The authors would like to thank Alex Volkerling, Martin Engel, and Rachel Bleach for their assistance in developing the protocol and feedback on the manuscript.

3D Bioprinted Cocultures of iPSC-Derived Neurons and Astrocytes

<ol>
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CW, NC, and JB are employees of Merck Sharp &amp; Dohme (UK) Limited, London, UK. YH is an employee of Merck Sharp &amp; Dohme LLC, a subsidiary of Merck &amp; Co., Inc., Rahway, NJ, USA.
Figure one was created using Biorender.com.

The need for accurate models of the CNS has never been higher, and limitations of two-dimensional (2D) traditional cell culture models have driven a generation of complex CNS models in recent years19. However, many complex 3D models that represent interactions between neural cell types and the ECM have limitations that would prevent the application of these models in industrial processes6,20,21. In this protocol, we develop a 3D coculture model of human iPSC-derived neurons and astrocytes, which aims to resolve some of these limitations using 3D bioprinting technology to create a bioactive hydrogel scaffold in 96-well and 384-well formats.
The methodology for developing these models has been simplified through the plate map design software, auto-generated print protocols, and guided print process from the bioprinter. However, due to the sensitive nature of the sensitive iPSC-derived cell types used in this protocol care should be taken with the following critical steps in thawing and culture. Firstly, the inclusion of ROCK inhibitor (ROCKi) has several benefits throughout the bioprinting process and during early culture. Cell thawing is a critical point in which the neurons can experience a stress response, and improper thawing protocols can decrease the chances of survival22. It is typically recommended to thaw cells, add media, and raise cells to incubator temperature as efficiently as possible23. However, during the bioprinting process described in this protocol, it is necessary that neurons and astrocytes are resuspended in an activator solution rather than media, and cells will not be raised above room temperature until the end of the print run (up to 30 minutes post-thaw). Thus, the addition of ROCKi to the media immediately after thawing and including this during the two centrifugation steps (steps 2.1--2.7 and 1.3.15-1.3.20) is imperative to inhibit cell stress pathways, which would result in lower viability levels24. Furthermore, ROCKi has been shown to promote neurite outgrowth and improve neuronal maturation25. Thus, ROCKi supplementation is continued for 48 h post-bioprinting. However, it is imperative to remove ROCKi supplementation after 48 h to ensure complete wash-off during the subsequent media changes before cells are used for assay.
A further step that requires critical attention is during post-print media addition and media changes (steps 2.8-2.13). The bioprinted hydrogel scaffold has an equivalent biomechanical stiffness of only 1.1 kPa, equivalent to grey matter. As described in step 2.10, it is critical to pipette gently into the side of the well during media addition and aspiration to prevent disturbance. This is of particular relevance to 384-well plates, where the gel level takes up a higher proportion of total well volume. This method should also be used in 2D control wells to prevent edge lifting of cells and shearing of neurite outgrowths. The authors would also like to highlight the importance of sterile technique within the bioprinter, which should be treated with equivalent caution to that of a biosafety cabinet used for iPSC-derived cell cultures. This includes sterile filtering 70% EtOH and dH2O used in the greenlighting and printing procedures, keeping lids on the cartridges and plates while moving hands in and out of the bioprinter, and decontaminating surfaces inside the bioprinter with 70% ethanol wipes before and after printing.
The bioprinted hydrogel scaffold, formed from bioink and activator solutions, selected to develop this model is selected from a range of bioinks and activator solutions developed by Inventia Life Science for use within the RASTRUM bioprinter. Laminin and hyaluronic acid were identified as molecules of relevance to iPSC-derived neuronal maturation due to their role in axonal guidance, synapse formation, and formation of the perineuronal net26,27. Furthermore, a biomechanical stiffness of 1.1 kPa was selected, as lower-density hydrogels have been shown to enable better neurite outgrowth from neurons12. If modifications are made to the protocol by using neurons and astrocytes that have been differentiated in-house or from a different commercial supplier, it would be recommended to do a matrix selection test to determine the most supportive hydrogel scaffold15. Furthermore, cell density may also need to be optimized if changes to cell sources are made to ensure optimal viability and prevent hydrogel overcrowding. For all modifications and troubleshooting related to the bioprinter function, the authors recommend contacting manufacturers and referencing manufacturer protocols.
The CNS contains a broad range of neuronal subtypes and glial cells, all of which exist in different brain niches and have specific roles contributing to neural function28. Within the context of this broad scope, this model represents only the two most abundant cell types (astrocytes and excitatory glutamatergic neurons). Important cell types such as microglia, oligodendrocytes, and blood-brain barrier-forming endothelial cells are omitted from this system. The inclusion of microglia could be of relevance in focus on neuroimmune interactions, and oligodendrocytes could be of interest in diseases that affect central myelination. In addition to their role in pathology, cells such as blood-brain barrier-forming endothelial cells excrete drug-metabolizing enzymes, which could affect the use of this model for pharmacokinetic assays29. A further limitation of the model may be the ratio of astrocytes to neurons; the ratio of astrocytes to neurons varies greatly between brain regions, with suggested values of between 1:1 and 1:330,31. This model has an approximate ratio of 1:1.5 astrocytes to neurons; thus, this model might not be of relevance to model brain areas where astrocytes are more abundant, such as in white matter areas30.
Other protocols to develop 3D bioprinted coculture models have been published in recent years. A publication by Sullivan et al., 2021, presented a 3D bioprinted neural model using iPSC-derived neural progenitor cells, which demonstrates high post-print viability and enhancement of neuronal function compared to 2D cultures32. However, in this protocol, neural progenitor cells were used as a cell source and were maintained in culture for 4 weeks. In this protocol, commercially available iPSC-derived glutamatergic neurons and astrocytes were used. This allows a 3D network of co-cultured cells to be established in as little as 7 days; as demonstrated by neurite growth analysis, neurite outgrowth begins within 24 h and continues in a linear fashion throughout the 156 h period for which cell growth was monitored. The rapid establishment of these networks can be partly attributed to the use of glutamatergic neurons that use optimized doxycycline-inducible gene expression of NGN2, which shows expression of mature neuronal subtype markers within 7 days, even in 2D culture33. The shortening of this growth period using this technique is important to implementing models within the biopharmaceutical industry, as assay development requires rapid turnaround and development of cell models15.
In conclusion, this model shows potential for a 3D model of neurons and astrocytes, which is established quickly and conveniently for screening purposes. Future applications for this model type could be for drug discovery efforts across different CNS diseases, with the opportunity to expand to different diseases using patient or gene-edited disease iPSC lines. Furthermore, the use of doxycycline-inducible NGN2 expression iPSC-derived glutamatergic neurons allows cells to reach maturity in less time, which can be utilized for developing models of the aging brain for neurodegeneration research. This system could also be expanded through the use of additional cell types in coculture, including microglia and oligodendrocytes.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/65856">Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications</a>. The Authors section was updated from:
Chloe Ann Whitehouse1 
Yufang He2 
Janet Brownlees1 
Nicola Corbett1 
1MSD R&#38;D Innovation Centre Ltd 
2Merck &#38; Co., Inc.
to:
Chloe Ann Whitehouse1 
Yufang He2 
Janet Brownlees1 
Nicola Corbett1 
1MSD Research Laboratories, London, UK 
2Merck &#38; Co., Inc., Rahway, NJ, USA

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/65856">Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications</a>. The Authors section was updated.

Erratum: Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

Research into central nervous system (CNS) diseases in the drug discovery industry is expanding1. However, many prevalent CNS diseases such as epilepsy, schizophrenia, and Alzheimer&#39;s disease still have no curative treatments2,3,4. The lack of effective therapeutics across CNS diseases can, at least in part, be attributed to a lack of accurate in vitro models of the brain5. This has resulted in a translational gap between current in vitro models and in vivo data and a subsequent bottleneck in research efforts.
Driven by this translational gap, there has been a significant increase in the development of novel 3D cell models within recent years, including neural organoids, neurospheroids, and scaffold-based models6. The 3D structure of these models aids in recapitulating the neural microenvironment, including biomechanical stresses, cell-cell contacts, and the brain extracellular matrix (ECM)7. The brain ECM is a dynamic element of neurophysiology that occupies the space between neural cell types, including neurons, astrocytes, oligodendrocytes, and the neurovascular unit7. Recapitulation of the brain ECM has been shown to affect neuronal morphology and neuronal firing, and many complex 3D models of the brain have demonstrated deposition of ECM proteins by neural cell types8,9,10,11. Scaffold-based models consist of mature neural cocultures suspended in a porous synthetic or biological hydrogel matrix which represents the brain ECM12. Unlike organoid and spheroid systems, scaffold-based 3D models allow the customization of ECM proteins present and have the added benefit of tunability of hydrogel stiffness to mimic biomechanical stresses13,14.
Although an overwhelming majority of 3D neural models demonstrate an increased recapitulation of the brain microenvironment, not all models are well suited for implementing drug discovery applications15. For a 3D model to be implemented into industrial processes, the system must meet throughput requirements for screening applications and have a relatively short development time16. 3D Bioprinting is a novel technology that offers the potential to create 3D scaffold-based neural models with rapid development time, increased throughput, and higher levels of precision control, alongside the removal of variability caused by human error17. This protocol presents a 3D coculture model of human iPSC-derived glutamatergic neurons and astrocytes in a hydrogel scaffold. This hydrogel scaffold contains physiologically representative bioactive peptides (RGD, IKVAV, YIGSR) and ECM proteins within a mimetic biomechanical stiffness. These full-length ECM proteins include full-length laminin-211 and hyaluronic acid, abundant in the human cortex, with a stiffness of 1.1 kPa in line with in vivo measurements18. This model is designed with practicality for drug discovery, and is&#160;created using a 3D bioprinter in a 96-well or 384-well plate format suitable for screening analysis using imaging techniques with cell dyes and antibodies, alongside neurite outgrowth assays. Cells show expression of neural cell type markers and growth of neurite and astrocytic projections within 7 days of culture. Thus, this protocol will present the methodology to develop a high-throughput 3D neural coculture model for use in drug discovery applications.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65856/65856fig01.jpg" /> 
Figure 1: Illustrative overview of methodology used to 3D bioprint cocultures. Human iPSC-derived neurons and astrocytes are combined with activator and bioink solutions containing bioactive peptides and are bioprinted to hydrogel scaffolds in 96-well or 384-well formats using drop-on-demand bioprinting technology. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65856/65856fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>2-mercaptoethanol</td><td>Thermofisher</td><td>31350010</td><td></td></tr><tr><td>384-well plate</td><td>PerkinElmer</td><td>6057300</td><td></td></tr><tr><td>96-well plate</td><td>PerkinElmer</td><td>6055300</td><td></td></tr><tr><td>Activator fluid F299</td><td>Inventia Life Science</td><td>N/A</td><td></td></tr><tr><td>Activator fluid F3</td><td>Inventia Life Science</td><td>N/A</td><td></td></tr><tr><td>B27 (50x) minus Vit A</td><td>Thermofisher</td><td>12587010</td><td></td></tr><tr><td>Bioink fluid F261</td><td>Inventia Life Science</td><td>N/A</td><td></td></tr><tr><td>Bioink fluid F32</td><td>Inventia Life Science</td><td>N/A</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma Aldrich</td><td>D5207</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Thermofisher</td><td>35050061</td><td></td></tr><tr><td>Goat anti-mouse IgG H&amp;amp;L Alexa Fluor 647</td><td>Abcam</td><td>ab150115</td><td></td></tr><tr><td>Goat anti-rabbit IgG H&amp;amp;L Alexa Fluor 488</td><td>Abcam</td><td>ab150077</td><td></td></tr><tr><td>Hoechst</td><td>Abcam</td><td>ab228551</td><td></td></tr><tr><td>Human BDNF Recombinant Protein</td><td>Thermofisher</td><td>PHC7074</td><td></td></tr><tr><td>Human NT3 Recombinant Protein</td><td>Thermofisher</td><td>PHC7036</td><td></td></tr><tr><td>iCell Astrocytes</td><td>Fujifilm CDI</td><td>1434</td><td></td></tr><tr><td>INCell Analyser 6500HS</td><td>Molecular Devices</td><td>N/A</td><td>high content imaging system</td></tr><tr><td>Incucyte S3</td><td>Sartorius&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>ioGlutamatergic Neurons (Large vial)</td><td>Bit.bio</td><td>e001</td><td></td></tr><tr><td>Laminin (1 mg/mL)</td><td>Sigma Aldrich</td><td>L2020</td><td></td></tr><tr><td>Live/dead kit (Calcein-AM, Ethidium homo-dimer-1)</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Mouse anti-BIII tubulin NL637 conjugated</td><td>R&amp;amp;D systems</td><td>SC024</td><td></td></tr><tr><td>Neurobasal media</td><td>Thermofisher</td><td>21103049</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Abcam</td><td>ab7475</td><td></td></tr><tr><td>NucBlue Live (Hoechst 33342)</td><td>Thermofisher</td><td>R37605</td><td></td></tr><tr><td>Opti-MEM</td><td>Thermofisher</td><td>11058021</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>PEI 50% in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>181978</td><td></td></tr><tr><td>Pierce Borate Buffer 20x</td><td>Thermofisher</td><td>28341</td><td></td></tr><tr><td>Prism</td><td>GraphPad</td><td></td><td>Data analysis software</td></tr><tr><td>Rabbit anti-ionotropic glutamatre receptor 2 (GluR2)</td><td>Abcam</td><td>ab206293</td><td></td></tr><tr><td>RASTRUM(TM) Bioprinter</td><td>Inventia Life Science</td><td>N/A</td><td>Bioprinter</td></tr><tr><td>RASTRUM(TM) Bioprinter Cartridges</td><td>Inventia Life Science</td><td>N/A</td><td>Bioprinter Cartridges</td></tr><tr><td>RASTRUM(TM) Targeting plate</td><td>Inventia Life Science</td><td>N/A</td><td>Targeting plate</td></tr><tr><td>Rho kinase (ROCK) inhibitor</td><td>Abcam</td><td>ab120129</td><td></td></tr><tr><td>Sheep anti-GFAP NL493 conjugated</td><td>R&amp;amp;D systems</td><td>SC024</td><td></td></tr><tr><td>Signals Image Artist&amp;nbsp;</td><td>PerkinElmer&amp;nbsp;</td><td>N/A</td><td>Image analysis platform</td></tr><tr><td>Triton X-100</td><td>Thermofisher</td><td>HFH10</td><td></td></tr><tr><td>Zeiss Axio Observer</td><td>Zeiss</td><td>N/A</td><td>Inverted microscope platform&amp;nbsp;</td></tr></tbody></table>

three-dimensional bioprinting of human ipsc-derived neuron-astrocyte cocultures for screening applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models&#160;do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

Neurite growth analysis 
In this protocol, iPSC-derived glutamatergic neurons and astrocytes were bioprinted in coculture into a hydrogel matrix using the 3D bioprinter. Over the first 7 days post-printing, cells were imaged every 12 h using a live cell microscope. Post-bioprinting, cells should have a rounded morphology and should be dispersed throughout the hydrogel matrix, gradually changing to form smaller cell clusters with few protrusions over the first few days of culture (see Supplementary&#160;Video 1 for representative healthy cell growth). By day 4, healthy cells will migrate throughout the gel to form larger clusters, which are connected through neurite outgrowths. By day 7, almost no single cells should remain, the interconnecting bundles of neurites and astrocytic projections should appear fortified, and many smaller neurite outgrowths can be seen forming from the clusters (Figure 2A). Using a series of live cell brightfield images taken over the 7-day growth period, an analysis of neurite outgrowth was performed as detailed in section 3. This analysis demonstrated that neurite outgrowth increases in a near linear fashion (R2 value = 0.84) between 12 h and 156 h (Figure 2B). During this period of neurite outgrowth, cell body clusters also increase in size (see Supplementary&#160;Video 1), which is indicative of cell migration throughout the hydrogel.
Cell viability and population ratio 
In this protocol, a concentration of 20 million cells/mL, comprising 15 million neurons/mL and 5 million astrocytes/mL, is used for bioprinting the cell models. Using live cell staining with calcein-AM (live cells), ethidium homodimer-1 (dead cells) and a nuclear stain, the number of cells surviving over a 7-day period can be calculated as per section 4 (Figure 3A). Cell viability results for representative cultures are shown for day 4, where 72% &#177; 1% (mean &#177; SEM) of total cells are live and show staining for Calcein-AM, while 29% &#177; 2% (mean &#177; SEM) total cells are dead and show staining with ethidium homodimer-1 (Figure 3B). Representative images of cell staining with Calcein-AM and ethidium homodimer-1 can be seen in Supplementary Figure 1. It should be noted that cell survival values for 3D cultures cannot be directly compared to 2D cultures, as dead cells are retained in the hydrogel and will not be removed during cell feeding processes.
Using the immunofluorescent staining for &#914;-III tubulin and GFAP, as described in section 5 and in Figure 4, image analysis can be carried out to determine cell population ratios between neurons and astrocytes (Figure&#160;3A). Of total cells per model in representative cultures, &#914;-III tubulin positive neurons represent 49% &#177; 3% (mean &#177; SEM), while GFAP positive astrocytes represent 30% &#177; 4% (mean &#177; SEM). This gives a ratio of 1:1.5, astrocytes to neurons, respectively. This leaves a remainder of 21% total cells per model, which do not stain for either cell marker. As cell viability analysis demonstrated that a mean value of 29% of cells are not viable at day 4, it is likely that these cells are dead within the hydrogel.
Expression of cell markers 
Morphology of the bioprinted neurons and astrocytes was assessed through immunostaining for neuronal cell type marker (&#914;-III tubulin) and astrocytic marker (GFAP). In the representative cultures shown, immunostaining is localized to individual cell types, showing healthy cell morphology, with both cell types exhibiting outgrowth of cellular protrusions (Figure 4A,B). As the hydrogel and cell structures are three-dimensional, each image represents only one slice through the structure in Figure 4A,B. Figure 4C shows a merged stack of images throughout the hydrogel, demonstrating views of cell localization in the X, Y, and Z planes. Figure 4D shows immunostaining for &#914;-III tubulin only; highlighting finer neurite outgrowths from the cell body clusters. To further examine the phenotype of the glutamatergic neurons, immunostaining for the glutamatergic ionic receptor marker, GluR2, can be carried out. Within Figure 4E, area 4E.1 (inset) has been highlighted to show higher resolution punctate staining along the neurite bundles. This therefore, confirms that neurons in this coculture have a glutamatergic phenotype. Across all immunostaining images, immunofluorescent stained non-cell structures can be observed surrounding the cell clusters and neurites. It is likely that these structures represent debris retained within the hydrogel in combination with minor amounts of non-specific antibody binding to the hydrogel. This is expected in bioprinted cultures, as within 3D scaffold models, dead cells and debris are not removed during cell feeding. A representative negative control immunostaining image is shown in Supplementary Figure 2 for a demonstration of hydrogel non-specific binding of secondary antibodies.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65856/65856fig02.jpg" /> 
Figure 2: Glutamatergic neurons and astrocytes were bioprinted into the hydrogel matrix using the bioprinter and were imaged every 12 h using a brightfield microscope. (A) An example brightfield image taken of cell cultures during analysis. The image represents time point 156 h, and the scale bar represents 400 &#181;m. (B) The average length of neurite outgrowths (&#181;m) from the cultures measured using the NeuronJ package for ImageJ. Each data point is n = 3 neurites, and data is shown as mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65856/65856fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65856/65856fig03v2.jpg" /> 
Figure 3: A concentration of 20 million cells/mL of activator solution was used for bioprinting the cell models. (A) Cell viability was calculated using live/dead cell dyes (Calcein-AM and ethidium homodimer-1, respectively). Values show 72% &#177; 1% (mean &#177; SEM, n = 3) of total cells per well are live and 29% &#177; 2% (mean &#177; SEM, n = 3) of cells are dead of total cell population per well at Day 4. Values shown represent mean &#177; SEM. (B) Cell populations percentage of neurons and astrocytes per well were calculated through image analysis of staining shown in Figure 4. Neurons represent the percentage of cells staining positive for &#914;-III tubulin at Day 7 (49% &#177; 3%, mean &#177; SEM, n = 3), while astrocytes represent the percentage of cells staining positive for GFAP at Day 7 (30% &#177; 4%, mean &#177; SEM, n = 3). Values shown represent mean &#177; SEM. All imaging for calculations shown in Figure 3 was performed on a confocal imaging system, and all analyses were performed on the image analysis platform and GraphPad Prism as per methods. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65856/65856fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65856/65856fig04.jpg" /> 
Figure 4: Expression of neural cell type markers in 3D bioprinted cocultures of glutamatergic neurons and astrocytes at day 7. (A,B) Immunofluorescent staining of neuronal marker &#914;-III tubulin and astrocyte marker GFAP, imaged on an inverted microscope platform at 10x magnification. Scale bars represent 100 &#181;m. (C) Immunofluorescent staining of neuronal marker &#946;-III tubulin and astrocyte marker GFAP co-stained with Hoechst, shown in XYZ plane view, imaged on a confocal imaging system at 10x magnification. Created on the image analysis platform. The scale bar represents 100 &#181;m. (D) Immunofluorescent staining of neuronal marker &#946;-III tubulin co-stained with Hoechst, imaged on a confocal imaging system at 20x magnification. Scale bar represents 100 &#181;m. (E) Immunofluorescent staining of glutamatergic marker GluR2 co-stained with Hoechst, imaged on an inverted microscope platform at 10x magnification. Box 3E.1 shows highlighted areas of GluR2 staining. The scale bar represents 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65856/65856fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: Glutamatergic neurons and astrocytes were bioprinted into the hydrogel matrix using the bioprinter and were imaged every 12 h using a brightfield microscope. Video of brightfield images taken of cell cultures during analysis, time points are indicated in the bottom right corner, and scale bars represent 400 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65856/Supplementary Video 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 1: Example images of live/dead analysis of bioprinted neurons and astrocytes on Day 4. Calcein-AM stain shown in green (488 nm), and ethidium homodimer stain shown in red (647 nm). The image is shown in XYZ plane view, created on the image analysis platform . The scale bar represents 100 &#181;m. (A) Imaging was carried out using a confocal imaging system at 4x magnification. (B) Imaging was carried out using a confocal imaging system at 10x magnification <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65856/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Example of negative control image after immunofluorescent staining. Primary antibodies were omitted, and green (488 nm) and red (647 nm) secondary antibodies were used as per immunostaining protocols. The image is shown in XYZ plane view, created on the image analysis platform. The scale bar represents 100 &#181;m. Imaging was carried out using a confocal imaging system at 10x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65856/Supplementary Figure 2.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancing 3D Cell Modeling – A High-Throughput Approach for Neural Cocultures at JoVE.com

Author Spotlight: Advancing 3D Cell Modeling &#8211; A High-Throughput Approach for Neural Cocultures 

Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development ...

three-dimensional-bioprinting-human-ipsc-derived-neuron-astrocyte

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

4.2K Views.  MSD Research Laboratories, London, UK. Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

Video: Author Spotlight: Advancing 3D Cell Modeling – A High-Throughput Approach for Neural Cocultures 

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Developmental Biology

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

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.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Bioengineering

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...

Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

Source: Cvetkovic, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a procedure for co-culturing the astrocytes and neurons to form 3D spheres. These 3D spheres provide a distinct platform for examining synapse formation and neural network integration, thereby serving as a potential model for research in developmental neuroscience, neurodegenerative diseases, and neuropharmacological interventions.

1. Preparation and Maintenance of 3D Sphere Cocultures

	Dissociate hAstros from 3D aggregates in suspension.&#8203;
	
		Gently dissociate human astrocyte ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

A Method for 3D Bioprinting Murine Cortical Astrocytes

Source: de Melo, B. A. G., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62691">3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue.</a> J. Vis. Exp. (2021)
The video demonstrates the process of creating a 3D bioprinted construct using astrocytes suspended in a bioink solution. The bioprinted construct, after crosslinking, facilitates astrocyte adhesion and their spreading, forming neural-like tissue.

1. Synthesis of gelatin methacryloyl (GelMA)

	Weigh 10 g of gelatin obtained from porcine skin and dissolve in 100 mL of PBS by letting the solution stir ...