Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Summary

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Abstract

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

Introduction

Dendritic cells (DCs) are essential mediators of the innate and adaptive immune systems. They function to induce primary immune responses and facilitate the development of immunological memory. These cells are primarily responsible for antigen capture, migration and T cell stimulation and are therefore referred to as professional antigen presenting cells (APCs) ¹.Manipulation of DCs could be utilized across a wide variety of research fields and in the clinical setting to treat different inflammatory diseases such as HIV ^6,7, cancer ⁸, autoimmune diseases ⁹, and allergic responses ¹⁰. DCs are also being used for substance abuse research in order to solve unknown mechanisms and pathways such as those associated with alcohol dependence ^11-14, drug dependence ^13,15, and the combination of HIV infection and substance abuse ^16-19. These ongoing studies and future research studies in the field of immunology make in vitro generation of DCs extremely important for research. However, there are several difficulties associated with isolating DCs from human blood as they only constitute 0.1 - 1% of total blood mononuclear cells ²⁰.

To date, some of the well-established methods for the generation of DCs in vitro consists of plastic or glass adherence of monocytes ^21,22, density gradient centrifugation ²³, specific marker based separation such as magnetic activated cell sorting ²², fluorescent activated cell sorting ²⁴, positive selection of CD14+ monocytes using dextran-coated magnetic nanoparticles ²⁵, and rapid isolation of highly purified monocytes using fully automated negative cell selection ²⁶. However, the best method of choice remains controversial. Therefore, to improve DC generation techniques, several methods have been developed in which the purity of these cells can be greatly increased by differentiation from purified CD34+ progenitor cells and monocytes isolated from peripheral blood mononuclear cells (PBMCs) ²⁷. As mentioned prior, a widely used and popular method for generating monocyte derived dendritic cells (MDDCs) is to explore the ability of monocytes to adhere to glass or plastic (adherence method) ^21,22,27. The adherence method is a rapid and straightforward method that does not require the use of complex equipment. However, some disadvantages of this process include lymphocyte contamination, low flexibility, and monocyte transient manipulation ²⁸. An alternative method to the adherence method is the magnetic isolation of monocytes from total PBMCs , particularly with the use of a human monocyte enrichment kit, which is designed to isolate monocytes from PBMCs by negative selection ²⁶. During this procedure, unwanted cells are targeted for removal with tetrameric antibody complexes and dextran-coated magnetic particles. The advantage of this isolation method is that the unwanted labeled cells are separated using a magnet while target cells can be freely poured off into a new tube without the need for columns. To date, with the availability of specific monoclonal antibodies that can label unique cell populations, the magnetic separation technique has become not simply an additional method, but a necessity for the isolation of rare cells in the field of immunology. For instance, techniques such as magnetic cell sorting with commercially available paramagnetic MACS-nanoparticles have facilitated the development of new approaches for research and clinical applications ^22,29. Furthermore, recent research studies comparing DC generation from monocyte adherence and MACS technology methods have demonstrated a higher DC purity and viability using MACS separated monocytes ^22,30.

The current study presents a comparison between two methods for the generation of human DCs from monocytes isolated from PBMCs: 1) monocyte isolation by adherence and 2) monocyte isolation by negative selection using a commercial human monocyte enrichment kit. This study provides evidence to show that the negative selection magnetic separation procedure to isolate monocytes generates the highest yield of monocytes with no significant differences in monocyte viability when compared with monocytes isolated by adherence method. In turn, after seven days, the monocytes isolated by magnetic separation differentiated into MDDCs with significantly higher proliferative capacity and higher amount of cells expressing double positive (CD11c+/CD14+) phenotype without affecting MDDC viability. Overall, the current study differs from the previous studies referenced above since it demonstrates the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into CD11c+ MDDCs (> 70%) after seven days in culture without compromising their viability. In addition, the current approach provides for the first time characterization of different CD11c/CD14 MDDCs populations by imaging flow cytometry.

In summary, since DCs play a focal role regarding research in the field of immunology, different parameters must be taken into consideration when considering how they are derived and what methods are used to isolate and culture them in vitro. Therefore, this study aims to provide insights on two different methods of monocyte isolation and how these methods differentially affect monocyte viability and yield eventually affecting dendritic cell viability, proliferation, and phenotype. These findings will contribute greatly to the field of immunology and will provide a detailed protocol of DC isolation, purification, and characterization.

