Dieses Projekt wurde durch die Graduate Student Studium im Ausland Programm (NSC-101-2917-I-007-010) unterstützt; die biomedizinische Technik-Programm (NSC-101-2221-E-007-032-MY3); und die Nanotechnologie National Program (NSC-101-2120-M-007-001-), National Science Council der r.o.c., Taiwan. Die Autoren möchten Prof. Ali Khademhosseini, Gulden Camci-Ünal, Arghya Paul und Ronglih Liao an der Harvard Medical School danken für die gemeinsame Nutzung der Hydrogel und Zelle Kapselung-Technologie.

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 per second) using a pipette. Let it react at 60 &#176;C for 3 h.</li>
	<li>Add pre-warmed DPBS (40 &#176;C) to the flask to a final volume of 500 mL and allow this mix well for 15 min to stop the reaction.</li>
	<li>Meanwhile, cut a dialysis membrane (14 kDa cut-off molecular weight) into several 25 cm-long tubes. Immerse them in deionized (DI) water for 15 min and make a knot to close one end of the dialysis tubes.</li>
	<li>Load the appropriate amount (30 - 60 mL) of the polymer solution into the dialysis tubes and close the other end. Place them in a 5-L plastic beaker with DI water for a week. Renew the DI water twice a day and maintain the solution at 40 - 50 &#176;C during the dialysis process.</li>
	<li>Collect the solution from the tube in a 500-mL glass bottle. Pour ~450 - 500 mL of solution in a 500-mL filter cup (pore size of 0.22 &#181;m) and apply a vacuum to the filter cup to force the solution to pass through the filter membrane for sterilization.</li>
	<li>Transfer the sterilized polymer into several 50-mL sterilized tubes and store them in a -80 &#176;C freezer for 3 - 5 days.</li>
	<li>Freeze-dry the -80 &#176;C polymer for 1 week using a freeze dryer to form GelMA. Store the GelMA in a -80 &#176;C freezer.</li>
</ol>
2. 3-(Trimethoxysilyl)propyl Methacrylate (TMSPMA) Modification
<ol>
	<li>&#160;Cut commercial glass slides into two small pieces (25 mm x 37.5 mm) and immerse them in 0.5 M NaOH solution for 4 h. Wash the slides with a large amount of DI water.</li>
	<li>Place the slide on a rack inside a glass container with 95% ethanol and clean using an ultrasonicator at 43 kHz for 15 min. Air dry the glass slide.</li>
	<li>Immerse the glass slides in 5% TMSPMA in 99.5% ethanol for 1 h.</li>
	<li>Wash the slides in 95% ethanol, air dry the slides, and anneal the TMSPMA coating in an oven at 80 &#176;C for 2 h.</li>
</ol>
3. Chip Fabrication
<ol>
	<li>Take one 2 mm- and one 0.3 mm-thick polymethylmethacrylate (PMMA) plate, apply double-sided tape to one side of the PMMA surface, and release the liner on one side. Leave two 2 mm-thick PMMA plates and one 1 mm-thick plate without double-sided tape.</li>
	<li>Laser-cut a 2 mm-thick PMMA plate without double-sided tape to 42 mm x 30 mm to make the bottom plate. Cut a 2 mm-thick PMMA plate with double-sided tape to make the boundary frame, with outer dimensions of 42 mm x 30 mm and inner dimensions of 37.5 mm x 25 mm.</li>
	<li>Laser-cut the 0.3 mm-thick PMMA plate with double-sided tape into a 12-mm center circle with two 2 mm-wide and 8 mm-long flow channels on opposite sides of the circle (Figure 1a).</li>
	<li>To prepare the PMMA mold for casting the PDMS cover, assemble the three pieces of PMMA components from steps 3.2-3.3 (Figure 1b) using double-sided tape.</li>
	<li>Laser-cut a 2 mm-thick PMMA plate with double-sided tape into a 5 cm x 5 cm piece with a 3 x 3 array of 8.5 mm x 8.5 mm hollow rectangles. Cut another 5 cm x 5 cm piece with a 3 x 3 array of 4-mm hollow circles. Laser-cut a 1 mm-thick PMMA plate without double-sided tape into a 5 cm x 5 cm PMMA bottom plate.</li>
