The work presented here is supported by the Singapore National Research Foundation under CREATE program (NRF-Technion): The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research Program and by the Public Sector Funding (PSF) 2012 from the Science and Engineering Research Council (SERC) under the Agency for Science, Technology and Research (A*STAR).

NOTE: This protocol describes the use of a back-pressure assisted robotic dispensing system (Figure 1A) as a surface etching (Figure 1B) and extrusion-based bioprinter (Figure 1C)10.
1. Modification of a Polystyrene Surface
<ol>
	<li>Use 1 mm polystyrene sheets since polystyrene tissue culture plates tend to bow upwards in the center, ruining the height consistency of both etching and printing. 
	NOTE: As the polystyrene sheets are not modified for cell adhesion, plasma treatment is performed.</li>
	<li>Oxygen plasma treatment.
	<ol>
		<li>First, select a pressure of 2 bar on the regulator of the oxygen cylinder connected to the plasma machine. Then switch the plasma machine on and input the following conditions into the machine&#39;s control panel: 150 W, 30 sscm of oxygen, 10 min (for power, gas flow and time respectively).</li>
		<li>Place the polystyrene substrate into the plasma chamber, seal the door and press the start button on the control panel. Soak the oxygen plasma treated polystyrene substrate in fetal bovine serum and incubate at 37 &#176;C for 2 hr before washing three times 1x phosphate buffered saline (PBS).</li>
	</ol>
	</li>
</ol>
2. Programming Print Patterns
<ol>
	<li>Use the Program to First Etch the Surface with a Stylus or Scalpel.
	<ol>
		<li>When using a stylus, insert a 1.5 mm diameter textile needle (from the inside with great care) into the nozzle of a dispensing syringe (either 5 or 10 ml) until it becomes wedged and secured. If using a scalpel, choose a round handled scalpel so that it may be fastened into the clamp mechanism of the printing arm. 
		NOTE: The stylus etches curved patterns better than the scalpel blade.</li>
		<li>When first attempting to create a bioprinted arrangement, sketch the desired pattern on graph paper with numbered axes to generate the X-Y coordinates. Then, input the coordinates of etched/bioprinted pattern into the spreadsheet software (Figure 2). 
		NOTE: The &#34;desired pattern&#34; can be in many shapes such as linear, S-shaped, or circular. The choice depends on the experimental model required, such as parallel linear lines for the differentiation of MSCs to cardiomyocytes as demonstrated here. The scatter graph function allows the visualization of the proposed plot pattern.</li>
		<li>Open the print/dispensing software. Select &#34;Program &#62; Add Program&#34; followed by &#34;Edit &#62; Add Point&#34; to set up the program. Export the x and y coordinate values obtained from the spreadsheet into the print/dispensing software using the &#34;Copy and Paste&#34; function.</li>
		<li>Calibrate the &#34;z&#34; height of the robot before each run in order to place the stylus/print nozzle onto the surface.
		<ol>
			<li>In the print/dispensing software, select the &#34;Robot&#34; option, click on &#34;Changing Mode&#34; and enable the &#34;Teaching mode&#34; option. Once selected, the software enables the &#34;Jog&#34; function of the robot.</li>
			<li>To Jog, first initialize the robot to its default position by selecting the following commands from the menu bar; &#34;Robot &#62; Meca Initialize&#34;, then select &#34;Robot &#62; Jog&#34;. Input the numerical values (in mm) in the &#34;X and Y slots&#34; required to place the stylus exactly on the origin of the pattern.</li>
			<li>Next, input a numerical value (in mm) in the &#34;Z slot&#34; to place the stylus or printing nozzle in contact with the surface but not flex or indent the surface. This point is designated as &#34;Z&#34; starting value. 
			NOTE: The depth of each groove can be varied easily using the Z-height of the system. Grooves of 40, 80, and 170 &#181;m are demonstrated to cut into the surface of 1 mm thick polystyrene sheets (Figure 4 and Table 1).</li>
		</ol>
		</li>
		<li>Select the print instruction for each of the coordinate points, i.e., &#34;Start of Line Dispense&#34; to define the first point and print initiation, &#34;Line Passing&#34; to designate the intermediate points and &#34;End of Line Dispense&#34; to signal to the robot to terminate the print run.</li>
		<li>Communicate the program to the robot by selecting the follow commands from the top menu bar: &#34;Robot &#62; Send C&#38;T Data&#34;.</li>
		<li>Initiate the etching/print run, by changing the robot to the &#34;Run&#34; mode. Do this by selecting &#34;Robot &#62; Changing Mode &#62; Switch Run Mode&#34; from the top menu bar.</li>
		<li>Start the printing procedure by pressing the green &#34;start button&#34; on the robot dispenser console.</li>
	</ol>
	</li>
</ol>
3. Preparation and Printing of Cell-containing Gelatin Bioink
<ol>
	<li>Dissolve 2% gelatin in Minimum Essential Medium Alpha Medium (&#945;MEM) (supplemented with 10% FBS and 2% antibiotic/antimycotic) at 60 &#176;C for 2 hr to prepare the bioink solutions.</li>
	<li>Pre-culture the Red Fluorescent Protein expressing Mesenchymal Stem Cells (RFP-MSCs) to 70% confluence in 10 cm tissue culture dishes using &#945;MEM/10% FBS. Release the attached cells into suspension by removing the medium and coating with 1x trypsin-EDTA solution for 5 min at 37 &#176;C.</li>
	<li>Pellet the cells by centrifugation at 1,000 x g for 5 min and remove the supernatant. Resuspend the cell pellet in 0.5 ml of 1x PBS and count the cell density using a hemocytometer.</li>
	<li>After allowing the bioink to cool to room temperature, gently mix in a sufficient volume to the suspension of RFP-MSCs in the bioink to achieve a final concentration of 5 x 106 cells ml&#8722;1.</li>
	<li>Pour the cell bearing bioink into a printing syringe with the nozzle sealed. Chill the loaded syringe to 4 &#176;C in order to attain a printable viscosity.</li>
	<li>Place the loaded syringe on the automated robotic dispensing system and attach the air pressure lines. Remove the syringe Luer seal and attach the print nozzle.</li>
	<li>Extrude the cellularized bioinks into thin lines using 0.05 MPa back pressure, at 5 mm/sec writing speed from a 10 ml syringe via a 27 G needle/nozzle (tapered recommended over cylindrical) onto a polystyrene film surface, following the pre-programmed deposition pattern described in Step 2.1 to place the cellularized bioink onto the pre-etched grooves.</li>
	<li>After 1 hr incubation at room temperature, add 10 ml growth media (supplemented with 10% FBS and antibiotics) and incubate the cells for 24 hr (to allow the cells to sense and react to the etched features) before viewing using an inverted fluorescence microscope at 10X magnification.</li>
</ol>

<ol>
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The authors declare they have no competing financial interest.

The critical step of this procedure is the actual bioprinting delivery of the stem cells as the process must allow cell sedimentation to the features, print without bioink spreading/bleeding, deliver cells without shear stress cell death and not trigger differentiation towards unwanted lineage.
If the expected cell alignment fails to occur, then the bioink viscosity should be assessed for its suitability for printing. It is important that the bioink allows the cells to sediment to the patterne...

The deliberate patterning of cell placement enables the formation of cultures that mimic in vivo cellular organization1. Indeed, research into the interaction between multiple cell types can be assisted by organizing their spatial placement2,3. Most patterning systems rely on surface modification procedures for promoting or preventing cell adhesion with subsequent passive cell deposition. Bioprinting offers spatial and temporal control over cell distributions1. In addition to these functions, bioprinting has been described as being a technically straightforward, rapid and cost effective method for generating geometrically complex scaffolds4. It utilizes computer design software and allows the introduction of cells into the fabrication process4.
Bioprinting systems have been categorized based on their working principles as laser based, inkjet-based or extrusion-based4. Extrusion bioprinting has been described as the most promising as it allows the fabrication of organized constructs of clinically relevant sizes within a realistic time frame4-6. It is performed by either mechanical or back pressure assisted extrusion of a cell bearing hydrogel bioink. In the method presented here, back pressure was employed. As mentioned, the cells are delivered in a cytocompatible bioink. Such a bioink should support the delivery of cells without producing deleterious shear stress, and be of a sufficient viscosity to retain the integrity of the printed trace, without collapsing or spreading (referred to as &#34;ink bleed&#34;)7-10.
The interaction of cells with their adherent surface is known to influence cellular behavior. The surface topography can control the cell shape, orientation11, and even the phenotype. In particular, the fabrication of grooves and channels have been demonstrated to induce a stretched, elongated morphology on multiple cell types. The adoption of this morphology has been found to influence the phenotype of multipotent and pluripotent cells. For example, when aligned on grooves, mesenchymal stem cells (MSC) show evidence of differentiation towards cardiomyocytes12,13 and vascular smooth muscle cells adopt the contractile phenotype over the synthetic10,14-17.
The cell aligning channels or grooves can be generated on a polymeric surface via a number of methods, for example, deep reactive ion etching, electron beam lithography, direct laser printing, femtosecond laser, photolithography and plasma dry etching18. These approaches are often time-consuming, require complex apparatus and can be limiting in the shape of the pattern generated. In addition, they do not synchronize patterning with bioprinting and do not allow for immediate cellularization. The coordinately controlled movement of an automated dispensing system can follow complex patterns for the deposition of solutions. Here we demonstrate how the microscale-controlled movement can be exploited to create channels for cell orientation. A sharpened stylus or scalpel is attached to the print head in place of the extrusion syringe and the equipment can then etch the polymer surface under the guidance of the plotting software. The method offers versatility in pattern design and is applicable to polymeric materials commonly used in bioengineering such as polystyrene, PTFE, and polycaprolactone. As a subsequent step to the etching, cells can be bioprinted directly to the scratched grooves. The gelatin bioink utilized here was able to both maintain the trace and allow the deposited cells to sense the etched features. Mesenchymal stem cells bioprinted to the etched grooves were demonstrated to elongate along them in distinct lines.

<table border="0" cellpadding="0" cellspacing="0" width="448">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Equipment</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:151px;">Robotic Dispensing System</td>
			<td style="width:131px;">Janome</td>
			<td style="width:104px;">2300N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Plasma Machine</td>
			<td style="width:131px;">Femto Science</td>
			<td style="width:104px;">Covance</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">USB Microscope</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Optical Microscope</td>
			<td style="width:131px;">Olympus</td>
			<td style="width:104px;">IX71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Software</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Spreadsheet</td>
			<td style="width:131px;">Excel</td>
			<td style="width:104px;">Excel</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:151px;">Printing Co-ordinate Software</td>
			<td style="width:131px;">Janome</td>
			<td style="width:104px;">JR C-Points</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:151px;">Imaging Software</td>
			<td style="width:131px;">National Institutes of Health (NIH)</td>
			<td style="width:104px;">ImageJ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Equipment</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Stylus (Blade)</td>
			<td style="width:131px;">OLFA</td>
			<td style="width:104px;">AK-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">5ml printing syringe</td>
			<td style="width:131px;">San-ei Tech</td>
			<td style="width:104px;">SH10LL-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">30G printing needle</td>
			<td style="width:131px;">San-ei Tech</td>
			<td style="width:104px;">SH30-0.25-B</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:151px;">1mm polystyrene sheets</td>
			<td style="width:131px;"></td>
			<td style="width:104px;">Purchased locally</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Fetal bovine serum</td>
			<td style="width:131px;">Invitrogen&#160;</td>
			<td style="width:104px;">10270-098</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:151px;">Phosphate buffered saline</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:151px;">Gelatin from porcine skin, Gel strength 300, Type A</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td style="width:104px;">9000-70-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">&#945;MEM</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:104px;">41061-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:151px;">Antibiotc antimycotic</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td style="width:104px;">A5955-100ML</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:151px;">Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)</td>
			<td style="width:131px;">Cyagen Biosciences Incorporation</td>
			<td style="width:104px;">RASMX-01201</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated robotic dispensing technique for surface guidance and bioprinting of cells

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

The representative results demonstrate that the backpressure assisted robotic dispensing system can be used as an extrusion-based bioprinter for performing both surface etching and bioink printing (Figure 1 A). It can be used for the generation of etched grooves into polymer surfaces, and to subsequently print a cell bearing bioink directly to the features (Figure 1 B and C).
Both the etching an...

Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

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

automated-robotic-dispensing-technique-for-surface-guidance

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.3K Views.  Nanyang Technological University. This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Video: Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

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

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

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

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.