Questo progetto è stato sostenuto dal Graduate Student studio all'estero programma (NSC-101-2917-I-007-010); il programma di ingegneria biomedica (NSC-101-2221-E-007-032-MY3); e il programma nazionale di nanotecnologia (NSC-101-2120-M-007-001-), Consiglio nazionale della scienza della Repubblica di Cina, Taiwan. Gli autori vorrei ringraziare Prof Ali Khademhosseini, Unal-Gulden Carafa, Arghya Paul e Ronglih Liao presso la Harvard Medical School per la condivisione della tecnologia di incapsulamento di idrogel e cella.

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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In questa carta, segnaliamo su un approccio semplice per confrontare il comportamento di allineamento delle cellule dopo idrogel forma di orientamento e di allungamento a trazione. Una membrana flessibile di PDMS crea una curvatura a forma di cupola per la generazione di diverse altezze di idrogeli circolari concentrici. Dopo aver rilasciato la pressione, la membrana PDMS applica automaticamente la forza per il micro-fantasia idrogeli per formare gradiente ceppo/allungamento, con un massimo al centro e un minimo al confine esterno. Come la formazione del ceppo gradiente è progettata dalla membrana flessibile di PDMS e la gestione del chip fluidico, ci sono diversi parametri importanti che dovrebbero essere seguiti: membrana (i) un controllo preciso dello spessore del PDMS è fondamentale per regolare il valore del gradiente di deformazione. Se la membrana è troppo spessa, anche il volume di iniezione massima del prepolimero cella non sarà in grado di generare una curva convessa corretta nella membrana PDMS per reticolazione un'altezza gradiente di idrogeli carichi di cella. Al contrario, una membrana PDMS troppo sottile non può applicare forza sufficiente nel caso degli idrogeli. Si prega di controllare che il peso di non polimerizzato PDMS nello stampo copertura PDMS è circa 1.6-2.0 g / chip. (ii) la contaminazione prevenzione è molto importante durante la manipolazione di crosslink dell'idrogel di cella-carichi nel chip fluidico. Prima cella coltura nell'incubatrice, accuratamente lavaggio con PBS sterile nel canale fluidico e con 75% etanolo per pulire la superficie del chip può contribuire ad per evitare il problema di contaminazione. (iii) la concentrazione di fotoiniziatore e il dosaggio dell'esposizione UV dovrebbe essere attenti controllata e nella gamma di ~0.1% - 2% (0,5% è raccomandato). Over-reticolazione l'idrogel di iperdosaggio l'irradiazione di ultravioletti comporterà la vitalità cellulare basso. (iv) lo spessore della linea dell'idrogel di fantasia non dovrebbe essere troppo grande. In caso contrario, il tasso di sostituzione dei nutrienti nel caso degli idrogeli spessa non sarà in grado di sostenere la proliferazione delle cellule. Di solito, meno di 300 µm è raccomandato. La spaziatura tra due cerchi di idrogel può essere variata, e un 50% duty cycle è raccomandato. (v) durante il lavaggio o il canale di flusso con soluzione di riempimento, deve essere evitata la formazione di bolle. Pipettaggio delicatamente la soluzione nel chip può contribuire a rimuovere le bolle.
Il concetto di gradiente ceppo generato da PDMS-deforme curvatura può essere ulteriormente aggiornato all'applicazione dinamiche gradiente ceppi e può essere integrato con stimolazione biochimica, che possa beneficiare di molti studi sulla rigenerazione dei tessuti funzionali. Il modulo semplice iniezione fluidico con una spina PDMS può essere sostituito da qualsiasi sistema fluidico avanzato per controllo sperimentale esteso. Lo stampo PMMA può essere sostituito anche da una muffa microfabbricati SU-8 o una muffa del silicone inciso a massa.
Questo chip di gradiente ceppo con un idrogel carichi di cella circolare può generare la forza di compressione statica sull'idrogel 3D senza macchine meccaniche o elettriche esterne. Di conseguenza, fornisce una piattaforma di screening rapido per indagare i comportamenti di cella in una serie di condizioni di sforzo, senza il rischio di problemi di contaminazione causato tramite il funzionamento di macchine esterne. Tuttavia, ceppo temporizzato stimolazione non è realizzabile, perché la membrana PDMS genera ceppi fino la degradazione dell'idrogel.