	<li>Prepare the PMMA mold for casting the PDMS plug by assembling the three pieces of PMMA components from step 3.5 (Figure 1c) using double-sided tape.</li>
	<li>Prepare the PDMS cover and PDMS plug by properly mixing 30 g of PDMS elastomer and 3 g of PDMS curing agent; degas the mixture under a vacuum chamber for 1 h.</li>
	<li>Cast 1.8-2.0 g of the mixture into the PMMA mold for the PDMS cover and use the appropriate amount to fill each cavity of the PMMA mold for the PDMS plug. Cast 10 g of uncured PDMS mixture in a blank 10-cm plastic plate. Put these molds in a vacuum chamber to degas for 30 min.</li>
	<li>Cure the PDMS for 2 h at 80 &#176;C. After cooling down the cured PDMS on the mold, detach the PDMS covers and the PDMS plugs from the PMMA molds.</li>
	<li>Punch two holes with diameters of about 3 mm at the ends of flow channels of the PDMS covers.</li>
	<li>Cut the PDMS sheet molded from the 10-cm plastic plate into many 1 cm x 1 cm cubes and punch a 3-mm hole in each PDMS cube. Glue two uncured 1 cm x 1 cm PDMS cubes onto the openings of the PDMS cover (to serve as medium reservoirs and to aid in the curing process of the gradient circular hydrogel patterns) and cure for 1 h at 80 &#176;C.</li>
	<li>Bond the PDMS covers with the two PDMS reservoirs onto a TMSPMA-coated slide by pre-treating the bonding side of the PDMS cover and the TMSPMA-coated slide under an oxygen plasma machine (30 W RF power (high mode) and 600 mTorr compressed air) or a high-frequency electronic corona generator (115 V, 50/60 Hz, 0.35 A) for 90 s of O2 plasma treatment.</li>
	<li>Contact the plasma-treated surface of the PDMS covers and the TMSPMA slide and press them closely for permanent bonding through the formation of an Si-O-Si bond. 
	NOTE: Placing the chip in an oven at 80 &#176;C for 1 h can further enhance the bonding strength.</li>
	<li>After cooling, immerse the chips in 95% ethanol for 15 min and air dry. Then, sterilize the chips under UV irradiation for 1 h and store them in a box wrapped in aluminum foil in the laminar hood.</li>
</ol>
4. Static Gradient Strain on the Cell-laden Hydrogel
<ol>
	<li>Print and cut a piece of photomask, 25 mm x 37.5 mm in size, by printing the layout in Figure 2b on a transparent film. Adjust the printed size of Figure 2b to match the dimension in Figure 2a.</li>
	<li>Prepare 100 mL of DMEM medium with 10% FBS, 1% Pen-Strep, and 250 mL of DPBS in a 37 &#176;C water bath to use as the cell culture medium.</li>
	<li>Weigh 25 mg of freeze-dried GelMA into 0.3 mL of prewarmed (37 &#176;C) cell culture medium in a 1.5-mL black microcentrifuge tube. Put the microcentrifuge tube on a laboratory stirrer/hot plate until the GelMA dissolves in the medium.</li>
	<li>Weigh 50 mg of photoinitiator into 1 ml of DPBS in a microcentrifuge tube and place it in an 80 &#176;C oven for 15 min or until the photoinitiator has dissolved.</li>
	<li>Take 25 &#181;L of the 10% photoinitiator from step 4.4 and add it to the microcentrifuge tube from step 4.3. Pipette several times to mix well.</li>
	<li>Count 3 x 106 NIH 3T3 cells using an automated cell counter. Centrifuge the suspension at 200 x g for 5 min, discard the supernatant, and re-suspend the cells in 175 &#181;L of cell culture medium.</li>
	<li>Add the cell solution from step 4.6 to the microcentrifuge tube from step 4.5 to get a prepolymer cell solution of 5% GelMA, 0.5% photoinitiator, and ~6 x 106 3T3 cells/mL. After mixing, load 100 &#181;L of cell prepolymer in a 100-&#181;L micro syringe.</li>
