This work was supported by Government Support under Grant No. EPS-0903795 awarded by the National Science Foundation, NIH NIDCR R01-DE019355 (MJY PI), and Grant 8P20 GM103444 (YM PI).

1. Preparation of Gelatin Containing Substrate for Three-Dimensional Bioprinting of Alginate Hydrogels
<ol>
	<li>Prepare the calcium/gelatin substrate following the calcium/gelatin substrate method described by Pataky et al11 to avoid reduced viability associated with high content. The calcium/gelatin substrate method is listed below.
	<ol>
		<li>Combine calcium chloride dehydrate (1.5 wt%), sodium chloride (0.9 wt%), and porcine gelatin (2 wt%) in distilled water and boil for 2 min&#160;to create a 100 mM gelatin solution.</li>
	</ol>
	</li>
	<li>Pour 5 ml&#160;of the gelatin/calcium solution into 100 mm standard petri dishes, swirl the solution around to make an even coating on the surface, and place on a flat surface in the fridge to gel O/N (allow to gel at least 8 hr&#160;before use).</li>
	<li>To increase the opacity of the substrate surface, add titanium dioxide (0.3 wt%) to the gelatin/CaCl2 solution. Stir for 10 min. Autoclave the gelatin/TiO2 solution on the liquids cycle for 30 min&#160;to sterilize it.
	<ol>
		<li>Add 3 ml&#160;of the gelatin/TiO2 solution to the surface of the previously prepared gelatin plates. Swirl the mixture to ensure it is spread evenly across the surface. Allow to gel in the 4&#160;&#176;C&#160;fridge O/N (allow to gel at least 8 hr&#160;before use). The substrates must be used within 3 days.</li>
	</ol>
	</li>
</ol>
2. Alginate Oxidation
<ol>
	<li>Oxidize the sodium alginate bioink following the method for partially oxidized alginate by Bouhadir et al30&#160;described below.
	<ol>
		<li>To make a 5% oxidized alginate solution, dissolve 1 g of sodium alginate in 100 ml&#160;of distilled water. Add an aqueous solution of sodium periodate (0.25 M, 0.25 mmol), the oxidizing agent, to produce a 5% oxidation solution. Stir for 19 hr at RT. Add 40 ml&#160;ethylene glycol to the solution after 24 hr to end the reaction.</li>
		<li>Dissolve 2.5 g of sodium chloride in the solution. Add an excess amount of ethyl alcohol (2:1 ratio) to precipitate the oxidized alginates. Centrifuge the solution at 1,000 x g to collect the precipitates and re-dissolve them in distilled water. Repeat the ethanol wash.</li>
		<li>Freeze-dry the oxidized alginate pellets and store at -20 &#176;C&#160;until ready for use.</li>
	</ol>
	</li>
	<li>Determine the degree of oxidation by measuring the percentage of sodium periodate consumed before being terminated by the ethylene glycol.
	<ol>
		<li>Prepare a potassium iodide solution (20% w/v, pH 7.0 sodium phosphate buffer) and a thyodene solution (10% w/v, pH 7.0 sodium phosphate buffer). Mix the two solutions with the oxidized alginate at RT.</li>
		<li>Gradually drop the reacted alginate and sodium periodate solution into the mixture of potassium iodide and theodyne solutions. Measure the absorbance of the mixture spectrophotometrically at 426 nm. When it has reached a maximum, record the used volume of alginate and sodium periodate solution as V1.</li>
		<li>The reaction is <img alt="Equation 1" fo:content-width="0.8in" src="/files/ftp_upload/53156/53156eq1.jpg" width="120" />. The amount of unreacted sodium periodate is <img alt="Equation 1.5" fo:content-width="1.2in" src="/files/ftp_upload/53156/53156eq1.5.jpg" width="150" /></li>
		<li>Subtract the amount of unreacted sodium periodate from the original concentration to determine the amount of sodium periodate consumed. Using the previous formula, determine the final oxidation degree of the alginate.</li>
	</ol>
	</li>
</ol>
3. Alginate Peptide Conjugation
<ol>
	<li>Conjugate ligands with an exposed arginine-glycine-aspartate sequence ( peptide) into the previously prepared oxidized alginate by following the RGD-Alginate conjugation method by Rowley et al31described below to promote cell attachment and spreading.</li>
	<li>Use aqueous carbodiimide chemistry with G4RGDSPto conjugate31.</li>
	<li>Dissolve 1 g of 5% oxidized alginate in a 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer, pH = 4. Add 1-ethyl-(dimethylaminopropyl) carbodiimide (EDC, 0.54 mmol) and N-Hydroxysuccinimide (NHS, 0.27 mmol) at 2:1 ratio to form amide intermediate.</li>
