简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究
Summary
Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.
Abstract
Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 – 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn’t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.
Introduction
大多数组织在体内是软的粘弹性材料具有的杨氏模量为0.1千帕为脑至100千帕软软骨，然而，大多数体外细胞研究的是组织培养聚苯乙烯（TCP）的进行，其具有〜1京帕的模量1，本刚度匹配极大地影响方式细胞所处的环境做出反应。越来越多的研究机构是这样的专用于阐明对各种细胞类型，2,3-包括干细胞。4，结果的命运衬底刚度的效果，多水凝胶已经开发了在刚性依赖的细胞的帮助理解生物学包括聚丙烯酰胺（PA），5-7聚乙二醇（PEG），8,9-二甲基硅氧烷（PDMS），10和藻酸盐。11虽然这种衬底刚度对细胞命运产生重大影响的证据正在增长，大多数研究的进行上小规模用少量第amples。在基板刚度的细胞类型或环境条件是罕见的数组效果系统，多维的研究。12
几个有前途的高通量凝胶技术已被开发出来，其中包括基于PEG的微阵列，用于生产琼脂糖凝胶微珠，14或微和纳米棒，其中刚度由微米棒的直径和高度调制13的微流体装置15不过的技术来制备这样的基体是复杂的，并提供给限于实验室的数目。许多研究涉及刚度调制细胞应答使用聚丙烯酰胺（PA）的凝胶，这是不仅便宜和简单的实现，但也显示出杨氏模量，即0.3的生理学相关范围- 300千帕16-22但是，现有的方法来制造的PA凝胶为细胞培养是劳动密集型的，因此制备ARED小批量。从该凝胶中必须准备的规定部分与PA的凝胶作为细胞底物的制备相关的困难茎：1）在不存在氧，以允许完全聚合，2）具有平坦和光滑的表面，以允许均匀的细胞附着和铺展，和3）永久地固定在细胞培养皿为了防止浮动的底部。
几个小组已尝试产生的PA的凝胶为细胞培养在大批量。塞姆勒等人制备的厚片的PA凝胶中，将其“切断”用打孔器并置于96孔板中。23然而，该方法仅限于更硬的凝胶， 即 > 1千帕在杨氏模量，因为较软的凝胶是“粘性”，难以切割，且容易损坏。 MIH 等人开发出了更复杂的技术，它允许对凝胶直接聚合在玻璃底多孔板。 <s达> 6这是通过浇注胶解决方案集成到功能化玻璃底板和“夹”他们自定义的玻璃罩阵列形成凝胶达到6虽然非常有前途的，轻微的边缘效应仍在使用这种技术观察。此外，该技术需要定制设计的阵列不立即向许多实验室以及昂贵玻璃底多孔板访问。
本文介绍了一种简单而廉价的方式来组装PA凝胶中，可以很容易地通过任何实验室多孔板。这里，一个挠性塑料支撑被利用，其中有两个侧边 – 一个疏水性的，这是斥PA凝胶，和亲水性的，在沉积该共价结合的PA的凝胶。一旦PA凝胶片被淀积并永久地固定到柔性塑料支撑，它使得能够处理任何厚度或硬度的凝胶和切削成任何所需的形状。这APPRoach不仅产生定制的塑料“盖玻片'的尺寸不否则市售，也省却了必要的预治疗玻璃表面，无论是玻璃盖玻片或昂贵的玻璃底多孔板的孔中，具有PA的粘合溶液，这是一个繁琐和耗时的工序。最后，均匀的PA的凝胶片材可以在大批量制备并存储的去水合数月。
总之，这里介绍的测定法是一种改进的几个方面的现有的方法。首先，多孔板组装的过程中是有效的，并且所需要的材料的总成本是低的。其次，水凝胶生产大批量在一个单一的均匀的凝胶膜。最后，仅是商业上可用的材料是必需的。该测定的效用是通过探索基板刚度对细胞形态的影响，传播区域所示。
Protocol
Representative Results
Discussion
聚丙烯酰胺凝胶，最初开发用于电泳28现在常规用作细胞培养基材来研究衬底刚度对细胞形态，运动性，并且除其他细胞特征的通信3,24,29的影响。聚丙烯酰胺允许操纵衬底刚度以包含在身体的所有软组织的刚度（0.3 – 300千帕）1，在聚合物前体浓度的简单改变（ 图2，表1中 ，也见参考文献17,25,26）。耦合到一个事实，即PA的凝胶是相当便宜和简单的制备…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President’s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.
Materials
| Reagents | |||
| 40% Acrylamide | Bio-Rad | 161-0140 | |
| 2% Bis-acrylamide | Bio-Rad | 161-0142 | |
| Ammonium Persulfate | Bio-Rad | 161-07000 | |
| TEMED | Sigma Aldrich | T9281 | |
| Sulfo-SANPAH | Thermo Scientific | 22589 | |
| Collagen Type 1, from Rat tail, 3.68 mg/mL | BD Biosciences | 354236 | |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific | BP231-100 | |
| Hydrophobic solution – Repel Silane | GE Healthcare Bio-Sciences | 17-1332-01 | |
| PBS (1x), pH 7.4 | HyClone | SH30256.01 | |
| Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit] | Elsworth Adhesives | 3097358-1004 | |
| Tyrpsin/EDTA (10x) | Sigma Aldrich | 44174 | |
| RPMI-1640 Medium (1x) | HyClone | SH30027-02 | |
| Fetal Bovine Serum | HyClone | SH30073-03 | |
| Penicillin Streptomycin | MP Biomedicals | 1670046 | |
| Detergent – Triton-X | Sigma Aldrich | T8787 | |
| Formaldehyde 37% Solution | Sigma Aldrich | F1635 | |
| Bovine Serum Albumin (BSA) | Sigma Aldrich | A2153 | |
| BSA-based cell adhesion blocking kit – ECM Cell Adhesion Array Kit | Chemicon International | ECM540 | |
| Disposable lab equipment | |||
| flexible plastic support – GelBond PAG Film for Polyacrylamide Gels | GE Healthcare Bio-Sciences | 309819 | |
| Glass Plates | Slumpys | GBS4100SFSL | |
| 50 mL Conicals | Fisher Scinetific | 3181345107 | |
| 15 mL Conicals | FALCON | 352097 | |
| Micro centrifuge tubes | Fisher Scinetific | 2 mL: 02681258 | |
| 96-well plate (flat bottom) | Fisher Scinetific | 12565501 | |
| Disposable Pipettes (1 mL, 2mL, 5mL, 10mL, 25 mL, 50mL) | Fisher Scinetific | 1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F | |
| Glass Transfer Pipettes | Fisher Scinetific | 5 3/4": 1367820A, 9":136786B | |
| Pipette Tips (1-200uL, 101-1000uL) | Fisher Scinetific | 2707509 | |
| Plastic Standard Disposable Transfer Pipettes | Fisher Scientific | 13-711-9D | |
| Parafilm | PARAFILM | PM992 | |
| Powder Free Examination Gloves | Quest | 92897 | |
| Silicone spacers – Silicone sheet, 0.5 mm thick/13 cm x 18 cm | Grace Bio-Labs | JTR-S-0.5 | |
| Large/non-disposable lab equipment | |||
| Light and Flourescent Microscope (Axiovert 200M) | Zeiss | 3820005619 | |
| Microscope Software | Zeiss | AxioVision Rel. 4.8.2 | |
| UV oven | UVITRON | UV1080 | |
| Vacuum chamber/degasser | BelArt | 999320237 | |
| Vacuum pump for degasser | KNF Lab | 5097482 | |
| Tissue Culture Hood | NUAIRE | NU-425-600 | |
| Chemical Fume Hood | KEWAUNEE | 99151 | |
| Inverted Microscope (Axiovert 25) | Zeiss | 663526 | |
| Incubator | NUAIRE | NU-8500 | |
| Pipette Aid | Drummond Scientific Co. | P-76864 | |
| Hemacytometer | Bright-Line | 383684 |
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