Name: initial 3d cell cluster control in a hybrid gel cube device for repeatable pattern formations
Uploaded: 2019-03-21T14:30:00
Description: Watch this Scientific Journal Video about Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations at JoVE.com

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% a...

<ol>
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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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">12-well-plate</td>
			<td style="width:124px;">Corning&#160; Inc.</td>
			<td align="right" style="width:132px;">3513</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:251px;">2-Methoxy-1-methylethyl Acetate</td>
			<td style="width:124px;">FUJIFILM Wako Pure Chemical Co.</td>
			<td style="width:132px;">130-10505</td>
			<td>PGMEA, CAS: 108-65-6&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">4% paraformaldehyde</td>
			<td style="width:124px;">FUJIFILM Wako Pure Chemical Co.</td>
			<td style="width:132px;">161-20141</td>
			<td>CAS: 30525-89-4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:251px;">Agarose, low gelling temperature&#160; BioReagent</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:132px;">A9414</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Alexa fluor 488 phalloidin</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:132px;">A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">AZ1512</td>
			<td>Merck</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">BEGM bullet kit</td>
			<td style="width:124px;">Lonza</td>
			<td style="width:132px;">CC-3170</td>
			<td>Specialized medium for NHBE cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Bovine Serum Albumin solution (10 %)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:132px;">A1595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">EGM-2 bullet kit</td>
			<td style="width:124px;">Lonza</td>
			<td style="width:132px;">CC-3162</td>
			<td>Specialized medium for endothelial cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Lipidure</td>
			<td style="width:124px;">NOF co.</td>
			<td style="width:132px;"></td>
			<td>MPC polymer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Matrigel growth factor reduced basement membrane matrix</td>
			<td style="width:124px;">Corning&#160; Inc.</td>
			<td style="width:132px;">354230</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Normal Goat Serum (10%)</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:132px;">50197Z</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Normal human bronchial epithelial cells</td>
			<td style="width:124px;">Lonza</td>
			<td style="width:132px;">CC-2541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">SILPOT 184 W/C</td>
			<td style="width:124px;">Dow Corning Co.</td>
			<td style="width:132px;">3255981</td>
			<td>Base resin and catalyst for PDMS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">SUEX D300</td>
			<td style="width:124px;"></td>
			<td style="width:132px;">DJ MicroLaminates, Inc</td>
			<td>Thick negative photoresist (thichness: 300 mm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Triton X-100 (1%)</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:132px;">HFH10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:132px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">TWEEN 20</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:132px;">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 ...

This protocol uses a simple method for enhancing the reliability of a 3D cell culture model without the use of any special equipment. The main advantage of this technique is that we can conduct the repeatable 3D culture experiment by controlling the initial culture condition within the cubic device. After preparing a five by five millimeter polycarbonate cubic frame, place the frame on a pre cooled slide and an ice box and use a pipette to add 12 microliters of preheated 1.5%agarose from the top face of the cubic frame to the bottom surface.Spread the agarose to obtain a flat surface and allow the polymer to cure. Use tweezers to slide the frame to the edge of the glass and rotate the frame so that the open side is facing down before placing the frame back onto the slide. After filling the next two surfaces of the frame with more agarose, as just demonstrated, drop the agarose into a container from an open face to form an agarose wall on the fourth and fifth sides.To set up an initial cell cluster shape inject an appropriate extracellular matrix for the cell culture of interest into the hybrid gel cube culture space and set a fabricated micro mold onto the cube. Then place the cube in a carbon dioxide incubator for 25 minutes at 37 degrees Celsius. When the extracellular matrix is cured, carefully lift out the micro mold so that the matrix does not deteriorate.A pocket in the desired mold shape will be fabricated in the extracellular matrix. To load the cells concentrate the experimental cell suspension after centrifugation in the appropriate experimental cell culture medium and inject the cells into the pocket within the extracellular matrix. Place the cube into the carbon dioxide incubator for 20 minutes at 37 degrees Celsius to allow the cells to fall into the extracellular matrix pocket, filling the space created by the micro mold.At the end of the incubation, place the cube into one well of a 24 well plate and add 100 microliters of the appropriate cell culture medium to the well. Inject additional extracellular matrix to close the pocket and return the cube to the incubator for 25 minutes. When the extracellular matrix is cured drop approximately 10 microliters of 20 degree Celsius agarose onto the top surface to the cube to close the surface and cure the drop for 10 to 20 minutes at 37 degrees Celsius.Then cover the entire cube with fresh medium to promote osmotic pressure for facilitating the transfer of nutrition to the cells within the cube. For noninvasive 3D shape recognition by multidirectional observation, place the plate onto a microscope stage and obtain images of each side of the cube by bright field or phase contrast microscopy, using tweezers to rotate the cube service to be imaged between captures. For immunofluorescent imaging by multidirectional observation, first aspirate the supernatant from the well containing the cube and fix the cube in 4%paraformaldehyde for 20 minutes at room temperature.At the end of the fixation, wash the cube two times with PBS for 10 minutes per wash. Impermeabilize the cube with 0.5%triton x-100 and PBS for 10 minutes at four degrees Celsius. Next, wash the cube three times in PBS for 10 minutes per wash, before blocking any nonspecific bonding with goat serum and immunofluorescence buffer for 60 minutes at room temperature.Then stain the cells with the appropriate primary antibody of interest according to standard antibody staining protocols. Then image all six sides of the cube on a laser or fluorescent microscope as just demonstrated. In this representative experiment multidirectional imaging of hybrid gel cubes, cultured with normal human bronchial epithelial cells, demonstrates the generation of an initial cylindrical or prism shaped cell culture by phase contrast and immunofluorescent imaging of the cultures.Here immuno staining of normal human epithelial cells initially controlled to a cylindrical shape in the hybrid gel cube, after generation of the bronchial tree is shown. The branches demonstrate the perpendicular to the cylindrical axis, as revealed by multidimensional imaging. It is important to inject a high density of cells into the extracellular matrix pocket, as a low density of cells may result in deterioration of that cell quality after incubation.This method pattern is the repeatable formation of a 3D cellular pattern with a quantitative measurement by multidirectional imaging to study the mechanism of dish development.

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