Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

Summary

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Abstract

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

Introduction

Study of cultured macrophages is a useful model to understand the function of these cells in inflammation such as occurs in atherosclerotic plaques. When the research focus is on human diseases involving macrophages, it is useful to study primary human macrophages rather than non-human macrophages to avoid species differences. Also, the effects of cell transformation can be avoided by using primary macrophages rather than macrophage cell lines. For this purpose, macrophages differentiated from monocytes isolated from human blood serve as a means of obtaining primary human macrophages.

Tissue macrophages may be either resident within tissues or may be derived from monocytes that migrate into the tissue and differentiate into macrophages ¹. Two types of human monocyte-derived macrophages have been defined that differ not only in morphology but also gene expression and cell function ². These two types are obtained from monocytes that are differentiated into macrophages in the presence of M-CSF + fetal bovine serum (FBS) and monocytes that are differentiated into macrophages in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) + FBS ^2-6. In our experience (unpublished observation), use of human serum rather than FBS generates the GM-CSF type regardless of whether M-CSF or GM-CSF is included in the differentiation medium. M-CSF type macrophages tend to be more elongated than GM-CSF type macrophages, which resemble fried eggs in their morphology. Investigators should recognize that human M-CSF and GM-CSF monocyte-derived macrophages are not the same as so called M1 and M2 mouse bone marrow-derived macrophages ⁶.

During research using human monocyte-derived M-CSF differentiated macrophages, we experienced difficulty related to the availability of monocytes to initiate experiments and variation in the obtained cell densities of macrophages during differentiation of monocytes into macrophages. To overcome these problems, we have developed the following protocol in which monocytes are frozen until required for use, and monocytes are differentiated in bulk into macrophages that can then be removed from culture flasks and plated at desired cell densities to obtain more uniform cultures from experiment to experiment.

Protocol

Leukapheresis was carried out under a human subject's research protocol approved by a National Institutes of Health institutional review board.

1. Isolation and Cryopreservation of Monocytes

1. Obtain mononuclear cells by leukapheresis of human donors, and enrich monocytes by continuous counter-flow centrifugation elutriation of mononuclear cells as described in the references ^7,8. Obtain approximately 100 x 10⁶ elutriated cells (approximately 80 - 90% monocytes) in elutriation buffer specified by the manufacturer. Count cells using a hemocytometer.
2. Centrifuge monocytes in a 15 ml polypropylene tube at 300 x g for 5 min at room temperature.
3. Remove the supernatant and gently resuspend the cells in FBS followed by dimethyl sulfoxide to achieve a final concentration of 90% FBS/10% dimethyl sulfoxide and 50 x 10⁶ cells/ml.
4. Add each ml of cell suspension to an individual cryovial.
5. Place the cryovials in a cell freezing container and transfer to a -80 °C freezer for 24 hr before transferring to a liquid nitrogen cryovial storage tank for long-term storage.

2. Differentiation of Monocytes into Macrophages

1. Thaw a cryovial of cells by quickly transferring to a 37 °C water bath and then immediately removing when the cell suspension has thawed about 70%.
2. Immediately upon complete thawing at room temperature, transfer the 1 ml cryovial contents into 50 ml of 37 °C warmed Roswell Park Memorial Institute (RPMI) 1640 medium with 2 mM L-glutamine, 50 ng/ml M-CSF, 25 ng/ml interleukin-10 (IL-10), and 10% FBS (complete medium).
3. Transfer 25 ml of monocyte suspension into each of two 75 cm² plastic cell culture flasks.
4. Incubate cultures in a 37 °C cell culture incubator with 5% CO₂ / 95% air for 48 hr.
5. Rinse the cultures 3 times with 10 ml RPMI 1640 medium (pre-warmed to 37 °C), gently removing the culture medium to avoid dislodging any loosely attached cells.
6. Following rinsing, add fresh complete medium, changing medium every 2 days until monocytes differentiate and proliferate sufficiently to become confluent. This requires about one week of culture.

3. Harvesting Macrophages to Initiate Experiments

1. Rinse differentiated macrophages in flask 3 times with 10 ml pre-warmed 37 °C Dulbecco's phosphate-buffered saline without Ca²⁺ and Mg²⁺ before adding 10 ml pre-warmed 37 °C 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution.
2. Place flask in cell culture incubator for 10 - 15 min at which time approximately 90% of the macrophages should have rounded and detached. Verify this by microscopic examination of the flask.
3. Add 10 ml of RMPI 1640 medium containing 10% FBS to stop trypsinization.
4. Transfer the macrophage cell suspension from the flask into a 50 ml polypropylene tube, centrifuge 300 x g for 5 min, and discard the supernatant.
5. Gently resuspend the macrophages into 1 ml of complete medium.
6. Mix 10 µl of macrophage cell suspension with 10 µl Trypan Blue solution and determine macrophage cell number and viability (typically > 95%) using a hemocytometer.
7. Based on the cell count (usually about 15 - 18 x 10⁵ macrophages), add additional complete medium to achieve the desired seeding cell density. Note: 1 x 10⁵ macrophages in 1.5 ml medium per well of a 12-well plate will produce a near-confluent culture.
8. Following seeding, incubate macrophages overnight in a 37 °C cell culture incubator with 5% CO₂ / 95% air to allow cell attachment. Then, begin desired experiments.

Results

The viability of fresh or cryopreserved monocytes was greater than 95% as determined with Trypan Blue staining ⁹. Figure 1 and Figure 2 compare at lower and higher magnifications, respectively, the progress of fresh and cryopreserved (i.e., frozen) monocyte differentiation into macrophages. Note that the fresh compared with cryopreserved monocytes show a subpopulation of differentiating monocytes that do not spread but...

Discussion

Generating defined macrophage types can clarify some of the conflicting results obtained by investigators when studying macrophage biology. The use of various culture conditions and differentiation factors to generate primary human macrophages can lead to very different macrophage types, a fact that may not be appreciated by the researcher. For example, macrophages sometimes are generated from human monocytes using no serum, human serum alone, human serum supplemented with M-CSF, FBS alone, or FBS containing M-CSF or GM-...

Materials

Name	Company	Catalog Number	Comments
Cellbind 12-well culture plate	Corning	3336
CELLSTAR, T-75 flask, tissue culture treated	Greiner Bio-One North America	658157
RPMI 1640 culture medium	Cellgro Mediatech	15-040-CM	warmed to 37 °C
L-Glutamine	Cellgro Mediatech	25-005-CI
Fetal bovine serum	Gibco	16000-036
M-CSF	PeproTech	300-25
GM-CSF	PeproTech	300-03
IL-10	PeproTech	200-10
DMSO	Sigma	D2650
Cryovial	Thermo Scientific	375418
DPBS without Ca2+ and Mg2+	Corning cellgro	21-031-CV
0.25% Trypsin-EDTA	Gibco	25200-056
50 ml polypropylene conical tube	Falcon	352070
Trypan Blue	Lonza	17-942E
Neubauer-improved bright light hemocytometer	Paul Marienfeld GmbH & Co. KG	610031	http://www.marienfeld-superior.com/index.php/counting-chambers/articles/counting-chambers.html
CoolCell LX cell freezing container	BioCision	BCS-405	other freezing containers also should  be adequate for this step
Liquid Nitrogen Storage System, CryoPlus 1	Thermo Scientific	7400	any liquid nitrogen tank should be adequate