&#65279;The research was primarily funded by the BrainMatTrain European Union Horizon 2020 Programme (No. H2020-MSCA-ITN-2015) under the Marie Sk&#322;odowska- Curie Initial Training Network and Grant Agreement No. 676408. C.R. and J.U.L. would like to gratefully acknowledge the Lundbeck Foundation (R250-2017-1425) and the Independent Research Fund Denmark (8048-00050) for their support.&#160;We gratefully acknowledge funding for the HORIZON-EIC-2021-PATHFINDEROPEN-01 Project 101047177 OpenMIND.

1. Preparation of the buffers and reagents
<ol>
	<li>Prepare cell growth medium by adding the following supplements to DMEM/F12 with L-alanyl-L-glutamine dipeptide: 30 mM glucose, 5 &#181;M HEPES, 0.5% w/v lipid-rich bovine serum albumine, 40 &#181;M L-alanine, 40 &#181;M L-asparagine monohydrate, 40 &#181;M L-aspartic acid, 40 &#181;M L-glutamic acid, 40 &#181;M L-proline, 1% N2 supplement, 1% penicillin-streptomycin, and 20 ng/L each of epidermal growth factor (EGF) and fibroblast growth factor (FGF). Perform these steps in a laminar air-flow (LAF) bench.</li>
	<li>Prepare 1% w/v alginate solution in ultrapure water by stirring vigorously for 4 h at 60 &#176;C. Sterile-filter the dissolved, hot alginate solution with a 0.45 &#181;m pore-size filter in a&#160;LAF bench. Store it at 4 &#176;C. 
	NOTE: If the temperature of the alginate solution is below 60 &#176;C, it will not be possible to pass it through the filter.</li>
	<li>Prepare a 37 g/L NaHCO3 solution in ultrapure water. Adjust the pH of the solution to 9.5 using NaOH, and filter-sterilize in a&#160;LAF bench. Store the solution at 4 &#176;C.</li>
</ol>
2. SHAPE composite material preparation
<ol>
	<li>Alginate microparticle generation
	<ol>
		<li>Prepare a 2 mg/mL CaCO3 solution in ultrapure water in a sterile beaker. Mix it 1:1 with the alginate solution, and stir for 1 h at room temperature using a magnetic stirrer.</li>
		<li>Add acetic acid in a 1:500 ratio, and leave stirring overnight at 650 rpm. 
		NOTE: The alginate solution will start gelling immediately. Make sure to use a stirring magnet with the same length as the diameter of the glassware used to ensure optimal, homogeneous stirring of the forming gel. The next day, the solution generated via stirring during gelation will appear viscous (Figure 2A).</li>
		<li>Mechanically fragment the gelled alginate solution into microparticles by homogenizing it at 15,000 rpm for 10 min with a homogenizer placed in a LAF bench (Figure 2B).</li>
		<li>Centrifuge the microparticles at 18,500 &#215; g for 20 min (Figure 2C) at room temperature.</li>
		<li>Carefully discard the supernatant inside the LAF bench, resuspend the particles in DMEM containing 2 mM NaOH and 1% P/S, and incubate overnight at 4 &#176;C (Figure 2D). 
		NOTE: The color of the suspension should go back to red. If the suspension stays yellow, add NaOH dropwise, and mix until it turns red (Figure 2E).</li>
		<li>Homogenize the particles at 15,000 rpm for 3 min, and centrifuge at 18,500 &#215; g for 10 min at room temperature.</li>
		<li>Observe the pellet (Figure 2F), and remove the supernatant carefully. 
		NOTE: The pellet of tightly packed alginate microparticles can be stored at 4 &#176;C until further use. If the alginate solution was not filter-sterilized, the microparticles should be used up within 1 week.</li>
	</ol>
	</li>
	<li>SHAPE composite formulation
	<ol>
		<li>The day before printing, resuspend the alginate microparticle pellet in twice the volume of growth medium containing 4% HEPES (1 M stock) and 4% NaHCO3 solution in a&#160;LAF bench, and incubate it overnight at room temperature.</li>
		<li>Centrifuge the microgel suspension at 18,500 &#215; g for 10 min at room temperature, and discard the supernatant.</li>
		<li>Neutralize the collagen to be mixed with the alginate microparticles in a LAF bench. Dilute the collagen stock solution to reach a final concentration of 1 mg/mL, and neutralize it by adding 4% HEPES and 4% NaHCO3. For example, for 3 mL of SHAPE composite, mix 2 mL of alginate microparticles with 1 mL of neutralized collagen (0.12 mL of HEPES, 0.12 mL of NaHCO3, 0.16 mL of growth medium, and 0.6 mL of collagen). 
		NOTE: All the materials being mixed with collagen need to be handled on ice to avoid collagen polymerization. As soon as the collagen is neutralized, polymerization will slowly start.</li>
		<li>Generate the SHAPE composite by mixing the alginate microparticle pellet with the diluted and neutralized collagen in a 2:1 ratio. Mix the gel thoroughly by pipetting slowly up and down on ice in a&#160;LAF bench. 
		NOTE: Do not vortex, as this will introduce bubbles into the support material.</li>
		<li>In a LAF bench, transfer the generated composite material into a cooled microwell plate or any&#160;suitable printing container, and use within 30 min (Figure 3E, F).</li>
	</ol>
	</li>
</ol>
3. hNSC culture and bioink preparation
<ol>
	<li>Culture the cells in a basement membrane extract-coated T75 flask in growth medium. Passage the cells at least two times after thawing before using them for a 3D printing experiment.</li>
	<li>Before printing, dissociate the cells enzymatically using a 0.025% trypsin solution for 5 min at 37 &#176;C, neutralize the trypsin with growth medium, and centrifuge the cell suspension at 400 &#215; g for 5 min at room temperature. Following the centrifugation, aspirate the supernatant, and resuspend the pellet in 2-3 mL of growth medium.</li>
	<li>Perform a cell count, centrifuge the cells at 400 &#215; g, and resuspend the pellet in growth medium supplemented with 0.1% xanthan gum (to prevent the cells from sedimenting) at a final concentration of 9 &#215; 106 cells/mL.</li>
	<li>Use a 21 G blunt metal needle to load 100 &#181;L of tightly packed alginate microparticles into a syringe (Figure 3A). 
	NOTE: Creating a plug with the alginate microparticles serves two purposes: it helps maintain extrusion stability during printing and allows for the complete extrusion of the loaded cellular ink (no dead volume).</li>
	<li>Using the same needle, load the prepared cell suspension into the syringe (Figure 3B). 
	NOTE: Take extra care not to introduce any air bubbles during the syringe-loading steps. Air bubbles will cause instabilities during printing.</li>
	<li>Replace the loading needle with a 27 G blunt metal needle, which will be used for printing (Figure 3C).</li>
</ol>
4. Embedded 3D printing
<ol>
	<li>Design a structure to print using the referenced software (see the Table of Materials). 
	NOTE: Make sure to adjust the initial height of the printed structure to within the depth of the SHAPE composite support. Design suggestions can be found in Figure 4A (spiral and woodpile designs).</li>
	<li>Generate a G-Code by clicking on Generate.</li>
	<li>Insert the cell-laden glass syringe into a volumetric extrusion head on an extrusion-based bioprinter (Figure 3D).</li>
	<li>Measure the needle length of the cell-laden glass syringe by clicking on Needle Length Measurement.</li>
	<li>Place the microwell plate or container loaded with the SHAPE composite onto the printer. 
	NOTE: Keep the cell plate or container at 4 &#176;C until printing to prevent the premature crosslinking of the support.</li>
	<li>Measure the surface height of an empty well within the same microwell plate or container in which the SHAPE gel is loaded by clicking on SHM (surface height measurement). 
	NOTE: Alternatively, determine the surface height manually by measuring the height of the well with the needle.</li>
	<li>Set the extrusion rate to 3.6 &#181;L/min and the feed rate to 0.3 mm/s. 
	NOTE: Make sure to test the extrusion before printing. Cell sedimentation can cause nozzle clogging and can be avoided by pre-extruding a small volume before the actual embedded printing or by adding a retraction volume to the volumetric syringe. In some cases, the feed rate needs to be kept below 0.5 mm/s when the needle is being inserted into or exiting the SHAPE gel to avoid the dragging of the ink.</li>
	<li>Load the G-Code into the user interface of the printer. 
	NOTE: A new G-Code needs to be generated every time changes are made to the designed structure.</li>
	<li>Initiate the print procedure by clicking on Run (Figure 3G).</li>
	<li>Immediately after printing, place the SHAPE gel at 37 &#176;C in a cell culture incubator for 30 min for annealing.</li>
	<li>Add growth medium gently on top of the annealed SHAPE gel support.</li>
	<li>The next day, replace the growth medium with differentiation medium formulated as follows: DMEM/F12 with L-alanyl-L-glutamine dipeptide with 30 mM glucose, 5 &#181;M HEPES, 0.5% w/v lipid-rich bovine serum albumine, 40 &#181;M L-alanine, 40 &#181;M L-asparagine monohydrate, 40 &#181;M L-aspartic acid, 40 &#181;M L-glutamic acid, 40 &#181;M L-proline, 1% N2 supplement, 1% penicillin-streptomycin, 100 &#181;M dibutyryl-cyclic adenosine monophosphate (dibutyryl-cAMP), and 2 ng/mL glial cell-line derived neurotrophic factor (GDNF). 
	NOTE: Make sure not to damage the hydrogel during the medium changes. Tilt the plate, and remove the old medium gently. Add fresh medium dropwise to the wall of the well containing the gel rather than directly on top of the gel. Do not use an aspirator to remove the medium.</li>
	<li>Refresh the differentiation medium every 2 days until the experimental endpoint.</li>
</ol>
5. Live-cell fluorescence imaging
<ol>
	<li>Remove excess medium from the gel.</li>
	<li>Add an equal volume of 20 &#181;M Calcein AM (diluted in differentiation medium from the stock solution) to the volume of the support gel.</li>
	<li>Incubate for 40 min at 37 &#176;C.</li>
	<li>Remove the Calcein AM solution, and add an appropriate volume of fresh differentiation medium.</li>
	<li>Transfer the plate to the microscope for imaging.</li>
</ol>
6. Immunocytochemistry
<ol>
	<li>Remove the excess medium from the gel.</li>
	<li>Using a small spatula, transfer the gel to a larger container containing DPBS.</li>
	<li>Wash three times for 20 min each time with DPBS, and transfer the plate to a fume hood.</li>
	<li>Remove the DPBS, add enough 4% formaldehyde solution to cover the gel, and incubate for 1 h at room temperature.</li>
	<li>Wash three times with DPBS for 20 min each time.</li>
	<li>Prepare blocking solution consisting of 5% donkey serum, 0.25% detergent, and 0.02% sodium azide in DPBS. 
	NOTE: Prepare three times the volume needed to cover the gel; it will be used later as the basis for the primary and secondary antibody solutions.</li>
	<li>After washing with DPBS, add the blocking solution to the gel, and incubate for 6 h at room temperature to prevent unspecific binding. Rock the plate gently.</li>
	<li>Prepare the primary antibody solution by diluting the TUBB3 antibody in blocking solution at a ratio of 1:1,000.</li>
	<li>Remove the blocking solution from the gel, add the primary antibody solution, and incubate for 48 h at 4 &#176;C. Rock the plate gently.</li>
	<li>Wash three times with DPBS for 20 min each time.</li>
	<li>Prepare the secondary antibody solution by diluting 4&#39;,6-diamidino-2-phenylindole (DAPI, 1:1,000) and the secondary antibody (1:200) in blocking solution.</li>
	<li>After washing with DPBS, incubate the gel in the secondary antibody solution for 24 h at 4 &#176;C. Rock the plate gently.</li>
	<li>Wash three times with DPBS for 20 min each time, and store at 4 &#176;C until imaging.</li>
	<li>Before imaging, transfer the stained gel with a spatula to a dish or a well-plate with a thin imaging bottom.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printing+in+suspension+baths:+Keeping+the+promises+of+bioprinting+afloat.">3D printing in suspension baths: Keeping the promises of bioprinting afloat.</a> Trends in Biotechnology. 38 (6), 584-593 (2020).</li><li>Kajtez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embedded+3D+printing+in+self-healing+annealable+composites+for+precise+patterning+of+functionally+mature+human+neural+constructs.">Embedded 3D printing in self-healing annealable composites for precise patterning of functionally mature human neural constructs.</a> Advanced Science. 9 (25), 2201392 (2022).</li><li>Rommel, D., Vedaraman, S., Mork, M., de Laporte, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interlinked+macroporous+3D+scaffolds+from+microgel+rods.">Interlinked macroporous 3D scaffolds from microgel rods.</a> Journal of Visualized Experiments. 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The authors declare no conflicts of interest.

