Assessment and Comparison of Viability Assays for Cellular Products

Abstract

Background aims

Accurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) or peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs, and cultured chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered -T cell products.

Methods

Viability assays, including manual trypan blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining, or cell surface markers staining in conjunction with 7AAD, Cellometer acridine orange (AO)/PI staining, and Vi-Cell BLU Analyzer, were evaluated. A viability standard was established using live and dead cell mixtures to assess the accuracy of these assays. Furthermore, precision assessment was conducted to determine the reproducibility of the viability assays. Additionally, the viability of individual cell populations from cryopreserved PBSC or PBMC apheresis products was examined.

Results

All methods provided accurate viability measurements and generated consistent and reproducible viability data. The assessed viability assays demonstrated to be reliable alternatives when evaluating the viability of fresh cellular products. However, cryopreserved products exhibited variability among the tested assays. Additionally, analyzing the viability of each subset of cryopreserved PBSC or PBMC apheresis products revealed that T cells and granulocytes were more susceptible to the freeze-thawing process, showing decreased viability.

Conclusions

The study demonstrates the importance of careful assay selection, validation, and standardization, particularly for assessing the viability of cryopreserved products. Given the complexity of cellular products, choosing a fit-for-purpose viability assay is essential.

Introduction

Cell viability is a critical quality attribute measured throughout the manufacturing process of cellular products, from the collection of starting materials to in-process and final product release testing, as well as the analysis of post-thaw cryopreserved products[1, 2]. Cell viability assays quantify the number of live and healthy cells in a given sample, a fundamental criterion for characterizing and releasing cellular products, thereby ensuring their quality, consistency, and safety[3]. Determining cell viability also helps in establishing suitable dosage during manufacturing and administration to patients. Moreover, the evaluation of viability in cryopreserved products after thawing is imperative for product release as viability often decreases following cryopreservation[4–7]. Low viability might also indicate unapparent manufacturing errors or starting material deficiencies, potentially impacting the efficacy and safety of the cellular product. Additionally, a substantial number of dead cells in the final product could trigger adverse immune responses upon patient infusion.

However, selecting an appropriate cell viability assay can be challenging due to the complexity of cellular products, limited quantities of sample, need for rapid resulting, assay cost, and assay availability. Traditional methods such as manual trypan blue (TB) exclusion are valued for their simplicity, cost-effectiveness, and versatility. However, they have inherent limitations, including subjectivity, a narrow dynamic range requiring sample dilution, and a small number of events for concentration calculation. Furthermore, these methods lack audit-proof documentation, as neither photographs nor electronic counts are recorded[8, 9]. In contrast, flow cytometry-based viability assays, utilizing nucleic acid-binding dyes such as 7-aminoactinomycin D (7-AAD) and propidium iodide (PI), provide a more objective and high-throughput approach to assess cell viability. In this method, live cells with intact membranes exclude both dyes, resulting in low fluorescence intensity, while dead or dying cells with damaged membranes uptake the dyes, yielding high fluorescence intensity[10]. Additionally, flow cytometry enables the simultaneous analysis of multiple parameters, facilitating the evaluation of both cell viability and other cellular markers[11]. This method is particularly useful for characterizing cellular product phenotypes, especially for products with non-homogeneous cell populations. In recent years, modern automated instrumentation has emerged as a complement to traditional TB counting for cell viability assessment. These instruments employ computerized technology, advanced imaging systems, and automated sample handling to enhance efficiency and reproducibility. The Cellometer and Vi–Cell BLU Cell Viability Analyzer, as two representatives of image-based instruments, are both used for the viability evaluation in cellular product manufacturing. The Cellometer acridine orange (AO)/PI staining method uses an automated cell counter that integrates fluorescence imaging and software to provide rapid and accurate cell viability measurements. Live cells stained with AO appear green while dead cells stained with PI appear red[12].

On the other hand, the Vi–Cell BLU Cell Viability Analyzer is based on the trypan blue exclusion principle and is an automated system that measures cell viability and concentration.

Previous studies have compared various viability assays for cellular products. Automated methods demonstrated comparable results to the manual assay, offering efficiency for high sample volumes [13, 14]. However, assessing viability in cryopreserved cellular products poses challenges due to debris, dead cells and other factors, potentially impacting accuracy [1]. Furthermore, methods effective for fresh cells might differ in cryopreserved samples, highlighting the necessity for comprehensive viability assay evaluation, especially in the context of post-thaw cryopreserved cellular products.

