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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Cryobiology. 2021 Jan 21;99:78–87. doi: 10.1016/j.cryobiol.2021.01.011

Human serum albumin and chromatin condensation rescue ex vivo expanded γδ T cells from the effects of cryopreservation

Rebecca E Burnham 1,2, Donald Tope 1, Gianna Branella 1,3, Erich Williams 1, Christopher B Doering 1, H Trent Spencer 1
PMCID: PMC7941345  NIHMSID: NIHMS1665325  PMID: 33485898

Abstract

Clinical applications of gamma delta (γδ) T cells have advanced from initial interest in expanding γδ T cells in vivo to the development of a manufacturing process for the ex vivo expansion. To develop an “off-the-shelf” allogeneic γδ T cell product, the cell manufacturing process must be optimized to include cryopreservation. It is known that cryopreservation can dramatically reduce viability of primary cells and other cell types after thawing, although the exact effects of cryopreservation on γδ T cell health and functionality have not yet been characterized. Our aim was to characterize the effects of a freeze/thaw cycle on γδ T cells and to develop an optimized protocol for cryopreservation. γδ T cells were expanded under serum-free conditions, using a good manufacturing practice (GMP) compliant protocol developed by our lab. We observed that cryopreservation reduced cell survival and increased the percentage of apoptotic cells, two measures that could not be improved through the use of 5 GMP compliant freezing media. The choice of thawing medium, specifically human albumin (HSA), improved γδ T cell viability and in addition, chromatin condensation prior to freezing increased cell viability after thawing, which could not be further improved with the use of a general caspase inhibitor. Finally, we found that cryopreserved cells had depolarized mitochondrial membranes and reduced cytotoxicity when tested against a range of leukemia cell lines. These studies provide a detailed analysis of the effects of cryopreservation on γδ T cells and provide methods for improving viability in the post-thaw period.

Keywords: Gamma delta T cells, cryopreservation, human serum albumin, chromatin condensation, allogeneic cell therapy

Introduction

Gamma delta (γδ) T cells are a unique subset of T cells that provide a promising avenue for the development of an allogeneic cell immunotherapy for use against both hematological and solid tumors. Unlike αβ T cells, γδ T cells have the ability to recognize malignant cells independently of Human Leukocyte Antigen (HLA) molecules on antigen-presenting cells [15; 19; 31; 47], and thus, present a low risk of graft versus host disease (GVHD) in an allogeneic setting [10]. The use of γδ T cells has been investigated in a number of clinical trials, yet significant challenges remain in regards [1] to the manufacturing protocol used for the expansion of these cells. Additionally, there is a lack of information detailing the effects of cryopreservation on γδ T cell health and functionality [25]. Cryopreservation is a critical process in the development of a commercial cell therapy and further investigation is necessary to ensure that the use of cryopreserved γδ T cells are a viable option for therapy.

The use of γδ T cells has clinical applications in a variety of cancers [4; 23; 27; 41; 49], yet only two studies have explored the impact of cryopreservation on γδ T cells [6; 49]. Although both reports found that γδ T cells expanded from peripheral blood mononuclear cells (PBMCs) remained cytotoxic after cryopreservation, neither provided an in depth characterization of the effects of cryopreservation. Additionally, neither used an αβ T cell-depleted γδ T cell product, a necessary step for the use of an allogeneic therapy, suggesting that these results might not translate to a clinically relevant allo-γδ T cell product. Patients enrolled in clinical trials using γδ T cells receive on average between three to ten doses of cells throughout the course of their treatment [5; 20; 32; 39], a strategy that poses many disadvantages when using fresh γδ T cells. Using a cryopreserved cell product could overcome disadvantages including the potential for delayed treatment if manufacturing is unsuccessful or time limitations on safety and quality control testing. The use of cryopreserved γδ T cells would eliminate these disadvantages, providing an “off-the-shelf” immunotherapy product.

In this study, we assessed the effects of cryopreservation on γδ T cell health and functionality, hypothesizing that the negative effects of cryopreservation could be reduced once a better understanding was achieved. We used a protocol, readily adaptable to good manufacturing practice (GMP) guidelines, developed by our lab to expand γδ T cells [44]. Initially, we characterized the viability of cryopreserved cells post-thaw, then aimed to optimize the cryopreservation protocol using a variety of GMP-compliant freezing and thawing media. To further reduce the effects of cryopreservation, we tested the effectiveness of chromatin condensation and caspase inhibitors on γδ T cell viability post-thaw. Using an optimized cryopreservation protocol, we identified a potential mechanism for the initiation of apoptosis after thawing and tested the functionality of cryopreserved γδ T cells against a range of leukemia cell lines.

