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&#...

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 border="0" cellpadding="0" cellspacing="0" width="903">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Hyaluronic acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">53747</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Gelatin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">G6144</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">410896</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:340px;">Hyaluronic acid and gelatin hydrogel kit (HyStem-HP)</td>
			<td style="width:108px;">ESI-BIO</td>
			<td style="width:119px;">GS315</td>
			<td style="width:336px;">Kit contains the components Heprasil (thiolated and heparinized hyaluronic acid), Gelin-S (thiolated gelatin), and Extralink (PEGDA)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">PEG 8-Arm Alkyne, 10 kDa</td>
			<td style="width:108px;">Creative PEGWorks</td>
			<td>PSB-887</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Primary human hepatocytes</td>
			<td style="width:108px;">Triangle Research Labs</td>
			<td>HUCPM6</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Primary human liver stellate cells</td>
			<td style="width:108px;">ScienCell</td>
			<td style="width:119px;">5300</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Primary human Kupffer cells</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:119px;">HUKCCS</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Hepatocyte Basal Media (HBM)</td>
			<td style="width:108px;">Lonza</td>
			<td style="width:119px;">CC-3199</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:340px;">Hepatocyte Media Supplement Kit</td>
			<td style="width:108px;">Lonza</td>
			<td style="width:119px;">CC-3198</td>
			<td style="width:336px;">HCM SingleQuot Kits (contains ascorbic acid, 0.5mL; bovine serum albumin [fatty acid free], 10 mL; gentamicin sulfate/amphotericin B, 0.5mL; hydrocortisone 21-hemisuccinate, 0.5 mL; insulin, 0.5 mL; human recombinant epidermal growth factor, 0.5 mL; transferring, 0.5 mL)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Triton X-100</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td style="width:336px;">Other manufacturers are ok.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Ammonium hydroxide</td>
			<td style="width:108px;">Fischer Scientific</td>
			<td style="width:119px;">A669</td>
			<td style="width:336px;">Other manufacturers are ok.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Fresh porcine cadaver tissue</td>
			<td style="width:108px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Lyophilizer</td>
			<td style="width:108px;">any</td>
			<td style="width:119px;">n/a</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Freezer mill</td>
			<td style="width:108px;">any</td>
			<td style="width:119px;">n/a</td>
			<td style="width:336px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:340px;">Bioprinter</td>
			<td style="width:108px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:336px;">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 height="21">
			<td height="21" style="height:21px;width:340px;">Hanging drop cell culture plate</td>
			<td style="width:108px;">InSphero</td>
			<td>CS-06-001</td>
			<td style="width:336px;">InSphero GravityPlus 3D Culture Platform</td>
		</tr>
	</tbody>
</table>

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 so...

Watch this Scientific Journal Video about Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink at JoVE.com

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

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

16.4K Views.  Wake Forest Univeristy Health Sciences. 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 commercial...