Bu proje Lisansüstü Öğrenci Çalışma yurtdışında Program (NSC-101-2917-I-007-010 tarafından) destek verdi; Biyomedikal Mühendisliği programı (NSC-101-2221-E-007-032-MY3); ve nanoteknoloji Ulusal Program (NSC-101-2120-M-007-001-), R.O.C., Tayvan Ulusal Bilim Konseyi. Yazarlar Prof. Ali Khademhosseini, gülden Camci-Ünal, Arghya Paul ve Ronglih Liao, Harvard Tıp Okulu hidrojel ve hücre kapsülleme teknoloji paylaşımı için teşekkür etmek istiyorum.

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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Yazarlar ifşa gerek yok.

Bu yazıda, hücre hizalama davranışını hidrojel şekil rehberlik ve gerilme streç sonra karşılaştırmak için basit bir yaklaşım üzerinde rapor. Esnek PDMS membran konsantrik dairesel hydrogels farklı yükseklikte üretmek için bir kubbe seklinde eğrilik oluşturur. Basınç bırakmadan sonra PDMS membran degrade zorlanma/uzama, en merkezi ve en azından dış sınır oluşturmak için mikro-desenli hydrogels için kuvvet otomatik olarak uygular. Degrade zorlanma oluşumu esnek PDMS membran ve sıvı çip işlenmesi tarafından tasarlandığı gibi katıldı bazı önemli parametreler bulunur: (i) PDMS kalınlığı hassas kontrol membran degrade zorlanma değeri ayarlamak önemlidir. Membran çok kalın ise, hücre prepolymer bile en fazla enjeksiyon hacmi PDMS membran çapraz hücre yüklü hydrogels degrade yüksekliğini için uygun bir dışbükey eğri oluşturmak mümkün olmayacaktır. Buna ek olarak, bir çok ince PDMS membran hydrogels için yeterli kuvvet uygulayamazsınız. İyileşmemiş PDMS PDMS kapak kalıp içinde ağırlığı yaklaşık 1.6 2.0 g çipi başına olup olmadığını kontrol edin. (ii) kirlilik önleme sıvı yongası hücre yüklü hidrojel crosslink işlenmesi sırasında çok önemlidir. Kuluçka makinesine kültür, iyice akışkan kanaldaki sterilize PBS ile yıkama ve % 75 kullanarak hücre önce çip yüzeyini silmek için etanol kirlilik sorunu önlemek için yardımcı olabilir. (III) photoinitiator konsantrasyonu ve UV Işınlarına maruz kalma doz dikkatli olmalıdır kontrollü ve aralıktaki (% 0.5 önerilir) ~0.1% - %2. Bitti-crosslinking hidrojel ve UV ışınlama doz aşımı düşük hücre canlılığı içinde sonuçlanacaktır. (iv çizgi genişliğini desenli hidrojel çok büyük olmamalıdır. Aksi takdirde, kalın hydrogels besin değiştirme ücretine hücre çoğalması desteklemek mümkün olmayacaktır. Genellikle, daha az 300 µm önerilir. İki hidrojel daire arasındaki boşluğu değiştirilebilir ve % 50 iş hacmi önerilir. (v) yıkama veya çözüm ile akışı kanal dolumu, kabarcık oluşumu kaçınılmalıdır. Yavaşça çip çözümde pipetting kabarcıklar kaldırmak için yardımcı olabilir.
Degrade zorlanma PDMS deforme eğriliği tarafından oluşturulan dinamik degrade suşları uygulamak için daha fazla yükseltilebilir ve fonksiyonel doku rejenerasyonu üzerine birçok çalışma yararlanabilir biyokimyasal stimülasyon ile entegre. Basit sıvı enjeksiyon modülü bir PDMS fiş ile genişletilmiş deneysel kontrol için herhangi bir gelişmiş sıvı sistemi tarafından değiştirilebilir. PMMA kalıp da bir microfabricated SU-8 kalıp veya bir toplu kazınmış silikon kalıp tarafından değiştirilebilir.
Bu degrade zorlanma çip ile dairesel bir hücre yüklü hidrojel dış mekanik veya elektrikli makineler olmadan 3D hidrojel statik basınç gücü oluşturabilir. Bu nedenle, bu hücre davranışları dış makineleri işlemi tarafından neden olduğu kirlilik sorunları riski olmadan zorlanma koşulları bir dizi araştırma için bir hızlı tarama platformu sağlar. Ancak, PDMS membran suşları hidrojel bozulması kadar oluşturduğu için zaman kontrollü zorlanma stimülasyon ulaşılabilir değildir.

