Skip to main content
NCBI home page
Biomolecules & Therapeutics logoLink to Biomolecules & Therapeutics
. 2025 Oct 14;33(6):963–974. doi: 10.4062/biomolther.2025.144

Glucose-Enhanced Cryopreservation of hCAR-T Cells: Improved Recovery and Reduced Apoptosis

Sang-Eun Jung 1,2,†, Youngchae Moon 1,2,†, Minji Lim 1,2, Hyungwoo Jeong 1,2, Hyungseok Seo 1,2,*
PMCID: PMC12580645  PMID: 41084238

Abstract

Human chimeric antigen receptor T (hCAR-T) cells are highly potent cellular therapeutics, but their clinical utility depends on stable long-term preservation due to high production costs and lengthy manufacturing processes. Cryopreservation is essential for ensuring the quality and logistics of these therapies. However, current commercial cryoprotectants such as CellBanker® are limited by high cost, undisclosed composition, and lack of flexibility for optimization. This study aimed to evaluate defined sugar-based cryoprotectants—trehalose, sucrose, and glucose—as potential alternatives for hCAR-T cell preservation. hCAR-T cells were cryopreserved using various concentrations of the three sugars in combination with DMSO. Post-thaw evaluations included viability, recovery, apoptosis, proliferative capacity, and immunophenotypic analysis. At 18 h after thawing, glucose 50 mM significantly improved recovery (1.03 ± 0.29 vs. 1.59 ± 0.20×10⁶ cells) and reduced apoptosis (52.58 ± 7.31% vs. 39.50 ± 2.16%) compared with DMSO alone. These results were comparable to, and in some cases exceeded, those obtained with the commercial product CellBanker®. Moreover, glucose at 50 mM exhibited approximately 1.9-fold higher cell proliferation after three days of culture compared to CellBanker®, while preserving a stable CD4+/CD8+ ratio and central memory T cell (TCM) profile. These findings indicate that sugar-based cryoprotectants, particularly glucose at 50 mM, can support post-thaw survival and function of hCAR-T cells. Given their defined composition, lower cost, and comparable efficacy, sugar-based formulations represent promising alternatives to commercial cryopreservation agents for advanced cell therapies.

Keywords: Human CAR-T cells, Cryopreservation, Sugar-based cryoprotectants, Glucose

INTRODUCTION

Human chimeric antigen receptor T (hCAR-T) cells represent a highly advanced form of adoptive cell therapy, offering potent antitumor activity and antigen specificity against hematologic malignancies and, increasingly, solid tumors (Hong et al., 2020; Labanieh et al., 2018). However, the production of hCAR-T cells involves complex genetic engineering and prolonged ex vivo activation, resulting in significant manufacturing costs and time (Abdo et al., 2025; Cliff et al., 2023). Consequently, effective cryopreservation strategies are essential to ensure stable long-term storage, facilitate quality-controlled distribution, and maximize clinical utility (Watanabe et al., 2022).

Currently available commercial cryopreservation solutions, such as CellBanker®, offer acceptable levels of viability and preservation efficiency. Nevertheless, their use is limited by high cost and proprietary, undisclosed compositions, which hinder mechanistic optimization and rational improvement (Miki et al., 2016). Furthermore, cryopreservation efficiency may vary depending on cell type and handling conditions, and in certain cases, such formulations may be insufficient to maintain the functional quality required for therapeutic applications (Esmeryan et al., 2025; Wilsher et al., 2025). This underscores the need for alternative cryoprotective agents with clearly defined compositions and broader applicability across diverse cell types.

In this context, sugar-based cryoprotectants have been proposed as promising alternatives due to their ability to mitigate cryo-induced damage via multiple protective mechanisms, including membrane stabilization, free water immobilization, and attenuation of osmotic stress (Deng et al., 2025; Gokulanathan et al., 2024; Izaguirre-Pérez et al., 2025; Kim et al., 2015; Lee et al., 2014). For instance, trehalose stabilizes membrane structures by forming hydrogen bonds with phospholipid bilayers (Jung et al., 2020; Murray et al., 2024), whereas glucose reduces cell shrinkage and membrane damage by increasing extracellular osmolarity and limiting intracellular water retention (Oldenhof et al., 2013; Robinson, 1960; Steiner et al., 2000). Similarly, sucrose helps suppress intracellular ice crystal formation by modulating extracellular osmotic pressure, and alleviates osmotic shock during thawing, thereby protecting membrane integrity (Ferrero and Zaritzky, 2000; Mahdjoub et al., 2006; Strauss and Hauser, 1986). Although these disaccharides cannot penetrate the cell membrane, they can serve as co-solutes that complement the intracellular protective effects of dimethyl sulfoxide (DMSO) by regulating the extracellular osmotic environment (Gao and Critser, 2000). Given that the efficacy of these sugars varies depending on concentration, structural properties, and cellular characteristics, systematic screening and cell-specific validation are required to define optimal conditions, particularly for metabolically active and structurally complex cells such as hCAR-T.

Importantly, hCAR-T cells are known to be more vulnerable to cryoinjury than peripheral blood mononuclear cells (PBMCs), likely due to their high metabolic demand and activation status (Xu et al., 2018). In addition, cryopreservation-induced cellular damage may not be immediately evident after thawing, but can manifest hours later as delayed cell death (Baust et al., 2022; Gonzalez-Martinez and Gibson, 2023; Yu et al., 2024). Thus, conventional assessments at a single post-thaw time point may underestimate the extent of cryodamage. To obtain an accurate evaluation of cryopreservation efficacy, it is necessary to implement multi-time-point assessments that account for post-thaw survival, functional recovery, and phenotypic integrity.

In this study, we aimed to systematically compare the cryoprotective effects of three well-characterized sugar-based formulations—trehalose, sucrose, and glucose—on hCAR-T cells. We conducted a comprehensive evaluation encompassing not only post-thaw viability but also apoptosis, proliferative capacity, and T cell phenotypic composition. The overarching goal was to determine whether sugar-based cryoprotectants could serve as viable alternatives to commercial solutions by preserving both the survival and functionality of hCAR-T cells in a cost-effective and mechanistically transparent manner.

