Isolation and freezing of human peripheral blood mononuclear cells from pregnant patients

Summary To analyze immune cell populations accurately, a large number of Peripheral Blood Mononuclear Cells (PBMCs) must be obtained from blood samples. Traditional manual isolation and SepMateTM isolation of PBMCs consistently yield blood-stained plasma layers and overall low numbers of CD4+ and CD8+ cells. Here, we describe an optimized protocol, using PBS with EDTA to increase PBMC yield from pregnant patients. This protocol enables analysis of CD4+, CD8+, and Regulatory T Cells and is potentially applicable to any immune cell population. For complete details on the use and execution of this protocol, please refer to the SepMateTM website https://www.stemcell.com/products/brands/SepMateTM-pbmc-isolation.html.


SUMMARY
To analyze immune cell populations accurately, a large number of Peripheral Blood Mononuclear Cells (PBMCs) must be obtained from blood samples. Traditional manual isolation and SepMate TM isolation of PBMCs consistently yield bloodstained plasma layers and overall low numbers of CD4+ and CD8+ cells. Here, we describe an optimized protocol, using PBS with EDTA to increase PBMC yield from pregnant patients. This protocol enables analysis of CD4+, CD8+, and Regulatory T Cells and is potentially applicable to any immune cell population. For complete details on the use and execution of this protocol, please refer to the SepMateTM website https://www.stemcell.com/products/brands/SepMateTMpbmc-isolation.html.

BEFORE YOU BEGIN
The protocol below describes the specific steps for isolating and freezing PBMCs from pregnant patients. The challenge of the procoagulant state of pregnancy (Sanches et al., 2020) (including an increase in clotting factors, reduced anticoagulant and fibrinolytic activity) made density centrifugation difficult, as the plasma layer was stained with blood. We found that use of EDTA in PBS to the blood before centrifugation improved separation and yield. Though blood is collected in EDTA lined tubes as standard, without dilution with further EDTA, the plasma layer remained stained. This protocol was developed to analyze CD4+, CD8+ and Regulatory T Cell (Treg) populations, though this protocol may be useful for the analysis of any immune cell line in pregnancy. Alternatives: The SepMate TM -50 (Stemcell Technologies, Cat No: 154500) tubes were used for efficiency and time saving, though we found that their use did not significantly increase the number of PBMCs isolated. Manual separation, whereby layering the diluted blood and removal of the buffy coat is done by hand, may be performed. Proper technique is required to prevent contamination during layering and avoid disturbing the gradient when manually isolating the buffy coat layer after centrifugation.

Isolation of PBMCs from peripheral blood
Timing: 1 h This step describes how to isolate PBMCs from whole blood using SepMate TM Tubes (Blood et al., 2016) 1. Invert Ficoll-Paque PLUS several times. 2. Place 17 mL Ficoll-Paque PLUS medium into the SepMateä-50 tube by carefully pipetting it through the central hole of the SepMateä-50 insert (Ficoll to be just above the insert). 3. Transfer the blood to a separate 50 mL Falcon tube in a sterile manner, i.e., working in the hood, using sterile pipetting technique. 4. Dilute the blood 1:1 in PBS-EDTA with 2% FBS, close the tube and mix by careful inversion a. (In our study): add 16.5 mL blood +16.5 mL PBS-EDTA 2% FBS to reach a total of 33)mL; if less than 16.5 mL of blood is available, you can mix it with PBS-EDTA in a 1:1 ratio b. SepMateä-50 is designed to process 4-17 mL of an initial blood sample. 5. Keeping the SepMateä tube vertical, add the diluted sample by pipetting it down the side of the tube. The sample will mix with the density gradient medium above the insert. Take care not to pipette the diluted sample directly through the central hole. 6. Centrifuge at 12003g for 20 min at room temperature, with the brake on 7 (out of 9).
Note: Each tube contains four layers after the centrifugation, the yellow plasma layer on the top, the white MNC (mononuclear cells) layer, the Ficoll layer, and the red cell layer on the bottom.
7. Remove some of the plasma layer without removing the interface with the PBMCs, to allow better washing of the cells. 8. Pour off the top layer, which contains the enriched MNCs, into a new 50 mL Falcon tube. Do not hold the SepMateä tube in the inverted position for more than 2 s as this may disrupt the filter within the SepMate TM tube, which ensures the Ficoll and red cell layer stay at the bottom. 9. Register the cryovials using the barcode and record them as PBMCs. 10. Fill up the 50 mL falcon tube with RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 733-1690) and carefully mix. 11. Centrifuge at 3003g at 20 C for 8 min and discard the supernatant. 12. Repeat steps 10 and 11 using 5-10 mL PBS-EDTA with 2% FBS. 13. Centrifuge for 8 min at 3003g at 20 C and remove as much of the PBS-EDTA with 2%FBS as possible by pouring quickly without disturbing the pellet.

Freezing of PBMCs
Timing: $ 24 h 19. Leave the cryo boxes in a À80 C freezer for 12-24 h (max four days), then transfer the cryovials into the liquid nitrogen tank.

