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;C). Typically, prepare 200 mL of 10% gelatin solution with 12 g of methacrylic anhydride; the volume depends on the needs of the study.</li>
	<li>Transfer the mixed solution containing gelatin and methacrylic anhydride to a 50 mL sterile tube.</li>
	<li>Centrifuge the mixed solution at 3,500 x g. It usually takes 3-5 min to obtain two layers. Collect the upper layer (GelMA) and discard the bottom layer (unreacted methacrylic anhydride).</li>
	<li>Dilute the upper layer solution obtained in step 1.5 with two volumes of deionized water (40&#8722;50 &#176;C).</li>
	<li>Dialyze the solution obtained in step 1.6 with a 12-14 kDa molecular weight cutoff dialysis membrane against deionized water for 5&#8722;7 days (40&#8722;50 &#176;C). Change the water twice every day.</li>
	<li>Collect and freeze the GelMA solution at &#8722;80 &#176;C overnight.</li>
	<li>Lyophilize the GelMA solution for 3&#8722;5 days in a freeze dryer with the temperature set to -45 &#176;C and the pressure set to 0.2 mbar.</li>
	<li>Dissolve the lyophilized GelMA (degree of substitution of approximately 75%) in 1x PBS containing 10% FBS (fetal bovine serum, v/v), 25 mM HEPES (N-2-hydroxyethylpiperazine-N-ethane-sulfonic acid), and photoinitiator (0.5%, w/v) to obtain the GelMA bioink preparation. 
	NOTE: The degree of substitution of GelMA can be calculated by a ninhydrin assay3.</li>
	<li>Mix the GelMA solution (10%, w/v) with different volumes of initial SF solution (5%, w/v) and different volumes of 1x PBS to obtain SF-GelMA bioinks with different concentrations of SF. The proportion per 1 mL of GelMA and SF solution in the different bioinks is presented in Table 1. 
	NOTE: All bioinks must contain a final concentration of 5% (w/v) GelMA, but the concentration of SF varies: 0.5, 0.75, 1.0, 1.25, and 1.5% (w/v) in the different SF-GelMA bioinks. Use SF-M-Layered-GelMA to term the GelMA bioink modified with SF in a layer-by-layer manner. Use SF-X-GelMA to term those GelMA modified with SF in a homogeneous manner (e.g., GelMA with modification of 1% SF was termed SF-1-GelMA).</li>
	<li>Sonicate all bioinks for 10-20 min.</li>
	<li>Grow NIH3T3 cells using DMEM (Dulbecco&#39;s modified Eagle medium) containing 10% FBS and 1% penicillin-streptomycin in an incubator (37 &#176;C with 5% CO2). Passage the cells at a ratio of 1:3 when the density reaches 80%.</li>
	<li>Centrifuge the suspension of NIH3T3 cells at 1500 rpm for 5 min. Remove the supernatant with suction and resuspend the cell pellet with fresh bioink solution in a 15 mL sterile tube. 
	NOTE: The concentration of the encapsulated cells in the bioink should be 1 x 106 cells/mL, and the concentration needs to be calculated with a cytometer.</li>
	<li>Use 2 mL of different SF-GelMA bioinks to suspend the cell pellets to obtain various cell-laden bioinks.</li>
</ol>
2. Loading, reheating and bioprinting of the SF-M-Layered-GelMA
<ol>
	<li>Load 0.4 mL of cell-laden SF-0.5-GelMA into the bottom layer of the syringe. 
	NOTE: Use a 2 mL syringe as the bioink reservoir in this study. Load different SF-GelMA bioinks into the syringe in a layer-by-layer manner.</li>
	<li>Place the syringe in ice water bath (0 &#176;C) for 5 min to cause the bottom-layer bioink to transform into a gel state.</li>
	<li>Load 0.4 mL of cell-laden SF-0.75-GelMA bioink above the bottom layer.</li>
	<li>Place the syringe with the two layers of bioinks in an ice water bath (0 &#176;C) for 5 min to cause the two layers of bioinks to transform into a gel state.</li>
	<li>Cycle the loading and cooling steps another 3 times with the remaining 3 kinds of bioinks (SF-1-GelMA, SF-1.25-GelMA, SF-1.5-GelMA) to obtain a multilayered bioink system with different concentrations of SF in the different layers. The volume used for the loading of all bioinks should be 0.4 mL.</li>
	<li>Reheat the multilayered bioink by placing the syringe in an incubator (37 &#176;C) for 30 min prior to bioprinting.</li>
	<li>Use a 2 mL syringe as the bioink reservoir with a 27 G printing nozzle. Set the speed of flow at 50 &#956;L/min, the moving speed of the nozzle at 2 mm/s, and the height of the nozzle at 1 mm. Perform the bioprinting procedure at room temperature (approximately 20 &#176;C).
	<ol>
		<li>Print the tissue construct in an extrusion manner using a custom-made bioprinter under the parameters adapted from our previous studies11,12.</li>
	</ol>
	</li>
	<li>Use ultraviolet light (365 nm, 800 mW) for 40 s to crosslink the bioprinted tissue construct.</li>
	<li>Culture the crosslinked tissue construct in DMEM (Dulbecco&#39;s modified Eagle medium) containing 10% FBS and 1% penicillin-streptomycin in an incubator (37 &#176;C with 5% CO2). The medium was changed every 8 h during the first 2 days and every 2-3 days thereafter.</li>
</ol>

<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-multilayered-hydrogel-bioink-three-dimensional-bioprinting-for

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6.4K Views.  Fudan University. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

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

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

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

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

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

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

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

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

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

Scalable Generation of Uniform Cell Spheroids Using a 3D Acoustic Assembly Device

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

Author Spotlight: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...

IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular matrix (osteoid) express the Pdpn gene to form cellular dendrites and transform into preosteocytes. Later, preosteocytes express the Dmp1 gene to promote matrix mineralization and thereby transform into mature osteocytes.This process is called osteocytogenesis. IDG-SW3 is a well-known cell line for in vitro studies of osteocytogenesis. Many previous methods have used collagen I as the main or the only component of the culturing matrix. However, in addition to collagen I, the osteoid also contains a ground substance, which is an important component in promoting cellular growth, adhesion, and migration. In addition, the matrix substance is transparent, which increases the transparency of the collagen I-formed gel and, thus, aids the exploration of dendrite formation through imaging techniques. Thus, this paper details a protocol to establish a 3D gel using an extracellular matrix along with collagen I for IDG-SW3 survival. In this work, dendrite formation and gene expression were analyzed during osteocytogenesis. After 7 days of osteogenic culture, an extensive dendrite network was clearly observed under a fluorescence confocal microscope. Real-time PCR showed that the mRNA levels of Pdpn and Dmp1 continually increased for 3 weeks. At week 4, the stereomicroscope revealed an opaque gel filled with mineral particles, consistent with the X-ray fluorescence (XRF) assay. These results indicate that this culture matrix successfully facilitates the transition from osteoblasts to mature osteocytes.

Here, we present a protocol for culturing IDG-SW3 cells in a three-dimensional (3D) extracellular matrix.

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular ...