The authors would like to acknowledge Chris Morrow for technical assistance. Funding was provided by NIH SBIR (R43) grant 1R43GM101859-01 (MPI) GRANT10940803.

1. Cell Culture Preparation
<ol>
	<li>Store NIH/3T3 cell stocks in liquid nitrogen until ready for use.</li>
	<li>Prepare complete media for NIH/3T3 cells using Dulbecco&#8217;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 10 mM HEPES buffer, 50 units/ml penicillin, and 50 &#956;g/ml streptomycin.</li>
	<li>Thaw cells for 2-3 min in a shaking water bath at 37 &#176;C.</li>
	<li>Resuspend cells in 5 ml of complete media and centrifuge at 1,500 x g for 3 min.</li>
	<li>Remove the cell supernatant without disturbing the cell pellet.</li>
	<li>Resuspend the cells in 5 ml of media and count cells using a hemocytometer.
	<ol>
		<li>Remove 10 &#956;l of the cell suspension and place in a 0.5 ml microcentrifuge tube.</li>
		<li>Add 10 &#956;l of Trypan Blue to the cell suspension and stir with the tip of the pipette.</li>
		<li>Pipette 10 &#956;l of the stained cells into a hemocytometer chamber.</li>
		<li>Count cells in hemocytometer at 10x magnification.
		<ol>
			<li>Count the live cells (small, white balls) for 4 of the 9 large squares. Only count the live cells that do not appear dark blue from the Trypan Blue stain.</li>
			<li>Calculate the average number of cells per large square, resulting in the number of cells x 104 cells/ml in the original cells suspension.</li>
			<li>Seed cells at a density of 1 x105 cells/ml with a total of 5 ml in each T25 flask.</li>
		</ol>
		</li>
		<li>Culture cells to between 70% and 80% confluency before beginning experiments. Refresh media every 2-3 days or as needed.</li>
	</ol>
	</li>
</ol>
2. Printing Surface Preparation
<ol>
	<li>Mark a rectangle with marker on the bottom of a tissue-culture-treated polystyrene (TCTPS) 12 well plate the size of the print head (7 mm x 19 mm).</li>
	<li>Pipette 50 &#956;l of fetal bovine serum into the rectangular area and spread it over the entire region with the tip.</li>
	<li>Leave the Petri dish in a sterile biosafety hood O/N to allow the serum spot to dry completely. These plates should be prepared no more than 2 days prior to use.</li>
</ol>
3. Submerged Printing
<ol>
	<li>Rinse the print head out with distilled water.
	<ol>
		<li>Fill a 60 mm Petri dish with distilled water and dock the print head onto the surface.</li>
		<li>Flow distilled water through each of the lines for 2 min at 150 &#956;l/min using a pneumatic pump.</li>
		<li>Discard the water from the Petri dish and fill it with clean water.</li>
		<li>Move the print head up and down in the water 3 times.</li>
	</ol>
	</li>
	<li>Dock the print head center over the serum-coated spot in the pre-prepared 12 well plate.</li>
	<li>Prime the print head with pre-warmed complete media, 300 &#956;l per channel.</li>
	<li>Print suspended cells at a concentration of 50,000 cells/ml onto the surface and 60 &#956;l/min, 100 &#956;l per channel.</li>
	<li>Place the print head, left docked against the surface, and the manifold in a cell culture incubator set to 5% CO2 and 37&#160;&#176;C for 2 hr.</li>
	<li>After the 2 hr incubation, remove the print head and place the lid on the culture dish. The time at which the print head removed is considered the 0 hr time point.</li>
</ol>
4. Standard Cell Culture
<ol>
	<li>Add 1 ml of pre-warmed media to one of the serum-spotted TCTPS 12-well plate prepared in step 2.</li>
	<li>Take 1 ml of the remaining cell suspension from step 2 and pipette into a single well of a 12-well TCTPS plate. This will produce a culture containing 50,000 cells in the dish.</li>
	<li>Place the cell culture plate in an incubator at 37 &#176;C for 2 hr.</li>
	<li>After the 2 hr incubation begin timing; for consistency between printed and seeding samples, this is considered the 0 hr time point.</li>
</ol>
5. Culture Visualization
<ol>
	<li>After 0, 2, 24, and 48 hr take cells from each of the two methods described above and image using a standard inverted microscope.
	<ol>
		<li>Stain the cells using propidium iodide, a red fluorescent stain that is only incorporated into the nucleus of dead cells.
		<ol>
			<li>Gently rinse the cells 3 times with 1 ml of phosphate buffered saline with calcium and magnesium (PBS++) to remove any debris.</li>
			<li>Add 1 ml of pre-warmed PBS++ to each culture vessel.</li>
			<li>Add 1 &#956;l of propidium iodide stock solution (1 mg/ml) to each culture vessel.</li>
			<li>Incubate the cells stained with propidium iodide at 37 &#176;C&#160;for 10 min.</li>
		</ol>
		</li>
		<li>Image the cells using an inverted microscope at 10x and 40x magnification.</li>
	</ol>
	</li>
	<li>Discard the cells in an appropriate biohazard container.</li>
</ol>

