This work was financially supported by JSPS KAKENHI (18H04765) and the Program to Disseminate Tenure Track System, MEXT, Japan.

1. Fabrication of hybrid gel cube device
<ol>
	<li>Prepare a polycarbonate (PC) cubic frame either by using a machining process or a 3D printer. The size of the PC frame depends on the sample size. In this study, we used a frame of 5 mm on each side so that the branches had sufficient space to elongate during culturing.</li>
	<li>Place the PC frame on a pre-cooled glass slide or another smooth surface in an ice box (&#60;0 &#176;C) (Figure 1A).</li>
	<li>Add 12 &#181;L of pre-heated 1.5% agarose (w/v) from the top face of the cubic frame to the bottom surface with a pipette. Stroke the pipette so that the agarose spreads to create a flat surface (Figure 1B).</li>
	<li>Within minutes, the agarose will be cured. Pick up the PC frame by sliding it to the edge of the glass slide using tweezers. Note that vertically picking up the frame from the glass slide may result in detaching of the agarose from the cubic frame owing to the adhesive force between the agarose and the glass slide (Figure 1C).</li>
	<li>Rotate the cubic frame to make the open face down, and then place on the glass slide again (Figure 1D).</li>
	<li>Repeat steps 1.3&#8211;1.5 until three surfaces are filled with agarose (Figure 1E).</li>
	<li>To form an agarose wall on the fourth and fifth faces, drop the agarose in from an open face (Figure 1F). Once an agarose wall is formed on the fifth face, the preparation of the HGC is completed.</li>
</ol>
2. Fabrication of micromolds by photolithography or a machining process
<ol>
	<li>Fabrication by photolithography
	<ol>
		<li>Clean a silicon (Si) wafer via ultrasonic cleaning with acetone, isopropyl alcohol (IPA), then distilled (DI) water for 5 min each. After nitrogen blowing the Si wafer with nitrogen, dehydrate in an oven at 140 &#176;C for 20 min (Figure 2A(i)).</li>
		<li>Spin-coat a sacrificial layer such as LOR and Az resists on the Si wafer (2,000 rpm, 30 s) by using a spin-coater under yellow light. Immediately after spin-coating, heat the wafer on a hot plate at 90 &#176;C for 3 min (Figure 2A(ii)). The desired thickness of the sacrificial layer is more than 1 &#181;m so that the lift off process can be carried out easily, but an accurate thickness is not necessary.</li>
		<li>Place the wafer on a hot plate at 70 &#176;C, and then laminate a thick negative photoresist sheet with the desired thickness on the sacrificial layer using a hand roller (Figure 2A(iii))</li>
		<li>Five minutes after lamination, place the wafer on a mask aligner for UV exposure. Set a photomask with the desired pattern on the sheet resist and expose with UV light for an appropriate time duration. The exposure time is determined by the thickness of photoresist and the intensity of the UV light. Subsequently, place the wafer on a hot plate for a post-exposure bake at 60 &#176;C for 5 min, followed by baking at 90 &#176;C for 10 min. (Figure 2A(iv))</li>
		<li>Develop a negative photoresist by immersing the wafer in propylene glycol monomethyl ether acetate (PGMEA) until the unexposed area is completely dissolved (approximately 20 min) (Figure 2A(v)).</li>
		<li>Dissolve the sacrificial layer by using IPA for ultrasonic cleaning to lift off the microstructure. Once the microstructure is peeled off from the wafer, rinse with DI water by ultrasonic cleaning for 10 min (Figure 2A(vi)).</li>
		<li>Immerse the mold in 30 &#181;L of the MPC polymer for 30 min at room temperature (approximately 20 &#176;C) to completely dry it out. Figure 2B shows the fabricated prism mold.</li>
	</ol>
	</li>
	<li>Fabrication of a cylinder mold
	<ol>
		<li>Fabricate a steel cylinder mold with desired size by a machining process. Alternatively, use commercially available stainless steel. (In this study, a &#966; 600 &#181;m cylinder is used.)</li>
		<li>Immerse the cylinder steel in 30 &#181;L of the MPC polymer for 30 min at room temperature (approximately 20 &#176;C) to completely dry it out.</li>
		<li>Prepare liquid polydimethylsiloxane (PDMS) by mixing base resin and its catalyst (see Table of Materials) in a 10:1 ratio followed by degassing with a vacuum degassing system for 20 min. Then pour the PDMS in a 12-well-plate.</li>
		<li>Set the steel cylinder perpendicular to the datum surface and fixed to a linear z-stage so that it can be perpendicularly inserted in the uncured PDMS surface in a 12-well-plate placed on the same datum surface by moving the z-stage. Incubate the PDMS with the cylinder steel in an oven for 20 min at 90 &#176;C for curing. Cut the PDMS to form a flank surface so that the pipet can be inserted in the following process (Figure 2C).</li>
	</ol>
	</li>
</ol>
3. Controlling initial cell cluster shape in a hydrogel
<ol>
	<li>Inject an appropriate ECM for the desired cell culture, such as collagen and artificial basement membrane, into the HGC to fill the culture space (Figure 3A).</li>
	<li>Set the fabricated micromold on the HGC directly or indirectly with an appropriate holder, as shown in Figure 3B. Subsequently, place the HGC in a CO2 incubator for 25 min at 37 &#176;C for curing it.</li>
	<li>Carefully lift out the micromold so that the ECM does not deteriorate (Figure 3C). The pocket, as the desired mold shape, will be fabricated in the ECM.</li>
	<li>Harvest cells from the cultured dish by trypsin&#8211;EDTA or its equivalent depending on the cell types. Subsequently, centrifuge the cells to obtain highly concentrated cells (for NHBE culture, apply 0.25% trypsin-EDTA and incubate cells for 3 min, then neutralize with FBS-containing medium and centrifuging at 300 x g for 4 min).</li>
	<li>After centrifuging, remove the supernatant medium to condense the cells, and inject highly concentrated cells (3.0 &#215; 104 cells/&#181;L for NHBE cells) into the pocket in the ECM (Figure 3D).</li>
	<li>Incubate the HGC with cells in a CO2 incubator for 20 min at 37 &#176;C so that the cells fall into the ECM pocket to fill the space created by the micromolds. If excessive medium or cells are present on the top surface, carefully remove using a pipette.</li>
	<li>Place the HGC on a 24-well-plate and add 100 &#181;L of appropriate medium (Figure 3E). For NHBE cells, use a 1:1 mixture of commercially available specialized medium for NHBE and endothelial cells (see Table of Materials).</li>
	<li>Inject additional ECM to close the pocket in the ECM, and then incubate at 37 &#176;C for 25 min.</li>
	<li>Cool down preheated 1.5% agarose to room temperature (approximately 20 &#176;C). Drop approximately 10 &#181;L of the agarose onto the top surface of the HGC to close the surface and prevent the ECM from falling out of the HGC during culturing and imaging (Figure 3F). Then, incubate the HGC for another 20 min at 37 &#176;C to cure the agarose.</li>
	<li>Add medium to cover the entire HGC so that the osmotic pressure can help in providing nutrition to the cells inside the HGC.</li>
</ol>
4. Multi-directional imaging
<ol>
	<li>Non-invasive 3D shape recognition by multi-directional observation
	<ol>
		<li>Place the culture dish or well plate containing the HGC on a microscope and orient the HGC to the camera frame. Then, obtain a sample image by bright field or phase contrast.</li>
		<li>Pick up and rotate the HGC to place a different surface down.</li>
		<li>Repeat steps 4.1.1 and 4.1.2 until images from all six sides are obtained.</li>
	</ol>
	</li>
	<li>Immuno-fluorescent imaging by multi-directional observation
	<ol>
		<li>For fixation, apply 4% paraformaldehyde to the sample over the HGC at room temperature (approximately 20 &#176;C) for 20 min, followed by two rinses of PBS for 10 min each.</li>
		<li>Permeabilize the HGC with PBS containing 0.5% Triton X-100 for 10 min at 4 &#176;C, then wash 3x for 10 min with PBS.</li>
		<li>Incubate the HGC with 10% goat serum in IF-buffer (0.2% Triton X-100, 0.1% bovine serum albumin, and 0.05% Tween-20 in PBS) for 60 min at room temperature for a primary blocking step.</li>
		<li>For staining of a specific molecule, use the appropriate antibody. To observe the collective cell geometry, Alexa Fluor 488 Phalloidin can be used to stain actin.</li>
		<li>Place the HGC on a glass bottom dish under a laser or fluorescent microscope and perform scanning from six sides to obtain the entire sample image.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Baker, B. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+third+dimension+-+how+3D+culture+microenvironments+alter+cellular+cues.">Deconstructing the third dimension - how 3D culture microenvironments alter cellular cues.</a> Journal of Cell Science. 125 (13), 3015-3024 (2012).</li><li>Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosystems+dynamics+in+self-organization+of+tissue+architecture.">Cytosystems dynamics in self-organization of tissue architecture.</a> Nature. 493 (7432), 318-326 (2013).</li><li>Shamir, E. R., Ewald, A. J. <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+organotypic+culture:+Experimental+models+of+mammalian+biology+and+disease.">Three-dimensional organotypic culture: Experimental models of mammalian biology and disease.