Este projecto foi apoiado pela graduação aluno estudo no exterior programa (NSC-101-2917-I-007-010); o programa de Engenharia Biomédica (NSC-101-2221-E-007-032-MY3); e o programa nacional de nanotecnologia (NSC-101-2120-M-007-001-), Conselho Nacional de ciência, do R.O.C, Taiwan. Os autores gostaria de agradecer a Prof Ali Khademhosseini, Gulden Camci-Unal, Arghya Paul e Ronglih Liao na Harvard Medical School para compartilhar a tecnologia de encapsulamento de hidrogel e célula.

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>

<ol>
	<li>Simmons, C. S., Petzold, B. C., Pruitt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsystems+for+biomimetic+stimulation+of+cardiac+cells.">Microsystems for biomimetic stimulation of cardiac cells.</a> Lab Chip. 12 (18), 3235-3248 (2012).</li><li>Aubin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+3D+cell+alignment+and+elongation+in+microengineered+hydrogels.">Directed 3D cell alignment and elongation in microengineered hydrogels.</a> Biomaterials. 31 (27), 6941-6951 (2010).</li><li>Guan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stimulation+of+the+cardiac+differentiation+of+mesenchymal+stem+cells+in+tissue+constructs+that+mimic+myocardium+structure+and+biomechanics.">The stimulation of the cardiac differentiation of mesenchymal stem cells in tissue constructs that mimic myocardium structure and biomechanics.</a> Biomaterials. 32 (24), 5568-5580 (2011).</li><li>Wan, C. R., Chung, S., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+into+cardiomyocytes+in+a+compliant+microfluidic+system.">Differentiation of embryonic stem cells into cardiomyocytes in a compliant microfluidic system.</a> Ann Biomed Eng. 39 (6), 1840-1847 (2011).</li><li>Huh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+organ-level+lung+functions+on+a+chip.">Reconstituting organ-level lung functions on a chip.</a> Science. 328 (5986), 1662-1668 (2010).</li><li>Li, X., Chu, J. S., Yang, L., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+effects+of+mechanical+strain+on+neural+crest+stem+cells.">Anisotropic effects of mechanical strain on neural crest stem cells.</a> Ann. Biomed. Eng. 40 (3), 598-605 (2012).</li><li>Butcher, J. T., Barrett, B. C., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equibiaxial+strain+stimulates+fibroblastic+phenotype+shift+in+smooth+muscle+cells+in+an+engineered+tissue+model+of+the+aortic+wall.">Equibiaxial strain stimulates fibroblastic phenotype shift in smooth muscle cells in an engineered tissue model of the aortic wall.</a> Biomaterials. 27 (30), 5252-5258 (2006).</li><li>Ramon-Azcon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin+methacrylate+as+a+promising+hydrogel+for+3D+microscale+organization+and+proliferation+of+dielectrophoretically+patterned+cells.">Gelatin methacrylate as a promising hydrogel for 3D microscale organization and proliferation of dielectrophoretically patterned cells.</a> Lab Chip. 12 (16), 2959-2969 (2012).</li><li>Park, S. H., Sim, W. Y., Min, B. H., Yang, S. S., Khademhosseini, A., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chip-Based+Comparison+of+the+Osteogenesis+of+Human+Bone+Marrow-+and+Adipose+Tissue-Derived+Mesenchymal+Stem+Cells+under+Mechanical+Stimulation.">Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation.</a> PLoS One. 7 (9), e46689 (2012).</li><li>Gould, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+Strain+Anisotropy+Regulates+Valvular+Interstitial+Cell+Phenotype+and+Tissue+Remodeling+in+3D+Culture.">Cyclic Strain Anisotropy Regulates Valvular Interstitial Cell Phenotype and Tissue Remodeling in 3D Culture.</a> Acta Biomater. 8 (5), 1710-1719 (2012).</li><li>Kurpinski, K., Chu, J., Hashi, C., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proc+Anisotropic+mechanosensing+by+mesenchymal+stemcells.">Proc Anisotropic mechanosensing by mesenchymal stemcells.</a> Natl Acad Sci USA. 103 (44), 16095-16100 (2006).</li><li>Sim, W. Y., Park, S. W., Park, S. H., Min, B. H., Park, S. R., Yang, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatic+micro+cell+chip+for+the+differentiation+of+human+mesenchymal+stem+cells+under+mechanical+stimulation.">A pneumatic micro cell chip for the differentiation of human mesenchymal stem cells under mechanical stimulation.</a> Lab Chip. 7 (12), 1775-1782 (2007).</li><li>Vader, D., Kabla, A., Weitz, D., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-Induced+Alignment+in+Collagen+Gels.">Strain-Induced Alignment in Collagen Gels.</a> PLoS One. 4 (6), e5902 (2009).</li><li>Aguado, B. A., Mulyasasmita, W., Su, J., Lampe, K. J., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+viability+of+stem+cells+during+syringe+needle+flow+through+the+design+of+hydrogel+cell+carriers.">Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers.</a> Tissue Eng Part A. 18 (7-8), 806-815 (2012).</li><li>Wan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic-Based+Synthesis+of+Hydrogel+Particles+for+Cell+Microencapsulation+and+Cell-Based+Drug+Delivery.">Microfluidic-Based Synthesis of Hydrogel Particles for Cell Microencapsulation and Cell-Based Drug Delivery.</a> Polymers. 4 (2), 1084-1108 (2012).</li><li>Moraes, C., Wang, G., Sun, Y., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfabricated+platform+for+high-throughput+unconfined+compression+of+micropatterned+biomaterial+arrays.">A microfabricated platform for high-throughput unconfined compression of micropatterned biomaterial arrays.</a> Biomaterials. 31 (3), 577-584 (2010).</li><li>Keung, A. J., Kumar, S., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presentation+Counts:+Microenvironmental+Regulation+of+Stem+Cells+by+Biophysical+and+Material.+Cues.">Presentation Counts: Microenvironmental Regulation of Stem Cells by Biophysical and Material. Cues.</a> Annu Rev Cell Dev Biol. 26, 533-556 (2010).</li><li>Segers, V. F., Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-cell+therapy+for+cardiac+disease.">Stem-cell therapy for cardiac disease.</a> Nature. 451 (7181), 937-942 (2008).</li><li>Hsieh, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient+static-strain+stimulation+in+a+microfluidic+chip+for+3D+cellular+alignment.">Gradient static-strain stimulation in a microfluidic chip for 3D cellular alignment.</a> Lab Chip. 14 (3), 482-493 (2014).</li></ol>