In this study, we compared the accuracy, precision, and suitability of four commonly used viability assays at our institution: manual TB exclusion method, flow cytometry-based assays using 7-AAD/PI staining, image-based assays using AO/PI staining by Cellometer, and the Vi–Cell BLU Cell Viability Analyzer. These assays were evaluated using three cellular therapy products collected or manufactured at our institution: peripheral blood stem cell (PBSC) or peripheral blood mononuclear cell (PBMC) apheresis samples, purified PBMCs, and cultured chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered -T cell products. We first assessed the accuracy and reproducibility of tested assays in measuring viability. Next, we compared the viability results obtained from fresh and cryopreserved samples using the tested assays. Additionally, we investigated the impact of cryopreservation on different cell populations. Ultimately, the results of this study may contribute in selecting a fit-for-purpose cell viability assay that delivers accurate, reliable, and robust cell viability measurements, meeting the requirements of cellular therapy manufacturing.

Materials and Methods

Specimens and study design:

The study included samples collected or manufactured at the Center for Cellular Engineering (CCE), NIH Clinical Center, between November 2020 and April 2023. A total of eighteen fresh samples, including six each from PBSC or PBMC apheresis products, purified PBMCs, and cultured CAR/TCR-T cell products, were included, along with twenty-six cryopreserved products, consisting of eight PBSC or PBMC apheresis samples, six purified PBMCs samples, and twelve cultured CAR/TCR-T cell samples. White blood cell count and concentration were measured using ADVIA 120 hematology analyzer (Siemens, Erlangen, Germany), and viability was assessed by manual TB exclusion assay, flow cytometry, Cellometer by AO/PI staining, or Vi-Cell BLU Cell Viability Analyzer. All measurements were performed in triplicate by the same operator and completed within 1.5 hours. Additionally, sixteen cryopreserved PBSC or PBMC apheresis products were collected to analyze the viability of different cell populations using flow cytometry staining for cell surface markers in conjunction with 7AAD. All subjects signed an informed consent approved by a NIH institutional review board. In this study, our main focus was to evaluate the performance of viability assays on both fresh and cryopreserved cellular products. Therefore, we did not conduct a comparison analysis of cell concentrations.

Manual trypan blue exclusion assay:

Samples were appropriately diluted in Hank’s Balanced Salt Solution (HBSS, ThermoFisher Scientific, Waltham, MA, USA) based on the concentrations obtained from ADVIA 120 hematology analyzer. The diluted samples were stained with 0.4% trypan blue (TB) solution (Lonza, Morristown, NJ, USA). To determine cell viability, the stained samples were loaded onto a disposable C-Chip Hemocytometer (InCyto, South Korea) and examined using a light microscope at 40X magnification. The total number of cells, including both unstained (viable) and blue-stained (non-viable) cells, was counted. The percentage of cell viability was calculated using the following formula: (number of viable cells/total cells) X 100.

Flow cytometry-based viability assay:

Two different flow cytometry-based viability assays were utilized in this study to assess the viability: 7-AAD/PI direct staining and cell surface markers staining in conjunction with 7AAD. For the 7-AAD/PI direct staining assay, samples were stained directly with 7-AAD (BD Biosciences, Franklin Lakes, NJ, USA) or PI (ThermoFisher Scientific) at room temperature. After an incubation period of 10 minutes for 7-AAD staining or 5 minutes for PI staining, the samples were acquired on a BD FACSCanto 10-Color flow cytometer (BD Biosciences) without washing. In the surface staining assay, PBSC or PBMC apheresis samples, purified PBMCs samples, and cultured CAR/TCR-T cell samples were subjected to staining with fluorochrome-labeled anti-human antibodies. PBSC or PBMC apheresis samples were stained with antibodies targeting CD34, CD3, CD19, CD56, CD14, CD16, CD15, and CD45 (BD Bioscience, except CD15 which was obtained from BioLegend, San Diego, CA, USA). Purified PBMCs samples were stained with antibodies targeting CD3, CD19, CD56, CD14, CD16, and CD45. Cultured CAR/TCR-T cell samples were stained with antibodies targeting CD3 and CD45. All samples were also stained with 7-AAD. After a 20-minute incubation at 4 °C, the samples were washed using the BD FACS^™ Lyse Wash Assistant (LWA, BD Biosciences) with either a washing-only program or an ACK (Ammonium-Chloride-Potassium) red blood cell (RBC) lysing program with ACK lysis buffer (ThermoFisher Scientific) if necessary. Subsequently, the samples were acquired on a BD FACSCanto 10-Color flow cytometers (BD Biosciences).