Che serve come un blocco di materiale che imita un microambiente nativo, un idrogel contenenti matrice extracellulare (ECM) può ri-costruire impalcature tessuto biomimetici per supportare la crescita delle cellule. Per possedere le funzioni di un tessuto, cellula organizzata allineamento è un requisito essenziale. Vari 2D (cioè, cellule coltivate su una superficie) e 3D (ad esempio, le celle incapsulate in un idrogel) gli allineamenti delle cellule sono stati raggiunti da coltura o incapsulamento di cellule in o su substrati flessibili con micro- o nano-pattern1. Allineamento della cella 3D in microarchitettura è più attraente, come il microambiente è più vicino al tessuto nativo costrutto2,3,4. Un approccio comune per l'allineamento di cella 3D è il cue geometrico di idrogel forma2,3. A causa dello spazio limitato per proliferazione delle cellule nella direzione dell'asse corto, celle obiettivo allineare lungo la direzione di assi lunghi in un idrogel di micro-fantasia. Un altro approccio consiste nell'applicare a trazione Allungamento a idrogel per raggiungere celle allineamento parallelo alla direzione stretch4,5.
Stimolazione biofisica su idrogeli di ECM, come deformazione compressiva o un campo elettrico, può regolare le funzioni delle cellule per integrazione tissutale adeguata, proliferazione e differenziazione1,2,3. Gran parte della ricerca è stato fatto per studiare il comportamento cellulare mediante l'applicazione di una condizione di sforzo alla volta utilizzando più controllo meccanico unità4,6,7,8,9. Ad esempio, l'uso di motori passo-passo meccanico spremuto o allungato su un idrogel di collagene 3D incapsulato delle cellule è stato un approccio comune7,10. Tuttavia, tale apparecchiatura di controllo richiede spazio aggiuntivo e affronta il problema della contaminazione nell'incubatore7,9,11,12. Inoltre, lo strumento di grandi dimensioni non può dare un ambiente di controllo preciso di fornire elevata riproducibilità13.
Considerando che carichi di cella idrogeli sono in genere impiegati su micro-scala per applicazioni biomediche, è vantaggioso per combinare le tecniche MEMS per generare una gamma di stimolazione di ceppo/tratto contemporaneamente studiare i comportamenti delle cellule in 3D biomimetici costrutti in vitro2,14,15,16,17,18. Ad esempio, utilizzando la pressione del gas per deformare la membrana PDMS in chip microfluidici possa dar luogo a vari ceppi, guidando la differenziazione delle cellule a diversi lignaggi9,16. Tuttavia, ci sono molte sfide tecniche, quali i processi di fabbricazione di complicati chip in una camera pulita e il software di integrazione di controllo dei motori, pompe, valvole e gas compressi.
In questo lavoro, dimostriamo un approccio semplice per ottenere un chip di gradiente statico-ceppo microfluidici autosufficiente con l'ausilio di un modello di idrogel circolari concentrici e una membrana flessibile di PDMS. Diversamente dalla maggior parte degli approcci esistenti, la nostra piattaforma è un dispositivo portatile e USA e getta in miniatura che può essere fabbricato fuori una camera gialla e che possiede auto-generazione di gradienti ceppi su concentrici idrogeli incapsulato in cella, senza attrezzature meccaniche esterne durante l'incubazione. Comportamenti di cellulare dei fibroblasti 3T3 influenzata da una combinazione di forma idrogel e una varietà di segnali di orientamento elasticizzato trazione sono stati dimostrati durante l'osservazione di allineamento della cella all'interno di ambienti 3D ECM-mimetici nel chip gradiente di deformazione per 3 giorni.

<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

Guida artificiale per allineamento cellulare è un tema caldo nel campo dell'ingegneria tissutale. La maggior parte della ricerca precedente ha studiato singolo allineamento cellulare indotta da sforzo su un idrogel carichi di cella utilizzando processi sperimentali complessi e controllo impianti di massa, che sono associate solitamente con problemi di contaminazione. Così, in questo articolo, vi proponiamo un approccio semplice per creare un gradiente ceppo statico utilizzando un chip fluidico con una copertura di plastica PDMS e un substrato di vetro trasparente UV per la stimolazione del comportamento cellulare in un idrogel 3D. Sovraccarico prepolimero cella foto-derivativazione nella camera di fluidica può generare una membrana PDMS curva convessa sulla copertina. Dopo reticolazione UV, attraverso un micropattern circolare concentrico sotto la membrana PDMS curvilinea e tampone di lavaggio, un microambiente per inquirente cella comportamenti sotto una varietà di ceppi di gradiente è self-consolidata in un singolo chip fluidico, senza strumenti esterni. Le cellule NIH3T3 sono state dimostrate dopo aver osservato il cambiamento di tendenza allineamento cellulare sotto la Guida di geometria, in collaborazione con stimolazione del ceppo, che varia da 15-65% su idrogel. Dopo 3 giorni di incubazione, la geometria di idrogel dominato l'allineamento della cella sotto sforzo di compressione basso, dove le cellule allineati lungo la direzione di allungamento di idrogel sotto alta deformazione compressiva. Tra questi, le cellule hanno mostrato allineamento casuale a causa della dissipazione del Consiglio radicale di idrogel allungamento e la Guida di geometria dell'idrogel di fantasia.