EXPECTED OUTCOMES
The described protocol provides a reproducible method for isolating PBMCs, including lympho-

cytes (T cells, B cells, and Natural Killer cells), monocytes, and dendritic cells in pregnant patients.
After thawing, the number of PBMCs obtained were counted using CountBright Absolute Counting Beads (Thermo Fisher Scientific, C36950) as per the manufacturer's instructions, using the BD Fortessa cell analyzer (BD Biosciences) combined with the BD FACSDiva software (BD Biosciences). Our protocol yielded an average of 53 3 10 6 PBMCs per healthy pregnant patient, though there will be biological variability in each individual.
Protocols for isolation of PBMCs have been optimized in non-pregnant patients, and there is no current standard method for isolation of PBMCs in pregnant patients. The standard protocol of dilution of collected blood with PBS alone yielded low numbers of CD4+ and CD8+ T cells in our pregnant population. We noted that after density centrifugation, the plasma layer was blood-stained indicating potential contamination of the PBMCs. This can be seen in Figure 1, which demonstrates a comparison of diluted blood samples with PBS without EDTA ( Figure 1A) and the addition of ll OPEN ACCESS 1 mM of EDTA ( Figure 1B). We considered the differences in haemostatic profile in pregnant women vs healthy controls -during pregnancy, the significant increase in clotting factors, reduced quantity of anticoagulants and reduced fibrinolytic activity creates a state of hypercoagulability (Sanches et al., 2020). This likely makes density centrifugation more complicated, and why the anticoagulant EDTA helped reduce blood contamination. The PBMCs obtained were counted using CountBright Absolute Counting beads and compared between the two conditions. In our case, the addition of EDTA to PBS increased the number of viable PBMCs obtained by an average of 15 3 10 6 cells (PBS: 38 3 10 6 G 16 3 10 6 , EDTA + PBS:53 3 10 6 G 8 3 10 6 cells) (Figure 2). The difference was not significant (p=0.2208), though upon analysis with flow cytometry, the addition of EDTA to PBS allowed a higher frequency of CD4+ T cells and CD8+ T cells to be isolated compared to PBS alone. (Figure 3)

LIMITATIONS
We recognize this protocol has limitations. An area of consideration is the effect of cryopreservation on the viability and function of the PBMCs. In studies comparing frozen PBMCs vs fresh PBMCs, fresh PBMCs yield more viable cells (Weinberg et al., 2009). Ficoll isolation and cryopreservation affect the ratio of plasmacytoid to conventional dendritic cell subsets and their chemokine receptor expression. Some studies have also found frozen PBMCs to spontaneously secrete more IL-6, IFN-g, TNF-a. (Mallone et al., 2011) While there is likely reduced viability and altered functioning, processing and freezing is sometimes necessary and allow for batch analysis of PBMCs at single laboratories, reducing confounding factors. Though there are differences in the absolute number of viable cells, results of assays performed using frozen PBMCs correlate well with fresh PBMCs, and differences when present are consistent across subjects. (Weinberg et al., 2009) Another line of caution is that EDTA may not be the preferred anticoagulant for PBMC isolation for some applications. For example, it has been shown that natural killer cytotoxicity is dramatically TROUBLESHOOTING Problem 1 Incomplete separation of whole blood into layers (step 6).

Potential solution
Ensure whole blood is fresh and uncoagulated (use of EDTA tubes, appropriate agitation, and storage at room temperature) Reduce ejection speed of pipette and tilt SepMate TM tube. This will help to layer the whole blood onto the Ficoll slowly.

Problem 2 Decreased viability after freezing the cells (step 14)
Potential solution When you cryopreserve cells, make sure that you work quickly and efficiently  . Density centrifugation was completed using the SepMate TM tubes, and samples were frozen. Patient samples isolated using PBS or PBS containing EDTA were later thawed and analyzed using flow cytometry. Single live cells contained within samples were analyzed using the expression of CD4 and CD8 in the PBS sample to gate for CD4+ and CD8+ T cells and in an EDTA with PBS sample.

OPEN ACCESS
Always work on ice and avoid leaving the cells in the freezing medium at room temperature after resuspension.

Problem 3
Decreased viability after thawing the cells (when using own protocol)

Potential solution
When thawing, make sure that you work fast and efficient Submerge the cryovial with the PBMCs vial in a 37 C water bath for about 1 min.
Using the medium of your choice, add 1 mL of warmed (37 C) medium in the cryovial with the thawed PBMCs using a transfer pipette.
The thawed PBMCs can be poured in a 15 mL conical Falcon TM tube (catalog number 11507411) with 5 mL of warmed (37 C) medium.
Rinse the cryovials once with 1 mL of medium.
Incubate for 5 min, in the 15 mL conical Falcon TM tube (catalog number 11507411), in the 37 C water bath.
Centrifuge for 5 min at 3003g at room temperature.
Pour off the supernatant and proceed to further downstream applications.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr Panicos Shangaris, Email: panicos.shangaris@kcl.ac.uk

Materials availability
This study did not generate new unique reagents.

Data and code availability
The published article includes all datasets generated or analyzed during this study.