This material is based on work supported by the U.S. Department of Energy, Materials Sciences and Engineering Division (Award No. DEFG-02-07ER46471) and the DOE Energy Research Center on Light-Materials Interactions in Energy Conversion (Award No. DE-SC0001293), and benefitted from access to the Center for Microanalysis of Materials within the Frederick Seitz Materials Research Laboratory (FSMRL).

1. Introduction
<ol>
	<li>This paper demonstrates the 1D, 2D, and 3D printing of conductive microelectrodes via direct-write assembly.</li>
	<li>Direct-write assembly is a method of building up 1D-to-3D printed structures by the deposition of concentrated inks through fine nozzles.</li>
	<li>Our system consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for imaging (Figure 1).</li>
	<li>Direct-write assembly is a filamentary printing approach in which concentrated inks are extruded through cylindrical nozzles, whose diameters range from 0.1 to 250 &mu;m (Figure 2). Notably, due to the viscoelastic ink characteristics, direct-write assembly enables self-supporting spanning features (Figure 3). To date, a wide range of inks, including those composed of ceramic6,7, organic8-10, metallic11-15, polymeric16,17, and sol-gel18,19 materials have been developed for this printing approach (Figure 4).</li>
</ol>
2. Preparation of highly concentrated silver nanoparticle inks
<ol>
	<li>Silver nanoparticle inks are prepared by first dissolving a blend of 5,000 and 50,000 molecular weight of poly(acrylic acid) in a mixture of 50 g water and 40 g diethanolamine (Video 2.1).</li>
	<li>The polymer acts as a capping agent to control the size of the silver nanoparticles.</li>
	<li>Next, an aqueous solution of silver nitrate is injected into the polymer solution. After addition, a light yellow transparent solution is obtained (Video 2.2).</li>
	<li>After stirring for 24 hrs at room temperature, the solution develops a reddish-brown color (Video 2.3), that coincides with the formation of silver nanoparticles of 5 nm diameter, as determined by transmission electron microscopy.</li>
	<li>Next, the solution is sonicated at 65 &deg;C in a water bath for 2 hrs for further particle growth (Video 2.4).</li>
	<li>After sonication, the solution is transferred to a 500 ml beaker and cooled to room temperature. Then, 300 ml of ethanol is titrated at a rate of 30 ml / min. Since ethanol is a poor solvent for the poly(acrylic acid) capping agent, the particles rapidly coagulate and precipitate from solution (Video 2.5).</li>
	<li>After decanting the supernatant, the precipitate is collected into a centrifuge tube and centrifuged at 9000 rpm for 20 minutes (Video 2.6).</li>
	<li>After this step, a highly concentrated silver nanoparticle ink with a solids loading of ~85 wt% is obtained (Video 2.7).</li>
	<li>Further control over ink viscosity and elastic modulus can be achieved by dilution, followed by homogenization. For an example, a humectant solution, such as ethylene glycol, can be added to the ink and then homogenized at 2000 rpm for 3 minutes using a Thinky homogenization mixer. After this process, a uniform ink of a bluish to magenta color is obtained (Video 2.8).</li>
	<li>The TEM image shows silver nanoparticles obtained by this synthesis procedure (Figure 5_left). The particles have a mean diameter of 20 nm with a size distribution of 5-50 nm. Printed structures require post-annealing to enhance their conductivity. After annealing at 250&deg;C for less than 30 min, the silver nanoparticles form conductive microelectrodes with an electrical resistivity approaching to 10-5 &Omega;&bull;cm (Figure 5_bottom right). The microstructural evolution of the printed silver microelectrodes as a function of annealing temperature is shown in Figure 5_top right. As the temperature increases from 150&deg; to 550&deg;C, the microelectrodes undergo densification with a total volumetric shrinkage of ~30%.11</li>