Yerel bir microenvironment taklit eden bir blok malzeme olarak hizmet veren, hücre dışı Matriks (ECM) içeren bir hidrojel yeniden hücre büyümesini desteklemek için biomimetic doku iskele inşa edebilirsiniz. Bir doku fonksiyonları sahip olmak, organize hücre hizalama temel bir gereksinimdir. Çeşitli (Yani, bir yüzeye kültürlü hücreleri) 2D ve 3D (Yani, bir hidrojel kapsüllenmiş hücreleri) kültür veya hücreleri veya mikro esnek yüzeyler üzerinde Kapsüllenen tarafından elde hücre hizalamaları-ya da nano kalıpları1. Microenvironment yerel doku yapısı2,3,4' e daha yakın olduğu gibi 3D hücre hizalama mikro mimarisi içinde daha çekicidir. 3D hücre hizalama için bir ortak yaklaşım hidrojel şekil2,3geometrik işaretimiz. Nedeniyle kısıtlı alan kısa eksenli yönde hücre çoğalması için hücreleri mikro desenli hidrojel uzun ekseni yönünde boyunca Hizala hedefliyoruz. Çekme dayanımı streç hücre hizalama streç yön4,5' e paralel elde etmek için hydrogels uygulamak için başka bir yaklaşımdır.
Biyofiziksel stimülasyon basınç zorlanma gibi ECM hydrogels üzerinde veya bir elektrik alanı, hücre fonksiyonları için uygun doku Tümleştirme, yayılması ve farklılaşma1,2,3düzenleyen. Hücresel davranış birden çok mekanik denetim birimleri4,6,7,8,9kullanarak bir defada bir yük durumu uygulayarak araştırmak için çok araştırma yapılmıştır. Örneğin, mekanik adım motorlar kullanımı sıkılmış veya 3D cep kapsüllenmiş kollajen hidrojel üzerinde gerilmiş ortak bir yaklaşım7,10oldu. Ancak, bu tür kontrol araçlar ilave alanı gerektirir ve kirlenme kuluçka makinesi7,9,11,12sorunu karşı karşıya. Buna ek olarak, büyük araç yüksek tekrarlanabilirlik13sağlamak için bir hassas kontrol ortamı veremem.
Hücre yüklü hydrogels genellikle Biyomedikal uygulamalar için mikro ölçekte istihdam edilmektedir göz önüne alındığında, MEMS teknikleri zorlanma/streç stimülasyon aynı anda 3D biomimetic yapıları vitro2,14,15,16,17,18cep davranışları araştırmak için bir dizi oluşturmak için birleştirmek için avantajlıdır. Örneğin, gaz basıncı PDMS membran mikrosıvısal fiş deforme kullanarak çeşitli suşları, hücre farklılaşması farklı soy9,16' sürüş için çıkmasına neden olabilir. Ancak, karmaşık çip üretim süreçlerinde bir temiz oda ve yazılım denetim Tümleştirme motor, pompa, vanalar ve sıkıştırılmış gazlar gibi pek çok teknik sorun vardır.
Bu çalışmada, biz kendi kendini idame ettiren bir degrade statik yük mikrosıvısal çip bir eş merkezli dairesel hidrojel desen ve esnek bir PDMS membran istihdam ederek elde etmek için basit bir yaklaşım göstermek. Varolan yaklaşımlar en aksine, bizim bu sarı odanın dışında sahte olduğu ve kuluçka sırasında dış mekanik aletler olmadan konsantrik hücre kapsüllü hydrogels üzerinde gradyan suşları kendini üreten sahip bir taşınabilir ve tek kullanımlık minyatür aygıt platformudur. Çekme dayanımı streç rehberlik ipuçları çeşitli degrade zorlanma yongası 3D ECM mimetic ortamlar içinde hücre hizalama 3 gün boyunca gözlem sırasında gösterdi ve 3T3 fibroblast hücre davranışları hidrojel şekil kombinasyonu tarafından etkiledi.

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

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

Mekanik varyasyonları arasında tamamlanan degrade zorlanma stimülasyon yongası dairesel her hidrojel karşılaştırmak için iki enjeksiyon miktarlar 0 µL (şekil 4a) ve 40 µL (şekil 4b) ile aynı fiş dairesel her hidrojel çizgi genişlikleri sırasıyla şiddetindeydi. Her daire, yüzde elongations karşılık gelen hydrogels 0 µL enjekte yongası (şekil 4 c) çizgi genişlikleri 40 µL enjekte yongası elongations bölünerek hesaplanır. Dikey sözleşme zorlanma yanal uzama zorlanma için eşdeğer olur, böylece hidrojel incompressible bir malzeme var. Bu nedenle, basınç zorlanma için dönüştürülebilir 15-%65 uzama yongası 40-µL enjeksiyon hacmi yüzde elongations göster (bkz. ek dosya 1).
Floresan hücre çekirdeği ve F-aktin DAPI ve phalloidin, sırasıyla, boyama hücresel hizalama analiz etmek için yapıldı. Veri üzerinde nükleer yönlendirme sağlanan DAPI boyama ve phalloidin boyama hücre yayılan değerlendirmek için uygulanır. Şekil 5a -c hücre hizalama degrade zorlanma yongasında gösterir. Hat 1, 3T3 hücreler Radyal yönde uyumlu. Hidrojel 7, hücrelerin rasgele hizaladım ve line 12 dairesel yönde boyunca hücreleri uyumlu. Resim Boyama floresan uygun olarak, hat 1 (Radyal yönde en fazla hidrojel uzama) 3T3 hücrelerde hücre hizalamasını açısı 90 ° vardiya uzun eksen hizalama 12 (en düşük fiyat hidrojel uzama Radyal yönde) doğrultusunda hücrelerle hizalama açısı için keşfedilmiştir.
Önceki araştırma2,9' da 200 µm çizgi desenli hydrogels hücrelerde hidrojel uzun eksen yönünü Hizala hedefliyoruz. Ancak, bu çalışmada, kısa-ekseni yönünde 200 µm hydrogels üzerinde uzun streç başka bir faktör etkiler ve hücre hizalama hidrojel üzerinde yük yüzdesini denetleyerek hakim için sağlanan görülmektedir. Hat %1 65 strain için radikal hizalama hidrojel uzama streç hücre hizalama hakim oldu. Line %12 15 strain için dairesel hizalama uzun eksen etkisi hücre hizalama hakim oldu. Hidrojel %7 40 strain için hücreleri rastgele geometri rehberlik ve zorlanma etkisini nötralize nedeniyle hizalı.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/53715/53715fig1.jpg"> 
Resim 1 . PMMA anne kalıp PDMS levha ve fiş imalat için. (a) alt plaka, sınır çerçeve ve akışı kanal gibi PMMA kalıp ayrı bileşenleri. Çift taraflı bant ile derleme sonra (b) PMMA anne kalıp akış tablosu için oluşturulur. (c) PDMS fiş için başka bir PMMA kalıp monte edilir. Kırmızı derinliği temsil eder. (birim: mm) <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/53715/53715fig2.jpg"> 
Resim 2 . Plastik Photomask tasarım için hücre yüklü hidrojel Micropattern. Üçgen şekiller (2 mm alt satırında ve 6.5 mm içinde yükseklik) ile iki açıklıklar vardır taze hücre kültür ortamı sağlamak için akış kanala bağlanma. (a) plastik photomask boyut ile etiketlenir. Eşmerkezli daire 400 dönemidir nm, iş hacmi ise % 50. Orta daireyi çapı 2 mm. olduğunu (b) photomask düzen etiketleri lazer baskı plastik şeffaf film almak için olmadan. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/53715/53715fig3.jpg"> 
Şekil 3. İmalat süreçleri geçişin zorlanma hücre yüklü hidrojel Radyal yönde boyunca PDMS sıvı yongasında. (a) bir plastik photomask uyumlu ve TMSPMA kaplı bir sıvı kanalı ile belgili tanımlık küçük parça altında sıkışmış. Bir mikro şırınga prepolimer hücre çözümü çip giriş eklenen ve akışı kanal doldurmak için yaklaşık 50 µL enjekte için kullanılan. (b) akışı kanal çıkış ile kapalı PDMS takın ve prepolimer hücre çözümü bir ek 40 µL enjekte edilir. UV-30 için desenli cam alt s eş merkezli dairesel hidrojel akışı yongası imal etmek. (c) çıkış çıkararak sıvı basıncı akışı kanalda yayınlandı ve BM-crosslinking karışımı DPBS ile yıkanır. (d) bir çip statik degrade zorlanma ile hücre kültürü çalışmalarının için hazır konsantrik hücre yüklü hydrogels geçerlidir. (E) hidrojel yarıçapı boyunca sürekli olmayan bir degrade yüksekliğini UV crosslinking işlemi sırasında oluşur. (f) sonra çıkış prizden, PDMS membran düz olur ve degrade stres hücre kapsüllü hydrogels geçerlidir. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/53715/53715fig4.jpg"> 
Şekil 4. Saf hidrojel uzama degrade. Uzama çizgi genişliği ve hücre kapsülleme degradedeki olmadan saf hidrojel yüzdesi çipte 3. gün (a) 0-µL ile (kontrol grubu) ve (b) 40-µL enjeksiyon birimleri süzüyorlar. (c) uzama yüzde 40 µL ve 0 µL arasındaki çizgi genişliği fark değerini satır genişliği 40 µL. tarafından bölünerek hesaplanır <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig4large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/53715/53715fig5.jpg"> 
Şekil 5 . Floresan aktin-çekirdek leke görüntülerini 3T3 hücreleri Encapsulated degrade fiş gün 3. (A-c) çizgi 1, (d-f) satır 7 ve (g-ı) line 12 hücre hizalama yönde ortaya Radyal hizalama, rasgele hizalama ve dairesel hizalama, anılan sıraya göre. Yeşil ve mavi renkler aktin ve çekirdek lekeleri, sırasıyla gösterir. Noktalı beyaz çizgi hidrojel sınırlarını temsil eder. Ölçek çubuğu: 200 µm. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/53715fig5large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
Ek resim 1. Hesaplama kubbe seklinde PDMS kavisi. F: dışbükey PDMS eğrisi; H0: öncesi ve sonrası deformasyon PDMS kubbe arasındaki maksimum yükseklik farkı; r: kubbe yarıçapı; V: aşırı enjeksiyon hacmi bir kubbe gibi PDMS deformasyona neden olur mavi bölge. Ayrıntılı bilgi için bkz: ek dosya 1 . <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/Supplementary_Figure_1.tif" target="_blank">Bu rakam indirmek için buraya tıklayınız.</a>
Ek dosya 1. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53715/Supplementary_File_1-SI.docx" target="_blank">Bu dosyayı indirmek için buraya tıklayınız.</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

