Multiple Sclerosis (MS) In Vitro Modeling Service

Multiple Sclerosis (MS) is a chronic autoimmune disorder characterized by the degeneration of the myelin sheath surrounding nerve fibers in the central nervous system, leading to a spectrum of neurological symptoms and disability. In vitro modeling plays a crucial role in understanding the complex pathophysiology of MS, providing a controlled environment to study cellular mechanisms, immune interactions, and therapeutic responses without the ethical constraints of in vivo studies. These models primarily focus on key cellular components, including oligodendrocytes, neurons, microglia, and astrocytes, as well as immune cells such as T cells and B cells.

At Creative Biolabs, we offer various modeling approaches, including a diverse array of cell types sourced from primary cell cultures, immortalized cell lines and human iPSC-derived cells, which enable researchers to explore disease progression, identify potential biomarkers, and test novel treatment strategies, ultimately advancing understanding and management of MS.

Key Cellular Models in MS Research

• Oligodendrocytes and Myelination Defects

Oligodendrocytes are the primary target in MS, as their dysfunction leads to demyelination and impaired axonal conduction. Primary oligodendrocyte precursor cells (OPCs) isolated from rodent brains are widely used to study remyelination processes. However, human iPSC-derived OPCs have gained prominence due to their ability to mimic human-specific disease mechanisms. For example, iPSC-derived OPCs can be exposed to pro-inflammatory cytokines like TNF-α or IFN-γ to inhibit their differentiation into mature myelinating oligodendrocytes, effectively mimicking a demyelination, followed by testing the efficacy of remyelination-promoting compounds.

• Microglia and Neuroinflammation

Microglia, the resident immune cells of the central nervous system (CNS), are central to MS pathology. Primary microglia and immortalized microglial cell lines, such as BV-2, N9, or HMC3, are often used to study neuroinflammatory responses. These cells can be stimulated with lipopolysaccharide (LPS) or myelin debris to mimic the inflammatory environment observed in MS. Additionally, iPSC-derived microglia offer a more physiologically relevant platform for investigating the role of genetic variants, such as those in the TREM2 gene, in MS progression.

• Neurons and Axonal Degeneration

Neurons are critical in MS research due to their vulnerability to axonal damage and neurodegeneration, which underlie progressive disability in patients. Primary cortical neurons or iPSC-derived neurons can be used to model axonal injury and synaptic loss induced by inflammatory cytokines (e.g., IL-6, TNF-α) or demyelination. For example, microfluidic devices enable the study of axonal transport deficits in compartmentalized neuron cultures exposed to myelin-reactive T cells or oxidative stress. Additionally, iPSC-derived neurons from MS patients with specific genetic variants can provide insights into disease mechanisms and potential therapeutic targets.

• Astrocytes and Glial Scarring

Astrocytes contribute to MS pathology through the formation of glial scars, which inhibit remyelination and axonal regeneration. Primary astrocytes or iPSC-derived astrocytes can be activated with TGF-β or IL-1β to study their role in scar formation and secretion of inhibitory molecules like chondroitin sulfate proteoglycans (CSPGs). Co-culture systems with neurons or OPCs are often employed to assess the impact of astrocyte activation on neuronal survival and remyelination.

• Immune Cell Interactions

MS is fundamentally an autoimmune disease, making immune cell interactions a critical focus. Co-culture systems involving T cells and iPSC-derived neural cells are used to model the autoimmune attack on the CNS. For instance, myelin-reactive CD8+ T cells can be co-cultured with oligodendrocytes or neurons to study demyelination and axonal damage. Similarly, B cell-derived cytokines like IL-6 can be tested for their effects on OPC differentiation and survival.

• Blood-Brain Barrier (BBB)-Related Cells

The BBB plays a crucial role in MS pathogenesis, as its disruption allows immune cells to infiltrate the CNS and exacerbate neuroinflammation. In vitro BBB models typically consist of brain endothelial cells, pericytes, and astrocytes co-cultured in transwell systems. These models can be used to study the effects of pro-inflammatory cytokines (e.g., IL-17, IFN-γ) on BBB integrity, as well as the migration of immune cells across the barrier. For instance, iPSC-derived brain endothelial cells can be combined with astrocytes to create patient-specific BBB models, enabling researchers to test the efficacy of drugs designed to restore barrier function or prevent immune cell infiltration.

MS In Vitro Assays

1. Demyelination and Remyelination Assays

• Myelin Basic Protein (MBP) Staining

MBP is a major component of myelin, and its expression is commonly used as a marker for myelination. Immunocytochemistry (ICC) or immunofluorescence (IF) staining of MBP in oligodendrocytes or co-cultures with neurons can quantify myelination efficiency. For example, iPSC-derived OPCs can be differentiated into mature oligodendrocytes, and MBP staining can be used to assess the impact of pro-inflammatory cytokines or remyelination-promoting compounds.