	<li>Manually align (see Figure 3a) 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 DI water in between. Connect the 100-&#181;L micro-syringe loaded with prepolymer cell solution to the inlet of the chip.</li>
	<li>Place (see Figure 3b) 50 &#181;L of prepolymer cell solution in the flow channel using the micro-syringe and then plug the outlet using a PDMS plug. Inject an extra 40 &#181;L of solution to create a convex bulge in the circular PDMS membrane.</li>
	<li>Move the chip with the photomask (bottom), micro-syringe (inlet), and PDMS plug (outlet) from step 4.9 under a UV lamp (365 nm, 9 mW/cm2) and expose it for 30 - 45 s to crosslink the concentric circular hydrogel in the fluidic chamber.</li>
	<li>Remove the PDMS plug and the micro-syringe to release the liquid pressure (see Figure 3c). Use a 1-mL syringe loaded with prewarmed DPBS to wash out uncrosslinked resins 3 times by flushing from the inlet to the outlet.</li>
	<li>Fill the flow channel with about 100 &#181;L of cell culture medium.</li>
	<li>Place the chip in a sterilized culture dish and culture in a 5% CO2 atmosphere at 37 &#176;C for a week. Refresh the medium every day.</li>
	<li>Take images of three chips on day 0 after 4 h of incubation, as a control group, and three chips on day 3, as the experimental set, using a microscope with a 20X objective. Measure the line width of each hydrogel from line 1 to line 12 using software (e.g., ImageJ) to calculate the compress strains ( Figure 4). 
	NOTE: The elongation percentage is calculated by dividing the value of the line width difference between 40 &#181;L and 0 &#181;L by the line width at 40 &#181;L.</li>
</ol>
5. Cell Staining for Alignment Analysis
<ol>
	<li>Use a syringe to inject 4% paraformaldehyde (PFA) in DPBS at RT into the flow chip for 15 min for the fixation of the cell-laden hydrogel. 
	NOTE: Caution. PFA is toxic and should be handled with care.</li>
	<li>Replace the solution with 0.5% cell membrane permeating solution in DPBS for 10 min to permeabilize the cell membrane at RT.</li>
	<li>PBS wash the samples 3 times with 5- to 10-min interval between washes (using a loaded syringe, as in step 5.1).</li>
	<li>Load 1% BSA solution into the fluidic channel for 45-60 min at RT (using a loaded syringe through the inlet port).</li>
	<li>Add 1.5 mL of methanol into the vial with Alexa Fluor 488 phalloidin to yield a final concentration of 6.6 &#181;M stock solution.</li>
	<li>Take 5 &#181;L of Alexa Fluor 488 phalloidin from step 5.5 and dilute it in 200 &#181;L of DPBS with 0.1% BSA to form a final concentration of 0.165 &#181;M Alexa Fluor 488 phalloidin.</li>
	<li>Add 200 &#181;L of the mixture solution to the fluidic channel using a micropipette and incubate the chip at 37 &#176;C for 45 - 60 min for actin staining. PBS wash (as above) the samples 3 times.</li>
	<li>Prepare 1 &#181;g/mL DAPI in PBS, flow it through the chip, and stain cell nuclei at 37 &#176;C for 5 min.</li>
	<li>Pipette DPBS into the fluidic channel to wash out the staining solution and refill the fluidic chamber with PBS solution to take images with a fluorescence microscope.</li>
	<li>Capture the fluorescent images of the 3T3 cells in the hydrogels using an inverted fluorescence microscope under 40X magnification with a CCD detector and filter sets of ex/em at 488/520 nm and 358/461 nm for Alexa Fluor 488 phalloidin (actin) and DAPI (nucleus), respectively.</li>
</ol>

