The research leading to these results has received funding from the European Research Council under grant agreement ERC-2010-StG-260991.

The detailed procedure for operating with the volumetric hand-held optoacoustic probe is described below. This procedure is performed according to approved institutional regulations regarding animal and human experiments.
1. System Preparation
<ol>
	<li>Switch on the laser for a warm-up period of ~15&#160;min prior to operation for stabilizing the output light beam.</li>
	<li>Place the water enclosing part with the isolating membrane that is in contact with the skin (Figure 1). 
	NOTE: The distance between the isolating membrane (in contact with the skin) and the region with maximum sensitivity of the transducer (center of the spherical probe) establishes the effective imaging depth.</li>
	<li>Fill the whole volume of approximately 100&#160;ml between the isolating membrane and the surface of the transducer with deionized water by means of a pump.</li>
	<li>Ensure that no water leakage nor air bubbles are present. Alternatively, avoid air bubbles by providing recirculation of water.</li>
	<li>Perform the experiments at RT and ensure the coupling medium (water) is maintained at this temperature.</li>
</ol>
2. Imaging Preparation
<ol>
	<li>Human imaging preparation.</li>
	<li>Remove hair from the to-be-imaged part with a depilation lotion in order to avoid an undesired background in the images (this step is optional).</li>
	<li>Apply ultrasound gel onto the skin around the region to be imaged in order to provide efficient acoustic coupling. Place the optoacoustic probe in the region of interest. Ensure that no air bubbles are present in the ultrasound coupling gel.</li>
	<li>Animal imaging preparation.</li>
	<li>Ensure that the care and experimental procedures with animals are in agreement with institutional and government rules and regulations.</li>
	<li>Remove the fur of the animal in the region to be imaged with a shaving lotion. Protect the eyes of the animal with vet ointment, which prevents dryness and damage from exposure to intense pulsed laser radiation.</li>
	<li>Anesthetize the animal by using intraperitoneal injection (IP) of ketamine/xylazine (100&#160;mg/kg KG Ketamine + 5&#160;mg/kg KG Xylazine) prior to the experiment or use isoflurane anesthesia (2-3&#160;% (by volume) with 0.9&#160;l/min gas flow) during the experiment. Confirm anesthetization by checking the reflex of the hind limb of the animal.</li>
	<li>Apply ultrasound gel onto the skin around the region to be imaged in order to provide efficient acoustic coupling and place the optoacoustic probe in the region of interest. Ensure that no air bubbles are present in the ultrasound coupling gel.</li>
</ol>
3. Pre-view Operation Mode
<ol>
	<li>Establish the imaging wavelength(s) between 690&#160;nm and 900&#160;nm and the pulse repetition rate between 10 and 50&#160;Hz. Select the parameters for the acoustic data acquisition system &#8211; 1&#160;M&#937; input impedance. Acquire&#160;2030 samples for each laser pulse at a sampling rate of 40&#160;megasamples per second and 12&#160;bit vertical resolution. Trigger the acquisition with Q-switch output of the laser.</li>
	<li>Ensure that both the operator and the patient use protective goggles adapted to the optical excitation wavelength(s). Set the laser power so that the light fluence at the tissue surface is maintained below 20&#160;mJ/cm2 during the experiment for near-infrared wavelengths in order to satisfy safety exposure limits for human experiments19 and to prevent thermal strain and skin damage in animals.</li>
	<li>Start the pre-view software with a GPU implementation of processing algorithms to allow visualizing three-dimensional images at a frame rate corresponding to the pulse repetition rate of the laser.</li>
	<li>Move the probe and/or the object to be imaged in order to optimize visualization performance and localize the structures of interest.</li>
</ol>
4. Data Acquisition
<ol>
	<li>Data acquisition for scanning (hand-held) mode.</li>
	<li>If necessary, inject a contrast agent prior to acquisition to enrich the contrast in the region of interest. 
	NOTE: In our experiments we have not performed contrast-enhanced human imaging. Yet, various contrast agents can be potentially used for this purpose. Indocyanine Green (ICG) is one example of clinically-approved optical contrast agent that can be used for contrast enhancement at maximal recommended dose of 2 mg/kg of body weight in adults.</li>
	<li>Start the hardware for data acquisition with the parameters described in 3.1 maintaining the execution of the preview software. Gently move the probe around the imaged region to track the structures of interest. 
	NOTE: When images at multiple laser wavelengths are acquired simultaneously, the speed of probe motion in the hand-held mode has to be decreased significantly (preferably below 2&#160;mm/sec for a laser pulse repetition rate of 50&#160;Hz) in order to avoid motion-related artifacts in the spectrally unmixed images.</li>
	<li>Data acquisition for stationary mode.</li>
	<li>Mount the imaged object (e.g., animal) and the hand-held probe onto the holder and start the acquisition with the parameters described in 3.1 maintaining the execution of the pre-view software.</li>
	<li>Maintain the optoacoustic probe and the imaging part in the same position during the experiment to visualize dynamic biological events in the region of interest.</li>
	<li>Inject a contrast agent to track its dynamic distribution in the region of interest. 
	NOTE: In our mouse experiments, Indocyanine Green (ICG) was used for contrast enhancement. As a general guideline, an amount of 10 nmol or 0.4 mg/kg of ICG has to be introduced into the mouse circulation in order to create detectable contrast with multi-spectral optoacoustics in&#160;vivo. 
	NOTE: The contrast agent must be approved for human and/or animal use by the respective authority.</li>
</ol>
5. Finalizing the Experiment
<ol>
	<li>Stop the laser.</li>
	<li>Remove the optoacoustic probe from the imaged region. For animal study, stop the anesthesia supply.</li>
	<li>Position the animal under an infrared heater to keep it warm and prevent the contact with other animals until it fully recovered from the anesthesia. Do not leave the animal unattended during recovery from the anesthesia.</li>
</ol>
6. Off-line Data Processing
<ol>
	<li>Load the file(s) containing the acquired optoacoustic signals in the software application used for data processing.</li>
	<li>Use a reconstruction algorithm to obtain a three-dimensional matrix array corresponding to a volumetric image of the optical absorption for each frame and each wavelength. 
	NOTE: For the reconstruction it is preferable to use an algorithm accounting for distorting factors, such as heterogeneities and attenuation in the imaged object, effects of final bandwidth and geometrical shape of the detection elements and light fluence variations, in order to obtain a more quantitative representation of the distribution of the absorbed energy.</li>
	<li>Use an unmixing algorithm to obtain, from each multi-wavelength frame, a new set of three-dimensional matrix arrays representing the optical absorption for each absorbing substance present in the sample.</li>
	<li>If needed, further process the matrix arrays representing the optical absorption distribution to facilitate visualization and reading of biologically relevant parameters.</li>
</ol>

