Optimisation of an In Vitro Macrophage Model to Investigate the Interactions with Adipose Tissue-derived Stromal Cells and Extracellular Matrix
Translated title
Optimering af en in vitro makrofag model med henblik på at undersøge interaktionerne med fedtvævs-deriverede stromale celler og ekstracellulær matrix
Author
Lethager, Laura Lykke
Term
4. term
Publication year
2023
Pages
61
Abstract
Background: Adipose‑derived stromal cells (ASCs) are being explored as a cell therapy because they can modulate immune responses and influence other immune cells such as macrophages. Macrophages are flexible cells that can either promote or resolve inflammation. Prolonged inflammation can lead to fibrosis—excess, scar‑like connective tissue that impairs tissue function. ASCs communicate with neighboring cells through secreted signals (paracrine effects) and direct contact, but the exact mechanisms in their interactions with macrophages remain unclear. This study examined how ASCs affect the fibrotic actions of macrophages. Methods: We established a co‑culture of macrophages and ASCs. To set macrophage activation, two immune stimulators—lipopolysaccharide (LPS) and interferon‑γ—were tested, and optimal doses were identified by measuring secreted TNF‑α. The two cell types were then separated using magnetic beads targeting CD90, and macrophages were seeded onto an extracellular matrix produced by TGF‑β‑stimulated fibroblasts. After six days, collagen types I and III were measured as indicators of extracellular matrix deposition. Fibroblast seeding density was optimized to create a dense matrix, confirmed by Sirius Red staining. Results and conclusion: A robust macrophage‑ASC co‑culture model was established, with optimal activation at LPS 100 ng/mL and interferon‑γ 20 ng/mL. After six days with fibroblasts, ASC‑treated macrophages led to significantly lower collagen I and III levels, indicating that macrophages can influence extracellular matrix deposition. The clearest difference between ASC‑treated and mature macrophages was observed at a macrophage seeding density of 6,250 M1/cm². Overall, the findings suggest that ASCs can shift macrophage behavior to reduce fibrotic matrix deposition in this model.
Baggrund: Fedtvævs‑afledte stromale celler (ASCs) bliver undersøgt som potentiel celleterapi, fordi de kan påvirke immunforsvaret og regulere andre immunceller som makrofager. Makrofager er fleksible celler, der både kan fremme og dæmpe betændelse. Langvarig betændelse kan føre til fibrose—en overdreven dannelse af arvævs‑lignende bindevæv, som forringer vævets funktion. ASCs kommunikerer med andre celler via udskilte signalstoffer (parakrin sekretion) og direkte kontakt, men de præcise mekanismer i samspillet med makrofager er ikke fuldt klarlagt. Formålet var at undersøge, hvordan ASCs påvirker makrofagers fibrotiske effekter. Metoder: Vi opbyggede en samkultur af makrofager og ASCs. For at finde passende aktivering af makrofagerne blev to immunstimuli—lipopolysakkarid (LPS) og interferon‑γ—afprøvet, og de optimale koncentrationer blev bestemt ved at måle udskilt TNF‑α. Derefter blev cellerne adskilt med magnetiske perler rettet mod CD90, og makrofagerne blev placeret på en ekstracellulær matrix dannet af TGF‑β‑stimulerede fibroblaster. Efter seks dage målte vi kollagen type I og III som mål for aflejring af ekstracellulær matrix. Udsætningsdensiteten af fibroblaster blev desuden optimeret for at skabe en tæt matrix, bekræftet med Sirius Red‑farvning. Resultater og konklusion: Vi etablerede en funktionsdygtig makrofag‑ASC‑samkultur og fandt de optimale koncentrationer til aktivering (LPS 100 ng/mL og interferon‑γ 20 ng/mL). Efter seks dages samkultur med fibroblaster medførte ASC‑behandlede makrofager signifikant lavere niveauer af kollagen I og III, hvilket tyder på, at makrofager kan påvirke aflejring af ekstracellulær matrix. Den tydeligste forskel mellem ASC‑behandlede og modne makrofager sås ved en udsætningsdensitet på 6.250 M1/cm². Samlet peger resultaterne på, at ASCs kan ændre makrofagers adfærd, så fibrotisk matrixaflejring reduceres i denne model.
[This apstract has been rewritten with the help of AI based on the project's original abstract]