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Updated: Jan 24, 2026
A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells
Published on: May 24, 2011
A simple culture method for liver and intestinal tissue-resident macrophages from neonatal mice
Yu Shimizu1, Naoya Sakuragi1,2, Kiminori Nakamura1,2
1Innate Immunity Laboratory, Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.
Abstract:
The liver and intestine contain a remarkably large portion of tissue-resident macrophage cells representing a phenotype that downregulates inflammation and initiates tissue repair. Here, liver and intestinal tissues obtained from neonatal mice were minced, enzymatically digested, and incubated in RPMI1640-based media. In a 2-wk culture, spherical floating cells emerged on a fibroblastic sheet. These cells showed phagocytic activity and F4/80+-CD11b+-CD206+-Arg1+-iNOS--CD209a- phenotype, suggesting that these cells are tissue-resident macrophages. These macrophages proliferated in the co-culture system in the presence of fibroblastic feeder cell layer and absence of supplemental cytokines; the co-culture system did not cause a significant change in the phenotype of cells grown in a 4-wk culture. On the feeder cells, macrophage density was approximately 1.5 × 104/cm2 and the doubling time was approximately 70 h. Based on these observations, we present a simple method for the isolation and propagation of tissue-resident macrophages resembling M2 macrophage from neonatal mice, and this method provides a useful platform for in vitro studies of tissue-resident macrophages.
Insights
Researchers developed a simple method to isolate and grow tissue-resident macrophages from neonatal mice. These macrophages downregulate inflammation and promote tissue repair, offering a valuable tool for in vitro studies.
Area of Science:
- Immunology
- Cell Biology
- Tissue Engineering
Background:
- Liver and intestinal tissues host a significant population of tissue-resident macrophages.
- These macrophages possess a unique phenotype that suppresses inflammation and facilitates tissue repair.
Purpose of the Study:
- To establish a straightforward method for isolating and propagating tissue-resident macrophages from neonatal mouse tissues.
- To characterize the phenotype and proliferative capacity of these isolated macrophages in a co-culture system.
Main Methods:
- Neonatal mouse liver and intestinal tissues were minced and enzymatically digested.
- Cells were cultured in RPMI1640-based media on a fibroblastic feeder layer.
- Phenotypic analysis was performed using flow cytometry (F4/80+, CD11b+, CD206+, Arg1+, iNOS-, CD209a-).
Main Results:
- Spherical, phagocytic cells with a macrophage phenotype (F4/80+, CD11b+, CD206+, Arg1+, iNOS-, CD209a-) emerged within 2 weeks.
- Macrophages proliferated on the feeder layer without added cytokines, maintaining their phenotype for up to 4 weeks.
- Observed macrophage density was approximately 1.5 x 10^4/cm^2 with a doubling time of ~70 hours.
Conclusions:
- A simple, cytokine-independent method for isolating and propagating M2-like tissue-resident macrophages from neonatal mice has been developed.
- This method provides a valuable in vitro platform for studying the functions of these crucial immune cells.
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