<ol>
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The authors have nothing to disclose.

The unique advantages offered by optoacoustic imaging techniques in small animal research have created strong motivation for translating the technology into clinical practice, with a number of diagnostics and treatment monitoring applications envisioned e.g., in breast and skin cancer, inflammation or peripheral vasculature diseases. However, as opposed to mice or smaller animals, which can be surrounded by a sufficient number of illumination sources and detection elements to create an effective tomographic imag...

Optoacoustic (photoacoustic) imaging attracts growing interest from the biological and medical research communities, as manifested by the ever increasing number of publications encompassing variety of new applications that exploit the unique advantages offered by the technology 1-5. In particular, the capacity to image spectrally distinctive photo-absorbing agents with high spatio-temporal resolution at depths far beyond the diffusive limit of light opens unprecedented capabilities for functional and molecular imaging 6-10.
Indeed, translation of the optoacoustic technology into clinical practice comes with promising prospects in diagnostics and treatment monitoring of many diseases. Yet, the limited propagation of photons in optically scattering and absorbing tissues and the generally weak responses associated with the optoacoustic phenomenon limit the applicable depth of the method. As a result, hand-held optoacoustic probes have been attempted to image parts accessible from outside of the body 11,12 while endoscopic systems are used to provide images from within the body by inserting them via natural orifices 13. Some low absorbing parts of human body, such as female breast, are also accessible by tomographic optoacoustic scanners 14,15. Of particular interest is the hand-held approach as it enables large versatility, similarly to ultrasonography. Here, adaptation of the common ultrasound linear array probes for optoacoustic imaging remains challenging, mainly due to fundamental differences in tomographic imaging requirements between ultrasound and optoacoustics. While high frame rates in standard ultrasonography are enabled by sequential transmit-receive schemes employing high pulse repetition frequencies in the kHz range, real-time three-dimensional optoacoustic imaging is achieved by simultaneous collection of volumetric tomographic data from a single interrogating laser pulse. Thus, high quality optoacoustic imaging implies acquisition of three-dimensional data from the largest possible solid angle around the imaged object.
Recently, we introduced the first handheld optoacoustic probe for three-dimensional (volumetric) imaging in real time16. The system is based on a two-dimensional array of 256 piezoelectric elements arranged upon a spherical surface (blue dots in Figure&#160;1A) covering an angle of 90&#176;. The size of the individual elements of approximately 3 x 3&#160;mm2, as well as their orientation and frequency bandwidth (approximately 2-6&#160;MHz) guarantee effective signal collection from a centimeter-scale volume surrounding the center of the sphere (black cube in Figure&#160;1A). Optical excitation of the imaging region is provided with a fiber bundle inserted through a central cylindrical cavity of the array, so that any wavelength susceptible of being transmitted through the fiber bundle can be used for imaging. An actual picture of the array of transducers along with the optical fiber bundle is shown in Figure&#160;1B. The efficient excitation and simultaneous detection of signals allows deep-tissue imaging with single-shot excitation (one laser pulse), so that real-time imaging at a frame rate determined by the pulse repetition frequency of the laser is further enabled with a graphics-processing-unit (GPU) implementation of the reconstruction procedure 17. A cylindrical casing with a transparent polyethylene membrane (Figure&#160;1C) is attached to the transducer array to enclose an acoustically transmitting liquid medium (water). The membrane is further coupled to the tissue by means of acoustic gel. A picture of the optoacoustic probe as being used in hand-held operation mode is shown in Figure&#160;1D.
The demonstrated three dimensional hand-held optoacoustic imaging combined with the real time functional imaging capacity come with important advantages for clinical diagnostics and a number of potential applications are envisioned for various indications, such as peripheral vascular disease, lymphatic system disorders, breast cancer, skin lesions, inflammation or arthritis 18. Furthermore, the fast imaging capacity enables visualization of dynamic biological events with the probe arranged in a stationary position. Combined with fast wavelength-tuning optical parametric oscillator (OPO) laser technology, this approach allows for real-time imaging of biodistribution of photo-absorbing agents. Thereby, new possibilities may equally emerge in small animal imaging applications, e.g., in studying tissue hemodynamics, in vivo cell tracking, visualization of pharmacokinetics, organ perfusion, targeted molecular imaging of tumors and cardiovascular system, or neuroimaging.
In this work we provide a detailed description of the experimental imaging protocol to operate with the spherical array optoacoustic hand-held probe and showcase performance in several typical clinical and small animal imaging scenarios.

