Name: fabrication of a multiplexed artificial cellular microenvironment array
Uploaded: 2018-09-07T13:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array at JoVE.com

We thank Prof. N. Nakatsuji at iCeMS, Kyoto University, for providing human ES cells. We also thank Prof. A. Maruyama at Tokyo Institute of Technology for his support in the use of the atomic force microscope. Funding was generously provided by the Japan Society for the Promotion of Science (JSPS; 22350104, 23681028, 25886006, and 24656502); funding was also provided by the New Energy and Industrial Technology Development Organization (NEDO) and the Terumo Life Science Foundation. The WPI-iCeMS is supported by the World Premier International Research Centre Initiative (WPI), the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. A part of this work was supported by Kyoto University Nanotechnology Hub and the AIST Nano-Processing Facility in "Nanotechnology Platform Project" sponsored by MEXT, Japan.

1. Fabrication of MACME Array
Note: All materials and equipment are listed in the Materials Table.
<ol>
	<li>Preparation of masks for a nanofiber array and a mold for a microfluidic structure
	<ol>
		<li>Create three-dimensional (3D) images of masks used for the nanofiber arrays and molds for microfluidic structures using 3D-computer graphics software packages (Table 1). 
		Note: The 3D images are read and printed by a 3D printer. The printed masks and mold have the same dimensions with 3D images defined using the graphics software at step 1.1.1.</li>
		<li>Print masks and a mold based on these designs using a 3D printer. 
		Note: In this protocol, an ink-jet-like 3D printer with UV-curable resin was used30. The printer resolution was 635 &#215; 400 dpi and 15 &#181;m in X, Y and Z, respectively, however, the actual X, Y resolution was approximately four times lower.</li>
	</ol>
	</li>
	<li>Nanofiber-array preparation (Figure 3A)
	<ol>
		<li>Prepare polymer solutions for electrospinning.
		<ol>
			<li>Dilute 13% PMGI liquid with tetrahydrofuran by 9% (w/v)22.</li>
			<li>Dissolve 0.08 g of PS (Number-average molecular weight (Mn): 130,000) in THF:dimethylformamide (1:1 volume ratio)21, and add THF:dimethylformamide to bring the volume up to the final 1 mL (8% (w/v) PS solution).</li>
			<li>Dissolve 0.1 g of GT in water:acetic acid:ethyl acetate (1:1.6:2.1 volume ratio), and add water:acetic acid:ethyl acetate to bring the volume up to the final 1 mL (10% (w/v) GT solution) as described23,31.</li>
		</ol>
		</li>
		<li>Use a magnetron sputtering machine, deposit a thin platinum layer with a 5-nm thickness on a polystyrene (PS) baseplate (127.7 &#215; 85.5 mm), which serves as a cathode in the electrospinning setup.</li>
		<li>Load each polymer solution into a 5-mL syringe equipped with a 23-G stainless-steel blunt needle.</li>
		<li>Place and anchor the syringe with the needle on a syringe pump with 12-cm apart from the collector of the electrospinning device.</li>
		<li>Connect the syringe needle to a high-voltage power supply, and set to apply to 11 kV.</li>
		<li>Set the pumping rate of 0.2 mL/h.</li>
		<li>Keep temperature and humidity at 30 &#176;C and &#60; 30% (v/v).</li>
		<li>Put the mask on the baseplate prepared at step 1.2.2.</li>
		<li>Fabricate nanofibers on the baseplate through the holes of mask with distinct densities by changing the electrospinning time (e.g., 20, 60, 90, and 180 s).</li>
		<li>Remove the mask from the baseplate. 
		Note. Ethanol can be sprayed on the electrospun GT nanofibers before removing the mask to avoid peeling off GT nanofibers together with the mask.</li>
		<li>Repeat step 1.2.4-1.2.10 until the completion of nanofiber-array fabrication.</li>
		<li>Place the nanofiber array in a desiccator at 25 &#176;C for 16 h to evaporate the remaining solvent.</li>
		<li>Crosslink GT nanofibers with 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.2 M N-hydroxysuccinimide (NHS) in ethanol at 25 &#176;C for 4 h23.</li>
		<li>Rinse GT nanofibers twice with 99.5% (v/v) ethanol and vacuum-dry at 25 &#176;C for 16 h.</li>
	</ol>
	</li>
	<li>Fabrication of microfluidic structures (Figure 3B)
	<ol>
		<li>Mix 2 g of PDMS curing agent and 20 g of PDMS base (1:10 weight ratio; Pre-PDMS). 24</li>
		<li>Pour the pre-PDMS mixture onto the mold fabricated in step 1.2.</li>
