"Isolation of Whole Mononuclear Cells from Peripheral Blood and ...

Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood

UNIT 7.1

Ivan J. Fuss,1 Marjorie E. Kanof,2 Phillip D. Smith,3 and Heddy Zola4 1

Mucosal Immunity Section, National Institute of Health, Bethesda, Maryland GAO, U.S. Government Accountability Office, Department of Health and Human Services, Washington, D.C. 3 National Institute of Dental Research/NIH, Bethesda, Maryland 4 Child Health Research Institute, North Adelaide, Australia 2

ABSTRACT Peripheral blood is the primary source of lymphoid cells for investigation of the human immune system. Its use is facilitated by Ficoll-Hypaque density gradient centrifugation— a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. Thus, cells are distributed in the solution in layers based on the differences in their density/size. Additional purification methods can be employed as the mononuclear cell sample can be purified from monocytes by adherence or by exposure to L-leucine methyl ester; these methods are described for both procedures. Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, which can result in significant contamination of the mononuclear cell layer, and removal of these cells requires additional steps that are described. The isolation procedures presented here can also be applied to cell populations derived from tissues. Curr. Protoc. Immunol. C 2009 by John Wiley & Sons, Inc. 85:7.1.1-7.1.8. Keywords: mononuclear cell r Ficoll-Hypaque r density gradient r peripheral blood

INTRODUCTION The ability to isolate purified lymphoid cells has greatly advanced the analysis of the functional immune system. The isolation procedure employed is a Ficoll-Hypaque gradient (Ferrante and Thong, 1980; Vissers et al., 1988). Ficoll-Hypaque is composed of sodium diatrizoate (3.5-bis acetylamine-2.4.6 tri-iodo benzoic acid; hypo-opaque, iso-opaque) and Ficoll. Ficoll is a high-molecular-weight sucrose polymer (specific gravity, 1.076 to 1.078), whereas Hypaque is a dense iodinated organic compound most commonly used in a radiographic contrast medium. Ficoll contributes viscosity to the solution and allows rouleaux formation of the RBCs, whereas Hypaque increases solely the viscosity of the solution. When prepared, a Ficoll-Hypaque solution has a specific gravity of 1.077 at room temperature and is denser than lymphocytes, monocytes, and platelets but less dense than granulocytes and RBCs, allowing for the successful separation of these cell populations. Therefore, mononuclear cells and platelets collect on top of the Ficoll-Hypaque layer because they have a lower density; in contrast, red blood cells (RBC) and granulocytes have a higher density than Ficoll-Hypaque and collect at the bottom of the FicollHypaque layer. Platelets are separated from the mononuclear cells by subsequent washing or by centrifugation through a fetal bovine serum (FBS) cushion gradient that allows penetration of mononuclear cells but not platelets. The mononuclear cell sample can be purified from monocytes by adherence or by exposure to L-leucine methyl ester (see Support Protocol 1 and Support Protocol 2). Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, which can result in significant

Current Protocols in Immunology 7.1.1-7.1.8, April 2009 Published online April 2009 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142735.im0701s85 C 2009 John Wiley & Sons, Inc. Copyright

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contamination of the mononuclear cell layer. Removal of these cells requires additional steps (see Support Protocol 3 and Ridings et al., 1996). The isolation procedures described here can also be applied to cell populations derived from tissues. CAUTION: When working with human blood, cells, or infectious agents, biosafety practices must be followed. NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. BASIC PROTOCOL

ISOLATION OF MONONUCLEAR CELLS BY FICOLL-HYPAQUE GRADIENT CENTRIFUGATION This protocol describes the procedure used for the preparation and isolation of purified human mononuclear cell populations from peripheral blood.

