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 p...

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 border="0" cellpadding="0" cellspacing="0" style="width:700px;" width="699">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polystyrene (PS)</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">#182435</td>
			<td style="width:231px;">Average Mw: 290,000, average Mn: 130,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polymethylglutarimide (PMGI)</td>
			<td style="width:135px;">MicroChem</td>
			<td style="width:119px;">G113113</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelatin (GT)</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">G2625</td>
			<td style="width:231px;">From porcine skin, type A</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:135px;">Doe Corning Toray</td>
			<td style="width:119px;">#1064291</td>
			<td style="width:231px;">PDMS curing agent and silicone elastomer base are components of this kit.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:216px;">OpenSCAD</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;">This is a free 3D computer graphics software (http://www.openscad.org/) used for designing the mold of the microfluidic device.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">AutoCAD 2014</td>
			<td style="width:135px;">Autodesk</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">3D printer, AGILISTA-3000</td>
			<td style="width:135px;">Keyence</td>
			<td style="width:119px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UV-curable resin, AR-M2</td>
			<td style="width:135px;">Keyence</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">This is used for 3D printing by Agilista.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetic acid</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">#338826</td>
			<td style="width:231px;">&#8805;99.99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethyl acetate</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">#270989</td>
			<td style="width:231px;">Anhydrous, 99.8%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetrahydrofuran (THF)</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">#401757</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MSP-30T</td>
			<td style="width:135px;">Vacuum Device</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">Magnetron sputtering machine</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Nunc OmniTray</td>
			<td style="width:135px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">#242811</td>
			<td style="width:231px;">This is a polystyrene baseplate on which the nanofiber array is created. This plate size is typically 127.7 x 85.5 mm.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Gun-type corona discharge machine</td>
			<td style="width:135px;">Shinko Electric &#38; Instrumentation</td>
			<td style="width:119px;">CFG-500</td>
			<td style="width:231px;">This handy device is used to generate corona for activation of the bottom surface of the PDMS layer at step 1.5 &#34;Assembly of the MACME arrays&#34; in the protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5 mL syringe</td>
			<td style="width:135px;">Terumo</td>
			<td style="width:119px;">SS-05SZ</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stainless-steel blunt needle (23-gauge)</td>
			<td style="width:135px;">Nipro</td>
			<td style="width:119px;">#2166</td>
			<td style="width:231px;">Outside diameter and length are 0.6 and 32 mm, respectively.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High-voltage power supply</td>
			<td style="width:135px;">TechDempaz</td>
			<td style="width:119px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride</td>
			<td style="width:135px;">Dojindo</td>
			<td style="width:119px;">W001</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N-Hydroxysuccinimide</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">#56480</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Matrigel hESC-Qualified Matrix</td>
			<td style="width:135px;">Corning</td>
			<td style="width:119px;">#354277</td>
			<td style="width:231px;">This protein is refered as basement membrane gel matrix in the protocol.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellAdhere Vitronectin, Human, Solution</td>
			<td style="width:135px;">STEMCELL Technologies</td>
			<td style="width:119px;">#07004</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TeSR-E8</td>
			<td style="width:135px;">STEMCELL Technologies</td>
			<td style="width:119px;">#05940</td>
			<td style="width:231px;">Feeder-free, xeno-free culture medium for maintenance of human ES and iPS cells</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Y-27632</td>
			<td style="width:135px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">#253-00513</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TrypLE Express Enzyme (1X), phenol red</td>
			<td style="width:135px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">#12605028</td>
			<td style="width:231px;">This ia a recombinant trypsin-like protease for dissociation of adherant mammalian cells.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Click-iT EdU Imaging Kit with Alexa Fluor 647 Azides</td>
			<td style="width:135px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C10086</td>
			<td style="width:231px;">The fluorescent labeling of proliferating cells in on-plate fluorescent staining was performed along the product manual of this kit.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Annexin V, Alexa Fluor 594 conjugate</td>
			<td style="width:135px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A13203</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:135px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oct-3/4 Antibody (C-10)</td>
			<td style="width:135px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-5279</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Donkey Anti-Mouse IgG H&#38;L (DyLight 488)</td>
			<td style="width:135px;">abcam</td>
			<td style="width:119px;">ab96875</td>
			<td style="width:231px;">This is a secondary antibody used in on-plate fluorescent cell staining.</td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">ECLIPSE Ti-E</td>
			<td style="width:135px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">This is an inverted fluorescence microscope equipped with a CFI Plan Fluor 4&#215;/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 height="63">
			<td height="63" style="height:63px;width:216px;">NIS-Elements Advanced Research</td>
			<td style="width:135px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">This is a microscope imaging software used for automatic image acquisition.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">CellProfiler, Version 2.1.0</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;">This is a free open software for cell image analysis (http://cellprofiler.org/).</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">R</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;">SOM analysis is performed by kohonen package of this software. This is freely available (https://www.r-project.org/).</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Cluster 3.0</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;">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 height="105">
			<td height="105" style="height:105px;width:216px;">Java TreeView</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;">This open source software (http://jtreeview.sourceforge.net/) is used to visualize clustering data as a heatmap and a dendrogram.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">H9 human embryonic stem cell</td>
			<td style="width:135px;">WiCell Stem Cell Bank</td>
			<td style="width:119px;">WA09</td>
			<td style="width:231px;"></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 ...

This method can help with a key question in the stem cell engineering field such as, how cellular microenvironment alter the cellular phenotypes and function. The main advantage of this technique is it provides the multiple artificially created environment for the cells in a single plate which you could not perform with a conventional method in micro-contact plate demonstrating this procedure will be Koki Yoshimoto and Risako Sakai technicians from our laboratory. After creating 3D images of masks for nano fiber arrays and molds from microfluidic structures according to the text protocol.Use a 3D printer to print the masks and mold. To prepare polymer solutions for electro spinning, dilute 13 percent PMGI liquid with tetrahydrofuran to nine percent. Dissolve 0.08 grams of PS in a one to one volume ratio of THF to dimethylformamide and use a liquid regent to bring the volume up to the final one milliliter of eight percent weight per volume PS solution.Dissolve 0.1 gram of GT in water, acidic acid and ethyl acetate and use the mixed solvent solution to bring the volume up to the final one milliliter of 10 percent weight by volume GT solution. Next, using a magnetron sputtering machine, deposit a five nanometer thick platinum layer on a polystyrene base plate which serves as a cathode in the electro spinning setup. Load each polymer solution into a five milliliter syringe equipped with a 23 gauge stainless steel blunt needle.Then anchor the syringes on a syringe pump 12 centimeters apart from the collector of the electro spinning device. Connect the syringe needle to a high voltage power supply and set it to 11 kilovolts. Then set the pumping rate to 20 milliliters per hour and keep the temperature and humidity at 30 degrees Celsius and less than 30 percent by volume, respectively.Now put the mask on the base plate. Then through the holes of the mask, fabricate nano fibers on the base plate with distinct densities by changing the electro spinning time. Remove the mask from the base plate and repeat the nano fiber fabrication.When the array is complete, place it in a desiccator at 25 degrees Celsius for 16 hours to evaporate the remaining solvent. To cross link the GT nano fibers, treat them with 0.2 molar EDC and 0.2 molar NHS in ethanol. And incubate the samples at 25 degrees Celsius for four hours.To rinse the GT nano fibers, use 99.5 percent ethanol twice and vacuum dry the plates at 25 degrees Celsius for 16 hours. To fabricate microfluidic structures, mix 2 grams of PDMS curing agent and 20 grams of PDMS base. Then pour the pre-PDMS mixture on to the fabricated mold.In a desiccator degas the pre PDMS mixture for 30 minutes. Then cure the pre PDMS mixture in an oven at 65 degrees Celsius for 16 hours. To assemble Macme arrays, peel off the cured PDMS structure from the mold and use 70 percent ethanol to clean it.Then discharge atmospheric corona on the bottom side of the PDMS structure. Quickly assemble the PDMS structure with the nano fiber array. Then oven bake the assembly at 65 degrees Celsius for two days.Using DPBS wash H9HESCs cultured on a 35 millimeter dish. Then add 0.5 milliliters of recombinant trypsin light protease to the dish and incubate it at 37 degrees Celsius for one minute. Carefully aspirate on the supernatant protease mixture.Immediately add HPSC medium and gently dispense the medium against the dish surface repeatedly to dissociate cells. Then transfer the detached cells to a 15 milliliter conical tube. Centrifuge the tube a 200 times gravity for three minutes.Aspirate the supernatant and suspend the cells in pre warmed HPSC medium. Then pipette 12 microliters of the cell suspension into each microfluidic chamber. After fluorescently labeling the cells according to the text protocol, peel off the PDMS microfluidic structure from the Macme array.Then remove the residual PBMS from the Macme array add 90 percent glycerol in PBS to the stained cells and apply a cover slip. Finally, set the plate upside down on the stage of an inverted fluorescence microscope. And use the microscope imaging software to acquire 12 bit color images.Shown here are immunofluorescence images of H9HPSCs cultured at high initial seeding density on gelatin nano fibers and stained with 3 cellular phenotypic markers. SOM analysis was used to convert high dimensional multi parametric data sets into low dimensional 2D maps for comparing homogeneity and heterogeneity of expression levels for the four phenotypic markers in each data set and unsupervised hierarchical clustering was performed for the SOM nodes. As indicated here, all groups exhibited higher expression of OCT4 than was observed on basement membrane gel matrix, or MG.Group one showed high EdU signals, indicating that most cells were actively proliferating.Thus, microenvironments in group one were suitable for HPSC maintenance because undifferentiated HPSCs proliferate rapidly when cell cycle gap phases were shortened. Group two included cells seeded at an insufficient initial density and cells grown on 2D GT scaffolds that did not support HPSC self renewal. For example, this sample's EdU signal was lost and annexin V levels were slightly increased indicating that the cells had lost their stemness and had gradually become apoptotic.PMGI MidNF HighCD from group three which represents the microenvironments comprising PMGI nano fiber matrices showed the larger variations in OCT4 and EdU signals compared with the other conditions. After it's development, this technique paved the way for the researcher in the field of STEM cell to explore the tissue engineering and advanced screening.

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Bioengineering

8.3K visualizações.  Kyoto University. This method can help with a key question in the stem cell engineering field such as, how cellular microenvironment alter the cellular phenotypes and function. The main advantage of this technique is it provides the multiple artificially created environment for the cells in a single plate which you could not perform with a conventional method in micro-contact plate demonstrating this procedure will be Koki Yoshimoto and Risako Sakai technicians from our laboratory. After creating 3D images of ...