This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) [NRF-2015R1D1A1A01060397] and Bio &#38; Medical Technology Development Program of the NRF funded by the Ministry of Science, ICT &#38; Future Planning [NRF-2017M3A9C6029563].

This study was approved by the Institutional Review Board at Korea University Anam Hospital (IRB No. ED170495). All procedures were carried out in accordance with the Helsinki Declaration and its later amendments.
1. Preparation of Aluminum (Al) Substrate by Electropolishing
Caution: Electropolishing solution is corrosive and toxic. Wear personal protective equipment including nitrile gloves, goggles and lab coat. Perform this step in a fume hood.
<ol>
	<li>Prepare electropolishing solution by mixing ethyl alcohol (C2H5OH, 99.9%) and perchloric acid (HClO4, 60%) in a 1 L double jacket beaker (C2H5OH:HClO4 = 4:1).</li>
	<li>Connect the double jacket beaker to a circulator and set the temperature at 7 &#176;C. Put the double jacket beaker on a magnetic stirrer. Leave the solution under stirring for at least 30 min until the temperature drops to 7 &#176;C.</li>
	<li>Immerse the ultrapure Al plate (99.999%, 20 &#215; 50 &#215; 1 mm3) and carbon counter electrode into the electropolishing solution using crocodile clips and copper wire. Adjust the position of the Al plate and the carbon counter electrode to face each other. 
	Note: It is necessary to install the clips carefully so as not to touch the solution. When in contact with the solution, impurities flowing out from the clips can contaminate the Al plate.</li>
	<li>Connect the Al plate to the positive terminal, and carbon counter electrode to the negative terminal, of a direct current (DC) power supply.</li>
	<li>Turn off the magnetic stirrer and apply a voltage of 20 V. Maintain this state for 4 min.</li>
	<li>Turn on the magnetic stirrer, and allow the current to flow for another 6 min. 
	Note: The static condition removes most of the impurities and roughness from the Al plate, and the dynamic condition improves the final quality of electropolishing.</li>
	<li>Turn off the power supply and manually separate the Al plate from the clip. Rigorously wash the Al plate with deionized water to remove all remaining solution.</li>
	<li>Dry the Al plate with nitrogen and store under inert gas. Conduct this drying process at the end of all experiments in step 1 and 2. 
	Note: When injecting compressed air, make air flow from the polished part to the opposite side. In the opposite case, impurities can escape from the non-polished part and contaminate the Al plate.</li>
</ol>
2. Fabrication of Gradient AAO Mold with Phosphoric Acid Electrolyte
Caution: Methyl alcohol and its fume are ocular toxic. Continuous exposure to chromium can lead to serious chromium poisoning. Perform this step in a fume hood.
<ol>
	<li>Primary anodization
	<ol>
		<li>To prepare 1 L of phosphoric acid electrolyte, load 590 mL of deionized water into a glass bottle.</li>
		<li>Add 400 mL of methyl alcohol (CH3OH, 99%) and 10 mL of phosphoric acid (H3PO4, 85%) into the glass bottle. Mix the solution well by shaking manually or with magnetic stirring.</li>
		<li>Pour 500 mL of the electrolyte into a 1 L double jacket beaker. Immerse the polished Al plate and carbon counter electrode into the solution with crocodile clips and copper wire.</li>
		<li>Pour additional electrolyte to locate the boundary of electropolishing 2-3 mm above the surface of the solution. 
		Note: If anodization is carried out with the boundary of electropolishing touching the solution, the Al plate tends to burn during the anodization.</li>
		<li>Install a vertical shaft on a U-shape pedestal. Attach an overhead stirrer, and impeller using metal clamps. Position the propeller of the impeller near the lower end of both electrodes. Set the rotation speed of the overhead stirrer between 200 and 300 rpm.</li>
		<li>Connect the double jacket beaker to a circulator and set the temperature at -10 &#176;C. Leave the system for at least 1 h under stirring until the temperature drops to -10 &#176;C. 
		Note: Do not use water as the coolant. Because the water freezes at low temperatures, the circulation will not work normally. It is recommended to use a mixture of water and ethyl alcohol at a ratio of 1:1 as the coolant.</li>
		<li>Apply a voltage of 195 V under stirring for 16 h. 
		Note: If the current rises more than 50 mA within 2 or 3 h after applying the voltage, the probability that burning will occur is very high. Immediately stop the process and replace the Al plate with a new one. If this problem occurs repeatedly, check that there is no abnormality in the temperature control of the circulator. If there is no abnormality, change the electrolyte.</li>
		<li>Stop the power supply and manually separate the Al plate from the clip. Wash the anodized Al plate with deionized water to remove all remaining solution.</li>
	</ol>
	</li>
	<li>Alumina etching
	<ol>
		<li>Prepare chromic acid etching solution by dissolving 9.0 g of chromium oxide (CrO3) and 20.3 mL of phosphoric acid (H3PO4, 85%) in 500 mL of deionized water.</li>
		<li>Pour the etching solution into the 1 L double jacket beaker. Set the temperature at 65 &#176;C.</li>
		<li>Immerse the anodized Al plate in the etching solution for at least 10 h under stirring. Seal the top of the beaker with aluminum foil.</li>
		<li>Rinse the etched Al plate several times with deionized water.</li>
	</ol>
	</li>
	<li>Secondary anodization
	<ol>
		<li>Set the same experimental conditions as step 2.1.1 to 2.1.7.</li>
		<li>Load the etched Al plate to the anode of the system and apply a voltage of 195 V for 6 h. Then, turn off the power supply and manually separate the Al plate from the clip. Immediately wash the anodized Al plate with deionized water. 
		Note: When the time of washing is delayed, the pore size of AAO increases unintentionally owing to the residual phosphoric acid.</li>
	</ol>
	</li>
	<li>Gradient pore widening
	<ol>
		<li>Prepare a pore widening solution by dissolving 5.765 g of phosphoric acid in 500 mL of deionized water. Pour the solution into a 1 L double jacket beaker.</li>
		<li>Connect the double jacket beaker to the circulator and set the temperature at 30 &#176;C. Place a linear moving stage vertically next to the beaker. Attach a bracket to the moving part of the linear stage.</li>
		<li>Set the moving part of the linear stage to the home position. Attach the clip to the bracket and load the anodized Al plate.</li>
		<li>Manipulate the position of the Al plate manually so that the Al plate will come right above the surface of the solution. 
		Note: In this step, the Al plate should not touch the solution. The pore widening process begins as soon as the Al plate reaches the solution.</li>
		<li>Gradually immerse the Al plate into the solution at a speed of 4.86 &#181;m/s for 120 min to make a 35 mm AAO mold with a size gradient from 120 nm to 200 nm (GP 120/200). Start a stopwatch at the moment the Al plate touches the surface of the solution.</li>