Materials Heparinized blood (APPENDIX 3F) or heparinized cord blood Phosphate-buffered saline (PBS; APPENDIX 2A) Ficoll-Hypaque solution (density 1.077 g/liter; see recipe) Hanks’ balanced salt solution (HBSS; APPENDIX 2A) Fetal bovine serum (FBS; e.g., HyClone), with heat inactivation (1 hr, 56◦ C; APPENDIX 2A) Complete RPMI-1640 medium (APPENDIX 2A) Pipets, sterile 15- or 50-ml conical centrifuge tubes Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent temperature-controlled centrifuge) Additional reagents and equipment necessary for counting cells and trypan blue exclusion for determining viability (APPENDIX 3A & 3B), and depletion of contaminating cells from mononuclear fractions (see Support Protocol 3; optional) 1. Place fresh heparinized blood into 15- or 50-ml conical centrifuge tubes. Using a sterile pipet, add an equal volume of room-temperature 1 × PBS. Mix well. 2. Pellet the leukocyte/RBC fraction by centrifuging the cells 15 min at 200 × g, room temperature. The platelet cell fraction will remain in the upper suspension. Using a sterile pipet, remove the supernatant suspension containing this platelet cell fraction. When isolating cells from a leukapheresis donor, dilute blood with PBS (1:4 blood/PBS). Cord blood is readily available in 10-ml volumes; larger volumes can be obtained if required. Cord blood is prone to clotting and it is therefore helpful to add to each 10-ml aliquot 1 ml PBS containing 250 U heparin to supplement the heparin already contained in heparin-coated tubes. The blood should immediately be gently mixed, and should be examined when it reaches the laboratory for small blood clots. As the clotting reaction releases proteins that can affect lymphocyte phenotype and function, samples containing clots should be discarded.

3. Using a sterile pipet, add an equal volume of room-temperature 1× PBS suspension (if utilizing a 15-ml conical tube) or bring the leukocyte/RBC cell suspension to a final volume of 40 ml with 1× PBS (if utilizing a 50-ml conical tube). Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood

4. Slowly layer the Ficoll-Hypaque solution underneath the leukocyte/RBC/PBS mixture by placing the tip of the pipet containing the Ficoll-Hypaque at the bottom of the

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peripheral blood

add Ficoll-hypaque

Figure 7.1.1 Ficoll-Hypaque density isolation of mononuclear cells. Ficoll-Hypaque is underlayered onto whole blood or cord blood. The sample is centrifuged allowing the separation of lymphocyte cell population from other blood elements based upon density gradient.

peripheral blood

Ficoll-hypaque solution

sample tube. Use 3 ml Ficoll-Hypaque per 10 ml blood/PBS mixture. Use a maximum of 10 ml Ficoll-Hypaque per 40 ml leukocyte/RBC/PBS mixture. To maintain the Ficoll-Hypaque/blood interface, it is helpful to hold the centrifuge tube at a 45◦ angle. Alternatively, the leukocyte/RBC/PBS mixture may be slowly layered over the FicollHypaque solution. See Figure 7.1.1.

5. Centrifuge 20 to 30 min in a GH-3.7 rotor at 2000 rpm (900 × g), 18◦ to 20◦ C, with no brake. After a centrifugation with Ficoll-Hypaque, platelets (specific gravity, 1.040) and plasma (specific gravity, 1.025 to 1.029) are located above the Ficoll-Hypaque, lymphocytes (specific gravity, 1.070), and some platelets are found at the plasma-Ficoll-Hypaque interface. Granulocytes (specific gravity, 1.087 to 1.092) and RBCs (specific gravity, 1.093 to 1.096) form a cell pellet at the bottom of the tube (see Fig. 7.1.2 and Nisperos, 1990).

