Name: Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink (Video) | JoVE Journal
Uploaded: 2016-04-21T14:00:00.000+00:00
Duration: 8 min 34 s
Description: Learn to design hydrogel bioinks for bioprinting functional 3D tissue constructs using modular commercial components. This method enables precise control of mechanical properties for extrusion and supports applications in drug testing and disease modeling across multiple tissue types.

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5 minutes

The authors gratefully acknowledge funding by the Defense Threat Reduction Agency (DTRA) under Space and Naval Warfare Systems Center Pacific (SSC PACIFIC) Contract No. N6601-13-C-2027. The publication of this material does not constitute approval by the government of the findings or conclusions herein.

1. Hydrogel Bioink Formulations and Preparation
<ol>
	<li>In order to provide tissue-specific biochemical profiles, prepare tissue-specific ECM digest solutions as previously described for liver.20 
	Note: In general, this ECM digest will comprise 40% of the final hydrogel bioink volume that is employed. Several hundred milliliters of ECM digest solution can be prepared, aliquoted, and frozen at -80 &#176;C for future use.</li>
	<li>Prior to hydrogel formulation, dissolve a photoinitiator, 2-hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone, in water at 0.1% w/v. 
	Note: Volumes in the 50-100 ml range can be prepared ahead of time and stored shielded from light at 4 &#176;C for several months.</li>
	<li>To form hydrogel bioinks, first dissolve the base material components from the hyaluronic acid (HA) hydrogel kits in the water-photoinitiator solution.
	<ol>
		<li>Dissolve the thiolated HA and thiolated gelatin separately in water-photoinitiator solution (step 1.2) to make 2% w/v solutions.</li>
		<li>Dissolve polyethylene glycol diacrylate (PEGDA), the crosslinker in the hydrogel kits, in water-photoinitiator solution (step 1.2) to make an 8% w/v solution.</li>
		<li>Dissolve polyethylene glycol (PEG) 8-Arm alkyne (10 kDa MW) in water-photoinitiator solution (step 1.2) to make an 8% w/v solution.</li>
	</ol>
	</li>
	<li>In general, form hydrogels using the following scheme, although additional customization is possible.
	<ol>
		<li>Combine 4 parts 2% thiolated HA, 4 parts 2% thiolated gelatin, 1 part crosslinker 1, 1 part crosslinker 2 with 8 parts tissue ECM solution and 2 parts hepatocyte culture media (HCM) (or 10 parts water as a generic non-tissue-specific hydrogel). 
		Note: Additional unmodified HA or gelatin can be added to make the bioink extrude more smoothly. This is described below.</li>
	</ol>
	</li>
	<li>Vortex the resulting mixture on high (speed 10 out of 10) for 10 sec to mix prior to use.</li>
	<li>Use of hydrogel bioink
	<ol>
		<li>For extrusion or bioprinting testing, transfer the mixture into a syringe or printer cartridge and allow it to crosslink spontaneously for 30 min (stage 1 crosslinking) at 37 &#176;C.</li>
		<li>For rheological measurements, transfer the mixture into a 35 mm Petri dish and allow it to crosslink for 30 min. 
		Note: The mixture immediately begins to crosslink through thiol-acrylate bond formation and will begin to increase in viscosity. The mixture should be transferred to a syringe, print cartridge, or target location within 10 min to avoid clogging of a pipette or syringe during transfer.</li>
		<li>When secondary crosslinking (stage 2) is desired, irradiate the stage 1-crosslinked gels with ultraviolet light (365 nm, 18 W/cm2) to initiate a thiol-alkyne polymerization reaction. 
		Note: Duration of irradiation is dependent on the surface area of the material. In general, a square centimeter of material only requires 1-2 sec of UV exposure at this UV power.</li>
	</ol>
	</li>
</ol>
2. Printer Compatibility Testing
<ol>
	<li>Prior to integration testing with bioprinting devices, test extrusion characteristics on the laboratory bench with simple extrusion tests using standard syringes and small gauge needle tips (20-30 gauge).
	<ol>
		<li>Push the bioink through a standard syringe to achieve smoothly extruded filaments of hydrogel with few or no bumps. Extruding of lines or simple patterns is sufficient to determine success.</li>
	</ol>
	</li>
	<li>For bioprinter integration, load bioink preparations by pipetting them into printer cartridges, and allow 30 min for the bioink to undergo spontaneous stage 1 crosslinking within the cartridge. 
	Note: Volume of bioink depends on the specific application and should be determined by the user. Printer cartridges may resemble or be syringes that are compatible with the bioprinter device.</li>
	<li>Evaluate extrusion compatibility for bioprinting by printing a simple pattern using the bioink. For example, print a 7 x 7 mm pattern comprised of parallel lines. Apply pressure (e.g., 20 kPa pneumatic pressure) while the printhead moves in the X-Y plane at a velocity of approximately 300 mm/min. 
	Note: Printhead nozzle diameters of various sizes can be used, but conical nozzles with 400-500 &#181;M diameter openings are optimal for printing spheroids in the 250-350 &#181;m range.
	<ol>
		<li>If the extruded materials are lumpy or irregular, see step 2.4, or reduce the amount of PEGDA to soften the stage 1 crosslinked material. Properly prepared bioink formulations extrude smoothly, allowing precise deposition in desired patterns or architectures. 
		Note: The bioprinting procedures described use a custom 3-D bioprinting device designed in house specifically for tissue construct printing.32 As such, specific printing parameters vary dramatically for each type of device and require testing by the user.</li>
	</ol>
	</li>
	<li>To improve extrusion properties, supplement unmodified HA and gelatin to the bioinks (1.5 mg/ml and 30 mg/ml, respectively).</li>
</ol>
3. Validation by Bioprinting with Primary Liver Constructs
<ol>
	<li>Prepare 3-D primary cell liver spheroids by hanging drop methods as the cellular component33 
	Note: Bioprinting can be performed without spheroids, but instead with individual cells suspended in the hydrogel bioinks as well. Spheroids are employed here to accelerate cell-cell interactions and construct functionality. The number of spheroids or cells employed depends on the specific application and should be determined by the user. These steps should be performed under sterile conditions, using sterile supplies.
	<ol>
		<li>Prepare HCM by adding the thawed contents of the HCM supplement component kit to the hepatocyte basal media (HBM) and sterile filtering.
		<ol>
			<li>Thaw the supplement components until liquid.</li>
			<li>Add the supplement components (ascorbic acid, 0.5 ml; bovine serum albumin [fatty acid free], 10 ml; gentamicin sulfate/amphotericin B, 0.5 ml; hydrocortisone 21-hemisuccinate, 0.5 ml; insulin, 0.5 ml; human recombinant epidermal growth factor, 0.5 ml; transferring, 0.5 ml) to the 500 ml HBM.</li>
			<li>Sterile filter through a 0.45 &#956;m or 0.22 &#956;m filter using a bottle-top filter unit or a syringe tip filter.</li>
		</ol>
		</li>
		<li>Determine the cell density of primary human hepatocytes, Kupffer cells, and stellate cells by counting on a hemocytometer after each cell type has been thawed according to manufacturers&#39; instructions.</li>
		<li>Combine primary human hepatocytes, Kupffer cells, and stellate cells in an 80:10:10 ratio by cell number in HCM media that has been warmed to 37 &#176;C in a conical tube. 
		Note: The volume of media to be used depends on the overall cell number specific to the application and should be determined by the user.</li>
		<li>Centrifuge the cell suspension for 5 min at 520 x g at 20 &#176;C.</li>
		<li>Aspirate the supernatant, leaving behind the cell pellet.</li>
		<li>Resuspend the cell pellet in HCM media to yield a cell suspension containing 1,000 cells per 40 &#181;l media. Total volume is dependent on the number of spheroids being produced.</li>
		<li>Transfer the cell suspension to 96-well format hanging drop plates. Add a total of approximately 1,000 cells to each well in HCM and maintain at 37 &#176;C, in 5% CO2 for 3 days during which multicellular spheroids form.</li>
		<li>Collect liver spheroids from the hanging drop plate using a pipettor. Transfer to a sterile 15 ml conical tube.</li>
	</ol>
	</li>
	<li>Bioprint liver spheroids in liver-specific hydrogel bioink
	<ol>
		<li>Prepare a formulation of liver ECM-containing hydrogel bioink as described in Step 1, employing 8% PEGDA and 8% 8-Arm PEG alkyne as crosslinkers. Use this combination for its capability in resulting in a hydrogel close in shear elastic modulus to native liver tissue.</li>
		<li>Let the spheroids settle to the bottom of the conical tube in which they were placed in Step 3.1.7. This varies based on spheroid size and density, but generally occurs within 1-2 min. Remove all media by carefully aspirating or with a pipettor.</li>
		<li>Transfer the desired volume of freshly prepared hydrogel bioink solution to the conical tube containing the spheroids. Generally, an appropriate volume is 10%-25% greater than the volume of the 3-D construct to be printed. Carefully pipette up and down to resuspend the spheroids in the hydrogel bioink solution. Transfer to a bioprinter cartridge using a pipettor or serological pipette.</li>
		<li>Inside the bioprinter cartridge, allow the solution to undergo the first crosslinking stage (thiol-acrylate reaction) for 30 min. 
		Note: Depending on spheroid size, the cartridge may need to be slowly rotated or the contents may need to be mixed with a sterile spatula to keep the spheroids distributed throughout the bioink during the stage 1 crosslinking. This is less of a necessity for bioinks prepared with suspended cells instead of spheroids. 
		Note: Following stage 1 crosslinking, users have an operating window of several hours. However, it is recommended to perform the bioprinting process quickly to improve cell viability.</li>
		<li>Following stage 1 crosslinking, use a bioprinting device to create desired hydrogel structures containing the primary liver spheroids (or other cells). 
		Note: This technology provides a system for biofabricating a wide variety of structures. Parameters such as total volume, number of cells or spheroids, the printed structure geometry, and substrate on which constructs are printed are highly dependent on the goals of the user.</li>
		<li>After deposition into the desired configuration, administer UV light for 2-4 sec to initiate the secondary crosslinking mechanism, stabilizing the constructs and increasing stiffness to the desired level. 
		Note: The concentration of PEG-alkyne, and thus the overall final crosslinking density, primarily controls the final construct stiffness.</li>
		<li>Repeat the steps 3.2.4 and 3.2.5 in order to create multi-layered constructs.</li>
	</ol>
	</li>
</ol>