		<li>To make GP 200/280 and GP 280/360 molds, immerse the entire area of GP 120/200 mold in the pore widening solution for 2 or 4 h, respectively.</li>
		<li>Rinse the gradient Al plate several times with deionized water.</li>
	</ol>
	</li>
</ol>
3. Deposition of Anti-Sticking Layer on Gradient AAO Mold with Self-Assembled Monolayer
Note: Perform steps 3.2.1 to 3.3.3 in a glove box. Connect a vacuum pump and dry nitrogen gas injector to the glove box. Place all samples, reagents, and apparatuses in the glove box prior to the dehumidification process. Repeat the evacuation and nitrogen gas injection cycle more than three times to adequately remove moisture from the glove box. Let the dry nitrogen flow through the experiment.
<ol>
	<li>Hydroxyl modification of AAO mold with Piranha treatment
	<ol>
		<li>Pour 140 mL of sulfuric acid (H2SO4, 95%) in a polytetrafluoroethylene (PTFE) beaker. Add 60 mL of hydrogen peroxide dropwise (H2O2, 30%). Leave it for 30 min until the solution has cooled down. 
		Caution: Be extremely careful when making Piranha solution. From the moment of adding hydrogen peroxide, the solution vigorously boils and generates high temperatures. Perform the experiment in a fume hood.</li>
		<li>Immerse the AAO mold in the solution for 30 s using a nonmetallic tweezer.</li>
		<li>Rinse the AAO mold several times with deionized water. Soak the mold in deionized water for 30 min and put it in methyl alcohol for another 30 min. 
		Caution: When discarding the used Piranha solution, dilute the solution with a copious amount of tap water.</li>
		<li>Completely dry the AAO mold under vacuum for 3 h.</li>
	</ol>
	</li>
	<li>Preparation of 20&#215; HDFS stock solution 
	Note: HDFS is an abbreviation of (heptadecafluoro-1,1,2,2, -tetrahydrodecyl) dimethylchloro-silane
	<ol>
		<li>Fill one-third of a 1 L n-hexane bottle with molecular sieves. Seal the bottle with paraffin film and store it under dry nitrogen for 24 h.</li>
		<li>Filter the 200 mL of n-hexane using a glass syringe and 0.2 &#181;m PTFE syringe filter.</li>
		<li>Pour the 80 mL of filtered n-hexane into a 100 mL glass bottle. Add 2 mL of HDFS.</li>
	</ol>
	</li>
	<li>Self-assembled monolayer deposition
	<ol>
		<li>Load 57 mL of filtered n-hexane in a 100 mL beaker. Immerse the AAO mold into the solvent.</li>
		<li>Add 3 mL of HDFS stock solution to the n-hexane to make 2.9 mM HDFS solution. Wait 10 min for the reaction to proceed.</li>
		<li>Transfer the AAO mold to fresh n-hexane. Close and seal all reagent bottles. Close the nitrogen valve, then pull out samples from the glove box. 
		Note: HDFS is extremely sensitive to moisture. Store all reagents that contain HDFS in a desiccator.</li>
		<li>Soak the AAO mold in 60 mL of methoxynonafluorobutane (C10H6F18O2, 99%). Ultrasonically clean the AAO mold in a beaker for 10 s per one time to remove physically adsorbed HDFS molecules. Repeat the cleaning procedure 10 times. 
		Note: Exposing the AAO mold to ultrasonic for a long time without intervals will damage the oxide layer.</li>
		<li>Completely dry the AAO mold under vacuum for 24 h. 
		Note: A successfully deposited anti-sticking layer is super water repellent and stable for months when stored in a desiccator. The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
4. Fabrication of Gradient Nanopattern Plates by Thermal Imprinting
Note: Perform steps 4.2 to 4.7 in a clean room.
<ol>
	<li>Cut a 1.1 mm thick polystyrene sheet to a size of 2.9 mm wide by 3.7 mm long using a printed circuit board (PCB) cutter.</li>
	<li>Cut the AAO mold 35 mm from the bottom using a nipper. Mark the sample name and date of manufacture on the back.</li>
	<li>Clean an 8.0&#34; wafer with a small dose of ethyl alcohol. Put the polystyrene sheets on the wafer, then mount the AAO mold.</li>
	<li>Put the bottom film in the drawer of a thermal imprinter. Attach the top film to the gasket.</li>
	<li>Put the wafer on the bottom film. Place the gasket on the wafer. Make sure that there is no dust on the wafer.</li>
	<li>Close the drawer of the thermal imprinter. Apply 165 &#176;C of heat and 620.52&#8239;kPa of pressure for 100 s.</li>
	<li>Cool the sample to room temperature. Gently twist the polystyrene sheet to release the AAO mold.</li>
</ol>
5. Sterilization and Hydrophilic Modification of Gradient Nanopattern Plates
<ol>
	<li>Place the nanopattern plates on a square dish. Pour 100 mL of 70% ethyl alcohol and expose to ultraviolet light for 30 min.</li>
	<li>Dry the nanopattern plates with nitrogen. Transfer the nanopattern plates to an oxygen plasma generator.</li>
	<li>Evacuate the chamber until it reaches 1.33 Pa. Then, inject oxygen at a rate of 30 cm3/min. Apply a radio frequency power of 60 W. Maintain the oxygen plasma for 90 s. 
	Note: It is recommended to use plasma-treated nanopattern plates within 14 days.</li>
	<li>Attach the nanopattern plate to the bottom of a cell culture dish using a drop of toluene.</li>
	<li>Seal the samples in a pouch and sterilize with low-temperature plasma sterilizer.</li>
</ol>
6. Cultivation of hECFCs
Note: Conduct all centrifuging procedures at 4 &#176;C unless otherwise noted.
<ol>
	<li>Before isolating the peripheral blood mononuclear cells (PBMCs), incubate a collagen-coated 12-well culture dish at 37 &#176;C for 1 h.</li>
	<li>Wash the 12-well culture dish using 1&#215; sterile phosphate buffered saline (PBS).</li>
	<li>Collect 50 mL of human blood using a heparin inhibitor tube.</li>
	<li>Put 6 mL of hydrophilic polysaccharide solution in a 15 mL tube and add 8 mL of blood to the hydrophilic polysaccharide solution. 
	Note: Slowly pipette the blood into the hydrophilic polysaccharide solution.</li>
	<li>Centrifuge the tube at 1020 x g for 20 min to pellet the cells.</li>
	<li>Harvest the opaque cell layer, and transfer to a new 15 mL tube.</li>
	<li>Add 10% fetal bovine serum (FBS)-contained PBS and centrifuge the tube at 1020 x g for 10 min to pellet the cells.</li>
	<li>Remove the supernatant and add red-blood-cell lysis buffer. Keep on ice for 5 min.</li>
	<li>Add 10% FBS-contained PBS and centrifuge the tube at 1020 x g for 5 min to pellet the cells.</li>
	<li>Remove the supernatant and add 10% FBS-contained PBS. Centrifuge the tube at 1020 x g for 5 min to pellet the cells.</li>
	<li>Remove the supernatant and add 1 mL of endothelial cell expansion medium supplemented with 10% FBS. Seed 8.0 &#215; 106 cells per well for each collagen-coated dish.</li>
	<li>Change the culture medium every day for 1 week. Then, change the culture medium every 2 days. 
	Note: hECFCs can be found after culture day 10-14. hECFCs show typical cobblestone-like morphology and form a colony which can be observed in phase contrast microscopy. Immunofluorescence staining of vascular endothelial cadherin (CD144) and von Willebrand factor (vWF) can be also performed for the characterization of the hECFCs after further cell expansion.</li>
	<li>To cultivate the hECFCs, use endothelial cell expansion medium supplemented with 5% FBS and penicillin/streptavidin at 37 &#176;C in a humidified atmosphere containing 5% CO2. Replace the medium once a day.</li>
	<li>After hECFC induction, grow the cells to passage 7 for further experiments.</li>
</ol>
7. Cell Seeding and Culture on the Gradient Nanopattern Plates
Note: Step 7 describes the culture of hECFCs on the gradient nanopattern plate, but other cell sources also can be used.
<ol>
	<li>Coat the gradient nanopattern plates with 1 mL of 0.1% protein coating solution in PBS for 10 min at room temperature. 
	Note: The 0.1 % protein coating does not cover the nanopillar features.</li>
	<li>Make a hECFCs suspension from the culture dish by a standard cell-splitting method using trypsin.</li>
	<li>Dilute the cell suspension to achieve the desired confluence. Seed the hECFCs onto the gradient nanopattern plates and flat control (e.g., 1.0 &#215; 104 cells/cm2). 
	Note: When hECFCs are seeded at 1.0 &#215; 104 cells/cm2 on gradient nanopattern plates, the cell confluency usually reaches to 70 - 80 % after 2 days.</li>
	<li>Culture the cells on flat or gradient nanopattern plates for 2 days in the incubator.</li>
</ol>
8. Observation and Analysis
<ol>
	<li>Scanning electron microscope (SEM) imaging
	<ol>
		<li>Fix hECFCs cultured on flat or gradient nanopattern plates with 2.5% glutaraldehyde at 4 &#176;C for overnight .</li>
		<li>Treat 1% osmium tetroxide for 1 h at room temperature. Wash samples with PBS.</li>
		<li>Dehydrate samples with ethyl alcohol from low to high concentration (50%, 70%, 80%, 90%, and 100%) for 10 min per each step.</li>
		<li>Treat hexamethyldisilazane (HMDS) for 15 min at room temperature. After that, wash samples with fresh HMDS and leave samples under the fume hood at least 1 day until the residual HMDS evaporates completely.</li>
		<li>Coat the samples with platinum sputter coater for 5 min and examine the samples with an SEM.</li>
	</ol>
	</li>
	<li>Transmission electron microscope (TEM) imaging
	<ol>
		<li>Fix hECFCs cultured on flat or gradient nanopattern plates with 2% paraformaldehyde and 2.5% glutaraldehyde mixture in PBS at 4 &#176;C for overnight.</li>
		<li>Perform post-fixation using 1% osmium tetroxide and dehydrate samples using the method in 8.1.3.</li>
		<li>Embed samples in epoxy resin. Make 60 nm thick sections from blocks and place a section on a TEM grid.</li>
		<li>Stain the section with the mixture of 10 mg uranyl acetate in 100 mL methyl alcohol and 0.1 mg lead citrate in 100 mL distilled water. Obtain images with a TEM.</li>
	</ol>
	</li>
	<li>Fluorescence staining
	<ol>
		<li>Fix hECFCs cultured on flat or gradient nanopattern plates with 4% paraformaldehyde at room temperature for 15 min.</li>
		<li>Permeabilize and block samples with 5% goat serum and 0.1% octylphenol ethoxylate in PBS (PBST) at room temperature for 30 min.</li>
		<li>Incubate samples with anti-human vinculin primary antibody (1:500 in PBST) at room temperature for 2 h. Rinse samples three times with PBST.</li>
		<li>Incubate samples with fluorescence-conjugated phalloidin (1:1,000 in PBST), fluorescence-conjugated secondary antibody (1:1,000 in PBST) at room temperature for 2 h. Rinse samples three times with PBST.</li>
		<li>Incubate samples with 4&#39;, 6-diamidino-2-phenylindole (DAPI, 1:1,000 in PBST) at room temperature for 5 min.</li>
		<li>Drop 10 &#181;L of mounting medium on the surface of gradient nanopattern. Place a coverslip on the mounting medium.</li>
		<li>Place the sample upside down on the sample stage and acquire images using confocal fluorescence microscope.</li>
	</ol>
	</li>
	<li>Image analysis
	<ol>
		<li>Transfer captured images of the hECFCs to an image analysis system.</li>
		<li>Choose the random field of phalloidin staining image. Adjust the threshold and create a binary image in which cells are distinct from the background.</li>
		<li>Draw contours around cells to measure the cell area and perimeter and manually count the filopodia number per cell.</li>
		<li>Choose the random field of vinculin staining image. Adjust the threshold and create a binary image of focal adhesion areas. 
		Note: Adjust image threshold appropriately to avoid artificial over- or under-filling of focal adhesion areas.</li>
		<li>Count the number of focal adhesion of each cell.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Dalby, M. J., Gadegaard, N., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+nanotopography+and+integrin-matrix+interactions+to+influence+stem+cell+fate.">Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate.</a> Nature materials. 13 (6), 558-569 (2014).</li><li>Qian, W., Gong, L., Cui, X., Zhang, Z., Bajpai, A., Liu, C., Castillo, A., Teo, J. C., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopographic+Regulation+of+Human+Mesenchymal+Stem+Cell+Osteogenesis.">Nanotopographic Regulation of Human Mesenchymal Stem Cell Osteogenesis.</a> ACS Applied Materials &amp; Interfaces. 9 (48), 41794-41806 (2017).</li><li>Naganuma, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+cell+adhesion+force+activation+on+nano/micro-topographical+surfaces+and+temporal+dependence+of+cell+morphology.">The relationship between cell adhesion force activation on nano/micro-topographical surfaces and temporal dependence of cell morphology.</a> Nanoscale. 9 (35), 13171-13186 (2017).</li><li>Han, J., Lin, K. H., Chew, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+regulation+of+focal+adesions+and+cortical+actin+by+matrix+nanotopography+in+3D+environment.">Study on the regulation of focal adesions and cortical actin by matrix nanotopography in 3D environment.</a> Journal of Physics: Condensed Matter. 29 (45), 455101 (2017).</li><li>Liu, X., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+nano-biointerface+as+a+new+platform+for+guiding+cell+fate.">Three-dimensional nano-biointerface as a new platform for guiding cell fate.</a> Chemical Society Reviews. 43 (8), 2385-2401 (2014).</li><li>Turner, L. A., Dalby, 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=Nanotopography-potential+relevance+in+the+stem+cell+niche.">Nanotopography-potential relevance in the stem cell niche.</a> Biomaterials science. 2 (11), 1574-1594 (2014).</li><li>Driscoll, M. K., Sun, X., Guven, C., Fourkas, J. T., Losert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+contact+guidance+through+dynamic+sensing+of+nanotopography.">Cellular contact guidance through dynamic sensing of nanotopography.</a> ACS nano. 8 (4), 3546-3555 (2014).</li><li>Yim, E. K., Darling, E. M., Kulangara, K., Guilak, F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopography-induced+changes+in+focal+adhesions,+cytoskeletal+organization,+and+mechanical+properties+of+human+mesenchymal+stem+cells.">Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.