Methods

Expansion of γδ T cells

Whole blood was collected from healthy donors through the Emory Children’s Clinical and Translational Discovery Core (IRB0010797) and peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll-Paque Plus (GE Healthcare Life Sciences) density centrifugation. PBMCs were cultured in OpTmizer media supplemented with OpTmizer T-cell expansion supplement (Life Technologies), 1% penicillin/streptomycin, and 2 mM L-glutamine. Cell counts were performed using a Cellometer (Nexelcom) and cells were resuspended in fresh media at 1.5×106 cells/mL every 3 days. To selectively expand γδ T cells from PBMCs, 5 μM of Zoledronate and 500 IU/mL of IL-2 were added on day 0 and 3 of expansion. αβ T cells were depleted from the culture on day 6 of expansion using a GMP-compliant protocol (Miltenyi Biotec). Briefly, cells were washed in autoMACS Rinsing Solution containing 0.5% BSA (Miltenyi Biotec), incubated with Anti-TCRα/β-Biotin for 10 minutes at 4°C, washed in autoMACS Rinsing Solution and then filtered through a 0.4μM filter. Cells were then incubated with Anti-Biotin Microbeads (Miltenyi Biotec) for 15 minutes at 4°C, washed in autoMACS Rinsing Solution and passed through an LD Column (Miltenyi Biotec). Post-depletion, 1000 IU/mL of IL-2 was added on day 6 and 9 of expansion.

Cryopreservation of γδ T cells

On day 12 of expansion, γδ T cells were washed and prepared for cryopreservation. For studies comparing different GMP-grade freezing media, γδ T cells were washed and resuspended at 1 × 107 cells/mL in one of the 5 media tested: Synth-a-Freeze Cryopreservation Medium (ThermoFisher Scientific), GIBCO Recovery Cell Culture Freezing Medium (ThermoFisher Scientific), STEM-CELLBANKER (amsbio), Nutrifreez D10 Cryopreservation Medium (Biological Industries), or 5% human albumin (2.5g human albumin in 50 mL aqueous diluent, Grifols Therapeutics) with 10% Me2SO. For all subsequent experiments, γδ T cells were cryopreserved in 5% human albumin (2.5g human albumin in 50 mL aqueous diluent, Grifols Therapeutics) with 10% Me2SO. Cells were frozen at a rate of −1°C per minute. When the cells reached −80°C, they were promptly moved to liquid nitrogen.

Thawing of γδ T cells

γδ T cells were removed from liquid nitrogen storage and thawed in a water bath (37°C). When the cells were nearly thawed, they were removed from the water bath and washed with 10X OpTmizer media pre-warmed to 37°C. Cells were spun at 300 x g and resuspended in thawing medium with 1,000 IU/mL of IL-2. As noted, OpTmizer media was used initially as the thawing medium to characterize the effects of cryopreservation on γδ T cells. Additional thawing media tested include human platelet lysate (HPL, Emory University Hospital), 5% human albumin (HSA) (2.5g human albumin in 50 mL aqueous diluent, Grifols Therapeutics), and human serum collected from the blood and pooled after the isolation of γδ T cells from whole blood. For studies comparing the effects of thawing into 1% HSA and 2.5% HSA, HSA was added to OpTmizer media at the appropriate concentrations. For all subsequent studies, γδ T cells were thawed into 5% HSA with IL-2 (1,000 IU/mL).

Flow cytometry

To prepare cells for flow cytometry, cells were washed with 10 X phosphate buffer saline (PBS) and spun at 300 x g. Cells were stained with eBioscience Fixable Viabililty dye eFluor780 (ThermoFisher) for 30 minutes. After incubation with the viability dye, cells were washed in 10 X PBS and resuspended with the appropriate antibodies. Antibodies from BD Biosciences include: BV421 Mouse Anti-Human CD3 (Clone UCHT1, 5 µL/100 µL sample), PE Mouse Anti-Human γδ TCR (Clone 11F2, 5 µL/100 µL sample for a final concentration of 1.25 µg/mL), and APC-R700 Mouse Anti-Human CD56 (Clone NCAM16.2, 5 µL/100 µL sample). APC Annexin V was acquired from Biolegend. Cells were analyzed using an Aurora (CYTEK) flow cytometer.

Chromatin Condensation

Chromatin condensation was induced by incubating γδ T cells in hyperosmotic media [12] OpTmizer media has an osmolarity of 290 mOsm. To raise the osmolarity of OpTmizer to 570 mOsm, the osmolarity required to induce chromatin condensation according to the literature, 1 mL of 20 X PBS (2.8 mM NaCl, 54 mM KCl, 30 mM KH2PO4, 120 mM Na2PO4, adjusted to pH 7.4) was added to 19 mL of OpTmizer. γδ T cells were incubated in the hyperosmotic media for 12 min, then spun down at 300 x g and prepared for cryopreservation

Caspase Inhibitor Treatment

The pan-caspase inhibitor Z-VAD-FMK (TOCRIS) was used for all caspase inhibitor studies. For studies in which cells were treated with Z-VAD-FMK prior to cryopreservation, cells were spun down for 5 min at 300 x g and resuspended in OpTmizer containing 50 μM Z-VAD-FMK for 30 min at 37°C. After this incubation, cells were spun down and immediately prepared for cryopreservation. For studies in which cells were treated with Z-VAD-FMK after cryopreservation, cells were thawed as described above and resuspended in 5% HSA containing 50 μM of Z-VAD-FMK and 1,000 IU/mL of IL-2.

