Optimisation of an In Vitro Macrophage Model to Investigate the Interactions with Adipose Tissue-derived Stromal Cells and Extracellular Matrix

Abstract

Formål: I de senere år stromale celler fra fedtvæv (ASCer) blevet undersøgt som celleter- api på grund af deres immunmodulatoriske egenskaber, og fordi de kan regulere funktio- nen af immunceller, såsom makrofager. Makrofager er plastiske celler med både pro- og anti-inflammatoriske funktioner. Disse egenskaber gør ASCerne i stand til at mindske in- flammation og forebygge fibrose. Fibrose kan opstå som følge af vedvarende inflammation og medfører nedsat funktionalitet af vævet. ASCer kan interagere med de omkringliggende celler gennem både parakrine udskillelser af for eksempel proteiner og ved celle-celle kon- takt. Den præcise virkningsmekanisme bag interaktionen mellem ASCer og makrofager er dog stadig ukendt. Derfor var formålet med dette studie at undersøge effekten af ASCer på makrofagernes fibrotiske effekt. Metoder: For at udvikle en makrofag-ASC-kokultur model blev den optimale koncen- tration af lipopolysakkarid og interferon-γ bestemt ved at måle koncentrationen af tumor necrosis factor-α, som blev udskilt af makrofagerne. Derefter blev makrofager isoleret fra ASCerne ved hjælp af Dynabeads med CD90 antistof og herefter sået ud i en extracellulær matrix, som var dannet af transforming growth factor-β-stimulerede fibroblaster. Efter seks dage blev niveauerne af collagen I og III bestemt som et mål for udskillelsen af ek- stracellulær matrix. Optimering af fibroblasternes optimale densitet fik dem til at danne en tæt ekstracellulær matrix, hvilket blev bestemt ved farvning med Sirius Rød (0,1%) i pikrinsyre. Resultater og konklusion: Der blev etableret en makrofag-ASC-kokultur model, hvor makrofagerne blev aktiveret med 100 ng/mL lipopolysakkarid og 20 ng/mL interferon-γ. Efter seks dage i kokultur med fibroblaster havde de ASC-behandlede makrofager forårsaget signifikant nedsatte niveauer af kollagen I og kollagen III, hvilket tyder på, at makrofagerne er i stand til at påvirke udskillelsen af ekstracellulær matrix. Den optimale koncentration af makrofager var 6250 M1/cm2, idet denne koncentration resulterede i den største signifikante og numeriske forskel mellem de ASC-behandlede makrofager og en kontrol med aktiverede makrofager

Aim: In recent years, adipose-derived stromal cells (ASCs) have been investigated for use as a cellular therapy, due to their immunomodulatory properties and their ability to regu- late the function of immune cells, such as macrophages. Macrophages are plastic cells with both pro- and anti-inflammatory functions. These features enable them to reduce inflam- mation and prevent fibrosis. Fibrosis can arise as a result of prolonged inflammation and this leads to decreased tissue functionality. The ASCs can interact with the surroundings cell through both paracrine secretion and cell-cell contact. However, the exact mechanisms of action for the interactions between ASCs and macrophages are still unknown. There- fore, the aim of this study was to investigate the effect of ASCs on the fibrotic effects of macrophages. Methods: To develop a macrophage-ASC-coculture, the optimal concentration of activat- ing stimuli, lipopolysaccharide and interferon-γ, was determined by measuring the concen- tration of secreted tumor necrosis factor-α by the macrophages. Hereafter, macrophages were isolated from the ASCs using Dynabeads coated with CD90 antibody, and then seeded onto an extracellular matrix derived from transforming growth factor-β-stimulated fibrob- lasts. After six days, the levels of collagen I and III were determined as a measure of deposition of extracellular matrix. Optimisation of fibroblast seeding density allowed them to create a dense extracellular matrix, which was determined through staining with Sirius Red (0.1%) in Picric Acid. Results and Conclusion: A macrophage-ASC-coculture model was established, and the optimal concentrations of lipopolysaccharide and interferon-γ were found to be 100 ng/mL and 20 ng/mL, respectively. After six days of coculture with fibroblasts, the ASC-treated macrophages had resulted in significantly decreased levels of collagen I and III, which suggests that macrophages are able to affect the deposition of extracellular matrix. The optimal seeding density of the macrophages was 6250 M1/cm2 at which concentration the greatest significant and numerical difference between the ASC-treated macrophages and the mature macrophages was found.

A master thesis from Aalborg University

Optimisation of an In Vitro Macrophage Model to Investigate the Interactions with Adipose Tissue-derived Stromal Cells and Extracellular Matrix

[Optimering af en in vitro makrofag model med henblik på at undersøge interaktionerne med fedtvævs-deriverede stromale celler og ekstracellulær matrix]