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Updated: Aug 20, 2026
Isolation and Culture of Bone Marrow-Derived Macrophages from Mice
Published on: June 23, 2023
Macrophage population dynamics within fetal mouse fibroblast cultures derived from C57BL/6, CD-1, CF-1 mice and
Neil C Talbot1, Max Paape, Eun Jung Sohn
1U.S. Department of Agriculture, Agricultural Research Service, ANRI, Biotechnology and Germplasm Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland 20705, USA.
Abstract:
In vitro models of macrophage growth, differentiation, and function are needed to facilitate the study of their biology as important immune facilitator cells and as frequent targets of bacterial and viral infection. A simple method for the selective expansion and continuous culture of mouse macrophages from primary explant cultures of mouse embryonic tissue is described. Culture in Dulbecco modified Eagle medium (DMEM) low-glucose (1 g/L) formulation (DMEM/L) inhibited fibroblast growth. In contrast, macrophages continued to proliferate in the presence of DMEM/L when in contact with the fibroblasts. Alternating growth in high-glucose DMEM with DMEM/L produced a 1.16- to 2.1-fold increase (depending on mouse strain) in the percentage of macrophages within the cell culture in comparison with culturing in DMEM with high glucose exclusively. Macrophage yields of over 1 million cells/T12.5 flask were achieved by passages 3-4, and, thereafter, declined over the next 5-10 passages. The peak percentage of macrophages within a culture varied depending on the strain of mouse (C57BL/6, CD-1, and CF-1 and two knockout C57BL/6 strains deficient in either interleukin-6 [IL-6] or granulocyte colony stimulating factor [GCSF]). The GCSF (-/-)-derived cultures had the lowest peak macrophage content (30%) and CD-1 the highest content (64.9%). The IL-6 (-/-) and CD-1 cultures appeared to spontaneously transform to create cell lines (IL6MAC and CD1MAC, respectively) that were composed of 50-75% macrophages. The macrophages were phagocytic and were positive for CD14, acetylated low-density lipoprotein receptors, and F4-80 antigen. Light and electron microscopy showed that the cultured macrophages had in vivo-like morphological features, and they could be plated to high purity by differential attachment to petri dishes in serum-free medium.
Insights
A new method enables selective expansion and continuous culture of mouse macrophages using a low-glucose medium formulation. This technique facilitates macrophage research by improving cell yields and purity for immune studies.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Macrophage research requires effective in vitro models for studying immune responses and infection.
- Existing methods for macrophage culture present challenges in selective expansion and maintaining purity.
Purpose of the Study:
- To describe a simple method for selective expansion and continuous culture of mouse macrophages.
- To optimize macrophage yields and purity for in vitro studies.
Main Methods:
- Utilized Dulbecco modified Eagle medium (DMEM) low-glucose (DMEM/L) to inhibit fibroblast growth while promoting macrophage proliferation.
- Employed alternating culture in high-glucose DMEM and DMEM/L to increase macrophage percentage.
- Analyzed macrophage content across different mouse strains, including knockout models for IL-6 and GCSF.
Main Results:
- DMEM/L selectively supported macrophage proliferation over fibroblasts.
- Alternating DMEM formulations increased macrophage percentage by 1.16- to 2.1-fold compared to high-glucose DMEM alone.
- Achieved high macrophage yields (>1 million cells/T12.5 flask) by passages 3-4.
- Identified variations in peak macrophage percentage based on mouse strain, with CD-1 yielding the highest (64.9%).
- Observed spontaneous transformation into macrophage cell lines (IL6MAC, CD1MAC) in IL-6 (-/-) and CD-1 cultures.
- Confirmed macrophage phagocytic activity and expression of CD14, LDL receptors, and F4-80 antigen.
- Demonstrated in vivo-like morphology and high-purity plating via differential attachment.
Conclusions:
- The described method provides a robust approach for generating pure, functional in vitro macrophage cultures.
- This technique is valuable for immunological research, particularly for studying macrophage roles in infection and immunity.
- The development of spontaneously transforming cell lines offers a novel resource for long-term macrophage studies.
