A 3D in vitro model of the human subcutaneous tissue

Abstract

Macrophages play a huge role in eliciting the immune response. Macrophages are heterogenous and can switch between polarisation states from pro-inflammatory M1 and anti-inflammatory M2 in response to environmental cues. For this project, an in vitro model of the human subcutaneous tissue was prepared from collagen and hyaluronic acid to form a polymer network to replicate the ECM to study drug absorption as an alternative to animal models.
Hydrogels were initially seeded with THP-1 cells followed by primary monocytes and adipocytes to form a co-culture transwell system to determine if hydrogels could be developed as a mechanism to study cell and drug delivery systems in adipose tissue. Mechanical testing concluded Collagen hydrogels prepared without HA and EDC crosslinking were unable to maintain structural integrity which reinforced the key role of HA in the ECM.
Findings from this project suggest successful adherence of THP-1 cells to the hydrogel surface but not primary macrophages. Characterisation assays using ELISA and immunofluorescent staining were performed to assess cell morphology and activation status of THP-1 cells and macrophages with THP-1 cells used a model of primary macrophages. These assays were unable to confirm the successful polarisation of THP-1 cells and primary macrophages to M1 and M2 phenotypes on Collagen-HA hydrogels.
The transwell co-culture system using macrophages and adipocytes requires further optimisation. Oil red O staining confirmed the successful differentiation of hMSC's to mature adipocytes however, the macrophage seeding protocol requires further optimisation to successfully polarise PBMC’s to M1 and M2 phenotypes. Elevated levels of IL-10 were detected by ELISA in M0 cells which suggest cells have an anti-inflammatory response to Collagen-HA hydrogels however, specific cell markers and techniques such as flow cytometry and qPCR could be explored to quantify cell populations.

Date of Award	19 Mar 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Asme Boussahel (Supervisor)