Flow cytometry data analysis was performed using the BD FACSDiva^™ software and FlowJo software (BD Biosciences). In the 7-AAD/PI direct staining assay, viable cells were identified by gating from 7-AAD/PI negative population. In the surface staining assay, cells were first gated from CD45 positive population and then viable cells were gated from 7-AAD negative population. To further assess the viability within each specific cell population, CD45 positive cells were characterized based on their respective cell population markers. The viable cells of each cell population were then analyzed by gating from the 7-AAD negative population. The gating strategy for analyzing the viability of CD3 positive T cells was shown in Supplemental Figure 1 as an example, where the 7-AAD negative population was gated from CD45 positive CD3 positive cells.

Cellometer using AO/PI viability assay:

The Cellometer Auto2000 system (Nexcelom Bioscience, Lawrence, MA, USA) was used in this study. Prior to sample analysis, the instrument was calibrated using Cellometer Check Validation Bead Solution according to the manufacturer’s instructions. Sample concentrations were adjusted with HBSS (ThermoFisher Scientific) to fit within the measurement range. Samples were stained with AO/PI dyes, loaded into the counting slide, and analyzed using the appropriate immune cell assay (high or low RBCs). Viability was reported based on the staining results.

Vi–Cell BLU Cell Viability Analyzer’s automated TB exclusion assay:

The Vi–Cell BLU Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA) was used to measure cell viability. Prior to analysis, the instrument was validated using quality control beads (4M concentration control and a 50% viability control) provided by the manufacturer. Sample concentrations were adjusted with HBSS (ThermoFisher Scientific) to fall within the measurement range. Viability measurements were then conducted accordingly.

Our preliminary data showed that the Vi–Cell BLU Cell Viability Analyzer’s automated TB exclusion method produced inaccurate viability measurements in the presence of RBCs (data not shown). Therefore, we only used this method for cultured CAR/TCR-T cell products, as apheresis products contain RBCs, and PBMCs are susceptible to RBC contamination during separation.

Generation of viability standard:

To generate dead cells, fresh purified CD4/CD8 T cells were fixed with 4% Paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, USA) for 10 minutes at room temperature, followed by permeabilization with 0.2 % Triton-X-100 (Millipore Sigma, Burlington, MA, USA). To create a range of viability standards, dead cells were mixed with live cells in serial dilutions (0%, 25%, 50%, 75%, and 100%). Viability was assessed using manual TB exclusion assay, flow cytometry, Cellometer with AO/PI staining, and the Vi-Cell BLU Cell Viability Analyzer.

Statistical analysis:

Unless otherwise indicated, all quantitative data are presented as mean ± standard deviation (SD). The precision assessment was performed by calculating the coefficient of variation (CV) using the equation: CV = (SD / Mean) X 100. Pearson correlation coefficient analysis was conducted using GraphPad Prism software to compare the viability standard with the theoretical value. A p values less than 0.05 was considered significant. Additionally, the Euclidean distance was calculated in R using the formula $\sqrt{{{\sum }_{i=1}^n\left(x_i-y_i\right)}^2}$ to quantitatively assess the agreement or discrepancy between the values obtained from each tested assay and the theoretical value. In this context, x_i represents the actual value obtained by each viability assay, while y_i represents the corresponding theoretical value. The variable n represents the number of dilutions.

RESULTS

Determination of the accuracy of viability assays

As accuracy is critical when measuring viability, our primary objective was to evaluate the accuracy of viability assays. To this end, we generated a viability standard using purified CD4/CD8 T cells. By mixing fresh cells with dead cells in serial dilutions (0%, 25%, 50%, 75%, and 100%), we obtained a range of viability levels. The viability was then measured using the manual TB exclusion assay, flow cytometry, Cellometer with AO/PI staining, and the Vi–Cell BLU Cell Viability Analyzer. To determine the correlation between the viability values obtained from these tested methods and the theoretical value, we calculated the Euclidean distance and Pearson correlation coefficient (r).