	<li>Add 0.28 mmol peptide, coupling to the backbone of the alginate polymer via the terminal amine. Stir at RT O/N.</li>
	<li>Stop the coupling reaction by adding 2.5 g sodium chloride to the solution. Add an excess amount of ethyl alcohol (2:1 ratio) to precipitate the oxidized alginates. Centrifuge the mixture at 4,000 x g for 5 min&#160;to collect the precipitates. Aspirate the media in the cell culture hood and re-dissolve the precipitates in distilled water. Repeat the ethanol wash.</li>
	<li>Freeze-dry the precipitates until it becomes completely dried (will appear as a white powdery substance) and store in the -20&#160;&#176;C&#160;fridge for later use.</li>
</ol>
4. Human Adipose Tissue Stromal Cells (hADSC&#8217;s) Cell Culture
<ol>
	<li>Culture human adipose tissue stromal cells (hADSC&#8217;s) in 75 cm treated cell culture flasks (T75 flasks), covered with 15 ml&#160;low glucose DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin, 1% glutamine, and 1% antimycin. Change the media, in the cell culture hood, every two days until they have reached confluency (80-90%).</li>
	<li>Once confluent, transfer the T75 flasks to the cell culture hood and suspend the hADSC&#8217;s using the trypsin enzyme digestion method.
	<ol>
		<li>In the hood, aspirate all of the cell culture media off of the cells. Rinse with 5 ml&#160;of Dulbecco&#8217;s Phosphate-Buffered Saline with calcium and magnesium (DPBS ++). Aspirate the DPBS++ off of the cells.</li>
		<li>While in the hood, make a solution of trypsin and DPBS++ by mixing 1 ml&#160;trypsin and 4 ml&#160;DPBS++. Each flask requires 5 ml&#160;of the solution, so make the appropriate volume for the number of confluent flasks. Add 5 ml&#160;of the trypsin/DPBS++ to each flask and put them in the incubator for 2 min.</li>
		<li>After 2 min, remove the flasks and lightly tap the sides of them to loosen the cells from the bottoms. Look at each flask under a microscope to ensure the cells are suspended. Place the flasks back in the cell culture hood and add 3 ml&#160;of appropriate cell culture media to each flask. This ends the trypsin reaction.</li>
		<li>Transfer the cell-laden media from each flask and put in a 50 ml&#160;conical. Centrifuge them at 1,000 x g for 5 min. The cells should appear as a little white pellet in the bottom of the conical. Transfer back to the cell culture hood and aspirate the media. Resuspend the cells in 2 ml&#160;of cell culture media.</li>
		<li>Count the cells using a hemocytometer under the microscope. Once the cells have been counted, in the culture hood, aliquot the amount of media containing ~1.3 million cells and transfer to a 15 ml&#160;conical. Centrifuge the 15 ml&#160;conical containing the cells again for 5 min&#160;at 1,000 x g.</li>
		<li>In the culture hood, reseed the remaining cells in multiple T-75 flasks, adding a concentration of ~350,000 cells to each flask. Add 15 ml&#160;of DMEM media and return to the incubator until confluent again.</li>
		<li>Once the centrifuge cycle is complete, return the 15 ml&#160;conical to the cell culture. Aspirate the media from the cell pellet, and resuspend the cells in aqueous alginate solution at a concentration of 1.3 million cells per milliliter of bioink, terteriating the solution often so there is a homogeneous distribution of cells throughout the bioink. Load the cell-laden solution into a sterile printer-compatible 3 ml&#160;syringe and screw on the sterile 22 G plastic tip.</li>
	</ol>
	</li>
</ol>
5. Bioprinter Setup
<ol>
	<li>Turn on the bioprinter, each of the dispenser computers, and the recirculating water bath.
	<ol>
		<li>Manually set the recirculating water bath temperature to for the gelation mechanism.</li>
		<li>Manually set printing parameters for each dispenser on the correlating dispenser computer. Set the dispense volume to 230 nl, number of backsteps to 0, and the dispense rate to 10&#956;l &#8211;sec.</li>
	</ol>
	</li>
	<li>Open the design software and the program for viewing the USB camera&#8217;s display on the computer.
	<ol>
		<li>Using the software, manually enter the coordinates for a 5 x 5 dot array with 2.4 mm spacing between drops.</li>
		<li>Set the printing parameters to be: distance between tip end and substrate surface = 0.1 mm; height syringe is lifted between depositions = 20 mm; the amount of time per deposition = 1 sec.</li>
