(1) Background: Placental immune cells are playing a very important role in a successful placentation and the prevention of pregnancy complications. Macrophages dominate in number and relevance in the maternal and the fetal part of the placenta. The evidence on the polarization state of fetal and maternal macrophages involved in both, healthy and pregnancy-associated diseases, is limited. There is no representative isolation method for the direct comparison of maternal and fetal macrophages so far. (2) Material and Methods: For the isolation of decidual macrophages and Hofbauer cells from term placenta, fresh tissue was mechanically dissected and digested with trypsin and collagenase A. Afterwards cell enrichment was increased by a Percoll gradient. CD68 is represented as pan-macrophage marker, the surface markers CD80 and CD163 were further investigated. (3) Results: The established method revealed a high cell yield and purity of the isolated macrophages and enabled the comparison between decidual macrophages and Hofbauer cells. No significant difference was observed in the percentage of single CD163⁺ cells in the distinct macrophage populations, by using FACS and immunofluorescence staining. A slight increase of CD80⁺ cells could be found in the decidual macrophages. Considering the percentage of CD80⁺CD163^- and CD80^-CD163⁺ cells we could not find differences. Interestingly we found an increased number of double positive cells (CD80⁺CD163⁺) in the decidual macrophage population in comparison to Hofbauer cells. (4) Conclusion: In this study we demonstrate that our established isolation method enables the investigation of decidual macrophages and Hofbauer cells in the placenta. It represents a promising method for direct cell comparison, enzyme independently, and unaffected by magnetic beads, to understand the functional subsets of placental macrophages and to identify therapeutic targets of pregnancy associated diseases.

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