Name: automated robotic dispensing technique for surface guidance and bioprinting of cells
Uploaded: 2016-11-18T13:00:00
Description: Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

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 shee...

<ol>
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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>
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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...

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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 ...

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Bioengineering

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