&#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>

<ol>
	<li>Wu, W., DeConinck, A., Lewis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omnidirectional+printing+of+3D+microvascular+networks.">Omnidirectional printing of 3D microvascular networks.</a> Advanced Materials. 23 (24), H178-H183 (2011).</li><li>Bhattacharjee, T., 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=Writing+in+the+granular+gel+medium.">Writing in the granular gel medium.</a> Science Advances. 1 (8), e1500655 (2015).</li><li>Hinton, T. 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=Three-dimensional+printing+of+complex+biological+structures+by+freeform+reversible+embedding+of+suspended+hydrogels.">Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.</a> Science Advances. 1 (9), e1500758 (2015).</li><li>Skylar-Scott, M. A., 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=Biomanufacturing+of+organ-specific+tissues+with+high+cellular+density+and+embedded+vascular+channels.">Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels.</a> Science Advances. 5 (9), (2019).</li><li>Highley, C. B., Rodell, C. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+3D+printing+of+shear-thinning+hydrogels+into+self-healing+hydrogels.">Direct 3D printing of shear-thinning hydrogels into self-healing hydrogels.</a> Advanced Materials. 27 (34), 5075-5079 (2015).</li><li>Romanazzo, S., 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=Synthetic+bone-like+structures+through+omnidirectional+ceramic+bioprinting+in+cell+suspensions.">Synthetic bone-like structures through omnidirectional ceramic bioprinting in cell suspensions.</a> Advanced Functional Materials. 31 (13), 2008216 (2021).</li><li>Noor, N., 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=3D+printing+of+personalized+thick+and+perfusable+cardiac+patches+and+hearts.">3D printing of personalized thick and perfusable cardiac patches and hearts.</a> Advanced Science. 6 (11), 1900344 (2019).</li><li>Lee, A., 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=3D+bioprinting+of+collagen+to+rebuild+components+of+the+human+heart.">3D bioprinting of collagen to rebuild components of the human heart.</a> Science. 365 (6452), 482-487 (2019).</li><li>LeBlanc, K. 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=Stability+of+high+speed+3D+printing+in+liquid-like+solids.">Stability of high speed 3D printing in liquid-like solids.</a> ACS Biomaterials Science and Engineering. 2 (10), 1796-1799 (2016).</li><li>Prendergast, M. E., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+modeling+and+experimental+characterization+of+extrusion+printing+into+suspension+baths.">Computational modeling and experimental characterization of extrusion printing into suspension baths.</a> Advanced Healthcare Materials. 11 (7), 2101679 (2022).</li><li>Shapira, A., Noor, N., Oved, H., Dvir, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+support+media+for+high+resolution+3D+printing+of+volumetric+cell-containing+ECM+structures.">Transparent support media for high resolution 3D printing of volumetric cell-containing ECM structures.</a> Biomedical Materials. 15 (4), 45018 (2020).</li><li>McCormack, A., Highley, C. B., Leslie, N. R., Melchels, F. P. 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. (184), e64010 (2022).</li><li>Griffin, D. R., Weaver, W. M., Scumpia, P. O., di Carlo, D., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+wound+healing+by+injectable+microporous+gel+scaffolds+assembled+from+annealed+building+blocks.">Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks.</a> Nature Materials. 14 (7), 737-744 (2015).</li><li>Mirdamadi, E., Muselimyan, N., Koti, P., Asfour, H., Sarvazyan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+slurry+as+a+support+medium+for+bioprinting+and+culturing+freestanding+cell-laden+hydrogel+constructs.">Agarose slurry as a support medium for bioprinting and culturing freestanding cell-laden hydrogel constructs.</a> 3D Printing and Additive Manufacturing. 6 (3), 158-164 (2019).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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&#38;D Systems</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>R&#38;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&#38;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&#160;</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...

Watch this Scientific Journal Video about Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs at JoVE.com

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 ...

microgel-extracellular-matrix-composite-support-for-embedded-3d

Research

JoVE Journal

Bioengineering

1.2K 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

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...