Per confrontare le variazioni meccaniche tra ogni idrogel circolare nel chip stimolazione ceppo gradiente completato, abbiamo misurato la larghezza di riga di ogni idrogel circolare in due i chip stesso, con volumi di iniezione di 0 µ l (Figura 4a) e 40 µ l (Figura 4b), rispettivamente. Il percentuali allungamenti al ogni cerchio sono stati calcolati dividendo gli allungamenti nel chip 40 µ l-iniettato per la larghezza di riga nel caso degli idrogeli corrispondente nel chip 0 µ l-iniettato (Figura 4c). L'idrogel è un materiale incomprimibile, così il ceppo contraente verticale sarà equivalente al ceppo di allungamento laterale. Di conseguenza, il percentuali allungamenti nel chip con il volume di iniezione di 40-µ l Visualizza 15-65% di allungamento, che può essere convertito al ceppo alla compressione (Vedi supplementari File 1).
Macchiatura fluorescente dei nuclei delle cellule e F-actina con DAPI e falloidina, rispettivamente, allo scopo di analizzare l'allineamento cellulare. La colorazione DAPI fornito dati sull'orientamento nucleare, e la colorazione falloidina è stata applicata per valutare la diffusione delle cellule. Figura 5a -c Mostra l'allineamento della cella nel chip gradiente di deformazione. Nella riga 1, le cellule 3T3 allineati lungo la direzione radiale. In idrogel 7, le celle allineate in modo casuale, e cellule allineati lungo la direzione circolare nella riga 12. In conformità con la colorazione immagini di fluorescente, è stato scoperto uno spostamento di 90 ° dall'angolo di allineamento delle cellule delle cellule 3T3 in linea 1 (idrogel massimo allungamento in direzione radiale) per l'angolo di allineamento delle celle con l'allineamento di assi lunghi in linea 12 (minima idrogel allungamento in direzione radiale).
Nella precedente ricerca2,9, cellule a 200 µm con motivi linea idrogeli mirano a allineare lungo la direzione di assi lunghi dell'idrogel. Tuttavia, in questo studio, abbiamo osservato che il tratto allungato l'idrogel di 200 µm nella direzione dell'asse corto fornito un altro fattore per influenzare e dominare l'allineamento cellulare controllando la percentuale del ceppo sull'idrogel. Per il ceppo di 65% sulla linea 1, l'allineamento radicale ha dimostrato che il tratto di allungamento dell'idrogel domina l'allineamento della cella. Per il ceppo di 15% sulla linea 12, l'allineamento circolare ha dimostrato che l'effetto di lungo-asse dominato l'allineamento della cella. Per il 40% di deformazione su idrogel 7, le cellule allineati in modo casuale dovuto la neutralizzazione dell'effetto geometria di orientamento e di sforzo.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/53715/53715fig1.jpg"> 
Figura 1 . PMMA madre stampo per fabbricazione di spina e lastra PDMS. (a) i componenti separati dello stampo PMMA, tra cui una piastra inferiore, frame di contorno e canale di flusso. Dopo il montaggio con nastro biadesivo, (b) la muffa di madre PMMA per il foglio di flusso è formata. (c) stampo un altro PMMA viene assemblato per collegare il PDMS. Il numero rosso rappresenta la profondità. (unità: mm) <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/53715/53715fig2.jpg"> 
Figura 2 . Plastica Photomask Design per il Micropattern di idrogel carichi di Cell. Ci sono due aperture con forme di triangolo (2mm presso la linea di fondo e 6,5 mm di altezza) di collegamento ai canali di flusso per la fornitura di terreno di coltura di cellule fresche. (a) strato di fotoresist plastica è etichettata con la dimensione. Il periodo del cerchio concentrico è di 400 nm e il duty cycle è del 50%. Il diametro del cerchio di centrocampo è 2mm (b) il layout di fotomaschere, senza etichette per stampa laser su una pellicola di plastica trasparente. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/53715/53715fig3.jpg"> 
Nella figura 3. Processi di fabbricazione della sfumatura ceppo idrogel cella-carichi lungo la direzione radiale in un Chip fluidico PDMS. (a) un photomask plastica è allineato e bloccato sotto il chip con un canale fluidico rivestite con TMSPMA. Una micro-siringa con la soluzione di cella prepolimero viene inserita nell'ingresso del chip e usata per iniettare circa 50 µ l per riempire il canale di flusso. (b) l'uscita del canale di flusso è chiuso con il PDMS spina e un ulteriori 40 µ l di soluzione di cella prepolimero viene iniettato. Il fondo di vetro è UV-fantasia per 30 s per fabbricare l'idrogel circolari concentrici nel chip flusso. (c) la pressione del liquido viene rilasciata nel canale di flusso staccando la presa e la miscela di ONU-reticolazione è lavata fuori con DPBS. (d) un chip con ceppo gradiente statico si applica concentrici idrogeli carichi di cellula che sono pronti per la coltura delle cellule. Durante il processo di reticolazione UV, è formata (e) un'altezza di gradiente discontinuo di idrogel lungo il raggio. (f) dopo aver staccato la presa, la membrana PDMS diventa pianeggiante e si applica gradienti stress nel caso degli idrogeli incapsulato in cella. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig3large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/53715/53715fig4.jpg"> 
Nella figura 4. Il gradiente di allungamento di idrogel puro. La larghezza della riga di allungamento e la percentuale di idrogel puro senza incapsulamento cella del gradiente di deformazione chip il giorno 3 con la (a) 0-µ l (gruppo di controllo) e (b) 40-µ l iniezione volumi. (c) la percentuale di allungamento è calcolata dividendo il valore della differenza di larghezza di linea tra 40 µ l e 0 µ l per lo spessore della linea di 40 µ l. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig4large.jpg" target="_blank">per favore clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/53715/53715fig5.jpg"> 
Figura 5 . Immagini di actina-nucleo macchia fluorescente del 3T3 celle incapsulate nei chip gradiente il giorno 3. La direzione di allineamento delle cellule in linea (a-c) 1 (d-f) linea 7 e linea (g-i) 12 rivelare allineamento radiale, casuale e allineamento circolare, rispettivamente. I colori verdi e blu indicano le macchie dell'actina e nucleo, rispettivamente. La linea bianca tratteggiata rappresenta il limite dell'idrogel. Barra della scala: 200 µm. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig5large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
Complementare nella figura 1. Calcolo della curvatura a forma di cupola PDMS. H (x): la curva PDMS convessa; H0: la differenza di altezza massima tra la cupola PDMS prima e dopo la deformazione; r: il raggio della cupola; V: il volume di iniezione eccessiva della regione blu, che causa la deformazione di PDMS come una cupola. Per dettagli, vedere complementare File 1 . <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/Supplementary_Figure_1.tif" target="_blank">Per favore clicca qui per scaricare questa figura.</a>
File supplementari 1. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/Supplementary_File_1-SI.docx" target="_blank">Per favore clicca qui per scaricare questo file.</a>