Protocol

Overall human blood studies have been reviewed and approved by the Institutional Review Board (IRB) of FIU, IRB protocol approval # IRB-13-0440. Human leukopaks were purchased from the community blood bank in Miami, FL.

1. Isolation of PBMCs by Standard Density Gradient Technique

1. Perform a 1:1 dilution of blood with 1x-phosphate-buffered saline in a T75 flask.
2. Pipette 15 ml of density gradient solution into 50 ml centrifuge tubes and carefully layer (25 - 30 ml/tube) of the diluted blood over this gradient.
3. Centrifuge for 20 min at 1,200 x g with acceleration of 1 and deceleration of 0.
4. After centrifugation, collect the interface layer (white blood cells) into a new 50 ml centrifuge tube and wash cells twice in PBS (3 min at 1,080 x g). Discard supernatant each time.
5. Treat cells with ammonium-chloride-potassium lysing (ACK) buffer (to lyse red blood cells). Add 10 ml of buffer and incubate for 15 min at 4 °C.
6. Wash cells twice with PBS, centrifuge for 3 min at 1,080 x g and discard supernatant each time.
7. Save the pellet, which will contain the PBMCs.
8. Proceed with monocyte purification method of choice.

2. Monocyte Purification by Adherence Method

1. Prepare complete cell culture medium containing L-glutamine (300 mg/ml) and supplemented with penicillin (50 U/ml)-streptomycin (50 µg/ml) and 10% fetal bovine serum.
2. Allow PBMCs to adhere for approximately 2 hr by culturing them in a T75 flask at a concentration of 5 x 10⁷ cells per 10 ml of complete medium at 37 °C and 5% CO₂ in a humidified incubator.
3. After incubation, remove non-adherent floating cells from culture flask and gently wash adherent cells twice with PBS.
4. Incubate adherent cells in complete cell culture medium supplemented with 2 µl/ml of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) stored at a stock concentration of 10 µg/ml.
5. Change half of the medium and replenish cytokines every 48 hr.
6. Allow 5 - 7 days for the differentiation of monocytes into MDDCs .

3. Monocyte Purification by Magnetic Separation Method

1. Collect PBMCs from standard density gradient technique, pour into a 5 ml polystyrene tube and re-suspend in PBS buffer at a concentration of cells/ml.
2. Add human monocyte enrichment cocktail using 50 µl/ml cells, mix well and incubate at 4 °C for 10 min.
3. After incubation, add magnetic particles using 50 µl/ml cells, mix well and incubate at 4 °C for 5 min.
4. Bring cell suspension up to a total volume of 2.5 ml by adding PBS buffer, mix well by pipetting up and down 2 - 3 times.
5. Place the polystyrene tube without a cap into the magnetic device and set aside at RT for 2.5 min.
6. After incubation and in one continuous motion, pick up the magnet and pour the desired purified monocyte fraction into a clean 50 ml centrifugation tube.
7. Incubate adherent cells in complete cell culture medium supplemented with 2 µl/ml of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) stored at a stock concentration of 10 µg/ml.
8. Every 48 hr, change half of the medium, spin down at 1,080 x g for 5 min and re-suspend pellet with new media (CRPMI) and cytokines.
9. Allow 5 - 7 days for the differentiation of monocytes into MDDCs.

4. Trypan Blue Exclusion Viability Assay

Note: Use this technique to obtain cell yield and viability of PBMCs, monocytes, and MDDCs.

1. Harvest cells using standard trypsin-EDTA and perform washes of the flasks and the cell pellet using PBS. Depending on the size of the pellet, resuspend in 5 to 10 ml of PBS to dissolve the pellet.
2. In a micro-centrifuge tube, perform a 1:1 dilution of trypan blue reagent with diluted cell pellet.
3. From this mix, aliquot 10 µl into a cell counting slide and read results using an automated cell counter according to manufacturer's protocol. If an automated cell counter is not available, a similar cell count is possible using a hemocytometer or a manual counter.