</a> Nature Reviews Molecular Cell Biology. 15 (10), 647-664 (2014).</li><li>Frantz, C., Stewart, K. M., Weaver, V. 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+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> Journal of Cell Science. 123 (24), 4195-4200 (2010).</li><li>Choquet, D., Felsenfeld, D. P., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+rigidity+causes+strengthening+of+integrin-+cytoskeleton+linkages.">Extracellular matrix rigidity causes strengthening of integrin- cytoskeleton linkages.</a> Cell. 88 (1), 39-48 (1997).</li><li>Hannezo, E., Prost, J., Joanny, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+epithelial+sheet+morphology+in+three+dimensions.">Theory of epithelial sheet morphology in three dimensions.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (1), 27-32 (2014).</li><li>Tawk, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mirror-symmetric+cell+division+that+orchestrates+neuroepithelial+morphogenesis.">A mirror-symmetric cell division that orchestrates neuroepithelial morphogenesis.</a> Nature. 446 (7137), 797-800 (2007).</li><li>Eiraku, M., 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472 (7341), 51-58 (2011).</li><li>Zegers, M. M. P., O&#8217;Brien, L. E., Yu, W., Datta, A., Mostov, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+polarity+and+tubulogenesis+in+vitro.">Epithelial polarity and tubulogenesis in vitro.</a> Trends in Cell Biology. 13 (4), 169-176 (2003).</li><li>Affolter, M., Bellusci, S., Itoh, N., Shilo, B., Thiery, J. P., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tube+or+not+tube:+Remodeling+epithelial+tissues+by+branching+morphogenesis.">Tube or not tube: Remodeling epithelial tissues by branching morphogenesis.</a> Developmental Cell. 4 (1), 11-18 (2003).</li><li>Denk, W., Strickler, J., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Huisken, J., 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=Optical+Sectioning+Deep+Inside+Live+Embryos+by+Selective+Plane+Illumination+Microscopy.">Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy.</a> Science. , 1007-1009 (2004).</li><li>Hagiwara, M., Kawahara, T., Nobata, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+in+Cube:+In+Vitro+3D+Culturing+Platform+with+Hybrid+Gel+Cubes+for+Multidirectional+Observations.">Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations.</a> Advanced Healthcare Materials. 5 (13), 1566-1571 (2016).</li><li>Hagiwara, M., Nobata, R., Kawahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+Scale+Imaging+by+Fine+Spatial+Alignment+of+Multi-Scanning+Data+with+Gel+Cube+Device.">Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device.</a> Applied Sciences. 8 (235), (2018).</li><li>Kolesky, D. B., Homan, K. A., Skylar-Scott, M. A., Lewis, J. A. <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+bioprinting+of+thick+vascularized+tissues.">Three-dimensional bioprinting of thick vascularized tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (12), 3179-3184 (2016).</li><li>Mironov, V., Visconti, R. P., Kasyanov, V., Forgacs, G., Drake, C. J., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+Tissue+spheroids+as+building+blocks.">Organ printing: Tissue spheroids as building blocks.</a> Biomaterials. 30 (12), 2164-2174 (2009).</li><li>Onoe, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metre-long+cell-laden+microfibres+exhibit+tissue+morphologies+and+functions.">Metre-long cell-laden microfibres exhibit tissue morphologies and functions.</a> Nature Materials. 12 (6), 584-590 (2013).</li><li>Miller, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+casting+of+patterned+vascular+networks+for+perfusable+engineered+three-dimensional+tissues.">Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues.</a> Nature Materials. 11 (9), 768-774 (2012).</li><li>Hagiwara, M., Nobata, R., Kawahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+repeatability+from+3D+experimental+platform+for+quantitative+analysis+of+cellular+branch+pattern+formations.">High repeatability from 3D experimental platform for quantitative analysis of cellular branch pattern formations.</a> Integrative Biology. 10, 306-312 (2018).</li><li>Tainaka, K., 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=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159 (4), 911-924 (2014).</li><li>Murray, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+Proteomic+Imaging+for+High-Dimensional+Profiling+of+Intact+Systems.">Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems.</a> Cell. 163 (6), 1500-1514 (2015).</li></ol>

The authors and the Osaka Prefecture University and Kyushu Institute of Technology have filed a patent application for a hybrid gel cube device, and Nippon Medical and Chemical Instruments Co. Ltd, Japan has recently commercialized the cube. The company did not affect any of the design, process, and methods described.

The method presented in this paper is simple and can be performed without high-technology equipment. Concurrently, a precise cell cluster shape control result in the 3D space of hydrogel can be obtained. After the initial control, the cells can grow in the HGC as much as they are cultured on a dish. The multi-directional imaging is performed by rotating the sample with the HGC using any microscopy system, and it significantly enhances the imaging quality. The choice of the materials for the HGC frame and micromold is fle...

The importance of a 3D culture, which better mimics biological environments than does a 2D culture, is considerably emphasized in cell/tissue culture1,2,3. The interaction between the cells and extracellular matrix (ECM) provides important cues regarding morphogenesis4,5. Many tissue formations can emerge only under 3D environments, such as the folding process6,7, invagination8, and tubular formation9,10. However, numerous difficulties prevent researchers from shifting to 3D experiments from 2D experiments on a dish. One of the major difficulties in 3D experiments is the issue of imaging 3D samples. Compared with planar experiments, acquisition of appropriate 3D images is still challenging in many cases. In particular, obtaining an appropriate 3D image is a difficult task when the sample size reaches the millimeter range owing to the large focal depth of low-magnification lenses. For example, the focal depth reaches more than 50 &#181;m when a 10x magnification lens is used while the size of the single cell is normally less than 10 &#181;m. To enhance the imaging quality, high-technology microscopy systems are being developed (e.g., two-photon microscopy11 and light-sheet microscopy system12), but their availability is limited owing to their expensive price. As an alternative, we have previously developed a hybrid gel cube (HGC) device13. The device consists of two types of hydrogels: agarose as a support gel and an ECM such as collagen or Matrigel as a culture gel. The HGC allows us to collect the sample during culturing and rotate the cube to achieve multi-directional imaging, which addresses the focal depth problem14.
Another difficulty in 3D experiments is their low repeatability owing to the poor controllability of the 3D environments. Unlike a planar culture on a plastic dish, variations in the initial culture conditions easily occur in a 3D space surrounded by a soft material. A significant variation in the experimental results deteriorates the following analysis and masks the underlying mechanisms. Many engineering technologies have been developed to spatially align single cells, such as bioprinting15,16, fiber weaving17, and scaffolding18, but they require complex preprocessing or specifically designed equipment. In contrast, we have developed a methodology for achieving 3D cell alignment in an HGC19.
In this protocol, we illustrated a simple procedure with commonly used equipment for controlling the 3D initial cell cluster shape in an HGC. First, the fabrication process of the HGC was demonstrated. Then, micromolds fabricated by photolithography or a machining process were placed in the HGC to produce a pocket with an arbitrary shape in an ECM. Subsequently, highly dense cells after centrifugation were injected into the pocket to control the initial cell cluster shape in the HGC. The precisely controlled cell cluster could be imaged from many directions because of the HGC. Normal human bronchial epithelial (NHBE) cells were used to demonstrate the control of the initial cell cluster shape and imaging of the branches from multiple directions for enhancing the imaging quality.