Bu makalede sürekli sigara degrade statik suşlar doku mühendisliği için hücre hizalama düzenleyen konsantrik bir hücre yüklü hidrojel Tarih sağlamak için basit bir yaklaşım sunar.

Hücre yüklü hidrojel uyarıcı hücresel davranışları için degrade zorlanma çip

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

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JoVE Journal

Bioengineering

7.8K Görüntüleme.  National Tsing Hua University. Bu makalede sürekli sigara degrade statik suşlar doku mühendisliği için hücre hizalama düzenleyen konsantrik bir hücre yüklü hidrojel Tarih sağlamak için basit bir yaklaşım sunar.

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.

Kalp Doku Mühendisliği Fibrin Hidrojel kardiyomiyositlerinin Kapsülleme

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

Biyomühendislik

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).

Hücresel Lipid Ekstraksiyon Hedefli Kararlı İzotop Seyreltme Sıvı Kromatografi-Kütle Spektrometresi Analizi

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.

ICPMS Numune Giriş için mikroakışkan Chip

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.

Bir Çok Kanallı Çift elektrik alan mikroakışkan Chip kullanarak Akciğer Kanseri Hücreleri Electrotaxis Çalışmaları

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.

Micropatterned Makro ölçekli 3D Hücresel Mimarlık Modüler Hidrojel Levhaların İnşaatı

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.

Geliştirilmiş 3D hidrojel Neuroinflammation Vitro modelleme için birincil gliyal hücreler kültürleri

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.

Kimyasal epigenetik değiştiriciler ile hücresel makine yeniden yönlendirerek Gen transkripsiyonu bastırmak

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.

Bir böbrek korteks ekstraselüler matrisi elde edilen hidrojel imalatı

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.

Dinamik Sıkıştırma ile Chondrocytes Uyarıcı için Bir Mikroakışkan Platform

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.

3D Hücre Yüklü Pankreas Dokusu Yapıları Baskı için Pankreas Doku Türetilmiş Ekstrasellüler Matrix Bioink

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