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

Summary

This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.

Abstract

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This detailed protocol describes the equipment, supplies, and steps for isolating, cryopreserving, and thawing high-quality and highly viable PBMCs from whole blood cells suitable for downstream applications such as flow cytometry and RNA-sequencing. Protocols for processing plasma and buffy coat from the whole blood in parallel and concurrently with PBMCs are also described. This easy-to-follow step-by-step protocol, which utilizes density gradient centrifugation to isolate PBMCs, is accompanied by a checklist of supplies, equipment, and preparation steps. This protocol is suitable for individuals with any prior experience with laboratory techniques and can be implemented in clinical or research laboratories. High-quality cell viability and RNA sequencing resulted from PBMCs collected by operators with no prior laboratory experience using this protocol.

Introduction

This protocol demonstrates an accessible method and workflow for the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood. This protocol is especially targeted towards novice research technicians, students, and clinical lab staff with the objective of facilitating the collection and cryopreservation of PBMCs without assuming prior training in laboratory techniques.

This protocol utilizes centrifugation to separate the components of whole blood by density. Whole blood consists of four main components listed in order of decreasing density: red blood cells/erythrocytes (~45% of volume), white blood cells (<1% of volume), platelets (<1% of volume), and plasma (~55% of volume)^1,2,3,4,5,6. White blood cells can be subdivided into two categories based on their nuclei characteristics: round or multi-nucleated⁶. PBMCs are defined as white blood cells with round nuclei and consist of the following cell types: lymphocytes (T cells, B cells, NK cells), dendritic cells, and monocytes⁶. Multi-nucleated white blood cells include granulocytes, which consist of the following cell types: neutrophils, basophils, and eosinophils⁶. Multi-nucleated white blood cells are denser than PBMCs⁶. The densities of each component of whole blood are detailed in Table 1.

In this protocol, whole blood is collected in density gradient centrifugation tubes. These tubes contain a pre-packed density gradient medium that has a density of 1.077 g/mL. Following centrifugation, denser cells, including multi-nucleated white blood cells and erythrocytes, are separated from PBMCs and platelets by the density gradient medium (Figure 1A)^6,7. The PBMC and platelet fraction is then collected, washed, and centrifuged to remove platelets. The resulting purified PBMCs are collected and stored at -80 °C or in liquid nitrogen. Cryopreserved PBMCs may be viably thawed and directly used in downstream analyses or additionally processed to isolate specific component cell types.

This protocol has been optimized for high-quality RNA sequencing from highly viable PBMCs. In this article, PBMCs were isolated and cryopreserved from patients in an outpatient clinic. Subsequently, monocytes were isolated from PBMCs by FACS and analyzed via RNA-sequencing. However, the protocol can be widely adapted to other experimental needs such as cell culture, gene editing, ex-vivo functional studies, single-cell analyses, phenotyping by flow cytometry or cytometry by time of flight, isolation of DNA/RNA or proteins, slides for immunohistochemistry, amongst others^{8,9,10,11,12,13,14,15,16,17,18}.

In addition to PBMC collection from density gradient centrifugation tubes, this protocol reviews how to collect plasma and buffy coat via centrifugation using an EDTA tube. After centrifugation, whole blood is separated into plasma, erythrocytes, and a thin interface layer termed buffy coat containing leukocytes (Figure 1B)⁶. The buffy coat is commonly used for DNA extraction and subsequent genomic analyses^19,20. The plasma layer contains the cell-free components of whole blood and can be used for biomarker assays^21,22.

Protocol

The study protocol was approved by the UCSD and KUMC Human Protections Program and conforms to the Declaration of Helsinki. All individuals provided informed consent for participation and blood collection.