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<li>Flynn, L., Prestwich, G. D., Semple, J. L., Woodhouse, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose+tissue+engineering+with+naturally+derived+scaffolds+and+adipose-derived+stem+cells%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Adipose tissue engineering with naturally derived scaffolds and adipose-derived stem cells.</a> Biomaterials. 28, 3834-3842 (2007).</li>
<li>Flynn, L., Prestwich, G. D., Semple, J. L., Woodhouse, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose+tissue+engineering+in+vivo+with+adipose-derived+stem+cells+on+naturally+derived+scaffolds%5BTitle%5D)+AND+%22J+Biomed+Mater+Res+A%22%5BJournal%5D)">Adipose tissue engineering in vivo with adipose-derived stem cells on naturally derived scaffolds.</a> J Biomed Mater Res A. 89, 929-941 (2009).</li>
<li>Duflo, S., Thibeault, S. L., Li, W., Shu, X. Z., Prestwich, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+a+synthetic+extracellular+matrix+on+vocal+fold+lamina+propria+gene+expression+in+early+wound+healing%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Effect of a synthetic extracellular matrix on vocal fold lamina propria gene expression in early wound healing.</a> Tissue Eng. 12, 3201-3207 (2006).</li>
<li>Duflo, S., Thibeault, S. L., Li, W., Shu, X. Z., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vocal+fold+tissue+repair+in+vivo+using+a+synthetic+extracellular+matrix%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Vocal fold tissue repair in vivo using a synthetic extracellular matrix.</a> Tissue Eng. 12, 2171-2180 (2006).</li>
<li>Liu, Y., Shu, X. Z., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteochondral+defect+repair+with+autologous+bone+marrow-derived+mesenchymal+stem+cells+in+an+injectable%2C+in+situ%2C+cross-linked+synthetic+extracellular+matrix%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Osteochondral defect repair with autologous bone marrow-derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix.</a> Tissue Eng. 12, 3405-3416 (2006).</li>
<li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accelerated+repair+of+cortical+bone+defects+using+a+synthetic+extracellular+matrix+to+deliver+human+demineralized+bone+matrix%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">Accelerated repair of cortical bone defects using a synthetic extracellular matrix to deliver human demineralized bone matrix.</a> J Orthop Res. 24, 1454-1462 (2006).</li>
<li>Zhang, J., Skardal, A., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+extracellular+matrices+with+cleavable+crosslinkers+for+cell+expansion+and+easy+cell+recovery%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Engineered extracellular matrices with cleavable crosslinkers for cell expansion and easy cell recovery.</a> Biomaterials. 29, 4521-4531 (2008).</li>
<li>Serban, M. A., Scott, A., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unit+10.14%2C+Use+of+hyaluronan-derived+hydrogels+for+three-dimensional+cell+culture+and+tumor+xenografts%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Unit 10.14, Use of hyaluronan-derived hydrogels for three-dimensional cell culture and tumor xenografts.</a> Curr Protoc Cell Biol. Chapter 10, (2008).</li>
<li>Xu, X., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+tumor+growth+and+angiogenesis+by+a+lysophosphatidic+acid+antagonist+in+an+engineered+three-dimensional+lung+cancer+xenograft+model%5BTitle%5D)+AND+%22Cancer%22%5BJournal%5D)">Inhibition of tumor growth and angiogenesis by a lysophosphatidic acid antagonist in an engineered three-dimensional lung cancer xenograft model.</a> Cancer. 116, 1739-1750 (2010).</li>
<li>Liu, Y., Shu, X. Z., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+engineering%3A+orthotopic+cancer+models+in+mice+using+cell-loaded%2C+injectable%2C+cross-linked+hyaluronan-derived+hydrogels%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Tumor engineering: orthotopic cancer models in mice using cell-loaded, injectable, cross-linked hyaluronan-derived hydrogels.</a> Tissue Eng. 13, 1091-1101 (2007).</li>
<li>Skardal, A., Devarasetty, M., Rodman, C., Atala, A., Soker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver-Tumor+Hybrid+Organoids+for+Modeling+Tumor+Growth+and+Drug+Response+In+Vitro%5BTitle%5D)+AND+%22Ann+Biomed+Eng%22%5BJournal%5D)">Liver-Tumor Hybrid Organoids for Modeling Tumor Growth and Drug Response In Vitro.</a> Ann Biomed Eng. , (2015).</li>
</ol>

Authors have nothing to disclose.

There are several components that are critical to consider when attempting to biofabricate 3-D tissue constructs, for eventual use in humans or for in vitro screening applications. Employing the appropriate cellular components determines the end potential functionality, while the biofabrication device itself determines the general methodology for reaching the end construct. The third component, the biomaterial, is equally important, as it serves dual roles. Specifically, the biomaterial component must be compati...

In recent years, a variety of technologies have become available that addresses the need for alternative sources of functional organs and tissues by seeking to manufacture, or biofabricate, them. Bioprinting has emerged as one of the most promising of these technologies. Bioprinting can be thought of as a form of robotic additive fabrication of biological parts, that can be used to build or pattern viable organ-like or tissue-like structures in 3 dimensions.1 In most cases, bioprinting employs a 3-dimensional (3-D) printing device that is directed by a computer to deposit cells and biomaterials into precise positions, thereby recapitulating anatomically-mimicking physiological architectures.2 These devices print a &#34;bioink&#34;, which can take the form of cell aggregates, cells encapsulated in hydrogels or viscous fluids, or cell-seeded microcarriers, as well as cell-free polymers that provide mechanical structure or act as cell-free placeholders.3,4 Following the bioprinting process, the resulting structure can be matured into functional tissue or organ structures, and used for its intended end application.5,6 To date, a complete fully functional human-sized organ has not been printed, but it remains the primary long-term goal of bioprinting research and development.2 However, small-scale &#34;organoid&#34; tissue constructs are currently being implemented in a number of applications, including pathology modeling, drug development, and toxicology screening.
One of the main hurdles that researchers have encountered in applying bioprinting technology is that very few materials have been developed for the explicit purpose of bioprinting. To effectively succeed in bioprinting, a biomaterial must meet 4 basic requirements. The biomaterial needs to have 1) the appropriate mechanical properties to allow deposition (be it extrusion through a nozzle as a gel or an inkjet as a droplet), 2) the ability to hold its shape as a component of a 3-D structure after deposition, 3) the capability for user control of the 2 prior characteristics, and 4) a cell friendly and supportive environment at all phases of the bioprinting procedure.7 Historically, bioprinting work has often tried to employ existing traditional biomaterials in bioprinting devices without consideration for their compatibility, instead of designing a biomaterial to have the properties necessary for bioprinting and subsequent post-printing applications.
A variety of bioinks have been developed recently to better interface with the deposition and fabrication hardware. Standard hydrogel systems pose significant problems because they generally exist as either precursor fluid solutions with insufficient mechanical properties, or polymerized hydrogels that if printed can clog nozzles or become broken up upon the extrusion process. Our team, as well as others, have explored various hydrogel formulations to address these bioprinting problems, including cell spheroid printing into hydrogel substrates,5,8 cell and hydrogel filament extrusion from microcapillary tubes,9-11 extrudable hyaluronic acid (HA)-gold nanoparticle hydrogels with dynamic crosslinking properties,12 temporal control of hydrogel stiffness using photopolymerizable methacrylated HA and gelatin,13 fibrinogen-thrombin-based crosslinking,14,15 ionic exchange alginate-collagen gels,16 and recently rapid polymerizing ultraviolet light (UV)-initiated crosslinking,17
These examples demonstrate the feasibility of generating materials that can by bioprinted effectively. However, in addition to integration with hardware, to successfully generate viable and functional 3-D tissue constructs, biomaterials must contain biochemical and mechanical cues that aid in maintaining cellular viability and function. These additional factors, biochemical and mechanical profiles, can have a significant influence on the successful function of bioprinted tissue constructs.
Both cells and the native extracellular matrix (ECM) are responsible for presenting a wide range of signaling molecules such as growth factors and other cytokines to other cells. The combination of these signals varies from tissue to tissue, but can be extremely potent and influential in regulating cell and tissue behavior.18 Employing tissue-specific ECM components from different organs and implementing as a hydrogel or as part of a hydrogel has been explored with success.19-21 This approach, which is comprised of decellularizing a given tissue, pulverizing it, and dissolving it, can be used to produce tissue-specific biochemical signals from any tissue and can be incorporated in 3-D hydrogel constructs.22
Additionally, it is widely documented that tissues in the body occupy a wide range of stiffnesses.23 As such, the ability to tune the mechanical properties of biomaterials, such as elastic modulus E&#39; or shear elastic modulus G&#39;, is a useful tool in tissue engineering. As described above, control over bioink mechanical properties allows for extrusion-based biofabrication using a soft gel, which can then further manipulated by secondary crosslinking at a later point, at which elastic modulus levels can be attained that match that of the target organ type. For example, biomaterials could be customized to match a stiffness of 5-10 kPa like a native liver,23 or match a stiffness of 10-15 kPa like native cardiac tissue,24,25 in theory increasing the ability of these organoids to function in a similar manner to their native tissue counterparts. The influence of environmental stiffness on cell phenotype has been explored in recent years, particularly with respect to stem cells. Engler et al. demonstrated that substrate elasticity aided in driving mesenchymal stem cells (MSC) towards lineages with tissue elasticity matching that of the substrate.25 This concept has been further explored for differentiation into muscle, cardiac function, liver phenotype, hematopoietic stem cell proliferation, and maintenance of stem cell therapeutic potential.24,26-29 Being able to tune a hydrogel to different elastic moduli is an important feature of a biomaterial that will be used to biofabricate tissue constructs.30
Here we describe a protocol that represents a versatile approach used in our laboratory to formulate a hydrogel system that can be extrusion bioprinted, and customized to 1) contain the biochemical profile of a particular tissue type and 2) mimic the elastic modulus of that tissue type. By addressing these requirements, we aim to provide a material that can recapitulate the physiochemical and biological characteristics of in vivo tissue.31 The modular hydrogel composite system described herein takes advantage of a multi-crosslinking approach to yield extrudable bioinks, and allows a secondary crosslinking to stabilize and increases the stiffness of the end products to match a range of tissue types. Biochemical customization is met by using tissue-specific ECM components. As a demonstration, we employ a liver-specific variety of this hydrogel system to bioprint functional liver organoid constructs. The protocol described uses a custom 3-D bioprinting device. In general, this protocol can be adapted to most extrusion-based printers, specific printing parameters vary dramatically for each type of device and require testing by the user.

