Background Resident macrophages (Mø) originating from yolk sac Mø and/or foetal monocytes colonise tissues/organs during embryonic development. They persist into adulthood by self-renewal at a steady state, independent of adult monocyte inputs, except for those in the intestines and dermis. Thus, many resident Mø can be propagated in vitro under optimal conditions; however, there are no specific in vitro culture methods available for the propagation of resident Mø from diverse tissues/organs. Results We provided a simple method for propagating resident Mø derived from the liver, spleen, lung, and brain of ICR male mice by co-culture and subculture along with the propagation of other stromal cells of the respective organs in standard culture media and successfully demonstrated the propagation of resident Mø colonising these organs. We also proposed a simple method for segregating Mø from stromal cells according to their adhesive property on bacteriological Petri dishes, which enabled the collection of more than 97.6% of the resident Mø from each organ. Expression analyses of conventional Mø markers by flow cytometry showed similar expression patterns among the Mø collected from the organs. Conclusion This is the first study to clearly provide a practical Mø propagation method applicable to resident Mø of diverse tissues and organs. Thus, this novel practical Mø propagation method can offer broad applications for the use of resident Mø of diverse tissues and organs.
Tissue-resident macrophages (Mø) originating from foetal precursors are maintained by self-renewal under tissue/organ-specific microenvironments (niches). We recently developed a simple propagation method applicable to tissue-resident Mø by co-culturing. Here, we examined the properties of lung tissue-resident Mø propagated by co-culturing with lung interstitial cells. The intracardially and intratracheally perfused lung from BALB/c and C57BL/6 mice could minimise the contamination of alveolar Mø and lung monocytes. Lung tissue-resident Mø could be largely propagated under standard culture media along with the propagation of lung interstitial cells demonstrating a fibroblastic morphology. Propagated lung Mø showed characteristic expression properties for Mø/monocyte markers: high expressions of CD11b, CD64 and CD206; substantial expressions of Mertk; and negative expressions of Ly6C, MHC II and Siglec-F. These properties fit with those of lung interstitial Mø of a certain population that can undergo self-renewal. Propagated fibroblastic cells by co-culturing with lung Mø possessed niche properties such as Csf1 and Tgfb1 expression. Propagated lung Mø from both the mouse types were polarised to an M2 phenotype highly expressing arginase 1 without M2 inducer treatment, whereas the M1 inducers significantly increased the iNOS-positive cell percentages in C57BL/6 mice relative to those in BALB/c mice. This is the first study to demonstrate fundamental properties of lung tissue-resident Mø propagated by co-culturing. Propagated lung Mø showing features of lung interstitial Mø can serve as an indispensable tool for investigating SARS-CoV-2 diseases, although lung interstitial Mø have gained little attention in terms of their involvement in SARS-CoV-2 disease pathology, in contrast to alveolar and recruited Mø.
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