1. Processing and cryopreservation of PBMCs

1. One day prior to the blood collection, print out the checklist of materials and reagents listed in Table 2 and prepare and label accordingly.
   NOTE: This protocol was designed for the collection of 6 density gradient centrifugation tubes. Each tube yields roughly 12 million PBMCs, of which approximately 50% and 20% will be live lymphocytes and monocytes respectively. Scale accordingly for experimental needs. Note that the absolute number of cells may differ significantly between patients or treatment conditions.
2. Using the standard phlebotomy technique, collect whole blood in 6 density gradient centrifugation tubes and 1 EDTA tube until filled, approximately 6 mL of volume.
   NOTE: If samples from multiple patients or treatment conditions are required, the collected tubes may be stored up to 4 h and then processed simultaneously.
3. After collection, centrifuge all tubes at 1,800 x g for 20 min at room temperature.
   NOTE: Refer to Supplemental Video 1 for a demonstration of how to operate a centrifuge.
4. 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 caution, leaving excess plasma rather than discarding PBMCs. Repeat for all tubes.
   NOTE: 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.
5. 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.
6. After resuspension, pipette the resuspended contents of the tube into a labeled 50 mL conical tube. Repeat for all tubes.
7. Pour Solution 1 (RPMI Medium) into the 50 mL tube until it reaches the 50 mL line.
8. Repeat steps 1.4 to 1.7 for each patient or treatment condition as needed.
   NOTE: Ensure that different samples are not mixed and use a new transfer pipette to avoid contamination.
9. Centrifuge the 50 mL tube at 300 x g for 10 min at room temperature.
   NOTE: The plasma and buffy collection protocol detailed in Section 2 may be completed during this time.
10. Discard as much supernatant as possible. Tap as needed to remove residual drops.
    NOTE: Following centrifugation, the pellet contains PBMCs, and the supernatant contains platelets and residual plasma.
11. Pour a 15 mL tube of Solution 2 (RPMI Medium + 12.5% Human Serum Albumin + 1 µM Flavopiridol) into the 50 mL conical tube containing the PBMC pellet. Gently invert the tube to resuspend the cell pellet.
12. Aspirate 15 mL of Solution 3 (RPMI Medium + 11.25% Human Serum Albumin + 1 µM Flavopiridol + 10% DMSO) using a transfer pipette. Add dropwise to the 50 mL tube.
    NOTE: Solution 3 contains DMSO, which is toxic to cells at room temperature. Do not add Solution 3 until it is ready to be immediately processed.
13. Invert the tube gently 3 times to mix.
14. Fill each pre-labeled and pre-chilled cryovial with 1 mL of PBMCs using a multi-dispense pipette.
    NOTE: Refer to Supplemental Video 2 for a demonstration of how to operate a multi-dispense pipette.
15. Place the cryovials inside a pre-chilled freezing container. Then, move the container to a -80 °C freezer.
16. Repeat steps 1.9 to 1.13 for each patient or treatment condition as needed.
17. Keep the cryovials inside the freezing container for a minimum of 12 h, then transfer them to a labeled storage box at -80 °C.
    NOTE: Alternatively, transfer the frozen cryovials to liquid nitrogen (<-135 °C) for long-term storage.

2. Processing of plasma and buffy coat from EDTA tubes

1. Centrifuge at 1,800 x g for 10 min at room temperature. 
   NOTE: After centrifugation, the top layer consists of plasma, the bottom contains erythrocytes, and the interface will be the buffy coat. See Figure 1B for a visualization.
2. 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 prelabeled cryovials. Aspirate the buffy coat layer and transfer it to the appropriately labeled cryovial.
   NOTE: Some plasma and erythrocytes may contaminate the buffy coat collection.
3. Repeat step 2.2 for each patient or treatment condition as needed.
4. After collection, store the cryovials in a -80 °C freezer.
   NOTE: Alternatively, store in liquid nitrogen (<-135 °C) for long-term storage.

3. Thawing PBMCs

1. One day before thawing, print out a checklist of materials and reagents listed in Table 3 and prepare and label accordingly.
   NOTE: This protocol was designed for the thawing of 1 1.5 mL tube containing 1 mL of PBMCs. Each tube yields approximately 2 million PBMCs, of which approximately 50% and 20% will be live lymphocytes and monocytes, respectively. Scale accordingly for experimental needs. Note that the absolute number of cells may differ significantly between patients or treatment conditions.
2. Transfer frozen PBMCs from storage using dry ice and thaw in a 37 °C incubator for 4 min, or just prior to the last ice crystal thawing.
   NOTE: If desired, an aliquot can be taken for cell counting and viability measurements, as detailed in Section 4.
3. After thawing, add 1 mL of Solution 4 (PBS + 2 mM EDTA) to each cryovial. 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.
4. Place the cell strainer on the empty 50 mL tube and then pour the cell suspension through the cell strainer.
   NOTE: If needed, lightly tap to break the surface tension.
5. Centrifuge each tube containing the strained cells at 400 x g for 7 min at room temperature.
6. After centrifugation, pour out the supernatant containing DMSO. Tap as needed to remove residual drops.
7. Resuspend the cell pellet in the desired medium appropriate for downstream experimental needs (e.g., cell culture media, flow cytometry antibody cocktail, etc.).

