在单元格拉丹水凝胶中刺激细胞行为的应变梯度芯片

摘要

这篇文章介绍了一个简单的方法，对提供非连续梯度静态菌株对同心单元格拉丹水凝胶来调节单元格对齐方式为组织工程。

摘要

人工指导细胞对齐方式是在组织工程领域的热点话题。大部分的先前的研究已经进行单个单元格拉丹的水凝胶应变诱导细胞对准使用复杂的实验过程和质量控制系统，这是通常与污染问题。因此，在本文中，我们提出一种构建流控芯片用塑料的聚二甲基硅氧烷盖和紫外透明的玻璃基板，刺激的 3D 水凝胶的细胞行为的静态应变梯度的简单方法。超载的照片; 细胞预聚物中射流室可以生成凸曲线的 PDMS 膜在封面上。紫外光交联后通过弯曲的聚二甲基硅氧烷膜和缓冲区洗，调查细胞微环境下的同心圆形优缺点行为下个变种的梯度是自建在单一的流控芯片，没有外部文书。观察到的几何在指导下，在合作与应变刺激，相差 15-65%的水凝胶细胞对齐趋势变化后论证了 NIH3T3 细胞。后 3 天孵化，水凝胶几何主导下低的压应变，单元格对齐方式哪里细胞沿水凝胶伸长率高的压缩应变下的方向排列。这些，之间细胞细胞随机对齐方式由于耗散的激进指导的水凝胶伸长和图案的水凝胶的几何指导。

引言

作为模仿一个本机的微环境块材料，含细胞外基质 (ECM) 水凝胶可以重新生成仿生支架支持细胞的生长。拥有组织的职能，组织单元格对齐方式是一项基本要求。各种 （即，细胞表面） 2D 和 3D （即，封装在水凝胶的细胞） 培养或封装细胞中或与微型柔性衬底上取得了单元格对齐方式-或纳米模式¹。在微体系结构的三维单元格对齐方式是更有吸引力，因为微环境是更接近于自然组织构造^2，，34。三维单元格对齐方式的一个常用方法是水凝胶形状^2，3的几何提示。由于细胞增殖在短轴方向的受限空间，细胞目的沿微图案的水凝胶的长轴方向对齐。另一种方法是适用于凝胶来实现单元格对齐方式平行于拉伸方向^4，5拉抻。

生物物理刺激对 ECM 的水凝胶，如压缩应变或电场的作用，能调节细胞功能的适当组织一体化、 增殖和分化^1，，23。很多研究已经通过使用多个机械控制单位^{4，6，7，，89}一次应用一个应变条件探讨细胞行为。例如，机械步进电动机使用挤压或拉伸对 3D 封装细胞胶原凝胶一直共同的办法^7，10。然而，这种控制的设备需要额外空间并面临孵化器^{7，9，，1112}中受污染的问题。此外，大型仪器不能精确的控制环境，以提供高重复性¹³。

考虑到单元格拉丹水凝胶通常受雇在微尺度生物医学应用，它有利于结合 MEMS 技术来生成一个范围的应变/拉伸刺激同时探讨细胞行为在 3D 仿生构造体外^{2，14，15，16，，1718}。例如，利用气体压力变形聚二甲基硅氧烷膜在微流控芯片可以引起不同品系，开车去不同的谱系^9，16细胞分化。然而，有许多技术难题，如在一个干净的房间和软件控制集成的电机、 泵、 阀门和压缩的气体的复杂的芯片制造工艺。

在这项工作，我们演示了一个简单的方法来获得自我维持的梯度静态应变微流控芯片采用同心圆形水凝胶模式和灵活的 PDMS 膜。不同于大多数现有的方法，我们的平台是便携式和一次性使用的微型设备，可以制作黄色房间外面并拥有自生梯度菌株对同心细胞封装水凝胶，无需外部机械设备孵化期间。3T3 成纤维细胞细胞行为受水凝胶形状组合和期间的观察 3 天内 3D ECM 拟态环境梯度应变片在单元格对齐方式显示出各种拉伸弹力的指导线索。

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研究方案

1. GelMA Synthesis

1. Weigh 10 g of gelatin powder and add it to a glass flask with 100 mL ofDulbecco's phosphate-buffered saline (DPBS). Put a magnetic stir bar into the flask and place the flask on a stirring hot plate.
2. Cover the flask with aluminum foil to avoid water evaporation. Set the hot plate temperature to 50-60 °C and the stirrer at 100 rpm for 1 h to dissolve the gelatin powder well.
3. 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 °C for 3 h.
4. Add pre-warmed DPBS (40 °C) to the flask to a final volume of 500 mL and allow this mix well for 15 min to stop the reaction.
5. 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.
6. 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 °C during the dialysis process.
7. 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 µm) and apply a vacuum to the filter cup to force the solution to pass through the filter membrane for sterilization.
8. Transfer the sterilized polymer into several 50-mL sterilized tubes and store them in a -80 °C freezer for 3 - 5 days.
9. Freeze-dry the -80 °C polymer for 1 week using a freeze dryer to form GelMA. Store the GelMA in a -80 °C freezer.

2. 3-(Trimethoxysilyl)propyl Methacrylate (TMSPMA) Modification

1.  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.
2. 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.
3. Immerse the glass slides in 5% TMSPMA in 99.5% ethanol for 1 h.
4. Wash the slides in 95% ethanol, air dry the slides, and anneal the TMSPMA coating in an oven at 80 °C for 2 h.

3. Chip Fabrication

1. 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.
2. 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.
3. 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).
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. Cure the PDMS for 2 h at 80 °C. After cooling down the cured PDMS on the mold, detach the PDMS covers and the PDMS plugs from the PMMA molds.
10. Punch two holes with diameters of about 3 mm at the ends of flow channels of the PDMS covers.
11. 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 °C.
12. 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.
13. 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 °C for 1 h can further enhance the bonding strength.
14. 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.

4. Static Gradient Strain on the Cell-laden Hydrogel

1. 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.
2. Prepare 100 mL of DMEM medium with 10% FBS, 1% Pen-Strep, and 250 mL of DPBS in a 37 °C water bath to use as the cell culture medium.
3. Weigh 25 mg of freeze-dried GelMA into 0.3 mL of prewarmed (37 °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.
4. Weigh 50 mg of photoinitiator into 1 ml of DPBS in a microcentrifuge tube and place it in an 80 °C oven for 15 min or until the photoinitiator has dissolved.
5. Take 25 µL 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.
6. 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 µL of cell culture medium.
7. 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 µL of cell prepolymer in a 100-µL micro syringe.
8. 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-µL micro-syringe loaded with prepolymer cell solution to the inlet of the chip.
9. Place (see Figure 3b) 50 µL 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 µL of solution to create a convex bulge in the circular PDMS membrane.
10. 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.
11. 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.
12. Fill the flow channel with about 100 µL of cell culture medium.
13. Place the chip in a sterilized culture dish and culture in a 5% CO₂ atmosphere at 37 °C for a week. Refresh the medium every day.
14. 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 µL and 0 µL by the line width at 40 µL.

5. Cell Staining for Alignment Analysis

1. 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.
2. Replace the solution with 0.5% cell membrane permeating solution in DPBS for 10 min to permeabilize the cell membrane at RT.
3. PBS wash the samples 3 times with 5- to 10-min interval between washes (using a loaded syringe, as in step 5.1).
4. Load 1% BSA solution into the fluidic channel for 45-60 min at RT (using a loaded syringe through the inlet port).
5. Add 1.5 mL of methanol into the vial with Alexa Fluor 488 phalloidin to yield a final concentration of 6.6 µM stock solution.
6. Take 5 µL of Alexa Fluor 488 phalloidin from step 5.5 and dilute it in 200 µL of DPBS with 0.1% BSA to form a final concentration of 0.165 µM Alexa Fluor 488 phalloidin.
7. Add 200 µL of the mixture solution to the fluidic channel using a micropipette and incubate the chip at 37 °C for 45 - 60 min for actin staining. PBS wash (as above) the samples 3 times.
8. Prepare 1 µg/mL DAPI in PBS, flow it through the chip, and stain cell nuclei at 37 °C for 5 min.
9. 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.
10. 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.

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结果

若要比较每个循环的水凝胶在完成的应变梯度刺激芯片之间的机械变化，我们分别测量中的两个相同的芯片，具有 0 微升 (图 4a) 注射量和 40 µ L (图 4b)，每个循环水凝胶的线条宽度。%伸长率每个圆圈都伸长 40 µ L 注射芯片中的除以相应的水凝胶在 0 微升注射芯片 (图 4 c) 的线宽。水凝胶是一种不可压缩的?...

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讨论

在本文中，我们报告一个简单的方法来比较水凝胶形状指导和拉伸拉伸后的单元格对齐方式行为。灵活的 PDMS 膜创建用于生成不同高度的同心圆形水凝胶圆顶形的曲率。后释放压力，PDMS 膜自动适用力于微图案水凝胶形成梯度应变延伸，与中心的最大值和最小值外, 边界处。应变梯度的形成由灵活的 PDMS 膜和处理的流控芯片设计的正应参加的几个重要参数: (i) 精确控制厚度的聚二甲基硅氧烷膜是关...

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材料

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