<ol>
	<li>Fernandes, T. G., Diogo, M. M., Clark, D. S., Dordick, J. S., Cabral, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cellular+microarray+platforms:+applications+in+drug+discovery,+toxicology+and+stem+cell+research.">High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research.</a> Trends in Biotechnology. 27, 342-349 (2009).</li><li>Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A., Roda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+assays:+fuelling+drug+discovery.">Cell-based assays: fuelling drug discovery.</a> Anal Bioanal Chem. 398, 227-238 (2010).</li><li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+microfluidic+devices+for+in+vitro+cell+culture+for+cell-biology+research.">Recent developments in microfluidic devices for in vitro cell culture for cell-biology research.</a> TrAC Trends in Analytical Chemistry. 35, 150-164 (2012).</li><li>Geysen, H. M., Schoenen, F., Wagner, D., Wagner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+compound+libraries+for+drug+discovery:+an+ongoing+challenge.">Combinatorial compound libraries for drug discovery: an ongoing challenge.</a> Nature Reviews Drug Discovery. 2, 222-230 (2003).</li><li>Xu, Y., Shi, Y., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+approach+to+stem-cell+biology+and+regenerative+medicine.">A chemical approach to stem-cell biology and regenerative medicine.</a> Nature. 453, 338-344 (2008).</li><li>Khademhosseini, A., Langer, R., Borenstein, J., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+technologies+for+tissue+engineering+and+biology.">Microscale technologies for tissue engineering and biology.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 2480-2487 (2006).</li><li>Bhadriraju, K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cellular+microenvironments+to+improve+cell-based+drug+testing.">Engineering cellular microenvironments to improve cell-based drug testing.</a> Drug Discovery Today. 7, 612-620 (2002).</li><li>Castel, D., Pitaval, A., Debily, M. -. A., Gidrol, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+microarrays+in+drug+discovery.">Cell microarrays in drug discovery.</a> Drug Discov. Today. 11, 616-622 (2006).</li><li>Tsui, J. H., Lee, W., Pun, S. H., Kim, J., Kim, D. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-assisted+in+vitro+drug+screening+and+carrier+production.">Microfluidics-assisted in vitro drug screening and carrier production.</a> Advanced Drug Delivery Reviews. , (2013).</li><li>Gottwald, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chip-based+platform+for+the+in+vitro+generation+of+tissues+in+three-dimensional+organization.">A chip-based platform for the in vitro generation of tissues in three-dimensional organization.</a> Lab on a Chip. 7, 777-785 (2007).</li><li>Liu, R., Lee, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Integrated Biochips for DNA Analysis. , (2008).</li><li>Natarajan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous-flow+microfluidic+printing+of+proteins+for+array-based+applications+including+surface+plasmon+resonance+imaging.">Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging.</a> Analytical Biochemistry. 373, 141-146 (2008).</li><li>Natarajan, S., Hatch, A., Myszka, D. G., Gale, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+Conditions+for+Protein+Array+Deposition+Using+Continuous+Flow.">Optimal Conditions for Protein Array Deposition Using Continuous Flow.</a> Anal. Chem. 80, 8561-8567 (2008).</li><li>Keighley, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+for+high+throughput+kinetics+early+in+the+drug+discovery+process.">The need for high throughput kinetics early in the drug discovery process.</a> , (2011).</li><li>Xu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+in+vitro+ovarian+cancer+coculture+model+using+a+high-throughput+cell+patterning+platform.">A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform.</a> Biotechnol J. 6, 204-212 (2011).</li><li>Tuckwell, D. S., Weston, S. A., Humphries, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins:+a+review+of+their+structure+and+mechanisms+of+ligand+binding.">Integrins: a review of their structure and mechanisms of ligand binding.</a> Symposia of the Society for Experimental Biology. 47, 107 (1993).</li><li>Roberts, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mixed+self-assembled+monolayers+presenting+RGD+and+(EG)+3OH+groups+to+characterize+long-term+attachment+of+bovine+capillary+endothelial+cells+to+surfaces.">Using mixed self-assembled monolayers presenting RGD and (EG) 3OH groups to characterize long-term attachment of bovine capillary endothelial cells to surfaces.</a> Journal of the American Chemical Society. 120, 6548-6555 (1998).</li><li>Chandra, R. A., Douglas, E. S., Mathies, R. A., Bertozzi, C. R., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+cell+adhesion+encoded+by+DNA+hybridization.">Programmable cell adhesion encoded by DNA hybridization.</a> Angewandte Chemie. 118, 910-915 (2006).</li><li>Hsiao, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell+surface+modification+with+DNA+for+the+capture+of+primary+cells+and+the+investigation+of+myotube+formation+on+defined+patterns.">Direct cell surface modification with DNA for the capture of primary cells and the investigation of myotube formation on defined patterns.</a> Langmuir. 25, 6985-6991 (2009).</li><li>Black, C. B., Duensing, T. D., Trinkle, L. S., Dunlay, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Screening+Using+High-Throughput+Flow+Cytometry.">Cell-Based Screening Using High-Throughput Flow Cytometry.</a> ASSAY and Drug Development Technologies. 9, 13-20 (2011).</li><li>Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting:+inkjet+printing+proteins+and+hybrid+cell-containing+materials+and+structures.">Bioprinting: inkjet printing proteins and hybrid cell-containing materials and structures.</a> J. Mater. Chem. 18, 5717-5721 (2008).</li><li>Onoe, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Microfabrication:+Observing+Intercellular+Interactions+Using+Lithographically-Defined+DNA+Capture+Sequences.">Cellular Microfabrication: Observing Intercellular Interactions Using Lithographically-Defined DNA Capture Sequences.</a> Langmuir. 28, 8120-8126 (2012).</li><li>Hsiao, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-Coated+AFM+Cantilevers+for+the+Investigation+of+Cell+Adhesion+and+the+Patterning+of+Live+Cells.">DNA-Coated AFM Cantilevers for the Investigation of Cell Adhesion and the Patterning of Live Cells.</a> Angewandte Chemie International Edition. 47, 8473-8477 (2008).</li></ol>

The authors are employee and shareholders of Wasatch Microfluidics that produces reagents and/or instruments used in this manuscript.

The 3D microfluidic printing technology described here is uniquely capable of microfluidically printing arrays of cells into a liquid filled well, i.e. a submerged surface. By printing onto submerged surfaces, cell microarrays can be produced that maintain the physiologically relevant cellular phenotype of cells as well as the ability to multiplex cells in the bottom of a single well Figure 4. The results of this study show that microfluidically printing cells results in cell attachment with com...

Recent advances in the pharmaceutical industry have led to increased interest in using cellular microarrays in the drug discovery process for drug screening and cytotoxicological analysis1,2,3. The development of in vitro high-throughput assays and screening methods using cell microarrays would facilitate the rapid and cost-effective development of drug candidates as well as advance the fundamental understanding of the cell1,4. The traditional approach to screening with cells uses conventional well-plate platforms; however this approach is limited due to the high cost, limited throughput, and limited ability for quantitative information on cell function1,5. Due to these limitations, research in cellular microarray technologies is burgeoning for molecular biological characterization, tissue engineering, and drug screening1,6. The advantages of cellular microarrays include smaller sample use, minimal effects of cellular phenotype heterogeneity masking information, and most importantly the ability to automate assays for more high-throughput applications1,7,8.
The pharmaceutical industry currently utilizes high-throughput cell-based screening assays with 2D cell monolayer cultures for drug screening in microtiter well plates9. Multiplexing cells in wells of microtiter plates offers the potential for higher throughput with unique experimentation options. Further, the current technologies for cellular microarrays allow the cells to dry which could dramatically alter the phenotype of the cells from in vivo10,11. In order to overcome these problems, the MFCA was engineered and is shown in Figure 1. The design of the MFCA 3D fluidics enables the printhead tip in Figure 1 to be lowered into a bath and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and forms a reversible seal. The MFCA technology is uniquely suited to interface with submerged surfaces, which is required for both cell cultures and tissue slice systems, and is difficult or impossible with most other approaches. Pins or ink-jet printing will not work, and 2D microfluidic devices are not suited for deposition or interfacing with large arrays of discrete spots. Further, by miniaturizing and localizing the experiment - the cellular microarray - the MFCA overcomes the major problems associated with high-throughput cell-based screening assays.
The CFM uses 3D channel networks to cycle small volume fluid samples over microscopic spot locations on a surface12,13. By printing with flow, biomolecules, cells, and other reagents are maintained in a liquid environment throughout the printing process, enabling the printing of sensitive biomolecules and cells without exposure to air, which hinders the current cell printing techniques. It is also possible to print directly from crude material such as hybridoma or supernatants provided there is a capture mechanism on the array surface. The objective of this manuscript is to explain in detail the submerged printing of two cell types onto a surface.

<table border="0" cellpadding="0" cellspacing="0" width="571">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Continuous Flow Microspotter</td>
			<td style="width:71px;">Wasatch Microfluidics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NIH/3T3 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dubbleco&#39;s Modified Eagle Medium</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">11965-092</td>
			<td>base media for cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">HEPES buffer</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">15630-080</td>
			<td>cell media additive (control pH)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Sodium pyruvate</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">11360-070</td>
			<td>cell media additive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>cell media additive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Trypan blue</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">15250-061</td>
			<td>stain cell sfor counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Haemocytometer</td>
			<td style="width:71px;">Fisher</td>
			<td align="right" style="width:119px;">267110</td>
			<td>cell chamber to count cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Nikon Eclipse TS100</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Used to check on cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Nikon Eclipse TE2000-U</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Used for collecting images</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Phosphate Buffered Saline (with calcium and magnesium)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">14040-133</td>
			<td>rinsing cells before passaging and before staining with PI</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">TrypLE Express&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A12177-01</td>
			<td>used to remove cells from surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the submerged printing of cells onto a modified surface using a continuous flow microspotter

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

The fibroblast cell line NIH/3T3 cells were printed or seeded onto a submerged surface. The cells were grown to a density of 5 X 104 cells per ml. Cells were seeded using traditional cell culture techniques. Cells were printed using a large-format, twelve-flow cell printhead where the channels are larger (~500 &#181;m) than the CFM used for proteins and other biomolecules. The cells were printed or seeded onto a serum coated surface. The printing process is shown in Figure 1.
<p class="jov...

Watch this Scientific Journal Video about The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter at JoVE.com

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

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

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

the-submerged-printing-cells-onto-modified-surface-using-continuous

Research

JoVE Journal

Bioengineering

8.7K vistas.  Wasatch Microfluidics. This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

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

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

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 ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

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 ...

Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by scoring MN in once-divided, cytokinesis-blocked binucleated cells or fully divided mononucleated cells. Historically, light microscopy has been the gold standard method to score the assay, but it is laborious and subjective. Flow cytometry has been used in recent years to score the assay, but is limited by the inability to visually confirm key aspects of cellular imagery. Imaging flow cytometry (IFC) combines high-throughput image capture and automated image analysis, and has been successfully applied to rapidly acquire imagery of and score all key events in the MN assay. Recently, it has been demonstrated that artificial intelligence (AI) methods based on convolutional neural networks can be used to score MN assay data acquired by IFC. This paper describes all steps to use AI software to create a deep learning model to score all key events and to apply this model to automatically score additional data. Results from the AI deep learning model compare well to manual microscopy, therefore enabling fully automated scoring of the MN assay by combining IFC and AI.

The micronucleus (MN) assay is a well-established test for quantifying DNA damage. However, scoring the assay using conventional techniques such as manual microscopy or feature-based image analysis is laborious and challenging. This paper describes the methodology to develop an artificial intelligence model to score the MN assay using imaging flow cytometry data.

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by ...

Quantitative Characterization of Bioink Properties for Continuous DLP Printing

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.

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.

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