	<li>The ink rheology, which strongly depends on its solids loading, determines its printability. The ink viscosity increases with increasing solids loading (Figure 6). Because dilute inks with low viscosity result in a significant lateral spreading, concentrated inks with a solids loading ranging from 70 to 85 wt% are required for printing of planar and spanning ink filaments.</li>
	<li>The ink elastic modulus increases with increasing solids loading (Figure 7). In the linear viscoelastic region, the elastic modulus rises nearly three orders of magnitude as the solids loading increases 60 to 75 wt%. A minimum elastic modulus of 2000 Pa is required to produce self-supporting or spanning features.</li>
</ol>
3. Direct-write assembly
<ol>
	<li>Direct-write assembly is carried out by first loading the ink into a syringe barrel. After attaching a deposition nozzle, the ink-loaded syringe barrel is mounted onto the 3-axis printing stage (Video 3.1).</li>
	<li>Using a computer program, arbitrary designs, including linear, planar, and complex three-dimensional structures can be easily generated (Video 3.2).</li>
	<li>Next, the nozzle height is adjusted with the aid of telescope lens with a 10x zoom (Video 3.3).</li>
	<li>After applying pressure using an air-powered fluid dispensing system, the ink is deposited onto the substrate with a controlled printing speed (Video 3.4). The required pressure depends upon ink rheology, nozzle diameter, and printing speed, but typical values range from 10-100 psi at 20-500 &mu;m/s. This printing is performed in air at room temperature. Using this printing procedure, the printing of silver microelectrodes in different layouts and size scales is demonstrated.</li>
	<li>For an example, the printing of conductive silver grids with a center-to-center line spacing of 100 &mu;m, patterned by a 5-&mu;m nozzle on a silicon wafer substrate is demonstrated (Video 3.5).</li>
	<li>In addition, this video shows how to create a high aspect ratio cylindrical structure by a 30-&mu;m nozzle using a layer-by-layer printing method (Video 3.6).</li>
	<li>Furthermore, the omnidirectional printing of silver microelectrodes between two glass substrates offset by a 1-mm height difference is demonstrated using a 30-&mu;m nozzle (Video 3.7).</li>
	<li>Completely free-standing, vertically printed silver microspikes can be created by a 30-&mu;m nozzle on a Si wafer substrate (Video 3.8).</li>
	<li>Finally, this video shows direct writing of a spanning silver microelectrode using a 10-&mu;m nozzle (Video 3.9). The printed feature can span distances up to one centimeter with minimal drooping or buckling.</li>
</ol>
4. Representative results:
We prepared a highly concentrated silver nanoparticle inks and demonstrated printed conductive features in planar and 3D motifs for electronic and optoelectronic applications with printing resolution ~ 2 - 30 &mu;m. For an example, Figure 8 exhibits the printing resolution of this technique. Printed features with minimum electrode width of ~ 2 &mu;m (1.4 &mu;m thick) are obtained in a single pass using a 1-&mu;m nozzle11.
Figure 9 shows transparent conductive silver grids, patterned by a 5 &mu;m nozzle on a flexible polyimide film12. The texts underneath the printed grids are clearly visible. These transparent silver grids could be attractive alternatives for transparent conducting oxide (TCO) materials.
Conformal printing onto a non-planar substrates is also enabled by this method. Figure 10 demonstrates the conformal printing of a 3D electrically small antenna. A 100 &mu;m metal nozzle is used to print meander-line patterns on the surface of a glass hemisphere13. This approach may find several applications including implantable and wearable antennas, electronics, and sensors.
