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Passage culture of human monocyte/macrophage lineage cells using a temperature‐responsive culture dish

MONOCYTE/MACROPHAGE lineage cells have diverse functions and are key regulators not only in immunity but also in tissue repair and regeneration (1–3). The cells have traditionally been obtained and used with adherent cultures of mononuclear cells from peripheral blood (4). However, there have been no established methods of passage culture for monocyte/macrophage lineage cells, probably because the cells become strongly adherent to the culture dish, making it difficult to detach them (5). The strong adherence also makes it difficult to analyze the cells by flow cytometry. In this study, we took advantage of a temperature-responsive culture dish coated with poly-(N-isopropylacrylamide) (6). On lowering to room temperature, the culture surfaces change from hydrophobic to hydrophilic, and this weakens the cells’ attachment to the dishes (7). The temperature-responsive culture dishes have been used for cell sheets of various kinds of cells in basic research (8) and clinical applications (9). We established a novel passage culture of monocyte/macrophage lineage cells with the temperatureresponsive culture dishes, RepCell VR (CellSeed, Tokyo, Japan), and examined the characteristics of the passaged cells (Fig. 1A) (Methods in Supporting Information). Among the cytokines examined (M-CSF, GM-CSF, INFc, IL-4, SDF-1, MCP-1), M-CSF and GM-CSF had a proliferative effect on mononuclear cells, M-CSF showing a greater effect than GM-CSF (Fig. 1B). M-CSF was further examined for the dose-dependency of its proliferative effect, and the concentration of 20 ng/ml was found to be most proliferative (Fig. 1C). The following passage culture was performed with M-CSF (20 ng/ml). Spindle-shaped adherent cells were grown at 378C in the temperature-responsive culture dishes. At passage, the cells changed in morphology to round under room temperature and were easily detached by pipetting (Fig. 1D). We examined cell viability with trypan blue staining at the time of passage. >90% of the cells were viable after detachment. Fresh mononuclear cells were composed of lymphocytes and monocyte/ macrophage lineage cells, and flow cytometry showed that both cell populations were positive for CD45 (data not shown). The monocyte/macrophage lineage cells were distinguished as CD11b1/CD141 cells (Fig. 2A). The rate of monocyte/macrophage lineage cells increased by passage: 7.3 6 3.0%, 73.0 6 8.6%, 96.7 6 1.0%, 95.5 6 1.3% in fresh, passage 1, passage 2, and passage 3 cells (Figs. 2B and 2C). Although the number of mononuclear cells in peripheral blood varies in donors, 10 to 15 ml of peripheral blood was enough to start primary culture in a 3.5 cm dish used in our study. After three passages, we obtained eight dishes of the expanded cells. We examined the expression of CD206, a mannose receptor, on cultured cells. All the monocyte/macrophage lineage