		<li>Save the program and send it to the robot.</li>
	</ol>
	</li>
	<li>Place the gelatin/TiO2-containing Petri dish on the 4 &#176;C&#160;printer stage. Close and lock the chamber door.</li>
	<li>Use the PLC to initialize the ultraviolet light sources, and sterilize the chamber for 90 sec.</li>
	<li>Once sterilization is complete, open the chamber and load the syringe containing hADSC&#8217;s suspended in alginate into Gun 1. Close and lock the chamber door.</li>
	<li>Use the PLC to turn on the fan system, wait 30 sec for equilibrium internal pressure.</li>
	<li>On the computer, run the program containing the geometrical pathway and printing parameters.</li>
	<li>Throughout the printing process, watch the USB camera&#8217;s display on the computer to confirm accurate and uniform printing.</li>
	<li>Once printing has finished, allow the constructs to gel for 40 min.</li>
</ol>
6. Cell Viability Assessment
<ol>
	<li>Cover the constructs that are not going to be imaged immediately post-printing in DMEM and store in the incubator until time of imaging.</li>
	<li>To quantify the viability of the constructs, stain them using a fluorescent-based viability/cytotoxicity assay, and image using confocal microscopy.
	<ol>
		<li>Following the kit instructions, prepare a staining solution containing calcein AM and ethidium homodimer-1. To make 10 ml&#160;of staining solution, add 20 &#956;l&#160;of the ethidium homodimer-1 and 5 &#956;l of the calcein am to 10 ml&#160;of sterile, tissue culture-grade Dulbecco&#8217;s Phosphate-Buffered Saline (+magnesium, +calcium; DPBS++).</li>
		<li>Immerse the bioprinted constructs in the stain solution for 15 min&#160;in the dark.</li>
		<li>Image the stained constructs using a confocal microscope system at days 0 and 8. Take multiple pictures of each bioprinted construct, using Z-stack parameters of 30 optical slices over a 300 &#956;m depth, and manually count the cells. If cells appear yellow or green count them as alive, and if red, count them as dead.</li>
	</ol>
	</li>
	<li>Calculate the cell viability percentage as the number of live cells divided by the total number of cells in the construct; Cell Viability = number of live cells (green+yellow)/ number of total cells (green+yellow+red) x 100%.</li>
	<li>Calculate the amount of cell proliferation for each sample as the cell number of day 8 divided by the cell number on day 0; Cell Proliferation = live cell count on day 8/ live cell count on day 0 x 100%.</li>
</ol>
7. RGD Peptide Conjugation Analysis
<ol>
	<li>To analyze the success of RGD peptide conjugation on the alginate, compare RGD-conjugated alginate and non-conjugated alginate. To do this, image the printed constructs using (4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride) (DAPI) and phalloidin stains.
	<ol>
		<li>Make the phalloidins working solution by diluting 5 &#956;l&#160;of the methanolic stock solution with 200 &#956;l&#160;of DPBS++. Store at -20&#160;&#176;C&#160;until use.</li>
		<li>Make a 300 &#956;M stock solution of the DAPI stain following the equation: (0.10509 g/L)/(350.3 g/mol)=3 &#215; 10-4 M=0.0003 M=0.300 mM=300 &#956;M. Make the DAPI working solution by diluting the stock solution 1:100 in DPBS++ to obtain 3 &#956;M solution. Store at -20 &#176;C&#160;until use.</li>
	</ol>
	</li>
	<li>Completely submerge the sample in &#160;4% paraformaldehyde. Incubate for 1 hr&#160;at RT. Wash three times with DPBS++, allowing the solution to sit for 5 min&#160;each wash. Transfer the gel sample from the well to a glass slide, flipping the gel over in the process. Immerse the gel in 0.1% Triton X-100 (0.1 g/ 100 ml) in DPBS++ for 10 min. Wash three times with DPBS++, allowing 5 min&#160;for each wash.</li>
	<li>Stain the printed constructs with phalloidin by immersing them in the working solution. Cover with foil and incubate for 4 hr. Remove the phalloidin stain and wash three times with DPBS++. The first wash should be fast, the latter washes should sit for 5 min&#160;each.</li>
	<li>Stain the printed constructs with DAPI by immersing them in the DAPI working solution. Cover with foil and incubate at RT for 30 min. Wash three times with DPBS++, allowing each wash to sit for 5 min. Observe and image the samples on a confocal microscope system.</li>
</ol>