Applications of spanning silver microelectrodes in three-dimensional photovoltaics and light-emitting diodes are demonstrated (Figure 11-14).
First, Figure 11 is an example of silicon spherical shell. This tenuous film with a 2-&mu;m thickness can be wire-bonded to an external circuit by omnidirectional printing14. This method uses minimal contact pressure, which is highly advantageous for delicate devices.
Next, Figure 12 demonstrates an example of printing a spanning interconnect for a silicon solar microcell array in which silicon microribbon elements are separated by 33-&mu;m gap15.
Next, Figure 13 shows silver interconnects for the gallium arsenide-based LED array with 4-by-4 pixels, where each pixel (500 x 500 x 2.5 &mu;m3) is spaced 200 &mu;m apart11. The bottom image displays the LED array, emitting uniform red light under an applied bias of 6 V from a single pixel. The ability to print spanning electrodes enables multilayer interconnection without the use of supporting or sacrificial layers (top images).
As a final demonstration, Figure 14 shows SEM images for the complex 3D microperiodic silver lattice printed by a 5&mu;m nozzle.
<img alt="Figure 1" src="/files/ftp_upload/3189/3189fig1.jpg" /> 
Figure 1. Optical image of direct ink writing apparatus.
<img alt="Figure 2" src="/files/ftp_upload/3189/3189fig2.jpg" /> 
Figure 2. Direct ink writing of a filamentary feature.
<img alt="Figure 3" src="/files/ftp_upload/3189/3189fig3.jpg" /> 
Figure 3. Direct ink writing of self-supporting spanning features.
<img alt="Figure 4" src="/files/ftp_upload/3189/3189fig4.jpg" /> 
Figure 4. Ink designs for direct ink writing. A wide range of concentrated viscoelastic inks have been developed for direct writing of planar and complex 3D structures with microscale features.
<img alt="Figure 5" src="/files/ftp_upload/3189/3189fig5.jpg" /> 
Figure 5. (Left) Transmission electron microscopy (TEM) image of silver nanoparticles. (Top right) SEM images of silver microelectrodes patterned with a 15-&mu;m nozzle as a function of annealing temperature. (Bottom right) Electrical resistivity of silver microelectrodes as a function of annealing temperature and time.
<img alt="Figure 6" src="/files/ftp_upload/3189/3189fig6.jpg" /> 
Figure 6. Apparent viscosity (&eta;) of the silver nanoparticle inks as a function of solids loading.
<img alt="Figure 7" src="/files/ftp_upload/3189/3189fig7.jpg" /> 
Figure 7. Shear elastic modulus (G') as a function of shear stress for silver nanoparticle inks of varying solids loading.
<img alt="Figure 8" src="/files/ftp_upload/3189/3189fig8.jpg" /> 
Figure 8. SEM images of planar arrays of silver microelectrodes patterned on a Si wafer with a 1-&mu;m nozzle.
<img alt="Figure 9" src="/files/ftp_upload/3189/3189fig9.jpg" /> 
Figure 9. Optical image of transparent conductive silver grids (left) and SEM images of the grids printed as a function of line pitch (right).
<img alt="Figure 10" src="/files/ftp_upload/3189/3189fig10.jpg" /> 
Figure 10. Optical image captured during conformal printing of electrically small antennas on a hemispherical glass substrate.
<img alt="Figure 11" src="/files/ftp_upload/3189/3189fig11.jpg" /> 
Figure 11. Optical image obtained during printing of spanning silver microelectrodes onto a thin (2-&mu;m) silicon spherical shell.
<img alt="Figure 12" src="/files/ftp_upload/3189/3189fig12.jpg" /> 
Figure 12. SEM image of a spanning silver microelectrode printed onto a silicon solar microcell array.
<img alt="Figure 13" src="/files/ftp_upload/3189/3189fig13.jpg" /> 
Figure 13. SEM images (top) and optical image (bottom) of a 4-by-4 LED chip array interconnected by silver microelectrodes.
<img alt="Figure 14" src="/files/ftp_upload/3189/3189fig14.jpg" /> 
Figure 14. SEM image of 3D microperiodic silver lattice.

