A simple culture method for liver and intestinal tissue-resident macrophages from neonatal mice

  • Yu Shimizu
  • Naoya Sakuragi
  • Kiminori Nakamura
  • Toshio Taira
  • Tokiyoshi Ayabe
  • Akimasa FukuiEmail author


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.


Intestinal macrophage Tissue-resident macrophage Simple culture method Feeder cell 



The authors thank Kazuya Furusawa and Naoki Sasaki for the discussions and suggestions.


This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 17K11661 to KN and 18H02788 to TA), the Center of Innovation Program from the Japan Science and Technology Agency (to NS, KN, and TA), and a Chuo University Personal Research Grant (to AF).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11626_2019_359_MOESM1_ESM.tif (108 kb)
Fig S1 FACS analysis of CD206 expression on macrophage-like cells. Macrophage-like cells stained anti-isotype control (red) or anti-CD206 (blue). CD206+ cells were observed in macrophage-like cells derived from the liver and small intestine. (TIF 108 kb)
11626_2019_359_Fig7_ESM.png (394 kb)

High Resolution Image (PNG 394 kb)


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Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  1. 1.Innate Immunity Laboratory, Graduate School of Life ScienceHokkaido UniversitySapporoJapan
  2. 2.Innate Immunity Laboratory, Department of Cell Biological Science, Faculty of Advanced Life ScienceHokkaido UniversitySapporoJapan
  3. 3.Primary Cell Division, Cosmo Bio Co., Ltd.OtaruJapan
  4. 4.Laboratory of Tissue & Polymer Sciences, Graduate School of Life ScienceHokkaido UniversitySapporoJapan
  5. 5.Laboratory of Tissue Morphogenesis, Biological Sciences, Faculty of Science and EngineeringChuo UniversityTokyoJapan

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