Name: gradient strain chip for stimulating cellular behaviors in cell-laden hydrogel
Uploaded: 2017-08-08T12:30:00
Description: Watch this Scientific Journal Video about Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel at JoVE.com

This project was supported by the Graduate Student Study Abroad Program (NSC-101-2917-I-007-010); the Biomedical Engineering Program (NSC-101-2221-E-007-032-MY3); and the Nanotechnology National Program (NSC-101-2120-M-007-001-); and the Ministry of Science and Technology (MOST-104-2221-E-007-072-MY3), National Science Council of the R.O.C., Taiwan. The authors would like to thank Prof. Ali Khademhosseini, Gulden Camci-Unal, Arghya Paul, and Ronglih Liao at Harvard Medical School for sharing the hydrogel and cell encapsulation technology.

1. GelMA Synthesis
<ol>
	<li>Weigh 10 g of gelatin powder and add it to a glass flask with 100 mL ofDulbecco&#39;s phosphate-buffered saline (DPBS). Put a magnetic stir bar into the flask and place the flask on a stirring hot plate.</li>
	<li>Cover the flask with aluminum foil to avoid water evaporation. Set the hot plate temperature to 50-60 &#176;C and the stirrer at 100 rpm for 1 h to dissolve the gelatin powder well.</li>
	<li>After the gelatin has dissolved, add 8 mL of methacrylic anhydride very slowly (one drop ...

<ol>
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In this paper, we report on a simple approach to compare cell alignment behavior after hydrogel shape guidance and tensile stretch. A flexible PDMS membrane creates a dome-shaped curvature for generating different heights of concentric circular hydrogels. After releasing the pressure, the PDMS membrane automatically applies force to the micro-patterned hydrogels to form gradient strain/elongation, with a maximum at the center and a minimum at the outer boundary. As the formation of the gradient strain is designed by the ...

Serving as a block material that mimics a native microenvironment, a hydrogel containing extracellular matrix (ECM) can re-build biomimetic tissue scaffolds to support cell growth. To possess the functions of a tissue, organized cell alignment is an essential requirement. Various 2D (i.e., cells cultured on a surface) and 3D (i.e., cells encapsulated in a hydrogel) cell alignments have been achieved by culturing or encapsulating cells in or on flexible substrates with micro-or nano-patterns1. 3D cell alignment in microarchitecture is more attractive, as the microenvironment is closer to the native tissue construct2,3,4. One common approach for 3D cell alignment is the geometric cue of hydrogel shape2,3. Because of the restricted space for cell proliferation in the short-axis direction, cells aim to align along the long-axis direction in a micro-patterned hydrogel. Another approach is to apply tensile stretch to the hydrogels to achieve cell alignment parallel to the stretch direction4,5.
Biophysical stimulation on ECM hydrogels, such as compressive strain or an electrical field, can regulate cell functions for proper tissue integration, proliferation, and differentiation1,2,3. Much research has been done to investigate cellular behavior by applying one strain condition at a time using multiple mechanical control units4,6,7,8,9. For example, the use of mechanical step motors squeezed or stretched on a 3D cell-encapsulated collagen hydrogel has been a common approach7,10. However, such controlling equipment requires extra space and faces the issue of contamination in the incubator7,9,11,12. In addition, the large instrument cannot give a precise control environment to provide high reproducibility13.
Considering that cell-laden hydrogels are usually employed on the micro-scale for biomedical applications, it is advantageous to combine MEMS techniques to generate a range of strain/stretch stimulation to simultaneously investigate cell behaviors in 3D biomimetic constructs in vitro2,14,15,16,17,18. For example, using gas pressure to deform the PDMS membrane in microfluidic chips can give rise to various strains, driving cell differentiation to different lineages9,16. However, there are many technical challenges, such as complicated chip fabrication processes in a clean room and the software control integration of motors, pumps, valves, and compressed gases.
In this work, we demonstrate a simple approach to obtain a self-sustaining gradient static-strain microfluidic chip by employing a concentric circular hydrogel pattern and a flexible PDMS membrane. Unlike most of the existing approaches, our platform is a portable and disposable miniature device that can be fabricated outside a yellow room and that possesses self-generating gradient strains on concentric cell-encapsulated hydrogels, without external mechanical equipment during the incubation. 3T3 fibroblast cell behaviors influenced by a combination of hydrogel shape and a variety of tensile stretch guidance cues were demonstrated during the observation of cell alignment within 3D ECM-mimetic environments in the gradient strain chip for 3 days.

