Name: Video: Generating Plate Map and Coating of 2D Control on Plastic Wells for 3D Bioprinting of Human iPSC-Derived Cells
Uploaded: 2023-09-29T12:00:00
Duration: 1 min 48 s
Description: 298 Views. Generating Plate Map and Coating of 2D Control on Plastic Wells for 3D Bioprinting of Human iPSC-Derived Cells

generating plate map and coating of 2d control on plastic wells for 3d bioprinting of human ipsc-derived cells

3D Bioprinted Cocultures of iPSC-Derived Neurons and Astrocytes

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/files/ftp_upload/65856/65856fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

Watch this Scientific Journal Video about Generating Plate Map and Coating of 2D Control on Plastic Wells for 3D Bioprinting of Human iPSC-Derived Cells at JoVE.com

Generating Plate Map and Coating of 2D Control on Plastic Wells for 3D Bioprinting of Human iPSC-Derived Cells  

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Research

JoVE Journal

Neuroscience

298 Views. Generating Plate Map and Coating of 2D Control on Plastic Wells for 3D Bioprinting of Human iPSC-Derived Cells

Video: Generating Plate Map and Coating of 2D Control on Plastic Wells for 3D Bioprinting of Human iPSC-Derived Cells  

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.

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.

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