</a> Biomaterials. 31 (6), 1299-1306 (2010).</li><li>Davis, G. E., Senger, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+extracellular+matrix.">Endothelial extracellular matrix.</a> Circulation research. 97 (11), 1093-1107 (2005).</li><li>Nakayama, K. H., Surya, V. N., Gole, M., Walker, T. W., Yang, W., Lai, E. S., Ostrowski, M. A., Fuller, G. G., Dunn, A. R., Huang, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+patterning+of+extracellular+matrix+alters+endothelial+function+under+shear+stress.">Nanoscale patterning of extracellular matrix alters endothelial function under shear stress.</a> Nano letters. 16 (1), 410-419 (2015).</li><li>Bettinger, C. J., Zhang, Z., Gerecht, S., Borenstein, J. T., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+in+vitro+capillary+tube+formation+by+substrate+nanotopography.">Enhancement of in vitro capillary tube formation by substrate nanotopography.</a> Advanced materials. 20 (1), 99-103 (2008).</li><li>Katz, B. Z., Zamir, E., Bershadsky, A., Kam, Z., Yamada, K. M., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+state+of+the+extracellular+matrix+regulates+the+structure+and+molecular+composition+of+cell-matrix+adhesions.">Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions.</a> Molecular biology of the cell. 11 (3), 1047-1060 (2000).</li><li>Deanfield, J. E., Halcox, J. P., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+function+and+dysfunction.">Endothelial function and dysfunction.</a> Circulation. 115 (10), 1285-1295 (2007).</li><li>Tajima, S., Chu, J., Li, S., Komvopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+endothelial+cell+adhesion,+spreading,+and+cytoskeleton+on+low-density+polyethylene+by+nanotopography+and+surface+chemistry+modification+induced+by+argon+plasma+treatment.">Differential regulation of endothelial cell adhesion, spreading, and cytoskeleton on low-density polyethylene by nanotopography and surface chemistry modification induced by argon plasma treatment.</a> Journal of Biomedical Materials Research Part A. 84 (3), 828-836 (2008).</li><li>Mohiuddin, M., Pan, H. A., Hung, Y. C., Huang, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+growth+and+inflammatory+response+of+macrophages+and+foam+cells+with+nanotopography.">Control of growth and inflammatory response of macrophages and foam cells with nanotopography.</a> Nanoscale research letters. 7 (1), 394 (2012).</li><li>Kyle, D. J., Oikonomou, A., Hill, E., Bayat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+functional+evaluation+of+biomimetic+silicone+surfaces+with+hierarchical+micro/nano-topographical+features+demonstrates+favourable+in+vitro+foreign+body+response+of+breast-derived+fibroblasts.">Development and functional evaluation of biomimetic silicone surfaces with hierarchical micro/nano-topographical features demonstrates favourable in vitro foreign body response of breast-derived fibroblasts.</a> Biomaterials. 52, 88-102 (2015).</li><li>Seo, H. R., Joo, H. J., Kim, D. H., Cui, L. H., Choi, S. C., Kim, J. H., Cho, S. W., Lee, K. B., Lim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopillar+Surface+Topology+Promotes+Cardiomyocyte+Differentiation+through+Cofilin-Mediated+Cytoskeleton+Rearrangement.">Nanopillar Surface Topology Promotes Cardiomyocyte Differentiation through Cofilin-Mediated Cytoskeleton Rearrangement.</a> ACS Applied Materials &amp; Interfaces. 9 (20), 16803-16812 (2017).</li><li>Hwang, J. H., Lee, D. H., Byun, M. R., Kim, A. R., Kim, K. M., Park, J. I., Oh, H. T., Hwang, E. S., Lee, K. B., Hong, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopological+plate+stimulates+osteogenic+differentiation+through+TAZ+activation.">Nanotopological plate stimulates osteogenic differentiation through TAZ activation.</a> Scientific Reports. 7 (1), 3632 (2017).</li><li>Bae, D., Moon, S. H., Park, B. G., Park, S. J., Jung, T., Kim, J. S., Lee, K. B., Chung, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopographical+control+for+maintaining+undifferentiated+human+embryonic+stem+cell+colonies+in+feeder+free+conditions.">Nanotopographical control for maintaining undifferentiated human embryonic stem cell colonies in feeder free conditions.</a> Biomaterials. 35 (3), 916-928 (2014).</li><li>Cui, L. H., Joo, H. J., Kim, D. H., Seo, H. R., Kim, J. S., Choi, S. C., Huang, L. H., Na, J. E., Lim, I. R., Kim, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+the+response+of+human+endothelial+colony-forming+cells+by+focal+adhesion+assembly+using+gradient+nanopattern+plates.">Manipulation of the response of human endothelial colony-forming cells by focal adhesion assembly using gradient nanopattern plates.</a> Acta biomaterialia. 65, 272-282 (2017).</li><li>Lee, W., Park, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+anodic+aluminum+oxide:+anodization+and+templated+synthesis+of+functional+nanostructures.">Porous anodic aluminum oxide: anodization and templated synthesis of functional nanostructures.</a> Chemical reviews. 114 (15), 7487-7556 (2014).</li><li>Masuda, H., Fukuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+metal+nanohole+arrays+made+by+a+two-step+replication+of+honeycomb+structures+of+anodic+alumina.">Ordered metal nanohole arrays made by a two-step replication of honeycomb structures of anodic alumina.</a> science. 268 (5216), 1466 (1995).</li><li>Masuda, H., Satoh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+gold+nanodot+array+using+anodic+porous+alumina+as+an+evaporation+mask.">Fabrication of gold nanodot array using anodic porous alumina as an evaporation mask.</a> Japanese Journal of Applied Physics. 35 (1B), L126 (1996).</li><li>Zaraska, L., Sulka, G. D., Jasku&#322;a, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+n-alcohols+on+porous+anodic+alumina+formed+by+self-organized+two-step+anodizing+of+aluminum+in+phosphoric+acid.">The effect of n-alcohols on porous anodic alumina formed by self-organized two-step anodizing of aluminum in phosphoric acid.</a> Surface and Coatings Technology. 204 (11), 1729-1737 (2010).</li><li>Yang, K. Y., Kim, J. W., Byeon, K. J., Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+deposition+of+the+silver+nano-particles+using+patterned+the+hydrophobic+self-assembled+monolayer+patterns+and+zero-residual+nano-imprint+lithography.">Selective deposition of the silver nano-particles using patterned the hydrophobic self-assembled monolayer patterns and zero-residual nano-imprint lithography.</a> Microelectronic engineering. 84 (5), 1552-1555 (2007).</li><li>Park, B. G., Lee, W., Kim, J. S., Lee, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superhydrophobic+fabrication+of+anodic+aluminum+oxide+with+durable+and+pitch-controlled+nanostructure.">Superhydrophobic fabrication of anodic aluminum oxide with durable and pitch-controlled nanostructure.</a> Colloids and Surfaces A: Physicochemical and Engineering Aspects. 370 (1), 15-19 (2010).</li></ol>

