Establishment of a Standardised in vitro Assay of Human Monocyte-derived Macrophages to Investigate the Immunomodulatory Potential of Adipose Tissue-derived Mesenchymal Stromal Cells
Translated title
Etablering af et Standardiseret in vitro Assay for Human Monocyt-deriverede Makrofager til at Undersøge det Immunmodulatoriske Potentiale af Fedtvævs-deriverede Mesenkymale Stromale Celler
Author
Hansen, Stine Bangsgaard
Term
4. term
Publication year
2021
Submitted on
2021-05-28
Pages
69
Abstract
Macrophages are key players in both inflammation and tissue repair and display high plasticity. This thesis aimed to establish and standardize an in vitro assay using human monocyte-derived macrophages to evaluate the immunomodulatory effects of adipose tissue-derived mesenchymal stromal cells (AT-MSC). Monocytes were differentiated with GM-CSF or M-CSF into pro- and anti-inflammatory precursors and subsequently activated into mature mM1 (LPS+IFNγ), M2a (IL-4), and M2c (IL-10) macrophages. During activation, macrophages were co-cultured with AT-MSC, and phenotype and function were assessed by flow cytometry and functional assays, including mixed lymphocyte reaction and phagocytosis. The developed protocol reproducibly generated distinct macrophage phenotypes and proved useful as an assay to assess the immunomodulatory properties of AT-MSC. Co-culture with AT-MSC decreased maturation marker expression on mature M1 macrophages and significantly suppressed their ability to induce allogeneic lymphocyte proliferation, while enhancing anti-inflammatory M2a and M2c phenotypes. These findings indicate the assay’s utility for characterizing MSC-driven immunomodulation and suggest that shifting M1 toward less pro-inflammatory and more M2-like states may help resolve inflammation in disease settings.
Makrofager er centrale for både inflammation og vævsreparation og udviser høj plasticitet. Denne afhandling havde til formål at etablere og standardisere et in vitro-assay med humane monocyt-deriverede makrofager til at undersøge den immunmodulatoriske effekt af fedtvævs-deriverede mesenkymale stromale celler (AT-MSC). Monocytter blev differentieret med GM-CSF eller M-CSF til pro- og anti-inflammatoriske udgangsfænotyper og dernæst aktiveret til modne mM1 (LPS+IFNγ), M2a (IL-4) og M2c (IL-10) makrofager. Under aktiveringen blev makrofager co-kultiveret med AT-MSC, og fænotype og funktion blev vurderet med flowcytometri og funktionelle assays, herunder mixed lymphocyte reaction og fagocytose. Den udviklede protokol genererede reproducerbart distinkte makrofagfænotyper og var anvendelig som assay til at måle AT-MSC’s immunmodulatoriske egenskaber. Co-kultur med AT-MSC reducerede ekspressionen af modenhedsmarkører på modne M1-makrofager og hæmmede signifikant deres evne til at inducere proliferation af allogene lymfocytter, samtidig med at AT-MSC forstærkede de anti-inflammatoriske M2a- og M2c-fænotyper. Disse fund peger på, at assayet kan bruges til at karakterisere MSC-baseret immunmodulation, og at et skift fra M1 mod mindre pro-inflammatoriske og mere M2-prægede tilstande potentielt kan understøtte opløsning af inflammation i sygdomskontekster.
[This apstract has been generated with the help of AI directly from the project full text]