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>

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 ...

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

Research

JoVE Journal

Bioengineering

16.6K Views.  Wake Forest Univeristy Health Sciences. 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.

Video: Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

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.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

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.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

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 ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

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

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

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.

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 ...