MATERIALS AND METHODS

Ethical approval and consent to participate

All procedures involving human samples, including the use of peripheral blood mononuclear cells (PBMCs), were approved by the Institutional Review Board (IRB) of Seoul National University (Approval No. 2406/002-003) on May 20, 2024. Written informed consent was obtained from all donors prior to sample collection. The collected samples were used exclusively for the approved research purpose and stored under controlled conditions. The approved project was titled “Establishment of optimal cryopreservation conditions to enhance the freezing efficiency of human-derived immune cells”.

Isolation of peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated using density gradient centrifugation with Lymphocyte Separation Medium (LSM; Corning, 25-072-CV, Corning, NY, USA). Fresh peripheral blood samples were diluted 1:1 with Dulbecco’s phosphate-buffered saline (DPBS; Gibco, 14190-250, Waltham, MA, USA) and carefully layered over the LSM in 15 mL conical tubes. The samples were centrifuged at 400×g for 30 min at room temperature (25°C) without brake. The PBMC layer was collected from the interface between the plasma and LSM, transferred to new tubes, and washed twice with DPBS by centrifugation at 300×g for 5 min. The final cell pellet was counted using an automated cell counter (Countess 3; Thermo Fisher Scientific, Waltham, MA, USA), and then either cryopreserved in cryovials (Corning, 430488) with various cryoprotectant formulations or used immediately for hCAR-T cell generation.

Isolation and activation of human T cells

Pan T cells were isolated from PBMCs using the EasySep™ Human T Cell Isolation Kit (StemCell Technologies, 17951, Vancouver, BC, Canada), according to the manufacturer’s instructions. Briefly, thawed PBMCs were washed and resuspended in MACS buffer (PBS supplemented with 2% FBS and 2.5 mM EDTA) at a concentration of 5×10⁷ cells/mL. The isolation cocktail (50 μL/mL) was added and incubated at room temperature (20-25°C) for 5 min. Afterward, RapidSpheres (40 μL/mL) were added to the cell suspension, and the total volume was adjusted to 10 mL with MACS buffer. The tube was then placed in The Big Easy EasySep™ magnet (StemCell Technologies, 18001) and incubated for 3 min at room temperature (20-25°C). The enriched pan T cells were collected by pouring the supernatant into a new 15 mL tube. Isolated cells were subsequently washed and counted using an automated cell counter (Countess 3; Thermo Fisher Scientific).

For activation, pan T cells were co-cultured with anti-CD3/CD28 Dynabeads (Human T-Expander; Gibco, 11141D) at a 1:3 cell-to-bead ratio (0.5×10⁶ cells in 500 μL T cell culture medium (TCM) per well in a 24-well plate). The TCM consisted of RPMI 1640 (Gibco, 22400105) supplemented with 10% heat-inactivated FBS (Gibco, 430488), 1× non-essential amino acids (Gibco, 11140-050), 1× sodium pyruvate (Gibco, 11360-070), 1× GlutaMAX (Gibco, 35050-061), 1× HEPES (Gibco, 15630-080), and 55 μM 2-mercaptoethanol (Gibco, 21985-023).

Lentivirus production and transfection

Lentiviral particles were produced in Lenti-X 293T cells cultured in DMEM (Gibco, 11995-073) supplemented with 10% FBS (Gibco, 430488). On Day 0, 6×10⁶ cells were seeded in a 100 mm dish. On Day 1, cells were transfected using Lipofectamine 2000 (Invitrogen, 11668-019, Carlsbad, CA, USA) with four plasmids: pRSV-Rev (8 μg), pMDLg/pRRE (8 μg), pVSV-G (3.3 μg), and the CAR-encoding transfer vector (10 μg). DNA and Lipofectamine were each diluted in Opti-MEM (Gibco, 51985034) and incubated separately for 5 min before being mixed and incubated for an additional 20 min at room temperature (20-25°C). The transfection mixture was added dropwise to the cells. At 12-18 h post-transfection, the medium was replaced with fresh DMEM supplemented with 10% FBS. Viral supernatants were collected at 24-30 h post-replacement, centrifuged at 300×g for 5 min, and filtered through a 0.45 μm PES syringe filter (VWR, 514-1257, Radnor, PA, USA). The virus was concentrated using Lenti-X Concentrator (Clontech, 631232, Mountain View, CA, USA) at a 3:1 ratio (supernatant:Lenti-X) by incubating at 4°C for at least 30 min, followed by centrifugation at 1,500×g for 45 min at 4°C. The viral pellet was resuspended in DMEM and stored at –80°C in aliquots.

Human CAR-T cell generation

On the day following activation (Day 1), clustered T cells were transduced with concentrated lentivirus at a multiplicity of infection (MOI) of 5 by gently adding the viral suspension to each well, taking care not to disrupt the cell clusters. After overnight incubation at 37°C in a 5% CO2 incubator, 1 mL of pre-warmed TCM was carefully added to each well. On Day 3, the transduced cells were transferred to a T25 flask along with three volumes of fresh TCM. As the cells expanded, they were transferred to T75 flasks and cultured in TCM, maintaining a cell density of approximately 0.5×10⁶ cells/mL. Transduction efficiency was assessed on Day 7 by flow cytometry using CAR-specific staining and T cell markers (CD3, CD4, CD45RA, and CCR7). Final expansion was continued until Day 9 or later. When the average cell size dropped below 0.4 pL, as measured using the Scepter 3.0 handheld cell counter (Merck Millipore, PHCC360KIT, Burlington, MA, USA), the cells were harvested and the magnetic beads were removed using the EasySep™ magnet. The hCAR-T cells were then cryopreserved in designated freezing media at a final concentration of 2×10⁶ cells/mL.