The authors have nothing to disclose.

Fabrication of an AAO often suffers from defects such as cracks, irregular shapes of pores, and burning. The main reason for these defects is called an electrolytic breakdown, which is strongly affected by the nature of the metal substrates being anodized and the resistivity of the electrolyte21. Since the resistivity of the electrolyte varies depending on its temperature, eliminating heat continuously from electrodes is the critical point to maintain the locational temperature of the electrolyte as stable in such a high-voltage anodizing condition. In this protocol, we fabricated AAO molds by modifying Masuda&#39;s two-step anodization22,23. Replacing almost half the electrolyte with methyl alcohol not only prevents the electrolyte from freezing at low temperatures but also deprives the electrode of heat by evaporation at the bottom of the pore24. The reason for using an overhead stirrer and impeller instead of a magnetic stirrer is also to ensure the temperature control. The typical magnetic stirrer has a high probability of idling owing to the accidental misalignment of the magnetic bar during long-term operation. This problem induces the improper circulation of the electrolyte, raising its temperature, and leading to burning.
To give a size gradient to the AAO mold, we gradually immersed the AAO in a phosphoric acid etching solution. The pores are widened in proportion to the time the AAO stays in the etching solution, as shown in Figure 1. Under this condition, the average pore-widening rate is 0.67 nm/min. Manipulating the immersing speed and time will change the slope of the size gradient and maximum pore diameter of the AAO. The maximum possible pore diameter is 400 nm. When the pore diameter exceeds 400 nm, the spacing wall between pores becomes so thin that it cannot tolerate the pressure during the thermal imprinting process.
An HDFS self-assembled monolayer is known to be able to add a hydrophobic property to the material surface25. The HDFS molecule makes a strong covalent bond with the hydroxyl groups on the AAO surface introduced by the Piranha treatment26. Therefore, as shown in Supplementary Figure 1, a solid self-assembled monolayer can be formed when a large number of hydroxyl groups are present on the substrate surface. For optimum quality with an HDFS self-assembled monolayer, we suggest conducting the reaction in a dehumidified atmosphere. When HDFS is exposed to moisture, a side reaction with water forms dimers, trimers, and even oligomers of silane molecules, and decreases the overall quality of the self-assembled monolayer.
Upon using the AAO mold with a size gradient in thermal nanoimprinting, cell culture plates with a gradient of nanopillars were obtained Figure 2 and Figure 3. The thermal nanoimprinting method has an advantage in that it can produce a large quantity of identical nanopattern plates by using one AAO master mold. Supplementary Figure 2 shows that there is no difference in the quality of the nanopattern produced, even after fifteen imprinting processes, compared to the nanopattern produced by the new AAO mold. The reason for choosing polystyrene as a material for transferring a nanopattern is that the polystyrene has an appropriate glass transition temperature (100 &#176;C) for thermal imprinting, and it is widely used in commercial cell culture dishes owing to its transparent and nontoxic properties.
Fabricating nanostructures with high aspect ratio using thermal nanoimprinting method is difficult. Because, when process conditions, such as pressure and time increase, the mold becomes deeply embedded into the polymer substrate, making it hard to separate the mold from the substrate. Therefore, it is very challenging to produce nanopatterns having an aspect ratio of 1:3 or more using our nanoimprinting technique. However, according to TEM images we observed Figure 5C, we have found an interesting fact that the membrane of cells on nanopatterns was not in contact with the bottom of the nanopattern. In other words, all physical stimuli that can manipulate a response of cells were originated from the top parts of the nanopattern. Therefore, in this study, we did not have to consider the aspect ratio in studying responses of the cell to the nanopattern. As shown in Supplementary Figure 3, 0.1% protein coating solution had no influence on the attachment of hECFCs on the nanopillar surfaces. Furthermore, these nanoscaled stimuli from the gradient nanopattern plates decreased the cell area and perimeter of hECFCs and increased filopodial outgrowth Figure 5.
In summary, we established gradient nanopattern plates by manipulating a response of hECFCs in vitro. To know exactly what size of nanostructures the cells are greatly affected, a future work is required to design a cell-specific-sized nanopillar by adjusting the slope of the size gradient and minimum-maximum diameter of the nanopillars. We expect this gradient nanopattern to be a fascinating tool to screen the interaction between cells and nanostructures as well as provide advanced cell niches in which nanostructures can be freely tuned in size.

