Name: microgel-extracellular matrix composite support for the embedded 3d printing of human neural constructs
Uploaded: 2023-05-05T15:00:00
Description: Watch this Scientific Journal Video about Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs at JoVE.com

&#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)...

<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 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 ...

Embedded 3D Printing within SHAPE composites overcomes traditional biofabrication limitations to enable accurate patterning of mechanically-sensitive tissues inside hydrogels that resemble the native extracellular environment. SHAPE toolbox relies on modular biomaterial design, allowing easy changes in printing support formulation. It also leverages low cost materials and accessible equipment for simple adaptation by other research groups.This approach can be applied to create spatially-defined human neural tissue models for examining neuronal development and communication in neurodegenerative disorders such as Parkinson's. Begin by preparing calcium carbonate solution in ultrapure water at two milligrams per milliliter concentration. Mix it with the alginate solution at an equal ratio and magnetically stir it at 650 RPM for one hour at room temperature.Then add acetic acid to this mixture at a ratio of one to 500 and stir again overnight. The next day, mechanically fragment the jelled alginate solution into microparticles at 15, 000 RPM for 10 minutes using a homogenizer. Then centrifuge to obtain the microparticles.Carefully discard the supernatant and resuspend the particles in the DMEM, containing two millimolar sodium hydroxide and 1%penicillin streptomycin. The color of the suspension should change back to red. Incubate the suspension overnight at four degrees Celsius.After incubation, homogenize the suspension for three minutes at 15, 000 RPM and centrifuge at 18, 500 G for 10 minutes. After centrifuging, remove the supernatant and observe the pellet for tightly-packed alginate microparticles. To generate the SHAPE composite, mix the alginate microparticles pellet with diluted and neutralized collagen at a ratio of two to one and pipette it up and down on ice.Transfer the generated composite material into a cooled 24 well plate. Before printing, dissociate the previously cultured human neural stem cells or hNSCs using a 0.025%trypsin solution for five minutes at 37 degrees Celsius. Then neutralize the trypsin with the growth medium and centrifuge the cell suspension at 400 G for five minutes at room temperature.After removing the supernatant, resuspend the cell pellet in two to three milliliters of growth medium. Using a 21 gauge blunt metal needle, load 100 microliters of tightly-packed alginate microparticles into the syringe. Then load the prepared cell suspension into a syringe.For printing, replace the loading needle with a 27 gauge blunt metal needle. Using referenced software, once the structure to print is designed, click on generate for a G code generation. Insert the cell loaded syringe into a volumetric extrusion head on an extrusion-based bioprinter and record the needle length by clicking on start needle length measuring process.Then place the 24 well plate loaded with SHAPE composite onto the printer, and click on SHM to measure the empty well surface height. Select pH 2 followed by wrench symbol. Then under the parameter set, change the volume flow rate to 3.6 microliters per second and press Apply Parameter, followed by Save and Done.Load the G code into the printer user interface by clicking an icon for open G code. Select the icon for the run program to initiate the print procedure. Immediately after printing, incubate the SHAPE gel at 37 degrees Celsius for 30 minutes for annealing.Then gently add growth medium to the annealed SHAPE gel support. For immunostaining, remove the excess medium from the gel, and using a spatula, transfer the gel to a container containing DPBS. Once the plate is placed in the fume hood, remove the DPBS.Cover the gel with 4%formaldehyde solution and incubate for one hour at room temperature. After washing with DPBS, add the blocking solution to the gel. Rock the plate gently and incubate for six hours at room temperature to prevent unspecific binding.Once the blocking solution is removed, add the primary antibody to the gel and gently rock it. Incubate the plate for 48 hours at four degrees Celsius. After DPBS wash, similarly treat the gel with the secondary antibody solution for 24 hours.Place the stained gel with a spatula into a well plate with a thin imaging bottom before imaging. In the present study, the hNSC ink printed a filament of cells with a diameter of around 200 micrometers. The printed strands were rich with viable cells with around morphology.After 30 days of printing, the cells exhibited neural morphology with small cell bodies and long thin processes, which indicated the successful differentiation of hNSCs. Staining with tubulin beta three allowed visualization of the generated neural networks with maintained geometry. And during the differentiation process, the cells did not migrate out of the printed strands.SHAPE composite can be used to produce perfusible channels by printing sacrificial ink instead of cells. Moreover, the support can include oxygen sensitive beads to monitor oxygen tension in 3D printed structures over time and space.

Research

JoVE Journal

Bioengineering

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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 ...

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 ...

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) ...

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 ...

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 ...

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 ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...