Cryopreservation and thawing of PBMCs and hCAR-T cells

Human CAR-T cells with an average cell volume below 0.4 pL were resuspended at a concentration of 2×10⁶ cells/mL, and PBMCs were adjusted to 1-3×10⁶ cells/mL for cryopreservation. Cells were mixed with a two-fold concentrated sugar solution (100, 200, or 400 mM trehalose, sucrose, or glucose in DPBS; T0167, S1888, or G8270; Sigma–Aldrich, St. Louis, MO, USA) and an equal volume of cryoprotective solution containing 20% dimethyl sulfoxide (DMSO) and 20% fetal bovine serum (FBS; v/v) in DPBS. By mixing the two solutions at a 1:1 ratio (v/v), the final cryopreservation medium contained 50, 100, or 200 mM sugar, 10% DMSO, and 10% FBS in DPBS. The prepared cell suspensions were aliquoted into 1.8 mL cryovials (Corning, 430488), placed in a Mr. Frosty™ freezing container (Nalgene, Rochester, NY, USA) filled with isopropyl alcohol, and stored at –80°C overnight to achieve a controlled cooling rate of approximately –1°C/min. Cryovials were transferred to a liquid nitrogen (LN2) tank the following day for long-term storage. Thawing was performed after a minimum of one month in LN2 storage. Cryovials were thawed in a 37°C water bath for 2.5 min and then gradually diluted with TCM. The cells were centrifuged at 300×g for 5 min and subsequently processed for downstream analyses.

Assessment of recovery and proliferation rate

Following centrifugation at 300×g for 5 min, cell viability was assessed using the trypan blue exclusion method with an automated cell counter (Countess 3; Thermo Fisher Scientific). The post-thaw survival rate (%) was calculated using the following equation:

Survival rate (%)=(Number of viable cells/Total number of cells)×100

For hCAR-T cells, since all replicates were cryopreserved at a consistent density of 2×10⁶ cells per vial, the number of recovered cells was reported directly instead of calculating a recovery rate. For PBMCs, due to donor-dependent variation in the number of cryopreserved cells (1-3×10⁶ cells/vial), recovery rate was calculated as follows:

Recovery rate (%)=(Number of recovered cells after thawing/ Number of frozen cells)×100

Thawed PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS, whereas thawed hCAR-T cells were cultured in TCM supplemented with 50 IU/mL recombinant human IL-2 (rhIL-2). After 18 h of incubation, cell survival, recovery rate, and apoptosis were evaluated using the same formulas as above. To evaluate the proliferative capacity of hCAR-T cells post-thaw, cells were cultured for an additional 3 days in TCM containing 50 IU/mL rhIL-2. The proliferation rate (%) was calculated using the following formula:

Proliferation rate (%)=(Number of harvested cells/Number of seeded cells)×100

Apoptosis analysis

Apoptosis was evaluated using the FITC Annexin V Apoptosis Detection Kit with Propidium Iodide (PI) (BioLegend, 640914, San Diego, CA, USA), following the manufacturer’s protocol. Cells were washed twice with cold Dulbecco’s phosphate-buffered saline (DPBS; Gibco, 14190-250) and resuspended in Annexin V Binding Buffer (BioLegend, 422201) at a concentration of 0.5×10⁶ cells/mL. A 100 µL aliquot of the cell suspension was transferred to a 5 mL polystyrene tube, followed by the addition of 2 µL of FITC-conjugated Annexin V.

To differentiate apoptotic from dead cells, 0.5 µL of propidium iodide (PI; BioLegend, 421301) was added. The samples were gently vortexed and incubated for 15 min at room temperature (25°C) in the dark. After incubation, 400 µL of Annexin V Binding Buffer was added to each tube to stabilize the cells prior to acquisition. Flow cytometric analysis was performed immediately, and cell populations were classified as viable (Annexin V–/PI–), early apoptotic (Annexin V+/PI–), or late apoptotic/necrotic (Annexin V+/PI+).

Flow cytometry

Cells were collected and resuspended in FACS buffer (PBS with 2% FBS and 2.5 mM EDTA). For surface marker staining, cells were incubated with fluorochrome-conjugated antibodies (1:200 dilution in FACS buffer) for 30 min at 4°C in the dark. After staining, cells were washed twice with FACS buffer to remove excess antibodies and resuspended for analysis. Samples were acquired using a Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, CA, USA), and data were analyzed using FlowJo software (v10.10.0; FlowJo LLC, Ashland, OR, USA). The gating strategy used for analysis is shown in Supplementary Fig. 1. A complete list of antibodies and fluorochromes used is provided in Supplementary Table 1.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (version 10.1.0; GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± standard deviation (SD). Group comparisons were conducted using one-way ANOVA followed by Dunnett’s multiple comparisons test, which compares each treatment group to the control (DMSO only). The number of biological replicates (n) and significance levels are indicated in the figure legends.

RESULTS

Effect of sugar-based cryoprotectants on the cryopreservation efficiency of hPBMCs

The cryoprotective efficacy of sugar-based cryoprotectants, including trehalose, sucrose, and glucose, was evaluated in human PBMCs by assessing cell viability and recovery immediately and 18 h following thawing (Fig. 1A). At the immediate post-thaw time point, no significant differences in either viability or recovery were observed among the sugar-treated groups when compared to the DMSO-only control or the commercial cryoprotectant CellBanker® (Fig. 1B, 1C). While viability measured immediately after thawing reflects acute physical damage, intracellular injury induced by cryogenic stress may not be fully manifested until several hours later, often leading to delayed apoptosis. To account for this delayed effect, a secondary assessment was conducted at 18 h post-thaw. At this later time point, cell viability remained statistically comparable across all groups (Fig. 1D). However, the recovery rate in the glucose 50 mM group was significantly higher than that in the DMSO-only group (Fig. 1E; DMSO only vs. Glucose 50 mM, 74.49 ± 35.06% vs. 99.15 ± 40.03%). These findings indicate that glucose at 50 mM may confer a protective effect against cryoinjury in hPBMCs, possibly by enhancing post-thaw cell stability. In contrast, trehalose and sucrose exerted only marginal effects on post-thaw recovery under the same conditions.

Fig. 1.

Fig. 1

Open in a new tab

Evaluation of cryopreservation efficiency in human peripheral blood mononuclear cells (hPBMCs) using sugar-based cryoprotectants (trehalose, sucrose, and glucose). (A) Experimental scheme for freeze-thaw process and evaluation of hPBMC cryopreservation efficiency. (B, C) Cryopreservation efficiency of hPBMCs immediately after thawing: (B) post-thaw survival rate and (C) post-thaw recovery rate relative to the initial frozen cell number across different cryoprotectant conditions. (D, E) Cryopreservation efficiency at 18 h post-thawing: (D) post-thaw survival rate and (E) post-thaw recovery rate. DMSO-only (negative control) and CellBanker® (positive control) were included for comparison with sugar-based cryoprotectants. Data represent the mean of three independent experiments (gray bars), with each dot indicating an individual biological replicate (n=3). Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05.

