工程三维上皮组织细胞外基质中嵌入
Summary
该原稿描述了软基于光刻技术工程师三维（3D）的均匀阵列由细胞外基质包围限定的几何形状的上皮组织。这种方法是适合于多种细胞类型和实验的上下文，并允许对于相同重复的高通量筛选。
Abstract
The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.
Introduction
支上皮组织，称为分支形态的发展，是由细胞衍生的，物理的和环境因素调节。在乳腺，分支形态是通过该引导集体细胞迁移创建树形结构一个迭代过程。第一步是从乳管主芽的形成，其次是分支起始和延长1,2。分支的侵入周围基质是由类固醇激素在青春期的全身性释放诱导。新的主芽，然后从现有分行的两端发起，而这个过程继续创造上皮树3。虽然许多重要的生化信号已经确定，指导这个复杂的发展过程，目前缺乏细胞生物学机制的全面了解。此外，在特定的线索的影响机理研究难以从experi解构体内 ments，因为精确时空扰动和测量常常是不可能的。
三维（3D）培养技术，例如全器官培养，主要类器官和细胞培养模型，是系统地研究组织形态4-6的基本机制的有用工具。这些可以用于确定特定因素的影响单独，例如机械力和生化信号，对各种细胞的行为，包括迁移，增殖和分化的是特别有用的。6工程化细胞培养模型，特别是容易使扰动的单个细胞和它们的微环境。
一个这样的养殖模式使用基于微细加工的方法来工程师控制的三维结构模型的乳腺上皮组织在与一个感应一贯和可重复形成集体迁移分支机构ppropriate生长因子。模型的主要优点是能够精确地操纵和测量的物理和生化因素，如机械应力的模式，具有很高的统计置信的影响的能力。该技术中，用计算模型一起，已经被用来确定物理和生化信号的乳腺上皮组织和其它支上皮7-11的正常发展的指导的相对贡献。这里介绍的是用于构建这些模型的组织，它可以被容易地扩展到其它类型的细胞和细胞外基质（ECM）凝胶的详细协议，其用作治疗剂的测试的潜在工具。
Protocol
Representative Results
Discussion
The protocol described above outlines a method to produce identical epithelial tissues of pre-defined shape, enabling spatial control of the mechanical stress experienced by cells in the tissue. An elastomeric mold is used to create cavities in type I collagen that are then filled with epithelial cells and covered with an additional collagen layer such that cells are completely encapsulated in a 3D collagen matrix environment. Further culture of these tissues and treatment with growth factors to induce branching from the…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由来自美国国立卫生研究院（HL118532，HL120142，CA187692），大卫和露西尔·帕卡德基金会，卡米尔和亨利德雷福斯基金会和巴勒斯补助部分支持欢迎基金。 ASP是由夏洛特伊丽莎白宝洁尊敬的奖学金的部分资助。
Materials
| Polydimethylsiloxane (PDMS) | Ellsworth Adhesives | Sylgard 184 | |
| PDMS curing agent | Ellsworth Adhesives | Sylgard 184 | |
| Lithographically patterned silicon master | self-made | N/A | |
| Plastic weigh boat | Fisher Scientific | 08-732-115 | |
| 100-mm-diameter Petri dishes | BioExpress | D-2550-2 | |
| Ethyl Alcohol 200 Proof | Pharmco-Aaper | 111000200 | Make a 70% EtOH (v:v) solution by mixing with dH2O |
| Razor blade | American Safety Razor | 620179 | |
| 1:1 Dulbecco’s Modified Eagle’s Medium : Ham’s F12 Nutrient Mixture (DMEM/F12) (1:1) | Hyclone | SH30023FS | |
| Fetal Bovine Serum (FBS) | Atlanta Biologicals | S11150H | |
| 10x Hank’s balanced salt solution (HBSS) | Life Technologies | 14185-052 | |
| Insulin | Sigma Aldrich | I6634-500MG | |
| Gentamicin | Life Technologies | 15750-060 | |
| 10X Phosphate-buffered saline (PBS) | Fisher Scientific | BP399-500 | |
| Sodium hydroxide (NaOH) | Sigma Aldrich | 221465-500G | |
| Bovine type I collagen (non-pepsinized) | Koken | IAC-50 | |
| Albumin from bovine serum (BSA) | Sigma Aldrich | A-7906 | |
| Curved stainless steel tweezers | Dumont | 7 | |
| 35-mm-diameter tissue culture dishes | BioExpress | T-2881-6 | |
| 15 mL conical tubes | BioExpress | C-3394-2 | |
| 1.5 mL Eppendorf Safe-Lock Tube | USA Scientific | 1615-5500 | |
| Circular #1 glass coverslips, 15-mm in diameter | Bellco Glass Inc. | Special order | |
| 0.05% 1X Trypsin-EDTA | Life Technologies | 25300-054 | |
| Paraformaldehyde | VWR | 100503-916 | |
| Triton X-100 | Perkin Elmer | N9300260 | Detergent |
| HGF | Sigma Aldrich | H 9661 | Resuspended in dH2O at 50 mg/mL |
| Rabbit anti-mouse FAK antibody | Life Technologies | AMO0672 | |
| Goat anti-rabbit Alexa 488 antibody | Life Technologies | A-11034 | |
| Adobe Photoshop | Adobe | N/A | Used for color-coding pixel frequency maps. |
| FIJI (ImageJ) | NIH | N/A | Free image analysis software used for thresholding, registering, and overlaying images to create a pixel frequency map. The StackReg plugin was used for registering binary images. |
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