<table><tbody><tr><td>Hyaluronic acid</td><td>Sigma</td><td>53747</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G6144</td><td></td></tr><tr><td>2-Hydroxy-4&amp;prime;-(2-hydroxyethoxy)-2-methylpropiophenone</td><td>Sigma</td><td>410896</td><td></td></tr><tr><td>Hyaluronic acid and gelatin hydrogel kit (HyStem-HP)</td><td>ESI-BIO</td><td>GS315</td><td>Kit contains the components Heprasil (thiolated and heparinized hyaluronic acid), Gelin-S (thiolated gelatin), and Extralink (PEGDA)</td></tr><tr><td>PEG 8-Arm Alkyne, 10&amp;nbsp;kDa</td><td>Creative PEGWorks</td><td>PSB-887</td><td></td></tr><tr><td>Primary human hepatocytes</td><td>Triangle Research Labs</td><td>HUCPM6</td><td></td></tr><tr><td>Primary human liver stellate cells</td><td>ScienCell</td><td>5300</td><td></td></tr><tr><td>Primary human Kupffer cells</td><td>Life Technologies</td><td>HUKCCS</td><td></td></tr><tr><td>Hepatocyte Basal Media (HBM)</td><td>Lonza</td><td>CC-3199</td><td></td></tr><tr><td>Hepatocyte Media Supplement Kit</td><td>Lonza</td><td>CC-3198</td><td>HCM SingleQuot Kits (contains ascorbic acid, 0.5&amp;nbsp;ml; bovine serum albumin [fatty acid free], 10&amp;nbsp;ml; gentamicin sulfate/amphotericin B, 0.5&amp;nbsp;ml; hydrocortisone 21-hemisuccinate, 0.5&amp;nbsp;ml; insulin, 0.5&amp;nbsp;ml; human recombinant epidermal growth factor, 0.5&amp;nbsp;ml; transferring, 0.5&amp;nbsp;ml)</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td>Other manufacturers are ok.</td></tr><tr><td>Ammonium hydroxide</td><td>Fischer Scientific</td><td>A669</td><td>Other manufacturers are ok.</td></tr><tr><td>Fresh porcine cadaver tissue</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Lyophilizer</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Freezer mill</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Bioprinter</td><td>n/a</td><td>n/a</td><td>The bioprinter described herein was custom built in-house. In general, other devices are adequate provided they support computer controlled extrusion-based printing of hydrogel materials.</td></tr><tr><td>Hanging drop cell culture plate</td><td>InSphero</td><td>CS-06-001</td><td>InSphero GravityPlus 3D Culture Platform</td></tr></tbody></table>

bioprinting cellularized constructs using a tissue-specific hydrogel bioink

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.

When the procedures described above are followed correctly, hydrogels should contain a biochemical profile specific to the target tissue type,20 allow for a high degree of control over bioprinting and final elastic modulus,34 and support viable functional cells in tissue constructs.
Hydrogel Customization 
To best mimic native liver, the hydrogel bioink was supplemented by liver ECM solu...

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 ...

