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>

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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 flexible as long as they are biocompatible. A 3D printer may be used to produce the HGC frame or micromold if the accuracy of the 3D printer is sufficient for their applications. The proposed method is compatible with many image enhancing technologies such as transparency reagents20,21 and light-sheet microscopy systems. By employing these technologies, the image quality can be improved.
The volume of the agarose, ECM, and highly dense cells to be injected depends on the HGC and mold size. A careful manual operation that does not inject air bubbles is required, otherwise the bubbles formed will deteriorate the accuracy of the control, cell growth, and imaging quality.
The fabrication process of the micromolds will be determined by the desired shape to control. Photolithography enables fabrication of precise 2D shapes with a certain depth, such as a prism shape on the order of micrometers; however, it is not effective with 3D shapes, such as a cylinder. The machining process allows us to fabricate the 3D shape, but in general, the dimensional accuracy is lower than photolithography. After the fabrication of the micromolds, coating of the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer on the molds is required to prevent adhesion on the ECM.
By taking advantage of an HGC, microscopic imaging with or without laser exposure can be performed in multiple directions. A 3D sample shape can be approximately recognized by multi-directional observation without fluorescent labeling; this method does not cause any cell invasiveness during observation. In contrast, fluorescent imaging is required for obtaining a more accurate 3D sample shape and molecular expression.
The constraint of the presented method for initial cell control is that it cannot be applied to a mold with a complex 3D shape. The mold has to be removed by picking up without deteriorating the ECM. Thus, the mold is limited to a simple straight or tapered shape. To obtain more complex shape control, further development of the protocol is needed.
The proposed methodology has broad utility and can be easily conducted in most of the laboratory environments. This simple procedure can overcome the limitations of 3D culture, imaging, and repeatability, and contribute to the further development required for 3D culture.

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 border="0" cellpadding="0" cellspacing="0" style="width:673px;" width="673">
	<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 presented. The cells are suitably aligned to yield the desired 3D shape in the ECM environment. Subsequently, the cells were cultured for four days to form a specific shape and then were analyzed by multi-directional imaging. The large sample size makes it quite difficult to capture the entire tissue image from one direction; however, the HGC allowed imaging from up to six sides, revealing the whole tissue shape. Figure 5 shows the immunostaining results of the bronchial tree developed from the NHBE cells. The NHBE cells were initially controlled to a cylindrical shape in the HGC. Then, most of the branches were directed perpendicular to the cylindrical axis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59214/59214fig1.jpg" /> 
Figure 1: Fabrication process of hybrid gel cube device. (A) The polycarbonate cubic frame is set on a precooled ice box. (B) Agarose (1.5%) is injected to cover the bottom surface. (C) The cube is slid to be collected. (D) The cube is rotated to make another surface down. (E) Agarose (1.5%) is injected to form another agarose wall. (F) After an agarose wall is produced for three faces, agarose is injected through an open face. Scale bar: 5 mm. <a href="https://www.jove.com/files/ftp_upload/59214/59214fig1large.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/59214/59214fig2.jpg" /> 
Figure 2: Fabrication process of micromolds. (A) Fabrication process of a micromold by photolithography. (B) Complete form of a fabricated prism shape micromold by photolithography. (C) Fabricated micromold with cylinder steel. Scale bar: (B) 2 mm, (C) 10 mm. <a href="https://www.jove.com/files/ftp_upload/59214/59214fig2large.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/59214/59214fig3.jpg" /> 
Figure 3: Fabrication process of initial cell cluster control. (A) ECM (e.g., collagen and Matrigel) is injected into the HGC. (B) The micromold is set on the cubic frame with or without a holder. (C) After the ECM is cured, the micromold is removed. (D) After centrifuging, highly dense cells are injected and incubated. (E) Medium is applied. (F) Additional ECM followed by agarose are injected to cover the top surface of the HGC. Scale bar: 5 mm. <a href="https://www.jove.com/files/ftp_upload/59214/59214fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59214/59214fig4.jpg" /> 
Figure 4: Results of initial cell control with NHBE cells. (A) Phase-contrast images of the cylindrically controlled NHBE cells taken for the bottom and lateral surface. The entire shape could be recognized by multi-directional observation without requiring fluorescent imaging. (B) Fluorescence images of the cylindrically controlled NHBE cells staining F-actin. (C) Phase-contrast images of the NHBE cells controlled in a triangular prism shape captured from the bottom and lateral surfaces. (D) Fluorescence images of the NHBE cells controlled in a triangular prism shape. Scale bar: 100 &#181;m. 10x magnification lens (0.3 numerical aperture) was used. <a href="https://www.jove.com/files/ftp_upload/59214/59214fig4large.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/59214/59214fig5.jpg" /> 
Figure 5: Projected fluorescent images captured by multi-directional scanning. The NHBE cells were initially controlled to produce a cylindrical shape and cultured in the HGC for 4 days. Subsequently, actin was stained by phalloidin. The branches were elongated from the initial cylinder along the x axis, and the length, size, and angle from the axis were equally developed. Lower right image shows the schematic of the x-y-z axis and the sample shape. Scale bar: 100 &#181;m. A 10x magnification lens (0.3 numerical aperture) was used. <a href="https://www.jove.com/files/ftp_upload/59214/59214fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

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

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

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 ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

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

Summary

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Chapters in this video

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

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

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Micropatterning and Assembly of 3D Microvessels

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk