Peripheral blood samples from three adult volunteers were obtained at a single time point using three 8mL Cell Preparation Tubes (CPT) with sodium heparin (BD Biosciences) and three 10mL Vacutainer plastic blood collection tubes with sodium heparin (BD Biosciences). Each sample was processed within 30 minutes of collection and processed in parallel by three different operators who each isolated PBMCs from each donor using two methods: isolation by CPT tubes and isolation using LP tubes (Axis-Shield) (see below). De-identified blood samples were obtained from healthy adult volunteers following the guidelines of the Environmental Health and Safety Biosafety program of Stanford University. Donors provided informed consent in accordance with IRB protocols approved by the Stanford University Administrative Panel on Human Subjects Research.
PBMC isolation with CPT
Whole blood was collected into an 8mL sodium heparinized CPT vacutainer and inverted multiple times to ensure homogenization of the sodium heparin anti-coagulant and blood. The vacutainer was centrifuged at 1700 x g for 20 minutes at 21°C, resulting in the separation of contents into layers: the upper layer containing plasma with a cloudy band of PBMCs, the middle layer containing a think polyester resin, and the lower layer containing erythrocytes and granulocytes. After centrifugation, the CPT was gently inverted 10 times to resuspend PBMCs and plasma, then decanted into a 15mL conical tube pre-filled with 8mL of Dulbecco’s Phosphate-Buffered Saline (PBS, Thermo Scientific). The capped 15mL conical tube was mixed by inversion and centrifuged at 300 x g for 15 minutes at 4°C. The supernatant was carefully aspirated, the cell pellet was resuspended in 10mL of fresh PBS, and centrifuged for a subsequent 10 minutes at 300 x g at 4°C. Following this centrifugation step, the supernatant was again carefully aspirated without disturbing the cell pellet, and the pelleted PBMCs were resuspended in 3mL of fetal bovine serum (FBS, HyClone). 500µL of cells were aliquoted into six 2mL cryovials, each pre-filled with 500µL of freezing medium composed of FBS and 20% DMSO (Sigma Hybri-Max D2650) for a final DMSO concentration of 10%. The filled cryovials were placed in CoolCell freezing containers (Biocision) at -80°C for 24 hours prior to transfer to liquid nitrogen for long-term storage. Residual volume of PBMCs in FBS were retained and used for counting and viability assessment.
PBMC isolation with Lymphoprep Tubes
In preparation for PBMC isolation, Lymphoprep tubes were first centrifuged at 500 x g for 1 minute at 21°C to ensure displacement of the pre-filled Lymphoprep media to the bottom of the tube. Whole blood was collected by phlebotomy into a 10mL sodium heparin vacutainer and inverted to ensure mixing of blood with anti-coagulant. Nine milliliters of blood were transferred into a 50mL conical tube pre-filled with 10mL PBS for a ~1:1 dilution. This diluted blood was decanted into the upper chamber of a Lymphoprep tube, separated by a porous polyethylene insert from the lower Ficoll-Paque-containing chamber. The filled Lymphoprep tube was centrifuged at 800 x g for 20 minutes 21°C with no brake. Centrifugation resulted in an upper layer of plasma with a cloudy band of PBMCs and a lower layer of erythrocytes and polymorphonuclear cells, separated by the polyethylene insert. After centrifugation, the cloudy PBMC band was collected using a sterile transfer pipette and added to a 15mL conical tube pre-filled with 10mL of PBS. The capped 15mL conical tube was mixed by inversion and centrifuged at 300 x g for 15 minutes at 4°C. The supernatant was carefully aspirated, the cell pellet was resuspended in 10mL of fresh PBS, and centrifuged for a subsequent 10 minutes at 300 x g at 4°C. Following this centrifugation step, the supernatant was again carefully aspirated without disturbing the cell pellet, and PBMCs were resuspended in 3mL of FBS. 500µL of cells were aliquoted into six 2 mL cryovials each pre-filled with 500µL of freezing medium composed of FBS and 20% DMSO for a final DMSO concentration of 10%. The filled cryovials were placed in CoolCell freezing containers at -80°C for 24 hours prior to transfer to liquid nitrogen for long-term storage. Residual volume of PBMCs in FBS were retained and used for counting and viability assessment.
