Using Cryopreserved Cells for Cultures of Human Primary Macrophages

Primary human macrophages (Mϕ) are hard to separate from tissue in adequate amounts with a homogeneous phenotype, and are unable to proliferate in culture.

Highly pure human monocyte-derived macrophages (hMDM) are easily available and offer a superior alternative, even though their differentiation in vitro is time-intensive. Nevertheless, the distinct cell loss observed at the time of detachment of this adherent cell type and the low rate of re-attachment after subculture considerably affects experimental design and flexibility in the day-to-day routine. There is a growing demand for easy-to-handle and less time-consuming applications since macrophage research is gaining continuous importance.

Cryopreserved human macrophages are a part of PromoCell’s distinctive range of macrophage culture products and now come as a reliable source of standardized cells in a ready-to-use format, enabling flexibility of the entire experiment. In PromoCell’s well-proven M1/M2- Macrophage Generation Media DXF, the frozen macrophages are produced and are available as fully qualified M1- (hMDM-GMCSF(-)) or M2- (hMDM-MCSF(-)) polarized cells. It is possible to add the macrophages into all types of multiwell-plates, flasks, and dishes. The macrophages can be preserved as biologically functional, adherent cultures (see Figures 1 and 2) for a number of weeks, after plating. If necessary, user-customizable activation of the cells can be conducted (refer to the culture protocol, Figure 3 and Table 1).

Morphology of PromoCell frozen macrophages in culture. Image shows hMDM-GMCSF(-) 24 hours after plating at 100.000 cells/cm2. A healthy culture with the typical cellular morphology of non-activated mature Mϕ-M1 is established from the cryopreserved cells, 200x magnification.

Figure 1. Morphology of PromoCell frozen macrophages in culture. Image shows hMDM-GMCSF(-) 24 hours after plating at 100.000 cells/cm2. A healthy culture with the typical cellular morphology of non-activated mature Mϕ-M1 is established from the cryopreserved cells, 200x magnification.

“All you can eat”: PromoCell frozen macrophages in action. Demonstration of the strong phagocytic activity of hMDM- MCSF(-) using pHrodo™-labeled E. coli. Red fluorescence indicates large numbers of bacteria ingested by the macrophages. Plating density: 140.000 cells/cm2, 200x magnification.

Figure 2. “All you can eat”: PromoCell frozen macrophages in action. Demonstration of the strong phagocytic activity of hMDM- MCSF(-) using pHrodo™-labeled E. coli. Red fluorescence indicates large numbers of bacteria ingested by the macrophages.
Plating density: 140.000 cells/cm2, 200x magnification.

The culture of PromoCell cryopreserved human macrophages. The macrophages can be directly used for experiments after thawing and plating. Optional: Activation of polarized M1/M2 macrophages before assay. See Table 1 for more activation details.

The culture of PromoCell cryopreserved human macrophages. The macrophages can be directly used for experiments after thawing and plating. Optional: Activation of polarized M1/M2 macrophages before assay. See Table 1 for more activation details.

Figure 3. The culture of PromoCell cryopreserved human macrophages. The macrophages can be directly used for experiments after thawing and plating. Optional: Activation of polarized M1/M2 macrophages before assay. See Table 1 for more activation details.

A versatile and complete solution for macrophage-related research is now available, thanks to a broad array of PromoCell’s macrophage media that is compatible with cryopreserved cells.

Cultures of Cryopreserved Human Macrophages

Materials

  • 70% Ethanol
  • Human Fibronectin Solution (C-43060)
  • Cryopreserved hMDM-MCSF(-) (C-12915, C-12917), or hMDM-GMCSF(-) (C-12914, C-12916)
  • PBS w/o Ca2+/Mg2+ (C-40232)
  • M1- or M2-Macrophage Generation Medium DXF (C-28056 or C-28055)
  • Human serum AB for long-term cultures of hMDM-MCSF (see II.D.3), if necessary

Culture Protocol

  1. Coating the culture vessel with human fibronectin
  1. The culture vessel is coated with human fibronectin as specified in the instruction guidebook of the fibronectin solution. Around 100µL of the diluted fibronectin solution (10µg/mL) is used per cm2 of culture surface.

Note: In addition, commercially available fibronectin-coated plastic ware can be used.

  1. Preparation of the complete medium and pre-equilibration in the coated culture vessel
  1. As specified in the instruction guidebook, the PromoCell M1/M2-Macrophage Generation Medium DXF is prepared.

Note: The freshly prepared medium is used to achieve the best results. Researchers must ensure that Macrophage Generation Medium DXF with added cytokines used for plating cryopreserved macrophages is not older than one week during plating. Medium older than two weeks must be discarded completely.