As shown in Figure 1A–1F, all tested viability assays exhibited a strong correlation with the theoretical value. The Pearson correlation coefficients (r) were notably high, with values of 0.9982 for the manual TB exclusion assay, 0.9813 for flow cytometry staining for cell surface markers in conjunction with 7AAD, 0.9985 for flow cytometry 7-AAD direct staining, 0.9991 for flow cytometry PI direct staining, 0.9985 for Cellometer, and 0.9971 for Vi–Cell BLU Cell Viability Analyzer. The p-values obtained for these correlations were all less than 0.01.

Figure 1. Determination of the accuracy of viability assays.

A viability standard was generated by mixing live T cells with dead T cells in serial dilution (0%, 25%, 50%, 75%, and 100%). Viability measurements were obtained using manual trypan blue (TB) exclusion assay (A), flow cytometry staining for cell surface markers in conjunction with 7-aminoactinomycin D (7-AAD) (B), flow cytometry with 7-AAD direct staining (C), flow cytometry with propidium iodide (PI) direct staining (D), Cellometer with acridine orange (AO)/PI staining (E), and the Vi-Cell BLU Cell Viability Analyzer (F). All measurements were performed in duplicate. Data are shown as the correlation between the values obtained from each tested assay (red line) and the theoretical value (grey line). The Pearson correlation coefficients (r) and p value were calculated. (G) The Euclidean distance was calculated between the values obtained from each tested assay and the theoretical value and presented as mean ± SD.

Additionally, the Euclidean distance was calculated as a measure of correlation quality between the actual and theoretical viabilities. The Euclidean distance measures the length of a line segment between two points in Euclidean space. A smaller Euclidean distance indicates a better correlation. Analysis revealed that the manual TB exclusion assay had the lowest Euclidean distance, followed by flow cytometry 7-AAD/PI direct staining, flow cytometry staining for cell surface markers in conjunction with 7AAD, Vi–Cell BLU Cell Viability Analyzer, and Cellometer (Figure 1G).

Overall, these findings indicate that all tested viability assays are capable of accurately measuring viability, as demonstrated by the high correlation values and low Euclidean distances. The results affirm the reliability and suitability of these methods for viability assessments.

Evaluation of the precision of viability assays

A crucial aspect in evaluating viability assays is to ensure that they generate consistent and reliable viability data. To address this, we conducted an analysis to assess the reproducibility of the viability assays on both fresh and cryopreserved cellular products. Each assay was performed in triplicate, and the CV was calculated as a measure of precision for each tested assay.

The CV values obtained from all the assays were found to be below 5%. However, when comparing the CV values among the assays, the manual TB exclusion assay and Cellometer exhibited slightly higher CV values compared to the other tested methods. Specifically, the manual TB exclusion assay showed the highest CV, followed by Cellometer and the flow assays on fresh products. Similarly, the Cellometer assay had the highest CV when assessing the cryopreserved products, followed by the manual TB exclusion assay and the flow assays. In addition, the CV value measured by the Vi-Cell Blu analyzer on CAR/TCR-T cell products was below 1%. A summary of the mean CV values for each assay across all samples was provided in the Table 1.

Table 1.

Comparison of Coefficient of Variance (CV) among Different Viability Assays for Cellular Product Viability Measurements

	Manual TB	Flow Cytometry Staining for Cell Surface Markers in Conjunction with 7-AAD	Flow Cytometry 7-AAD Direct Staining	Flow Cytometry PI Direct Staining	Cellometer AO/PI Staining	Vi-Cell Blu Analyzer
Fresh PBSC or PBMC Apheresis (n=6)	1.14	0.26	0.04	0.08	0.71	NA
Fresh purified PBMCs (n=6)	2.01	0.20	0.26	0.19	0.92	NA
Fresh CAR/TCR-T cell (n=6)	1.40	0.17	0.26	0.16	1.39	0.93
Cryo PBSC or PBMC Apheresis (n=8)	2.51	0.55	0.41	0.47	3.52	NA
Cryo PBMCs (n=6)	2.10	0.35	0.49	0.55	2.88	NA
Cryo CAR/TCR-T cell (n=12)	2.02	1.17	0.61	0.45	2.68	0.73