5. MTT Assay

1. Plate cells at a concentration of 4 x 10⁴/100 µl of complete media into each well in a 96-well plate. Allow 24 hr for cells to adjust to culture.
2. Incubate and perform readings at 0 (day 0), 36 (day 3) and 84 (day 7) hr respectively.
3. At completion of desired incubation, remove media and replace with 100 µl of PBS alone.
4. Prepare 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (MTT) (5 mg/ml) by adding 10 ml of dimethyl sulfoxide (DMSO) to 1 g of sodium dodecyl sulfate (SDS).
5. Add 10 µl (5 mg/ml) of MTT solution to each well and incubate at 37 °C for 2 additional hours.
6. After incubation, remove supernatants which contain PBS and unconverted MTT mixture (yellow-color solution).
7. Add 100 µl of SDS-DMSO solution to each well and incubate for one additional hour.
8. Read absorbance at 540 nm using a microplate reader.

6. Cell Surface Staining Analysis by Imaging Flow Cytometry

1. After 7 days of differentiation from purified monocytes, dispense 1x10⁶ cell aliquots of the monocyte-derived dendritic cells into the appropriate number of 1.5 ml tubes.
2. Start cell surface staining by blocking cells using 50 µl of heat inactivated (HI) human serum, incubate at 4 °C for 10 min.
3. After incubation, centrifuge cells at 720 x g for 5 min, discard supernatant.
4. To cell pellet, add respective fluorochrome-conjugated antibodies (e.g., anti-CD11c or anti-CD14) and incubate at 4 °C for 20 min protected from light. In separate tubes, prepare single fluorochrome-stained samples/ml) since they are needed for compensation.
5. After incubation, wash cells twice in 1 ml of PBS buffer and centrifuge each time at 720 x g for 5 min.
6. Keeping cells protected from light, re-suspend in PBS buffer at a concentration of cells/100 µl for flow cytometry analysis.
7. In order to gate on viable cells, add 1 µl of DAPI to each tube prior to acquiring the cells on the single cell imaging flow cytometry instrument according to manufacturer's instructions.

7. Statistical Analysis

1. Input all data in a spreadsheet.
2. Compare results using the student's t-test and/or one-way ANOVA as appropriate.
3. Consider differences to be statistically significant if p < 0.05.

Results

Monocyte Yield by Magnetic Separation is Higher Compared to Monocyte Yield by Adherence Method

Data presented in Figure 1 display PBMC and monocyte cell counts by the trypan blue exclusion method at the day of isolation of PBMCs and separation of monocytes. On average, monocytes isolated by the adherence method accounted for approximately 6.2 percent of total PBMCs while monocytes isolated by ma...

Discussion

Based on the known difficulties of isolating and generating MDDCs from human blood, the present study aimed to provide a comprehensive comparison of two well-established methods for the generation of MDDCs. The first method compared is a well-established traditional method for generating MDDCs by exploiting the ability of monocytes to adhere to glass or plastic (adherence method) ^21,22,27. The adherence method is fast and cost effective, and does not require the use of complex equipment. However, some disadvan...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ficoll-Paque	GE Healthcare	17-5442-03	Must be used at RT
Phosphate-buffered saline (PBS)	Life Technologies	10010-023
ACK Lysing buffer	Quality Biological	118-156-101
RPMI 1640 medium	Life Technologies	22400-089
Antibiotic-Antimycotic (100x)	Life Technologies	15240-062
Fetal Bovine Serum (FBS)	Life Technologies	16000-044
RoboSep buffer	StemCell	20104
EasySep Human monocyte enrichment kit	StemCell	19059
TC20 Automated cell counter	Bio-Rad	145-0101
FITC-Dextran	Sigma Aldrich	FD4-100MG
Trypan blue stain (0.4%)	Life Technologies	15250-061
Synergy 2 multi-mode reader	Biotek	7131000
XTT Sodium salt bioreagent (XTT)	Sigma Aldrich	X4626-100MG
Dimethyl sulfoxide bioreagent (DMS)	Sigma Aldrich	D8418-500ML
Thiazolyl blue tetrazolium bromide (MTT)	Sigma Aldrich	m5655-500MG
Sodium Dodecyl Sulfate (SDS)	Bio-Rad	161-0302
Phenazine Methosulfate (DMSO)	Sigma Aldrich	P9626-1G
Inactivated (HI) Human Serum	Chemicon	S1-100ML
Accuri C6 Flow Cytometer	BD Accuri	653119
FlowSight Amnis Flow Cytometer	EMD Millipore	100300