Name: bioprinting cellularized constructs using a tissue-specific hydrogel bioink
Uploaded: 2016-04-21T14:00:00
Description: Watch this Scientific Journal Video about Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink at JoVE.com

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&#...

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 ...

Research

JoVE Journal

Bioengineering

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

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

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

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels (Video) | JoVE

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