<table border="0" cellpadding="0" cellspacing="0" width="1663">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Name of Material/ Equipment</td>
			<td style="width:389px;">Company</td>
			<td style="width:171px;">Catalog Number</td>
			<td style="width:777px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Optical parametric oscillator (OPO)-based laser</td>
			<td style="width:389px;">Innolas Laser GmbH, Krailling (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The laser provides laser pulses with a duration around 10ns and an energy up to 80mJ. 
			The wavelength is tunable between 680-950nm.</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:327px;">Spherical array of piezocomposite detectors</td>
			<td style="width:389px;">Imasonic SaS, Voray (France)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The array consists of 256 piezoelectric sensors distributed on a spherical surface. 
			Each element has dimensions 3x3mm2, a central frequency of 4MHz and a bandwidth of 100%.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data acquisition system (DAQ)</td>
			<td style="width:389px;">Falkenstein Mikrosysteme GmbH, Taufkirchen (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The DAQ simultaneously acquires 256 signals at 40 megasamples per second and 2030 samples. 
			The input impedance is 1MW.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Fiber bundle</td>
			<td style="width:389px;">CeramOptec GmbH, Bonn (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The bundle consists of 480 individual fibers randomly distributed in the input and output. 
			The numerical aperture of each individual fiber is 0.22.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Athymic Nude mouse</td>
			<td style="width:389px;">Harlan Laboratories (The Netherlands)</td>
			<td style="width:171px;">Athymic nude - Foxn1nu</td>
			<td style="width:777px;">The mouse was 8 weeks old (adult) at the time of the experiment. 
			The ethical protocol was approved by the Bavarian goverment (number 55.2.1.54-2632-102-11)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bepanthen cream</td>
			<td style="width:389px;">Bayer AG (Germany)</td>
			<td style="width:171px;">N/A</td>
			<td>Vet ointment to protect the eyes during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data processing software</td>
			<td style="width:389px;">Matlab (Mathworks, Natick, MA, USA)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The data processing software was devoped at our institute. 
			It allows reconstruction at each wavelength and multi-wavelength unmixing, as well as further data processing.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:327px;">Water-enclosing part</td>
			<td style="width:389px;">N/A</td>
			<td style="width:171px;">custom-made</td>
			<td>This part contains the water that acts as an acoustic coupling medium between skin and transducer elements</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Indocyanine green (ICG)</td>
			<td style="width:389px;">PULSION Medical Systems SE</td>
			<td style="width:171px;">N/A</td>
			<td style="width:777px;">ICG-PULSION (active ingredient: indocyanine green dye) is a drug used in cardiac, circulatory and micro-circulatory diagnostics, liver function diagnostics and ophthalmic angiography diagnostics.</td>
		</tr>
	</tbody>
</table>

universal hand-held three-dimensional optoacoustic imaging probe for deep tissue human angiography and functional preclinical studies in real time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

Representative results, demonstrating the capabilities of the described volumetric hand-held optoacoustic probe, are showcased in this section. In all cases, the light fluence on the skin surface was kept below the safety exposure limit of 20&#160;mJ/cm2 19.
The performance of the probe in real-time tracking peripheral human vasculature is showcased in Figure&#160;2. During the course of this experiment, the probe was slowly scanned along the hand of a he...

Watch this Scientific Journal Video about Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time at JoVE.com

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

universal-hand-held-three-dimensional-optoacoustic-imaging-probe-for

Research

JoVE Journal

Bioengineering

10.6K Views.  Helmholtz Zentrum München. We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Video: Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

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 ...

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.

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 ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

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 ...

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 ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

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 ...