6. Using a sterile pipet, remove the upper layer that contains the plasma and most of the remaining cell platelet fraction. Using another pipet, transfer the mononuclear lymphocyte cell layer to another centrifuge tube. This will appear as a white, cloudy band between the plasma and the Ficoll-Hypaque layers. Wash cells by adding HBSS (∼3 times the volume of the mononuclear cell layer) and centrifuging 10 min at 450


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plasma, platelets

Figure 7.1.2 Separation of blood components on a Ficoll-Hypaque gradient.

lymphocytes Ficoll-hypaque granulocytes, erythrocytes

to 600×g, 18◦ to 20◦ C. Remove supernatant, resuspend cells in HBSS, and repeat the wash once to remove any remaining platelets. The washing steps described above usually remove most of the platelets from the mononuclear cell suspension. There are certain disease states associated with increased platelet concentrations (mononuclear cell to platelet cell ratio >10:1) in the peripheral blood, and additional steps are needed to remove the extra platelets in these cases. Add 3 ml FBS to a centrifuge tube for each milliliter of mononuclear cells. Layer the cell suspension (1-2 × 107 cells/ml) over the FBS (alternatively, carefully layer the FBS under the cell suspension, which will rise as FBS is added). Centrifuge 15 min at 150 × g, 18◦ to 20◦ C. Discard the supernatant containing the platelets. Resuspend cell pellet in complete RPMI-1640 and proceed as in step 7.

7. Resuspend mononuclear lymphocyte cells in complete RPMI-1640. Count cells (APPENDIX 3A) and determine viability by trypan blue exclusion (APPENDIX 3B). If desired, purity of PBMC population can be determined by flow cytometry. Cord blood, and to a lesser extent peripheral blood from infants, gives a population of cells that is contaminated with erythrocytes and their precursors. Pure mononuclear cell populations may be obtained either by subjecting the cells to a second cycle of FicollHypaque gradient separation as described here, or by lysing the erythrocytes (see Support Protocol 3). SUPPORT PROTOCOL 1

DEPLETION OF MONOCYTES/MACROPHAGES FROM MONONUCLEAR CELLS USING ADHERENCE METHOD Approximately 40% of the isolated mononuclear cells obtained in the Basic Protocol are composed of monocytes and macrophages. Monocytes can be depleted from the isolated mononuclear cell suspension by taking advantage of the fact that monocytes adhere to plastic, whereas lymphocytes do not.

Additional Materials (also see Basic Protocol) Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood

Mononuclear cell suspension (see Basic Protocol) Complete RPMI-1640 medium (APPENDIX 2A), room temperature 150-cm2 tissue culture flasks, slanted-neck 37◦ C, 5% CO2 humidified incubator

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1. Centrifuge mononuclear cells 10 min at 450 to 600 × g, 18◦ to 20◦ C. Remove supernatant and resuspend cell pellet in complete RPMI-1640 to a final concentration of 2 × 106 cells/ml. Transfer 50-ml cell suspension to a 150-cm2 tissue culture flask. A biological-grade petri dish can be used, but adherence is increased when a tissue culture flask is used.

2. Incubate horizontally for 1 hr in a 37◦ C, 5% CO2 humidified incubator. 3. Decant nonadherent lymphocytes into a centrifuge tube. Rinse tissue culture flask gently with 37◦ C complete RPMI-1640; add this wash to the centrifuge tube. Centrifuge 10 min at 450 to 600 × g, 18◦ to 20◦ C. Repeat steps 1 to 3 one time. 4. Discard supernatant. Resuspend cells in 5 to 10 ml complete RPMI-1640. Count cells (APPENDIX 3A) and determine viability by trypan blue exclusion (APPENDIX 3B). Cells should be >90% viable. The adherent cells can be recovered using the procedures outlined in UNIT 7.6.

DEPLETION OF MONOCYTES/MACROPHAGES FROM MONONUCLEAR CELLS USING L-LEUCINE METHYL ESTER

SUPPORT PROTOCOL 2

Monocytes/macrophages can also be depleted from the PBMC suspension by using an additional procedure that takes advantage of their rich lysosomal enzyme content (Thiele et al., 1983). This procedure employs a lysosomotropic agent—L-leucine methyl ester— that is taken up by phagocytic and cytotoxic cells and is concentrated in lysosomes, where it is converted by lysosomal enzymes to L-leucyl-L-leucine methyl ester, which is toxic to the cells. B cells and most T cells (cells not rich in lysosomes) are unaffected by exposure to L-leucine methyl ester. This agent can also eliminate natural killer (NK) cells and cytotoxic T cells.

Additional Materials (also see Basic Protocol) Mononuclear cell suspension (see Basic Protocol) 1 mM L-leucine methyl ester (Sigma) in complete RPMI medium (serum-free and filter sterilized; APPENDIX 2A) Additional reagents and equipment for nonspecific esterase staining (APPENDIX 3C) or for flow cytometry (UNIT 7.9) 1. Incubate mononuclear cells (5 × 106 cells/ml) in 1 mM L-leucine methyl ester (prepared in complete serum-free RPMI medium) for 40 min at room temperature. 2. Wash cells twice in HBSS (see Basic Protocol, step 6) or 1× PBS and resuspend in complete RPMI-1640. 3. Confirm that remaining lymphocytes are not contaminated by monocytes (large cells with eccentric, crescented nuclei) by light microscopy or by nonspecific esterase staining (APPENDIX 3C), which results in staining of monocytes but not other cells. Alternatively, examine the remaining lymphocytes by flow cytometry (UNIT 7.9) for the presence of cells staining with monocyte-specific fluorescence-labeled antibodies.

DEPLETION OF CONTAMINATING CELLS FROM MONONUCLEAR CELL FRACTIONS FROM CORD OR INFANT PERIPHERAL BLOOD

SUPPORT PROTOCOL 3

When blood from young infants is fractionated under the same conditions as adult blood, the mononuclear cell layer is often visibly contaminated with red blood cells (see Fig. 7.1.3). In addition to this visible contamination, the mononuclear cell layer may contain nucleated red cell precursors. Flow cytometric analysis shows that the proportion of cells that fail to react with CD45 (a leukocyte-common marker) can exceed 50%,



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A

B

Figure 7.1.3 Separation of adult (A) and cord (B) blood under identical conditions. The cord blood sample shows significant contamination of the interface layer with red blood cells.

even after mature red cells have been excluded on the basis of light-scatter parameters. The contaminating cells can be removed from the mononuclear cell population by lysis in hypotonic ammonium chloride. This treatment does not affect the viability or most functions of mononuclear cells, but may affect antigen-processing function, because ammonium ions inhibit lysosomal function. For studies that may be adversely affected by hypotonic ammonium chloride, the contaminating cells may be removed almost as efficiently from the mononuclear cell population by two rounds of Ficoll-Hypaque density gradient separation. The improvement in purity produced by this second round of FicollHypaque density centrifugation suggests that the conditions (e.g., cell concentration and viscosity) in the first round do not allow all of the cells to reach their isopycnic position.

Additional Materials (also see Basic Protocol) Mononuclear cell suspension (see Basic Protocol) ACK lysing buffer (UNIT 3.1) 1. Centrifuge the cell suspension obtained from the Ficoll-Hypaque separation 10 min at 450 to 600 × g, room temperature, and remove the supernatant fluid. 2. Resuspend the cell pellet in ACK lysing buffer, using 5 ml solution per 10 ml of original blood volume. Allow to stand 5 min at room temperature, add 25 ml PBS, mix, and centrifuge 15 min at 200 × g, room temperature. Remove supernatant, resuspend cell mixture in 30 ml RPMI-1640 or PBS, and centrifuge once more 15 min at 200 × g, room temperature.


Alternatively, centrifuge the cell suspension obtained from the Ficoll-Hypaque separation 10 min at 450 to 600 × g, room temperature, remove the supernatant fluid, and resuspend the cell pellet in a volume of PBS equal to the original blood volume. Separate this suspension on a Ficoll-Hypaque density gradient (see Basic Protocol).

3. Determine the purity of the mononuclear cell population by flow cytometry using a CD45 (leukocyte-common antigen) monoclonal antibody (Chapter 5).

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REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see APPENDIX 5.