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The authors have nothing to disclose.

The primary focus of tissue engineering is to bridge the gap between organ shortages and transplantation needs by developing biological substitutes capable of restoring, maintaining, or improving native tissue functio. This has led to the direct fabrication of scaffolds with a complex, anatomically correct external geometry, and precise control over the internal geometr. Three-dimensional bioprinting is a methodology used for generating three-dimensional constructs of various sizes and shapes from a digital model using a layer-by-layer approac. The fabrication of three-dimensional biomimetic constructs plays an essential role in the advancement of tissue engineering.
There are critical aspects of the design process that impact the generated construct&#8217;s biomimetic functio. The ability to control the temperature of the biomaterial and substrate is essential for the gelling mechanism of the hydrogels and the maintenance of their mechanical properties, therefore influencing cell distribution, proliferation, and differentiation within the hydrogel. Organs consist of many cell types, so the multiple dispensers are critical for producing heterogeneous, tissue-like structures. The computer-aided design of the external architecture allows the production of custom tissue substitutes for distinct wounds or tissues. This is essential for the development of patient-specific replacements. The internal architecture is equally important because it affects the cell-cell relationships within the structure by placing the proper cells in intimate contact with each other and allowing them to form in vivo-like cell-cell junctions. Precise placement of cells determines how the cells communicate and network with each other to form vascular networks and mimic their bioactivity in native tissues. Three-dimensional bioprinting provides homogeneously dispersed cells within the bioink, as well as excellent precision on spatial placement of the cells . Generated scaffolds also have high local cell densities, which is essential for cell differentiation and the formulation of extracellular matrix.
Presented here is a 3D robotic bioprinter that reliably and consistently dispenses homogenous drops of individual cells or cells mixed with biomimetic hydrogel. Similar bioprinters have used a pre-fusion technology. In accordance with the differential adhesion hypothesis, individual cells when placed in a mold or similar device, will fuse and organize based on the concentration and distribution of adhesion molecules on the cell surfac. These devices create pre-fused rods or other geometric shapes that are then loaded into the dispenser and placed in close proximity to other pre-fused or pre-made rods, which then fuse into a larger geometric shap. End geometries are limited by what can be constructed from these pre-made entities. The bioprinter implemented here comprises a unique temperature controlled environment in which the cells and cell-hydrogel mixtures are not limited by the necessity of pre-fusion. Under these conditions, the bioprinter is not solely reliant on the differential adhesion hypothesis. The inclusion of hydrogel materials can help guide the cell distribution and allow the cells to fuse, or not, depending on the properties desired for specific experimentation. The selection of biomimetic hydrogels for cell-encapsulation also has a profound effect on cell phenotype. Materials are known to have an effect on cell attachment, as well as cell size and morpholog. The rheological characteristics, such as viscosity, of hydrogels dictate their influence on the cellular microenvironmen. Native alginate is inert and does not readily communicate with cells participating in the control of phenotype. However, using alginates that are chemically modified via peptide conjugation and oxidation, the resulting constructs display controlled degradability and increased cell attachment, migration, and proliferatio. Altering the physiochemical properties of a biomaterial can influence tissue developmen.
Three-dimensional bioprinting using a fluid-dispensing, direct-write machine is limited by the degree of resolution of printed constructs, the availability of hydrogel materials, initial cell death post-printing, and the ability to vascularize the biomimetic . An important feature of the bioprinting is its resolution. Every printing method is defined by the lower technical limit size of the smallest achievable details. There is a dynamic relationship between the lower limit size and attainable scale of the printed construct: the higher the resolution of the minute details, the smaller maximum construct . The bioprinter is capable of depositing volumes as small as 230 nl&#160;in highly specific and organized patterns, giving it a higher resolution than similar machines. Hydrogels have been commonly used in bioprinting due to their hydrophilicity, biocompatibility, structural similarity to the extracellular matrix, and ease of modificatio. The high water content of hydrogels improves their biocompatibility, but greatly reduces their mechanical strength and . There is a lack of optimal hydrogels with the appropriate mechanical properties for fluid delivery during bioadditive-manufacturing extrusion. Therefore, there is a large demand for developing hydrogels that are immunologically inert, have cytocompatible gelation mechanisms that can be successfully extruded using fluid delivery, and also produce a cell-laden matrix with an optimal range of mechanical . Before the printing process, the cell-laden hydrogel bioink must be stored in the syringes for an amount of time, compromising the cell&#8217;s . During the printing process, the shear stress induced on cells during extrusion can also be harmful to their . The bioprinter is able to produce highly viable (&#62;90%) constructs, therefore overcoming the issue of initial cell death. Vascularization plays a vital role in transmitting, supporting, or preserving the biomimetic function of bioprinted . The diffusion of oxygen is m; therefore in larger bioprinted constructs hypoxia is a . Conventional techniques are incapable of producing constructs with embedded vasculature, greatly limiting the size of producible scaffolds. The cell viability assessment of the bioprinter showed significant cell proliferation in the printed constructs over 8 days. Therefore, the technique proves its ability to generate scaffolds that allow cell growth, communication, and the formation of networks, requirements of vascularization.
The bioprinter provides the ability of using a variety of materials to quickly deposit cell-laden hydrogels in specific patterns. While this technique produces heterogeneous constructs with tunable properties, it is incapable of concurrent deposition and reactive mixing. For some biomaterials, this deposition method would enhance the gelation mechanism and shorten the time for scaffold productio. The addition of a multi-syringe dispenser could allow a broader range of biomaterials for the biofabrication technique. Investigation of cell activity in bioprinted constructs over a longer period of time would provide more information about hydrogel characteristics, cell network formation, and vascularization of the constructs.
The bioprinter&#8217;s deposition method described can further involve robotically positioning and driving the three dispensers to deposit a plurality of biological materials on top of previously deposited materials in a predetermined pattern. This step can be repeated using ascending patterns until a three-dimensional organ or tissue is produced. Therefore, the Palmetto Printer is suitable for reliably dispensing cell-laden hydrogels to create a three-dimensional construct that is capable of retaining vasculature and high cell viability, and could be used in tissue engineering applications.

Tissue engineering uses the principles of biology and engineering in the development of functional substitutes to maintain, restore, or enhance native tissue and . The capability of generating three-dimensional biomimetic constructs on demand would facilitate scientific and technological advances in tissue engineering as well as in cell-based sensors, drug/toxicity screening, tissue or tumor models, and other . The three-dimensional organization of tissue-engineered constructs is a fundamental component of the fabrication method because it must closely mimic the highly organized interaction of cells and extracellular matrix in native tissue.
Biodegradable and shape-forming three-dimensional scaffolds are critical factors in generating novel tissue constructs because cells migrate to form a two-dimensional layer of cells, but lack the ability to grow in favored three-dimensional . The scaffold serves as a temporary foundation for cell attachment and proliferation, so it must be constructed from materials with controllable porosity and biodegradability, and sufficient mechanical integrit. The scaffold materials should not be cytotoxic or create an adverse response from the host. Hydrogels have been commonly used in tissue engineering techniques, and due to their hydrophilicity, hydrogels permit fluid and gas exchange throughout the structur. By combining different hydrogels, the synthesized hydrogel&#8217;s properties are modifiable to meet distinct application requirement.
The conventional tissue engineering approach involves the creation of acellular porous sacrificial scaffolds that are seeded with cells post-fabricatio. Many techniques have been employed, such as fiber bonding, solvent casting, and melt molding, but proved to be minimally successful for tissue engineering applications. Fiber bonding methods allow fibers to be aligned in specific shapes, but they are only capable of producing very thin scaffold. Solvent casting methods produced highly porous constructs, however the largest produced membrane was only 3-mm thic. Therefore, creating three-dimensional constructs is not feasible using these techniques. Melt molding techniques proved successful in producing three-dimensional scaffolds, but such high temperatures are required that biological materials cannot be incorporated during the production proces. Scaffolds seeded post-fabrication are limited in their ability to meet the requirements of tissue engineering to produce three-dimensional scaffolds with pre-defined or controllable microstructures and . Another major issue with solid scaffold seeding technologies is the deficiency of vascularization and poor mechanical .
Bioprinting has since been extended to three dimensions through the use of nontoxic, biodegradable, thermo-reversible gels to overcome the disadvantages of conventional . A few of the solid freeform fabrication techniques currently being employed are laser-assisted bioprinting and inkjet printing. Laser-assisted bioprinting techniques use a pulsed laser source, a target plate, and a receiving substrate to generate three-dimensional . However, this technique is limited due to low throughput, low cell viability, and can only produce limited arrangements of fabricated structures because only photocrosslinkable prepolymers can be used to form a crosslinked hydrogel . Inkjet printing was developed as a non-contact methodology that reproduces digital image data on a substrate by depositing picoliter ink . However, inkjet printing does not produce a high-resolution construct, constructs experience rapid protein denaturation, and many of the cells are lysed during the deposition .
Currently, new additive manufacturing bioprinting methods have been developed. In these systems cells, proteins, growth factors, and biomimetic hydrogels are typically integrated into matrix materials during the fabrication process and concurrently deposited using computer-controlled actuators to generate three-dimensional scaffold-based cell-laden constructs that closely mimic the microarchitecture of native . The cell-laden hydrogels constitute the bioink, which can be heterogeneous, consisting of multiple cell types, or homogeneous. Additive manufacturing systems deposit bioink drop-by-drop or layer-by-layer via disposable syringes and tips onto a computer-controlled stage capable of moving in the x, y, and z directions. Through computer software, the architecture of printed scaffolds can be easily manipulated depending on requirements of the application. Unlike conventional techniques, three-dimensional medical technologies (magnetic resonance imaging, computer tomography) can be incorporated into the designs, generating patient-specific construct. These methods also allow the possibility of producing vascularized replacements because constructs are produced with a higher local cell density, allowing cell-cell interactions and improving the likelihood of post-implantation surviva.
The Palmetto Printer is a custom built three-dimensional multi-dispenser system that uses programmable robotic manufacturing methods to generate three-dimensional heterogeneous tissue constructs (Figure 1). It allows the use of a plurality of materials in unique combinations to produce heterogeneous structures. The initialization of the bioprinter is one of the most important steps in bioprinting because it allows you to set a variety of parameters to optimize the printability of the bioprinted constructs.
The bioprinter comprises a batch type process with startup, operation and shutdown sequences controlled by a programmable logic controller (PLC), which the user operates through an interactive touch screen control panel (Figure 1,&#160;A). To prevent contamination of biological materials the bioprinter is enclosed in a positively-pressured poly(methyl methacrylate) (PMMA) chamber with a high-efficiency particulate arrestance (HEPA)-filtered air circulation system (Figure 1, B,C). The interior of the printer can be sterilized using the built-in ultraviolet light sources (Figure 1, D). The central component of the bioprinter is a fully programmable positioning robot that can reproducibly place a dispenser tip with an accuracy of 10 micrometers (Figure 1, E). There are three dispensers, which are able to deposit volumes as small as 230 nl using a rotary screw (Figure 1, F). They are independently programmable using separate computers that govern printing parameters for each dispenser (Figure 1, G). Rotary-screw dispensing utilizes the rotation of a motor-driven screw to move bioink down a syringe and out of the syringe tip. These dispensers are mounted onto a pneumatically controlled Tool Nest (Figure 2A, B), allowing the robot to switch dispenser mounted onto the Z-axis robotic arm under programmed control (Figure 1, H).
The XYZ robot receives printing instructions from a computer running design software (Figure 1, I). Each program contains dispensing locations, calibration routines, and dispenser-changing protocols. The design of generated constructs primarily consists of the XYZ coordinates where each dispenser will deposit material. The bioprinter comprises two optical light sensors (Figure 2C) that determine the XYZ coordinates of the syringe tip end. These sensors send coordinate information to the robot, which uses these to calculate positions of the dispenser tip ends. There is an additional displacement laser (Figure 2D) that projects a 633 nm diode red laser beam of spot size 30 x 100 micrometers to measure distance with an accuracy of 0.1 micrometers. When the beam is highly focused the robot determines the Z distance of the printing surface. This measurement, and the optical light sensors measurement of the tip end in Z, allows calculation of accurate Z coordinates used to place the dispenser tip in relation to the printing surface. The dispenser tips move laterally and vertically through the X-axis oriented optical light sensor to find the Y and Z centers, and laterally through a Y-axis sensor to find the center of the X-axis. The printing surface is mapped using the formula for a flat plane in xyz space: ax + by +cz = d to determine where the surface is relative to the position of the dispensing tip end. The printer stage (Figure 1, J) holds a sample Petri dish up to 80 mm in diameter and uses a recirculating water bath to maintain the set temperature (Figure 1, K). Stage temperature can be set within a range of -20 and remains stable within . There is a USB camera mounted onto the robot Z-arm to provide a magnified view of the dispensing tip during the printing process (Figure 1, L). There is a second camera mounted towards the top of the chamber interior that provides a complete view of the bioprinter during the printing process (Figure 1, L).
A computer-aided design drawing software determines the deposition pattern and permits the user to generate incrementally spaced droplets and complex structures (Figure 3). Three-dimensional pathways can be manually coded into the printer-compatible design software or imported from a separate computer-aided design drawing software (Figure 4, Table 1). The printer-compatible software allows variations of printing parameters such as the deposition method (single droplet deposition or continuous pathway deposition), three-dimensional geometry of the pathways, deposition rate, distance between the syringe tip end and substrate printing surface, the amount of time to deposit an individual drop, and the height and speed the syringe is lifted between deposition of the drops. Each program contains XYZ dispensing locations, tip calibration routines, and dispenser-changing protocols to provide a sterile environment, without operator intervention, during printing. The programmable logic controller (PLC) of the robot receives instructions from the computer running the design software and controls the timing of events from the external controllers (e.g.,&#160;the dispensers). To do this, the PLC uses a looping mechanism to control the dispensers, robotic positioning device, and environmental factors.
Three-dimensional direct-write bioprinting utilizing a rotary-screw, liquid-dispensing system allows the process of depositing cells to be more efficient, accurate, and easier than previous methods. This study shows the custom built bioprinter is capable of generating cell-laden hydrogel constructs with high cell viability.

