Name: gradient strain chip for stimulating cellular behaviors in cell-laden hydrogel
Uploaded: 2017-08-08T12:30:00.000+00:00
Duration: 13 min 28 s
Description: Watch this Scientific Journal Video about Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel at JoVE.com

이 프로젝트 지원으로 대학원 학생 연구 해외 프로그램 (NSC-101-2917-I-007-010); 공학 프로그램 (NSC-101-2221-E-007-032-MY3); 그리고 나노기술 국가 프로그램 (NSC-101-2120-M-007-001-), R.O.C., 대만의 국가 과학 위원회. 저자는 교수 알리 Khademhosseini, 네델란드의 화 Camci-Unal, 대단하다 폴, 및 하버드의과 대학에서 Ronglih 리아 하이드로 겔 및 셀 캡슐화 기술을 공유 하기 위한 감사 하 고 싶습니다.

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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저자는 공개 없다.

이 논문에서 우리 히드로 모양 지도 인장 스트레치 후 셀 정렬 동작을 비교 하는 간단한 방법에 보고 합니다. 유연한 PDMS 막 동심 원형 hydrogels의 다른 고도 생성 하기 위한 돔 모양의 곡률을 만듭니다. 압력을 해제 한 후 PDMS 막 자동으로 그라데이션 변형/신장, 센터에서 최대와 최소 바깥 경계에 형성에 마이크로 패턴 hydrogels에 강제 적용 됩니다. 에 참석 해야 합니다 몇 가지 중요 한 매개 변수는 그라데이션 긴장의 형성 유연한 PDMS 막과 유체 칩의 처리에 의해 설계: (i)는 PDMS의 두께의 정확한 제어 막이 그라데이션 변형 값을 조정 하는 중요 한. 막 너무 두꺼운 경우 셀 prepolymer의 최대 사출 볼륨도 가교 셀 라덴 hydrogels의 그라데이션 높이 대 한 PDMS 막에서 적절 한 볼록한 곡선을 생성할 수 없습니다. 반면, 너무 얇은 PDMS 막은 hydrogels에 충분 한 힘을 적용할 수 없습니다. PDMS 커버 금형에서 uncured PDMS의 무게는 칩 당 약 1.6-2.0 g을 확인 하시기 바랍니다. (2) 오염 방지 유체 칩에 셀-라덴 히드로 crosslink 처리 하는 동안 매우 중요 하다. 인큐베이터에서 배양, 철저 하 게 유체 채널에서 소독된 PBS로 세척 하 고 75%를 사용 하 여 셀 전에 에탄올 칩의 표면을 닦아 오염 문제를 피하기 위해 도울 수 있다. (iii) photoinitiator의 농도 UV 노출의 복용량은 조심 한다 통제 및 범위 ~0.1%-2% (0.5% 권장). 오버-가교는 하이드로 겔 고 UV 방사선 도달 했 나 봐 낮은 세포 생존 능력에 발생 합니다. (패턴화 된 하이드로 겔의 선 폭 iv)는 너무 커서 해서는 안됩니다. 그렇지 않으면, 두꺼운 hydrogels에 영양 보충 속도 지원 세포 증식 수 없습니다. 일반적으로, 보다 적은 300 µ m 것이 좋습니다. 두 개의 히드로 동그라미 사이의 간격, 다양 하 고 50% 듀티 사이클 것이 좋습니다. (v) 동안 세척 또는 채우는 솔루션 흐름 채널, 거품 형성을 피해 야 한다. 부드럽게 칩에서 솔루션 pipetting 거품을 제거 시킬 수 있습니다.
그라데이션 변형 PDMS 변형 곡률에 의해 생성 된의 개념 더 업그레이드할 수 동적 그라데이션 긴장을 적용 하 고 기능 조직 재생에 대 한 많은 연구에 혜택을 받을 수 있는 생 화 확 적인 자극으로 통합 될 수 있다. PDMS 플러그와 간단한 유체 주입 모듈 확장된 실험 제어에 대 한 어떤 고급 유체 시스템으로 교체할 수 있습니다. PMMA 금형 ﹙ SU-8 형 또는 대량 에칭 실리콘 몰드로 교체할 수 있습니다.
원형 셀-라덴 히드로와 그라데이션 변형 칩이 외부 기계 또는 전기 기계 없이 3D 히드로에 정적 압축 힘을 생성할 수 있습니다. 따라서, 그것은 일련의 외부 컴퓨터의 작동에 의해 발생 하는 오염 문제의 위험 없이 변형 조건에 셀 행동 조사에 대 한 빠른 심사 플랫폼을 제공 합니다. 그러나 PDMS 막은 히드로의 저하까지 긴장을 생성 하기 때문에, 시간 제어 긴장 자극 달성 되지 않습니다.

