The authors acknowledge grants from the National Natural Science Foundation of China (81771971, 81970442, 81703470 and 81570422), National Key R&#38;D Program of China (2018YFC1005002), Science and Technology Commission of Shanghai Municipality (17JC1400200), Shanghai Municipal Science and Technology Major Project (Grant No. 2017SHZDZX01), and Shanghai Municipal Education Commission (Innovation Program 2017-01-07-00-07-E00027).

1. Preparation of cell-laden SF-GelMA
<ol>
	<li>Sterilize all the materials by using 0.22 &#956;m syringe filter units. Perform all the steps in a biological safety cabinet.</li>
	<li>Warm 1x PBS to 50 &#176;C, and dissolve gelatin in the heated 1x PBS with stirring. The final concentration of gelatin in PBS should be 10% (w/v).</li>
	<li>Add methacrylic anhydride into the gelatin solution (weight ratio of methacrylic anhydride to gelatin of 0.6 to 1) slowly with stirring, and mix the complex for at least 1 h (50 &#176...

<ol>
	<li>Khademhosseini, A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+progress+in+tissue+engineering.">A decade of progress in tissue engineering.</a> Nature Protocol. 11 (10), 1775-1781 (2016).</li><li>Heinrich, 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=3D+bioprinting:+from+benches+to+translational+applications.">3D bioprinting: from benches to translational applications.</a> Small. , 1805510 (2019).</li><li>Daniela, L., 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=Functionalization,+preparation+and+use+of+cell-laden+gelatin+methacryloyl-based+hydrogels+as+modular+tissue+culture+platforms.">Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms.</a> Nature Protocols. 11 (4), 727-746 (2016).</li><li>Pedde, R. D., 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=Emerging+biofabrication+strategies+for+engineering+complex+tissue+constructs.">Emerging biofabrication strategies for engineering complex tissue constructs.</a> Advanced Materials. 29 (19), (2017).</li><li>Holzl, K., Lin, S., Tytgat, L., Van Vlierberghe, S., Gu, L., Ovsianikov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioink+properties+before,+during+and+after+3D+bioprinting.">Bioink properties before, during and after 3D bioprinting.</a> Biofabrication. 8 (3), 032002 (2016).</li><li>Guillotin, B., Guillemot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+patterning+technologies+for+organotypic+tissue+fabrication.">Cell patterning technologies for organotypic tissue fabrication.</a> Trends in Biotechnology. 29 (4), 183-190 (2011).</li><li>Murphy, S. V., Atala, A. <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+tissues+and+organs.">3D bioprinting of tissues and organs.</a> Nature Biotechnology. 32, 773-785 (2014).</li><li>Dubbin, K., Hori, Y., Lewis, K. K., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-stage+crosslinking+of+a+gel-phase+bioink+improves+cell+viability+and+homogeneity+for+3D+bioprinting.">Dual-stage crosslinking of a gel-phase bioink improves cell viability and homogeneity for 3D bioprinting.</a> Advanced Healthcare Materials. 5 (19), 2488-2492 (2016).</li><li>Rutz, A. L., Hyland, K. E., Jakus, A. E., Burghardt, W. R., Shah, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multimaterial+bioink+method+for+3D+printing+tunable,+cell-compatible+hydrogels.">A multimaterial bioink method for 3D printing tunable, cell-compatible hydrogels.</a> Advanced Materials. 27 (9), 1607-1614 (2015).</li><li>Chahal, D., Ahmadi, A., Cheung, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+piezoelectric+cell+printing+accuracy+and+reliability+through+neutral+buoyancy+of+suspensions.">Improving piezoelectric cell printing accuracy and reliability through neutral buoyancy of suspensions.</a> Biotechnology and Bioengineering. 109 (11), 2932-2940 (2012).</li><li>Chen, 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=Hydrogel+bioink+with+multilayered+interfaces+improves+dispersibility+of+encapsulated+cells+in+extrusion+bioprinting.">Hydrogel bioink with multilayered interfaces improves dispersibility of encapsulated cells in extrusion bioprinting.</a> ACS Applied Materials &amp; Interfaces. 11, 30585-30595 (2019).</li><li>Zhu, K., 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=A+general+strategy+for+extrusion+bioprinting+of+bio-macromolecular+bioinks+through+alginate-templated+dual-stage+crosslinking.">A general strategy for extrusion bioprinting of bio-macromolecular bioinks through alginate-templated dual-stage crosslinking.</a> Macromolecular Bioscience. 18 (9), 1800127 (2018).</li><li>Nauman, J. V., Campbell, P. G., Lanni, F., Anderson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+insulin-like+growth+factor-I+and+ribonuclease+through+fibrin+gels.">Diffusion of insulin-like growth factor-I and ribonuclease through fibrin gels.</a> Biophysical Journal. 92 (12), 4444-4450 (2007).</li></ol>

The authors have nothing to disclose.