<table><tbody><tr><td>Positioning Robot (JR2000 XYZ)</td><td>Janome&amp;nbsp;</td><td></td><td></td></tr><tr><td>Dispensers: SDAV Linear Drive SmartDispensers</td><td>Fishman Corporation</td><td></td><td></td></tr><tr><td>Optical Light Sensors:&amp;nbsp;</td><td>Keyensce</td><td></td><td></td></tr><tr><td>Displacement Laser: OD Mini</td><td>SICK</td><td></td><td></td></tr><tr><td>Recirculating Water Bath: Polystat</td><td>Cole-Parmer</td><td>EW-12122-02</td><td></td></tr><tr><td>USB Cameras: Dino-Lite Premier 5MP</td><td>AnMo Electrionics/YSC Technologies</td><td>AD7013MT</td><td></td></tr><tr><td>Printer-Compatible Computer Design Software: JR-C Points</td><td>Janome</td><td></td><td>Comes with purchase of Janome Robot</td></tr><tr><td>Computer-Aided Design Drawing Software: Visual PathBuilder</td><td>RatioServ</td><td></td><td>Can be downloaded at: www.ratioserv.com/index.php/downloads</td></tr><tr><td>Printer 3 cc Syringes:&amp;nbsp;</td><td>Fishman Corporation</td><td>122051</td><td></td></tr><tr><td>22 G Dispenser Tips</td><td>Fishman Corporation</td><td>Z520122&amp;nbsp;</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>10035-04-8</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>Porcine Gelatin</td><td>Sigma-Aldrich</td><td>9000-70-8</td><td></td></tr><tr><td>Titanium Dioxide</td><td>Sigma-Aldrich</td><td>13462-67-7</td><td></td></tr><tr><td>Protanal LF 20/40 Alginate (Sodium Alginate)</td><td>FMC BioPolymer</td><td>9005-38-3</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Sigma-Aldrich</td><td>7647-01-0</td><td></td></tr><tr><td>Ethylene Glycol</td><td>Mallinckrodt Baker, Inc</td><td>9300-01</td><td></td></tr><tr><td>Sodium Periodate</td><td>Sigma-Aldrich</td><td>7790-28-5</td><td></td></tr><tr><td>hADSC</td><td>Lonza</td><td>PT-5006</td><td>Store in vials in liquid nitrogen until use.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Gibco Life Technologies</td><td>11965-092</td><td>Warm in 37 &amp;deg;C water before use.</td></tr><tr><td>Trypsin/EDTA</td><td>Lonza</td><td>CC-5012</td><td>Warm in 37 &amp;deg;C water before use.</td></tr><tr><td>Calcein AM</td><td>Gibco Life Technologies</td><td>C3100MP</td><td>Store in the dark at -80 &amp;deg;C until use.</td></tr><tr><td>Live/Dead Mammalian Viability Assay Kit</td><td>Invitrogen Life Technologies</td><td>L-3224</td><td>Store in the dark at -80 &amp;deg;C until use.</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich</td><td>M2933</td><td></td></tr><tr><td>N-Hydroxysuccinimide</td><td>Sigma-Aldrich</td><td>130672</td><td></td></tr><tr><td>1-ethyl-(dimethylaminopropyl) carbodiimide (EDC)</td><td>Sigma-Aldrich</td><td>E1769&amp;nbsp;</td><td>10 G</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, +Calcium, +Magnesium</td><td>Life Technologies</td><td>14040133</td><td>Warm in 37 &amp;deg;C water before use.</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, -Calcium, -Magnesium</td><td>Life Technologies</td><td>14190144</td><td>Warm in 37 &amp;deg;C water before use.</td></tr><tr><td>RGD Peptides</td><td>International Peptides</td><td></td><td></td></tr><tr><td>Alexa Fluor 546 Phalloidin Stain</td><td>Invitrogen Life Technologies</td><td>A22283</td><td>Store at -20 &amp;deg;C until use</td></tr><tr><td>(4&amp;rsquo;, 6-Diamidino-2-Phenylindole, Dihydrochloride) (DAPI) Stain</td><td>Life Technologies</td><td>R37606</td><td>Store at -20 &amp;deg;C until use</td></tr></tbody></table>

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.

The results demonstrate the bioprinter is capable of depositing cell-laden hydrogels in specific three-dimensional locations accurately and consistently using computer-aided software. These softwares determine the placement of each droplet and control many of the parameters for dispensing (Figure 3,4). The repeatability of the bioprinter to appropriately deposit biomaterials is fundamental to its success in tissue engineering applications.
Cell viability, one of the requirements of a successful bioprinting technique, was analyzed 1 hr and 8 days post-printing. High cell viability is essential for fabricating biomimetic constructs and is a direct representation of an adequate bioink. RGD peptide conjugation improves cell viability over extended periods of time by promoting cell spreading. Fluorescent microscopy was used to quantify cell viability in constructs after the printing process. Alginate bioink with a concentration of 15% and oxidation of 5% had a day 0 viability of 98%, day 4 of 96%, and day 8 of 95% (Figure 5). These results indicate the deposition technique of the direct-write bioprinter extrudes cells gently enough to produce constructs that remain viable during and after the printing process (Figure 1, 2). The high cell viability shows the 5% oxidation and 15% concentration alginate bioink was a suitable vehicle for cell deposition and provided an adequate environment for cell-survival. Similar cell counts in each of the areas showed a homogeneous cell distribution in the alginate bioink, a fundamental aspect of printing resolution.
Most tissues have complex combinations and gradients of extracellular matrix constituents, each with specific biological and mechanical influences. A biomaterial should be biomimetic of the native environment and facilitate cellular functions. The high porosity of the alginate scaffold allows the cells to communicate and network with each other, and may also facilitate the flux of nutrients and metabolites between the scaffold and its surrounding environment. Cell adhesion to the extracellular matrix is a preliminary phase of tissue formation that happens before cell proliferation and the organization of extracellular matrix molecules into functional tissue. The proliferation of cells plays a vital role in wound healing and tissue growth, and is therefore a very important factor when analyzing bioprinted constructs for tissue engineering applications. The RGD-conjugated alginate enhanced cell attachment in printed constructs, leading to improved cell spreading and proliferation. The proliferation of cells in the printed scaffolds was quantified by counting three separate areas on days 0 and 8 (Figure 6). The overall cell proliferation was found to be 219.674% after 8 days of culture. These results signify the scaffold has adequate biocompatibility to be used as a synthetic extracellular matrix for delivering cells to repair damaged or nonfunctional tissue.
To analyze the success of RGD peptide conjugation on the alginate bioink, a comparison experiment was performed using cell-laden, RGD-conjugated 15% concentration, 5% oxidation alginate bioink and cell-laden, non-conjugated 15% concentration, 5% oxidation alginate bioink. DAPI staining for nuclei and phalloidin staining for actin were used to analyze the cell spreading in printed constructs on day 8. Images of each sample (at least three random pictures per sample) were taking using a confocal microscope system using Z-stack parameters of 30 optical slices over a 300 depth (Figure 7). The cell spreading shown in the sample with RGD-conjugated alginate proves the successful incorporation of the peptide on the alginate. Cell migration is an important step in tissue development; therefore the conjugation of RGD peptides on alginate improves the likelihood of in vivo application using this bioink.
<img alt="Figure 1" src="/files/ftp_upload/53156/53156fig1.jpg" /> 
Figure 1. Palmetto Printer. Programmable Logic Controller coordinates the actions of all printer functions (A). An airtight containment chamber (B) with filtered intake (C) and exhaust (C) maintains a regulated internal positive pressure to reduce the chance of contamination. Dual UV lights (D) mounted in the chamber ceiling can be programmed to operate at safe intervals. A Janome 2300N XYZ Robot (E) is programmed and controlled by an integrated computer (I). Three dispenser controllers (G) are programmed to regulate the output of dispenser guns (F) available to be loaded onto the robot Z-axis arm (H) under computer control. The temperature of the robot sample holder (J) is set between 4 and 40 &#176;C by a water bath controller (K). Dual digital cameras (L) are available to monitor printer activity and sample formation. One camera is mounted onto the robot Z-axis arm and provides a magnified image of the dispenser tip of the loaded gun. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53156/53156fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53156/53156fig2.jpg" /> 
Figure 2. Tool Nest, Optic Sensors, and Displacement Laser of Bioprinter. A. Unloaded tool nest view from the front. B. Loaded tool nest view from the front. C. Optic Sensors measuring the dispensing tip end in three-dimensional space. D. Distance laser measuring the Z coordinate of the printing surface. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53156/53156fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53156/53156fig3.jpg" /> 