4. Cell counting and viability

1. Immediately after thawing frozen PBMCs in step 3.2, pipette 20 µL of cells into a 1.5 mL tube. Add an equal volume of 0.4% Trypan Blue solution and pipette up and down gently 3-4 times to mix.
2. Using an automated cell counter, follow the manufacturer's protocol to count cells and assess viability. Ensure that the dilution factor of 2 is accounted for.
   NOTE: As described elsewhere, a hemocytometer may be used as an alternative method for assessing cell count and viability²³.

Results

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 manufacturers' instructions (Figure 2A-F). A mean ± SD viability of PBMCs, monocytes, and lymphocytes of 94 ± 4.0%, 98 ± 1.1%, and 93 ± 5.6%, respectively, was achieved (Figure 2G). Viability measurement by Tr...

Discussion

This protocol for PBMC collection and cryopreservation has been successfully implemented by individuals with and without prior research laboratory training. In our application, FACS and RNA-sequencing of highly viable monocytes purified from stored PBMCs resulted in high-quality sequences.

A major strength of this protocol is its accessibility. The technique presented in the protocol utilizes tubes pre-packed with a solid-density gradient medium. As a result, whole blood can be collected direc...

Materials

Name	Company	Catalog Number	Comments
1000 µL Tips	Gilson	F174501	Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
15 mL tube	Biopioneeer	CNT-15	To hold Solutions 2/3 2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
2 mL Cryovials	Globe Scientific	3012	Approximately 40 cryovials needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
20 µL Tips	Gilson	F174201	For use in cell counting; volume needed is dependent on method of cell counting used. 1 tip is needed per 1 mL of unpooled frozen PBMCs to be defrosted
50 mL Conical Tube	CEM Corporation	50-187-7683	2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection 3 needed per 1 mL of unpooled frozen PBMCs to be defrosted
CD14 Antibody	Biolegend	325621
CD16 Antibody	Biolegend	360723
CD19 Antibody	Biolegend	363007
CD3 Antibody	Biolegend	300405
CD56 Antibody	Biolegend	318303
CD66b Antibody	Biolegend	305103
Cell Strainer	Biopioneeer	DGN258367	1 needed per 1 mL of unpooled frozen PBMCs to be defrosted
CPT Mononuclear Cell Preparation Tube	BD Biosciences	362753	6 tubes needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Cryo Freezer Box	Southern Labware	SB2CC-81	Holds 81 tubes 1 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Cryotube Rack	Fisherbrand	05-669-45	Holds up to 50 cryovials
DMSO	Invitrogen	15575020	Approximately 2.5 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
DNase/RNase-Free Distilled Water	Invitrogen	10977015	Approximately 2 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection Approximately 9 mL needed per 1 mL of frozen PBMCs to be defrosted
EDTA Tubes	BD Biosciences	366643	1 tube needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Flavopiridol	Sigma-Aldrich	F3055-5MG
HLA-DR Antibody	Biolegend	307609
Human Serum Albumin	GeminiBio	800-120	Approximately 25 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection
Human Trustain FcX	Biolegend	422301
Isopropanol	Sigma-Aldrich	W292912-1KG-K	For use in Mr. Frosty
Label Printer	Phomemo	M110-WH
Live-Dead Stain	Biolegend	423105
Mr. Frosty	Thermo Scientific	5100-0001	Holds up to 18 tubes. 2 Mr. Frostys needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Multidispense Pipette	Brandtech	705110
Multidispense Pipette Tips	Brandtech	705744	Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
P1000 Pipette	Gilson	F144059M
P20 Pipette	Gilson	F144056M	For use in cell counting; volume needed is dependent on method of cell counting used.
Printer Labels	Phomemo	PM-M110-3020	Approximately 45 labels needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection Approximately 5 labels needed per 1 mL of unpooled frozen PBMCs to be defrosted
RNase-Free EDTA (0.5 M)	Invitrogen	AM9260G	Approximately 40 µL needed per 1 mL of frozen PBMCs to be defrosted
RNase-Free PBS (10X)	Invitrogen	AM9625	Approximately 1 mL needed per 1 mL of frozen PBMCs to be defrosted
RNasin	Promega	N2111
RPMI	Corning	10-040-CV	Approximately 80 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection
Transfer Pipettes	Fisherbrand	13-711-7M	Approximately 5 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection
Tube Holder	Endicott-Seymour	14-781-15	Holds up to 80 CPT/EDTA Tubes