The overall goal of this protocol is to demonstrate a versatile approach for designing hydrogel bioinks that can be extruded through bioprinting devices. The bioinks can then be used to fabricate three dimensional tissue constructs. This method can help answer key questions in the bioprinting field, such as how to control the mechanical properties needed in order to provide a material that can be extruded using a bioprinter.The main advantage of this technique is that we use commercially available components combined in a modular fashion to create a simple and effective bioprintable hydrogel bioink. The applications of these technologies include the creation of 3D tissue organoids that can be used to accurately model the effects of drugs, toxins, and diseases. Although this method can provide a framework to bioprint 3D liver constructs, it can also be applied to other tissue types such as muscle, lung, and colon.Generally, individuals new to this method will struggle because there are a number of different reagents used to create the hydrogel bioink, but it is actually quite straightforward. Demonstrating the procedure will be Young-Joon Seol, a post-doc on our team. To begin, prepare a tissue-specific extracellular matrix digest to be used in the hydrogel formulation as described elsewhere.Then, dissolve a photoinitiator in water at a weight per volume ratio of 0.1%To form the hydrogel bioinks, first dissolve the base material components from the hyaluronic acid hydrogel kits into individual aliquots of the water photoinitiator solution. Then, combine the ECM solution, the 2%thiolated hyaluronic acid, the 2%thiolated gelatin, the crosslinkers, and hepatocyte culture media in the ratios shown here. To improve the extrusion properties of the bioink, add 1.5 milligrams per milliliter of unmodified hyaluronic acid and 30 milligrams per milliliter of gelatin to the mixture.Then, vortex the resulting mixture on high for ten seconds prior to use. Before testing the bioink in a bioprinting device, first test the extrusion characteristics on the laboratory bench. Using a standard syringe, drop a sample of the bioink and then attach a 20 to 30 gauge needle to the syringe.Allow the bioink to crosslink and then push the bioink through the needle to achieve smoothly extruded hydrogel filaments. If the formulation is able to create a filament with few or no bumps, then it is ready for bioprinting. To load the bioink preparations in a bioprinter, pipette the bioinks into sterilized printer cartridges.Allow them to sit for 30 minutes before extrusion as the bioink will undergo spontaneous stage one crosslinking within the cartridge. Next, load the cartridge into the print setup and connect a pneumatic pressure source to the cartridge. Prepare a simple pattern, like this seven by seven millimeter grid of parallel lines, to print in order to evaluate its extrusion compatibility.While the print head moves in the XY plane at a velocity of approximately 300 millimeters per minute, apply a pressure of 20 kilopascals to the cartridge to extrude the bioink. If the extruded materials are lumpy or irregular, reduce the amount of the crosslinker added in order to soften the stage one crosslinked material. A properly prepared bioink formulation should extrude smoothly, allowing for precise deposition and architectures.Prepare 3D primary cell liver spheroids in a 96 well plate using the hanging drop method as described in the accompanying text protocol. After three days in culture, collect the liver spheroids from the hanging drop plate using a pipette, and transfer them into a sterile, 15 milliliter conical tube. Let the spheroids settle to the bottom of the conical tube for one to two minutes.Then, carefully aspirate the media with a pipette. Transfer 110 to 125 percent of the desired printed 3D construct volume of the freshly prepared hydrogel bioink solution to the conical tube containing the spheroids. Then, carefully pipette the spheroids up and down to re-suspend them in the hydrogel bioink solution.Once evenly suspended, transfer the spheroid solution to a bioprinter cartridge using a pipette and allow the solution to undergo the first crosslinking stage for 30 minutes. Following stage on crosslinking, use a bioprinting device to create the desired hydrogel structures containing the primary liver spheroids. After each layer of deposition, expose the printed bioink to UV light for two to four seconds to initiate the secondary crosslinking mechanism.This will stabilize the constructs and increase the stiffness to the desired level. The concentration of PEG alkyne in the solution is what controls the overall crosslinking density, and therefore primarily controls the stiffness of the final construct. After bioprinting, high cell viability in the liver constructs was observed using confocal microscopy.Under optimal environmental conditions, viability should be above 85%Additionally, when the constructs were stained for markers indicative of liver tissue, positive expression of CYP3A4, a cytochrome P450 isoform, intracellular albumin, E-cadherin, an epithelial cell-cell adhesion protein, and DPP4, a protein expressed highly in the liver, were all observed. When the culture media was tested for levels of urea and albumin, it was found that the construct secreted both urea and albumin at constant levels over the 14 day time course. This further suggests that the tissue-specific hydrogel bioinks aid in maintaining the function of the liver cells.Once mastered, this technique can be done in about two hours from start to finish if it is performed properly. However, this is often dependent on the particular bioprinting device that is employed. While attempting this procedure, it's important to remember that the steps demonstrated must often be adapted to be compatible with other tissue types or bioprinting devices.Following this procedure, other hydrogel bioink formulations can be created in order to support the bioprinting of other tissue types. The development of these technologies helped pave the way for the creation of multi-organoid, body-on-a-chip platforms for the screening of drugs and modeling diseases. After watching this video, you should have a good understanding of how to begin designing materials that can be used for bioprinting 3D tissue constructs by using multi-step crosslinking.Don't forget that working with ultraviolet light can be extremely hazardous to one's vision, and precautions such as using UV protective goggles should always be taken while performing this procedure.

bioprinting-cellularized-constructs-using-tissue-specific-hydrogel

Research

JoVE Journal

Bioengineering

A bioprinter was used to create patterned hydrogels based on a sacrificial mold.  The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

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

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

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

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

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

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

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (FcεRI).

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Learn to design hydrogel bioinks for bioprinting functional 3D tissue constructs using modular commercial components. This method enables precise control of mechanical properties for extrusion and supports applications in drug testing and disease modeling across multiple tissue types.

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

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