The SHAPE composite material approach provides a versatile route for the formulation of annealable and biofunctional support baths for the embedded 3D printing of cellular inks. While this protocol provides an example of the 3D printing of neural constructs, the SHAPE toolbox could easily be adapted to biofabrication with other cell sources for the precise engineering of a range of target tissue types. The printing approach would also allow for the precise patterning of multiple cell types to study their interaction or t...

The precise and programmable 3D printing of cell-laden hydrogel constructs that mimic soft tissues in vitro presents a major challenge. For instance, attempts based on the direct extrusion of soft hydrogels are inherently problematic, as the poor mechanical properties required to recapitulate the in vivo microenvironment lead to a lack of structural integrity, deformations of the predefined features, or the complete collapse of the fabricated structures. A conventional workaround for this issue is to print a supporting scaffold from a stiffer biocompatible material that allows the final construct to maintain its shape. However, this approach greatly limits the design possibilities and requires careful rheological fine-tuning of the adjacent inks.
To overcome the limitations of the traditional layer-by-layer extrusion-based 3D printing, embedded 3D printing has emerged in recent years as a powerful alternative for soft material and tissue fabrication1,2,3,4,5,6. Instead of extruding the ink in ambient air on top of a surface, the ink is directly deposited through a syringe needle inside a support bath that is solid-like at rest but reversibly fluidizes around the moving needle tip to allow the precise deposition of soft cell-laden material. The deposited material is kept in place as the support resolidifies in the wake of the needle. As such, embedded 3D printing allows for the high-resolution freeform fabrication of intricate structures from soft biomaterials with expanded design possibilities7,8.
Granular gels have been extensively explored as support bath materials for embedded 3D printing, since they can be formulated to exhibit smooth, localized, and reversible solid-to-liquid transitions at low yield stresses9,10,11. While they show excellent rheological properties for high-resolution printing, granular gels have been restricted to a handful of biomaterials12. The lack of diversity in granular gel formulations, which is particularly evident if one considers the wide range of biomaterials available for bulk hydrogel formulations, is caused by the need for the cost-effective generation of a large number of microgels using simple chemistries. Due to the limited biomaterial landscape of granular gel supports, the tuning of the extracellular microenvironment provided by the printing support presents a challenge in the field.
Recently, a modular approach has been developed for the generation of embedded 3D printing supports, termed self-healing annealable particle-extracellular matrix (SHAPE) composites13. This approach combines the distinct rheological properties of granular gels with the biofunctional versatility of bulk hydrogel formulations. The presented SHAPE composite support consists of packed alginate microparticles (granular phase, ~70% volume fraction) with an increased interstitial space filled with a viscous collagen-based ECM pregel solution (continuous phase, ~30% volume fraction). It has further been shown that the SHAPE support facilitates the high-resolution deposition of human neural stem cells (hNSCs) that, after the annealing of the support bath, can be differentiated into neurons and maintained for weeks to reach functional maturation. Embedded 3D printing inside the SHAPE support bath overcomes some of the major limitations related to conventional techniques for neural tissue biofabrication while providing a versatile platform.
This work details the steps for the embedded 3D printing of hNSCs inside the SHAPE support and their subsequent differentiation into functional neurons (Figure 1). First, alginate microparticles are generated via shearing during internal gelation. This approach allows the easy generation of large volumes of microparticles without the need for specialized equipment and cytotoxic reagents. Furthermore, alginate is a widely available and economical material source for the formation of biocompatible hydrogel substrates for a diverse range of cell types. The generated alginate microparticles are combined with a collagen solution to form the SHAPE composite support material. Then, the hNSCs are harvested and loaded into a syringe as a cellular bioink for 3D printing. A 3D bioprinter is used for the extrusion-based embedded printing of hNSCs inside the SHAPE composite. The 3D-printed cells are differentiated into neurons to give rise to spatially defined and functional human neural constructs. Finally, the protocol describes how the generated tissue constructs can be characterized using live-cell imaging and immunocytochemistry. Additionally, tips for optimization and troubleshooting are provided. Notably, both the components of the granular and continuous phases could be exchanged with other hydrogel formulations to accommodate different biofunctional moieties, mechanical properties, and crosslinking mechanisms, as required by other cell and tissue types beyond neural applications.

