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>

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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 patterned polymer surface. The concentration of the bioink polymer can be reduced or its temperature increased (up to a maximum of 37 &#176;C), to reduce viscosity and promote cell sedimentation. However, these reductions may cause the bioink to spread/bleed post printing. Here we suggest using 2% gelatin (w/v) dissolved in 1x PBS. If the cell concentration post-printing appears reduced, then the shear stress induced by printing may have compromised the viability of the printed cells7. Check the cells in the bioink before and after printing using a viability assay such as the activation of resazurin fluoresence7. Additionally, if bioprinted stem cells contact the surface but do not align, then it is possible the shear stress during printing decreased their multipotency by triggering an undesired differentiation pathway. Post printing stemness can be assessed by Fluorescence-activated cell sorting (FACS) analysis using stem cell markers such as CD 2910,19,20. If it is found that the shear stress is having deleterious effects on the cells, then the bioink printing viscosity can be reduced by adding or replacing the bioink polymer with a shear thinning one, such as alginate, Pluronic F127, hyaluronic acid, gellan gum, gelatin methacrylate or polyethylene oxide19, 21-25.
Finally, other aspects that can be optimized include the print backpressure, bioink cell density, and nozzle diameter. The backpressure requires optimization to the viscosity of the bioink and can be altered to obtain a discrete unbroken trace. A high cell concentration may increase the bioink viscosity and clog the print head. However, this extrusion process does allow for printing of relatively higher cell densities than other bioprinting methods26,27. The selection of the print nozzle gauge should be considered since small diameter nozzles may produce a finer trace but will be prone to clogging. Tapered nozzles have been found to function better since the same flow rate can be achieved as a cylindrical nozzle but at a lower shear stress hence improved cell viability28.
The backpressure assisted deposition method, also referred to as extrusion bioprinting, has less resolution than other cell printing approaches such as ink jet and laser assisted methodology due to the minimum programmable step length and the spreading of the emerging bioink. However, the backpressure extrusion equipment is most readily modified for the inclusion of an etching stylus. Other methods of producing grooves such as micro-patterning using photolithography followed by PDMS molding have significantly more steps than the robotic etching and the method is less versatile at modifying existing patterns or producing new ones12,15,29-31.
This method offers a tool for in vitro study of cell interactions where the position, orientation, and arrangement of one or more cell types are important. Several cell types including mesenchymal stem cells, fibroblasts, and smooth muscle cells are known to align on surface grooves1,10,14,18. The etched patterns, presented here, can be modified and reprinted very quickly and with ease. This is of interest as surface patterning with grooves has been used in the study of cell adhesion, morphology, migration and stem cell differentiation. Furthermore, it has also been applied to neural and musculoskeletal tissue engineering18, 32,33. Since the grooves do play a role in cell differentiation or phenotype regulation for several cell types, this method can be used to create grooves, to aid differentiation and to deposit cells in discrete rows so that screens can be performed. Currently, we are assessing this method to promote anisotropic cell arrangement of cells for the production of cardiomyocyte cell sheets, as the cellular orientation is important for the function of cardiac tissue12-14,34.
The approach demonstrated here employs a soluble bioink that will eventually dissipate. However, if a more permanent hydrogel coverage is required, then the inclusion of microbial transglutaminase can cross-link and stabilize protein hydrogels such as gelatin based ones without adverse effects on the cells9, 35. Bioinks can also utilize methacrylated polymers for UV initiated crosslinking of cell bearing hydrogels in cytocompatible manner36. However, in our experience, it was found that the transglutaminase crosslinking conferred for an improved interaction between the hydrogel and polymer surface (less readily delaminated), and a more cytocompatible environment when compared to poly(ethylene glycol) diacrylate (PEGDA) hydrogel 37.

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><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Robotic Dispensing System</td><td>Janome</td><td>2300N</td><td></td></tr><tr><td>Plasma Machine</td><td>Femto Science</td><td>Covance</td><td></td></tr><tr><td>USB Microscope</td><td></td><td></td><td></td></tr><tr><td>Optical Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spreadsheet</td><td>Excel</td><td>Excel</td><td></td></tr><tr><td>Printing Co-ordinate Software</td><td>Janome</td><td>JR C-Points</td><td></td></tr><tr><td>Imaging Software</td><td>National Institutes of Health (NIH)</td><td>ImageJ</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stylus (Blade)</td><td>OLFA</td><td>AK-5</td><td></td></tr><tr><td>5 ml printing syringe</td><td>San-ei Tech</td><td>SH10LL-B</td><td></td></tr><tr><td>30 G printing needle</td><td>San-ei Tech</td><td>SH30-0.25-B</td><td></td></tr><tr><td>1 mm polystyrene sheets</td><td></td><td>Purchased locally</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen&amp;nbsp;</td><td>10270-098</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Gelatin from porcine skin, Gel strength 300, Type A</td><td>Sigma Aldrich</td><td>9000-70-8</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Invitrogen</td><td>41061-029</td><td></td></tr><tr><td>Antibiotc antimycotic</td><td>Sigma Aldrich</td><td>A5955-100ML</td><td></td></tr><tr><td>Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)</td><td>Cyagen Biosciences Incorporation</td><td>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 and printing are determined by the programmed coordinates (Figure 2) input into the print/dispensing software which enables printing of linear, curved and radial patterns as required for the application (Figure 3 A-C). The Z-axis settings allow for control of the etched groove depth (Figure 4 and Table 1).
After replacing the stylus with a hydrogel print head, the programming that coordinated the etching could be reused to enable the robotic dispensing system to deliver the cell bearing bioink directly to the etched grooves following the same design (Figure 3 D and E). As can be observed in Figure 5 A and B, the stem cells seeded by bioprinting within the bioink eventually sediment to the surface, and sense and elongate along the direction of the etching within discrete lines. The cells seeded in culture medium without bioprinting aligned in the direction of the features, however, they covered the entire surface (Figure 5 C), proving that the bioink constrains the cells to the printed trace. Without etched features, cells seeded in culture media show no orientation or alignment (Figure 5 D).
<img alt="Figure 1" src="/files/ftp_upload/54604/54604fig1.jpg" /> 
Figure 1: The automated robotic dispensing system. (A) The apparatus was customized with the inclusion of a sharpened stylus (secured in a syringe) central to the print head that etches concentric rings onto a polystyrene slide (B). The print head could then be converted to extrude cell bearing gelatin bioink, with back pressure assisted extrusion, and deposit on the previously etched pattern (C). This figure has been modified from Bhuthalingam et al. 2015 10.
<img alt="Figure 2" src="/files/ftp_upload/54604/54604fig2.jpg" /> 
Figure 2: Plotting x-y coordinates in a spreadsheet software. The screen grab shown here demonstrates that the plotted x-y coordinates created a sinusoidal wave pattern as shown by the graph plot. These coordinates were input into the plotting software using the copy/paste function.
<img alt="Figure 3" src="/files/ftp_upload/54604/54604fig3.jpg" /> 
Figure 3: Etched pattern versatility. The coordinates programmed into the plotting software were able to etch linear (A), sinusoidal (B) and concentric circles (C). The grooves etched into polystyrene could be directly printed onto with the hydrogel bioink during the subsequent step (D) and (E). This figure has been modified from Bhuthalingam et al. 2015 10.
<img alt="Figure 4" src="/files/ftp_upload/54604/54604fig4.jpg" /> 
Figure 4: Etched groove depth. The depth of the etched groove can be regulated by the z-axis control of the dispensing system. Images (A) to (C) show the overhead view and (D) to (E) show the cross section. The robot was programmed to etch the surface to a depth of 40 ((A) and (D)), 90 ((B) and (E)) and 180 &#181;m ((C) and (F)). The closeness of the programmed depth and actual etched depth is displayed in Table 1. This figure has been modified from Bhuthalingam et al. 2015 10.
<img alt="Figure 5" src="/files/ftp_upload/54604/54604fig5.jpg" /> 
Figure 5: Bioink delivered Mesenchymal stem cells. The RFP-MSCs printed in the bioink onto the etched patterns could sense the features as demonstrated by their elongated morphology and alignment with the grooves (A) and (B) as compared to cells seeded on the features without bioink (i.e., in culture medium) (C) and on a non-patterned surface (D). This figure has been modified from Bhuthalingam et al. 2015 10.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Programmed depth (&#181;m)</td>
			<td>Etched groove depth (&#181;m)</td>
		</tr>
		<tr>
			<td>40</td>
			<td>35 &#177; 7</td>
		</tr>
		<tr>
			<td>90</td>
			<td>81 &#177; 6</td>
		</tr>
		<tr>
			<td>180</td>
			<td>175 &#177; 3</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of programmed versus actual etching depth on polystyrene. This figure has been modified from Bhuthalingam et al. 2015 10.

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 San Sebastian

Research

JoVE Journal

Bioengineering

6.4K 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

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

Engineering

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

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 ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

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.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...