. doi:10.1007/s00018-019-03125-1
Nicotra, T., Desnos, A., Halimi, J., Antonot, H., Reppel, L., Belmas, T., et al. (2020). Mesenchymal stem/stromal cell quality control: validation of mixed lymphocyte reaction assay using flow cytometry according to ICH Q2(R1). Stem Cell Res. Ther. 11 (1), 426. doi:10.1186/s13287-020-01947-6
Nishigaki, T., Teramura, Y., Nasu, A., Takada, K., Toguchida, J., and Iwata, H. (2011). Highly efficient cryopreservation of human induced pluripotent stem cells using a dimethyl sulfoxide-free solution. Int. J. Dev. Biol. 55 (3), 305–311. doi:10.1387/ijdb.103145tn
Ntege, E. H., Sunami, H., Denda, J., Futenma, N., and Shimizu, Y. (2020). Effects of hydroxyapatite-coated nonwoven polyethylene/polypropylene fabric on non-mesodermal lineage-specific differentiation of human adipose-derived stem cells. BMC Res. Notes 13 (1), 471. doi:10.1186/s13104-020-05315-8
Ortiz Silva, N. A., Denis, S., Vergnaud, J., and Hillaireau, H. (2024). Controlled hydrogel-based encapsulation of macrophages determines cell survival and functionality upon cryopreservation. Int. J. Pharm. 650, 123491. doi:10.1016/j.ijpharm.2023.123491
Patel, T., Mešić, J., Meretzki, S., Bronshtein, T., Brlek, P., Kivity, V., et al. (2025). Therapeutic potential and mechanisms of mesenchymal stem cells in coronary artery disease: narrative review. Int. J. Mol. Sci. 26 (11), 5414. doi:10.3390/ijms26115414
Pilbauerova, N., Schmidt, J., Soukup, T., Prat, T., Nesporova, K., Velebny, V., et al. (2022). Innovative approach in the cryogenic freezing medium for mesenchymal stem cells. Biomolecules 12 (5), 610. doi:10.3390/biom12050610
Pola-Silva, L., Xerfan Nahas, F., Nascimento, F., Santos, T. R., Malinverni, A. M., Alves, A., et al. (2021). Technique for obtaining mesenchymal stem cell from adipose tissue and stromal vascular fraction characterization in long-term cryopreservation. J. Vis. Exp. JoVE 30 (178). doi:10.3791/63036
Polchow, B., Kebbel, K., Schmiedeknecht, G., Reichardt, A., Henrich, W., Hetzer, R., et al. (2012). Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks. J. Transl. Med. 10, 98. doi:10.1186/1479-5876-10-98
Pollock, K., Sumstad, D., Kadidlo, D., McKenna, D. H., and Hubel, A. (2015). Clinical mesenchymal stromal cell products undergo functional changes in response to freezing. Cytotherapy 17 (1), 38–45. doi:10.1016/j.jcyt.2014.06.008
Prickett, R. C., Marquez-Curtis, L. A., Elliott, J. A. W., and McGann, L. E. (2015). Effect of supercooling and cell volume on intracellular ice formation. Cryobiology 70 (2), 156–163. doi:10.1016/j.cryobiol.2015.02.002
Pu, Z., Zhang, L., Yang, H., Shao, T., Wang, D., Wang, J., et al. (2025). Vitrification of 3D-MSCs encapsulated in GelMA hydrogel: improved cryosurvival, reduced cryoprotectant concentration, and enhanced wound healing. Int. J. Biol. Macromol. 296, 139716. doi:10.1016/j.ijbiomac.2025.139716
Putra, I., Shen, X., Anwar, K. N., Rabiee, B., Samaeekia, R., Almazyad, E., et al. (2021). Preclinical evaluation of the safety and efficacy of cryopreserved bone marrow mesenchymal stromal cells for corneal repair. Transl. Vis. Sci. Technol. 10 (10), 3. doi:10.1167/tvst.10.10.3
Rall, W. F., and Fahy, G. M. (1985). Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature 313 (6003), 573–575. doi:10.1038/313573a0
Ramireddy, A., Brodt, C. R., Mendizabal, A. M., DiFede, D. L., Healy, C., Goyal, V., et al. (2017). Effects of transendocardial stem cell injection on ventricular proarrhythmia in patients with ischemic cardiomyopathy: results from the POSEIDON and TAC-HFT trials. Stem Cells Transl. Med. 6 (5), 1366–1372. doi:10.1002/sctm.16-0328
Rosell-Valle, C., Antúnez, C., Campos, F., Gallot, N., García-Arranz, M., García-Olmo, D., et al. (2021). Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products. Cytotherapy 23 (8), 740–753. doi:10.1016/j.jcyt.2020.12.004
