Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners

In this protocol, we aim to provide an easy-to-follow, step-by-step tutorial on how to collect and store high viability peripheral blood and mononuclear cells, or PBMCs, from whole blood. PBMCs include lymphocytes, dendritic cells, and monocytes. Bulk PBMCs can be processed to isolate individual cell types and are commonly used in a wide variety of experiments and assays, including RNA sequencing and flow cytometry.

In addition to PBMCs, we also demonstrate how to collect EDTA plasma and whole buffy coat. Prior to beginning this protocol, please prepare the materials and reagents listed in Table 2. Do note that this protocol is designed for the collection of PBMCs from six density gradient centrifugation tubes, each of which yields approximately 12 million cells.

Scale accordingly. Using standard phlebotomy technique, collect whole blood in six density gradient centrifugation tubes and one EDTA tube until filled. Centrifuge all tubes at 1800g for 20 minutes at room temperature.

If needed, collected tubes may be stored up to four hours and then processed simultaneously. Refer to supplemental video 1 for a demonstration of how to operate a centrifuge. After centrifugation of the tube containing the density gradient medium, plasma and PBMCs will be above the density gradient medium.

Erythrocytes and granulocytes will settle to the bottom. Refer to Figure 1A for a visualization. After centrifugation, carefully open the density gradient centrifugation tube.

Use a transfer pipette to remove and discard the translucent yellow layer containing plasma. Do not disturb the hazy layer of cells directly above the density gradient medium. Err on the side of leaving excess plasma rather than discarding PBMCs.

Repeat for all tubes. With a new transfer pipette, pipette the remaining volume containing cells and residual plasma in each tube up and down gently several times above the density gradient medium. After resuspension, pipette the resuspended contents of the tube into a labeled 50 mL conical tube.

Repeat for all tubes. Fill the 50 mL tube up to the 50 mL line with solution 1 by pouring. Centrifuge the 50 mL tube at 300g for 10 minutes at room temperature.

The plasma and buffy collection protocol detailed in Section 2 may be completed during this time. Following centrifugation, the pellet contains PBMCs and the supernatant contains platelets and residual plasma. Discard as much supernatant as possible.

Tap as needed to remove residual drops. Pour a 15 mL tube of solution 2 into the 50 mL conical tube containing the PBMC pellet. Gently invert the tube to resuspend the pellet.

Aspirate 15 mL of solution 3 using a transfer pipette. Add dropwise to the 50 mL tube. Solution 3 contains DMSO, which is toxic to cells at room temperature.

Do not add solution 3 until ready to be immediately processed. Invert the tube gently three times to mix. Fill each pre-labeled and pre-chilled cryo vial with 1 mL of PBMCs using a multi-dispense pipette.

Refer to supplemental video 2 for a demonstration of how to operate a multi-dispense pipette. Place the cryo vials inside a pre-chilled freezing container. Then, move the container to a negative 80 degree Celsius freezer.

Keep the cryo vials inside the freezing container for a minimum of 12 hours. Then, transfer to a labeled storage box at negative 80 degrees Celsius. Alternatively, transfer the frozen cryo vials to liquid nitrogen for long-term storage.

After centrifugation, the top layer consists of plasma, the bottom contains erythrocytes, and the interface will be the buffy coats. See Figure 1B for a visualization. Using a new transfer pipette, draw up as much plasma as possible without disturbing the buffy coat layer.

Then, dispense 0.5 mL aliquots into pre-labeled cryo vials. Aspirate the buffy coat layer and transfer it to the appropriately labeled cryo vial. Note that some plasma and erythrocytes may contaminate the buffy coat collection.

After collection, store the cryo vials in a negative 80 degree Celsius freezer. Alternatively, store in liquid nitrogen for long-term storage. Prior to starting the PBMC thawing protocol, please prepare the materials and reagents listed in Table 3.

This protocol is designed for the thawing of one 1.5 mL tube containing approximately 2 million cells. Scale accordingly. Do note that the absolute number of cells may differ significantly between different patients and different treatment conditions.

Transfer frozen PBMCs from storage using dry ice and thaw in a 37-degree Celsius incubator for about 4 minutes. If desired, an aliquot can be taken for cell counting and viability measurements as detailed in section 4 of the written protocol. After thawing, add 1 mL of solution 4 to each cryo vial.

Then, pour the contents into a pre-labeled 50 mL tube containing 10 mL of solution 4 for every PBMC tube thawed. Tap to remove residual drops. Place the cell strainer on the empty 50 mL tube and then pour the cell suspension through the cell strainer.

If needed, lightly tap to break the surface tension. Centrifuge each tube containing the strained cells at 400g for 7 minutes at room temperature. After centrifugation, pour out the supernatant containing DMSO.

Tap as needed to remove residual drops. Resuspend the cell pellet in the desired medium appropriate for downstream experimental needs, such as cell culture media or flow cytometry antibody cocktails. Proceed with your experiment.

Following PBMC collection and cryopreservation, viability of thawed PBMCs, monocytes, and lymphocytes from 56 unique samples was assessed by flow cytometry using reagents listed in Table 4 following manufacturer's instructions shown in Figures 2A through F.A mean and standard deviation viability of PBMCs, monocytes, and lymphocytes of 94 plus or minus 4%98 plus or minus 1.1%and 93 plus or minus 5.6%respectively, was achieved, shown in Figure 2G. Viability measurements by trypan blue exclusion yielded a mean viability of 88 plus or minus 7.5%and a cell count of 2.3 times 10 to the 6 plus or minus 1.9 times 10 to the 6. Flow cytometry analysis also demonstrated that live monocytes comprised 17 plus or minus 5.9%and lymphocytes made up of 53 plus or minus 13%of total PBMCs.

Subsequently, monocytes were sorted from thawed PBMCs from 59 unique samples by flow cytometry and submitted for library preparation and RNA sequencing as described elsewhere. A mean and standard deviation total sequence count of 18 plus or minus 16.3 million was achieved with a 93 plus or minus 6.3%read alignment and 49.6 plus or minus 1.4 GC content as shown in Figures 3A and B.A mean and standard deviation uniquely mapped reads percentage of 88 plus or minus 3.6%was found with a subset of 56 unique samples. These parameters demonstrate that highly viable PBMCs suitable for downstream applications including high-quality RNA sequencing can be obtained by operators with any level of laboratory training using this protocol.

When performing this protocol, it is critical to work quickly to avoid cell death, especially after addition of solution 3, which is toxic to cells at room temperature. In addition, the cells must also be handled gently to minimize damage. This protocol provides an efficient, easy-to-follow method for the isolation of PBMCs, EDTA plasma, and buffy coat.

These immune cells can be used for a broad range of experiments and assays focused on the role of the immune system in different biological contexts.