Ficoll-Hypaque solution (density 1.077 g/liter) 64.0 g Ficoll (molecular weight 400,000; e.g., Sigma) 99.0 g sodium diatrizoate (e.g., Sigma) 0.7 g NaCl Dissolve Ficoll and NaCl in 600 ml water using a magnetic stirrer at low speed. Add sodium diatrizoate. When all components are in solution, add water to 1 liter. Filter sterilize with a 0.22-μm filter unit (Nalgene). Store at 4◦ to 25◦ C and protect from direct light. Alternatively, Ficoll-Hypaque may be purchased already in solution (Lymphocyte separation medium; MP Biomedicals).

COMMENTARY Background Information Ficoll-Hypaque gradient centrifugation allows rapid and efficient isolation of mononuclear cells from human peripheral blood (Boyum, 1968). As such, this technique is the starting point of most studies of human lymphoid cells. In general, Ficoll-Hypaque centrifugation does not change either the phenotype or the function of the isolated mononuclear cell population. However, it may be best to verify this in studies of cells from patients with various diseases (Tse and Dutton, 1976; Kaplan et al., 1982; Tamul et al., 1995). Monocytes/macrophages are removed from the mononuclear cell population in order to study lymphocytic function in the absence of monocytes/macrophages, or monocyte function in the absence of lymphocytes. The mononuclear cell layer can be frequently contaminated with erythrocytes, usually visible as cell clumps sticking to the underside of the mononuclear layer. The use of a single Ficoll-Hypaque density separation gives variable results with cord blood and peripheral blood from infants. Flow cytometric analysis shows that even when mature erythrocytes are excluded on the basis of light scatter, a significant number of CD45-negative cells remain. The number of CD45-negative cells can vary between cord preparations, from ≤10% to up to 50%. Cord blood also contains a small number of CD34-positive stem cells. These cells are too few to affect phenotype significantly but may nonetheless affect functional cells. It should also be borne in mind that the number of CD45-negative cells is not fully accounted for by red cell precursors; this implies that there are uncharacterized additional cells in cord blood, which may affect functional cells.

Historically, initially erythrocyteaggregating chemicals were added to the blood to increase the efficiency of the cell separation process. This included chemicals such as dextran, polyvinyl-pyrolidon, methylcellulose, and phytohemagglutinin (Skoog and Beck, 1956). However, the advent of gradient centrifugation technique combined with purification of contaminating red cells by hypotonic lysis has eliminated this need and simplified this process.

Critical Parameters and Troubleshooting The yield of mononuclear cells from peripheral blood depends on many factors including the percentage of contaminating granulocytes and platelets and efficiency of erythrocyte removal (Thong et al., 1983). However, the most critical step is the under layer of Ficoll-Hypaque. A distinct and clear fraction between the Ficoll-Hypaque and leukocyte/RBC PBS mixture must be seen prior to centrifugation. If not, clear separation of the leukocyte layer will not occur and the mononuclear cell population may be lost. For maximum yield and purity after centrifugation, it is also essential to remove all the material at the Ficoll-Hypaque interface and to ensure that no Ficoll-Hypaque solution or supernatant is removed with the sample. Including parts of the Ficoll-Hypaque layer will increase the granulocyte contamination; including supernatant will increase the platelet contamination. Erythrocytes can aggregate and trap lymphocytes in the clumps. These clumps will sediment into the pellet and reduce the yield of lymphocytes. Diluting the blood with PBS before Ficoll-Hypaque centrifugation



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will reduce the clumping, as well as get rid of platelet contamination. In addition, the temperature of the centrifuge can affect the yield of lymphocytes. At low temperatures, lymphocyte yields are reduced because a longer centrifugation time is required. At high temperatures, lymphocyte viability is decreased and erythrocyte aggregation is increased. During the gradient centrifugation, the temperature should be maintained at 18◦ to 20◦ C. Occasionally, it is necessary to isolate mononuclear cells from clotted blood. This has been successfully accomplished by using streptokinase to dissolve the blood clot. Lymphocytes isolated form clotted blood function in some respects similarly to those isolated from heparinized blood, although the lymphocyte yield is only 60% of the yield from heparinized blood (Niku et al., 1987).

ence method, and 1 to 2 hr using L-leucine methyl ester. If the isolated mononuclear cells are frozen, further purifications can be done later.

