Macrophage retrieval from 3D biomaterials: A detailed comparison of common dissociation methods

• Published in: Journal of immunology and regenerative medicine Vol. 11; p. 100035
• Main Authors: Feuerer, Nora, Morschl, Johannes, Daum, Ruben, Weiss, Martin, Hinderer, Svenja, Schenke-Layland, Katja, Shipp, Christopher
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01-02-2021
• Subjects: Biomaterials; Electrospinning; Flow cytometry; Macrophage dissociation; Macrophage polarization; IFNγ; Flow cytometry; Macrophage polarization; M-CSF; PLA; PU; Electrospinning; EDTA; PBMC; LPS; ECM; Macrophage dissociation; Biomaterials; MFI; 7-AAD; MDM; FC
• ISSN: 2468-4988; 2468-4988
• DOI: 10.1016/j.regen.2020.100035

The immune system is widely recognized as a crucial determinant in implant biocompatibility and tissue integration. In order to assess macrophage response to biomaterials, commonly used analysis techniques require the removal of cells from the material. This process inherently has a negative impact on the cells, but few studies have comprehensively compared different dissociation methods in terms of impact on cell yield, viability, phenotype and function.
Cell scraping, EDTA at 4 °C, EDTA at 37 °C, trypsin, Accutase and the PromoCell macrophage detachment solution were compared in terms of cell yield and viability. The effect of Accutase on cell surface antigen conservancy and cell functionality was investigated. Lastly, effect of Accutase detachment of macrophages from electrospun biomaterials was analyzed.
The efficiency of common cell detachment protocols for human monocyte-derived macrophages (MDMs) varies significantly between enzymatic and non-enzymatic approaches. In terms of surface marker detection, we showed that enzymatic detachment not only selectively cleaves the M2 markers CD206 and CD163, but we also identified that this effect is variable across donors. Furthermore, we observed that the process of cell detachment impairs macrophage endocytic ability. Lastly, we tested the efficiency of enzymatic cell detachment on electrospun 3D matrices designed for tissue engineering.
Our results provide a thorough understanding of the advantages and disadvantages of commonly used cell dissociation methods and have important implications for studies investigating the macrophage response to biomaterials.