The stability of the multilayered system is a key point to perform this protocol successfully. We theoretically calculated the diffusion of SF molecules in the GelMA solution based on Nauman&#8217;s study13. It was found that the diffusion of proteins in solution was related to their molecular weight. The average molecular weight (MW) of bovine serum albumin (BSA) is 66.5 kDa, and its diffusion coefficient is 64-72 &#956;m2/s. The average MW of fibrinogen is 339.7 kDa, and its diffusion...

Three-dimensional (3D) bioprinting has been a promising method to manufacture complex architectural and functional replicas of native tissues in biofabrication and regenerative medicine1,2,3. The common strategies of bioprinting, including inkjet, extrusion, and stereolithography printing, have pros and cons from different perspectives4. Among these techniques, the extrusion procedure is most commonly used due to its cost-effectiveness. Bioink plays a key role in the process stability of extrusion bioprinting. The ideal cell-laden bioink should not only be biocompatible but also be suitable for mechanical properties5. Bioinks with low viscosity are typically presented as a liquid-like state. These bioinks can be easily and quickly deposited and avoid cell membrane damage induced by high shear stress during extrusion. However, in complex cases requiring long-term printing periods, low viscosity often gives rise to the inevitable sedimentation of the encapsulated cells in the bioink reservoir, which is usually driven by gravity and leads to an inhomogeneous cell dispersion in the bioink6,7. Consequently, a bioink with inhomogeneous cell dispersity hampers the in vitro bioprinting of a functional tissue construct.
Several recent studies focusing on bioinks have reported the promotion of homogenous dispersity of encapsulated cells. A modified alginate bioink based on dual-stage crosslinking was used for extrusion bioprinting8. An alginate polymer was modified with peptides and proteins in this study. Cells presented a more homogeneous distribution in this modified alginate than in the commonly used alginate due to the attachment sites provided by the peptides and the proteins. Alternatively, blended bioinks have been utilized to solve the sedimentation of cells in bioink. A blended bioink containing polyethylene glycol (PEG) and gelatin or gelatin methacryloyl (GelMA) with improved mechanical robustness was used in another study9. The encapsulated cells presented a homogeneous distribution mainly because the viscosity of the blended bioink was improved. In general, there are several factors influencing the dispersity of the encapsulated cells in the bioink, such as the viscosity of the bioink, the gravity of the cells, the density of the cells, and the duration of the working period. Among these factors, the gravity of cells plays a critical role in promoting sedimentation. The buoyancy and friction provided by the viscous bioink have been investigated as the main forces against gravity to date10.
Herein, we developed a novel strategy to promote homogeneous dispersity of the encapsulated cells in bioink by manipulating multiple liquid interfaces in the bioink reservoir. These liquid interfaces created by the multilayered modification of bioink can not only provide interfacial retention, which retards the sedimentation of cells, but also maintain a suitable biocompatibility and rheological behavior of the bioink. In practice, we modified aqueous GelMA solution (5%, w/v) with silk fibroin (SF) in a multilayered manner to longitudinally produce four interfaces, providing interfacial tensions in the blended bioink. As a result, the gravity loading on the cells was offset by the man-made interfacial tension, and a nearly homogeneous dispersion of the encapsulated cells in the bioink was obtained due to less sedimentation across the adjacent layers of cells. No similar protocol to slow down the sedimentation of encapsulated cells by manipulating interfacial retention in liquid bioinks has been reported to date. We present our protocol here to demonstrate a new way to solve cell sedimentation in bioprinting.

<table>
	<tbody>
		<tr>
			<td>2-Hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone (PI2959)</td>
			<td>TCI</td>
			<td>M64BK-QD</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Gibco</td>
			<td>10569044</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10091</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>V900863MSDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacrylic anhydride (MA)</td>
			<td>Sigma-Aldrich</td>
			<td>276685MSDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin antibiotics</td>
			<td>Gibco</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk fibroin</td>
			<td>Advanced BioMatrix</td>
			<td>5154</td>
			<td></td>
		</tr>
	</tbody>
</table>

using multilayered hydrogel bioink in three-dimensional bioprinting for homogeneous cell distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

A schematic of the preparation of cell-laden bioinks is shown in Figure 1. After preparation of the different bioinks, loading, reheating and bioprinting were performed (Figure 2). To evaluate the distribution of the encapsulated cells in the bioink reservoir, a bioprinting procedure was performed using three different cell-laden bioinks in three 96-well plates (Figure 3A). Two control groups (pristine GelMA and SF-1-GelMA bioinks) ...

