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. 2024 Jun 21;5(2):103061.
doi: 10.1016/j.xpro.2024.103061. Epub 2024 May 8.

Protocol to develop human alveolar macrophage-like cells from mononuclear cells or purified monocytes for use in respiratory biology research

Susanta Pahari  1 , Anna-Lena Neehus  2 , Bruce C Trapnell  3 , Jacinta Bustamante  4 , Jean-Laurent Casanova  5 , Larry S Schlesinger  6
Affiliations

Protocol to develop human alveolar macrophage-like cells from mononuclear cells or purified monocytes for use in respiratory biology research

Susanta Pahari et al. STAR Protoc. .

Abstract

Human alveolar macrophages are a unique myeloid subset critical for understanding pulmonary diseases and are difficult to access. Here, we present a protocol to generate human alveolar macrophage-like (AML) cells from fresh peripheral blood mononuclear cells or purified monocytes. We describe steps for cell isolation, incubation in a defined cocktail of pulmonary surfactant and lung-associated cytokines, phenotype analysis, and validation with human alveolar macrophages. We then detail procedures for quality control and technical readouts for monitoring microbial response. For complete details on the use and execution of this protocol, please refer to Pahari et al.1 and Neehus et al.2.

Keywords: cell biology; cell culture; cell isolation; flow cytometry; immunology.

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Conflict of interest statement

Declaration of interests A non-provisional patent application has been filed with the number 17/657,344 for an invention titled “Cell culture media and methods for generating human Alveolar Macrophage-Like cells.” The applicant is the Texas Biomedical Research Institute, located in San Antonio, Texas, USA. The inventors listed in the application are L.S.S. and S.P., both from San Antonio, Texas, USA. For more details, please refer to the following link: https://patentcenter.uspto.gov/applications/17657344.

Figures

None
Graphical abstract
Figure 1
Figure 1
PBMC isolation Peripheral blood is layered over Ficoll-Hypaque and subjected to density cushion centrifugation. Post centrifugation, blood components are separated into four layers containing plasma, buffy coat (lymphocytes, monocytes and few platelets and eosinophils), Ficoll-Hypaque, granulocytes and erythrocytes. Created with Biorender.com.
Figure 2
Figure 2
CD14 surface staining of isolated monocytes Representative flow cytometry staining on isolated monocytes to assess purity based on CD14 expression.
Figure 3
Figure 3
Schematic representation of the AML cell differentiation protocol PBMCs are isolated from heparinized blood and are either used directly or undergo monocyte isolation prior to AML cell differentiation. Cells can be cultured without the Infasurf, GM-CSF, TGF-β and IL-10 (ALL cocktail) to generate untreated (UT) MDMs or with the ALL cocktail to generate AML cells. Created with Biorender.com.
Figure 4
Figure 4
Upregulation of MARCO during AML cell differentiation (A) Monocytes were differentiated in the presence of ALL cocktail for six days. ALL cocktail treatment and flow cytometric analyses were performed on the indicated days. (B) Representative flow cytometry staining for CD14 and MARCO shows increased MARCO expression during AML differentiation. The highest expression of MARCO is visible after 6 days of ALL cocktail treatment.
Figure 5
Figure 5
Morphology of monocytes after 6 days of various treatments Monocytes were cultured for six days and either (A) left untreated, (B) treated with Infasurf, (C) treated with GM-CSF, TGF-β and IL-10 or (D) treated with Infasurf, GM-CSF, TGF-β and IL-10. Only treatment with Infasurf, GM-CSF, TGF-β and IL-10 (ALL cocktail) led to the characteristic rounded morphology. Scale bar 20 μm.
Figure 6
Figure 6
Characterization of AML cells after 6 days of differentiation (A) qRT-PCR data of AML cells or untreated (UT) MDMs for characteristic HAM genes. Data are expressed as mean ± SD of 2 biological replicates. (B) Representative flow cytometry staining of AML cells and UT MDMs for selected markers.
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References

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