Lab-specific variations are common in how cryopreserved PBMC are thawed and such variations have the potential to affect the performance of lymphocytes in downstream immune assays [1,13,14,17]. We formally assessed how the rapidity and temperature at which thawing is performed affects the viability and functionality of the cryopreserved PBMC. Specifically, cells were subjected to either “cold-” or “warm” processing. For “cold processing”, cryovials were thawed until the last ice crystals were visible, and ice-chilled media was added immediately to the ice-cold cells. For “warm processing”, the cryovials were incubated in a 37 °C bead bath for 10 minutes which raised the temperature of the cells in the cryovial to 37 °C. For both conditions, we also compared the impact of adding the media rapidly or slowly, as specified in the Experimental Section. Thus, a total of four thawing conditions were tested: (1) slow addition of 37 °C warm media to cells at 37 °C; (2) rapid addition of 37 °C media to cells at 37 °C; (3) slow addition of ice-chilled media to ice-cold cells, and; (4) rapid addition of ice-chilled media to ice-cold cells. PBMC derived from 7 donors were subjected in parallel to these thawing conditions and were evaluated for viability in initial experiments.

Data depicted in Figure 1 shows, of the four conditions tested, adding ice-chilled media rapidly to ice-cold cells strongly reduced the recovery of viable PBMC. Slow addition of ice-chilled media overcame this drop in viability. Therefore, in spite of the use of the cold media, its slow addition overcomes the drop in viability of the cells. Warm media, regardless of the speed of addition, provided comparably high levels of cell viability (Figure 1).

For further evaluation of thawing conditions, we chose to compare the least optimal condition (adding ice-chilled media rapidly to ice-cold cells) with an optimal warm condition (37 °C media added slowly to PBMC that have been warmed to 37 °C). The prior experiment was repeated using cryopreserved PBMC derived from ten additional donors.

In agreement with the original finding, the data from these ten donors also showed that the use of cold media significantly reduced (p = 0.002, Wilcoxon signed rank test) the viability of thawed PBMC (Figure 2A).

Next, we sought to establish if the different thawing conditions affected CD8 and CD4 cell functionality. To evaluate CD8 cell functionality, we used the CEF peptide pool comprising of defined Class I peptides from CMV, EBV, and influenza viruses that elicit recall CD8 responses in most donors [18,19] that elicited varying magnitudes of CEF responses among our donors. CEF-specific responses were assayed in 8 out of 10 donors due to paucity of PBMC from two donors (8 data points). To similarly test anamnestic CD4 cell responses, we first tested a variety of protein antigens including those derived from Mumps, Mosquito, Dust Mite, Candida, and PPD among the study subjects. In our cohort of 10 donors, most represented CD4 responses were toward mumps antigens (5 donors) and mosquito (6 donors) with lower number of donors responding to the other antigens. Only one donor had significant levels of candida antigen-specific responses. For further analysis of CD4 functionality, we concatenated responses from the most represented mosquito and mumps antigens (11 data points). Figure 2B shows while CD8 cells showed a tendency toward reduced functionality under “cold processing” conditions, this difference was not statistically significant. In stark contrast, CD4-driven responses directed against mumps and mosquito antigens were significantly impaired under “cold processing” conditions (Figure 2C). Specifically, when compared to “warm processing”, “cold processing” resulted in a 1.98-fold diminution in the median CD8 responses and 12.05-fold diminution in median CD4 responses.

Wash steps during the PBMC thawing procedure aim to remove the cryopreservant DMSO which is toxic for lymphoid cells [20,21] and reduces their functionality in bioassays [22]. While increasing the number of washes will dilute out the residual DMSO from thawed PBMC, it also increases the procedural time and leads to cell loss. Potentially, the prolonged processing may also affect the viability and functionality of the cells. To directly address if increasing the number of washing steps affects the viability and/or functionality of recovered PBMC, we thawed the cells under “warm processing” conditions and assessed viability and functionality of the cells following either 1 or 2 washes.

Data shown in Figure 3 suggests two washes significantly increase the viability of the recovered PBMC when compared to samples that underwent a single wash (Figure 3A). While functionality of the CD8 compartment (CEF peptide pool responses) was not significantly affected by the number of washes performed (Figure 3B) the functionality of CD4 cells was significantly impaired when the second wash was not implemented (Figure 3C).

Because prolonged exposure to DMSO is known to be toxic to varying lineages of cells [20,21,23] we sought to assess how long PBMC can be exposed to the DMSO once thawed and warmed to 37 °C before their viability and/or functionality is significantly affected. Following removal from liquid Nitrogen and thawing, the PBMC containing cryovials were warmed to 37 °C in the bead bath for 10 minutes, and kept at 37 °C for additional 0, 30 or 60 minutes before the cells were diluted and washed two times with warm media, counted and tested in ELISPOT assays.

