In vitro differentiation of Macrophages from Monocytes via M-CSF

Introduction

Tissue macrophages can be derived from monocytes. When isolated from blood and cultured in media with serum, adherent monocytes will differentiate into macrophages. For a pure macrophage culture, we recommend that you add factors such as M-CSF. Adding other factors including IL-4, IL-10, or TGF-β can help improve viability. The protocol below is for a T25 or T75 flask but can be scaled down for 100 mm culture dishes or plates.

Materials

T25 or T75 sterile flasks with vented caps (e.g., Nunc EasYFlask Cell Culture Flasks, T25, filter, Cat. No. 156367)
RPMI 1640 medium (e.g., BenchStable RPMI 1640 Medium, Cat. No. A4192301)
Fetal Bovine Serum (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140087, or Fetal Bovine Serum One Shot format, Cat. No. A3160401)
L-glutamine (e.g., 200 mM L-Glutamine, Cat. No. 25030149)
Optional: penicillin-streptomycin (e.g., Gibco Penicillin-Streptomycin, Cat. No. 15140148)
M-CSF purified protein (e.g., Gibco M-CSF Recombinant Human Protein, Cat. No. PHC9504)
Optional: IL-4 purified protein (e.g., IL-4 Recombinant Human Protein, Cat. No. A42602)
Phosphate buffered saline (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011044)
EDTA (e.g., UltraPure 0.5M EDTA, pH 8.0, Cat. No. 15575020)
50 mL conical tube (e.g., Nunc 50 mL conical sterile centrifuge tubes, Cat. No. 339652)
Flow cytometry staining buffer (e.g., eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26)

Procedure

Using aseptic techniques under sterile conditions, isolate PBMC from whole blood (See supplemental protocol A below).

Prepare complete RPMI 1640 medium by supplementing RPMI 1640 medium with fetal bovine serum to a final concentration of 10%, 2 mM L-glutamine (if using medium not currently supplemented with GlutaMAX). Bring medium to 37°C. Optional: Supplement media with 1% penicillin-streptomycin (5,000 units/mL).

Resuspend cell concentration to 2 x 10⁶ cells/mL in complete RPMI 1640 medium.

Transfer resuspended cell solution to a cell culture dish.

Incubate the culture dish for 24 hours in 5% CO₂ incubator at 37°C to allow the monocytes to adhere to dish.

In a sterile conical tube, prepare complete RPMI 1640 medium with M-CSF Recombinant Human Protein at a final concentration of 40-50 ng/mL. Optional: Add 20 ng/mL IL-4 Recombinant Human Protein.

Replace media in culture dish with the prepared media containing M-CSF and IL-4 (if using).

Incubate cells for 6 days in 5% CO₂ incubator at 37°C. Within the 6 days, replenish media with new complete RPMI 1640 medium supplemented with 40–50 ng/mL of M-CSF Recombinant Human Protein (Optional: and 20 ng/mL IL-4 Recombinant Human Protein) every 3–4 days. Check under microscope for cell health and confluence.

Cells are ready to harvest when cells exhibit more granules in the cytoplasm and are a bit elongated. In addition, cells should be more adherent to the culture plate. When cells are ready to harvest, discard old media, and rinse dish twice with 1X PBS, discarding PBS after each rinse.

Add 10 mL 10 mM EDTA to each culture dish, and let sit for 10 minutes, or until cells dissociate from dish at room temperature.

Collect cells in a 50 mL conical tube and centrifuge at 300–400 x g for 4–5 minutes at room temperature.

Discard the supernatant and rinse cells with 1X PBS.

Centrifuge cells at 300–400 x g for 4–5 minutes.

Discard the supernatant, and resuspend cells in flow cytometry staining buffer or desired medium.

Protocol tip:

Culturing monocytes on Nunc UpCell Dishes can help preserve cell viability and surface proteins during cell harvest. Nunc dishes with UpCell Surface enables harvesting of cells by temperature reduction without the need for dissociation enzymes (e.g., trypsin, EDTA), thereby maintaining cellular membrane and surface receptors.

Supplemental protocol A: isolation of PBMC from whole blood

Materials

Phosphate buffered saline (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011044)
15 mL or 50 mL conical tube (e.g., Nunc 15 mL conical sterile centrifuge tubes, Cat. No. 339650)
Ficoll-Paque^® density separation medium
Flow cytometry staining buffer (e.g., eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26)

Procedure

Dilute blood sample at least 1:1 with PBS in a conical tube.

Underlay the diluted sample with a volume of Ficoll-Paque^® medium that is equal to the original sample volume.

Centrifuge at 400 x g for 20 minutes at room temperature with the brake OFF.

Harvest PBMC located at the interface of the PBS and Ficoll-Paque^® medium layers into a fresh tube.

Fill the tube with PBS to wash the cells.

Centrifuge the cells at 300–400 x g for 4–5 minutes at 2–8°C. Discard supernatant.

Resuspend the cell pellet in an appropriate volume of flow cytometry staining buffer or buffer of choice, and perform a cell count and viability analysis.

Centrifuge cells as in step 6, and resuspend in appropriate volume of complete RPMI 1640 so that the final cell concentration is 2 x 10⁶ cells/mL.

Note:

As cells will be cultured, perform all steps using aseptic techniques, and use buffers that do not contain azide.

Protocol tip:

Significantly improve the accuracy of assessing cell health and concentration from freshly harvested peripheral blood mononuclear cells with automated counters. See detailed protocol on how to count PBMC using the Countess II FL Automated Cell Counter.

For Research Use Only. Not for use in diagnostic procedures.