  1. A suitable amount of medium (300–400µl/cm2, for example, 8mL per T-25 flask or 3mL per 6-well) is placed in the fibronectin-coated vessel and pre-equilibrated for at least 30 minutes at 37 °C and 5% CO2 before seeding the cells.
  1. Thawing the cryopreserved macrophages
  1. About 14 ml of fresh M1/M2-Macrophage Generation Medium DXF (tempered at 2 ° C to 8 ° C) is transferred to a 15mL conical tube and then placed under the laminar flow bench. This will be used as the thawing medium. Pre-warming should not be carried out.
  2. The cryovial is removed from the liquid nitrogen container and placed immediately on dry ice (even for brief transportation).
  3. Under a laminar flow bench, the cap is briefly twisted a quarter turn to relieve pressure and then re-tightened. The vial is immersed into a water bath (37 °C) just up to the screw cap for a period of 2 minutes. It must be guaranteed that no water enters the thread of the screw cap.
  4. The cryovial is thoroughly rinsed with 70% ethanol under a laminar flow bench. After that, the excess ethanol from the thread area of the screw cap is drawn out.

Note: Once the cells are thawed, work should be started quickly.

  1. The vial is opened and the cells are transferred to the 15mL tube containing the thawing medium using a 2mL serological pipette. During pipetting, care should be taken not to introduce air bubbles. Mixing is done by a single gentle inversion and the cells are allowed to recover for 20 minutes at room temperature under the laminar flow bench.
  2. The viable cell count is determined using the standard technique after the recovery period.
  3. The cells are centrifuged for 15 minutes at 350 x g at room temperature. The supernatant is aspirated except for 50–100µL. The cell pellet is loosened by holding the top of the tube and firmly flicking the bottom of it two or three times. After that, the cells are resuspended at 1 million cells/mL in a fresh ambient tempered M1/M2-Macrophage Generation Medium DXF using a serological pipette

Note: The specified centrifugation time and speed should be followed.

  1. Plating the thawed macrophages
  1. Soon after thawing, the cell suspension is plated at 100.000–200.000 cells/cm2 in the fibronectin-coated culture vessels containing the pre-equilibrated M1/M2-Macrophage Generation Medium DXF. The cells should be kept in the incubator to enable the attachment of the hMDM to the culture surface for a minimum of 4 hours (4–24 hours)

Note: A 70%–90% confluent cell monolayer of hMDM-GMCSF (Mϕ-M1) is continuously obtained with a seeding density of 100.000 cells/cm2, while the smaller-sized hMDM-MCSF (Mϕ-M2) need around 200.000 cells/cm2.

  1. The medium is changed 4–24 hours after plating using approximately 300–400µL fresh medium/cm2 of culture surface. Residual non-adherent cells should be discarded.
  2. Before starting the experiments, the hMDM is allowed to stay in culture for at least 24 hours after seeding.

Note: Optional activation of the polarized macrophages by the customer may be carried out 24 hours after thawing.

  1. In the subsequent time, the culture medium is changed every two to three days. For several weeks, the macrophages can be preserved in culture.

Note: M-CSF serves as a weaker long-term macrophage survival factor when compared to GM-CSF. Therefore, the integrity of M2 macrophage cultures (hMDM-MCSF) to be maintained for 1 week or more can be enhanced, if needed, by adding human serum AB from 5 to 7 days without impacting the polarization status of the cells. Next, 2% (v/v) of human serum AB is simply added to the fresh, complete M2-Macrophage Generation Medium DXF and filtered aseptically via a 0.22 µm filter so as to eliminate the serum-associated lipid precipitate.

Exemplary flow cytometric analysis of cryopreserved human M1/M2 macrophages. The M1/M2 macrophages were grown as an adherent culture on fibronectin-coated culture vessels in the corresponding M1- or M2-Macrophage Generation Medium DXF and were analyzed 1–3 days after thawing. The adherent cells were detached by using the Macrophage Detachment Solution DXF (C-41330). M1 macrophages exhibit a CD68+ (99,74%) and CD80+ (83,33%) marker expression profile, typical for M1 macrophages (upper row). M2 macrophages exhibit a CD68+ (99,72%) and CD163+ (94,91%) marker expression profile, typical for M2 macrophages (lower row)

Figure 4. Exemplary flow cytometric analysis of cryopreserved human M1/M2 macrophages. The M1/M2 macrophages were grown as an adherent culture on fibronectin-coated culture vessels in the corresponding M1- or M2-Macrophage Generation Medium DXF and were analyzed 1–3 days after thawing. The adherent cells were detached by using the Macrophage Detachment Solution DXF (C-41330). M1 macrophages exhibit a CD68+ (99,74%) and CD80+ (83,33%) marker expression profile, typical for M1 macrophages (upper row). M2 macrophages exhibit a CD68+ (99,72%) and CD163+ (94,91%) marker expression profile, typical for M2 macrophages (lower row)

Table 1. Human macrophage activation reference table according to the common framework consensus nomenclature [7] (see also Background). The published differentiation factor/activator combinations are listed to serve as basic guidelines. Specific effects of activation on macrophages should be tested in comparison to the most appropriate non-activated M(-)-baseline variant as a control. DEX = dexamethasone, IC = immune complexes, IFN = interferon, IgG = immunoglobulin G, GC = glucocorticoids, (G)M-CSF = (granulocyte/)macrophage colony stimulating factor, IL = interleukin, LPS = lipopolysaccharide, TAM = tumor-associated macrophages, TGF = transforming growth factor.