Effect of sugar-based cryoprotectants on the cryopreservation efficiency of hCAR-T cells

To determine whether the cryoprotective effects observed in hPBMCs could be reproduced in a homogeneous T cell population, hCAR-T cells were subjected to the same sugar-based cryopreservation conditions (Fig. 2A). Consistent with the hPBMC data, no significant differences in post-thaw viability or recovery were observed among the trehalose, sucrose, and glucose-treated groups when compared to the DMSO-only control or CellBanker® immediately after thawing (Fig. 2B, 2C). However, evaluation at 18 h post-thaw revealed more pronounced differences between cell types. While hPBMCs maintained stable viability over time (Fig. 1B, 1D), hCAR-T cells exhibited a decreasing trend in viability during the same interval (Fig. 2B, 2D), suggesting a heightened sensitivity of T cells to freeze–thaw-induced damage. In terms of recovery, the glucose 50 mM group demonstrated a significantly higher cell number than the DMSO-only control (Fig. 2E; DMSO only: 1.03 ± 0.29×10⁶ cells, Glucose 50 mM: 1.59 ± 0.20×10⁶ cells). Although statistical significance was not achieved, the glucose 50 mM group also exhibited a higher recovery than the CellBanker® group (1.11 ± 0.20×10⁶ cells). These results suggest that glucose at 50 mM may offer superior membrane stabilization and support for cellular recovery in hCAR-T cells compared to other tested sugars, particularly under post-thaw stress conditions.

Fig. 2.

Fig. 2

Open in a new tab

Evaluation of cryopreservation efficiency in human CAR-T (hCAR-T) cells using sugar-based cryoprotectants (trehalose, sucrose, and glucose) (A) Experimental scheme for freeze-thaw process and evaluation of hCAR-T cell cryopreservation efficiency. (B, C) Cryopreservation efficiency of hCAR-T cells immediately after thawing: (B) post-thaw survival rate and (C) recovered hCAR-T cell number (×106) determined using trypan blue exclusion assay across different cryoprotectant conditions. (D, E) Cryopreservation efficiency at 18 h post-thawing: (D) post-thaw survival rate and (E) recovered hCAR-T cell number (×106). DMSO-only (negative control) and CellBanker® (positive control) were included for comparison with sugar-based cryoprotectants. Data represent the mean of three independent experiments (gray bars), with each dot indicating an individual biological replicate (n=3). Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05.

Assessment of post-thaw apoptosis in hCAR-T cells

Given the observation that viability and recovery continued to change over time following thawing, particularly in hCAR-T cells, apoptosis was assessed at 18 h post-thaw to account for delayed cell death responses (Fig. 3A). Annexin V/PI staining was performed to quantify total apoptosis, including both early and late apoptotic populations. Among the tested conditions, the Trehalose 100 mM group exhibited a significantly lower apoptotic rate (38.04% ± 3.83%) compared to the DMSO-only control (52.58% ± 7.31%) (Fig. 3B, 3C). Glucose treatment resulted in a general reduction in apoptosis regardless of concentration, with both Glucose 50 mM (39.50% ± 2.16%) and Glucose 100 mM (38.30% ± 3.47%) showing significantly reduced apoptotic rates relative to DMSO. These values were comparable to that observed in the CellBanker® group (36.83% ± 3.69%). These results indicate that trehalose and glucose may confer post-thaw cytoprotection by attenuating freeze–thaw-induced apoptotic responses in hCAR-T cells. In particular, glucose demonstrated an anti-apoptotic effect that was comparable in magnitude to that of the commercial cryoprotectant.

Fig. 3.

Fig. 3

Open in a new tab

Apoptosis analysis of human CAR-T (hCAR-T) cells at 18 h post-thawing. (A) Experimental scheme for apoptosis assessment of hCAR-T cells. (B, C) Apoptosis analysis following freeze-thawing with sugar-based cryoprotectants at different concentrations: (B) representative flow cytometry plots of Annexin V and PI staining, and (C) quantification of early (Annexin V+/PI−) and late (Annexin V+/PI+) apoptotic populations in hCAR-T cells. Apoptosis was quantified as total apoptotic cells. DMSO-only (negative control) and CellBanker® (positive control) were included for comparison with sugar-based cryoprotectants. Data represent the mean ± SD of three independent experiments, with each dot indicating an individual biological replicate (n=3). Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05.

Post-thaw viability and proliferative capacity of hCAR-T cells during in vitro culture

To assess the functional recovery of hCAR-T cells following cryopreservation, cells were cultured for three days post-thaw, and both viability and proliferative capacity were evaluated (Fig. 4A). This assay was designed to complement initial viability measurements by evaluating the sustained metabolic activity and expansion potential of thawed cells, which serve as indicators of functional integrity beyond membrane survival. At the end of the 3-day culture period, cell viability was restored to comparable levels across all treatment groups, regardless of cryoprotectant type or concentration, and no statistically significant differences were observed (Fig. 4B). In contrast, proliferative capacity varied markedly among the groups. The Glucose 50 mM group demonstrated significantly enhanced proliferation, reaching 6.07 ± 0.64×10⁶ cells (proliferation rate, 382.95 ± 11.67%), compared to 2.46 ± 0.84×10⁶ cells (239.61 ± 39.67%) in the DMSO-only group (Fig. 4C). Although the increases observed in the Trehalose 50 mM (3.84 ± 0.89×10⁶ cells, 349.39 ± 86.83%) and Sucrose 100 mM (3.90 ± 0.90×10⁶ cells; 364.64 ± 73.60%) groups were not statistically significant, they exhibited a consistent upward trend. Moreover, the proliferative capacity of the Glucose 50 mM group was approximately 1.93-fold higher than that of the CellBanker® group (3.13 ± 0.87×10⁶ cells; 280.86 ± 62.20%). These findings suggest that, in addition to improving recovery, certain sugar-based cryoprotectants—particularly glucose—may enhance the post-thaw proliferative potential of hCAR-T cells, thereby supporting their suitability for downstream expansion and therapeutic application.

Fig. 4.

Fig. 4

Open in a new tab

Survival rate and proliferative capacity of human CAR-T (hCAR-T) cells after in vitro culture following cryopreservation with sugar-based cryoprotectants. (A) Experimental scheme for assessing hCAR-T cell survival and proliferation after 3-day in vitro culture post-thawing. (B) Survival rate and (C) proliferative capacity following culture. Proliferative capacity was calculated as the ratio of the number of hCAR-T cells recovered after 3 days of culture to the number of cells initially seeded post-thawing. DMSO-only (negative control) and CellBanker® (positive control) were included for comparison with sugar-based cryoprotectants. Data represent the mean of three independent experiments (gray bars), with each dot indicating an individual biological replicate (n=3). Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison test. **p<0.01.

Stability of CAR expression in hCAR-T cells following cryopreservation

As CAR expression is essential for antigen recognition and cytotoxic activity, its maintenance following cryopreservation is a critical indicator of functional preservation in hCAR-T cells. To evaluate this, CAR expression was assessed after three days of post-thaw culture (Fig. 5A). All sugar-based cryoprotectant groups exhibited CAR+ T cell frequencies that were comparable to those of both the DMSO-only control and the commercial cryoprotectant CellBanker® (Fig. 5B). In addition, the frequency of CAR+ cells in the sugar-treated groups was consistent with that observed in the fresh (non-frozen) group, indicating that neither the freeze–thaw process nor sugar-based cryoprotectant exposure adversely affected CAR expression. These results suggest that the structural integrity and expression of the CAR construct remain stable across all tested cryopreservation conditions, including those incorporating sugars. Consequently, the use of sugar-based cryoprotectants does not compromise the therapeutic identity of hCAR-T cells in terms of CAR expression.

Fig. 5.

Fig. 5

Open in a new tab

CAR expression in human CAR-T (hCAR-T) cells following cryopreservation with sugar-based cryoprotectants. (A) Representative histogram overlays of CAR expression in hCAR-T cells after thawing. CAR expression was assessed by detecting LNGFR, which was co-expressed as a marker linked to the CAR construct. (B) Quantification of CAR+ T cell populations. CAR expression was performed by flow cytometry after 3 days of in vitro culture. The fresh group (non-cryopreserved hCAR-T cells) was included to assess the impact of freeze-thawing on CAR expression. DMSO-only (negative control) and CellBanker® (positive control) were included for comparison with sugar-based cryoprotectants. Data represent the mean of three independent experiments (gray bars), with each dot indicating an individual biological replicate (n=3). No statistically significant differences were observed among the groups (p>0.05). UTD, untransduced human T cells.

Phenotypic assessment of hCAR-T cells following cryopreservation

The freeze–thaw process can influence not only the viability and function of T cells but also their phenotypic composition. In particular, the preservation of less differentiated subsets—such as central memory T cells (TCM)—is considered critical for long-term persistence and therapeutic efficacy in adoptive cell therapy. Accordingly, the phenotypic distribution of CD4+ and CD8+ T cell subsets was analyzed in hCAR-T cells following cryopreservation and three days of in vitro recovery (Fig. 6A, 6B). Analysis of the CD4+/CD8+ ratio revealed a trend toward decreased CD4+ and increased CD8+ proportions in frozen samples compared to the fresh (non-frozen) group. However, among the cryopreserved groups, no statistically significant differences were observed across the various sugar-based cryoprotectants, DMSO-only, or CellBanker® (Fig. 6A). Notably, the distribution of CAR+ T cell subsets remained consistent across all treatment groups, indicating that sugar-based cryoprotectants did not alter the phenotypic profile of CAR-expressing cells (Fig. 6B). Nevertheless, a subset-level analysis revealed that cryopreservation was associated with a reduction in the frequency of TCM (CD45RA–CCR7+) and an increase in effector memory T cells (TEM, CD45RA–CCR7–) within the CD4+ population. A similar trend was observed in CD8+ cells, with a significant decrease in TCM and an increase in terminal effector memory RA+ cells (TEMRA, CD45RA+CCR7–). These findings suggest that while cryopreservation may induce moderate shifts in T cell subset composition—particularly a decline in less differentiated memory phenotypes—sugar-based cryoprotectants do not appear to exacerbate this effect. Importantly, the overall phenotypic integrity of CAR+ T cells was maintained, supporting the functional viability of the cryopreserved product.

Fig. 6.

Fig. 6

Open in a new tab

Phenotypic distribution of thawed human CAR-T (hCAR-T) cells following cryopreservation with sugar-based cryoprotectants. (A) CD4+ and CD8+ T cell composition in thawed hCAR-T cells. Purple and yellow segments represent CD4+ and CD8+ populations, respectively. (B) Memory T cell subsets defined by CD45RA and CCR7 expression within each CD4+ or CD8+ T cell population. Naïve (CD45RA+CCR7+), central memory (CD45RA−CCR7+), effector memory (CD45RA−CCR7−), and TEMRA (CD45RA+CCR7−) subsets are shown in purple, yellow, red, and green, respectively. Phenotypic analysis was performed by flow cytometry after 3 days of in vitro culture. The fresh group (non-cryopreserved hCAR-T cells) was included to assess the impact of freeze-thawing on T cell phenotype. DMSO-only (negative control) and CellBanker® (positive control) were included for comparison with sugar-based cryoprotectants. Data are presented as mean – SD of three independent biological replicates (n=3) in stacked bar format. *p<0.05; **p<0.01. TEMRA, terminally differentiated effector memory cells re-expressing RA.

DISCUSSION

Human CAR-T cells are high-value, functionally sophisticated cell therapy products that require significant production costs and extended manufacturing time. Cryopreservation is essential for the stable storage and transport of these cells. However, if cell viability or functional integrity is compromised during the freeze-thaw process, the clinical utility of the therapy may be significantly diminished. Commercially available cryopreservation reagents, such as CellBanker®, offer a certain level of efficacy but are limited by their high cost, undisclosed composition, and restricted potential for mechanism-based optimization. Therefore, this study aimed to screen sugar-based cryoprotectant candidates with well-defined compositions and to verify their feasibility through multifaceted evaluations, including cell viability, functional recovery, and phenotypic stability.

In this study, three types of sugar-based cryoprotectants—glucose, trehalose, and sucrose—were tested for their ability to preserve hCAR-T cells. Although there were no significant differences in viability or recovery among treatment groups immediately after thawing, distinct effects were observed after 18 h, including increased recovery rates and reduced apoptosis in certain conditions. Consistent with previous studies, these findings suggest that freeze-thaw-induced damage may not manifest immediately but rather develop progressively as delayed cell death (Baust et al., 2022; Gonzalez-Martinez and Gibson, 2023; Yu et al., 2024). Consequently, relying solely on immediate post-thaw assessments may underestimate the actual extent of damage, highlighting the importance of including delayed timepoints for accurate evaluation of cryopreservation efficacy.

Moreover, hCAR-T cells exhibited a more pronounced decline in viability compared to hPBMCs under equivalent cryopreservation conditions. This observation is consistent with previous reports indicating that T cells are inherently more susceptible to cryoinjury than other leukocyte subsets (Pi et al., 2020; Xu et al., 2018). Similarly, activated T cells have been shown to experience substantial viability loss when exposed to suboptimal freezing and thawing protocols (Baboo et al., 2019). The heightened cryosensitivity of hCAR-T cells is likely attributable to their metabolically active state and structural modifications acquired during ex vivo expansion and CAR transgene expression. Collectively, these findings emphasize that cryoinjury susceptibility is not uniform across immune cell types but rather depends on cellular phenotype and activation status.

Sugar-based cryoprotectants are known to protect cells from cryoinjury through multiple mechanisms, including membrane stabilization, immobilization of free water, and mitigation of osmotic stress (Deng et al., 2025; Gokulanathan et al., 2024; Izaguirre-Pérez et al., 2025; Kim et al., 2015; Lee et al., 2014). Specifically, trehalose stabilizes phospholipid bilayers by forming hydrogen bonds, thereby preventing membrane disruption during freezing (Jung et al., 2020; Murray et al., 2024), while glucose increases extracellular osmotic pressure to facilitate water efflux and prevent membrane damage (Oldenhof et al., 2013; Robinson, 1960; Steiner et al., 2000). These protective mechanisms likely underlie the observed reductions in apoptosis and improvements in proliferative recovery, particularly in the glucose 50 mM group, which demonstrated comparable or superior performance to CellBanker® in terms of both recovery rate and post-thaw expansion. These findings suggest that glucose supplementation may offer a viable and cost-effective alternative in cryopreservation protocols for metabolically active immune cells.

Surprisingly, the cryoprotective effects of sugars did not follow a linear dose-response relationship. At low concentrations, sugars stabilized cell membranes, reduced ice formation, and alleviated osmotic stress, thereby enhancing preservation. In contrast, higher concentrations induced detrimental effects. Excessive sugar can cause osmotic toxicity, known as the “solution effect,” leading to cellular dehydration and impaired recovery (Hauptmann et al., 2018; Oldenhof et al., 2013). Consistently, our preliminary PBMC experiments showed reduced viability with 400 mM formulations (data not shown). Moreover, high sugar levels may disrupt molecular interactions essential for membrane and protein stabilization, as reported in previous studies where excessive supplementation impaired macromolecular stability and decreased gel strength in cryogel systems (Doyle et al., 2006; Roy et al., 2016). These findings indicate that moderate sugar supplementation, such as 50 mM glucose in our study, provides the optimal balance between protective and adverse effects, thereby explaining the superior preservation outcomes observed.

Phenotypic analyses revealed that sugar-based treatments maintained CD4+/CD8+ ratios and memory T cell subset distributions at levels comparable to those observed with CellBanker®. Notably, post-thaw cells exhibited a trend toward decreased TCM and increased TEMRA, which may reflect inherent differences in cryosensitivity among T cell subsets. Less differentiated subsets such as naïve and TCM cells are more susceptible to freeze-thaw stress, including membrane damage and osmotic imbalance, whereas TEMRA cells exhibit greater resistance (Geginat et al., 2003; Lemieux et al., 2016; Ludgate et al., 1983; Venkataraman and Westerman, 1986). Such subset shifts may influence long-term in vivo persistence and therapeutic efficacy, underscoring the need for subset-specific cryopreservation strategies in future protocol development.

Although this study systematically assessed the effects of sugar-based cryoprotectants on the preservation of hCAR-T cells, several limitations should be acknowledged. First, the proposed mechanisms of action—such as membrane stabilization, osmotic protection, and ROS attenuation—were inferred based on literature and functional outcomes, but were not directly validated through molecular or biochemical assays (e.g., ROS levels, membrane repair markers, signaling cascades). Second, functional recovery was assessed primarily by IL-2-driven proliferation assays. While such assays are widely used in cryopreservation research as a standardized measure of post-thaw cellular fitness (Elavia et al., 2017; Gramatzki et al., 1982), they do not directly reflect CAR-mediated, antigen-specific activity. We acknowledge that direct co-culture assays with target tumor cells would provide stronger evidence of functional efficacy. Nevertheless, consistent with previous reports (Abraham-Miranda et al., 2022; Brezinger-Dayan et al., 2022; Luo et al., 2017), our findings of stable CAR expression post-thaw suggest that cryopreservation under our conditions is unlikely to compromise antigen-specific function. Finally, as all experiments were conducted in vitro, future in vivo studies are warranted to validate the translational relevance of these findings.

In conclusion, this study conducted a systematic evaluation of well-defined sugar-based cryoprotectants for the preservation of hCAR-T cells, assessing parameters such as cell viability, apoptosis, proliferation, and phenotypic stability. Among the tested formulations, glucose 50 mM demonstrated superior or comparable performance to the commercial reagent CellBanker® across multiple endpoints. Sugar-based cryoprotectants present promising alternatives by offering transparency of composition, cost efficiency, and potential for mechanism-based optimization. These findings support the potential application of glucose-supplemented cryopreservation protocols in clinical and manufacturing settings, pending further validation in vivo and at process scale.

bt-33-6-963-supple.pdf (4.5MB, pdf)

ACKNOWLEDGMENTS

We thank all members of the Seo Lab for their valuable support and helpful discussions throughout this project. H.S. received funding from the Creative-Pioneering Researchers Program at Seoul National University. The work was also supported by the National Research Foundation of Korea (NRF) through grants RS-2023-00242443, RS-2024-00406325, RS-2023-00281471, RS-2023-00210035 and RS-2024-00440679, along with the Korea Drug Development Fund (KDDF); grant RS-2023-00282907, financed by the Korea government (MSIT).