While incubating the cryovials at 37 °C caused a significant decline in viability of thawed PBMC (Figure 4A), this diminution was minimal throughout the entire 60 minute incubation period in absolute terms. Specifically, while we observed a median 96.6% viability when DMSO was washed immediately post-thaw (zero time point), viability was at 94.2% when the washes were performed at 30 minutes (2.4% drop from zero time point) and at 93.0% when washes were performed at 60minutes (further 1.2% drop). Thus, the total decline in viability over a 60 minute period was only 3.6%.

When assessed for T cell functionality, we observed no significant reduction in the CD8 functionality over the entire 60 min incubation in the presence of DMSO (Figure 4B). Similarly, we found no significant reduction in CD4 functionality during the initial 30 minutes (median SFU of 144.6 at zero time point vs. 137.6 SFU per 10⁶ cells). However, further exposure to DMSO for up to 60 minutes (Figure 4C) resulted in significant reduction in CD4 cell functionality detected in downstream ELISPOT assays (median of 68.9 SFU / 10⁶ cells). These data suggests a safe window of up to 30 minutes may exist where DMSO exposure does not significantly affect either CD4 or CD8 functionality in ELISPOT assays with the CD8 positive cells being even more refractive to longer incubation in the presence of DMSO.

Cryopreserved PBMC were selected from a characterized library (CTL-CP1) available through Cellular Technologies Limited (CTL, Shaker Heights, Ohio). PBMC cryovials were transferred from liquid Nitrogen vapor phase to dry ice in styrofoam containers and were thawed by placing them for 10 minutes in a CTL Bead Bath™ (CTL-BB-001). Cryovials were inverted twice to resuspend the PBMC and the cells from a single cryovial (10 million cells) were transferred into a 15 mL Falcon tube utilizing a wide-bore 2 mL pipette. We found the use of nominal buffers such as PBS during washing resulted in suboptimal cell viability when compared to the use of CTL Anti-Aggregate Wash™ Medium (CTL-AA-005) which was specifically formulated to ensure optimal viability and functionality of thawed PBMC. This formulation includes supplementary nutrients and Benzonase (a DNAase shown to reduce clumping of cells during the thawing procedure). Cryovials were rinsed by adding 1mL CTL Anti-Aggregate Wash™ Medium to recover residual cells. For the “slow” wash, additional 8 mL CTL Anti-Aggregate Wash™ Medium was added at a rate of 1 mL/5 seconds. For “fast wash, the 8 mL wash medium was added in a shot. The CTL Anti-Aggregate Wash™ Medium was added either at 37 °C (“warm”) or ice-chilled (“cold”) as specified in the figures. An aliquot of diluted PBMC was counted by fluorescence microscopy using acridine orange and ethidium bromide to stain live and dead cells respectively. PBMC were washed by centrifugation at 330g for 10 min either once or twice in 10 mL of 37 °C or ice-chilled media. Cells were resuspended at a final concentration of 3 × 10⁶ PBMC/mL in CTL-Test™ Medium (CTLT-005).

Human Interferon-γ ImmunoSpot kits (CTL-HIFNG-1/5M) were acquired from C.T.L and the ELISPOT assay was performed according to the manufacturer’s recommendations. The membranes were not pre-wetted with ethanol as it is not required nor recommended for this kit. The following antigens were added first at appropriate concentrations in 100 µL/per well: CEF peptide pool (CTL-CEF-002; 2 µg/mL); mosquito antigen (Greer Labs, Lenoir, NC; 100 µg/mL) and mumps antigen (BioWhittaker, Walkersville, MD, USA, Lot# IV0094, 1:80 dilution). Plates were maintained at 37 °C in a CO₂ incubator until the cells were ready for plating. Thawed PBMC were adjusted in CTL Test Medium (CTLT-005) at 3 million PBMC/mL and the cells were plated at 100 µL/well (300,000 cells/well) using wide-bore pipette tips. Plates were gently tapped on each side to ensure even distribution of the cells and incubated for 24 hours at 37 °C in a CO₂ incubator. Following completion of the ELISPOT protocol according to kit recommendations, the plates were air dried in a laminar flow hood prior to analysis.

ELISPOT plates were scanned and analyzed using an ImmunoSpot S6 Core Reader (C.T.L). Spot Forming Units (SFU) were automatically calculated by the ImmunoSpot software for each antigen stimulation condition and the media (negative) control using the SmartCount™ and Autogate™ functions [35]. Data are presented as mean SFU per 10⁶ PBMC induced by the specified antigen minus the SFU count in the negative control. In all experiments, the negative control was less than 10 SFU per 10⁶ PBMC.