Anticipated Results

Nisperos, B. 1990. Density gradient isolation of peripheral blood lymphocytes. In ASHI Laboratory manual, 2nd ed. (A.A. Zachary and G.A. Teresi, eds.) pp. 23-27. American Society for Histocompatibility and Immunogenics, Lenexa, Kans.

When blood is obtained from a normal donor, the yield should be 1-2 × 106 mononuclear cells/ml of blood. Approximately 60% to 70% of the mononuclear cells are lymphocytes with a viability of >95%. The platelet count is 95%. After depletion using L-leucine methyl ester, >99% of the mononuclear cells are lymphocytes with a viability of >95%. Yields of cells from cord blood tend to be somewhat higher than those from adult peripheral blood, ranging from 0.7 to 4.2 × 106 cells per milliliter. Viabilities of cord blood cells range from 86% to 100%, with most samples giving 100%.

Time Considerations


Isolating mononuclear cells from peripheral blood takes ∼1 hr. This is dependent on the initial volume of blood obtained; the larger the blood volume, the longer the preparative time before the samples are ready for centrifuging. Lysis of erythrocytes followed by washing adds ∼20 min to the procedures, while the alternative procedure of introducing a second round of Ficoll-Hypaque separation adds 40 min. Decreasing the concentration of platelets from the isolated mononuclear cells takes 30 min. If the isolated mononuclear cells are to be stored, platelets should be removed before freezing the cells. Removing monocytes/macrophages from the mononuclear cell population takes 2 to 3 hr using the adher-

Literature Cited Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Invest. Suppl. 21:77-89. Ferrante, A. and Thong, Y. 1980. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the Hypaque-Ficoll method. J. Immunol. Methods 36:109. Kaplan, J., Nolan, D., and Ree, A. 1982. Altered lymphocyte markers and blastogenic response associated with 24 hour delay in processing of blood samples. J. Immunol. Methods 50:187191. Niku, S., Hoon, D., Cochran, A., and Morton, D. 1987. Isolation of lymphocytes from clotted blood. J. Immunol. Methods. 105:9-14.

Ridings, J., Weedon, H., Ioannu, C., Flego, L., Macadle, P., and Zola, H. 1996. Purification of cord blood lymphocytes. J. Immunol. Methods. 195:43-48. Skoog, W. and Beck, W. 1956. Studies of the fibrinogen, dextran and phytohaemagglutinin methods of isolating leukocytes. Blood 11:436. Tamul, K., Schmitz, J., Kane, K., and Folds, J. 1995. Comparison of the effects of Ficoll-Hypaque separation and whole blood lysis on results of immunophenotypic analysis of blood and bone marrow samples from patients with hematologic malignancies. Clin. Diag. Lab. Immunol. 2:337342. Thiele, D., Kurosaka, M., and Lipsky, P. 1983. Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: Delineation by its sensitivity to the lysosomotropic agent, L-leucine methyl ester. J. Immunol. 131:2282-2290. Thong, Y., Currell, J., and Rodwell, R. 1983. The rapid one-step gradient centrifugation procedure for simultaneous isolation of granulocytic and mononuclear leukocytes from human blood: biological, physical and chemical bases. Med. Hypotheses 12:103-111. Tse, H. and Dutton, R. 1976. Separation of helper and suppressor T lymphocytes on a Ficoll velocity sedimentation gradient. J. of Exp. Med. 143:1199-1210. Vissers, M., Jester, S., and Fantone, J. 1988. Rapid purification of human peripheral blood monocytes by centrifugation through FicollHypaque and Sepracell-MN. J. Immunol. Methods 13:203-207.

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