REFERENCES

  1. Abdo L., Batista-Silva L. R., Bonamino M. H. Cost-effective strategies for CAR-T cell therapy manufacturing. Mol. Ther. Oncol. 2025;33:200980. doi: 10.1016/j.omton.2025.200980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abraham-Miranda J., Menges M., Atkins R., Mattie M., Kanska J., Turner J., Hidalgo-Vargas M. J., Locke F. L. CAR-T manufactured from frozen PBMC yield efficient function with prolonged in vitro production. Front. Immunol. 2022;13:1007042. doi: 10.3389/fimmu.2022.1007042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baboo J., Kilbride P., Delahaye M., Milne S., Fonseca F., Blanco M., Meneghel J., Nancekievill A., Gaddum N., Morris G. J. The impact of varying cooling and thawing rates on the quality of cryopreserved human peripheral blood T cells. Sci. Rep. 2019;9:3417. doi: 10.1038/s41598-019-39957-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baust J. M., Snyder K. K., Van Buskirk R. G., Baust J. G. Assessment of the impact of post-thaw stress pathway modulation on cell recovery following cryopreservation in a hematopoietic progenitor cell model. Cells. 2022;11:278. doi: 10.3390/cells11020278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brezinger-Dayan K., Itzhaki O., Melnichenko J., Kubi A., Zeltzer L. A., Jacoby E., Avigdor A., Shapira Frommer R., Besser M. J. Impact of cryopreservation on CAR T production and clinical response. Front. Oncol. 2022;12:1024362. doi: 10.3389/fonc.2022.1024362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cliff E. R. S., Kelkar A. H., Russler-Germain D. A., Tessema F. A., Raymakers A. J. N., Feldman W. B., Kesselheim A. S. High cost of chimeric antigen receptor T-cells: challenges and solutions. Am. Soc. Clin. Oncol Educ. Book. 2023;43:e397912. doi: 10.1200/EDBK_397912. [DOI] [PubMed] [Google Scholar]
  7. Deng Y., Liu X., Jian X., Zhang Y., Hou Y., Hou S., Qi F., Xiao S., Deng C. A novel cryopreservation solution for adipose tissue based on metformin. Stem Cell Res. Ther. 2025;16:20. doi: 10.1186/s13287-025-04142-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doyle J. P., Giannouli P., Martin E. J., Brooks M., Morris E. R. Effect of sugars, galactose content and chainlength on freeze-thaw gelation of galactomannans. Carbohydr. Polym. 2006;64:391–401. doi: 10.1016/j.carbpol.2005.12.019. [DOI] [Google Scholar]
  9. Elavia N., McManus A., Highfill S. L., Ren J., Shah N. N., Fry T. J., Brudno J., Kochenderfer J. N., Stroncek D., Panch S. R. The post-thaw recovery of cryopreserved Chimeric Antigen Receptor (CAR) T-cells during manufacture is better than that of cryopreserved peripheral blood CD3+ cells. Blood. 2017;130:4475. [Google Scholar]
  10. Esmeryan K. D., Fedchenko Y. I., Chaushev T. A. Probing the impact of commercial cryoprotectants and freezing technique on the motility of human spermatozoa cryopreserved onto extremely water-repellent soot-coated surfaces. Cryobiology. 2025;118:105195. doi: 10.1016/j.cryobiol.2025.105195. [DOI] [PubMed] [Google Scholar]
  11. Ferrero C., Zaritzky N. E. Effect of freezing rate and frozen storage on starch-sucrose-hydrocolloid systems. J. Sci. Food Agric. 2000;80:2149–2158. doi: 10.1002/1097-0010(200011)80:14<2149::AID-JSFA759>3.0.CO;2-B. [DOI] [Google Scholar]
  12. Gao D., Critser J. K. Mechanisms of cryoinjury in living cells. ILAR J. 2000;41:187–196. doi: 10.1093/ilar.41.4.187. [DOI] [PubMed] [Google Scholar]
  13. Geginat J., Lanzavecchia A., Sallusto F. Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokines. Blood. 2003;101:4260–4266. doi: 10.1182/blood-2002-11-3577. [DOI] [PubMed] [Google Scholar]
  14. Gokulanathan A., Mo H.-H., Park Y. Glucose influence cold tolerance in the fall armyworm, Spodoptera frugiperda via trehalase gene expression. Sci. Rep. 2024;14:27334. doi: 10.1038/s41598-024-79082-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gonzalez-Martinez N., Gibson M. I. Post-thaw application of ROCK-inhibitors increases cryopreserved T-cell yield. RSC Med. Chem. 2023;14:2058–2067. doi: 10.1039/D3MD00378G. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gramatzki M., Strong D. M., Grove S. B., Bonnard G. D. Cryopreserved human cultured T cells as responder cells for the quantitative measurement of interleukin-2: improvement of the assay. J. Immunol. Methods. 1982;53:209–220. doi: 10.1016/0022-1759(82)90143-0. [DOI] [PubMed] [Google Scholar]
  17. Hauptmann A., Podgoršek K., Kuzman D., čič S., Sr, Hoelzl G., Loerting T. Impact of buffer, protein concentration and sucrose addition on the aggregation and particle formation during freezing and thawing. Pharm. Res. 2018;35:101. doi: 10.1007/s11095-018-2378-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hong M., Clubb J. D., Chen Y. Y. Engineering CAR-T cells for next-generation cancer therapy. Cancer Cell. 2020;38:473–488. doi: 10.1016/j.ccell.2020.07.005. [DOI] [PubMed] [Google Scholar]
  19. Izaguirre-Pérez N., Ligero G., Aguilar-Solana P. A., Carrillo-Ávila J. A., Rodriguez-Reyes C. R., Biunno I., Aguilar-Quesada R., Catalina P. Trehalose cryopreservation of human mesenchymal stem cells from cord tissue. Biopreserv. Biobank. 2025;23:374–382. doi: 10.1089/bio.2024.0025. [DOI] [PubMed] [Google Scholar]
  20. Jung S. E., Kim M., Ahn J. S., Kim Y. H., Kim B. J., Yun M. H., Auh J. H., Ryu B. Y. Effect of equilibration time and temperature on murine spermatogonial stem cell cryopreservation. Biopreserv. Biobank. 2020;18:213–221. doi: 10.1089/bio.2019.0116. [DOI] [PubMed] [Google Scholar]