Rui, K., Zhang, Z., Tian, J., Lin, X., Wang, X., Ma, J., et al. (2016). Olfactory ecto-mesenchymal stem cells possess immunoregulatory function and suppress autoimmune arthritis. Cell Mol. Immunol. 13 (3), 401–408. doi:10.1038/cmi.2015.82
Rusconi, G., Cremona, M., Gallazzi, M., Mariotta, L., Gola, M., Gandolfi, E., et al. (2024). Good manufacturing practice-compliant cryopreserved and thawed native adipose tissue ready for fat grafting. J. Clin. Med. 13 (11), 3028. doi:10.3390/jcm13113028
Santos, L. M., Shimabuko, D. Y., and Sipert, C. R. (2024). Dimethyl sulfoxide affects the viability and mineralization activity of apical papilla cells in vitro. Braz Dent. J. 35, e246054. doi:10.1590/0103-644020246054
Safwan, M., Bourgleh, M. S., and Haider, H. (2025). Clinical experience with cryopreserved mesenchymal stem cells for cardiovascular applications: a systematic review. World J. Stem Cells 17 (3), 102067. doi:10.4252/wjsc.v17.i3.102067
Seo, M. S., Kang, K. K., Oh, S. K., Sung, S. E., Kim, K. S., Kwon, Y. S., et al. (2021). Isolation and characterization of feline wharton’s jelly-derived mesenchymal stem cells. Vet. Sci. 8 (2), 24. doi:10.3390/vetsci8020024
Sharpe, P. T. (2016). Dental mesenchymal stem cells. Dev. Camb Engl. 143 (13), 2273–2280. doi:10.1242/dev.134189
Shi, Y., Wang, Y., Li, Q., Liu, K., Hou, J., Shao, C., et al. (2018). Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases. Nat. Rev. Nephrol. 14 (8), 493–507. doi:10.1038/s41581-018-0023-5
Spees, J. L., Lee, R. H., and Gregory, C. A. (2016). Mechanisms of mesenchymal stem/stromal cell function. Stem Cell Res. Ther. 7 (1), 125. doi:10.1186/s13287-016-0363-7
Stewart, M. P., Langer, R., and Jensen, K. F. (2018). Intracellular delivery by membrane disruption: mechanisms, strategies, and concepts. Chem. Rev. 118 (16), 7409–7531. doi:10.1021/acs.chemrev.7b00678
Strecanska, M., Sekelova, T., Smolinska, V., Kuniakova, M., and Nicodemou, A. (2025). Automated manufacturing processes and platforms for large-scale production of clinical-grade mesenchymal Stem/stromal cells. Stem Cell Rev. Rep. 21 (2), 372–389. doi:10.1007/s12015-024-10812-5
Svoradová, A., Vašíček, J., Zmrhal, V., Venusová, E., Pavlík, A., Bauer, M., et al. (2023). Mesenchymal stem cells of Oravka chicken breed: promising path to biodiversity conservation. Poult. Sci. 102 (8), 102807. doi:10.1016/j.psj.2023.102807
Swamynathan, P., Venugopal, P., Kannan, S., Thej, C., Kolkundar, U., Bhagwat, S., et al. (2014). Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton’s jelly derived mesenchymal stem cells? A comparative study. Stem Cell Res. Ther. 5 (4), 88. doi:10.1186/scrt477
Tan, Y., Salkhordeh, M., Wang, J. P., McRae, A., Souza-Moreira, L., McIntyre, L., et al. (2019). Thawed mesenchymal stem cell product shows comparable immunomodulatory potency to cultured cells in vitro and in polymicrobial septic animals. Sci. Rep. 9 (1), 18078. doi:10.1038/s41598-019-54462-x
Uberti, B., Plaza, A., and Henríquez, C. (2022). Pre-conditioning strategies for mesenchymal stromal/stem cells in inflammatory conditions of livestock species. Front. Vet. Sci. 9, 806069. doi:10.3389/fvets.2022.806069
Vajta, G., Holm, P., Kuwayama, M., Booth, P. J., Jacobsen, H., Greve, T., et al. (1998). Open pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev. 51 (1), 53–58. doi:10.1002/(SICI)1098-2795(199809)51:1<53::AID-MRD6>3.0.CO;2-V
Vymetalova, L., Kucirkova, T., Knopfova, L., Pospisilova, V., Kasko, T., Lejdarova, H., et al. (2020). Large-Scale automated hollow-fiber bioreactor expansion of umbilical cord-derived human mesenchymal stromal cells for neurological disorders. Neurochem. Res. 45 (1), 204–214. doi:10.1007/s11064-019-02925-y
Wang, H. Y., Lun, Z. R., and Lu, S. S. (2011). Cryopreservation of umbilical cord blood-derived mesenchymal stem cells without dimethyl sulfoxide. Cryo Lett. 32 (1), 81–88.
Wang, Y., Chen, X., Cao, W., and Shi, Y. (2014). Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nat. Immunol. 15 (11), 1009–1016. doi:10.1038/ni.3002
Wang, J., Zhao, G., Zhang, Z., Xu, X., and He, X. (2016). Magnetic induction heating of superparamagnetic nanoparticles during rewarming augments the recovery of hUCM-MSCs cryopreserved by vitrification. Acta Biomater. 33, 264–274. doi:10.1016/j.actbio.2016.01.026
Wang, J., Shi, X., Xiong, M., Tan, W. S., and Cai, H. (2022a). Trehalose glycopolymers for cryopreservation of tissue-engineered constructs. Cryobiology 104, 47–55. doi:10.1016/j.cryobiol.2021.11.004
Wang, W., Yan, M., Aarabi, G., Peters, U., Freytag, M., Gosau, M., et al. (2022). Cultivation of cryopreserved human dental pulp stem Cells-A new approach to maintaining dental pulp tissue. Int. J. Mol. Sci. 23 (19), 11485. doi:10.3390/ijms231911485
Wang, X., Wang, E., and Zhao, G. (2023). Advanced cryopreservation engineering strategies: the critical step to utilize stem cell products. Cell Regen. Lond Engl. 12 (1), 28. doi:10.1186/s13619-023-00173-8
Weng, L. (2023). Cell therapy drug product development: technical considerations and challenges. J. Pharm. Sci. 112 (10), 2615–2620. doi:10.1016/j.xphs.2023.08.001
Whaley, D., Damyar, K., Witek, R. P., Mendoza, A., Alexander, M., and Lakey, J. R. (2021). Cryopreservation: an overview of principles and cell-specific considerations. Cell Transpl. 30, 963689721999617. doi:10.1177/0963689721999617
Wiese, D. M., and Braid, L. R. (2020). Transcriptome profiles acquired during cell expansion and licensing validate mesenchymal stromal cell lineage genes. Stem Cell Res. Ther. 11 (1), 357. doi:10.1186/s13287-020-01873-7
Wiese, D. M., Wood, C. A., and Braid, L. R. (2022). From vial to vein: crucial gaps in mesenchymal stromal cell clinical trial reporting. Front. Cell Dev. Biol. 10, 867426. doi:10.3389/fcell.2022.867426
Windrum, P., Morris, T. C. M., Drake, M. B., Niederwieser, D., and Ruutu, T.EBMT Chronic Leukaemia Working Party Complications Subcommittee (2005). EBMT chronic leukaemia working party complications subcommittee. Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of EBMT centres. Bone Marrow Transpl. 36 (7), 601–603. doi:10.1038/sj.bmt.1705100
Wu, M., Han, Z. B., Liu, J. F., Wang, Y. W., Zhang, J. Z., Li, C. T., et al. (2014). Serum-free media and the immunoregulatory properties of mesenchymal stem cells in vivo and in vitro. Cell Physiol. Biochem. Int. J. Exp. Cell Physiol. Biochem. Pharmacol. 33 (3), 569–580. doi:10.1159/000358635
Wu, C., Mi, Y., Song, J., Zhang, M., and Wang, C. (2025). The regulatory effect of human umbilical cord mesenchymal stem cells on the gut microbiota in diabetic nephropathy rats. Iran. J. Biotechnol. 23 (1), e3975. doi:10.30498/ijb.2025.472772.3975
Yamatoya, K., Nagai, Y., Teramoto, N., Kang, W., Miyado, K., Nakata, K., et al. (2023). Dimethyl sulfoxide-free cryopreservation of differentiated human neuronal cells. Biopreservation Biobanking 21 (6), 631–634. doi:10.1089/bio.2022.0180
Yang, Y., Melzer, C., Bucan, V., von der Ohe, J., Otte, A., and Hass, R. (2016). Conditioned umbilical cord tissue provides a natural three-dimensional storage compartment as in vitro stem cell niche for human mesenchymal stroma/stem cells. Stem Cell Res. Ther. 7, 28. doi:10.1186/s13287-016-0289-0
Yi, J., Liang, X. M., Zhao, G., and He, X. (2014). An improved model for nucleation-limited ice formation in living cells during freezing. PloS One 9 (5), e98132. doi:10.1371/journal.pone.0098132
Zanata, F., Shaik, S., Devireddy, R. V., Wu, X., Ferreira, L. M., and Gimble, J. M. (2016). Cryopreserved adipose tissue-derived stromal/stem cells: potential for applications in clinic and therapy. Adv. Exp. Med. Biol. 951, 137–146. doi:10.1007/978-3-319-45457-3_11
Zhu, X., Wang, Y., Sun, Z., Cheng, W., Chen, K., Gao, X., et al. (2025). Mesenchymal stem cells attenuate podocyte injury in diabetic nephropathy through the promotion of type 2 macrophage polarization. Stem Cells Dev. 34 (11–12), 258–270. doi:10.1089/scd.2025.0038
Glossary
MSCs mesenchymal stem cells
CPAs cryoprotective agents
GVHD Graft-versus-Host Disease
QC quality control
IRI ice recrystallization inhibition
ECM extracellular matrix
DMSO dimethyl sulfoxide
FBS fetal bovine serum
HPL human platelet lysate
PEG polyethylene glycol
BSA bovine serum albumin
SCF stem cell factor
EG ethylene glycol
PVA polyvinyl alcohol
UCB-MSCs umbilical cord blood-derived MSCs
ISCT International Society for Cellular Therapy
AD-MSCs adipose-derived MSCs
hDPSCs human dental stem cells
BM-MSCs bone marrow-derived MSCs
LVEF left ventricular ejection fraction
L-MSCs lung-derived MSCs
UC-MSCs umbilical cord-derived mesenchymal stem cells
DN diabetic nephropathy
SOP standardized operating procedure
MLR mixed lymphocyte reaction
MPS microphysiological systems
IDO Indoleamine 2,3-dioxygenase
PGE2 Prostaglandin E2
SA/V Surface-area-to-volume ratio
IIF Intracellular ice formation
VILI Ventilator-induced lung injury
Keywords: “off-the-shelf” therapy, cryopreservation, mesenchymal stem cells (MSCs), potency assay, quality control, regenerative medicine
Citation: Wang Y, Yang K, Yuan S, Huo F, Yang C and Tian W (2026) Research advances in cryopreserved preparations of mesenchymal stem cells: technical innovations, application challenges, and quality control. Front. Bioeng. Biotechnol. 14:1717539. doi: 10.3389/fbioe.2026.1717539
Received: 02 October 2025; Accepted: 05 January 2026;
Published: 21 January 2026.
Edited by:
Aaron Goldstein, Virginia Tech, United StatesReviewed by:
Tracy Criswell, Wake Forest University, United StatesJaejin Cho, Seoul National University, Republic of Korea
Andreas Nicodemou, Comenius University, Slovakia
Copyright © 2026 Wang, Yang, Yuan, Huo, Yang and Tian. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Chao Yang, eWFuZ2NoYW8xMjA3QHFxLmNvbQ==; Weidong Tian, ZHJ0d2RAc2luYS5jb20=