<table><tbody><tr><td>1 mL Gastight Syringe 1001 TLL</td><td>Hamilton</td><td>81320</td><td></td></tr><tr><td>3DDiscovery 3D bioprinter</td><td>RegenHU</td><td></td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>AlbuMAX</td><td>ThermoFisher</td><td>11020021</td><td></td></tr><tr><td>Alexa Fluor 488 secondary antibody</td><td>ThermoFisher</td><td>A-11001</td><td>Goat anti-Mouse</td></tr><tr><td>Blunt Needle, Sterican (21 G)</td><td>Braun</td><td>9180109</td><td></td></tr><tr><td>Blunt Needle (27 G)</td><td>Cellink</td><td>NZ5270505001</td><td></td></tr><tr><td>BioCAD software</td><td>SolidWorks</td><td></td><td></td></tr><tr><td>Calcein AM</td><td>ThermoFisher</td><td>65-0853-39</td><td></td></tr><tr><td>Calcium carbonate</td><td>Sigma-Aldrich</td><td>C5929</td><td></td></tr><tr><td>Dibutyryl-cAMP sodium salt</td><td>Sigma-Aldrich</td><td>D0627</td><td></td></tr><tr><td>Cultrex Rat Collagen I (5 mg/mL)</td><td>R&amp;amp;D Systems</td><td>3440-100-01</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher</td><td>62248</td><td></td></tr><tr><td>DMEM/F-12, GlutaMAX</td><td>ThermoFisher</td><td>10565018</td><td></td></tr><tr><td>Donkey serum</td><td>Sigma-Aldrich</td><td>D9663</td><td></td></tr><tr><td>DPBS</td><td>ThermoFisher</td><td>14190094</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D Systems</td><td>236-EG</td><td></td></tr><tr><td>FGF</td><td>R&amp;amp;D Systems</td><td>3718-FB</td><td></td></tr><tr><td>Formaldehyde solution 4%, buffered, pH 6.9</td><td>Sigma-Aldrich</td><td>100496</td><td></td></tr><tr><td>GDNF</td><td>R&amp;amp;D Systems</td><td>212-GD</td><td></td></tr><tr><td>Geltrex</td><td>ThermoFisher</td><td>A1569601</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>HEPES Buffer (1 M)</td><td>ThermoFisher</td><td>15630080</td><td></td></tr><tr><td>L-Alanine</td><td>Sigma-Aldrich</td><td>5129</td><td></td></tr><tr><td>L-Asparagine monohydrate</td><td>Sigma-Aldrich</td><td>A4284</td><td></td></tr><tr><td>L-Aspartic acid</td><td>Sigma-Aldrich</td><td>A9256</td><td></td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>G1251</td><td></td></tr><tr><td>L-Proline</td><td>Sigma-Aldrich</td><td>P0380</td><td></td></tr><tr><td>Magnetic stirrer RET basic</td><td>IKA</td><td>3622000</td><td></td></tr><tr><td>N-2 Supplement</td><td>ThermoFisher</td><td>17502048</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher</td><td>15140122</td><td></td></tr><tr><td>S25N-10G dispersing tool</td><td>IKA</td><td>4447100</td><td></td></tr><tr><td>Sodium Alginate (80-120 cP)</td><td>FUJIFILM Wako</td><td>194-13321</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>T18 Digital ULTRA-TURAX homogenizer</td><td>IKA</td><td>3720000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Trypsin/EDTA Solution</td><td>ThermoFisher</td><td>R001100</td><td></td></tr><tr><td>TUBB3 antibody</td><td>BioLegend</td><td>801213</td><td>Mouse</td></tr><tr><td>Xanthan gum&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G1253</td><td></td></tr></tbody></table>

microgel-extracellular matrix composite support for the embedded 3d printing of human neural constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

Alginate microgel preparation via shear thinning during internal gelation followed by mechanical fragmentation yields alginate microgels that are polydispersed in size and flake-like in shape as seen in Figure 2G. The size of these irregular particles ranges from less than 1 &#181;m to approximately 40 &#181;m in diameter. When tightly packed, the microparticles form a transparent bulk material that is only slightly more opaque than the corresponding cell culture medium (<strong cla...

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Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

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Research

JoVE Journal

Bioengineering

1.3K Views.  University of Copenhagen. This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

Video: Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Cancer Research

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Neuroscience

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

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 ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

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Explore More Videos

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Embedded Bioprinting of Tissue-like Structures Using κ-Carrageenan Sub-Microgel Medium

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

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

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Interlinked Macroporous 3D Scaffolds from Microgel Rods