Os autores não têm nada para divulgar.

Neste trabalho, relatamos sobre uma abordagem simples para comparar o comportamento de alinhamento de célula depois de hidrogel forma de orientação e de estiramento elástico. Uma membrana flexível de PDMS cria uma curvatura em forma de cúpula para a geração de diferentes alturas de hidrogel circulares concêntricos. Após liberar a pressão, a membrana PDMS aplica automaticamente a força para a micromodelados de hidrogel para formar o gradiente de tensão/alongamento, com um máximo no centro e um mínimo no lim...

Servindo como um material do bloco que imita um microambiente nativo, um hidrogel contendo a matriz extracelular (ECM) pode re-construir andaimes de tecido biomimético para apoiar o crescimento de células. Para possuir as funções de um tecido, o alinhamento da célula organizada é um requisito essencial. Vários 2D (isto é, células cultivadas sobre uma superfície) e 3D (i.e., células encapsuladas em um hidrogel) alinhamentos de células que foram alcançados pelo cultivo ou encapsular células em ou em substratos flexíveis com micro- ou nano-padrões1. Alinhamento da célula 3D na microarquitetura é mais atraente, como o microambiente aproxima-se do tecido nativo construção2,3,4. Uma abordagem comum para alinhamento de célula 3D é o cue geométrico de hidrogel forma2,3. Por causa do espaço restrito para proliferação celular na direção do eixo curto, células visam alinhar ao longo da direção do eixo longo em um hidrogel micromodelados. Outra abordagem é aplicar o estiramento elástico para o hidrogel para atingir o alinhamento da célula paralelo à direção de estiramento4,5.
Estimulação biofísica em hidrogel de ECM, tais como tensão compressiva ou um campo elétrico, pode regular funções de células de tecido adequada integração, proliferação e diferenciação1,2,3. Muita pesquisa tem sido feita para investigar o comportamento celular através da aplicação de uma condição de tensão de cada vez usando múltiplo controle mecânico unidades4,6,7,8,9. Por exemplo, o uso de motores de passo mecânico espremido ou esticada sobre um hidrogel de colágeno de célula encapsulada 3D tem sido comum uma abordagem7,10. No entanto, tal equipamento controlador requer espaço extra e enfrenta a questão da contaminação na incubadora7,9,11,12. Além disso, o grande instrumento não pode dar um ambiente de controle preciso para fornecer grande reprodutibilidade13.
Considerando que o hidrogel célula-carregado é normalmente utilizadas na microescala para aplicações biomédicas, é vantajoso combinar técnicas de MEMS para gerar uma gama de tensão/estiramento estimulação para investigar simultaneamente comportamentos de célula em 3D biomimetic construções em vitro2,14,15,16,17,18. Por exemplo, usando a pressão do gás a deformar-se a membrana PDMS em chips microfluídicos pode dar origem a várias cepas, dirigindo a diferenciação celular de linhagens diferentes9,16. No entanto, existem muitos desafios técnicos, tais como processos de fabricação de chip complicado em um quarto limpo e a integração de software controle de motores, bombas, válvulas e gases comprimidos.
Neste trabalho, vamos demonstrar uma abordagem simples para obter um chip de gradiente estático-estirpe microfluidic auto-sustentável, empregando-se um padrão de hidrogel circulares concêntricos e uma membrana flexível de PDMS. Ao contrário da maioria das abordagens existentes, a nossa plataforma é um dispositivo portátil e descartável em miniatura que podem ser fabricados fora um quarto amarelo e que possui a auto-geração de cepas gradientes em hidrogel concêntricos de célula encapsulada, sem equipamentos mecânicos externos durante a incubação. Comportamentos de células de fibroblastos 3T3 influenciaram por uma combinação de hidrogel forma e uma variedade de pistas de orientação estiramento elástico foram demonstradas durante a observação de alinhamento de célula dentro de ambientes 3D de ECM-mimética no chip gradiente de tensão para 3 dias.

<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

Orientação artificial para alinhamento celular é um tema quente no campo da engenharia de tecidos. A maioria da pesquisa anterior investigou único alinhamento celular induzida por tensão em um hidrogel célula carregada por meio de complexos processos experimentais e massa em sistemas de controlo, que são normalmente associados com problemas de contaminação. Assim, neste artigo, propomos uma abordagem simples para a construção de uma estirpe de estática gradiente usando um chip fluídico com uma tampa plástica de PDMS e um substrato de vidro transparente de UV para a estimulação do comportamento celular em um hidrogel 3D. Pré-polímero de célula foto-patternable sobrecarga na câmara fluídico pode gerar uma membrana PDMS curva convexa na capa. Após a reticulação de UV, através de um micropattern circular concêntrico sob a membrana PDMS curvada e tampão de lavagem, um microambiente para investigar célula comportamentos sob uma variedade de cepas de gradientes é self estabelecida em um único chip fluídico, sem instrumentos externos. NIH3T3 células foram demonstradas após observar a mudança da tendência de alinhamento celular sob orientação de geometria, em cooperação com a estimulação de estirpe, que variou entre 15-65% no hidrogel. Após uma incubação de 3 dias, a geometria de hidrogel dominou o alinhamento da célula sob baixa tensão compressiva, onde células alinhadas ao longo da direção de alongamento de hidrogel sob alta tensão compressiva. Entre estas, as células mostraram alinhamento aleatório devido a dissipação da orientação radical de alongamento de hidrogel e a orientação de geometria do hidrogel estampado.