Figure 3. Visual PathBuilder Software. Image of computer-aided design drawing software used for designing the external architecture of bioprinted constructs. This program provides the ability to generate incrementally spaced droplets and complex geometries. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53156/53156fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53156/53156fig4.jpg" /> 
Figure 4. JR-C Points Software. Screenshot of the printer-compatible design software. This program allows the user to control the deposition method (i.e.,&#160;single drop deposition or continuous pathway deposition), deposition speed, distance between syringe tip end and printing substrate surface, the allotted time for deposition of each drop, and the three-dimensional placement of droplets (refer to Table 1). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53156/53156fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53156/53156fig5.jpg" /> 
Figure 5. Fluorescent Image of Stained hADSCs Post-Printing.&#160;Cell viability/cytotoxicity fluorescent images of hADSC&#8217;s in bioprinted construct taken using a confocal microscope system (Z-stack parameters of 30 optical slices over a depth) after 0 (A) and 8 (B) days. The hADSC&#8217;s were labeled post-printing using a mammalian cell viability/cytotoxicity assay. Live cells are stained green, and dead cells are stained red. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53156/53156fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53156/53156fig6.jpg" /> 
Figure 6. Quantified Viabilities of Bioprinted Constructs. The number of live and dead cells was quantified using a viability/cytotoxicity assay. The live/dead cell counts for Day 0 are shown in (A), and the counts for Day 8 in (B). The number of live cells counted for each area on days 0 and 8 are shown in (C) and were used to quantify cell proliferation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53156/53156fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53156/53156fig7.jpg" /> 
Figure 7. Comparison of Cell-Laden, Non-Conjugated and RGD-Conjugated Alginates. Fluorescent images of bioprinted hADSC&#8217;s in non-conjugated (A), and in RGD-conjugated (B) 15% concentration 5% oxidation alginate bioink taken using a confocal microscope system (Z-stack parameters of 30 optical slices over a depth). The hADSC&#8217;s were stained with phalloidin and DAPI stains to analyze the cell spreading in each of the constructs. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53156/53156fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Table of Commands</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Command</td>
			<td>Robot Response</td>
		</tr>
		<tr>
			<td>PTP Point</td>
			<td>Robotic arm moves to indicated posiiton in X, Y, Z space</td>
		</tr>
		<tr>
			<td>Find_Base_Z</td>
			<td>Uses the SICK laser to measure the printing surface of the substrate; The distance between the syringe tip end and substrate surface is manually set.&#160;</td>
		</tr>
		<tr>
			<td>Work Adj. No. (Work Adjustment Number)</td>
			<td>Commands robot to use SICK laser (for determining substrate surface), Gun 1, Gun 2, or Gun 3.</td>
		</tr>
		<tr>
			<td>Get_1</td>
			<td>Commands robot ot retrieve and load Gun 1</td>
		</tr>
		<tr>
			<td>Find_Tip1_YZ</td>
			<td>The robot finds the tip end of Gun 1 in the Y and Z directions</td>
		</tr>
		<tr>
			<td>Find_Tip1_X</td>
			<td>The robot finds the tip end of Gun 1 in the X direction</td>
		</tr>
		<tr>
			<td>Point Dispense</td>
			<td>The robot will dispense one droplet of bioink in the determined X, Y, Z position</td>
		</tr>
		<tr>
			<td>Pallet No. (Pallte Number)</td>
			<td>Incorporates a manually coded design for printing, e.g., an array.</td>
		</tr>
		<tr>
			<td>Dispense Time</td>
			<td>Is the time allotted for the deposition of each individual drop.</td>
		</tr>
		<tr>
			<td>Store_1</td>
			<td>Commands the robot to return Gun 1 to the toolnest, and return to home position: (0,0,0).</td>
		</tr>
	</tbody>
</table>
Table 1. Programmable Computer Software Commands. This chart outlines the programmable computer software commands, which are used to control the robotic arm and optimize printability parameters.

Watch this Scientific Journal Video about Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer at JoVE.com

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

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 ...

viability-bioprinted-cellular-constructs-using-three-dispenser

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.0K Views.  Medical University of South Carolina. 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.

Video: Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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 ...

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.

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 ...

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 ...

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 ...

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

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 ...

3D Bioprinting for Rapid Vascular Network Fabrication Using Vessels-on-a-Plate

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

Author Spotlight: Automated Bioprinting for High-Throughput Vascular Model Fabrication

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

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

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

Embedded Bioprinting of Tissue-like Structures Using κ-Carrageenan Sub-Microgel Medium

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

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

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility