The authors gratefully acknowledge the support provided by the National Natural Science Foundation of China (Grant Nos. 12125205, 12072316, 12132014), and the China Postdoctoral Science Foundation (Grant No. 2022M712754).

1. Theoretical preparation
<ol>
	<li>Define three parameters: liquid absorbance (Al), solid absorbance (As), and threshold time (tT)17.</li>
	<li>Rewrite the traditional Jacobs working curve using these three parameters17 according to Equation 1: 
	<img alt="Equation 1" src="/files/ftp_upload/65277/65277eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(Equation 1) 
	Here, tH is the curing time of one single layer, and H is the height of one single layer.</li>
</ol>
2. Parameter acquisition
<ol>
	<li>Measure the threshold time of the bioink using a rheometer equipped with an element for temperature control.
	<ol>
		<li>Use a 365 nm light source to expose the testing platform of the rheometer and make the light intensity at a certain value.</li>
		<li>Set the rheometer to get the Time-Moduli data during a period of 300 s, and take each data point every 0.3 s through the Time Settings options in the rheometer software.Click the Start Test button of the rheometer to start the test, and at the same time, click the Start Button of the light source.</li>
		<li>Counting from the start of exposure, when the storage modulus data is equal to the loss modulus data, the corresponding time is recognized as the threshold time. Record manually.</li>
	</ol>
	</li>
	<li>Build the absorbance test equipment as shown in the previous work17. Use two upper and lower glass slides to clamp the ring-shaped printed structure (5 mm inner diameter, 10 mm outer diameter) with a thickness of 500&#181;m so that the inner circle of the ring forms a chamber. Place the chamber on the test area of the light intensity meter and set the light source to expose the chamber area. 
	NOTE: Figure 1 shows the schematic diagram of photorheological test results and data processing results, and the absorbance testing equipment.
	<ol>
		<li>Measure the incident light intensity (Ii) when the test chamber is not filled with material from the absorbance test equipment by reading the display of the light intensity meter of the testing equipment.</li>
		<li>Fill the test chamber with 10 &#181;Lof bioink.</li>
		<li>Expose the test chamber with bioink to UV light at 365 nm. Obtain the light intensity (Ilh) from the absorbance test equipment by reading the display of the light intensity meter of the testing equipment.</li>
		<li>Obtain the light intensity when the bioink is cured (Ish) from the absorbance test equipment by reading the display of the light intensity meter of the testing equipment when the value no longer changes. This value is the solid absorbance, Ish.</li>
		<li>Calculate the liquid absorbance and solid absorbance using Equations 2 and 3: 
		<img alt="Equation 2" src="/files/ftp_upload/65277/65277eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;Equation 2 
		<img alt="Equation 3" src="/files/ftp_upload/65277/65277eq03.jpg" style="margin: auto;" />&#160; &#160; &#160;Equation 3</li>
	</ol>
	</li>
	<li>Obtain the Jacobs working curve according to the obtained parameters.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65277/65277fig01.jpg" /> 
Figure 1: Test results and equipment. (A) Schematic diagram of photorheological test results and data processing results. (B) Absorbance testing equipment. This figure has been modified with permission from Li et al.17. <a href="https://www.jove.com/files/ftp_upload/65277/65277fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Continuous DLP printing parameter settings
<ol>
	<li>Use DLP software to achieve DLP printing, and the set of printing parameters in the software as follows.</li>
	<li>Set the exposure time of the first single layer as the threshold time (tT) in the software&#39;s parameter settings.
	<ol>
		<li>Calculate the exposure time of curing 10 &#181;mthick materials according to Equation 1 and subtract the threshold time to obtain the real exposure time for curing a single layer.</li>
	</ol>
	</li>
	<li>Set the time interval between adjacent layers to 0 s in the software&#39;s parameter settings.</li>
	<li>Start the printer by clicking the Start button in the printing software. When the printing process ends, finish printing by clicking the Stop button in the printing software.</li>
</ol>