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Die Autoren haben nichts preisgeben.

In diesem Beitrag berichten wir über einen einfachen Ansatz, Zelle Ausrichtung Verhalten nach Hydrogel Form Führung und Bruchdehnung zu vergleichen. Eine flexible Membran PDMS schafft eine kuppelförmige Wölbung zur Generierung von unterschiedlichen Höhen der konzentrische kreisförmige Hydrogele. Nach der Veröffentlichung des Drucks, gilt die PDMS-Membran automatisch Kraft für die Mikro-gemusterten Hydrogele gradient Belastung/Dehnung, mit einem Maximum in der Mitte und einem Minimum an der äußeren Grenze bilden...

Dienen als ein Blockmaterial, das eine native Mikroumgebung imitiert, kann ein Hydrogel, die extrazellulären Matrix (ECM) enthalten biomimetische Gewebe Gerüste um Zellwachstum zu unterstützen wieder aufzubauen. Um die Funktionen eines Gewebes besitzen, ist organisierte Zellenausrichtung eine wesentliche Voraussetzung. Verschiedene 2D (d. h. Zellen kultiviert auf einer Oberfläche) und 3D (d. h. Zellen in einem Hydrogel gekapselt) Zelle Achsen durch Kultivierung oder Zellen in oder auf flexiblen Substraten mit Micro Kapseln erreicht wurden- oder Nano-Muster1. 3D Zellausrichtung in Mikroarchitektur ist attraktiver, da die Mikroumgebung näher an der Heimat Gewebe Konstrukt2,3,4. Ein gemeinsames Konzept für 3D Zellenausrichtung ist die geometrische Cue von Hydrogel Form2,3. Aufgrund der Platzverhältnisse für die Zellproliferation in Richtung der kurzen Achse sollen Zellen entlang der langen Achse Richtung in eine Mikro-gemusterten Hydrogel ausrichten. Ein weiterer Ansatz ist die Hydrogele zu Ausrichtung der Zellen parallel zur Strecke Richtung4,5Bruchdehnung zuweisen.
Biophysikalische Stimulation auf ECM Hydrogele, z. B. Druckspannung oder ein elektrisches Feld kann regulieren Zellfunktionen für richtige Gewebeintegration, Proliferation und Differenzierung1,2,3. Viel Forschung wurde durchgeführt, um zelluläre Verhalten zu untersuchen, durch die Anwendung einer Sorte Zustand zu einem Zeitpunkt über mehrere mechanische Steuerung Einheiten4,6,7,8,9. Zum Beispiel die Verwendung von mechanischen Schrittmotoren gequetscht oder gestreckt auf eine 3D Zelle verkapselt Kollagen Hydrogel wurde einen gemeinsamen Ansatz7,10. Jedoch solche Steuerungsvorrichtung erfordert zusätzlichen Platz und sieht sich das Problem der Kontamination in den Inkubator7,9,11,12. Darüber hinaus kann nicht das große Instrument eine präzise Steuerungsumgebung ermöglichen hohe Reproduzierbarkeit13geben.
Da die Zelle beladenen Hydrogele in der Regel auf der Mikro-Skala für biomedizinische Anwendungen beschäftigt sind, ist es vorteilhaft, MEMS Techniken um eine Reihe von Belastung/dehnen Stimulation zur Zelle Verhalten in 3D biomimetische Konstrukte in-vitro-2,14,15,16,17,18gleichzeitig untersuchen zu generieren zu kombinieren. Beispielsweise kann mit Gasdruck um zu verformen die PDMS-Membran in mikrofluidischen Chips zu verschiedenen Stämmen, fahren-Zell-Differenzierung auf verschiedenen Linien9,16führen. Allerdings gibt es viele technische Herausforderungen, wie kompliziert Chip Fertigungsprozesse in ein sauberes Zimmer und die Softwareintegration Steuerung von Motoren, Pumpen, Ventile und komprimierte Gase.
In dieser Arbeit zeigen wir Ihnen eine einfache Methode um eine autarke gradient statische Belastung mikrofluidischen Chip zu erhalten durch den Einsatz einer konzentrische kreisförmige Hydrogel-Muster und einer flexiblen Membran PDMS. Anders als die meisten der vorhandenen Ansätze ist unsere Plattform ein tragbar und Einweg-Miniatur-Gerät, vor einem gelben Zimmer hergestellt werden können und, besitzt selbst generierenden gradient Stämme auf konzentrischen Zelle verkapselt Hydrogele, ohne externe mechanische Ausrüstung während der Inkubation. 3 t 3 Fibroblast Zellen Verhalten beeinflusst durch eine Kombination von Hydrogel-Form und eine Vielzahl von Zug-Stretch Anleitung Hinweise wurden während der Beobachtung der Ausrichtung der Zellen innerhalb von ECM-mimetischen 3D-Umgebungen im Farbverlauf Stamm Chip für 3 Tage gezeigt.

