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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S., Khademhosseini, A., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chip-Based+Comparison+of+the+Osteogenesis+of+Human+Bone+Marrow-+and+Adipose+Tissue-Derived+Mesenchymal+Stem+Cells+under+Mechanical+Stimulation.">Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation.</a> PLoS One. 7 (9), e46689 (2012).</li><li>Gould, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfabricated+platform+for+high-throughput+unconfined+compression+of+micropatterned+biomaterial+arrays.">A microfabricated platform for high-throughput unconfined compression of micropatterned biomaterial arrays.</a> Biomaterials. 31 (3), 577-584 (2010).</li><li>Keung, A. J., Kumar, S., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presentation+Counts:+Microenvironmental+Regulation+of+Stem+Cells+by+Biophysical+and+Material.+Cues.">Presentation Counts: Microenvironmental Regulation of Stem Cells by Biophysical and Material. Cues.</a> Annu Rev Cell Dev Biol. 26, 533-556 (2010).</li><li>Segers, V. F., Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-cell+therapy+for+cardiac+disease.">Stem-cell therapy for cardiac disease.</a> Nature. 451 (7181), 937-942 (2008).</li><li>Hsieh, H. Y., 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=Gradient+static-strain+stimulation+in+a+microfluidic+chip+for+3D+cellular+alignment.">Gradient static-strain stimulation in a microfluidic chip for 3D cellular alignment.</a> Lab Chip. 14 (3), 482-493 (2014).</li></ol>

The authors have nothing to disclose.

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 ...