		<li>De-gas the pre-PDMS mixture in a desiccator for 30 min.</li>
		<li>Cure the pre-PDMS mixture in an oven at 65 &#176;C for 16 h.</li>
	</ol>
	</li>
	<li>Assembly of MACME arrays (Figure 3B)
	<ol>
		<li>Peel off the PDMS structure cured at step 1.3.4 from the mold and clean with 70% (v/v) ethanol.</li>
		<li>Treat atmospheric corona discharge on the bottom side of the PDMS structure32. 
		Note: In this protocol, the corona is discharged on the entire surface of a layer 3 or 4 times with a &#34;Handy&#34; corona discharge apparatus. Oxygen plasma treatment was replaced with atmospheric corona discharge to alter the surface adhesiveness.</li>
		<li>Assemble the PDMS structure with the nanofiber array quickly.</li>
		<li>Oven-bake at 65&#176;C for 2 days.</li>
	</ol>
	</li>
</ol>
2. Loading hESCs into MACME Array
<ol>
	<li>For the routine culture of H9 human embryonic stem cells (hESCs), maintain cells in xeno-free chemically defined culture medium for hPSC maintenance33 supplemented with 10 &#181;M Y-27632 ROCK inhibitor34 (named hPSC medium) in a 35-mm cell culture dish coated with basement membrane gel matrix (MG).</li>
	<li>Passage cells every 4 to 7 days using a recombinant trypsin-like protease.</li>
	<li>Sterilize a MACME array with 99.5% ethanol, by loading into the chambers for 10 min, and then remove the ethanol. Repeat this step 3 times.</li>
	<li>Introduce 12 &#181;L of MG, 10 &#181;g/mL vitronectin (VN), and 0.1% (w/v) GT into control culture chambers, and incubate the chambers at 37 &#176;C for 1 h to coat them on the chamber surfaces. 
	Note: In this protocol, MG, VN, and GT were selected as reference proteins for cell adhesion and culture. Because MG and VN are standard cell-culture matrices used for maintaining hPSC self-renewal, they are used as positive controls; two-dimensional GT coating (2DGT) or non-coating (NC) were used as negative controls.</li>
	<li>Introduce pre-warmed hPSC medium into all chambers of MACME array. Incubate the chambers for 30 min at 37 &#176;C.</li>
	<li>Wash H9 hESC cultured on a 35-mm dish with DPBS, add 0.5 mL of a recombinant trypsin-like protease to the dish and incubate at 37 &#176;C for 1 min and carefully aspirate only the supernatants mixed with protease. 
	Note: At the aspiration step, the cells are not detached.</li>
	<li>Immediately add hPSC medium, gently dispense the medium against the dish surface repeatedly to dissociate cells and transfer the detached cells to a 15-mL conical tube.</li>
	<li>Centrifuge at 200 x g for 3 min. Aspirate the supernatant and suspend cells in a pre-warmed hPSC medium.</li>
	<li>Introduce 12 &#181;L of the cell suspension (1.2 &#215; 106 cells/mL) into each microfluidic chamber. 
	Note: Introduce cells to one chamber from another using a micropipette. Because the number of cells seeded to chambers is proportional to the column density above the chamber floor, the initial cell-seeding density could be controlled even though samples from same cell suspension were used. Ensure pipetting is done gently. Remove excess medium from the chambers using a cotton-tipped stick after loading. The MACME array is compatible with both commonly used laboratory pipettes and automated pipette systems, and it does not require any special equipment.</li>
	<li>Change hPSC medium every 12 h and culture for 4 days. 
	Note: Constant volumes of the medium (1.6, 3.2, and 6.4 &#181;L) are maintained by using chambers of distinct sizes. Culture cells under a static condition to minimize shear stress except for the exchanging of the cell culture medium35,36.</li>
</ol>
3. Quantitative Single-cell Profiling
<ol>
	<li>On-plate fluorescent cell staining 
	Note: To analyze phenotypic changes of self-renewing hPSCs, this protocol selected three key phenotypic markers for this protocol: OCT4, 5-ethynyl-2&#39;-deoxyuridine (EdU), and Annexin V, to measure the status of pluripotency, proliferation, and apoptosis, respectively (Figure 2b). 4&#39;,6-diamidino-2-phenylindole (DAPI) is used to identify cell nuclei. OCT4 is one of the critical transcription factors of pluripotency.
	<ol>
		<li>Incubate H9 hESCs in MACME arrays in hPSC medium supplemented with 10 &#181;M EdU alkyne at 37 &#176;C for 30 min.</li>
		<li>After washing with annexin-binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2), stain the cells with an orange-fluorescent dye-Annexin V conjugate at 20 &#176;C for 15 min.</li>
		<li>Fix cells in PBS with 4% (v/v) paraformaldehyde at 20 &#176;C for 15 min, and then rinse cells with PBS twice.</li>
		<li>Permeabilize cells using PBS with 0.3% (v/v) Triton X-100 at 20 &#176;C for 16 h. 
		Note: Paraformaldehyde weakens the bond between a plastic base plate and a PDMS microfluidic structure. Thus, the detachment of the PDMS layer from the array at step 3.2.1 can also be performed right after this step.</li>
		<li>Incubate cells in Tris-based reaction buffer with a red-fluorescent dye azide at 20 &#176;C for 30 min.</li>
		<li>Incubate cells in blocking buffer (PBS with 5% (v/v) normal goat serum, 5% (v/v) normal donkey serum, 3% (w/v) bovine serum albumin, 0.1% (v/v) Tween-20, and 0.1% (w/v) N-dodecyl-&#946;-D-maltoside) at 4 &#176;C for 16 h.</li>
		<li>Incubate in PBS with 0.5% (v/v) Triton X-100 solution and human OCT3/4 antibody (mouse IgG) at 4 &#176;C for 16 h.</li>
		<li>Incubate in blocking buffer with green-fluorescent dye-labeled donkey anti-mouse IgG (H+L) at 37&#176;C for 60 min.</li>
		<li>Incubate in PBS with DAPI at 20 &#176;C for 30 min.</li>
		<li>Replace PBS-DAPI with PBS.</li>
		<li>Preserve the MACME array at 4 &#176;C until image acquisition.</li>
	</ol>
	</li>
	<li>Image acquisition (Figure 4)
	<ol>
		<li>Peel off the PDMS microfluidic structure from the MACME array.</li>
		<li>Remove the residual PBS from the MACME array, apply 90%(v/v) glycerol in PBS to the cells and place coverslips over the stained cells.</li>
		<li>Set the plate upside down on the stage of an inverted fluorescence microscope.</li>
		<li>Acquire 12-bit color images automatically using all motorized components (stage, filters, shutter, and focus systems) that were controlled by a microscope imaging software. 
		Note. In this protocol, exposure times are adjusted to reach near maximum intensity values (highest pixel intensity: 4096), but no saturation, for each image, DAPI, OCT4, Annexin V, and EdU.</li>
	</ol>
	</li>
	<li>Computer-guided image processing and fluorescence signal quantification (Figure 5) 
	Note: The following cell image analysis is performed based on a previously described method37.
	<ol>
		<li>Convert color images to grayscale.</li>
		<li>Calculate a single threshold value for each DAPI image with the Otsu method and classify pixels above the threshold as foreground and below as background. Ensure that the foreground value corresponds to the fluorescent intensity of DAPI. 
		Note: The image analysis process contains 5 steps; identification of primary objects in DAPI images, identification of secondary objects in OCT4, Annexin V, and EdU images, respectively, and measurement of object intensity. Figure 3 provides the images of graphical user interface (GUI) on the setting of image processing.</li>
		<li>Measure the fluorescent intensities of OCT4 and EdU on each image.</li>
		<li>Identify the region stained with Annexin V with the propagation method, and quantify the fluorescent intensity.</li>
	</ol>
	</li>
	<li>3.4. Statistical analysis to visualize individual cellular phenotypes on single-cell imaging
	<ol>
		<li>Normalize the input fluorescent intensities of each phenotypic marker by centering and scaling these variables to give them equal weight.</li>
		<li>Perform self-organizing map (SOM) analysis by using a software environment for statistical computing and graphics as described by previous studies38,39.
		<ol>
			<li>Create 25 SOM nodes with distinctive four &#34;codebook vectors&#34; corresponding to the fluorescent intensities of four markers, DAPI, EdU, Annexin V, and OCT4.</li>
			<li>Assign individual cells in each microenvironment to a &#34;winning&#34; (most similar) node, and plot as cell frequency, according to which the codebook vectors of the winning node are updated using a weighted average, where the weight is the learning rate (here, 0.05 as default). 
			Note: Omit data from chambers containing less than 1,000 cells because datasets containing a few cells did not provide statistically relevant SOM results.</li>
		</ol>
		</li>
		<li>Perform unsupervised hierarchical clustering with the SOM-node values using the average-linkage method based on Pearson correlation by a clustering software40.</li>
		<li>Render the clustering data into dendrogram and heatmap views41.</li>
	</ol>
	</li>
</ol>

This protocol demonstrates the first screening method to establish a robust culture system for the maintenance of qualified hPSCs. First, we described how to prepare a platform featuring diverse artificial ECMs and cell seeding densities by using a microfluidic device integrated with a nanofiber array, the MACME array. Second, quantitative image-based single-cell phenotyping was performed50 to evaluate individual cellular outcomes and behaviors altered by distinct biochemical and biophysical featu...

Human pluripotent stem cells (hPSCs)1,2 self-renew unlimitedly and differentiate into various tissue lineages, which could revolutionize drug development, cell-based therapies, tissue engineering, and regenerative medicine3,4,5,6. General culture dishes and microtiter plates, however, are not designed to enable precise physical and chemical cell manipulation at the cellular level with the range of nano- to micro-meters, which is a critical factor for cellular expansion, self-renewal, and differentiation. To address this drawback, studies have investigated the roles of cellular microenvironments in regulating cell-fate decisions and cell functions4. In recent years, an increasing number of studies have been conducted to reconstruct cellular microenvironments in vitro7,8. Nano- and micro-fabrication processes have established these microenvironments through the manipulation of chemical9,10,11,12,13,14,15,16,17 and physical18,19,20 environmental cues. Until now, there were no reports to systematically investigate the underlying mechanisms of chemical and physical environmental cues on cell-fate decisions and functions within a single platform.
Here, we introduce a strategy based on simple design principles to establish a robust screening platform (Figure 1). First, we describe the development procedure of an integrated platform for creating versatile, artificial cellular microenvironments by using a nanofiber array and a microfluidic structure: The Multiplexed Artificial Cellular MicroEnvironment (MACME) array (Figure 1A and 2A). The nanofiber array has 12 different microenvironments in varying combinations of nanofiber materials and densities. Electrospinning was used to fabricate nanofibers. The nanofiber materials, such as polystyrene (PS)21, polymethylglutarimide (PMGI)22, and gelatin (GT)23, were designed to test their chemical properties, which might affect cell adhesion and maintenance of pluripotency (Figure 2B). Nanofiber densities were varied by changing electrospinning time and the generated nanofibers were defined according to their densities (DNF, with D = XLow/Low/Mid/High). The microfluidic structure is composed of polydimethylsiloxane (PDMS) harboring 48 cell-culture chambers, which can be positioned along the standard dimensions of the 96-well microplate. PDMS is a biocompatible and gas-exchangeable polymer generally used to fabricate microfluidic devices24. Each microfluidic channel was designed to be 700-&#181;m wide and 8.4-mm long and had two inlets at its edges (Table 1). The chambers had different heights (250, 500, and 1000 &#181;m) to manipulate the initial cell-seeding densities (0.3, 0.6, and 1.2 &#215; 105 cells/cm2), which might correlate with survival, proliferation, and differentiation of hPSCs25 (Figure 2C). The number of cells seeded into a chamber is proportional to the column density above the chamber floor, and thus initial cell seeding density was controlled by introducing the same cell suspension into culture chambers with different heights. All channels were designed to be &#8805; 250-&#181;m-high26 to minimize the effects of low-oxygen tension27 and shear stress28 on the cells. Channel heights of 250, 500, and 1000 &#181;m are abbreviated here as XCD with X = Low, Mid, and High, respectively. The environments with distinct nanofiber densities and initial cell-seeding densities were shortened as &#34;Material_NF density_Cell density&#34; (e.g., GT_HighNF_HighCD: an environment characterized by high-density GT nanofibers and high initial cell-seeding density).
Subsequently, we describe how to perform single-cell analyses to systematically investigate cell behavior in response to environmental factors (Figure 1B). As a proof-of-concept, we identified the optimal cellular environment for hPSC self-renewal, which is a key function for hPSC maintenance (Figure 1B)29. Image-based cytometry, followed by statistical analyses, allows for quantitative interpretation of individual cellular phenotypic responses to cellular environments. Among a variety of cellular functions, this paper provides a detailed procedure to identify the optimal conditions for maintaining hPSC self-renewal.

<table><tbody><tr><td>Polystyrene (PS)</td><td>Sigma</td><td>#182435</td><td>Average Mw: 290,000, average Mn: 130,000</td></tr><tr><td>Polymethylglutarimide (PMGI)</td><td>MicroChem</td><td>G113113</td><td></td></tr><tr><td>Gelatin (GT)</td><td>Sigma</td><td>G2625</td><td>From porcine skin, type A</td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Doe Corning Toray</td><td>#1064291</td><td>PDMS curing agent and silicone elastomer base are components of this kit.</td></tr><tr><td>OpenSCAD</td><td></td><td></td><td>This is a free 3D computer graphics software (http://www.openscad.org/) used for designing the mold of the microfluidic device.</td></tr><tr><td>AutoCAD 2014</td><td>Autodesk</td><td></td><td>This is a 3D computer graphics software (https://www.autodesk.com/products/autocad/overview) used for design of the mask used on nanofiber-array preparation.</td></tr><tr><td>3D printer, AGILISTA-3000</td><td>Keyence</td><td></td><td></td></tr><tr><td>UV-curable resin, AR-M2</td><td>Keyence</td><td></td><td>This is used for 3D printing by Agilista.</td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>#338826</td><td>&amp;ge;99.99%</td></tr><tr><td>Ethyl acetate</td><td>Sigma</td><td>#270989</td><td>Anhydrous, 99.8%</td></tr><tr><td>Tetrahydrofuran (THF)</td><td>Sigma</td><td>#401757</td><td></td></tr><tr><td>MSP-30T</td><td>Vacuum Device</td><td></td><td>Magnetron sputtering machine</td></tr><tr><td>Nunc OmniTray</td><td>Thermo Fisher Scientific</td><td>#242811</td><td>This is a polystyrene baseplate on which the nanofiber array is created. This plate size is typically 127.7 x 85.5 mm.&amp;nbsp;</td></tr><tr><td>Gun-type corona discharge machine</td><td>Shinko Electric &amp;amp; Instrumentation</td><td>CFG-500</td><td>This handy device is used to generate corona for activation of the bottom surface of the PDMS layer at step 1.5 &quot;Assembly of the MACME arrays&quot; in the protocol.</td></tr><tr><td>5 mL syringe</td><td>Terumo</td><td>SS-05SZ</td><td></td></tr><tr><td>Stainless-steel blunt needle (23-gauge)</td><td>Nipro</td><td>#2166</td><td>Outside diameter and length are 0.6 and 32 mm, respectively.</td></tr><tr><td>High-voltage power supply</td><td>TechDempaz</td><td></td><td></td></tr><tr><td>1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride</td><td>Dojindo</td><td>W001</td><td></td></tr><tr><td>N-Hydroxysuccinimide</td><td>Sigma</td><td>#56480</td><td></td></tr><tr><td>Matrigel hESC-Qualified Matrix</td><td>Corning</td><td>#354277</td><td>This protein is refered as basement membrane gel matrix in the protocol.</td></tr><tr><td>CellAdhere Vitronectin, Human, Solution</td><td>STEMCELL Technologies</td><td>#07004</td><td></td></tr><tr><td>TeSR-E8</td><td>STEMCELL Technologies</td><td>#05940</td><td>Feeder-free, xeno-free culture medium for maintenance of human ES and iPS cells</td></tr><tr><td>Y-27632</td><td>Wako Pure Chemical Industries</td><td>#253-00513</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X), phenol red</td><td>Thermo Fisher Scientific</td><td>#12605028</td><td>This ia a recombinant trypsin-like protease for dissociation of adherant mammalian cells.</td></tr><tr><td>Click-iT EdU Imaging Kit with Alexa Fluor 647 Azides</td><td>Thermo Fisher Scientific</td><td>C10086</td><td>The fluorescent labeling of proliferating cells in on-plate fluorescent staining was performed along the product manual of this kit.</td></tr><tr><td>Annexin V, Alexa Fluor 594 conjugate</td><td>Thermo Fisher Scientific</td><td>A13203</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Oct-3/4 Antibody (C-10)</td><td>Santa Cruz Biotechnology</td><td>sc-5279</td><td></td></tr><tr><td>Donkey Anti-Mouse IgG H&amp;amp;L (DyLight 488)</td><td>abcam</td><td>ab96875</td><td>This is a secondary antibody used in on-plate fluorescent cell staining.</td></tr><tr><td>ECLIPSE Ti-E</td><td>Nikon</td><td></td><td>This is an inverted fluorescence microscope equipped with a CFI Plan Fluor 4&amp;times;/0.13 N.A. objective lens (Nikon), CCD camera (ORCA-R2, Hamamatsu), mercury lamp (Intensilight, Nikon), XYZ automated stage (Ti-S-ER motorized stage with encoders, Nikon), and filter cubes for four fluorescence channels (DAPI, GFP HYQ, TRITC, Cy5; Nikon)</td></tr><tr><td>NIS-Elements Advanced Research</td><td>Nikon</td><td></td><td>This is a microscope imaging software used for automatic image acquisition.</td></tr><tr><td>CellProfiler, Version 2.1.0</td><td></td><td></td><td>This is a free open software for cell image analysis (http://cellprofiler.org/).</td></tr><tr><td>R</td><td></td><td></td><td>SOM analysis is performed by kohonen package of this software. This is freely available (https://www.r-project.org/).</td></tr><tr><td>Cluster 3.0</td><td></td><td></td><td>This is the open source clustering software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). Unsupervised hierarchical clustering is performed with this software.</td></tr><tr><td>Java TreeView</td><td></td><td></td><td>This open source software (http://jtreeview.sourceforge.net/) is used to visualize clustering data as a heatmap and a dendrogram.</td></tr><tr><td>H9 human embryonic stem cell</td><td>WiCell Stem Cell Bank</td><td>WA09</td><td></td></tr></tbody></table>

fabrication of a multiplexed artificial cellular microenvironment array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

MACME arrays: Design and fabrication: In combination with nanofiber technology, we used microfluidic cell culture and screening techniques employed previously to identify optimal conditions for hPSC self-renewal or differentiation35,36 (Figure 1). This is well suited for establishing robust high-throughput cell-based assays because the cell culture chambers and conditions are precisel...

Watch this Scientific Journal Video about Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array at JoVE.com

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

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8.4K Views.  Kyoto University. Dit artikel beschrijft de gedetailleerde methodologie ter voorbereiding van een Multiplexed kunstmatige cellulaire communicatie (MACME)-array voor high-throughput manipulatie van fysische en chemische cues nabootsen in vivo cellulaire microenvironments en aan het identificeren van de optimale cellulaire omgeving voor menselijke pluripotente stamcellen (hPSCs) met eencellige profilering.

Video: Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

Chemistry

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Biology

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

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