3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue
July 16th, 2021
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Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.
Overview
This article presents a method for 3D bioprinting murine cortical astrocytes to create neural-like tissues. This approach aids in studying astrocyte functionality in the central nervous system and their role in neurological diseases.
Key Study Components
Area of Science
- Neuroscience
- Tissue Engineering
- Cell Biology
Background
- 3D bioprinting advances neuro tissue engineering.
- In vitro models help understand neurological disease mechanisms.
- Biofabrication mimics biochemical and mechanical tissue features.
- Protocol applicable to other soft tissues.
Purpose of Study
- To biofabricate neural-like tissues using astrocytes.
- To explore astrocyte functionality in health and disease.
- To investigate glial cell mechanisms in neurological conditions.
Methods Used
- Isolation of cortical tissue from euthanized mice.
- Cutting tissue into small pieces for processing.
- Washing tissue with HBSS solution.
- Incubating with trypsin to prepare for bioprinting.
Main Results
- Successful biofabrication of astrocyte-based tissues.
- Improved understanding of cell dynamics in neurological contexts.
- Potential applications in studying various neurological diseases.
- Demonstrated advantages of 3D bioprinting in tissue engineering.
Conclusions
- 3D bioprinting is a valuable tool for neuro tissue engineering.
- Biofabricated models can enhance research on astrocytes.
- This method may lead to better insights into neurological treatments.
Frequently Asked Questions
What is 3D bioprinting?
3D bioprinting is a technique used to create three-dimensional structures using living cells and biomaterials.
How does this method help in studying neurological diseases?
It allows researchers to create in vitro models that mimic the brain's environment, facilitating the study of disease mechanisms.
What are astrocytes?
Astrocytes are star-shaped glial cells in the brain that support neurons and maintain the blood-brain barrier.
Can this method be applied to other tissues?
Yes, the protocol can be adapted for biofabricating various soft tissues beyond neural tissues.
What are the advantages of using 3D bioprinting?
It allows for precise control over the structure and composition of the biofabricated tissues, closely mimicking natural tissues.
What role do glial cells play in the central nervous system?
Glial cells support and protect neurons, and are involved in processes such as neurotransmission and response to injury.
Executive Industry Relevance
This protocol enables the creation of physiologically relevant in vitro models of astrocytes, supporting mechanistic de-risking in early-stage neuroscience target validation. By providing a reproducible 3D microenvironment that maintains astrocytic phenotype and function, it enhances predictive confidence in preclinical studies of neurological disorders. The approach addresses a key discovery-stage challenge: the lack of translatable cellular models that recapitulate glial-neuronal interactions in health and disease.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of astrocyte-mediated pathways in neuroinflammation and neurodegeneration.
- Operational Value: Provides a standardized system for assessing target engagement in glial cells.
- Predictive Value: Supports phenotypic screening by maintaining GFAP expression and astrocytic morphology over time.
Screening & Assay Development
- Assay Readiness: Delivers a biomaterial platform compatible with live/dead viability and immunofluorescence readouts.
- Quantitative Output: Enables measurement of astrocyte viability (74.08% post-print, increasing to 83.54% at 7 days) as a biomarker of microenvironment suitability.
- Scalability: Uses extrusion-based bioprinting adaptable to multi-well formats for compound screening.
Translational & Preclinical Research
- Disease Relevance: Models astrocytic reactivity, a key feature in neurological injury and disease progression.
- Translational Continuity: Maintains stellate morphology and GFAP expression, supporting extrapolation to in vivo glial responses.
- Mechanistic De-risking: Allows evaluation of candidate compounds on astrocyte adhesion, spread, and network formation.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target hypothesis testing to lead optimization, particularly for CNS targets where glial modulation is relevant.
- Discovery Biology: Supports functional validation of astrocyte-specific targets in a 3D context.
- Screening: Enables standardized, reproducible plating of bioactive constructs for dose-response assays.
- Analytics: Provides confocal-compatible readouts for morphological and marker-based analysis (e.g., GFAP+ cell density).
- Translational Research: Bridges in vitro findings to preclinical models by preserving astrocyte phenotype.
- Enterprise Reuse: The bioink formulation (gelatin/GelMA/fibrinogen/laminin) is adaptable to other neural or soft tissue types.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in glial target validation through phenotype-stable, long-term culture.
- Operational Value: Ensures reproducibility via standardized cell sourcing (passage ≤3) and bioink mixing ratios.
- Strategic Value: Improves go/no-go decisions by enabling early assessment of off-target glial effects.
- Portfolio Impact: Facilitates risk-adjusted prioritization of CNS drug candidates with glial modulation potential.
Implementation Considerations
- Requires expertise in primary cell isolation and sterile 3D bioprinting techniques.
- Depends on extrusion bioprinter, UV crosslinking system, and confocal microscopy for validation.
- Necessitates standardization of bioink preparation across batches to ensure consistent mechanical and biochemical properties.
- Adaptation to human iPSC-derived astrocytes may require optimization of enzymatic dissociation and culture conditions.
- Limited by murine cell source; species translation should be validated for human-relevant applications.
Biopharma FAQs
Why is astrocyte viability measured post-bioprinting?
Viability assessment confirms the bioprinting process does not induce excessive cell stress, ensuring a suitable microenvironment for downstream functional assays. The protocol reports 74.08% viability immediately after printing, increasing to 83.54% at 7 days, indicating supportive bioink conditions.
How does GFAP expression support target validation in glial cells?
GFAP expression confirms retention of astrocytic phenotype, which is essential for validating targets specific to glial biology. Its sustained expression over 7 days indicates the model maintains functional relevance for mechanistic studies.
What role does fibrinogen play in the bioink for neural tissue engineering?
Fibrinogen enables enzymatic cross-linking with thrombin to stabilize the 3D construct post-printing, providing structural integrity without compromising cell viability. This supports long-term culture and assay reproducibility.
Why is laminin included in the astrocyte-laden bio-ink?
Laminin promotes astrocyte attachment, spreading, and morphological differentiation, which are critical for modeling physiologically relevant glial behavior. Its inclusion contributes to the observed stellate morphology and network formation.
How do replication requirements improve cross-functional collaboration in CNS drug discovery?
The protocol’s reproducibility—using defined cell passages, standardized bioink ratios, and consistent bioprinting parameters—enables reliable data sharing between discovery, preclinical, and translational teams. This reduces variability in target validation outcomes across sites.
Chapters in this video
0:05
Introduction
0:49
Astrocyte Isolation and Culture
2:06
Astrocytes-laden Gelatin/GelMA/Fibrinogen Bioink Preparation
4:01
Bioprinting Syringe Preparation and Bioprinting
4:58
Bioprinted Construct and Culture Crosslinking
5:40
Astrocytes Viability Assessment
6:22
Results: 3D Bioprinted Astrocyte Cell Viability and Morphology Assessment
8:15
Conclusion