Cytotoxicity assays

The in vitro cytotoxicity of fresh and cryopreserved γδ T cells was evaluated using a flow cytometry-based cytotoxicity assay. The target cell lines used in these studies include: K562, Jurkats, Kasumi-1, Nomo-1, Molt-4, MV411, and 697. The K562 and Jurkats cell lines were obtained from American Type Culture Collection (ATCC). The Nomo-1, Kasumi-1, 697, and MOLT-4 cell lines were kindly provided by the laboratory of Dr. Douglas Graham (Emory University). The MV411 cell line was kindly provided by the laboratory of Dr. Kevin Bunting (Emory University). Before the cytotoxicity assay, target cells were labelled with Violet Proliferation Dye 450 (VPD450). γδ T cells were mixed and incubated with target cells at a ratio of 1:1 or 5:1 and the cells were incubated together for 4 hours at 37°C. Target cell death was assessed via flow cytometry using eBioscience Fixable Viability Dye eFlour780 (Thermofisher) and the early apoptosis stain Annexin V (Biolegend). γδ T cell cytotoxicity was calculated by subtracting the background cell death of each target cell line from each experimental sample.

Mitochondrial Membrane Potential

The Δψm was measured by flow cytometry, using the Mitochondrial Membrane Potential Kit (Millipore Sigma) according to the manufacturer’s instructions. This kit uses JC-10, a cationic lipophilic dye that changes emission from 590 nm to 525 nm when mitochondria shift from a polarized to depolarized state. Briefly, 5 × 105 cells were spun down and resuspended in 500 μL of the JC-10 dye loading solution and incubated at 37 °C for 30 min in the dark. Cells were then spun down and resuspended in PBS containing antibodies for BV421 Mouse Anti-Human CD3 (Clone UCHT1) and PE Mouse Anti-Human γδ TCR (Clone 11F2) (BD Biosciences). Treatment with 10 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Millipore Sigma) for 30 min prior to incubation with the JC-10 dye loading solution was used as a positive control for mitochondrial membrane depolarization. Cells were analyzed using an Aurora (CYTEK) flow cytometer.

Statistical analysis

Figures and statistics were generated using GraphPad Prism Software, Version 8.2.1. Corresponding statistical tests and p-values are stated in the results. Student’s t tests were used when comparing two groups. One-way ANOVAs were used to compare three or more groups and two-way ANOVAs were used when comparing three or more groups across two variables. Post-hoc analysis was performed when the one- or two-way ANOVA produced significant results (p< 0.05). All data are reported as mean ± standard error of the mean (SEM).

Results

Characterization of γδ T cells after cryopreservation

To characterize the effects of cryopreservation on γδ T cells, we assessed changes in cell number and viability after thawing (post-thaw). Substantial cell loss was observed when γδ T cells were thawed into their standard culture medium, OpTmizer T Cell expansion medium. Two hours after thawing, 88.0% ± 5.3% of cells survived, which decreased to 68.5% ± 3.9% after four hours post-thaw. Twenty-four hours after thawing, half of the initial population of cells remained (51.6% ± 4.4%) and this percentage was stable throughout 48 hours post-thaw (47.8% ± 6.0%) (Fig. 1A). Viability, as assessed by Trypan Blue staining, was 68.9% ± 5.5% immediately post-thaw and 70.1% ± 5.7% at two hours post-thaw. However, viability decreased to 55.2% ± 3.5% at four hours post-thaw and 39.0% ± 3.0% at 24 hours post-thaw. γδ T cell viability remained stable between 24 and 48 hours (38.0% ± 2.3%) (Fig. 1B).

Figure 1.

Figure 1.

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Survival and viability of γδ T cells thawed into OpTmizer media. A) Cell counts were performed at 0, 2, 4, 24, and 48 hours post-thaw and the percent recovery was calculated by normalizing counts to the number of cells recovered at 0 hours post-thaw. B) Viability of γδ T cells was measured via Trypan Blue Staining at 0, 2, 4, 24, and 48 hours post-thaw. C) Representative flow cytometry plots visualizing the shift in cell populations up to 24 hours post-thaw by Annexin V and eFluor780 (Live/Dead viability stain) staining. D) Quantification of the percentage of γδ T cells in culture staining positively for Annexin V.