• Live-Cell Imaging of Myelination

Live-cell imaging techniques, such as time-lapse microscopy, can be used to monitor the dynamic process of myelination in real-time. Fluorescent dyes or genetically encoded reporters (e.g., MBP-GFP) can label myelin sheaths, allowing researchers to track changes in myelination under different experimental conditions.

• Electron Microscopy (EM)

EM provides ultra-high-resolution images of myelin sheaths and axonal structures, enabling detailed analysis of demyelination and remyelination at the ultrastructural level. For instance, EM can be used to quantify the thickness of myelin sheaths in iPSC-derived oligodendrocyte-neuron co-cultures.

2. Neuroinflammation Assays

• Cytokine Profiling

ELISA or multiplex cytokine assays can measure the levels of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β) in cell culture supernatants. These assays are particularly useful for studying the inflammatory response of microglia or astrocytes exposed to myelin debris or immune cells.

• Reactive Oxygen Species (ROS) Detection

ROS play a key role in MS-related neuroinflammation and oxidative stress. Fluorescent probes, such as DCFH-DA or MitoSOX, can be used to quantify ROS production in microglia, astrocytes, or neurons under inflammatory conditions.

• NF-κB Activation Assays

NF-κB is a key transcription factor involved in neuroinflammation. Reporter assays (e.g., luciferase-based) or immunostaining for phosphorylated NF-κB can be used to monitor its activation in microglia or astrocytes treated with pro-inflammatory stimuli.

3. Axonal Damage and Neuronal Viability Assays

• β-III Tubulin Staining

β-III tubulin is a neuronal marker, and its staining can be used to assess neuronal morphology and axonal integrity. For example, β-III tubulin staining can quantify axonal damage in neurons co-cultured with myelin-reactive T cells or exposed to oxidative stress.

• Lactate Dehydrogenase (LDH) Assay

The LDH assay measures cell membrane integrity and is commonly used to assess neuronal or oligodendrocyte viability under inflammatory or toxic conditions. Increased LDH release indicates cell death or damage.

• Microfluidic Axonal Transport Assays

Microfluidic devices can compartmentalize neuronal cell bodies and axons, allowing researchers to study axonal transport deficits in MS models. Fluorescently labeled vesicles or mitochondria can be tracked to quantify transport dynamics under conditions mimicking MS pathology.

4. Blood-Brain Barrier (BBB) Integrity Assays

• Trans-endothelial Electrical Resistance (TEER)

TEER measures the integrity of the BBB by quantifying the electrical resistance across a monolayer of brain endothelial cells. A decrease in TEER indicates BBB disruption, which can be induced by pro-inflammatory cytokines or immune cell interactions.

• Dextran Permeability Assay

Fluorescently labeled dextran is used to assess BBB permeability. Increased dextran leakage across a brain endothelial cell monolayer indicates compromised barrier function, a hallmark of MS pathology.

• Immune Cell Migration Assays

Transwell systems can be used to study the migration of immune cells (e.g., T cells, monocytes) across a BBB model. Fluorescent labeling or flow cytometry can quantify the number of immune cells that migrate through the endothelial layer in response to chemokines or cytokines.

5. High-Throughput Screening (HTS) Assays

• Remyelination Compound Screening

High-content imaging platforms can be used to screen large libraries of compounds for their ability to promote remyelination in iPSC-derived OPCs or oligodendrocyte-neuron co-cultures. Automated image analysis can quantify MBP expression or myelin sheath formation.

• Anti-Inflammatory Drug Screening

Microglia or astrocytes can be treated with pro-inflammatory stimuli (e.g., LPS, myelin debris) and screened for compounds that reduce cytokine production or ROS levels. Luminescence- or fluorescence-based readouts enable high-throughput screening.

6. Advanced Omics-Based Assays

• Single-Cell RNA Sequencing (scRNA-seq)

scRNA-seq can reveal the transcriptional profiles of individual cells in MS models, providing insights into cell-type-specific responses to inflammation or demyelination. For example, scRNA-seq of iPSC-derived microglia or oligodendrocytes can identify novel therapeutic targets.

• Metabolomics

Metabolomic profiling can identify changes in metabolic pathways associated with MS pathology, such as mitochondrial dysfunction or lipid metabolism alterations. This approach is particularly useful for studying the effects of inflammatory cytokines on neuronal or glial cell metabolism.

Related Services

Reference

1. Birmpili, Dafni et al. "The Translatability of Multiple Sclerosis Animal Models for Biomarkers Discovery and Their Clinical Use." Int J Mol Sci. 2022;23(19):11532. Published 2022 Sep 29. Distributed under Open Access License CC BY 4.0 without modification.