This work was supported by CREST JST (grant number JPMJCR14W4), Society for the Promotion of Science (JSPS) KAKENHI (grant number 25600060, 16K16386), The Center of Innovation Program from MEXT and JST, Project Focused on Developing Key Evaluation Technology from Japan Agency for Medical Research and Development, AMED, Mizuho Foundation for the Promotion of Sciences. Microfabrication was supported by Kyoto University Nano Technology Hub.

1. Fabrication of the Microfluidic Device Mold
<ol>
	<li>Design the pattern of the microfluidic device using commercially available software (Clewin5 or AutoCAD 2016, etc.). For the function of the Clewin5, see the user manual (http://manualzz.com/doc/7159150/clewin-user-s-manual). 
	NOTE: The design file is available in Supplementary File 1.</li>
	<li>Transfer the design file to a micro pattern generator and load the tool with a chromium mask coated with the positive photoresist.</li>
	<...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+And+Environment+Interactions+In+Tumor+Microregions+-+The+Multicell+Spheroid+Model.">Cell And Environment Interactions In Tumor Microregions - The Multicell Spheroid Model.</a> Science. 240 (4849), 177-184 (1988).</li><li>Rothbauer, M., Zirath, H., Ertl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+microfluidic+technologies+for+cell-to-cell+interaction+studies.">Recent advances in microfluidic technologies for cell-to-cell interaction studies.</a> Lab on a Chip. , (2017).</li><li>Matsuura, K., Utoh, R., Nagase, K., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sheet+approach+for+tissue+engineering+and+regenerative+medicine.">Cell sheet approach for tissue engineering and regenerative medicine.</a> Journal of Controlled Release. 190, 228-239 (2014).</li><li>Esch, E. 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A., Gibot, L., Lacroix, D., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Annual Review of Biomedical Engineering. 15, 177-200 (2013).</li><li>Inamori, M., Mizumoto, H., Kajiwara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Approach+for+Formation+of+Vascularized+Liver+Tissue+by+Endothelial+Cell-Covered+Hepatocyte+Spheroid+Integration.">An Approach for Formation of Vascularized Liver Tissue by Endothelial Cell-Covered Hepatocyte Spheroid Integration.</a> Tissue Engineering Part A. 15 (8), 2029-2037 (2009).</li><li>Kunz-Schughart, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+fibroblasts+to+regulate+the+formation+of+three-dimensional+vessel-like+structures+from+endothelial+cells+in+vitro.">Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro.</a> American Journal of Physiology-Cell Physiology. 290 (5), C1385-C1398 (2006).</li><li>Rouwkema, J., De Boer, J., Van Blitterswijk, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+assemble+into+a+3-dimensional+prevascular+network+in+a+bone+tissue+engineering+construct.">Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct.</a> Tissue Engineering. 12 (9), 2685-2693 (2006).</li><li>Kim, S., Lee, H., Chung, M., Jeon, N. 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Previous reports show that hLFs secrete a cocktail of multiple angiogenic factors, such as angiopoietin-1, angiogenin, hepatocyte growth factor, transforming growth factor-&#945;, tumor necrosis factor and some extracellular matrix proteins29,30. This assay relies on the angiogenic secretion from hLFs in a coculture spheroid, which is the limitation of the technique. Therefore, it is critical for a stable vascular formation to set a coculture spheroid at the bott...

Shifting from a monolayer (two-dimensional) culture to a three-dimensional culture is motivated by the need to work with culture models that mimic the cellular functions of living tissues1,2,3. Flat and hard plastic substrates commonly used in cell culture do not resemble most of the extracellular environments in the human body. In fact, many studies demonstrate that three-dimensional culture recreate tissue-specific architecture, mechanical and biochemical cues, and cell-cell communication, which have not been observed in conventional two-dimensional culture4,5,6,7,8.
