J Sep Sci. 2016 Aug;39(15):3062-71.

Single-step enrichment of basophils from human peripheral blood by a novel method using a Percoll density gradient.

Shiono H1, Matsui T1, Okada T1, Ito Y2.
  • 1Department of Physiology, Aichi Medical University School of Medicine, Yazako, Nagakute-city, Aichi, Japan.
  • 2Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Abstract

We have developed a novel continuous flow-through cell separation method using a Percoll density gradient. This method can continuously separate a large number of cells into five fractions according to their densities. To apply this method to the separation of basophils, Percoll density gradients were modified to improve basophil enrichment. When a set of Percoll density gradients was prepared (1.071, 1.075, 1.080, 1.084, and 1.090 g/mL) the basophils in a healthy volunteer were enriched by an average of 23.1 and 63.5% at Percoll densities of 1.075 (fraction 3) and 1.080 g/mL (fraction 4), respectively. On average, the yield of basophils was 1.66 × 10(5) cells in fraction 3 and 1.61 × 10(5) cells in fraction 4 from 9 mL of peripheral blood. The expression of CD203c (cluster of differentiation 203c) on separated basophils was upregulated by anti-immunoglobulin E stimulation similar to basophils in whole blood. Histamine release induced by calcium ionophore was also observed in the separated basophils. The present method will be useful for basophil enrichment since it preserves their function without using counterflow elutriation and immunological reagents, and this method will be effective as a preparative separation for cell purification by flow cytometry.

KEYWORDS:Basophils; Cell separation; Centrifugation; Density gradient; Human peripheral blood

PMID: 27293108; DOI:10.1002/jssc.201600329

 

Supplement

We developed a novel, continuous, flow-through, cell separation method using a Percoll density gradient that can continuously separate a large number of cells into five fractions according to their densities.

In the present study, we applied this method to the separation of basophils from peripheral blood in a single step without using counterflow elutriation or immunological reagents.

Diluted peripheral blood is typically continuously pumped into the most proximal inlet-1 at a higher flow rate (0.3 mL/min) than that of the gradient medium. Under a centrifugal force field, cells present in peripheral blood gradually migrate into the gradient until they reach medium with a density matching their own. The centrifuge is rotated at 1500 rpm (about 140 × g).

Approximately 10 mL of anticoagulated blood was diluted by about a factor of 2 so as to not disturb the density gradient, then pumped into the separation column through the proximal inlet-1 at 0.3 mL/min for about 70 min. The density gradient is concentric in the separation disk under the centrifugal force field and is not disturbed by the addition of diluted blood. Red blood cells flow toward the outlets and gradually migrate into the density gradient (Fig. 1), then flow along the periphery of the column and out through outlet-6. Each type of leukocyte migrated to medium whose density matched their own and flowed out through the corresponding outlet. Consequently, the cells were continuously separated and harvested according to their densities. The supernatant, diluted plasma flowed out from outlet-1.

IgE receptors on basophils enriched using the present method were well preserved. In the case of blood from a patient with atopic dermatitis (AD), the CD203c antigen level on the enriched basophils was upregulated after activation by anti-IgE, similar to that of basophils in whole blood. Although it was thought that basophils from the AD patient would be more sensitive to various stimulations, the IgE receptors on the enriched basophils were also well preserved after processing using the present method, showing that functionally viable basophils could be separated equally well from an AD patient and from a healthy volunteer. The present method will be useful for the preparative and gentle separation of basophils and other functional cells.

In another study, density gradient media with densities of 1.073, 1.079, 1.090, 1.095 and 1.102 g/mL were used in an attempt to separate basophils and eosinophils simultaneously. In this set of density media, the 1.079 g/mL medium was most effective for concentrating basophils in one fraction. Eosinophils comprise 1-4% of peripheral blood leukocytes, have a higher density than basophils, and participate in allergic reactions and in response to parasites. Hemolysis processing was indispensable for the refinement of leukocytes in each separated fraction (Fig. 2, 3) but was time consuming and could potentially damage the cells. 

Based on these results, we used the newly modified procedure to address the following two points:

  1. Minimize prolongation of the cell separation time caused by sample dilution.
  2. Minimize the use of hemolytic agents for refining the harvested leukocytes.

 

Figure 1. Stroboscopic observation near the inlet tubes.

black arrow : flow of density gradient medium;     white arrow: direction of centrifugal force and of cell migration

 

In order to address the first point, a new set of media with densities of 1.050, 1.074, 1.079, 1.090, 1.095 and 1.104 g/mL were made by diluting Percoll and then they were continuously pumped into inlets 1 through 6 under a centrifugal force field. It is important for this procedure that the 1.050 g/mL density medium flow through inlet-1. The specific gravity (relative density) of human peripheral blood is about 1.05-1.06 g/mL. Therefore, even if undiluted anticoagulated peripheral blood is introduced through proximal inlet-1 instead of density with a density of 1.050 g/mL, the density layers are not disturbed. Furthermore, the time taken for pumping the sample into the separation column at 0.3 mL/min is about half that required using the previous method.

