Microglia-containing Brain Organoid Modeling Service

Creative Biolabs is at the forefront of neuroscience, offering sophisticated human iPSC-derived brain organoid models robustly integrated with functional microglia. Our advanced 3D platforms provide an unprecedented, physiologically relevant system to investigate complex neuroimmune interactions, accelerating the discovery of novel therapeutics for a spectrum of neurological disorders.

Introduction

The human brain's intricate cellular landscape, involving a delicate interplay between neurons, macroglia, and resident immune cells, poses significant challenges for accurate disease modeling. Microglia, the brain's primary immune effector and homeostatic cells, are pivotal in brain development, synaptic plasticity, neuroinflammation, and the response to injury and disease. Growing evidence links microglial dysfunction to the pathogenesis of Alzheimer's disease (AD), Parkinson's disease (PD), autism spectrum disorders (ASD), neurotropic viral infections (e.g., Zika, HIV-1, SARS-CoV-2), and other severe neurological conditions.

Traditional in vitro 2D cell cultures struggle to replicate the brain's three-dimensional architecture and critical intercellular communications. While animal models have provided valuable insights, they often fail to fully recapitulate human-specific microglial functions and complex disease phenotypes. Standard brain organoid protocols, which primarily drive neuroectodermal differentiation, typically yield models devoid of microglia. This is because microglia originate from a distinct mesodermal lineage during embryonic development, an origin not usually supported by these standard protocols. This absence critically limits the utility of such organoids in studying neuroimmune interactions and inflammatory pathways central to many brain disorders.

Core Features of Our Microglia-Containing Human Brain Organoid (MC-HBOs) Service

Creative Biolabs provides a comprehensive and customizable suite of services, employing state-of-the-art strategies for robust microglia integration and in-depth characterization:

1. Versatile and Advanced Microglia Integration Strategies:

• Co-culture of Differentiated Microglia (iMGLs) with Established Brain Organoids:
  For studies requiring the introduction of a well-defined, mature microglial population into an already developed neural context, we provide services for integrating fully characterized, pre-differentiated iMGLs into established human brain organoids (HBOs). This is particularly advantageous for investigating the roles of mature microglia in later-stage disease processes, assessing the effects of specific microglial phenotypes (e.g., pro-inflammatory vs. homeostatic), or evaluating therapeutic interventions targeting existing microglial populations.
• Innate Microglia Development within Brain Organoids (Spontaneous Formation in HBOs):
  We leverage protocols that promote the in situ emergence of microglia from mesodermal progenitors within the developing HBOs. By carefully modulating culture conditions—such as adjusting heparin concentrations, modifying Matrigel embedding timelines, or avoiding strict dual-SMAD inhibition—we create an environment conducive to the spontaneous differentiation of iPSCs into mesodermal precursors that subsequently mature into microglia. This approach allows microglia to co-develop and integrate organically with neural tissues from early stages, offering a highly biomimetic system for studying neurodevelopmental processes and inherent neuroimmune crosstalk. Advanced techniques, including the targeted overexpression of key myeloid transcription factors like PU.1, can be employed to further enhance and guide this innate microglial generation.

Fig.1 Spontaneous formation of microglia in HBOs.¹

• Co-culture of Microglial Progenitor Cells (MPCs) with Pre-formed Brain Organoids:
  This strategy involves the initial generation of highly characterized iPSC-derived MPCs, such as erythro-myeloid progenitors (EMPs) or primitive macrophage progenitors (PMPs). These MPCs are then introduced to established, developing brain organoids. The intrinsic signaling milieu of the organoid subsequently guides the migration, integration, differentiation, and maturation of these MPCs into functional microglia. This method provides a valuable model for investigating how the neural environment shapes microglial identity, and for studying the impact of microglial integration at specific developmental stages of the organoid.
• Co-culture of Microglial Progenitor Cells (MPCs) with Neural Progenitor Cells (NPCs):
  To explore the earliest interactions between nascent neural and immune cell lineages, Creative Biolabs offers models generated by the co-culture of iPSC-derived MPCs directly with NPCs. These mixed progenitor populations are then allowed to self-assemble and differentiate together into a cohesive MC-HBO. This approach uniquely facilitates the study of parallel co-development and mutual influences from the very onset of organoid formation. It also provides an excellent platform for controlling initial cell ratios to investigate dose-dependent effects of microglia on neurogenesis, neuronal specification, and the establishment of the fundamental neuroimmune architecture.

2. Comprehensive Functional and Molecular Validation:

• Thorough Marker Expression Profiling: Our integrated microglia are validated for the expression of a comprehensive panel of canonical markers, including IBA1, TMEM119, P2RY12, CX3CR1, CD68, PU.1, CD11b, CD45, and others, confirming their identity and maturation state.
• Physiological Morphology and Dynamic Behavior: Microglia within our organoids exhibit characteristic morphologies, from ramified, surveying states to amoeboid, activated forms, and demonstrate essential functions such as robust phagocytosis of Aβ, apoptotic cells, cellular debris, and synaptic material, alongside active synaptic pruning. Functional responses, like calcium signaling to ADP, are also assessed.
• Authentic Immune Responsiveness: We meticulously verify microglial responses to a wide range of stimuli, including PAMPs (e.g., LPS), DAMPs, disease-associated proteins (e.g., Aβ oligomers and fibrils, pathological tau), and viral challenges, by analyzing cytokine and chemokine release profiles and alterations in gene expression.

3. Brain Region-Specific and Enhanced Long-Term Culture Systems:

• Regional Specificity: We can generate MC-HBOs that model distinct brain regions (e.g., cortex, dorsal/ventral forebrain, midbrain), enabling focused investigations into region-specific microglial functions and disease vulnerabilities.
• Superior Long-Term Culture and Maturation: Our innovative culture methodologies, including Adhesion Brain Organoids (ABOs) and specialized tubular organoid-on-a-chip systems, promote enhanced nutrient and oxygen perfusion, minimize necrotic cores, and support the extended survival (e.g., >1 year for ABOs) and functional maturation of both the neural and integrated microglial populations. These systems also facilitate the development of other glial types like astrocytes and oligodendrocytes.
• Pioneering Vascularization Strategies: We are actively advancing our models by incorporating iPSC-derived MPCs to establish rudimentary vascular-like networks, aiming to create even more physiologically complete and viable long-term organoid systems.

Tailored Modeling Solutions for Your Unique Research Objectives

Creative Biolabs offers a highly flexible and collaborative service framework:

1. Custom iPSC Lines: We can work with our extensive, well-characterized iPSC lines from healthy donors and various disease backgrounds, or utilize your specific patient-derived iPSC lines. This allows for modeling of specific genetic predispositions, such as APOE4 variants in AD or trisomy 21 in Down Syndrome.
2. Bespoke Organoid and Microglia Design: We collaborate with you to define brain region specificity, select the most appropriate microglia integration strategy (innate development, co-culture, transcription factor induction), and determine optimal culture duration and experimental endpoints.
3. Comprehensive Multi-Modal Readout Capabilities:

• Immunohistochemistry and Advanced Microscopy: Including multi-channel confocal imaging, super-resolution microscopy (e.g., STED), and 3D image reconstruction and analysis.
• In-depth Molecular Analyses: Single-cell and bulk RNA-sequencing, quantitative PCR, Western blotting, and ELISA for precise quantification of cytokines, chemokines, and other secreted factors.
• Diverse Functional Assays: Including detailed phagocytosis assays (targeting Aβ, synaptosomes, cellular debris, or beads), live-cell calcium imaging, electrophysiological assessments (e.g., MEA), cell viability and apoptosis assays, and microglia migration studies.
• Targeted Gene Manipulation: Offering CRISPR-Cas9 based gene editing (e.g., CRISPRi/a) in the source iPSCs before differentiation, enabling the study of specific gene functions (e.g., AD-risk genes like TREM2, CD33, SORL1, or IFNARs) in the context of the MC-HBO.

Diverse Applications Driving Neuroscience Breakthroughs

1. Neurodegenerative Disease Research:

• Elucidating the complex roles of microglia and neuroinflammation in AD (investigating Aβ and tau pathologies, APOE4 isoform effects, IFN-I signaling pathways).
• Investigating microglial involvement in Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), and Huntington's disease.
• Screening and validating novel therapeutic compounds targeting microglial activation, Aβ/tau clearance, microglial senescence, or promoting neuroprotection.

2. Neurodevelopmental Disorder Modeling:

• Investigating microglial contributions to ASD, Rett Syndrome, and Down Syndrome, including phenotypes like altered synaptic pruning.
• Studying the impact of specific genetic mutations on microglia-neuron interactions and overall brain development.

3. Neuroinfectious Disease Studies:

• Modeling viral neuropathogenesis (e.g., ZIKV, HIV-1, SARS-CoV-2), including mechanisms of viral entry, replication, microglial responses, and viral persistence.
• Assessing the role of microglia as CNS reservoirs and mediators of virus-induced inflammation and synaptic damage.

4. Neuroimmunology and Neuroinflammation Research:

• Dissecting the fundamental mechanisms of microglia activation, polarization (e.g., in response to LPS, opioids), and their intricate communication with other CNS cell types.
• Evaluating the efficacy and mechanisms of action of novel anti-inflammatory and immunomodulatory agents.

5. Drug Discovery and Advanced Toxicology Screening:

• Performing high-throughput screening of compound libraries for neuroprotective, anti-inflammatory, or pro-clearance effects in a human-relevant 3D multicellular system.
• Assessing potential neurotoxic or immunomodulatory side effects of novel drug candidates with greater predictive validity.

Our Streamlined Project Workflow

Elevate your neuroscience research and accelerate your therapeutic development with Creative Biolabs' state-of-the-art Microglia-Containing Brain Organoid Modeling Service.

Contact us today to schedule a consultation with our scientific team and explore how our advanced human brain models can empower your next breakthrough.

Reference

1. Samudyata et al. "SARS-CoV-2 promotes microglial synapse elimination in human brain organoids." Molecular psychiatry vol. 27,10 (2022): 3939-3950. doi:10.1038/s41380-022-01786-2. Distributed under Open Access License CC BY 4.0. The original image was modified.