Pre-Freeze Cell Counting and Viability
Residual volumes of PBMCs in FBS from each sample were used to assess the PBMC yield and viability prior to cryopreservation. PBMCs were stained with acridine orange (AO) and propidium iodine (PI) to identify all nucleated cells and dead nucleated cells, respectively. Equal volumes of PBMCs and Cellometer Via Stain AOPI staining Solution (Nexelom Biosciences) were combined for staining. 20µL of stained PBMCs were dispensed into a disposable Cellometer Counting Chamber and placed into a Cellometer K2 instrument (Nexelom Biosciences) for automated counting and viability determination. Where indicated, additional PBMC counts were obtained using a Sysmex XE-2100 automated hematology analyzer (Sysmex Corporation) according to manufacturer’s protocol.
Thawing of PBMCs
Frozen cryovials of PBMCs were taken from liquid nitrogen storage were placed in a 37°C water bath until thawed and the contents were transferred to a 15mL conical tube containing pre-warmed media composed of complete RPMI (cRPMI) (RPMI-1640 [Gibco] containing 10% FBS [HyClone] and Benzonase nuclease [³ 250 units/µL, Sigma]). Samples were then centrifuged at 300 x g for 5 minutes at room temperature and resuspended thoroughly in pre-warmed cRPMI and Benzonase. A small aliquot of the cell suspension was aliquoted for counting and viability staining. The remaining cells for antibody staining were centrifuged again at 300 x g for 5 minutes at room temperature, resuspended in cRPMI, and placed at room temperature until cell counting was complete. Cells were divided into two aliquots, one for surface antibody staining analysis and one for intracellular antibody staining analysis.
Post-Thaw Cell Counting and Viability
Leukocyte viability stain (detectable in near-IR channels) was added into each 100µL cell suspension aliquot and incubated at 21°C for 10 minutes. 3mL of FBS stain buffer (BD Biosciences) was added to each cell sample for washing and centrifuged at 300 x g for 5 minutes at 21°C. The supernatant was aspirated, and cells were resuspended in a solution containing a final concentration of 1025 beads/µL (123count beads, eBioscience) and 1.6% PFA (Electron Microscopy Sciences). Following a 10-minute incubation at 21°C, viability was assessed by running the samples through a flow cytometer. Where indicated, additional cell counts were obtained using a Sysmex XE-2100 hematology analyzer according to manufacturer’s protocol.
Surface staining of PBMCs
Cells were washed in 150µL of BSA Stain Buffer (BD Biosciences) in preparation of phenotype staining. 50µL of Fc block cocktail (composed of Fc Block [Biolegend], Live Dead Near IR Fixable Viability Stain (Thermo Fisher), BSA Stain Buffer) was added to each sample and incubated for 10 minutes at room temperature. After the incubation period, cells were stained with a monoclonal antibody cocktail containing CD19-PacBlue, CD45-PE, CD45RA-FITC, CD4-PerCP-Cy5.5, CD16-PE, CD56-PE, CD19-PE-Cy7, and CD3-APC. All antibodies were received from Biolegend and diluted at a 1:50 ratio in BSA stain buffer. Following a 30-minute staining period at 4°C, samples were washed by centrifuging at 400 x g for 5 minutes (room temperature) and resuspended in BSA stain buffer. Following the third centrifugation step, PBMCs were resuspended in BSA Stain Buffer and 16% PFA was added to each sample for a final concentration of 1.6% PFA. After a 10-minute fixation period at 21°C, cells were washed and resuspended in BSA Stain Buffer for flow cytometry acquisition.