In order to address the second point, harvested cells with densities of 1.079 g/mL and 1.090 g/mL were washed, and the cell pellets containing red blood cells were re-suspended into 1 mL of medium with a density of 1.050 g/mL. Each re-suspended cell sample (1 mL) was introduced through inlet-1 at 0.3 mL/min successively and separated again, at 1500 rpm for about 10 min. Harvested cells with densities of 1.079 g/mL and 1.090 g/mL were subjected to the second separation step using the same density gradient. After the second separation, the fractions with densities of 1.079 g/mL and 1.090 g/mL comprised more than 70% basophils and 95% neutrophils, respectively. Some red blood cells appeared in both fractions. Cell viabilities, measured by the trypan blue-exclusion test, were more than 97%. As a result, peripheral blood could be separated without dilution and hemolysis was not necessary. The total time required for cell separation was shortened even if the separation was repeated in order to further remove red blood cells. Basophils and neutrophils were well separated and few red blood cells were present after the second separation. 

Basophils are the least common leukocytes in human peripheral blood and are therefore difficult to harvest efficiently, which has hampered research on basophils. However, it was recently revealed that basophils produce IL-4 when stimulated with IL-33. IL-4 from basophils is required for Th2 differentiation and natural helper cells. It appears that basophils regulate the expression of asthma in lung inflammation, and behave as antigen presenting cells (APC) in the acquired immune system (1, 2). Thus, the function of basophils in the immune response has attracted the attention of many researchers, increasing the need to harvest basophils and other immune cells without using immunological reagents. 

In conjunction with the immune response, dendritic cells (DCs), which are representative APCs, play an important role in the regulation of both humoral and cellular immune responses. DCs attract attention as a promising method for cancer immunotherapy (3, 4). DCs have various types and are distributed in spleen, lymph nodes, bone marrow, epidermis, and various other tissues. Most studies on DCs are carried out in animals (5). Mouse DCs are typically isolated from spleen by collagenase digestion and are isolated from low-density cells by density gradient centrifugation. The DCs are enriched by immuno-magnetic cell sorting (MACS), then purified by fluorescence-activated cell sorting (FACS). The final purity of DCs following FACS is approximately 99% (6). However, some researchers prefer to isolate DCs without using antibodies (to the extent possible). Therefore, we investigated conditions suitable for isolating and concentrating mouse DCs using the present method and without antibodies. Our aim was to achieve preparations comprising more than 90% dendritic cells. 

We currently use two types of density media. One is Percoll, our most common medium and one which is well-referenced for the density gradient centrifugation of cells and subcellular particles. The other is OptiPrep, developed as an X-ray contrast medium and composed of Iodixianol, which is non-ionic, non-toxic to cells, and metabolically inert. Approximately 2×108 cells were isolated from each mouse spleen. DCs could not be identified by observing stained cells under a microscope and thus we determined that CD11c-positive cells were dendritic cells by FACS analysis. Use of a set of Percoll media with densities of 1.060, 1.070, 1.080, 1.090, and 1.100 g/mL provided unsatisfactory concentration and viability of DCs. However, a set of OptiPrep gradients with densities of 1.066, 1.071, 1.078, 1.085, and 1.095 g/mL under isosmotic conditions (290 mOsm/L) allowed the harvesting of CD11c-positive DCs in the 1.066 g/mL medium with high viability and concentration.

The present method does not require specific antibodies and will be useful for the separation of various labile functional cells using different types of density gradient media with various densities. Because cell separation and purification are the most important steps in cell research, and the present method does not require specific antibodies or special skills, we feel that our report will be of interest to researchers in cell biology.

Figure 2. Harvested leukocytes in the 1.079 g/mL density fraction after hemolysis.

Figure 3. Harvested leukocytes in the 1.102 g/mL density fraction after hemolysis.

 

References

1.       Motomura Y., Morita H., Moro K., Nakae S., Artis D., Endo A. T., Kuroki Y., Ohara O., Koyasu S., Kubo M. (2014) Basophil-derived Interleukin-4 controls the function of natural helper cells, a member of ILC2s, in lung inflammation. Immunity 40: 758-771.

2.      Nakanishi K. (2010) Basophil are potent antigen-presenting cells that selectively induce Th2 cells. Eur J Immunol 40: 1836-1842.

3.      Steinman, R. M., Cohn, Z. A. (1973) Identification of a novel cell type in peripheral lymphoid organs of mice. J Exp Med 137: 1142-1162.

4.      Kroczek R. A., Henn V. (2012) The role of XCR1 and its ligand XCL1 In antigen cross-presentation by murine and human dendritic cells. Front Immunol 3, 14. 10.3389/fimmu.2012.00014

5.      Collin, M., Bigley, V., Haniffa, M., Hambleton S. (2011) Human dendritic cell deficiency: the missing ID? Nat Rev Immunol 11: 575-583. 10.1038/nri3046

6.      Williamson, E., Bilsborough, J. M., Viney, J. L. (2002) Regulation of mucosal dendritic cell fubction by receptor activator of NF-κB (RANK)/RANK ligand interactions: Impact on tolerance induction. J Immunol 169: 3606-3612.

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann