We thank Deborah Studer for the help with the bioprinter.
The work was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n&deg;NMP4-SL-2009-229292.

1. Preparation of the Poloxamer 407 Solution
If available, perform the preparation of the poloxamer solution in a cold room (4 &deg;C). If not available, place a glass bottle in a beaker filled with ice-cold water. At higher temperatures the poloxamer will be above the gel point and will not dissolve properly.
<ol>
	<li>Add 60 ml of ice cold PBS solution into a glass bottle and stir vigorously using a magnetic stirrer.</li>
	<li>Weigh 24.5 grams of poloxamer and add it in small amounts to the cold PBS. Wait until the poloxamer has partially dissolved before adding more.</li>
	<li>Stir the solution until all poloxamer has dissolved.</li>
	<li>Add cold PBS until a final volume of 100 ml is reached. The final concentration will be 24.5% w/v.</li>
	<li>Stop stirring the solution and let it rest at 4 &deg;C until bubbles and foam in the solution have disappeared. Bubbles that are trapped within the gel will be transferred to the printer cartridge and will lead to defects in the printed sacrificial molds.</li>
	<li>Filter (0.22 &mu;m filter) the solution directly into the printing cartridge to remove any unwanted particles that could clog the needle. The filtering step should be performed in a cold room (or if not available with cooled tips, filter etc.) to avoid gelling of the poloxamer in the filter. Keep the loaded cartridge at 4 &deg;C until 30 min before the experiment.</li>
</ol>
2. Preparation of the 3D Printer
The 3D printer used in this work was the "BioFactory" from regenHU. The extrusion part of the system consists of several parts. A cartridge under pressure at the top is attached to a connector via a luer-lock adapter. The connector bridges the spaces between the outlet of the cartridge and the inlet of a solenoid valve. At the outlet of the solenoid valve, needles with different diameters can be used. The material is extruded onto a substrate that is held to a moving stage by vacuum. The major parts of the system are depicted in Figure 1. Other extrusion based systems can be used for the printing process, and the optimization process needs to be done for each system.
<ol>
	<li>Place the solenoid valve (nozzle diameter 0.3 mm) and the needle (inner diameter 0.15 mm) in separate 1.5 ml test tubes filled with ultrapure water and place them in a heated ultrasonic bath to clean for 30 min. Rinse the cleaned valves with ethanol and dry them with a nitrogen gun.</li>
	<li>Install the valve and needle in the printer as well as an empty, clean cartridge.</li>
	<li>Apply 3 bar pressure to the system and blow out any residual liquids from the installed valve and needle with compressed air. For small needle diameters, it is recommended to have a filter (common syringe filter, 0.45 &mu;m pore size) installed at the exit of the compressed air to avoid entry of small particles that could clog the needle.</li>
	<li>Turn the pressure off and install the cartridge loaded with the poloxamer. The cartridge should be taken out of the refrigerator approximately 30 min before mounting the cartridge so the poloxamer can reach room temperature and gel.</li>
	<li>Apply 3 bar pressure to the system and dispense poloxamer until it reaches the needle tip and is extruded in a continuous strand.</li>
</ol>
3. Optimization of the Printing Parameters
To create accurate 3D structures, the printing process has to be optimized for the chosen material and concentration. Depending on the viscosity and the 3D printing system each material will yield a specific dispensing volume and line thickness for a fixed set of parameters.
<ol>
	<li>With a suitable CAD software (able to create ISO files from the drawings), draw a single line about the same length as the structure that you intend to print.</li>
	<li>Place a microscope glass slide 25 mm x 75 mm x 1 mm or any other substrate in the printer and secure it by turning on the vacuum.</li>
	<li>In the printer software, set the solenoid valve to a high frequency of 50 Hz and set a high pressure of 3 bar.</li>
	<li>Print one layer of a single line with a stage speed of 300 mm/min.</li>