γδ T cell viability was further assessed by flow cytometry (Fig. 1C). Immediately after thawing, 50.5% ± 3.4% of γδ T cells stained positively for Annexin V. Two hours later, 63.4% ± 7.0% of γδ T cells stained positively for Annexin V, which increased slightly at four hours post-thaw (68.5% ± 6.4%) (Fig. 1D). Twenty-four hours post-thaw, 79.5% ± 5.8% of the culture was comprised of apoptotic cells (Fig. 1D). Based on these data, the aim of our following experiments was to determine if the viability of γδ T cells could be increased in the post-thaw period.

Comparison of good manufacturing practice (GMP) grade freezing media

In this study, five different good manufacturing practice (GMP)-grade freezing media were tested to determine if any provided benefit to cell viability after thawing compared to the standard freezing medium of 5% HSA 10% Me2SO. None of the GMP-grade freezing media tested, including Synth-a-Freeze, Recovery Cell Culture, Stem-Cell Banker, Nutrifreeze, or Cryostor CS10 increased the percentage of cells recovered immediately post-thaw (one-way ANOVA, p= 0.79) (Fig. 2A). Additionally, none of the GMP-grade freezing media provided benefit in terms of decreasing the percentage of Annexin V+ γδ T cells at 0, 2, or 4 hours post-thaw when compared to 5% HSA 10% Me2SO (mixed-effects analysis, p= 0.86) (Fig. 2B).

Figure 2.

Figure 2.

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Cyropreservation of γδ T cells in different GMP grade freezing media. In this experiment, γδ T cells were frozen in either Synth-a-Freeze, Recovery Cell Culture, Stem-Cell Banker, Nutrifreeze, Cryostor CS10, or 5% HSA and 10% Me2SO and thawed into OpTmizer media. A) The percentage of cells recovered post-thaw after γδ T cells were cryopreserved into different freezing media. B) The percentage of γδ T cells staining positively for Annexin V at 0, 2, and 4 hours post-thaw.

Thawing medium impacts γδ T cell viability post-thaw

To determine if the type of thawing media used could influence cell viability, γδ T cells were thawed into their culture medium, OpTmizer T cell medium, and three human-derived products: 100% human platelet lysate (HPL), 5% human albumin (HSA), and 100% human serum (Serum). Thawing γδ T cells into 5% HSA, but not HPL or Serum, significantly decreased the percentage of Annexin V+ γδ T cells up to four hours after thawing (Fig. 3A). There was a significant increase in the percentage of apoptotic γδ T cells at 2 and 4 hours post-thaw, as compared to 0 hours post-thaw, when cells were thawed into OpTmizer (one-way ANOVA and Tukey’s multiple comparisons test, p= 0.03and p= 0.03), HPL (one-way ANOVA and Tukey’s multiple comparisons test, p= 0.003 and p= 0.005), and 5% HSA (one-way ANOVA and Tukey’s multiple comparisons test, p=0.02 and p= 0.002) (Fig. 3B).

Figure 3.

Figure 3.

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Characterization of γδ T cells frozen in 5% HSA and 10% Me2SO and thawed into different solutions. A) Representative flow cytometry plots visualizing the percentage of live and apoptotic γδ T cells 4 hours post-thaw when thawed into OpTmizer media, human platelet lysate (HPL), 5% human albumin (HSA), and human serum (Serum). B) Quantification of the percentage of Annexin V+ γδ T cells thawed into OpTmizer, HPL, 5% HSA, and S at 0, 2, and 4 hours post-thaw. C) Comparison of the percentage of Annexin V+ γδ T cells in culture at 4 hours post-thaw when thawed into different solutions. D) The percentage of γδ T cells in culture, assessed by flow cytometry, over the course of 4 hours post-thaw. E) Comparison of the percentage of Annexin V+ γδ T cells at 0, 2, and 4 hours post-thaw when thawed into OpTmizer + 1% HSA (1% HSA), OpTmizer + 2.5% HSA (2.5% HSA), or 5% HSA.

Immediately post-thaw, there was no significant difference in the percentage of apoptotic γδ T cells for the various thawing media tested. However, there were significantly fewer apoptotic γδ T cells at 2 and 4 hours (Fig. 3C) post-thaw when cells were thawed into 5% HSA as compared to thawing into OpTmizer, HPL, and Serum (one-way ANOVA, p= 0.0003, p=0.003 and p= 0.0002).

In addition to the difference in the percentage of apoptotic cells in culture post-thaw, the choice of thawing medium also impacted the percentage of γδ T cells in culture after thawing. For cells thawed into OpTmizer, there was a significant decrease in the percentage of γδ T cells between 0 and 4 hours post-thaw and 2 and 4 hours post-thaw (one-way ANOVA and Tukey’s multiple comparisons test, p = 0.001 and p= 0.001). Cells thawed into HPL showed similar decreases in the percentage of γδ T cells in culture, with significant decreases between 0 and 4 hours post and 2 and 4 hours after thawing (one-way ANOVA and Tukey’s multiple comparisons test, p<0.0001 and p = 0.0002). There was no change in the percentage of γδ T cells in culture at 2 or 4 hours post-thaw for cells thawed into 5% HSA (one-way ANOVA, p= 0.09) or serum (one-way ANOVA, p=0.10) (Fig. 3D).

