Development of a microfluidic model of lymphoid-like follicles in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the Central Nervous System (CNS). MS is highly heterogeneous and progressive in nature, and there is no model in existence which can recapitulate the complexity of the disease. Due to the chronic inflammation caused by the disease, aggregations of cells have been noted in up to 40% of people with progressive MS. These aggregations of cells, known as lymphoid-like follicles (LLFs), are found around the CNS, in the meninges, and most often found in the sulci (folds) of the brain or on the surface. They are made up of several cell types and are believed to contribute to disease progression and severity. This thesis investigated the development of an in vitro model of the surface of the brain in order to address the gap in knowledge about the formation of LLFs. The principal aim of this thesis was the development of a microfluidic model of the meningeal brain barrier, which emulated the surface of the brain of people with MS in order to model this toxic niche. To our knowledge, this is the first study to translate physical dimensions of the surface of the brain and the sulci onto an organ-on-a-chip device. From this study, it was determined that there were no significant differences in the precentral and cingulate sulci of people with MS compared to healthy controls. The dimensions were resolved to a 3:1 (depth to width) ratio in order to translate them to device designs. As this project is the first of its kind in our research group, and indeed, one of the first in the University, it was necessary to optimise the materials used, the manufacturing methods and the culture of cells within the device. Commonplace materials in microfluidic manufacturing were assessed for this specific application and polydimethylsiloxane was chosen as the optimal material for device development. The provision of a collagen IV extracellular matrix to cells in the device enhanced adherence and metabolic activity of meningeal cells. The developed microfluidic model demonstrated the ability to emulate features of the meninges. From the recreation of a meningeal barrier to its response to inflammation, several in vivo observations were recapitulated in this model. As LLFs in vivo are composed of various cell types, it was important to include these in the model. Peripheral Blood Mononuclear Cells (PBMCs) contain most of the required cells, such as B-cells, T-cells and some dendritic cells. The stromal cell support observed in vivo is provided by the meningeal cells within the device. In order to mimic immune infiltration, PBMCs and meningeal cells were co-cultured. LLF-related cytokines, such as CXCL13, a B-cell chemoattractant implicated in LLF organisation and accumulation, were assessed. The levels produced by the co-culture on the chip (≅100 pg/ml) are within the physiological ranges seen in people with MS (5-100 pg/ml). A second innovative aspect of this thesis was the pilot development of a human immune-meningeal-cortical organoid. This secondary in vitro model was developed as a possible extension of the primary microfluidic model. The incorporation of meningeal cells in a cortical organoid revealed no changes in the structure and growth of the organoid, and strikingly, meningeal cells preferentially bound to the surface of the organoids. Following the addition of PBMCs in order to mimic an immune infiltration, the levels of CXCL13 were significantly higher than those of the control organoid (≅157 pg/ml vs 142 pg/ml). These levels were also within the range observed in the microfluidic device. In summary, this thesis demonstrates the feasibility and early validation steps of a microfluidic model of LLFs found in people with MS. A novel methodology was used to characterise the sulci, and no significant differences in the sulci of people with MS compared to healthy controls were found, a finding not previously reported. Both the microfluidic model and the organoid developed show promise for in vitro observations of LLF, contributing to the gap in knowledge surrounding LLFs and potentially advancing the steps of personalised medicine.

Funder

Research Ireland
Engineering and Physical Sciences Research Council (EPSRC)

Publisher

University of Galway

Rights

CC BY-NC-ND

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University of Galway Theses (PhD Theses)