Quantitative Characterization of Bioink Properties for Continuous DLP Printing

<ol><li>Berthiaume, F., Maguire, T. J., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+engineering+and+regenerative+medicine%3A+history%2C+progress%2C+and+challenges%5BTitle%5D)+AND+%22Annual+Review+of+Chemical+and+Biomolecular+Engineering%22%5BJournal%5D)">Tissue engineering and regenerative medicine: history, progress, and challenges.</a> Annual Review of Chemical and Biomolecular Engineering. 2 (1), 403-430 (2011).</li>
<li>Ng, W. L., Chua, C. K., Shen, Y. -F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Print+me+an+organ%21+Why+we+are+not+there+yet%5BTitle%5D)+AND+%22Progress+in+Polymer+Science%22%5BJournal%5D)">Print me an organ! Why we are not there yet.</a> Progress in Polymer Science. 97, 101145(2019).</li>
<li>Sun, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+bioprinting+roadmap%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">The bioprinting roadmap.</a> Biofabrication. 12 (2), 022002(2020).</li>
<li>Jiang, T., Munguia-Lopez, J. G., Flores-Torres, S., Kort-Mascort, J., Kinsella, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extrusion+bioprinting+of+soft+materials%3A+An+emerging+technique+for+biological+model+fabrication%5BTitle%5D)+AND+%22Applied+Physics+Reviews%22%5BJournal%5D)">Extrusion bioprinting of soft materials: An emerging technique for biological model fabrication.</a> Applied Physics Reviews. 6 (1), 011310(2019).</li>
<li>Ng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((L.cControlling+droplet+impact+velocity+and+droplet+volume%3A+Key+factors+to+achieving+high+cell+viability+in+sub-nanoliter+droplet-based+bioprinting%5BTitle%5D)+AND+%22International+Journal+of+Bioprinting%22%5BJournal%5D)">L.cControlling droplet impact velocity and droplet volume: Key factors to achieving high cell viability in sub-nanoliter droplet-based bioprinting.</a> International Journal of Bioprinting. 8 (1), 424(2021).</li>
<li>Yu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Printability+during+projection-based+3D+bioprinting%5BTitle%5D)+AND+%22Bioactive+Materials%22%5BJournal%5D)">Printability during projection-based 3D bioprinting.</a> Bioactive Materials. 11, 254-267 (2022).</li>
<li>Zhong, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioprinting+of+dual+ECM+scaffolds+encapsulating+limbal+stem%2Fprogenitor+cells+in+active+and+quiescent+statuses%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">Bioprinting of dual ECM scaffolds encapsulating limbal stem/progenitor cells in active and quiescent statuses.</a> Biofabrication. 13 (4), (2021).</li>
<li>Huh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combinations+of+photoinitiator+and+UV+absorber+for+cell-based+digital+light+processing+%28DLP%29+bioprinting%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">Combinations of photoinitiator and UV absorber for cell-based digital light processing (DLP) bioprinting.</a> Biofabrication. 13 (3), (2021).</li>
<li>Saed, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functionalized+poly+l-lactic+acid+synthesis+and+optimization+of+process+parameters+for+3D+printing+of+porous+scaffolds+via+digital+light+processing+%28DLP%29+method%5BTitle%5D)+AND+%22Journal+of+Manufacturing+Processes%22%5BJournal%5D)">Functionalized poly l-lactic acid synthesis and optimization of process parameters for 3D printing of porous scaffolds via digital light processing (DLP) method.</a> Journal of Manufacturing Processes. 56, 550-561 (2020).</li>
<li>Ng, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vat+polymerization-based+bioprinting-process%2C+materials%2C+applications+and+regulatory+challenges%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">Vat polymerization-based bioprinting-process, materials, applications and regulatory challenges.</a> Biofabrication. 12 (2), 022001(2020).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-fidelity+and+high-efficiency+additive+manufacturing+using+tunable+pre-curing+digital+light+processing%5BTitle%5D)+AND+%22Additive+Manufacturing%22%5BJournal%5D)">High-fidelity and high-efficiency additive manufacturing using tunable pre-curing digital light processing.</a> Additive Manufacturing. 30, 100889(2019).</li>
<li>Kelly, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Volumetric+additive+manufacturing+via+tomographic+reconstruction%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Volumetric additive manufacturing via tomographic reconstruction.</a> Science. 363 (6431), 1075-1079 (2019).</li>
<li>Tumbleston, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+liquid+interface+production+of+3D+objects%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Continuous liquid interface production of 3D objects.</a> Science. 347 (6228), 1349-1352 (2015).</li>
<li>Classens, K., Hafkamp, T., Westbeek, S., Remmers, J. J. C., Weiland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiphysical+modeling+and+optimal+control+of+material+properties+for+photopolymerization+processes%5BTitle%5D)+AND+%22Additive+Manufacturing%22%5BJournal%5D)">Multiphysical modeling and optimal control of material properties for photopolymerization processes.</a> Additive Manufacturing. 38, 101520(2021).</li>
<li>Gong, H., Beauchamp, M., Perry, S., Woolley, A. T., Nordin, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+approach+to+resin+formulation+for+3D+printed+microfluidics%5BTitle%5D)+AND+%22RSC+Advances%22%5BJournal%5D)">Optical approach to resin formulation for 3D printed microfluidics.</a> RSC Advances. 5 (129), 106621-106632 (2015).</li>
<li>Hofstetter, C., Orman, S., Baudis, S., Stampfl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combining+cure+depth+and+cure+degree%2C+a+new+way+to+fully+characterize+novel+photopolymers%5BTitle%5D)+AND+%22Additive+Manufacturing%22%5BJournal%5D)">Combining cure depth and cure degree, a new way to fully characterize novel photopolymers.</a> Additive Manufacturing. 24, 166-172 (2018).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Theoretical+prediction+and+experimental+validation+of+the+digital+light+processing+%28DLP%29+working+curve+for+photocurable+materials%5BTitle%5D)+AND+%22Additive+Manufacturing%22%5BJournal%5D)">Theoretical prediction and experimental validation of the digital light processing (DLP) working curve for photocurable materials.</a> Additive Manufacturing. 37, 101716(2021).</li>
<li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecularly+cleavable+bioinks+facilitate+high-performance+digital+light+processing-based+bioprinting+of+functional+volumetric+soft+tissues%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Molecularly cleavable bioinks facilitate high-performance digital light processing-based bioprinting of functional volumetric soft tissues.</a> Nature Communications. 13 (1), 3317(2022).</li>
</ol>

The authors have nothing to disclose.

The critical steps of this protocol are described in section 2. It is necessary to unify the light intensity used in the photorheology test and the printing light intensity in the actual tests. The absorbance testing equipment is the most important part. The shape of the test chamber should be the same as the photosensitive area of the light intensity meter. Due to the properties of the materials that continuously change during the whole UV light exposure process, the light intensity needs to continue to change<sup class...

Tissue engineering1 is important in the field of organ repair. Due to the lack of organ donation, some diseases, such as liver failure and kidney failure, cannot be cured well, and many patients do not receive timely treatment2. Organoids with the required function of the organs may solve the problem caused by the lack of organ donation. The construction of organoids depends on the progress and development of bioprinting technology3.
Compared with extrusion-type bioprinting4 and inkjet-type bioprinting5, the printing speed and printing accuracy of the digital light processing (DLP) bioprinting method are higher6,7. The printing module of the extrusion-type method is line-by-line, while the printing module of the inkjet-type method is dot-by-dot, which is less efficient than the layer-by-layer printing module of DLP bioprinting. The modulated ultraviolet (UV) light exposure to a whole layer of material to cure a layer in DLP bioprinting and the feature size of the image determines the accuracy of DLP printing. This makes DLP technology very efficient8,9,10. Due to overcuring of the UV light, the precise relationship between the curing time and the printing size is important for high-accuracy DLP bioprinting. Furthermore, continuous DLP printing is a modification of DLP printing method that can greatly improve the printing efficiency11,12,13. For continuous DLP printing, precise printing conditions are the most important factors.
The relationship between the curing time and the printing size is called the Jacobs working curve, which is widely used in DLP printing14,15,16. The traditional method to obtain the relationship is to expose the material for a certain time and measure the curing thickness to obtain a data point about the exposure time and curing thickness. Repeating this operation at least five times and fitting the data points obtains the Jacobs working curve. However, this method has obvious disadvantages; it needs to consume a lot of material to achieve the curing, the results are highly dependent on the printing conditions, the bioinks used in DLP bioprinting are expensive and rare, and the formability of the bioinks is usually not good, which can lead to inaccurate measurements of curing thickness.
This article provides a new method to obtain the curing relationship according to the physical properties of the bioink. Using this theory can optimize continuous DLP printing. This method can be used to obtain the curing relationship more quickly and accurately; the continuous DLP curing can therefore be better determined.

<table><tbody><tr><td>Brilliant Blue</td><td>Aladdin (Shanghai, China).</td><td>6104-59-2&amp;nbsp;</td><td></td></tr><tr><td>DLP software</td><td>Creation Workshop</td><td>N/A</td><td></td></tr><tr><td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate</td><td></td><td>N/A</td><td>LAP; synthesized</td></tr><tr><td>Light source</td><td>OmniCure</td><td>https://www.excelitas.com/product-category/omnicure-s-series-lamp-spot-uv-curing-systems</td><td>365 nm</td></tr><tr><td>Polyethylene (glycol) diacrylate</td><td>Sigma-Aldrich</td><td>455008</td><td>PEGDA Mw ~700</td></tr><tr><td>Rheometer&amp;nbsp;</td><td>Anton Paar, Austria</td><td>MCR302</td><td></td></tr></tbody></table>

quantitative characterization of liquid photosensitive bioink properties for continuous digital light processing based printing

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing parameters of digital light processing (DLP). However, the acquisition of working curves wastes materials and requires high formability of materials, which are not suitable for biomaterials. In addition, the reduction of cell activity due to multiple exposures and the failure of structural formation due to repeated positioning are both unavoidable problems in conventional DLP bioprinting. This work introduces a new method of obtaining the working curve and the improvement process of continuous DLP printing technology based on such a working curve. This method of obtaining the working curve is based on the absorbance and photorheological properties of the biomaterials, which do not depend on the formability of the biomaterials. The continuous DLP printing process, obtained from improving the printing process by analyzing the working curve, increases the printing efficiency more than tenfold and greatly improves the activity and functionality of cells, which is beneficial to the development of tissue engineering.

This article shows a new method to obtain curing parameters and introduces a new way to achieve continuous DLP printing, demonstrating the efficiency of this method in determining the working curve.
We used three different materials in DLP printing to verify the accuracy of the theoretical working curve obtained by the method introduced in this article. The materials are 20% (v/v) polyethylene (glycol) diacrylate (PEGDA), 0.5% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) with d...

Watch this Scientific Journal Video about Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing at JoVE.com

Author Spotlight: Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing  

This study uses temperature and material composition to control the yield stress properties of yield stress fluids. The solid-like state of the ink can protect the printing structure, and the liquid-like state can continuously fill the printing position, realizing the digital light processing 3D printing of extremely soft bioinks.

Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing ...

quantitative-characterization-liquid-photosensitive-bioink-properties

Research

JoVE Journal

Bioengineering

851 Views.  Zhejiang University. This study uses temperature and material composition to control the yield stress properties of yield stress fluids. The solid-like state of the ink can protect the printing structure, and the liquid-like state can continuously fill the printing position, realizing the digital light processing 3D printing of extremely soft bioinks.

Video: Author Spotlight: Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing  

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Digital Printing of Titanium Dioxide for Dye Sensitized Solar Cells

Silicon solar cell manufacturing is an expensive and high energy consuming process. In contrast, dye sensitized solar cell production is less environmentally damaging with lower processing temperatures presenting a viable and low cost alternative to conventional production. This paper further enhances these environmental credentials by evaluating the digital printing and therefore additive production route for these cells. This is achieved here by investigating the formation and performance of a metal oxide photoelectrode using nanoparticle sized titanium dioxide. An ink-jettable material was formulated, characterized and printed with a piezoelectric inkjet head to produce a 2.6 &#181;m thick layer. The resultant printed layer was fabricated into a functioning cell with an active area of 0.25 cm2 and a power conversion efficiency of 3.5%. The binder-free formulation resulted in a reduced processing temperature of 250 &#176;C, compatible with flexible polyamide substrates which are stable up to temperatures of 350 &#730;C. The authors are continuing to develop this process route by investigating inkjet printing of other layers within dye sensitized solar cells.

This paper investigates the suitability of inkjet printing for the manufacturing of dye-sensitized solar cells. A binder-free TiO2 nanoparticle ink was formulated and printed onto a FTO glass substrate. The printed layer was fabricated into a cell with an active area of 0.25 cm2 and an efficiency of 3.5%.

Silicon solar cell manufacturing is an expensive and high energy consuming process. In contrast, dye sensitized solar cell production is less ...

Engineering

Inkjet-printed Polyvinyl Alcohol Multilayers

Inkjet printing is a modern method for polymer processing, and in this work, we demonstrate that this technology is capable of producing polyvinyl alcohol (PVOH) multilayer structures. A polyvinyl alcohol aqueous solution was formulated. The intrinsic properties of the ink, such as surface tension, viscosity, pH, and time stability, were investigated. The PVOH-based ink was a neutral solution (pH 6.7) with a surface tension of 39.3 mN/m and a viscosity of 7.5 cP. The ink displayed pseudoplastic (non-Newtonian shear thinning) behavior at low shear rates, and overall, it demonstrated good time stability. The wettability of the ink on different substrates was investigated, and glass was identified as the most suitable substrate in this particular case. A proprietary 3D inkjet printer was employed to manufacture polymer multilayer structures. The morphology, surface profile, and thickness uniformity of inkjet-printed multilayers were evaluated via optical microscopy.

An inkjet printer was used to manufacture polyvinyl alcohol multilayers. Polyvinyl alcohol water-based ink was formulated, and the main physical properties were investigated.

Inkjet printing is a modern method for polymer processing, and in this work, we demonstrate that this technology is capable of producing polyvinyl alcohol ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

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

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

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

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 ...

Frugal Imaging Technique of Capillary Flow Through Three-Dimensional Polymeric Printing Powders

The development of novel imaging techniques of molecular and colloidal transport, including nanoparticles, is an area of active investigation in microfluidic and millifluidic studies. With the advent of three-dimensional (3D) printing, a new domain of materials has emerged, thereby increasing the demand for novel polymers. Specifically, polymeric powders, with average particle sizes on the order of a micron, are experiencing a growing interest from academic and industrial communities. Controlling material tunability at the mesoscopic to microscopic length scales creates opportunities to develop innovative materials, such as gradient materials. Recently, a need for micron-sized polymeric powders has been growing, as clear applications for the material are developing. Three-dimensional printing provides a high-throughput process with a direct link to new applications, driving investigations into the physio-chemical and transport interactions on a mesoscale. The protocol that is discussed in this article provides a non-invasive technique to image fluid flow in packed powder beds, providing high temporal and spatial resolution while leveraging mobile technology that is readily available from mobile devices, such as smartphones. By utilizing a common mobile device, the imaging costs that would normally be associated with an optical microscope are eliminated, resulting in a frugal-science approach. The proposed protocol has successfully characterized a variety of combinations of fluids and powders, creating a diagnostic platform for quickly imaging and identifying an optimal combination of fluid and powder.

The proposed technique will provide a novel, efficient, frugal, and non-invasive approach for imaging fluidic flow through a packed powder bed, yielding high spatial and temporal resolution.

The development of novel imaging techniques of molecular and colloidal transport, including nanoparticles, is an area of active investigation in ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

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 ...