To determine if HSA could be used as a supplement in OpTmizer after thawing, we compared the effects of 1% HSA in OpTmizer (1% HSA) and 2.5% HSA in OpTmizer (2.5% HSA) to 5% HSA between 0 and 4 hours post-thaw. There were significantly fewer apoptotic γδ T cells for cells thawed into 5% HSA as compared to 1% HSA at 0, 2, and 4 hours post-thaw (Tukey’s post-hoc multiple comparisons test, p= 0.002, p< 0.0001, and p< 0.0001, respectively). Cells thawed into 2.5% HSA had significantly lower levels of apoptosis at 2 and 4 hours post-thaw compared to cells thawed into 1% HSA (two-way ANOVA and Tukey’s post-hoc multiple comparisons test, p= 0.02 and p= 0.03). Additionally, cells thawed into 2.5% HSA had significantly higher levels of apoptosis when compared to cells in 5% HSA at 2 and 4 hours post-thaw (Tukey’s post-hoc multiple comparisons test, p= 0.001 and p= 0.0008) (Fig. 3E).

Chromatin condensation improves γδ T cell viability post-thaw

Previous studies using normal human foreskin fibroblasts (NHDF) cells showed benefits to cell survival if chromatin condensation, induced by incubating cells in a hypertonic solution, was performed prior to cryopreservation [12]. Therefore, chromatin condensation was performed prior to cryopreservation to determine if it could provide protection to γδ T cell viability post-thaw. Representative flow cytometry images show that chromatin condensation decreased levels of apoptotic γδ T cells immediately post-thaw (Fig. 4A). Chromatin condensation decreased the percentage of apoptotic γδ T cells by an average of 10% immediately post-thaw into 5% HSA (Tukey’s post-hoc multiple comparisons test, p= 0.0004); however there was no advantage at 2 or 4 hours post-thaw (Tukey’s post-hoc multiple comparisons test, p= 0.52 and p= 0.99). Of the chromatin condensed γδ T cells that were thawed into 5% HSA, only 11.5% ± 0.62% were Annexin V+, as compared to 44.4 ± 3.2% Annexin V+ γδ T cells when neither chromatin condensed nor thawed into OpTmizer (Fig. 1D).

Figure 4.

Figure 4.

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The effect of chromatin condensation on γδ T cell viability post-thaw when thawed into 5% HSA. A) Representative flow cytometry plots visualizing the difference in the percentage of Annexin V+ γδ T cells at 0 hours post-thaw. B) Quantification of the percentage of Annexin V+ γδ T cells at 0, 2, and 4 hours post-thaw when cryopreserved under normal conditions or after chromatin condensation.

Caspase inhibitor treatment provides no additional benefit post-thaw

γδ T cells were treated with a general caspase inhibitor (CI) either prior to freezing or post-thaw to determine if inhibiting the caspase pathway could reduce levels of apoptosis. As previously described, chromatin condensation prior to freezing decreased the percentage of apoptotic γδ T cells immediately post-thaw (one-way ANOVA and Sidak’s post-hoc multiple comparisons test, p= 0.003). γδ T cells treated with the CI prior to freezing had decreased levels of apoptosis immediately post-thaw when compared to cells frozen under non condensed conditions (one-way ANOVA and Sidak’s post-hoc multiple comparisons test, p= 0.01). Cells that underwent chromatin condensation and CI treatment prior to freezing also had a decrease in levels of apoptosis immediately post-thaw (one-way ANOVA and Sidak’s post-hoc multiple comparisons test, p= 0.003). However, there was no additional benefit of CI treatment prior to cryopreservation when compared to the chromatin condensation treatment (one-way ANOVA and Sidak’s post-hoc multiple comparisons test, p= 0.88) (Fig. 5A). Additionally, neither chromatin condensation nor CI treatment provided benefit as compared to non-condensed cryopreservation conditions at 2 or 4 hours post-thaw (Fig. 5B).

Figure 5.

Figure 5.

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The effects of a general caspase inhibitor (CI) on γδ T cell viability when treated with the CI prior to and after cryopreservation. A) Quantification of the percentage of Annexin V+ γδ T cells at 0 hours post-thaw when cryopreserved under normal conditions, chromatin condensed conditions, chromatin condensed + CI treatment, or CI treatment alone. B) Comparison of the effects of chromatin condensation and CI treatment at 0, 2, and 4 hours post-thaw. C) The effects of CI treatment post-thaw on γδ T cells cryopreserved after chromatin condensation.

Because the CI treatment prior to freezing provided no benefit in the post-thaw period, we treated γδ T cells with the CI immediately post-thaw as well. In this study, γδ T cells underwent chromatin condensation prior to freezing and immediately post-thaw were resuspended in 5% HSA containing the CI. There were no statistical differences in the percentages of apoptotic γδ T cells at 0, 2, or 4 hours post-thaw when the cells were treated with the CI as compared to the cells that were not treated with the CI (two-way ANOVA, p= 0.51).

Mitochondrial membrane depolarization post-thaw

The percentage of cells with depolarized mitochondrial membranes was investigated because of the role that mitochondria play in initiating apoptosis through the intrinsic apoptotic pathway. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control to determine maximum mitochondrial depolarization for γδ T cells. Prior to freezing, 21.1% of γδ T cells had mitochondria with depolarized membranes. Immediately after thawing, this increased to 56.9% of cells with depolarized mitochondrial membranes (two-way ANOVA and Dunnett’s post-hoc multiple comparisons test, p= 0.002). At 2 and 4 hours after thawing, 74.6% (two-way ANOVA and Dunnett’s post-hoc multiple comparisons test, p< 0.0001) and 72.8% (two-way ANOVA and Dunnett’s post-hoc multiple comparisons test, p= 0.0001) of γδ T cells had mitochondria with depolarized membranes, respectively (Fig. 6A). There was no difference in the percentage of γδ T cells with depolarized mitochondrial membranes at 0, 2, or 4 hours after thawing for cells that underwent chromatin condensation prior to freezing (Condensed) and cells that did not (Not Condensed) (Two-way ANOVA, p= 0.24) (Fig. 6B).

Figure 6.

Figure 6.

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Mitochondrial membrane depolarization post-thaw. A). The percentage of chromatin condensed cells staining positively for depolarized mitochondria at 0, 2, and 4 hours after thawing, as compared to their pre-freeze (fresh) values. B) Comparison of the percentage of cells with depolarized mitochondria between cells that underwent chromatin condensation prior to freezing (Condensed) and those that did not (Not Condensed).

Cytotoxicity of γδ T cells post-thaw

γδ T cell functionality after thawing was assessed using in vitro cytotoxicity assays in which the effector: target ratios were determined based on live cell numbers via Trypan Blue staining. When γδ T cells were tested in a cytotoxicity assay immediately after thawing against K562 cells, there was a trend towards decreased cytotoxicity at a 1:1 effector:target ratio (two- way ANOVA and Sidak’s post-hoc multiple comparisons test, p= 0.08) and a significant decrease in cytotoxicity at a 5:1 ratio (two-way ANOVA and Sidak’s post-hoc multiple comparisons test, p = 0.005) when compared to γδ T cells that recovered for 1-hour post-thaw (Fig. 7A). There was no difference in γδ T cell cytotoxicity against K562 cells when they were incubated in OpTmizer or 5% HSA for the 1-hour recovery period prior to being tested in the assay (two-way ANOVA, p= 0.37) (Fig. 7B), indicating that the choice of incubation medium does not impact the cytotoxicity of viable cells that survive the thawing process.

Figure 7.

Figure 7.

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γδ T cell cytotoxicity post-thaw. A) Cytotoxicity of γδ T cells against K562 cells when the cytotoxicity assay was initiated immediately post-thaw (0 Hrs PT) or after allowing the cells to recover for 1 hour (1 Hr PT). B) Cytotoxicity of γδ T cells against K562 cells when the γδ T cells were recovered for 1 hour in OpTmizer media or 5% HSA. C) γδ T cell cytotoxicity against a range of leukemia cell lines at a target:effector ratio of 1:1. D) Analysis of the percentage of live γδ T cells (Annexin-eFluor-) after a cytotoxicity assay against K562 cells when recovered in 5% HSA for 1 hour post-thaw.

γδ T cells were tested against a panel of leukemia cell lines at a 1:1 effector:target ratio, allowing for measurement of changes in cytotoxicity prior to freezing and after thawing. γδ T cells that did not undergo chromatin condensation prior to freezing exhibited decreased cytotoxicity post-thaw against K562 (Student’s t-test, p= 0.003), Jurkats (Student’s t-test, p= 0.04), Kasumi-1 (Student’s t-test, p=0.049), Molt-4 (Student’s t-test, p= 0.006), and 697 cells (Student’s t-test, p= 0.001). There was no difference in cytotoxicity at the 1:1 ratio when γδ T cells were tested against Nomo-1 (Student’s t-test, p= 0.83) or MV411 cells (Student’s t-test, p= 0.37). Similar trends were seen in chromatin condensed γδ T cells with decreased cytotoxicity post-thaw against K562 (Student’s t-test, p= 0.003), Jurkats (Student’s t-test, p= 0.004), Kasumi-1 (Student’s t-test, p=0.004), Molt-4 (Student’s t-test, p= 0.004), and 697 cells (Student’s t-test, p= 0.0001), with no difference in cytotoxicity when tested against Nomo-1 (Student’s t-test, p= 0.32) or MV411 cells (Student’s t-test, p= 0.28). Additionally, there was no difference in cytotoxicity between chromatin condensed γδ T cells and cells that did not undergo chromatin condensation for any of the cell lines tested (Fig. 7C).

As a measure of γδ T cell health, we assessed the percentage of live γδ T cells (Annexin-eFluor780-) in the cytotoxicity assay against K562 cells when the assay was complete. When tested prior to cryopreservation, 82.2% ± 0.7% of γδ T cells were alive at the 1:1 ratio and 84.0% ± 2.0% were alive at the 5:1 ratio. When assessed after cryopreservation, γδ T cells that were resuspended in 5% HSA for a 1-hour recovery period prior to the assay had an average of 55.7% ± 2.2% live γδ T cells at the 1:1 ratio and an average of 47.6% ± 4.9% live γδ T cells at the 5:1 ratio, which were significantly decreased as compared to pre-freeze values (Sidak’s post-hoc multiple comparisons test, p= 0.0004 and p< 0.0001) (7E).

Discussion

Cryopreservation is a critical process in the development and manufacturing of off-the-shelf cell therapies. The process of freezing and thawing cells can lead to cell damage, resulting in decreased cell viability and functionality. γδ T cells have the potential to be applied as an allogeneic cell therapy, making the cryopreservation process essential to the manufacturing protocol. This study is the first report of the effects of cryopreservation on γδ T cell health and functionality and provides an optimized protocol for good manufacturing (GMP) compliant cryopreservation of γδ T cells.

The impact of cryopreservation on T cells is currently not well understood. Post-thaw viabilities of CD3+ T cells, primarily comprised of αβ T cells and a small proportion of γδ T cells, can range from 27%−93% after cryopreservation [26; 43]. Additionally, a 2.9–30-fold increase in cell death can be seen in CD3+ T cells after thawing [42]. Our studies show that cryopreservation has a dramatic effect on γδ T cell health during the post-thaw period. We observed that the majority of cryopreserved γδ T cells undergo apoptosis within four hours post-thaw. By 24 hours post-thaw, the cells are primarily apoptotic, with only 51% of cryopreserved γδ T cells surviving up to the 24-hour time point. Cryopreservation leads to significant loss of the total cell product when γδ T cells are cultured ex vivo post-thaw. However, it is unknown if the cell losses seen in an overnight culture are representative of what would occur in vivo if the cell product was injected immediately upon thawing, which requires further, ongoing, studies.

The choice of freezing medium and cryoprotective agent (CPA) used for cryopreservation is an important consideration, as the correct combination can limit osmotic stress during the freeze/thaw cycle. Cryopreserving cells without cryoprotectant results in up to 90% cell death, which can be avoided through the use of reagents such as Me2SO, Trehalose, or the antifreeze fusion protein TrxA-ApAFP752 (AFP). Me2SO is the most effective cryoprotectant in reducing cell death after cryopreservation as it penetrates the nucleus, condenses chromatin, and reduces the nuclear envelope size. AFP and Trehalose are also used, however they are less effective at condensing chromatin and result in reduced cell viability after thawing [22]. In this study, we compared five GMP quality freezing media to the standard γδ freezing medium of 5% human albumin (HSA) and 10% dimethyl sulfoxide (Me2SO). Although each of the five reagents tested are effective at preserving other cell lines throughout the cryopreservation process [17; 30; 33; 34; 35; 40], none of the cryopreservation specific media improved post-thaw recovery or viability of γδ T cells in comparison to 5% HSA 10% Me2SO. The freezing medium used to cryopreserve γδ T cells requires further optimization that could be achieved with investigation into a formula that properly mimics the intracellular composition of γδ T cells to limit osmotic stress and cell damage.

While the majority of clinical trials using T cell therapies inject cryopreserved cells immediately upon thawing [8; 9; 14; 18; 37; 45], this protocol does not allow for any post-thaw manipulations, such as genetic engineering. In an effort to increase post-thaw viability, we compared the effects of thawing cryopreserved γδ T cells into three human derived products: human platelet lysate (HPL), 5% human albumin (HSA), and human serum. Thawing γδ T cells into 5% HSA increased cell viability over the first four hours in the post-thaw period compared to the other thawing media tested. HSA is known to have anti-oxidant effects and can both limit the production of free radicals and act as a free radical scavenger [13]. Antioxidant treatments have successfully decreased the effects of oxidative stress induced by cryopreservation in other cell lines and 5% HSA may be offering protection through this pathway [2; 3; 46]. Supplementing OpTmizer with HSA was not sufficient to rescue the viability of γδ T cells in the post-thaw period, suggesting that any manipulations made to the cells after thawing would need to be performed in 5% HSA for optimal viability. Based on these studies, clinical trials using unmodified γδ T cells should consider washing and resuspending the cells in 5% HSA before injection.

Cryopreservation can induce extensive physical damage to cells through the formation of intracellular ice crystals [28; 29]. The use of CPAs, such as Me2SO, protects cells during the cryopreservation process by decreasing the formation of intracellular ice crystals [11; 38]. Me2SO influences the ability of ice crystals to form as it disrupts the hydrogen-bond networks of water, which directly impacts the formation of ice in the solution. Compared to other CPAs, solutions cryopreserved via Me2SO form smaller ice crystals, which contributes to increased viability after thawing [21]. Me2SO also offers protection during cryopreservation by penetrating the cell nucleus and effectively condensing chromatin [12; 22]. A recent report found that artificial chromatin condensation prior to freezing improves the viability of cells after thawing, offering chromatin additional protection from the effects of ice formation and cellular dehydration during the freezing process [12]. In our study, we found that chromatin condensation prior to cryopreservation did indeed increase γδ T cell viability immediately after thawing, bringing the percentage of healthy γδ T cells up to 90%. The combination of chromatin condensation prior to cryopreservation and thawing into HSA increased viability dramatically compared to our initial preoptimized conditions. Although chromatin condensation prior to cryopreservation increased cell viability after thawing, there was no benefit of this method at later timepoints, such as 24 hours post-thaw, suggesting that further optimization is necessary. As Me2SO can condense chromatin on its own, it is possible that thawing into a combination of Me2SO and HSA could offer better protection to cell viability overall.

The use of caspase inhibitors to prevent apoptosis in cryopreserved cells has been widely investigated [7; 36; 48]. Treatment with the general caspase inhibitor, Z-FAD-FMK, both prior to and after cryopreservation decreased apoptosis in γδ T cells immediately post-thaw, however it provided no additional benefit over chromatin condensation in reducing apoptosis. Caspase release can be initiated through either the extrinsic apoptotic pathway, in which there is ligation of a death receptor that initiates the caspase cascade, or through the intrinsic apoptosis pathway, where release of cytochrome C from the mitochondria activates the caspase cascade [16]. We show here that cryopreservation induces mitochondrial dysfunction, as seen by high levels of mitochondria with depolarized membranes. Taken together, these data suggest that apoptosis is occurring through the extrinsic apoptotic pathway. Cryopreservation is likely damaging the mitochondria to such an extent that it is not possible to rescue the cells from apoptosis with the use of a caspase inhibitor. Surprisingly, the combination of chromatin condensation prior to freezing, which protects the chromatin from the effects of a freeze/thaw cycle, and caspase inhibitor treatment, which protects the cells from the effects of initiation of the caspase cascade, provided no additional benefit to γδ T cells. Because the viability of γδ T cells that undergo chromatin condensation prior to cryopreservation can be as high as 90% when thawed, it is possible that viability cannot be significantly increased. Additionally, the mitochondria may be fatally damaged during the cryopreservation process, which would not allow for either treatment to be effective hours after thawing.

Although γδ T cells displayed decreased cytotoxicity post-thaw, they remained functional against a range of leukemia cell lines. Immediately post-thaw, γδ T cell cytotoxicity was greatly reduced, but increased when the cells were allowed to rest for one hour after thawing. These results are similar to a study characterizing the expansion of Natural Killer (NK) cells, which showed that cryopreserved NK cells had increased cytotoxicity when allowed to recover before the cytotoxicity assay, although they were not able to regain full cytotoxic potential as compared to fresh NK cells [24]. Because viability quickly decreases in γδ T cells after thawing, this biological response to freezing can be overcome by increasing the administered cell dose, as the remaining cells are viable and functional.

Significant progress has been made regarding the improvement of ex vivo expansion and use of γδ T cells. Moreover, clinical trials have shown that the γδ T cell therapies are safe, supporting their use in a number of therapeutic settings. This study suggests that cryopreservation significantly impacts γδ T cell health, and thus, further optimization, including comparing the safety and efficacy of cryopreserved γδ T cells to fresh γδ T cells, is necessary. A convenient solution would be to increase the cell dose for patients treated with cryopreserved cells. However, further research is needed to determine if this is a feasible route. Herein, we report a GMP compliant method for the expansion and cryopreservation of γδ T cells in which cells from healthy donors are expanded in OpTmizer with Zoledronate and IL-2, depleted of αβ T cells on day 6 of expansion, and cryopreserved on day 12 in 5% HSA and 10% Me2SO after chromatin condensation. Additionally, we show that γδ T cells should be given a recovery period for at least one hour after thawing into 5% HSA to achieve optimal cytotoxicity.

Supplementary Material

1

Funding:

These studies were funded by Curing Kids Cancer, the NIH-NCI grant 5R21CA223300, and the Peachbowl Legacy Foundation.

Abbreviations:

γδ

Gamma delta

HSA

Human albumin

GMP

Good manufacturing practice

HLA

Human leukocyte antigen

GVHD

Graft versus host disease

PBMCs

Peripheral blood mononuclear cells

HPL

Human Platelet Lysate

Serum

Human Serum

Footnotes

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Conflict of interest disclosure statement: The authors declare no conflict of interest.

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