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

תרביות ביולוגיות תלת-ממדיות של נוירונים ואסטרוציטים שמקורם ב-iPSC

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>

מחבר Spotlight: Advanceancing 3D Cell Modeling – גישה בעלת תפוקה גבוהה עבור תרבויות קו-תרבותיות עצביות 

הדפסה ביולוגית תלת-ממדית של תרביות נוירון-אסטרוציטים אנושיות שמקורן ב-iPSC עבור יישומי סינון

3D cell modeling is a novel field that has exponentially expanded in the past decade. These models have been shown to both facilitate neuronal growth and more accurately represent disease phenotypes. However, we believe there is a shift towards making these models higher throughput and a necessity to embrace automation within development.Traditional methods of developing 3D cultures can be laborious and time-consuming to establish, but 3D bioprinting is a technology that can be applied to scale up these development processes. This technology allows for hundreds of identical models to be created efficiently and without human error. This protocol develops complex cultures because the neural cells are grown in 3D in biologically active hydrogel matrices.But critically, this protocol prioritizes speed and convenience in model development, which can be lacking in this field and can hinder the implementation into industry. This protocol defines a method to establish many 3D cocultures very efficiently with limited input from users. We hope this will remove barriers to using complex cell culture models within high throughput assays and facilitate further investigation of the effect of 3D culture on neural cell types.

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  

יצירת מפת לוחות וציפוי של בקרה דו-ממדית על בארות פלסטיק להדפסה ביולוגית תלת-ממדית של תאים אנושיים שמקורם ב-iPSC

כדי להתחיל, פתח את תוכנת מפת הלוחות ובחר את תאי התרבית באפשרות התלת-ממד. לאחר מכן, בחר מודל הדמיה עבור 96 לוחות באר. בחלון בחירת המטריצה, בחר HydroGel PX-2.53.הזן את סוגי התאים כמו נוירונים גלוטמטרגיים אסטרוציטים, ואת צפיפות התא כמו 20 מיליון תאים למיליליטר. עצבו את מפת הלוחות הרצויה על ידי הדגשת בארות על הצלחת. כלול לפחות שורה אחת של בקרה דו-ממדית על פלסטיק בעיצוב.לאחר מכן, ודא שדגם הלוח המופיע בחלק העליון של החלון השתנה לדגם הלוח המיועד לשימוש. באמצעות כפתורי הורדה, הורד את הפרוטוקול והדפס קובץ עבור מפת הלוחות. לאחר מכן, שמור אותם במחשב המקושר למדפסת ביולוגית.הכינו תמיסת פוליאתילנימין 0.1% בחיץ בוראט וסננו אותה דרך מסנן של 0.22 מיקרון. הוסיפו 100 מיקרוליטר של תמיסת פוליאתילנימין 0.1% לכל באר בקרה דו-ממדית של צלחת בעלת 96 בארות, ודגרו בחום של 37 מעלות צלזיוס למשך שעתיים. לאחר מכן, שטפו את הבארות חמש פעמים עם PBS.לאחר ייבוש הבארות, מוסיפים תמיסת למינין מדוללת לבארות ודגרים בחום של 37 מעלות צלזיוס למשך ארבע שעות.

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Research

JoVE Journal

Neuroscience

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

293 Views.  MSD Research Laboratories, London, UK. כדי להתחיל, פתח את תוכנת מפת הלוחות ובחר את תאי התרבית באפשרות התלת-ממד. לאחר מכן, בחר מודל הדמיה עבור 96 לוחות באר. בחלון בחירת המטריצה, בחר HydroGel PX-2.53.הזן את סוגי התאים כמו נוירונים גלוטמטרגיים אסטרוציטים, ואת צפיפות התא כמו 20 מיליון תאים למיליליטר. עצבו את מפת הלוחות הרצו?...

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

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.

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 ...

To begin open the plate map software and select the culture cells in the 3D option. Then, select imaging model for 96 well plates. In the matrix selection window, select HydroGel PX-2.53.Enter the cell types as glutamatergic neurons and astrocytes, and cell density as 20 million cells per milliliter. Design the desired plate map by highlighting wells on the plate. Include at least one row of 2D control on plastic in the design.Then, verify that the plate model listed at the top of the window is changed to the intended plate model for use. Using the Download buttons, download the protocol and print file for the plate map. Then, save them to the bio printer-linked computer.Prepare a 0.1%polyethylenimine solution in a borate buffer and filter it through a 0.22-micron filter. Add 100 microliters of 0.1%polyethylenimine solution to each 2D control well of a 96-well plate, and incubate at 37 degrees Celsius for two hours. Then, rinse the wells five times with PBS.Once the wells are dried, add diluted laminin solution into the wells and incubate at 37 degrees Celsius for four hours.

3D Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures

To begin, maintain sterility by cleaning gloves, cartridges, and culture plates with 70%ethanol. Next, switch on the compressor for the bioprinter and select the initialized button to initialize the printer. Wipe down surfaces inside the printer with 70%ethanol.Using the Print Run button, load the print file and open the Print Protocol PDF. Thaw bioink, activator fluids, and 50 milliliters of complete media at room temperature. Prepare the printing cartridge as instructed in the Print Protocol PDF, ensuring the correct volumes of reagents in specified compartments.Next, add 1.2 milliliters of F3 to C1, 1.2 milliliters of F32 to C2, and 200 microliters of F261 to C4.Remove the polyethylenimine and laminin coated plate from the incubator. Set the plate holder compartment to a low profile plate. Insert the plate in the right hand plate holder compartment of the printer.Remove the lid from the plate and place it in the lid holder. Place the print cartridge into the bioprinter and select the Print Inert Base button to start the print run. After thawing, centrifuge the glutamatergic neurons and astrocytes, aspirate the supernatant and resuspend both cell types separately in one milliliter of complete media.Mix 20 microliters of the cell suspension with the same volume of trypan blue and count the cells to determine viable cell concentration for each cell type. Next, combine 3 million glutamatergic neurons with 1 million astrocytes in a 15 milliliter tube, and add complete media to make a final volume of eight milliliters. Centrifuge the cell suspension and aspirate the supernatant without disturbing the pellet.Suspend the pellet in 200 microliters of activator fluid F299 by pipetting up and down. Place the lids on the cartridge and culture plate, and remove them from the bioprinter. In a biosafety cabinet, add 200 microliters of cell suspension to well C3 of the print cartridge.Reinsert the cartridge into the bioprinter and remove the lid as demonstrated previously. Initiate the print models stage of the print run. Concurrently, replace the laminate media from the 2D control plate with complete media.Insert the bioprinting targeting plate into the bioprinter and remove the lid. Begin the bioprint targeting process. When prompted, remove the targeting plate from the bioprinter.Use the guide to identify locations where droplets can be observed on the plate. Reinsert the targeting plate into the bioprinter to repeat the droplet printing and selection process. When prompted, replace the targeting plate with a cell culture plate.After printing, add complete media to all 3D co-culture wells, and incubate at 37 degrees Celsius and 5%carbon dioxide. Finally, dispose of cartridges and remaining liquids. according to lab protocols.After seven days of printing, almost no single cells remained and interconnecting bundles of neurites and astrocytic projections appeared fortified. Neurite outgrowth increased linearly between 12 to 156 hours. During neurite outgrowth, cell body clusters also increased in size.Cell viability analysis showed that approximately 72%of total cells were live on day four while 29%were dead. Immunostaining confirmed the healthy cell morphology of the bioprinted neurons and astrocytes with both cell types exhibiting outgrowth of cellular protrusions. The glutamatergic phenotype of the neurons was confirmed through immunostaining for glutamate receptor 2.