Recently, the response of cells by the physical stimulation of surface topography has been spotlighted in the field of cell engineering1,2,3,4. Therefore, more attention has been focused on three-dimensional nanostructures at the cell attachment surface5. It has been reported that the integrin, which is the surface recognition device of the cell, transmits the physical stimulus driven by the micro-nano structures of ECM through mechano-transduction6. This mechanical stimulation regulates cell behavior through contact guidance7 and induces cytoskeletal reorganization to change shape, in addition to focal adhesions and stiffness of cells8.
Human endothelial progenitor cells (hEPCs) in the body closely interact with the microenvironment of the surrounding ECM9. This indicates that the physical state of the ECM acts as an important parameter for specific cell-matrix adhesion complex formation as much as shear stress derived from blood flow10. It is reported that surface nanotopography enhances the in vitro formation of extensive capillary tube networks of hEPCs11 and that an ECM/bio soluble factor combined system enables hEPCs to recognize dysfunctional substrates and promotes wound healing12,13. Nonetheless, the relationship between ECM and hEPCs is not clearly understood.
Although many researchers tried to clarify the relationship between cell responses and physical cues from different substrates14,15,16, these studies used only the fixed size of a nanostructure or nanopatterns with irregular arrangements that have a limitation to elucidate the relationship between the size of the nanostructure and cell behavior. The problem here is a lack of suitable tools for screening cellular responses that can replace existing tedious and iterative approaches to find the optimum size of the nanostructure. Therefore, a straightforward technique is required for screening cell reactions on physical stimulations without repetition.
Here, we describe a method used in our previous reports17,18,19 to produce a gradient nanopattern in which the diameter of the arranged nanopillars gradually increases. In addition, we also described how to cultivate and analyze the behavior of hECFCs on gradient nanopattern plates to determine the effect of physical stimuli on the cells. A mild anodization, gradual etching, and anti-sticking layer coating method were used to fabricate gradient AAO mold. By adopting a thermal imprinting lithography technique, identical polystyrene gradient nanopatterns were produced in a cost-effective and facile way. Using gradient nanopatterns, it is feasible to determine which size of nanostructure has a great effect on cell behavior in one set of experiment. We expect that this gradient nanopattern will be helpful in understanding the interaction mechanisms between blood-derived hECFC or other cells and various sizes of nanostructures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Perchloric acid 60%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>6512-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol, absolute 99.9%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4118-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid 85%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>6532-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl alcohol 99.5%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>5558-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium(VI) oxide</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>2558-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid 95%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>7781-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4104-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>n-hexane 95%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>4081-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene 99.5%</td>
			<td>Daejung Chemicals &#38; Metals</td>
			<td>8541-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>(heptadecafluoro-1,1,2,2,-tetrahydrodecyl)dimethylchlorosilane</td>
			<td>Gelest</td>
			<td>SIH5840.4</td>
			<td>Moisture sensitive</td>
		</tr>
		<tr>
			<td>Methoxynonafluorobutane 99%</td>
			<td>Sigma aldrich</td>
			<td>464309</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen solution</td>
			<td>Stemcell</td>
			<td>#4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma aldrich</td>
			<td>G1890</td>
			<td>Protein coating solution</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Heathcare</td>
			<td>17-1440-03</td>
			<td>Hydrophilic polysaccharide solution</td>
		</tr>
		<tr>
			<td>EGM-2MV</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>Endothelial cell expansion medium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma aldrich</td>
			<td>G5882-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma aldrich</td>
			<td>201030-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane</td>
			<td>Sigma aldrich</td>
			<td>440191</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma aldrich</td>
			<td>X100-100ML</td>
			<td>Octylphenol ethoxylate&#160;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human vinculin primary antibody&#160;</td>
			<td>Sigma aldrich</td>
			<td>V9131</td>
			<td></td>
		</tr>
		<tr>
			<td>F-actin probe</td>
			<td>Molecular Probes</td>
			<td>A12379</td>
			<td>Fluorescence-conjugated phalloidin</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated anti-mouse&#160;IgG antibody</td>
			<td>Molecular Probes</td>
			<td>A11001</td>
			<td>Fluorescence-conjugated secondary antibody&#160;</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole&#160;</td>
			<td>Sigma aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human vWF primary antibody&#160;</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human&#160;CD144 primary antibody&#160;</td>
			<td>BD Biosciences</td>
			<td>#555661</td>
			<td></td>
		</tr>
		<tr>
			<td>Eponate 12&#8482; Embedding Kit, with BDMA</td>
			<td>Ted Pella</td>
			<td>18012</td>
			<td>Epoxy resin</td>
		</tr>
		<tr>
			<td>Uranyl Acetate, 25g</td>
			<td>Ted Pella</td>
			<td>19481</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead Citrate, Trihydrate, 10g</td>
			<td>Ted Pella</td>
			<td>19312</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra pure aluminum plate</td>
			<td>Goodfellow</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene sheet</td>
			<td>Goodfellow</td>
			<td>ST313120</td>
			<td></td>
		</tr>
		<tr>
			<td>8.0&#34; silicon wafer</td>
			<td>Siltron</td>
			<td>29-01024-03</td>
			<td>Single side polished, 725 &#181;m thick</td>
		</tr>
		<tr>
			<td>Vacuum desiccator, 4.4 L</td>
			<td>Kartell</td>
			<td>KA.230</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Vacuumer</td>
			<td>V3.VOP100</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Unicorntech</td>
			<td>UDP-3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>Daihan scientific</td>
			<td>SL.SMS03022</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhead stirrer</td>
			<td>Daihan scientific</td>
			<td>HT120DX</td>
			<td></td>
		</tr>
		<tr>
			<td>Circulator</td>
			<td>Daihan scientific</td>
			<td>WCR-P12</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear moving stage</td>
			<td>Zaber</td>
			<td>A-LSQ300A-E01-KT07</td>