The overall goal of this gradient strain chip is to provide a simple approach to generate non continuous gradient static strains on a 3D hydrogel for the investigation of cell behaviors under a series of strain conditions. This method can help answer key questions in the bioengineering field such as the stimulations of a stem cell differentiation and the regulation of cellular alignment for artificial tissue organ development. The main advantage of this technique is that various strains can be applied on the cell layer and hydrogels in the same micro environment for comparison of cell behavior.Visual demonstration of this method is critical as the chip preparation steps are difficult to learn since the light should be complete in three to four hours for base of experiment to ensure high cell variability. Weigh 10 grams of gelatin powder and add it to a glass flask with 100 milliliters of Dulbecco's phosphate buffered saline, or DPBS. Put a magnetic stir bar into the flask and place the flask on a stirring hot plate.Cover the flask with aluminum foil to avoid water evaporation. Set the hot plate temperature to 50 to 60 degrees and the stirrer at 100 RPM for one hour to dissolve the gelatin powder. After the gelatin has dissolved, add eight milliliters of methacrylic anhydride very slowly using a pipette.Let it react at 60 degrees Celsius for three hours. Then add prewarmed DPBS into the flask to a final volume of 500 milliliters. Allow the solution to mix well for 15 minutes to stop the reaction.Meanwhile cut a dialysis membrane into several 25 millimeter long tubes. Immerse them in deionized water for 15 minutes. And make a knot to close one end of the dialysis tubes.Load the appropriate amount of the polymer solution into the dialysis tubes and close the other end. Then place the filled tubes in a five liter plastic beaker with deionized water for a week. Renew the deionized water twice a day and maintain the solution at 40 to 50 degrees Celsius during the dialysis process.Following dialysis collect the solution from the tubes into a 500 milliliter glass bottle. Pour approximately 450 to 500 milliliters of solution into a 500 milliliter filter cup. And apply a vacuum to force the solution to pass through the filter membrane for sterilization.Then transfer the sterilized polymer into several 50 milliliter sterilized tubes. Transfer the sterilized polymer into several 50 milliliter sterilized tubes and store them in a minus 80 degrees Celsius freezer for three to five days. Freeze dry the minus 80 degrees Celsius polymer for one week using a freeze drier to form GelMA which can then be stored in a minus 80 degree Celsius freezer.For chip fabrication first prepare the polymethacrylate or PMMA, played and molded as described in the text protocol. Then prepare the PTMS cover and PTMS plug by properly mixing 30 grams of PTMS elastomer and three grams of PTMS curing agent. Degas the mixture under a vacuum chamber for one hour.Cast 1.8 to two grams of the mixture into the PMMA mold for the PTMS cover. Also use the appropriate amount to fill each cavity for the PMMA mold for the PTMS plug. In addition cast 10 grams of uncured PTMS mixture in a blank 10 centimeter plastic plate.Put these molds in a vacuum chamber to degas for 30 minutes. Cure the PTMS for two hours at 80 degrees Celsius. After cooling down the cured PTMS on the mold, detach the PTMS covers and the PTMS plugs from the PMMA molds.Next punch two holes with diameters of about three millimeters at the ends of the flow channels of the PTMS covers. Cut the PTMS sheet molded from the 10 centimeter plastic plate into many one centimeter by one centimeter cubes. And punch a three millimeter hole in each PTMS cube.Glue two uncured one centimeter by one centimeter PTMS cubes onto the openings of the PTMS cover to serve as medium reservoirs and to aid in the curing process of the gradient circular hydrogel patterns. Then cure for one hour at 80 degrees Celsius. Next bond the PTMS covers with the two PTMS reservoirs onto a TMSPMA coated slide.To do so, pretreat the bonding side of the PTMS cover and the TMSPMA coated slide under an oxygen plasma machine for 90 seconds of oxygen plasma treatment. Contact the plasma treated surface of the PTMS covers and the TMSPMA slide and press them closely for permanent bonding through the formation of the silicon oxygen silicon bond. Print a photomask by printing the established layout on a transparent film.Then cut the printed photomask to 25 millimeters by 37.5 millimeters in size. Weigh 25 milligrams of freeze dried GelMA into 0.3 milliliters of prewarmed cell culture medium in a 1.5 milliliter black microcentrifuge tube. Put the microcentrifuge tube on a laboratory stirrer hot plate until the GelMA dissolves in the medium.Add 50 milligrams of photoinitiator to one milliliters of DPBS in a microcentrifuge tube. And place it in an 80 degree Celsius oven for 15 minutes or until the photoinitiator has dissolved. Add 25 microliters of the 10%photoinitiator to the microcentrifuge tube.Pipette several times to mix well. Next count three million NIH3T3 cells using an automated cell counter. After centrifuging the suspension at 200 Gs for five minutes discard the supernatant and resuspend the cells in 175 microliters of cell culture medium.Add the cell solution to the microcentrifuge tube to get a pre polymer cell solution of 5%GelMA, 0.5%photoinitiator and about six million 3T3 cells per milliliter. After mixing load 100 microliters of cell prepolymer into a 100 microliter microsyringe. Manually align a piece of the photomask onto the bottom slide of the sterilized gradient strain chip.And simply fix the position using a small drop of deionized water in between. Connect the 100 microliter microsyringe, loaded with prepolymer cell solution to the inlet of the chip. Then place 50 microliters of prepolymer cell solution in the flow channel using the microsyringe and plug the outlet using a PTMS plug.Inject an extra 40 microliters of solution to create a convex bulge in the circular PTMS membrane. Move the chip with the photomask, microsyringe, and PTMS plug to under a UV lamp. And expose it for 30 to 45 seconds to cross link the concentric circular hydrogel in the fluidic chamber.After cross linking remove the PTMS plug and the microsyringe to release the liquid pressure. Use a one milliliter syringe loaded with prewarmed DPBS to wash out uncrosslinked resins three times by flushing from the inlet to the outlet. Next fill the flow channel with about 100 microliters of cell culture medium.Place the chip in a sterilized culture dish and culture in a 5%carbon dioxide atmosphere at 37 degrees Celsius for a week. Refresh the medium every day. Take images of three chips on day zero after four hours of incubation as the control group.And three chips on day three as the experimental set using a microscope with a 20 times objective. Measure the line width of each hydrogel from line one to line 12 using software to calculate the compress stains. Shown here are representative results of hydrogel patterns with zero microliter and 40 microliters over injection, after UV cross linking and four hour incubation.The line width of the 3D hydrogel in the non over injection control looks uniform as expected. However the hydrogel patterns with 40 microliters over injection gives a gradient line width of hydrogels decreasing from line one to line 12. After three days of incubation, the cells in the hydrogel extend to different directions.The cells in line one align along the radial direction due to the stimulation of hydrogel elongation. On the contrary the cells in line 12 align along the hydrogel shape where the geometry cue dominates the cellular alignment. There is no specific cell alignment in line 7 where the two cellular alignment cues of line one and line 12 balance each other out.Once measuring the micro environment of gradient strains on 3D hydrogel can be done by the gradient strain chip in two hours if it's performed properly. While attempting this procedure, it is important to remember to sterilize the gradient strain chip before loading the cell prepolymer solution. Also thoroughly wipe the surface of the chip with 75%ethanol before placing the chips into the cell culture incubator.After watching this video you should have a very good understanding of how to generate a gradient strain chip to investigate cell behaviors under different strains imperial.

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Research

JoVE Journal

Bioengineering

7.6K visualizações.  National Tsing Hua University. The overall goal of this gradient strain chip is to provide a simple approach to generate non continuous gradient static strains on a 3D hydrogel for the investigation of cell behaviors under a series of strain conditions. This method can help answer key questions in the bioengineering field such as the stimulations of a stem cell differentiation and the regulation of cellular alignment for artificial tissue organ development. The main advantage of this technique is that various strains can b...