Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

Summary

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.

Abstract

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 µm 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' 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.

Introduction

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 placement^2,3. 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 frame^4-6. 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 "ink bleed")^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 cardiomyocytes^12,13 and vascular smooth muscle cells adopt the contractile phenotype over the synthetic^10,14-17.

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.

Protocol

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

1. 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.
2. Oxygen plasma treatment.
   1. 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's control panel: 150 W, 30 sscm of oxygen, 10 min (for power, gas flow and time respectively).
   2. 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 °C for 2 hr before washing three times 1x phosphate buffered saline (PBS).

2. Programming Print Patterns

1. Use the Program to First Etch the Surface with a Stylus or Scalpel.
   1. 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.
   2. 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 "desired pattern" 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.
   3. Open the print/dispensing software. Select "Program > Add Program" followed by "Edit > Add Point" to set up the program. Export the x and y coordinate values obtained from the spreadsheet into the print/dispensing software using the "Copy and Paste" function.
   4. Calibrate the "z" height of the robot before each run in order to place the stylus/print nozzle onto the surface.
      1. In the print/dispensing software, select the "Robot" option, click on "Changing Mode" and enable the "Teaching mode" option. Once selected, the software enables the "Jog" function of the robot.
      2. To Jog, first initialize the robot to its default position by selecting the following commands from the menu bar; "Robot > Meca Initialize", then select "Robot > Jog". Input the numerical values (in mm) in the "X and Y slots" required to place the stylus exactly on the origin of the pattern.
      3. Next, input a numerical value (in mm) in the "Z slot" to place the stylus or printing nozzle in contact with the surface but not flex or indent the surface. This point is designated as "Z" 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 µm are demonstrated to cut into the surface of 1 mm thick polystyrene sheets (Figure 4 and Table 1).
   5. Select the print instruction for each of the coordinate points, i.e., "Start of Line Dispense" to define the first point and print initiation, "Line Passing" to designate the intermediate points and "End of Line Dispense" to signal to the robot to terminate the print run.
   6. Communicate the program to the robot by selecting the follow commands from the top menu bar: "Robot > Send C&T Data".
   7. Initiate the etching/print run, by changing the robot to the "Run" mode. Do this by selecting "Robot > Changing Mode > Switch Run Mode" from the top menu bar.
   8. Start the printing procedure by pressing the green "start button" on the robot dispenser console.

3. Preparation and Printing of Cell-containing Gelatin Bioink

1. Dissolve 2% gelatin in Minimum Essential Medium Alpha Medium (αMEM) (supplemented with 10% FBS and 2% antibiotic/antimycotic) at 60 °C for 2 hr to prepare the bioink solutions.
2. Pre-culture the Red Fluorescent Protein expressing Mesenchymal Stem Cells (RFP-MSCs) to 70% confluence in 10 cm tissue culture dishes using αMEM/10% FBS. Release the attached cells into suspension by removing the medium and coating with 1x trypsin-EDTA solution for 5 min at 37 °C.
3. 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.
4. 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⁻¹.
5. Pour the cell bearing bioink into a printing syringe with the nozzle sealed. Chill the loaded syringe to 4 °C in order to attain a printable viscosity.
6. 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.
7. 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.
8. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Robotic Dispensing System	Janome	2300N
Plasma Machine	Femto Science	Covance
USB Microscope
Optical Microscope	Olympus	IX71
Name	Company	Catalog Number	Comments
Software
Spreadsheet	Excel	Excel
Printing Co-ordinate Software	Janome	JR C-Points
Imaging Software	National Institutes of Health (NIH)	ImageJ
Name	Company	Catalog Number	Comments
Equipment
Stylus (Blade)	OLFA	AK-5
5 ml printing syringe	San-ei Tech	SH10LL-B
30 G printing needle	San-ei Tech	SH30-0.25-B
1 mm polystyrene sheets		Purchased locally
Fetal bovine serum	Invitrogen	10270-098
Phosphate buffered saline	Invitrogen
Gelatin from porcine skin, Gel strength 300, Type A	Sigma Aldrich	9000-70-8
αMEM	Invitrogen	41061-029
Antibiotc antimycotic	Sigma Aldrich	A5955-100ML
Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)	Cyagen Biosciences Incorporation	RASMX-01201