<table><tbody><tr><td>12-well-plate</td><td>Corning&amp;nbsp; Inc.</td><td>3513</td><td></td></tr><tr><td>2-Methoxy-1-methylethyl Acetate</td><td>FUJIFILM Wako Pure Chemical Co.</td><td>130-10505</td><td>PGMEA, CAS: 108-65-6&amp;nbsp;</td></tr><tr><td>4% paraformaldehyde</td><td>FUJIFILM Wako Pure Chemical Co.</td><td>161-20141</td><td>CAS: 30525-89-4</td></tr><tr><td>Agarose, low gelling temperature&amp;nbsp; BioReagent</td><td>Sigma-Aldrich</td><td>A9414</td><td></td></tr><tr><td>Alexa fluor 488 phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td></td></tr><tr><td>AZ1512</td><td>Merck</td><td></td><td></td></tr><tr><td>BEGM bullet kit</td><td>Lonza</td><td>CC-3170</td><td>Specialized medium for NHBE cells</td></tr><tr><td>Bovine Serum Albumin solution (10 %)</td><td>Sigma-Aldrich</td><td>A1595</td><td></td></tr><tr><td>EGM-2 bullet kit</td><td>Lonza</td><td>CC-3162</td><td>Specialized medium for endothelial cells</td></tr><tr><td>Lipidure</td><td>NOF co.</td><td></td><td>MPC polymer</td></tr><tr><td>Matrigel growth factor reduced basement membrane matrix</td><td>Corning&amp;nbsp; Inc.</td><td>354230</td><td></td></tr><tr><td>Normal Goat Serum (10%)</td><td>Thermo Fisher Scientific</td><td>50197Z</td><td></td></tr><tr><td>Normal human bronchial epithelial cells</td><td>Lonza</td><td>CC-2541</td><td></td></tr><tr><td>SILPOT 184 W/C</td><td>Dow Corning Co.</td><td>3255981</td><td>Base resin and catalyst for PDMS</td></tr><tr><td>SUEX D300</td><td></td><td>DJ MicroLaminates, Inc</td><td>Thick negative photoresist (thichness: 300 mm)</td></tr><tr><td>Triton X-100 (1%)</td><td>Thermo Fisher Scientific</td><td>HFH10</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr></tbody></table>

initial 3d cell cluster control in a hybrid gel cube device for repeatable pattern formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

Normal human bronchial epithelial (NHBE) cells were used to demonstrate the illustrated methodology and control the initial collective cell geometry to achieve a cylinder shape and a prism shape, respectively in an ECM environment. The multi-directional imaging results obtained by phase contrast as well as phalloidin staining of a cylinder shape (Figure 4A,B) and the prism shape (Figure 4C,D) are...

Watch this Scientific Journal Video about Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations at JoVE.com

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

initial-3d-cell-cluster-control-hybrid-gel-cube-device-for-repeatable

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Bioengineering

5.7K Views.  Osaka Prefecture University. We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

Video: Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

Microfabricated Platforms for Mechanically Dynamic Cell Culture

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

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

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

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Biology

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

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

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

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

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

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Microfabricated Platforms for Mechanically Dynamic Cell Culture

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Simple, Affordable, and Modular Patterning of Cells using DNA

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