Para comparar as variações mecânicas entre cada hidrogel circular no chip estimulação concluído gradiente de tensão, medimos as larguras de linha de cada hidrogel circular em dois dos chips mesmos, com volumes de injeção de 0 µ l (figura 4a) e 40 µ l (figura 4b), respectivamente. Os alongamentos por cento em cada círculo foram calculados dividindo os alongamentos no chip 40 µ l-injetado pelas larguras de linha do hid...

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

Este artigo apresenta uma abordagem simples para fornecer não-contínuo gradientes cepas estáticas em um hidrogel de célula-carregado concêntrico para regular o alinhamento da célula para a engenharia de tecidos.

Gradiente de pressão Chip para estimulantes comportamentos celulares em hidrogel de célula-carregado

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.8K visualizações.  National Tsing Hua University. Este artigo apresenta uma abordagem simples para fornecer não-contínuo gradientes cepas estáticas em um hidrogel de célula-carregado concêntrico para regular o alinhamento da célula para a engenharia de tecidos.

Vídeo: 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.

Encapsulamento de Cardiomyocytes em um hidrogel de fibrina para engenharia de tecidos cardíacos

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

Bioengenharia

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).

Extração de lipídeos celulares para Targeted diluição de isótopos estáveis ​​Líquido Análise Espectrometria de Massa-Cromatografia

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.

Um chip microfluídico para ICPMS Amostra Introdução

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.

Electrotaxis Estudos de pulmão células de câncer usando um Multichannel Dual-elétrico-campo 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.

Construção de Fichas de hidrogel modular para micropadronadas Macro-escalado 3D Cellular Arquitetura

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.

Melhorado o hidrogel 3D culturas de células gliais primárias para In Vitro modelagem de 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.

Reprimir a transcrição do Gene redirecionando maquinaria celular com modificadores epigenéticas químicas

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.

Fabricando um rim córtex Extracellular Matrix-derivado hidrogel

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.

Uma plataforma Microfluídico para estimular condrócitos com compressão dinâmica

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.

Bioink extracelular derivado da matriz de tecido sipâncreas para a impressão de construções de tecidopanpático 3D Carregadas de células

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

Gradiente de pressão Chip para estimulantes comportamentos celulares em hidrogel de célula-carregado

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Electrotaxis Estudos de pulmão células de câncer usando um Multichannel Dual-elétrico-campo Microfluidic Chip

Construção de Fichas de hidrogel modular para micropadronadas Macro-escalado 3D Cellular Arquitetura

Melhorado o hidrogel 3D culturas de células gliais primárias para In Vitro modelagem de Neuroinflammation

Reprimir a transcrição do Gene redirecionando maquinaria celular com modificadores epigenéticas químicas

Fabricando um rim córtex Extracellular Matrix-derivado hidrogel

Uma plataforma Microfluídico para estimular condrócitos com compressão dinâmica

Bioink extracelular derivado da matriz de tecido sipâncreas para a impressão de construções de tecidopanpático 3D Carregadas de células

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Capítulos neste vídeo

Encapsulamento de Cardiomyocytes em um hidrogel de fibrina para engenharia de tecidos cardíacos

Extração de lipídeos celulares para Targeted diluição de isótopos estáveis ​​Líquido Análise Espectrometria de Massa-Cromatografia

Um chip microfluídico para ICPMS Amostra Introdução

Electrotaxis Estudos de pulmão células de câncer usando um Multichannel Dual-elétrico-campo Microfluidic Chip

Construção de Fichas de hidrogel modular para micropadronadas Macro-escalado 3D Cellular Arquitetura

Melhorado o hidrogel 3D culturas de células gliais primárias para In Vitro modelagem de Neuroinflammation

Reprimir a transcrição do Gene redirecionando maquinaria celular com modificadores epigenéticas químicas

Fabricando um rim córtex Extracellular Matrix-derivado hidrogel

Uma plataforma Microfluídico para estimular condrócitos com compressão dinâmica

Bioink extracelular derivado da matriz de tecido sipâncreas para a impressão de construções de tecidopanpático 3D Carregadas de células

Extração de lipídeos celulares para Targeted diluição de isótopos estáveis Líquido Análise Espectrometria de Massa-Cromatografia