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

Questo articolo introduce un approccio semplice per fornire ceppi statici gradiente discontinuo su un idrogel di cella-carichi concentrici per regolare l'allineamento di cella per l'ingegneria tissutale.

Gradiente di deformazione Chip per comportamenti stimolanti cellulari in cella-carichi idrogel

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.8K Views.  National Tsing Hua University. Questo articolo introduce un approccio semplice per fornire ceppi statici gradiente discontinuo su un idrogel di cella-carichi concentrici per regolare l'allineamento di cella per l'ingegneria tissutale.

Video: 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.

Incapsulamento di cardiomiociti in un idrogel di fibrina per l&#39;ingegneria tissutale cardiaca

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

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Bioingegneria

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).

Estrazione dei lipidi cellulari per analisi mirate Stabile diluizione isotopica cromatografia liquida-spettrometria di massa

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

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

La coltivazione di cellule progenitrici neurali umane in un 3-dimensionale di auto-assemblaggio Peptide Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

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.

Electrotaxis Studi di polmone cellule tumorali usando un Multicanale Dual-campo elettrico Microfluidic 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.

Costruzione di fogli modulare idrogel per micropatterned macro-scala 3D Cellular Architettura

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.

Migliorata 3D idrogel culture di cellule gliali primari In Vitro modellazione del Neuroinflammation

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.

Reprimere la trascrizione genica reindirizzando macchinario cellulare con modificatori epigenetici chimici

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.

Fabbricando un rene corteccia extracellulare Matrix-derivato idrogel

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.

Una piattaforma microfluidica per stimolare i condrociti con la compressione dinamica

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.

Bioinchiostro a matrice extracellulare derivato dal tessuto pancreatico per la stampa di costrutti di tessuto pancreatico 3D Cell-Laden

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