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No conflicts of interest declared.

Conventional droplet-based printing approaches, such as inkjet printing, are limited to the fabrication of planar electrodes with low aspect ratio due to the dilute nature and low viscosity of the inks used. Recently, dip-pen nanolithography (DPN)20-22 and e-jet printing23-25 have been used to pattern conductive features. These routes also employ dilute, low viscosity inks. Pearton and co-workers used DPN to deposit a commercially available silver nanoparticle ink at write speeds of up to 1600 &mu;m s-1 and line widths of approximately 0.5 &mu;m22. However, fabrication of reproducible patterns over large areas has yet to be demonstrated by this approach. Silver nanoparticle inks have also been deposited by e-jet printing to form conductive traces with line widths of ~ 1.5 &mu;m25. However, as with inkjet printing, inhomogeneous printed features may arise due to satellite drop formation and non-uniform drop drying24,25.
As demonstrated above, direct-write assembly of concentrated silver nanoparticle inks overcomes these limitations through a filamentary-based printing approach. This technique enables the fabrication of conductive microelectrodes with a high aspect ratios (h/w &asymp; 1.0) in a single passes allowing the creation of 1D, 2D, and 3D architectures. The size of the printed features depends upon nozzle diameter, ink solids loading, applied pressure, and printing speed. To date, conductive traces as small as ~ 2 &mu;m have been patterned using a 1 &mu;m nozzle at modest speeds (&lt; 2 mm s-1). By tailoring the ink composition and nozzle geometry, maximum printing speeds in excess of 10 cm s-1 are possible. However, high speed printing of using fine nozzles (&lt; 5 &mu;m) remains a significant challenge.
To demonstrate applications of direct-write assembly, we fabricated conductive grids, electrically small antennas, solar cells, and light-emitting diodes with planar and spanning printed electrodes (Figure 8-14). Notably, our approach is not restricted to the creation of metallic structures. Using other ink designs, such as those based on silk fibroin, hydrogel and fugitive organic inks, we have constructed 3D scaffolds and microvascular networks for tissue engineering and cell culture via direct-write assembly26-30.
Looking towards the future, there are many opportunities and challenges. Further advances require new ink designs, better modeling of ink flow dynamics, and enhanced robotic and control systems. Large-area fabrication of 1D to 3D structures with high throughput and nanoscale resolution (&lt; 100 nm) remains a significant challenge.

<table border="1">
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments </td>
</tr>
<tr>
<td>Poly(acrylic acid)</td>
<td>Polysciences, Inc.</td>
<td>06519</td>
<td>m.w. 5,000 g/mol</td>
</tr>
<tr>
<td>Poly(acrylic acid)</td>
<td>Polysciences, Inc.</td>
<td>00627</td>
<td>m.w. 50,000 g/mol</td>
</tr>
<tr>
<td>Silver nitrate</td>
<td>Sigma-Aldrich</td>
<td>209139</td>
<td>Silver source</td>
</tr>
<tr>
<td>Diethanolamine</td>
<td>Sigma-Aldrich</td>
<td>D8885</td>
<td>Solvent/Reducing agent</td>
</tr>
<tr>
<td>Ethylene glycol</td>
<td>Sigma-Aldrich</td>
<td>102466</td>
<td>Humectant</td>
</tr>
<tr>
<td>Sonicater</td>
<td>Fisher Scientific</td>
<td>FS30H</td>
<td>-</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Beckman Coulter</td>
<td>AvantiTM J-25 I</td>
<td>-</td>
</tr>
<tr>
<td>Robotic stage</td>
<td>Aerotech Inc.</td>
<td>ABL 900010</td>
<td>3-axis motion</td>
</tr>
<tr>
<td>Syringe barrel</td>
<td>EFD Inc.</td>
<td>5109LBP-B</td>
<td>3 ml</td>
</tr>
<tr>
<td>Nozzle</td>
<td>EFD Inc.</td>
<td>-</td>
<td>i.d. = 0.1 - 250 &mu;m</td>
</tr>
<tr>
<td>Dispenser</td>
<td>EFD Inc.</td>
<td>800</td>
<td>Air-powered</td>
</tr>
<tr>
<td>Design software</td>
<td>Custom designed</td>
<td>-</td>
<td>Mingjie Xu</td>
</tr>
</table>

planar and three-dimensional printing of conductive inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Watch this Scientific Journal Video about Planar and Three-Dimensional Printing of Conductive Inks at JoVE.com

Planar and Three-Dimensional Printing of Conductive Inks

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

planar-and-three-dimensional-printing-of-conductive-inks

Research

JoVE Journal

Bioengineering

36.9K Views.  University of Illinois at Urbana-Champaign. Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Video: Planar and Three-Dimensional Printing of Conductive Inks

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...