PBSC: Peripheral blood stem cell

PBMC: Peripheral blood mononuclear cell

CAR/TCR-T cell: Chimeric antigen receptor (CAR) or T-cell receptor (TCR)-T cell

Cryo: Cryopreserved

TB: Trypan blue

7-AAD: 7-aminoactinomycin D

PI: Propidium iodide

AO: Acridine orange

NA: Not applicable

Taken together, these findings demonstrated that these assays, particularly the flow cytometry assay and the Vi-Cell Blu analyzer, have the capability to consistently generate reproducible viability data.

Examination of viability assays on fresh cellular products

Next, we evaluated these viability assays on fresh cellular products. In total, eighteen fresh samples were analyzed, including six samples each from PBSC or PBMC apheresis products, purified PBMCs and cultured CAR/TCR-T cell products. Most cells in these samples were healthy and alive, indicating high-quality products. The viability of fresh apheresis and PBMCs products, as measured by different assays, exceeded 90%, as shown in Figure 2A and 2B. However, the viability of some cultured CAR/TCR-T cell products was below 90%, likely due to the cell death during culture (Figure 2C). Among all the assays tested, Cellometer consistently yielded the lowest viability values across the three product types. Conversely, the flow cytometry 7-AAD direct staining method demonstrated the highest viability for fresh apheresis and purified PBMCs, while the manual TB exclusion assay produced the highest viability for fresh CAR/TCR-T cells. The mean and standard deviation (SD) of each assay in three fresh product types were summarized in the Supplemental Table 1.

Figure 2. Examination of viability assays on fresh cellular products.

A total of eighteen fresh samples were collected, including six samples each from peripheral blood stem cell (PBSC) or peripheral blood mononuclear cell (PBMC) apheresis products (A, D), purified PBMCs (B, E) and cultured chimeric antigen receptor (CAR) or T-cell receptor (TCR)-T cell products (C, F). Viability was measured using trypan blue (TB) exclusion assay, flow cytometry staining for cell surface markers in conjunction with 7-aminoactinomycin D (7-AAD), flow cytometry with 7-AAD direct staining, flow cytometry with propidium iodide (PI) direct staining, Cellometer with acridine orange (AO)/PI staining, and Vi-Cell BLU Cell Viability Analyzer. All measurements were performed in triplicate. Data are shown as mean ± SD (A, B, C). Additionally, the median value of all tested assays was calculated and the viability results obtained by each assay were compared with the median value. A viability value falling within ±15% of the median value was considered acceptable. The absolute difference between the values obtained from each tested assay and the median value was calculated and presented as mean ± SD (D, E, F).

To further validate these findings, we calculated the median value of all tested assays and compared the viability results obtained by each assay with the median value. A viability value falling within ±15% of the median value was considered acceptable. We found that all tested methods produced values within ±15% of the median value, meeting the acceptance criteria. Among them, the flow cytometry assay, using 7-AAD/PI direct staining for the apheresis products, as well as the Cellometer assay for purified PBMCs and the manual TB assay for cultured CAR/TCR-T cells, showed the greatest difference compared to the median value (Figure 2D–2F). Taken together, our results demonstrate that the assessed viability assays are reliable alternatives to each other when evaluating the viability of fresh cellular products.

Evaluation of viability assays on post-thaw cryopreserved cellular products

Cryopreservation is a commonly used to store starting material, intermediates or final products generated in the cellular manufacturing process. However, post-thaw viability is often affected, making it crucial to evaluate the viability of these products before further manufacturing or patient infusion[15]. In this study, we assessed the viability on a total of twenty-six cryopreserved products, including eight PBSC or PBMC apheresis samples, six purified PBMCs samples and twelve cultured CAR/TCR-T cell samples. The viability of the cryopreserved samples after thawing, as measured by different assays, exhibited variability across the tested products, ranging from 47.37% to 95.47% in apheresis, 56.73% to 97.33% in PBMCs, and 60.36% to 96.33% in CAR/TCR-T cell products. Consistent with the findings from fresh samples, the viability measured by the Cellometer assay yielded the lowest values for all three product types, while the highest viability values were obtained from flow cytometry 7-AAD direct staining for apheresis samples, flow staining for cell surface markers in conjunction with 7AAD for purified PBMCs samples, and the Vi-Cell Blu analyzer for CAR/TCR-T cell samples (Figure 3A–3C). Additional details regarding the mean and SD of each assay for the three cryopreserved product types were summarized in the Supplemental Table 2.

Figure 3. Evaluation of viability assays on post-thaw cryopreserved cellular products.

A total of twenty-six cryopreserved products were collected, including eight peripheral blood stem cell (PBSC) or peripheral blood mononuclear cell (PBMC) apheresis samples (A, D), six purified PBMCs samples (B, E) and twelve cultured chimeric antigen receptor (CAR) or T-cell receptor (TCR)-T cell samples (C, F). After thawing, viability was measured using trypan blue (TB) exclusion assay, flow cytometry staining for cell surface markers in conjunction with 7-aminoactinomycin D (7-AAD), flow cytometry with 7-AAD direct staining, flow cytometry with propidium iodide (PI) direct staining, Cellometer with acridine orange (AO)/PI staining, and Vi-Cell BLU Cell Viability Analyzer. All measurements were performed in triplicate. Data are shown as mean ± SD (A, B, C). Additionally, the median value of all tested assays was calculated and the viability results obtained by each assay were compared with the median value. A viability value falling within ±15% of the median value was considered acceptable. The absolute difference between the values obtained from each tested assay and the median value was calculated and presented as mean ± SD (D, E, F). The red dot indicates the value of the absolute difference that failed to meet the acceptance criteria.

We further compared the viability results obtained from each assay with the median value of all tested assays on the cryopreserved samples. In contrast to the fresh products, greater discrepancies were observed between the tested assays and the median value for the cryopreserved products, particularly for the CAR/TCR-T cell samples. As depicted in Figure 3D and 3E, the viability values obtained from Cellometer in one apheresis sample and one PBMCs sample exceeded the acceptable ±15% range compared to the median value. Additionally, three CAR/TCR-T cell samples measured by the manual TB exclusion assay, flow cytometry staining for cell surface markers in conjunction with 7AAD, and Vi-Cell Blu analyzer failed to meet the acceptance criteria (Figure 3F). Furthermore, the Cellometer assay for apheresis and PBMCs, as well as the Vi-Cell Blu analyzer for cultured CAR/TCR-T cells, had the greatest difference compared to the median value among all the assays. Collectively, these results indicate the increased variability among viability assays when assessing cryopreserved cellular products.

Viability assessment of individual cell populations in post-thaw cryopreserved products

The cellular products consist of a range of cell types. Previous studies have indicated that the sensitivity and recovery rate of different cell types can vary significantly following the cryopreservation and thawing processes[16–19]. Therefore, it is crucial to investigate the impact of cryopreservation on the viability of distinct cell populations. In this study, we specifically utilized cryopreserved PBSC or PBMC apheresis products for analysis, as they contain a majority of the cell types present in cellular products.

A total of sixteen cryopreserved PBSC or PBMC apheresis products were analyzed using flow cytometry staining for cell surface markers in conjunction with 7AAD. The cell types evaluated included hematopoietic stem cells, lymphocytes, natural killer (NK) cells, monocytes, and granulocytes. As shown in Figure 4A and Figure 4B, the average viability of total CD45⁺ population was 71.90% (ranging from 54.14% to 91.68%). Upon analyzing each cell population individually, it was observed that the viability percentages were as follows: CD34⁺ stem cells 87.27% (ranging from 68.51% to 96.86%), CD3⁺ T cells 52.14% (ranging from 24.86% to 78.84%), CD19⁺ B cells 87.44% (ranging from 49.07% to 98.66%), CD3⁻CD56⁺ NK cells 89.59% (ranging from 83.80% to 94.81%), CD3⁻CD56⁻CD14/CD16⁺ monocytes 88.34% (ranging from 60.56% to 98.64%), and CD15⁺ granulocytes 56.68% (ranging from 26.35% to 92.93%). To eliminate the possibility of inherent viability differences among cell subsets before cryopreservation, we examined six fresh apheresis products. Our findings revealed no significant differences in viability levels among individual cell subsets in these fresh samples, indicating that the disparities observed in the post-thaw products were caused by the cryopreservation and thawing process (data not shown).

Figure 4. Viability assessment of individual cell populations in post-thaw cryopreserved products.

(A) Viability was analyzed in a total of sixteen post-thaw cryopreserved peripheral blood stem cell (PBSC) or peripheral blood mononuclear cell (PBMC) apheresis products using flow cytometry staining for cell surface markers in conjunction with 7-aminoactinomycin D (7-AAD). The viability of specific cell populations, including CD45⁺ total white blood cells, CD34⁺ stem cells, CD3⁺ T cells, CD19⁺ B cells, CD3⁻CD56⁺ NK cells, CD3⁻CD56⁻CD14/CD16⁺ monocytes, and CD15⁺ granulocytes, was determined. (B) The average viability of each cell population was summarized. Data are shown as mean ± SD.

Our results demonstrate that T cells and granulocytes from PBSC or PBMC apheresis products are more susceptible to the freeze-thawing process, resulting in decreased viability. In contrast, other cell populations, such as stem cells, B cells, NK cells, and monocytes, appear to be more stable after thawing, displaying higher viability rates. These results emphasize the importance of considering cell type-specific responses after thawing to optimize the cryopreservation protocols and ensure the efficacy of cellular products.

Discussion

Measurement of cell viability is required as a release criterion for cellular products[20]. A simple, accurate and reproducible test can serve as a fundamental tool for the manufacturing of cell therapy products. At our institution, we typically assess the viability of PBSC or PBMC apheresis and purified PBMCs products using flow cytometry staining for cell surface markers in conjunction with 7AAD, while manual TB exclusion assay is the standard method for cultured products like CAR/TCR-T cells.

Additionally, our facility is equipped with automated instruments like the Cellometer and Vi–Cell BLU Cell Viability Analyzer, which provide alternative options for measuring viability. In this study, we aimed to evaluate the performance of these four viability assays in measuring cell viability for both fresh and cryopreserved cellular products. We conducted an extensive analysis, including eighteen fresh cellular products and twenty-six cryopreserved cellular products, ensuring a thorough and in-depth comparison study.

Given the crucial importance of accuracy in assessing viability, we first evaluated the accuracy of different test methods. In line with previous studies investigating viability assays, we developed a viability standard by creating serial dilutions of a mixture containing live cells and fixed dead cells[13, 21]. It is worth noting that the dead cells induced by fixation may not precisely mimic the dead cells in actual samples. However, we considered this viability standard to be an effective tool for evaluating the accuracy of the tested methods. Our findings demonstrated that all tested methods consistently provided accurate viability measurements, as evidenced by the strong correlation observed and the lower Euclidean distances between the viability value obtained from each tested assay and the theoretical value. These results strongly support the reliability and validity of the assessed methods in accurately determining cellular viability.

To determine the reproducibility of the viability assays, we conducted a precision assessment. The CV values were calculated for each assay, serving as an indicator of assay precision. A CV value below 5% is generally considered indicative of good reproducibility. Encouragingly, all the tested viability assays exhibited CV values below 5%, suggesting that they provide reliable and consistent results. However, consistent with previous reports, the manual TB exclusion assay demonstrated a higher CV value compared to the other assays, on both fresh and cryopreserved samples. This finding supports the notion that traditional TB assays have limitations due to their subjective nature, where the interpretation of results can be influenced by the observer[8]. Surprisingly, the Cellometer AO/PI assay also displayed a higher CV value, particularly when applied to cryopreserved samples. This observation raises the possibility that the stability of the AO/PI dyes used in the staining process may affect the assay’s precision. Furthermore, the Cellometer requires operators to adjust focus for image capture, introducing another source of variation. It is important to note that this study was conducted within our laboratory, and thus, the potential for inter-laboratory variation should be acknowledged in the future studies for a comprehensive evaluation of these assays’ reproducibility.

At our institution, PBSC or PBMC apheresis, purified PBMCs, and cultured CAR/TCR-T cells are three commonly collected or manufactured cellular products. In order to comprehensively evaluate the viability of these products, we conducted viability assessments on both fresh and cryopreserved samples. As expectedly, fresh products exhibited great viability above 90%, with only a few CAR/TCR-T cell products showing lower viability. In contrast, the cryopreserved samples demonstrated a range of viability across the tested sample. It is well known that cryopreservation can lead to the formation of ice crystals and this coupled with the toxicity of certain cryoprotectants, causing cell death[22, 23]. Our data further supported the notion that the cryopreservation process can have varying impacts on viability upon thawing. Notably, among the evaluated assays, the Cellometer AO/PI assay consistently yielded the lowest viability values for both fresh and cryopreserved sample across all three product types. This could be due to the specific parameters and algorithms used by Cellometer for viability assessment, which may result in a higher sensitivity to detecting non-viable cells.

To further validate our findings, we calculated the median value for all tested assays and compared the viability results obtained by each assay with the median value. The ±15% threshold was used to define acceptable variability values. Remarkably, for fresh products all the tested methods produced values within this range, meeting the predefined acceptance criteria. However, greater discrepancies for cryopreserved products were overserved compared to fresh samples. This discrepancy was especially pronounced in CAR/TCR-T cell samples, indicating that the variability among viability assays increases when assessing cryopreserved cellular products, potentially due to the impact of cryopreservation on cellular integrity and assay compatibility.

Furthermore, we identified that certain assays, such as the Cellometer assay for cryopreserved apheresis and PBMCs and the Vi-Cell Blu analyzer for cryopreserved cultured CAR/TCR-T cells, exhibited larger deviations from the median value than other assays. These assays may require additional evaluation and optimization for assessing viability in cryopreserved samples

Previous studies have revealed the heterogeneous responses of distinct cell types to cryopreservation and thawing, involving variations in both sensitivity and recovery rates[16–19]. In this study, we sought to identify vulnerable cell types that may require special considerations during cryopreservation. Moreover, understanding the stability of different cell populations after thawing can help optimize cryopreservation protocols and enhance the overall success of cellular-based therapies and research studies. Our results indicate a greater vulnerability of T cells and granulocytes in cryopreserved PBSC or PBMC apheresis products to the freeze-thawing process, leading to decreased viability. Conversely, we observed enhanced stability and higher viability rates in other cell populations, such as stem cells, B cells, NK cells, and monocytes, after thawing. The differences observed in the post-thaw product were due to the cryopreservation and thawing process as no significant differences in viability among individual cell subsets in fresh apheresis products were identified. Notably, these findings also provide an explanation for the discrepancies observed in the viability assessment of cryopreserved CAR/TCR-T cell products. It is well recognized that granulocytes have poor recovery after freezing, a trend consistent with our findings [24–26]. In our study, T cells exhibited an average viability of 52.14% (ranging from 24.86% to 78.84%), which was lower than the viability reported by Pt et al[27]. This disparity could be attributed to the diverse sample pool we utilized, including both healthy donors and patients, whereas Pt et al. exclusively used healthy donors for their study. Furthermore, our study involved PBSC or PBMC apheresis products, unlike Pt et al.’s use of PBMCs, which might contribute to the observed differences in viability assessments.

Additionally, it is worth noting that the viability of cryopreserved cell subsets can be impacted over time after thawing. Bahsoun et al. previously reported that the impaired viability of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) that recovered after 24 hours, suggesting that the recovery rate of each subset of cryopreserved products could potentially change over a period of thawing[28]. Furthermore, several studies have indicated that NK cells exhibit good viability immediately after thawing, but the recovery rate decreases after overnight culture[29–32]. Therefore, further investigation is warranted to explore the stability and recovery rate of cryopreserved products following thawing, which can greatly inform and refine the cryopreservation protocols to optimize the viability and functionality of cellular products.

Taken together, our study highlights the reliable alternatives provided by the assessed viability assays when evaluating the viability of fresh cellular products. However, the selection of viability assays for cryopreserved cellular products needs careful consideration. Given the intricacy of cellular products, factors such as sample type, time constraints, assay cost, and accessibility must be considered to choose viability assays that align with their unique characteristics. The advantages and disadvantages of each viability assay tested are summarized in the Supplementary Table 3.

Conclusions

In summary, our study comprehensively evaluated four commonly utilized viability assays within our institute for measuring viability in both fresh and cryopreserved cellular products. Considering the complexity of cellular products, it becomes essential to select a fit-for-purpose viability assay, particularly for cryopreserved products, which can deliver accurate, reliable, and robust cell viability measurements, effectively meeting the rigorous demands of cellular therapy manufacturing.