<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

Künstliche Anleitung für zelluläre Ausrichtung ist ein heißes Thema im Bereich des Tissue Engineering. Die meisten der bisherigen Forschung untersuchte einzelne Stamm-induzierte zellulären Ausrichtung auf eine Zelle beladenen Hydrogel mithilfe von komplexen Versuchsabläufe und Masse Controllingsystemen, die in der Regel verbunden mit Verunreinigung Probleme sind. Daher schlagen wir in diesem Artikel einen einfachen Ansatz zum Aufbau einer Gradienten statische Belastung mit einem fluidische Chip mit einer Kunststoffabdeckung PDMS und ein UV-transparentem Glas-Substrat für die Stimulation der zellulären Verhalten in einem 3D Hydrogel. Überlastung Foto-patternable Zelle Prepolymer in der fluidische Kammer erzeugen eine konvexe gekrümmte PDMS-Membran auf dem Cover. Nach UV-Vernetzung durch eine konzentrische kreisförmige Micropattern unter dem geschwungenen PDMS-Membran und Puffer waschen, eine Mikroumgebung für Ermittlungsbehörden Zelle Verhalten unter einer Vielzahl von gradient Stämme ist selbst in einem einzigen fluidische Chip, ohne externe Instrumente etabliert. NIH3T3 Zellen wurden demonstriert, nachdem er beobachtet die Veränderung der zellulären Ausrichtung Trend unter Leitung der Geometrie, in Zusammenarbeit mit Belastung Stimulation, die von 15-65 % auf Hydrogele variiert. Nach einer 3-tägigen Inkubation dominiert die Hydrogel-Geometrie die Zellausrichtung unter niedrigen Druckspannung, wo Zellen entlang Richtung Hydrogel Dehnung unter hoher Druckspannung ausgerichtet. Zwischen diesen zeigten die Zellen zufällige Ausrichtung durch die Dissipation der radikalen Leitlinien Hydrogel Dehnung und die Geometrie-Führung der gemusterten Hydrogel.

Um die mechanischen Unterschiede zwischen jeder Runde Hydrogel im abgeschlossenen gradient Belastung Stimulation Chip zu vergleichen, haben wir die Linienstärken jede Runde Hydrogel in zwei die gleichen Chips, Einspritzmengen von 0 µL (Abb. 4a) mit 40 µL (Abbildung 4 b), bzw. gemessen. Prozent Dehnungen in jedem Kreis waren die Dehnungen in der 40 µL injiziert Chip durch dividiert die Linienbreiten der entsprechenden Hydrogel...

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

Dieser Artikel stellt eine einfache Methode zur Bereitstellung von nicht-kontinuierlichen gradienter statische Belastungen auf eine konzentrische Zelle beladenen Hydrogel, Ausrichtung der Zellen für das Tissue Engineering zu regulieren.

Gradient Stamm Chip für anregende zellulären Verhaltensweisen in Zelle beladenen Hydrogel

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

gradient-strain-chip-for-stimulating-cellular-behaviors-cell-laden

Research

JoVE Journal

Bioengineering

7.8K Aufrufe.  National Tsing Hua University. Dieser Artikel stellt eine einfache Methode zur Bereitstellung von nicht-kontinuierlichen gradienter statische Belastungen auf eine konzentrische Zelle beladenen Hydrogel, Ausrichtung der Zellen für das Tissue Engineering zu regulieren.

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

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Verkapselung von Kardiomyozyten in einer Fibrin-Hydrogel für Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Forschung

Biotechnik

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Cellular Lipidextraktion für Targeted Stable Isotope Dilution Flüssigchromatographie-Massenspektrometrie-Analyse

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Eines mikrofluidischen Chips für ICPMS Probenaufgabe

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Electro Studium der Lungenkrebszellen unter Verwendung eines Mehrkanal-Zwei elektrischen Feldes Mikrofluidik-Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Bau von Modular Hydrogel Blätter für mikrostrukturierten Makro skaliert 3D Cellular Architektur

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

3D Hydrogel Kulturen von primären Gliazellen für In-vitro- Modellierung von Neuroinflammation verbessert

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Gentranskription durch Umleiten zellulären Maschinerie mit chemischen epigenetische Modifikatoren zu unterdrücken

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Herstellung einer Niere Cortex extrazelluläre Matrix abgeleitet Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Eine mikrofluidische Plattform zur Stimulierung von Chondrozyten mit dynamischer Kompression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Pankreasgewebe-abgeleitete extrazelluläre Matrix Bioink für den Druck 3D-Zell-Laden Pankreasgewebe Konstrukte

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...