	<li>Reduce the pressure until the desired line width is reached. You can also control the volume that is extruded via the opening time of the valve.</li>
	<li>Reduce the frequency of the valve until no continuous line can be printed anymore. Choose a frequency above this value.</li>
</ol>
Note: Once the desired line width and continuous lines are achieved, determine the optimal stage speed and layer thickness i.e. the lift of the needle after one printed layer.
<ol start="7">
	<li>Print several layers on top of each other and see if the needle is in the right position above the previous layer after several printed layers. Adjust the layer thickness (needle lift) so that each layer is printed on top of the next one (Figure 3).</li>
	<li>Decrease the stage speed of the stage from 300 mm/min step-wise so that extruded layers start and end at the same positions as the previous ones (Figure 4). Too high stage speeds cause the stage to be moving before the extruded material has touched the previous layer.</li>
	<li>For printing the pillar structures follow steps 3.1.-3.8., but instead of drawing a single line draw a single point. The parameters to focus on when printing the pillars are the pressure (regulates layer thickness and pillar diameter of poloxamer), the opening time of the valve (extruded volume) and the residence time of the print head at the position where the pillar should be deposited.</li>
	<li>When the parameters are optimized, printing several layers of a line should result in a solid wall, or in case of the points, a pillar. Save the parameters for later use.</li>
</ol>
4. Printing and Elution of the Reverse Mold
Use the parameters found during the optimization procedure from this point on.
<ol>
	<li>Print the inner structure (here it is a pillar array) on a glass microscope slide and let it dry overnight. This a) reduces the size and thickness of the structures and b) provides better adhesion between the structure and the substrate, so lift-off during the backfilling can be avoided.</li>
	<li>With the CAD software, draw a structure that consists of an outer wall surrounding the structure you intend to have eluted away and filled. Print the structure with poloxamer. The printing of the wall will take 6 min.</li>
</ol>
Attention: The wall has to be printed at least 3.5 mm away from the inner structure because of the dimensions of the needle. Otherwise the printing of the outer wall will destroy the inner structure
<ol start="3">
	<li>Prepare the solution you want to backfill your sacrificial mold with (here 1% agarose in deionized water). The agarose solution should have a temperature between 35 &deg;C and 45 &deg;C. Beneath this temperature, the agarose will solidify too quickly; above this temperature, it might destroy the printed pillars because the poloxamer structure will soften.</li>
	<li>Slowly fill the sacrificial mold with the backfilling solution using a pipette. This should be done slowly to avoid destruction of the structure inside the wall.</li>
	<li>Let the backfilled solution gel or crosslink it depending on the polymer used. In the case of agarose the solidification took place at 4 &deg;C for 10 min.</li>
	<li>Place the backfilled sacrificial mold in an ice bath for 10 min to elute the poloxamer structure.</li>
	<li>Blot the backfilled structure with a paper tissue and place it on a new glass microscope slide. Press the structure carefully onto the glass microscope slide to avoid leakage of the third hydrogel from the void into the space between the backfilled structure and the glass microscope slide.</li>
</ol>
5. Filling of the Voids
<ol>
	<li>To fill the voids left by the eluted poloxamer, fill the intended polymer solution into a syringe equipped with a 30 G needle. In this example, we used a 1% alginate methacrylate in 0.15 M NaCl solution with the addition of 0.05% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 2.5% v/v of Alexa-488 conjugated fibrinogen. The Alexa-488 conjugated fibrinogen was added for visualization purposes.</li>
	<li>Photopolymerize the polymer with a high intensity UV lamp (100 Watt, 365 nm, distance from substrate was 3.5 cm) for 5 min and image the construct using an epi-fluorescence or confocal microscope.</li>
</ol>

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The authors have nothing to declare.

Here we present, for the first time, the use of a thermoresponsive polymer for a sacrificial mold that can be quickly eluted in cold water due to the gel-sol transition of poloxamer of ~20 &deg;C. The speed of the entire process makes poloxamer interesting for the rapid creation of biopolymer structures which cannot be printed with adequate resolution. The technique described here can be used for patterning one hydrogel within another hydrogel or for the creation of microfluidic channels as has been previously reported f...

Tissue engineering approaches have made much progress over the last years with respect to regeneration of human tissues and organs19,20. However, until now, the focus of tissue engineering has been often limited to tissues that have a simple structure or small dimensions such as the bladder21,22 or the skin23-25. The human body, however, contains many complex three-dimensional tissues where cells and extracellular matrix are arranged in a spatially defined manner. To manufacture these tissues, a technique is required that can place cells and extracellular matrix scaffolding within a three-dimensional construct at specified positions. Bioprinting has the potential to be such a technique where the vision of manufacturing complex three-dimensional tissues can be realized10,11,26-28.
Bioprinting is defined as "the use of material transfer processes for patterning and assembling biologically relevant materials - molecules, cells, tissues, and biodegradable biomaterials - with a prescribed organization to accomplish one or more biological functions"4. It encompasses several different techniques that work at different resolutions and length scales, ranging from the sub-micron resolution of two-photon polymerization29 to a resolution of 150 &mu;m to 420 &mu;m for extrusion printing1,12,30. Not a single material or material combination will satisfy the requirements of each method31. For extrusion printing, the key parameters are viscosity and gelation time32, where high viscosity and rapid gelation are desirable.
3D printing is a technique which allows the easy creation of sacrificial molds for creating complex geometries30,33,34. This process is based on the construction of a mold using a rapid prototyping technique such as an extrusion bioprinter. The created sacrificial mold is used to form complex structures from materials which are difficult to print due to their low viscosity and slow gelation time. The method presented here involves the creation of a sacrificial mold consisting of a material that dissolves quickly at low temperature and can be extruded accurately. The block copolymer poly(ethylene glycol)99-poly(propylene glycol)67-poly(ethylene glycol)99 (also known as Pluronic F127 or poloxamer 407) fulfills these requirements. It has already been used in a modified version in extrusion printing1 but, to our knowledge, has never been used for printing in its unmodified version due to its instability in liquid environments. Poloxamer 407 also shows an inverse thermal responsive behavior18 i.e. it changes from a gel to a sol upon cooling. Most importantly, it can be printed into complex arbitrarily curved structures with very high fidelity. This allows the creation of a structured hydrogel from a low viscosity material, in this case slow gelling agarose, by pipetting the solution into the printed sacrificial mold. The combination of printing the sacrificial mold with high fidelity and its quick elution from the casted structured hydrogel makes it a fast and flexible method to create molds with different geometries without the use of a mask or a stamp as it is often required in lithographic methods. The casted structured hydrogel can be further filled with another material that is not suitable for extrusion printing due to its low viscosity. This is in our case a low viscosity alginate methacrylate solution. Here we present the method of thermoresponsive reverse sacrificial molds for hydrogel patterning using the example of a pillar array.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Poloxamer (Pluronic F127)</td><td>Sigma</td><td>P2443</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>&lt;u&gt;&lt;a href=&quot;http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;amp;productID=10010015&quot; target=&quot;_blank&quot;&gt;10010-015&lt;/a&gt;&lt;/u&gt;</td><td></td></tr><tr><td>CAD software</td><td>regenHU</td><td>BioCAD</td><td></td></tr><tr><td>Alginate methacrylate</td><td>Innovent e.V Technologieentwicklung Jena</td><td></td><td>Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td></tr><tr><td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td><td>Invitrogen</td><td>F13191</td><td></td></tr><tr><td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td><td>Innovent e.V Technologieentwicklung Jena</td><td></td><td>Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Bioprinter</td><td>regenHU</td><td>Biofactory</td><td></td></tr><tr><td>Valve</td><td>regenHU</td><td>300 &amp;#956;m Nozzel Diameter</td><td></td></tr><tr><td>Needle</td><td>regenHU</td><td>150 &amp;#956;m Inner Diameter</td><td></td></tr><tr><td>Zeiss Axioobserver with ApoTome</td><td>Zeiss</td><td></td><td></td></tr><tr><td>UV Light Source</td><td>UVP</td><td>Blak-Ray B-100AP High Intensity UV Lamp</td><td>100 W</td></tr></tbody></table>

printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

The representative results show that the reverse mold technique (depicted in Figure 2) will create a structured gel that can be filled with a second material. At the beginning of every printing process the printing parameters are first optimized. Step-wise adjustments of the parameters will result in printed multilayered constructs depicted in Figure 3 and Figure 4 when single lines are printed. If the layer thickness (the needle lift after one printed layer) is too low,...

Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

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

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.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

printing-thermoresponsive-reverse-molds-for-creation-patterned-two

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.0K Views.  Cartilage Engineering & Regeneration. 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.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Bioengineering

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

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 ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

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

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

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

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

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...