			<td></td>
		</tr>
		<tr>
			<td>Angle bracket, 90 degrees</td>
			<td>Zaber</td>
			<td>AB90M</td>
			<td>Accessory of the linear moving stage</td>
		</tr>
		<tr>
			<td>PMP forcep, 145 mm</td>
			<td>Vitlab</td>
			<td>67995</td>
			<td>Nonmetallic tweezer</td>
		</tr>
		<tr>
			<td>PTFE beaker, 250 mL</td>
			<td>Cowie</td>
			<td>CW007.25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>Branson</td>
			<td>B2510MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>PCB cutter</td>
			<td>Hozan Tool Industrial</td>
			<td>K-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoimprint device</td>
			<td>Nanonex</td>
			<td>NX-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen plasma generator</td>
			<td>Femto Science</td>
			<td>CUTE</td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature sterilizer</td>
			<td>Lowtem</td>
			<td>Crystal 50</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr>
			<td>Confoal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>JEOL</td>
			<td>JSM6701</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Hitachi</td>
			<td>H-7500</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of gradient nanopattern by thermal nanoimprinting technique and screening of the response of human endothelial colony-forming cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Figure 1 shows SEM images of the fabricated gradient AAO molds according to their type and position. Figure 2 shows SEM images of gradient nanopattern plates with regular-rounded nanopillars, and Figure 3 is quantification data of the nanopillar diameter. Table 1 lists the characteristics of the fabricated nanopillars.
Figure 4A shows the scheme of cultivation of blood-derived hECFCs. Figure 4B shows the phase contrast image of thecultivated hECFCs. Figure 4C shows hECFC markers stained with CD144 (green), vWF (red), and nucleus (blue). Figure 5A shows representative SEM images of hECFCs after 2 days of culture on the flat, GP 120/200, GP 200/280, and GP 280/360 plates. Figure 5B and C depict quantitative data on cell area and perimeter as compared to the flat. The quantification data revealed that cells grown on nanopillar surfaces of smaller size showed reduced cell area and perimeter. Figure 5D is the representative SEM images of hECFCs which show the morphology of existing filopodia. Figure 5E depicts the quantitative data on filopodia number as compared to the flat. Significant filopodial outgrowth was observed in cells cultured on GP 120/200, while no significant change was observed in GP200/280 or GP280/360 in comparison with the flat. Figure 5F shows representative TEM images of hECFCs after 2 days of culture on flat and gradient nanopattern plates.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57661/57661fig1.jpg" /> 
Figure 1. Gradient AAO mold. SEM images of gradient AAO molds for GP 120/200, GP 200/280, and GP 280/360 plates according to the position of molds. <a href="/files/ftp_upload/57661/57661fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57661/57661fig2.jpg" /> 
Figure 2. Gradient nanopattern plates. SEM images of pillar-type gradient nanopattern plates for GP 120/200, GP 200/280, and GP 280/360 plates according to position of plates. <a href="/files/ftp_upload/57661/57661fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57661/57661fig3.jpg" /> 
Figure 3. Size gradient of nanopillar diameter. Quantification data showing the gradient of nanopillar diameters for GP 120/200, GP 200/280, and GP 280/360 plates, respectively. Bars represent standard error. This figure was modified from [Acta Biomaterialia]20. <a href="/files/ftp_upload/57661/57661fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Young&#8217;s modulus (GPa)</td>
			<td>Nanopillar Diameter (nm)</td>
			<td>Nanopillar Pitch (nm)</td>
			<td>Center to Center Distance (nm)</td>
			<td>Nanopillar height (nm)</td>
			<td>Nanopillar stiffness (N/m)</td>
		</tr>
		<tr>
			<td>Flat</td>
			<td>2.43&#177;0.19</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
		</tr>
		<tr>
			<td>GP 120/200</td>
			<td>1.74&#177;0.11</td>
			<td>120&#8211;200</td>
			<td>320&#8211;240</td>
			<td>440</td>
			<td>276.9&#177;21.9</td>
			<td>9.35</td>
		</tr>
		<tr>
			<td>GP 200/280</td>
			<td>2.01&#177;0.05</td>
			<td>200&#8211;280</td>
			<td>240&#8211;160</td>
			<td>440</td>
			<td>268.1&#177;25.9</td>
			<td>55.78</td>
		</tr>
		<tr>
			<td>GP 280/360</td>
			<td>2.1&#177;0.13</td>
			<td>280&#8211;360</td>
			<td>160&#8211;80</td>
			<td>440</td>
			<td>293.6&#177;23.6</td>
			<td>134.15</td>
		</tr>
	</tbody>
</table>
Table 1. Characteristics of fabricated nanopillars. Quantified data showing Young&#39;s modulus, diameter, pitch, center to center distance, height, and stiffness of fabricated nanopillars. This table was modified from [Acta Biomaterialia]20.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57661/57661fig4.jpg" /> 
Figure 4. Characterization of hECFCs. (A) Schematic diagram showing schedule of hECFCs derived from adult peripheral blood. (B) Phase contrast image of hECFC colony, derived from adult peripheral blood on day 14. (C) Immunofluorescent images showing CD144 (green), vWF (red), and cell nucleus stained with DAPI (blue). This figure was modified from [Acta Biomaterialia]20. <a href="/files/ftp_upload/57661/57661fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57661/57661fig5.jpg" /> 
Figure 5. hECFCs cultured on flat and gradient nanopattern plates for 2 days. (A) Representative SEM image of hECFCs cultured on Flat, GP 120/200, GP 200/280, and GP 280/360 plates. (B and C) Quantification data of the area and perimeter of hECFCs (n&#8239;=&#8239;50). *p&#8239;&#60;&#8239;.05 as compared to the Flat. (D) Representative SEM images of the leading edge of hECFCs on flat and gradient nanopattern plates. (E) Quantification data of the number of filopodia per one hECFC (n&#8239;=&#8239;20). *p&#8239;&#60;&#8239;.05 as compared to the Flat. (F) Representative TEM image of hECFC cultured on Flat and gradient nanopattern plates. White arrows indicate the interaction points between hECFCs and substrate surface. This figure was modified from [Acta Biomaterialia]20. <a href="/files/ftp_upload/57661/57661fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1. Water contact angle of pristine AAO and HDFS treated AAO. Contact angle measurements showing hydrophobic properties of HDFS self-assembled monolayer (A) before and, (B) after treatment. <a href="/files/ftp_upload/57661/1.pdf" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2. SEM images of fabricated gradient nanopattern plates with same AAO mold. Comparative SEM image sets for verifying the durability of AAO mold. (A) Gradient nanopattern plate produced with freshly made mold. (B) Gradient nanopattern plate produced with AAO mold after imprinting 15 times. <a href="/files/ftp_upload/57661/2.tif" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 3. Influence of protein coating. SEM images of hECFCs that were cultured on GP 120/200, GP 200/280, or GP 280/360 gradient nanopattern plates (A) without or (B) with the protein coating. This figure was modified from [Acta Biomaterialia]20 <a href="/files/ftp_upload/57661/3.pdf" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells at JoVE.com

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

fabrication-gradient-nanopattern-thermal-nanoimprinting-technique

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.9K Views.  Korea University. Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

Video: Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

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

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

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

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in magnetic resonance imaging (MRI). While iron oxide nanoparticles provide constant negative contrast on MRI over typical experimental timeframes, MnO generates switchable positive contrast on MRI through dissolution of MnO to Mn2+ at low pH within cell endosomes to &#8216;turn ON&#8217; MRI contrast. This protocol describes a one-pot synthesis of MnO nanoparticles formed by thermal decomposition of manganese(II) acetylacetonate in oleylamine and dibenzyl ether. Although running the synthesis of MnO nanoparticles is simple, the initial experimental setup can be difficult to reproduce if detailed instructions are not provided. Thus, the glassware and tubing assembly is first thoroughly described to allow other investigators to easily reproduce the setup. The synthesis method incorporates a temperature controller to achieve automated and precise manipulation of the desired temperature profile, which will impact resulting nanoparticle size and chemistry. The thermal decomposition protocol can be readily adapted to generate other metal oxide nanoparticles (e.g., iron oxide) and to include alternative organic solvents and stabilizers (e.g., oleic acid). In addition, the ratio of organic solvent to stabilizer can be changed to further impact nanoparticle properties, which is shown herein. Synthesized MnO nanoparticles are characterized for morphology, size, bulk composition, and surface composition through transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy, respectively. The MnO nanoparticles synthesized by this method will be hydrophobic and must be further manipulated through ligand exchange, polymeric encapsulation, or lipid capping to incorporate hydrophilic groups for interaction with biological fluids and tissues.

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of ManganeseII Acetylacetonate

This protocol details a facile, one-pot synthesis of manganese oxide (MnO) nanoparticles by thermal decomposition of manganese(II) acetylacetonate in the presence of oleylamine and dibenzyl ether. MnO nanoparticles have been utilized in diverse applications including magnetic resonance imaging, biosensing, catalysis, batteries, and waste water treatment.

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...