<table border="0" cellpadding="0" cellspacing="0" width="1383">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">1.5-mL black microcentrifuge tube</td>
			<td style="width:225px;">Argos Technologies&#160;</td>
			<td style="width:156px;">03-391-161</td>
			<td style="width:665px;">This one can be replaced with a neutral color of 1.5-mL tube covered with aluminun foil</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">10X DPBS</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">56064C</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Alexa Fluor 488 phalloidin&#160;</td>
			<td style="width:225px;">Invitrogen</td>
			<td style="width:156px;">A12379&#160;</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">BSA</td>
			<td style="width:225px;">Sigma</td>
			<td style="width:156px;">A1595</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Calcein</td>
			<td style="width:225px;">Molecular Probe</td>
			<td style="width:156px;">C1430</td>
			<td style="width:665px;">For labeling viable cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">CCD</td>
			<td style="width:225px;">PCO. Imaging</td>
			<td style="width:156px;">Pixelfly qe</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Cell membrane permeating solution</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">X100</td>
			<td style="width:665px;">0.5% Triton X-100 for permeating cell membrane</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">DAPI</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">D8417</td>
			<td style="width:665px;">Cell nucleus staining</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Dialysis membrane</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">D9527</td>
			<td style="width:665px;">Molecular weight cut-off = 14,000</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">DMEM</td>
			<td style="width:225px;">Gibco</td>
			<td style="width:156px;">11995-065</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Double-side tape</td>
			<td style="width:225px;">3M</td>
			<td style="width:156px;">8003</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">FBS</td>
			<td style="width:225px;">Hyclone</td>
			<td style="width:156px;">SH30071.03</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Gelatin</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">G2500</td>
			<td style="width:665px;">gel strength 300, type A, from porcine skin</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">High frequency electronic corona generator</td>
			<td style="width:225px;">Electro-technic products</td>
			<td style="width:156px;">MODEL BD-20</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Methacrylic Anhydride</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">276685</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Micro syringe</td>
			<td style="width:225px;">Hamilton</td>
			<td style="width:156px;">80501</td>
			<td style="width:665px;">50 &#956;L&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Microscope</td>
			<td style="width:225px;">Olympus</td>
			<td style="width:156px;">IX71</td>
			<td style="width:665px;">Include two filter sets: LF405/LP-B-000 and LF488/LP-C-000 from Semrock</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Oxygen plasma machine</td>
			<td style="width:225px;">Harrick plasma</td>
			<td style="width:156px;">PDC-001</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Paraformaldehyde</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">P6148</td>
			<td style="width:665px;">For fixing cell</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">PDMS</td>
			<td style="width:225px;">DOW CORNING</td>
			<td style="width:156px;">Sylgard 184</td>
			<td style="width:665px;">Mixture for PDMS chip cast-molding fabrication</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Pen-Strep</td>
			<td style="width:225px;">Gibco</td>
			<td style="width:156px;">10378-016</td>
			<td style="width:665px;">penicillin/streptomycin</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Photoinitiator</td>
			<td style="width:225px;">CIBA</td>
			<td style="width:156px;">Irgacure 2959</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Propidium iodide</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">P4170</td>
			<td style="width:665px;">For labeling dead cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Sterile Filtration cup</td>
			<td style="width:225px;">Millipore</td>
			<td style="width:156px;">SCGPT05RE</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">TMSPMA</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:156px;">440159</td>
			<td style="width:665px;">For hydrogel immobilization</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Ultrasonicator</td>
			<td style="width:225px;">Delta</td>
			<td style="width:156px;">D150H</td>
			<td style="width:665px;">150W, 43kHz</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">UV light</td>
			<td style="width:225px;">DAIHAN</td>
			<td style="width:156px;">WUV-L10</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">Freeze Dryer</td>
			<td style="width:225px;">FIRSTEK</td>
			<td style="width:156px;">150311025</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">NIH3T3(fibroblast)</td>
			<td style="width:225px;">Food Industry Research and Development Institute(FIRDI)</td>
			<td style="width:156px;">08C0011</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:337px;">MOXI Z Mini Automated Cell Counter</td>
			<td style="width:225px;">ORFLO</td>
			<td style="width:156px;">MXZ001</td>
			<td style="width:665px;"></td>
		</tr>
	</tbody>
</table>

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.

To compare the mechanical variations between each circular hydrogel in the completed gradient strain stimulation chip, we measured the line widths of each circular hydrogel in two of the same chips, with injection volumes of 0 &#181;L (Figure 4a) and 40 &#181;L (Figure 4b), respectively. The percent elongations at each circle were calculated by dividing the elongations in the 40 &#181;L-injected chip by the line widths of the cor...

Watch this Scientific Journal Video about Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel at JoVE.com

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

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