A multicellular aggregate or spheroid, is one of the most promising techniques to realize this three-dimensional culture9,10. Cells secrete the extracellular matrix (ECM) and can interact with others in the spheroid. Although some other bioengineering approaches11,12,13,14, such as cell stacking, successfully replicate spatial complexity of the human body, these approaches have only two or three kinds of cells for the ease of analysis and focused on only one function of target organs. In contrast, cells in spheroids are exposed to different culture environments depending on their positions in the spheroid due to the heterogeneous supply of nutrients, oxygen, and paracrine and autocrine signaling molecules in the spheroid. This feature of spheroids partially mimics in vivo culture condition and enable the cells in spheroids to create much more complex, organized tissue structure in vitro than those cultured in stacking tissue9,15,16. Note that if a spheroid is comprised of a single kind of cells, the function of the cells in the spheroid is not uniform due to the heterogeneous environment in the spheroid. In the past few years, spheroid cultures allowed embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) or tissue-resident stem cells to mimic in vivo developmental sequences and recreate mini-organs such as the brain17, liver18and kidney19,20.
Despite significant progress in spheroid culture techniques, culturing large spheroids for a long time is still problematic. In a three-dimensional tissue, cells need to be located within 150-200 &#181;m of a blood vessel because of the limited supply of oxygen and nutrients21. Vascular networks within the spheroid are necessary to recapitulate exchanging substances between blood and tissues in vivo. To achieve this, other groups have co-cultured endothelial cells with target cells22,23,24 or induced the differentiation of pluripotent cells into CD31-positive cells20. Nevertheless, the reported vessel-like structures do not have the open ends of the lumina to supply oxygen and nutrients to the center of the spheroid. To mimic the vascular role to nourish cells in the three-dimensional culture, open-ended and perfusable vascular network must be developed in the spheroid.
During the past few years, some research groups in the microengineering field reported methods to construct a perfusable vascular network, spontaneously formed in a microfluidic device by utilizing angiogenic factors from cocultured fibroblast cells25,26. These vascular networks have a similar morphology to their in vivo counterparts and can be remodeled by environmental factors, making them suitable for mimicking vascular functions in a spheroid culture. The purpose of this protocol is to construct a perfusable vascular network in a spheroid using a microfluidic platform27. The microfluidic device is modified from the previously reported device25 so that a spheroid can be incorporated. By directing the angiogenic secretion from fibroblast cells in a spheroid to endothelial cells in microchannels, angiogenic sprouts from the microchannels anastomosed with the spheroid and formed a perfusable vascular network. This method allows a direct delivery of a wide range of substances, such as fluorescent molecules and micrometer-scale beads into the interior of a spheroid, which provides the framework for a long-term tissue culture with vascular networks.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AutoCAD 2017</td>
			<td>Autodesk</td>
			<td>AutoCAD 2017</td>
			<td></td>
		</tr>
		<tr>
			<td>A chromium mask coated with AZP 1350.</td>
			<td>CLEAN SURFACE TECHNOLOGY</td>
			<td>CBL2506Bu-AZP</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pattern generator</td>
			<td>Heidelberg</td>
			<td>uPG101</td>
			<td></td>
		</tr>
		<tr>
			<td>MF CD-26 developer</td>
			<td>Rohm and haas electronic materials</td>
			<td>-</td>
			<td>Developer in protocol 1.4</td>
		</tr>
		<tr>
			<td>S-Clean</td>
			<td>Sasaki Chemical</td>
			<td>S-24</td>
			<td>Chromium etchant in protocol 1.5</td>
		</tr>
		<tr>
			<td>Aceton</td>
			<td>Wako</td>
			<td>012-00343</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>Canosis</td>
			<td>SiJ-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>MIKASA</td>
			<td>1H-D7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Tokyo Ohka Kogyo</td>
			<td>H0089</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3050</td>
			<td>MicroChem</td>
			<td>-</td>
			<td>Negative photoresist in protocol 1.9</td>
		</tr>
		<tr>
			<td>UV Exposure</td>
			<td>Nanometric Technology Inc</td>
			<td>LA310s</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td>Developer for the negative photoresist in protocol 1.13</td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Wako</td>
			<td>163-04841</td>
			<td></td>
		</tr>
		<tr>
			<td>Surhace profiler</td>
			<td>Vecco</td>
			<td>Veeco Dektak XT-S</td>
			<td></td>
		</tr>
		<tr>
			<td>(Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td>
			<td>Sigma</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Toray</td>
			<td>184W/C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch (1.0mm)&#160;</td>
			<td>Kai Industries</td>
			<td>BP-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch (2.0mm)&#160;</td>
			<td>Kai Industries</td>
			<td>BP-20F</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma System</td>
			<td>Femto Science</td>
			<td>COVANCE</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>MATSUNAMI GLASS</td>
			<td>C024241</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Dishes</td>
			<td>Iwaki</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr>
			<td>RFP Expressing Human Umbilical Vein Endothelial Cell</td>
			<td>Angio Proteomie</td>
			<td>cAP-0001RFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human Lung Fibroblasts</td>
			<td>Lonza</td>
			<td>CC-2512</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Media Kits</td>
			<td>Lonza</td>
			<td>CC-3132</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Wako</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05w/v% Trypsin-0.53mmol/l EDTA&#8226; 4Na Solution with Phenol Red</td>
			<td>Wako</td>
			<td>204-16935</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Salts)</td>
			<td>Takara bio</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sumitomo bakelite</td>
			<td>631-21031</td>
			<td></td>
		</tr>
		<tr>
			<td>1000ul Chip</td>
			<td>NIPPON Genetics</td>
			<td>FG-402</td>
			<td></td>
		</tr>
		<tr>
			<td>200ul&#160; Chip</td>
			<td>NIPPON Genetics</td>
			<td>FG-301</td>
			<td></td>
		</tr>
		<tr>
			<td>10ul Chip</td>
			<td>NIPPON Genetics</td>
			<td>37650</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Model 370</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Expressing Human Umbilical Vein Endothelial Cell</td>
			<td>Angio Proteomie</td>
			<td>cAP-0001GFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H21492</td>
			<td>Fluorescent dye to stain nuclei in protocol 5.5</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Solution</td>
			<td>Wako</td>
			<td>163-25983</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>OLYMPUS</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Degital CCD Camera</td>
			<td>OLYMPUS</td>
			<td>ORCA-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Biological Microscope</td>
			<td>OLYMPUS</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>OLYMPUS</td>
			<td>IX-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran</td>
			<td>Sigma</td>
			<td>FD70S</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusable vascular network with a tissue model in a microfluidic device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

Figure 1 shows a design and photo of the microfluidic device. It has three parallel channels, in which channel 2 contains the spheroid well. Channels 1 and 3 are used for the HUVEC culture and channel 2 is for the spheroid. Each channel is separated by trapezoidal microposts designed to pattern PDMS. The microposts prevent the hydrogel in channel 2 from leaking into channels 1 and 3 by surface tension and allow exchanging substances between the spheroid and H...

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Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

The overall goal of this procedure is to use a microfluidic platform to construct a perfusable vascular network in a multicellular aggregate, or spheroid. This method can help address key questions in the alternate shape of regenerative medicine fields, such as how is a vascular network important in vivo and in vitro tissue models. The main advantage of this technique is that the route of drug and supplement administration can be simulated in vitro via delivery of regions of interest directly into the spheroids.Generally, individuals new to this method will struggle because of the difficulty in introducing the cells into the target area in the microfluidic device. After casting the PDMS prepolymer, degas the material in a vacuum chamber for two hours, followed by overnight curing at 80 degrees Celsius in an air-vented oven. The next morning, peel the PDMS from the silicon wafer, and use a two-millimeter-diameter hole punch to create holes in the material at the indicated positions.Use a one-millimeter-diameter punch to create the spheroid well, and use adhesive tape to clean the PDMS slab and a 24-by-24 millimeter glass cover slip. Treat the clean slab with air plasma for 40 seconds, and bond the PDMS slab onto the cover slip. Then, cure the PDMS at 80 degrees Celsius for at least 12 hours.Two to three days before seeding the microfluidic device, add three times 10 to the fifth freshly thawed hLF, RFP-HUVECs, and GFP-HUVECs to 10 milliliters of fresh endothelial medium in a 10-millimeter dish. When the hLFs and RFP-HUVECs reach sub-confluency, suspend the hLFs and RFP-HUVECs in the endothelial medium to final concentrations at 1.0 times 10 to the fifth and 2.5 times 10 to the fourth cells per millimeter, respectively. After two days in suspension culture, in a biosafety cabinet, add a 99-microliter droplet of freshly prepared master mix solution into the center of a 35-millimeter Petri dish on ice.To load the microfluidic device, use a modified 100-microliter micropipette tip to transfer a spheroid and 100 microliters of medium into a second, room-temperature Petri dish. Then, aspirate the spheroid in a minimum amount of medium, and turn the pipette upright so that the spheroid moves to the bottom of the pipette tip by gravity. Touch the pipette tip to the meniscus of the master mix droplet to eject the spheroid into the solution without depressing the pipette plunger.Next, quickly add one microliter of freshly prepared thrombin to the droplet, and gently mix the thrombin into the solution. With the pipette set to seven microliters, slowly transfer the spheroid into the spheroid well of the PDMS slab. The most critical step of the procedure is injecting the spheroid with enough gel to allow the spheroid to settle at the bottom of the device.After loading, remove the pipette tip gently to prevent leakage between the microposts. Incubate the spheroid at 37 degrees Celsius for 15 minutes. When the fibrin has solidified, slowly inject 20 to 30 microliters of endothelial medium into holes one A and three A to load channels one and three, respectively.Then, transfer the device onto a wet lab wipe in a 100-millimeter dish, and incubate the device at 37 degrees Celsius and 5%CO2 for 24 hours to facilitate the removal of any bubbles at the interface between the medium and the fibrin. The next day, add two milliliters of 0.05%trypsin-EDTA to the sub-confluent GFP-HUVECs, and stop the trypsin reaction with four milliliters of complete medium when the cells have completely detached. Collect the endothelial cells by centrifugation and resuspend the pellet in fresh endothelial medium at a five times 10 to the sixth cells per milliliter concentration.Next, inject 20 microliters of HUVECs into hole one B to load channel one, and place the device at 37 degrees Celsius for 30 minutes, tilted at a 90 degree angle to ensure that the HUVECs adhere to the fibrin in channel two. After loading channel three in the same manner, place the device in a new 100-millimeter dish containing a damp lab wipe in the cell culture incubator for seven to 14 days, replacing half of the medium in channels one and three daily. These representative images taken on day zero of HUVEC loading, show fibrin gel loaded into channel two only, without any leakage into channels one or three and with the HUVECs successfully attached to the sidewall of the fibrin gel.The spheroid can also be observed to be properly settled at the bottom of the device. Angiogenic sprouts are observed beginning on day one, with the longest sprout reaching the spheroid on day three in this experiment, and with most of the angiogenic sprouts reaching the spheroid by day seven. Optimally, after four days in the device, flow can be observed through the vascular lumens with the vascular angle defined as the direction of the vascular tip and the center of the spheroid from the vascular root.The vascular angles decrease in a time-dependent manner indicating migration of the angiogenic sprouts toward the spheroid. RFP and GFP-HUVECs can coordinately form a single vascular lumen, clearly indicating how angiogenic sprouts from channels one and three anastomose to RFP-HUVECs in the spheroid to form a continuous vascular network. Further, FITC-dextran injected into channel one flows into the constructed vascular network and interior of the spheroid, eventually reaching channel three, implying that the integrated vascular network could supply nutrients to and remove waste products from the spheroid.After its development, this technique paved the way for researchers in the organic electric field to explore the efficacy of drugs or supplements of interest in in vitro tissue models.

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13.7K vistas.  Kyoto University. The overall goal of this procedure is to use a microfluidic platform to construct a perfusable vascular network in a multicellular aggregate, or spheroid. This method can help address key questions in the alternate shape of regenerative medicine fields, such as how is a vascular network important in vivo and in vitro tissue models. The main advantage of this technique is that the route of drug and supplement administration can be simulated in vitro via delivery of regions of interest directly...