Stimulation of PBMCs for cytokine production
Stimulation of PBMCs using a CEF (cytomegalovirus, Epstein-Barr virus, influenza virus) peptide pool (Anaspec; 1µg/mL) and a Phorbol 12-myristtate 13-acetate (PMA; Sigma; 50ng/mL) + Ionomycin (Thermo Fisher; 1µg/mL) cocktail was performed and compared to an unstimulated control. Following the thawing and counting procedure, cells were centrifuged and resuspended in 350µL of fresh cRPMI. Cells were then transferred into a 96-well, round-bottom plate and rested at 37°C for up to 2 hours. During this period, a 2X CEF cocktail (4µL of 5000x CEF and 20µL of 1000X Golgi Plug in 10mL of cRPMI) and a 2X PMA-I cocktail (20µL of 100x PMA, 20µL of 1000x Ionomyocin, and 20µL of 1000x Golgi Plug in 10mL of cRPMI) were prepared. Following the resting incubation, 100µL of stimulation cocktail was added to the appropriate cell samples and placed in a 37°C incubator for 4 hours.
Live Stain and Fixation
Cells were transferred from the 96 well plate to a PCR plate (Thermo Fisher) and centrifuged at 400 x g for 5 minutes at room temperature, making sure to aspirate the resulting supernatant in between each transfer. Once all cells were transferred to the new PCR plate, cells were centrifuged and resuspended in FBS Stain Buffer (BD Biosciences). FC Block Cocktail (Human TruStain FcX Fc Receptor Blocking Solution [Biolegend] and FBS Stain Buffer) was added to each sample and incubated for 10 minutes at 21°C. Following the blocking incubation, Live Stain Cocktail, composed of Live/Dead-Aqua (1:100, Biolegend), CD8-PerCP-Cy5.5 (1:20, Biolegend), and CD14-PE-Dazzle594 (1:20, Biolegend) in FBS Stain Buffer, was added to each well, and the plate was incubated for a subsequent 30 minutes at 21°C. Samples were washed twice via centrifugation and resuspension in 150µL of FBS Stain Buffer, and finally resuspended in 60µL of FBS Stain Buffer. 16% PFA was added to each sample for a final concentration of 1.6% PFA and incubated for 10 minutes at 21°C to fix the cells. After fixation, cells were washed as before and resuspended in FBS Stain Buffer in preparation for intracellular staining.
Permeabilization and Intracellular Staining of PBMCs
Cold 1X Perm Buffer I (BD Biosciences) was added to each sample and mixed by pipetting. Samples were centrifuged, aspirated, and resuspended in 150µL of cold Perm Buffer I before a 10-minute incubation period at 4°C. Incubated samples were centrifuged and resuspended in an intracellular staining cocktail composed of CD3-APC (1:400, Biolegend), CD4-PE (1:200, Biolegend), CD8-PerCP-Cy5.5 (5µL, BD Biosciences), IFNγ-A488 (1:20, Biolegend), IL-4-BV421 (1:50, Biolegend), and IL-17-PE-Cy7 (1:50, Biolegend) diluted in Perm Buffer for 60 minutes at 4°C. Following incubation, samples were washed twice using Perm Buffer I and once using FBS Stain Buffer (centrifugation at 400 x g for 5 minutes at 21°C). After the final washing step, each sample was resuspended in cold FBS stain buffer and was characterized using flow cytometry.
Flow cytometry samples were acquired on a custom 4-laser BD LSRII flow cytometer equipped with the BD FACSDiva Software (BD Biosciences).
Cellular yield was defined as the number of cells per mL of input blood both before and after cryopreservation while cellular recovery was the ratio of cells present after cryopreservation compared to prior. The cell viability was calculated as the percentage of living cells compared to the total cell count. Analysis of flow cytometry data for comparison of immune cell populations and cytokine productions were conducted using Cytobank.
Statistical comparisons between CPT and LP tubes were performed using R. The Wilcoxon Signed Rank Test was used to compare the means of repeated measurements on a single sample. Three-way ANOVA tests were used to identify any significant differences due to donor, operator, or method variability. For each parameter, a sum square was calculated for each independent variable (donor, method, and operator) to identify the deviation of each observation from the mean. The sum square contribution of each independent variable is calculated as sum square divided by the total sum square. From each sum square, a calculated F value was used to determine if a relationship exists between the dependent variables and the donor, method, and operator. A p-value is associated with the F-value, and any p-values less than 0.05 were considered to be statistically significant.