Watch this Scientific Journal Video about Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution at JoVE.com

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Using our protocol, the multilayering of the liquid-like bioink dilutions does not only promote a homogeneous dispersity of the encapsulated cells but also maintains a cell-suitable bioink environment. We use the interfacial tension to stop the sedimentation of the encapsulated cells within the bioink reservoir, avoiding the high shear stress load to the cells during extrusion printing. Using this technology, we can generate 3D tissue constructs with a homogeneous cell distribution, providing a functional in vitro tissue mold for use in drug screening and regeneration medicine studies.When preparing the bioink, it is important to pay attention to the experimental temperature and the hygrogel loading procedure, as these factors can affect the quality of the ink. Before beginning the preparation, use 0.22-micrometer syringe filter units to sterilize all of the materials, and dissolve gelatin to a 10%final concentration in 50-degrees-Celsius PBS in a biological safety cabinet. Add methacrylic anhydride to the gelatin solution at a 0.6 to one weight ratio with slow stirring for at least one hour at 50 degrees Celsius.At the end of the incubation, transfer the mixed solution into a sterile 50-milliliter tube, and separate the solution into layers by centrifugation. Collect the upper GelMA layer, and dilute the collected solution with two volumes of 40 to 50-degrees-Celsius deionized water. Dialyze the GelMA solution with a 12 to 14-kilodalton molecular weight cutoff dialysis membrane against deionized water for five to seven days at 40 to 50 degrees Celsius, changing the water twice daily.At the end of the dialysis, freeze the GelMA solution at minus 80 degrees Celsius overnight. Next lyophilize the GelMA solution for three to five days in a freeze dryer at minus 45 degrees Celsius and 0.2 millibars of pressure. Then dissolve the lyophilized GelMA in PBS supplemented with 10%fetal bovine serum, 25 millimolar HEPES, and 0.5%photoinitiator.To obtain the silk fibroin GelMA bioinks, mix the GelMA solution with different volumes of initial silk fibroin solution and different volumes of PBS as suggested in the table. When all of the bioinks have been prepared, sonicate the solution for 10 to 20 minutes. While the inks are sonicating, collect the cells from an 80%confluent NIH/3T3 cell culture into one 15-milliliter conical tube per bioink solution, and pellet the cells by centrifugation.Then carefully aspirate the supernatants and resuspend the cells in each tube with two milliliters of bioink at a one times 10 to the six cells per milliliter of bioink solution concentration. To load the bioink for printing, add aspirate 400 microliters of cell-laden silk fibroin 0.5 GelMA to the bottom layer of a two-milliliter syringe. Place the syringe in a zero-degrees-Celsius ice water bath for five minutes to transform the bottom layer bioink into a gel state before loading 400 microliters of cell-laden silk fibroin 0.75 GelMA bioink over the layer of silk fibroin 0.5 GelMA.Then return the syringe to the ice bath for five minutes, continuing to add each additional concentration of cell-laden bioink to the syringe in the same manner. When a multilayered bioink system with different concentrations of silk fibroin within the different layers has been achieved, reheat the bioink in a 37-degrees-Celsius incubator for 30 minutes. At the end of the incubation, load a 27-gauge printing nozzle onto the syringe, and set the flow speed to 50 microliters per minute, the moving speed of the nozzle to two milliliters per second, and the height of the nozzle to one millimeter.Next print the tissue construct in an extrusion manner using a custom-made bioprinter at room temperature, and cross-link the bioprinted tissue construct with 365 nanometers of ultraviolet light for 40 seconds at 800 milliwatts. Then culture the cross-linked tissue construct in DMEM supplemented with 10%fetal bovine serum and 1%penicillin-streptomycin in a cell culture incubator, changing the medium every eight hours during the first two days and every two to three days thereafter. In this representative analysis, as the bioprinting procedure progressed, the density of cells in the target wells decreased in all of the groups.In the silk fibroin zero GelMA group, approximately 70%of the cells were deposited in the bottom layer. In the silk fibroin one GelMA group, approximately 40%of the cells were deposited in the bottom layer, and approximately 5%of the cells were deposited in the top layer. In the silk fibroin multilayer GelMA group, the deposition of encapsulated cells was more homogenous than that of the control groups.The most critical part of this protocol is the loading of the SF-GelMA bioink multilayers. This protocol may be applied to other extrusion bioprinting analysis that use liquid-like bioinks.

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6.4K visualizações.  Fudan University. Usando nosso protocolo, a multicamadas das diluições de bioink semelhantes a líquidos não só promove uma dispersão homogênea das células encapsuladas, mas também mantém um ambiente de bioink adequado para células. Utilizamos a tensão interfacial para impedir a sedimentação das células encapsuladas dentro do reservatório de bioink, evitando a alta carga de estresse de tesoura para as células durante a impressão de extrusão. Usando essa tecnologia, podemos gerar construções...