네이티브 microenvironment을 블록 자료로 제공, 세포 외 기질 (ECM)를 포함 하는 하이드로 겔 세포 성장을 지원 하기 위해 생체 모방 조직 건설 기계를 다시 구축할 수 있습니다. 조직 기능을 소유 하기 위하여 조직 된 셀 정렬 필수 요건 이다. 다양 한 2D (즉, 세포 표면에 경작) 및 차원 (즉, 셀에는 히드로) 셀 정렬 배양 하거나 캡슐화 셀 또는 마이크로 유연한 기판에 의해 달성 되었습니다-또는 나노 패턴1. 3 차원 셀 맞춤 마이크로아키텍처에는 microenvironment는 기본 조직 구조2,,34에 가까운 더 매력적 이다. 1 개의 일반적인 방법은 3D 셀 맞춤 히드로 모양2,3의 기하학적 큐입니다. 짧은 축 방향으로 셀 확산에 대 한 제한 된 공간 때문에 셀 마이크로 패턴 히드로에서 긴 축 방향을 따라 정렬 하고자 합니다. 또 다른 방법은 셀 맞춤 스트레칭 방향4,5에 평행을 달성 하기 위해 hydrogels 인장 스트레치를 적용할 것입니다.
압축 변형 등 ECM hydrogels에 생물 자극 또는 전기 필드를 적절 한 조직 통합, 확산 및 감 별 법1,2,3에 대 한 셀 함수를 조절할 수 있습니다. 많은 연구가 여러 기계적 제어 단위4,6,7,,89를 사용 하 여 한 번에 하나의 긴장 상태를 적용 하 여 세포 행동을 조사 하는 일 하고있다. 예를 들어 기계적 단계 모터를 사용 하 여 압착 또는 공통 접근7,10되었습니다 3D 셀 캡슐화 콜라겐 하이드로 겔에 뻗어. 그러나, 이러한 제어 장비 추가 공간이 필요 하 고 인큐베이터7,9,,1112에 오염 문제를 직면. 또한, 큰 악기는 정밀 하 게 제어 환경을 제공 하는 높은 재현성13을 드릴 수 없습니다.
셀-라덴 hydrogels 생물 의학 응용 프로그램에 대 한 마이크로 스케일에 일반적으로 고용 되어 고려 하 고, 동시에 3D biomimetic 구문을 생체 외에서2,14,15,,1617,18에 셀 행동을 조사 하기 위해 스트레인/스트레칭 자극의 범위를 생성 하는 MEMS 기술을 결합 하 여 유리 하다. 예를 들어 다른 계보9,16에 세포 분화를 유도 하는 다양 한 긴장을 증가 줄 수 있습니다 가스 압력을 사용 하 여 미세 칩에서 PDMS 막 변형. 그러나, 복잡 한 칩 제조 프로세스 클린 룸 및 모터, 펌프, 밸브, 압축된 가스의 소프트웨어 제어 통합 등 많은 기술적인 도전이 있다.
이 작품에서 동심 원형 히드로 패턴 그리고 유연한 PDMS 멤브레인을 채용 하 여 자립 그라데이션 정적 스트레인 미세 칩을 얻기 위해 간단한 방법을 보여 줍니다. 기존 접근법의 대부분과 달리 우리의 플랫폼은 휴대용 및 처분할 수 있는 소형 장치 그 자체는 인큐베이션 기간 동안 외부 기계 장비 없이 동심의 세포 캡슐화 hydrogels에 그라데이션 긴장 생성 보유 하 고 있는 노란 방 밖에 서 날조 될 수 있다. 3T3 fibroblast 세포 행동 히드로 모양의 조합에 의해 영향을 하 고 인장 스트레칭 지도 신호의 다양 한 일 동안 그라데이션 변형 칩에서 3D ECM 모방 환경에서 셀 정렬의 관찰 동안 시연 했다.

<table><tbody><tr><td>1.5 mL black microcentrifuge tube</td><td>Argos Technologies&amp;nbsp;</td><td>03-391-161</td><td>This one can be replaced with a neutral color of 1.5 mL tube covered with aluminun foil</td></tr><tr><td>10x DPBS</td><td>Sigma-Aldrich</td><td>56064C</td><td></td></tr><tr><td>Alexa Fluor 488 phalloidin&amp;nbsp;</td><td>Invitrogen</td><td>A12379&amp;nbsp;</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A1595</td><td></td></tr><tr><td>Calcein</td><td>Molecular Probe</td><td>C1430</td><td>For labeling viable cells</td></tr><tr><td>CCD</td><td>PCO. Imaging</td><td>Pixelfly qe</td><td></td></tr><tr><td>Cell membrane permeating solution</td><td>Sigma-Aldrich</td><td>X100</td><td>0.5% Triton X-100 for permeating cell membrane</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D8417</td><td>Cell nucleus staining</td></tr><tr><td>Dialysis membrane</td><td>Sigma-Aldrich</td><td>D9527</td><td>Molecular weight cut-off = 14,000</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>Double-side tape</td><td>3M</td><td>8003</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30071.03</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G2500</td><td>gel strength 300, type A, from porcine skin</td></tr><tr><td>High frequency electronic corona generator</td><td>Electro-technic products</td><td>MODEL BD-20</td><td></td></tr><tr><td>Methacrylic Anhydride</td><td>Sigma-Aldrich</td><td>276685</td><td></td></tr><tr><td>Micro syringe</td><td>Hamilton</td><td>80501</td><td>50 &amp;mu;L&amp;nbsp;</td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX71</td><td>Include two filter sets: LF405/LP-B-000 and LF488/LP-C-000 from Semrock</td></tr><tr><td>Oxygen plasma machine</td><td>Harrick plasma</td><td>PDC-001</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>For fixing cell</td></tr><tr><td>PDMS</td><td>DOW CORNING</td><td>Sylgard 184</td><td>Mixture for PDMS chip cast-molding fabrication</td></tr><tr><td>Pen-Strep</td><td>Gibco</td><td>10378-016</td><td>penicillin/streptomycin</td></tr><tr><td>Photoinitiator</td><td>CIBA</td><td>Irgacure 2959</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>P4170</td><td>For labeling dead cells</td></tr><tr><td>Sterile Filtration cup</td><td>Millipore</td><td>SCGPT05RE</td><td></td></tr><tr><td>TMSPMA</td><td>Sigma-Aldrich</td><td>440159</td><td>For hydrogel immobilization</td></tr><tr><td>Ultrasonicator</td><td>Delta</td><td>D150H</td><td>150W, 43kHz</td></tr><tr><td>UV light</td><td>DAIHAN</td><td>WUV-L10</td><td></td></tr><tr><td>Freeze Dryer</td><td>FIRSTEK</td><td>150311025</td><td></td></tr><tr><td>NIH3T3(fibroblast)</td><td>Food Industry Research and Development Institute(FIRDI)</td><td>08C0011</td><td></td></tr><tr><td>MOXI Z Mini Automated Cell Counter</td><td>ORFLO</td><td>MXZ001</td><td></td></tr></tbody></table>

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

세포 정렬에 대 한 인공 지침 조직 공학의 분야에서 뜨거운 주제입니다. 이전 연구의 대부분 복잡 한 실험 프로세스 및 대량 시스템 제어는 일반적으로 관련 된 오염 문제를 사용 하 여 셀 라덴 히드로에 단일 스트레인 유발 세포 맞춤을 조사 했다. 따라서,이 문서에서 3 차원 하이드로 겔에 세포 행동의 자극에 대 한 플라스틱 PDMS 커버와 UV 투명 유리 기판 유체 칩을 사용 하 여 그라데이션 정적 스트레인을 구축 하는 간단한 방법 제안 한다. 유체 챔버에 오버 로딩 사진 patternable 셀 prepolymer 표지에 볼록한 곡선된 PDMS 멤브레인을 생성할 수 있습니다. 자외선 가교 후 곡선된 PDMS 막 및 버퍼, 조사 셀 microenvironment 동심 원형 micropattern를 통해 다양 한 그라데이션 긴장 아래 행동은 외부 기기 없이 단일 유체 칩에 자체 설립. NIH3T3 세포 형상 지도 아래, 협력 hydrogels 15-65%에서 다양 한 긴장 자극 세포 맞춤 트렌드의 변화를 관찰 한 후 증명 했다. 3 일 배양 후 히드로 형상 낮은 압축 변형, 셀 정렬 셀 높은 압축 변형 히드로 신장 방향을 따라 정렬 지배. 이들, 사이 셀 하이드로 겔 신장 패턴화 된 하이드로 겔의 형상 지도의 급진적인 지침의 소산 인 임의의 맞춤을 보였다.

완성 된 그라데이션 변형 자극 칩에서 각 원형 히드로 사이 기계적인 유사 비교를 우리가 주입 양의 0 µ L (그림 4a)와 40 µ L (그림 4b), 같은 칩의 두 각 원형 하이드로 겔의 선 폭 각각 측정. 각 원형에 백분율 elongations 0 µ L 주입 칩 (그림 4c)에서 해당 hydrogels의 선 폭 40 µ L 주입 칩에서 elongations 나누어 계산 했다. 히드로 비압축 소재 이므로 수직 계약 부담 측면 신장 긴장에 해당 될 것입니다. 따라서, 40 µ L 주입 량과 칩에 백분율 elongations 15-65% 신장, 압축 변형으로 변환 될 수 있는 표시 ( 보충 파일 1참조).
각각, 세포 핵 및 F-말라 DAPI와 phalloidin, 형광 얼룩 세포 맞춤 분석 이루어졌다. 핵 방향에 데이터를 제공 하는 DAPI 얼룩 그리고 셀 확산을 평가에 적용 된 phalloidin 얼룩. 그림 5a -c 그라데이션 변형 칩에서 셀 맞춤을 보여 줍니다. 1 호선, 3T3 세포 반지름 방향에 따라 정렬. 하이드로 겔 7에 셀 무작위로, 정렬 하 고 셀 라인 12 원형 방향을 따라 정렬 합니다. 이미지를 얼룩이 지기 형광에 선 12 (최저 히드로 신장 레이디얼 방향에서)에서 긴 축 정렬 셀의 정렬 각도를 줄 1 (반지름 방향에서 최대 히드로 신장) 3T3 세포의 셀 정렬의 각도에서 90 ° 변화 발견 되었다.
이전 연구2,9, 셀 라인 꽃무늬 hydrogels 200 µ m에에서는 히드로의 긴 축 방향을 따라 정렬 하고자 합니다. 그러나,이 연구에서 우리 짧은 축 방향으로 200 µ m hydrogels에 길쭉한 스트레칭 또 다른 요소에 영향을 하 고는 히드로에 긴장의 비율을 조절 하 여 세포 정렬 지배 제공 관찰. 라인 1에 65% 부담에 대 한 급진적인 맞춤은 히드로의 신장 스트레치 셀 정렬 지배 입증 했다. 15%에 긴장 라인 12에 대 한 원형 맞춤 긴 축 효과 셀 정렬 지배 입증 했다. 히드로 7에 40% 부담, 셀 정렬 임의로 형상 지도 스트레인 효과의 무력화 때문.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/53715/53715fig1.jpg"> 
그림 1 . PDMS 시트 및 플러그 제조 PMMA 어머니 형. (a)는 분리 된 구성 요소는 바닥판, 경계 프레임, 그리고 흐름 채널을 포함 하 여 PMMA 형의. 더블 양면 테이프와 조립 후 (b) 흐름 시트 PMMA 어머니 형 형성 됩니다. (c)는 PDMS 플러그에 대 한 또 다른 PMMA 금형 조립 된다. 빨간색 숫자는 깊이 나타냅니다. (단위: mm) <a href="//ecsource.jove.com/files/ftp_upload/53715/53715fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/53715/53715fig2.jpg"> 
그림 2 . 셀-라덴 히드로 Micropattern에 대 한 플라스틱 포토 마스크 디자인. 삼각형 모양 (2 mm 결론에, 높이 6.5 m m)와 두 개의 구멍을 있다 신선한 세포 배양 공급 흐름 채널에 연결. (a) 플라스틱 포토 마스크 표시 됩니다 차원에서. 동심 서클의 기간은 400 nm, 듀티 사이클은 50%. 센터 서클의 직경은 2 m m. (b) 플라스틱 투명 필름에 레이저 인쇄 레이블 없이 포토 마스크 레이아웃. <a href="//ecsource.jove.com/files/ftp_upload/53715/53715fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/53715/53715fig3.jpg"> 
그림 3. 그라데이션 제작 공정 PDMS 유체 칩에 방사형 방향 따라 셀 라덴 히드로 스트레인. (a) 플라스틱 포토 마스크 정렬 이며 TMSPMA 코팅 유체 채널 칩 아래 붙어. 중합체 셀 솔루션 마이크로 주사기에 칩의 삽입 이며 흐름 채널을 채우기 위해 약 50 µ L를 주사 하는 데 사용. (b) 흐름 채널의 출구로 폐쇄는 PDMS 연결 하 고 중합체 세포 솔루션의 추가 40 µ L 주입. 유리 바닥은 30에 대 한 UV 꽃무늬 흐름 칩에 동심 원형 하이드로 겔을 조작 하는 s. 액체 압력은 발표 흐름 채널에 콘센트를 분리 하 여 (c) 와 유엔-가교 혼합물 DPBS와 씻. (d) 칩에 정적 그라데이션 변형 세포 배양에 대 한 준비가 동심 셀-라덴 hydrogels 적용 됩니다. 자외선 가교 과정 (e) 반경에 따라 하이드로 겔의 연속 되지 않은 그라데이션 높이 형성 된다. (f) 후 콘센트 뽑기, PDMS 막 평면 되 고 세포 캡슐화 hydrogels에 그라데이션 스트레스를 적용 합니다. <a href="//ecsource.jove.com/files/ftp_upload/53715/53715fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/53715/53715fig4.jpg"> 
그림 4. 순수 하이드로 겔 신장 그라데이션. 신장 선 너비와 셀에서에서 캡슐화 없이 순수 하이드로 겔의 백분율은 (는) 0 µ L와 함께 하루 3에 칩 (제어 그룹) 및 (b) 40 µ L 주입 볼륨 스트레인. (c) 신장 백분율은 선 두께 40 µ L.에 의해 선 폭 차이 40 µ L 0 µ L의 값을 나누어 계산 <a href="//ecsource.jove.com/files/ftp_upload/53715/53715fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/53715/53715fig5.jpg"> 
그림 5 . 3 일에 그라데이션 칩에서 3T3 세포 캡슐화의 형광 걸 핵 얼룩 이미지. 1 호선 (-c) , (d-f) 7, 선과 (g-i) 선 12 셀 정렬 방향을 공개 방사형 정렬, 임의의 정렬 및 원형 맞춤, 각각. 녹색 및 파랑 색상 각각 말라와 핵 얼룩을 보여줍니다. 점선된 흰색 라인은 하이드로 겔의 경계를 나타냅니다. 눈금 막대: 200 µ m. <a href="//ecsource.jove.com/files/ftp_upload/53715/53715fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
보충 그림 1. 돔 모양의 PDMS 곡률의 계산. H(x): 볼록 PDMS 곡선; H0: 최대 높이 차이 PDMS 돔 전후 변형; r: 반지름 돔; 된 돔으로 PDMS 변형 하면 파란 지역의 과다 주입 볼륨. 자세한 내용은 보충 파일 1 을 참조 하십시오. <a href="//ecsource.jove.com/files/ftp_upload/53715/Supplementary_Figure_1.tif" target="_blank">이 그림을 다운로드 하려면 여기를 클릭 하십시오.</a>
보조 파일 1. <a href="//ecsource.jove.com/files/ftp_upload/53715/Supplementary_File_1-SI.docx" target="_blank">이 파일을 다운로드 하려면 여기를 클릭 하십시오.</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

이 문서는 조직 공학에 대 한 셀 정렬을 조절 하는 동심 셀-라덴 히드로에 연속 되지 않은 그라데이션 정적 긴장을 제공 하는 간단한 방법을 소개 합니다.

그라데이션 변형 칩 셀 라덴 히드로에 자극 세포 행동에 대 한

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

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.9K 조회수.  National Tsing Hua University. 이 문서는 조직 공학에 대 한 셀 정렬을 조절 하는 동심 셀-라덴 히드로에 연속 되지 않은 그라데이션 정적 긴장을 제공 하는 간단한 방법을 소개 합니다.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

기계 다이나믹 셀 문화 Microfabricated 플랫폼

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

연구

생체공학

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

세포 생물학을위한 그라디언트 생성 Microfluidic 장치

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

생물학

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

단일 또는 공존 화학 / 전기 / 전단 응력 자극에서 세포 응답 유학을위한 미세 유체 장치 설계

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

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.

동적 압축으로 연골 세포 자극을 위한 미세 유체 플랫폼

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

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

화학적 집단 이동을 위한 미세 유체와 통합된 견인 현미경 검사법

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

라이브 현미경 이미징 에서 3D 하이드로겔의 제어 변형

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

뇌혈관화의 초기 이벤트를 모방하는 미세 유체 모델

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Chondrocyte - 아가로 오스 Hydrogels의 기계적 자극

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

마이크로패턴 견인 현미경을 위한 패턴 생성

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Studying Tissue Rigidity and Shear Stress Effects on Endothelial Cells(내피 세포에 대한 조직 강성 및 전단 응력 효과 연구)

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

저자 스포트라이트: 혈관 기계생물학에서 내피 세포 반응을 연구하기 위한 기판 강성 및 흐름 조건 통합

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.

내피 세포 반응을 연구하기 위해 기질 강성과 흐름을 통합하는 시험관 내 모델

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...