Activation state Former designation Differentiation factor (day 0+) Activator (day 1+) Activation process reference
M1 M(IFN-γ) M1 GM-CSF (or M-CSF) IFN-γ (50ng/ml) [1]
M(LPS+IFN-γ) M1 GM-CSF (or M-CSF) IFN-γ (50ng/ml) + LPS (10ng/ml) [1]
M(LPS) M1 GM-CSF (or M-CSF) LPS (100ng/ml) [1]
M(-) M1, non-activated GM-CSF - [2]
M(-) M0 / Mϕ 2% human AB serum - [1, 3]
M(-) M2, non-activated M-CSF - [2]
M(GC) M2c M-CSF DEX (100nM) [2]
M(TGFb) M2c M-CSF TGF-b1 (20ng/ml) [2]
M(IL-10) M2c M-CSF IL-10 (10ng/ml) [4]
M(IC+LPS) M2b M-CSF IgG (immobilized) + LPS (100ng/ml) [5]
M2 M(IL-4) M2a M-CSF IL-4 (20ng/ml) [4, 5]

TAM M2-like tumor microenvironment tumor microenvironment [6]

Background

Macrophage Nomenclature

Macrophages are essentially tissue-resident professional phagocytes and antigen-presenting cells (or APC), which differentiate from circulating peripheral blood monocytes. They carry out significant regulatory and active functions in both innate and adaptive immunity [8]. Macrophages are definitely involved in the manifestation of many diseases, for example, rheumatoid arthritis, autoimmune and allergic disorders, diabetes, atherosclerosis, cancer, and metabolic syndrome [9].

Conventionally, activated macrophages of several phenotypes have been divided into M1 and M2 macrophages. The “classically activated” M1 macrophages contain immune effector cells with an acute inflammatory phenotype. These are very hostile against bacteria and create huge amounts of lymphokines [10]. On the contrary, the “alternatively activated” anti-inflammatory M2 macrophages adhere to different regulatory functions of many types including maintenance of tolerance, regulation of immunity, and tissue repair/wound healing [8, 10].

In reality, it is now thought that the conventional M1/M2 model of macrophage polarization/activation is not fully adequate to reflect the entire complexity of activation states of this highly plastic cell lineage [11]. Therefore, a common framework proposal for macrophage-activation nomenclature [7] has been published by a group of international macrophage specialists. This latest system specifies the designation of in vitro macrophage activation states based on the stimulus used (for example, 20ng/mL recombinant human (rhu) IL-4) together with clear disclosure of differentiation factors used for hMDM generation (for example, 100ng/ml rhuM-CSF). See Table 1.

Defined, xeno-free, and easy-to-handle macrophage culture systems without ill-defined stimuli, in tandem with the published guiding principles for combined experimental standards for in vitro macrophage activation, represent important corner points for effective and determined advancement in macrophage-related research.

References

  1. Fujiwara, Y., et al., Guanylate-binding protein 5 is a marker of interferon-gamma-induced classically activated macrophages. Clin Transl Immunology, 2016. 5(11): p. e111.
  2. Zizzo, G., et al., Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction. J Immunol, 2012. 189(7): p. 3508-20.
  3. Vogel, D.Y., et al., Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation. J Neuroinflammation, 2014. 11: p. 23.
  4. Iqbal, S. and A. Kumar, Characterization of In vitro Generated Human Polarized Macrophages. Journal of Clinical & Cellular Immunology, 2015. 06(06).
  5. Graff, J.W., et al., Identifying functional microRNAs in macrophages with polarized phenotypes. J Biol Chem, 2012. 287(26): p. 21816-25.
  6. Sousa, S., et al., Human breast cancer cells educate macrophages toward the M2 activation status. Breast Cancer Res, 2015. 17: p. 101.
  7. Murray, P.J., et al., Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity, 2014. 41(1): p. 14-20.
  8. Murray, P.J. and T.A. Wynn, Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol, 2011. 11(11): p. 723-37.
  9. Wynn, T.A., A. Chawla, and J.W. Pollard, Macrophage biology in development, homeostasis, and disease. Nature, 2013. 496(7446): p. 445-55.
  10. Murray, P.J. and T.A. Wynn, Obstacles and opportunities for understanding macrophage polarization. J Leukoc Biol, 2011. 89(4): p. 557-63.
  11. Martinez, F.O. and S. Gordon, The M1 and M2 paradigm of macrophage activation: time for reassessment. Prime Rep, 2014. 6: p. 13

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Last updated: May 21, 2019 at 9:45 AM

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