  21. Kim K.-J., Lee Y.-A., Kim B.-J., Kim Y.-H., Kim B.-G., Kang H.-G., Jung S.-E., Choi S.-H., Schmidt J. A., Ryu B.-Y. Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing. Cryobiology. 2015;70:175–183. doi: 10.1016/j.cryobiol.2015.02.007. [DOI] [PubMed] [Google Scholar]
  22. Labanieh L., Majzner R. G., Mackall C. L. Programming CAR-T cells to kill cancer. Nat. Biomed. Eng. 2018;2:377–391. doi: 10.1038/s41551-018-0235-9. [DOI] [PubMed] [Google Scholar]
  23. Lee Y.-A., Kim Y.-H., Ha S.-J., Kim B.-J., Kim K.-J., Jung M.-S., Kim B.-G., Ryu B.-Y. Effect of sugar molecules on the cryopreservation of mouse spermatogonial stem cells. Fertil. Steril. 2014;101:1165–1175.e5. doi: 10.1016/j.fertnstert.2013.12.033. [DOI] [PubMed] [Google Scholar]
  24. Lemieux J., Jobin C., Simard C., Néron S. A global look into human T cell subsets before and after cryopreservation using multiparametric flow cytometry and two-dimensional visualization analysis. J. Immunol. Methods. 2016;434:73–82. doi: 10.1016/j.jim.2016.04.010. [DOI] [PubMed] [Google Scholar]
  25. Ludgate M. E., Dryden P. R., Weetman A. P., McGregor A. M. T-cell subset analysis of cryopreserved human peripheral blood mononuclear cells. Immunol. Lett. 1983;7:119–122. doi: 10.1016/0165-2478(83)90056-1. [DOI] [PubMed] [Google Scholar]
  26. Luo Y., Wang P., Liu H., Zhu Z., Li C., Gao Y. The state of T cells before cryopreservation: effects on post-thaw proliferation and function. Cryobiology. 2017;79:65–70. doi: 10.1016/j.cryobiol.2017.08.008. [DOI] [PubMed] [Google Scholar]
  27. Mahdjoub R., Chouvenc P., Seurin M. J., Andrieu J., Briguet A. Sucrose solution freezing studied by magnetic resonance imaging. Carbohydr. Res. 2006;341:492–498. doi: 10.1016/j.carres.2006.01.005. [DOI] [PubMed] [Google Scholar]
  28. Miki T., Wong W., Zhou E., Gonzalez A., Garcia I., Grubbs B. H. Biological impact of xeno-free chemically defined cryopreservation medium on amniotic epithelial cells. Stem Cell Res. Ther. 2016;7:8. doi: 10.1186/s13287-015-0258-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Murray A., Kilbride P., Gibson M. I. Trehalose in cryopreservation. Applications, mechanisms and intracellular delivery opportunities. RSC Med. Chem. 2024;15:2980–2995. doi: 10.1039/D4MD00174E. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Oldenhof H., Gojowsky M., Wang S., Henke S., Yu C., Rohn K., Wolkers W. F., Sieme H. Osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents. Biol. Reprod. 2013;88:68. doi: 10.1095/biolreprod.112.104661. [DOI] [PubMed] [Google Scholar]
  31. Pi C.-H., Hornberger K., Dosa P., Hubel A. Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants. Cytotherapy. 2020;22:291–300. doi: 10.1016/j.jcyt.2020.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Robinson J. R. Metabolism of intracellular water. Physiol. Rev. 1960;40:112–149. doi: 10.1152/physrev.1960.40.1.112. [DOI] [PubMed] [Google Scholar]
  33. Roy A., Dutta R., Kundu N., Banik D., Sarkar N. A comparative study of the influence of sugars sucrose, trehalose, and maltose on the hydration and diffusion of DMPC lipid bilayer at complete hydration: investigation of structural and spectroscopic aspect of lipid-sugar interaction. Langmuir. 2016;32:5124–5134. doi: 10.1021/acs.langmuir.6b01115. [DOI] [PubMed] [Google Scholar]
  34. Steiner A. A., Petenusci S. O., Brentegani L. G., Branco L. G. The importance of glucose for the freezing tolerance/intolerance of the anuran amphibians Rana catesbeiana and Bufo paracnemis. Rev. Bras. Biol. 2000;60:321–328. doi: 10.1590/S0034-71082000000200017. [DOI] [PubMed] [Google Scholar]
  35. Strauss G., Hauser H. Stabilization of lipid bilayer vesicles by sucrose during freezing. Proc. Natl. Acad. Sci. U. S. A. 1986;83:2422–2426. doi: 10.1073/pnas.83.8.2422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Venkataraman M., Westerman M. P. Susceptibility of human T cells, T-cell subsets, and B cells to cryopreservation. Cryobiology. 1986;23:199–208. doi: 10.1016/0011-2240(86)90045-3. [DOI] [PubMed] [Google Scholar]
  37. Watanabe N., Mo F., McKenna M. K. Impact of manufacturing procedures on CAR T cell functionality. Front. Immunol. 2022;13:876339. doi: 10.3389/fimmu.2022.876339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wilsher S., Ismer A., Grippo A., Hoogewijs M., Bussade P., Kovacsy S. A comparison of the efficacy of three commercial human embryo vitrification kits for cryopreservation of in vivo produced equine embryos. Equine Vet. J. 2025:doi: 10.1111/evj.14539. doi: 10.1111/evj.14539. [Online ahead of print] [DOI] [PubMed] [Google Scholar]
  39. Xu H., Cao W., Huang L., Xiao M., Cao Y., Zhao L., Wang N., Zhou J. Effects of cryopreservation on chimeric antigen receptor T cell functions. Cryobiology. 2018;83:40–47. doi: 10.1016/j.cryobiol.2018.06.007. [DOI] [PubMed] [Google Scholar]
  40. Yu M., Marquez-Curtis L. A., Elliott J. A. W. Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers. Cryobiology. 2024;115:104857. doi: 10.1016/j.cryobiol.2024.104857. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

bt-33-6-963-supple.pdf (4.5MB, pdf)

Articles from Biomolecules & Therapeutics are provided here courtesy of Korean Society of Applied Pharmacology

RESOURCES