制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代
Summary
Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.
Abstract
In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (μCP) is a versatile technique to pattern such grafted PEG brushes, but manual μCP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-μCP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal ‘click’ chemistries. Here, we describe in detail the workflow to manufacture such substrates.
Introduction
的PEG接枝表面显示共价结合的配体的生化，同时保持固有的不污性能的能力，使他们为工程定制微观环境对培养基质1,2,3的理想选择。通过配体结合的PEG介导刷的生物特异性相互作用使得在单个细胞的表型体内组织的微环境中的复杂的生物化学发现线索的作用还原论分析。此外，生物正交的“点击”化学方法可用来促进配体的定向固定化，使得它们在天然构象4-6给出。因此，PEG的微观空间的图案笔刷是一个多功能的工具来创建设计师在体外龛调查引起的固定生化线索6,7细胞信号转导。
产生生化立方米的空间格局的常用方法ES限嗣继承微接触印刷（μCP）镀金基板与PEG结合的烷基硫醇模式。然后，PEG-聚乙二醇化复合链烷硫醇的微图案化的自组装单分子层限制了生化分子， 例如，蛋白质的物理吸附，只在基板8,9的非图案区域。然而，通过这种技术产生的自组装是在长期的细胞培养介质中对氧化敏感。因此，μCP'd硫醇自组装膜通常还采用表面引发的原子转移自由基聚合（SI-ATRP），以增加该地区的不污稳定性10接枝的PEG聚合物刷。具体地，微接触的烷基硫醇聚合引发剂，ω-meraptoundecyl溴异丁酸乙酯，对金涂覆的表面，随后由SI-ATRP的聚（乙二醇）甲基醚甲基丙烯酸酯（PEGMEMA）单体生成具有微图案化的长期，稳定，非表面结垢PEG刷。此外，这些能够被进一步修改，以呈现不同的化学部分11。
以这个属性，沙等优势。人 。开发出一种方法来设计培养基质多组分PEGMEMA画笔呈现直角“点击”化学物质。在该方法中，他们使用了一系列的穿插顺序叠氮化钠，乙醇胺微接触/ SI-ATRP的步骤，和炔丙基胺的亲核取代来创建培养基材呈现多种固定化配体6的微型图案。而使用这样的化学品在与人工微接触结合来设计新的培养基材的电位是巨大的，它是由精度和准确度与多个微接触的步骤可以在一个单一的基片对齐的限制。将需要的精度和准确度水平高可再现地制造复杂中使用这些通用的技术体外龛。
e_content“>为了解决此限制，几个自动化和半自动化的微接触系统已经生成。轮穴等人开发出了微接触系统，其中定制邮票被放置在轨道系统和进入共形接触以使用金包被的载玻片一个计算机控制的气动致动器，然而，这种方法需要的定制邮票设计的精确制造，并报告有10微米的精度与执行多个微接触步骤12，当所取得的精确度的报告。最近，利用集成的运动耦合系统的方法小于1微米的精度报告使用单个图案，但是无法准确地对准多个图案由于缺少精确控制从模具印模特征到模具13上 。另外，两者的以前的方法所需要的基底图案形成步骤之间保持固定从而显著限制性的表面改性化学，可以是分集利用。在这里，我们描述了一种自动化的R-微接触系统能够对多个微接触步骤，准确和精确的对准，同时允许在邮票的设计和制造的灵活性最大。此外，图案化衬底可以重复从冲压件之间的体系中除去，从而允许使用各种不同的底物改性化学品，包括顺序的亲核取代。衬底使用这样的化学工程已被用于预先细胞培养由我们两人6,14和其他7。因此，我们已经合并的R-微接触和连续的亲核取代反应来开发用于培养基材的复杂和微图案化的生化线索可伸缩的制造方法。Protocol
Representative Results
Discussion
理想的基质用于组织工程将仿生并由此概括的天然组织中发现的关键的生物活性配体的空间分布。他们也将具有动态属性，使配体的时间调整，并且在它们出现，以允许定向组织形态发生和空间上的限制诱导细胞命运的分布模式。这种基材的制造需要在衬底上复杂和高度有序取向多种生化线索的固定。虽然模拟细胞的小生境所有的内源性因素在体外是不合理的，这里所描述的R-μCP系统允许?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.
Materials
| Name | Company | Catalog Number | Comments |
| SCARA | Epson | LS3-401ST | Higher end models with increased precision are available if desired. |
| (TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)TRICHLOROSILANE | Gelest | SIT8174.0 | CAUTION, Should only be handled in a chemical fume hood. When silanizing wafers no one should enter the hood until all silane has been evaporated. |
| Sylgard 184 Silicone Elastomer Kit | Ellsworth Adhesive Co | NC9020938 | Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable. |
| 24mm X 50 mm #1 Cover Glass Slides | Fisher Scientific | 48393106 | These can be purchased from a number of suppliers with varying dimensions to suit need. |
| CHA-600 Telemark Electron Beam Evaporator | Telemark | SEC-600-RAP | Requries specialized training. |
| EPSON LS3 SCARA | EPSON | LS3-401ST | |
| ω-mertcaptoundecyl bromoisobutyrate | Prochimia | FT 015-m11-0.2 | Store at -20°C. Other ATRP initiators may be used as this R-μCP platform is applicable to all micropatterning modalities. |
| Schlenk Tube Flask 50 mL | Synthware | 60003-078 | Requires rubber stoppers with diaphram. |
| Poly(ethylene glycol) methyl ether methacrylate | Sigma Aldrich | 447943 | Shipped containing MEHQ and BHT free readical inhibitors. |
| Methanol (Certified ACS) | Fisher Scientific | A412-4 | CAUTION, only handle in chemical fume hood. |
| Copper(II) Bromide | Sigma Aldrich | 437867 | CAUTION, limit exposure with surgical mask. |
| 2',2-Bipyridine | Sigma Aldrich | D216305 | CAUTION, limit exposure with surgical mask. |
| Sodium L-Ascorbate | Sigma Aldrich | A4034 | |
| 20mL Borosilicate Glass Scintillation Vials | Fisher Scientific | 03-340-4E | |
| Sodium Azide | Sigma Aldrich | S2002 | CAUTION, limit exposure with surgical mask. |
| N,N-dimethyformamide | Sigma Aldrich | 227056 | CAUTION, only handle in chemical fume hood. |
| Ethanolamine | Sigma Aldrich | 398136 | CAUTION, only handle in chemical fume hood. |
| Triethylamine | Sigma Aldrich | T0886 | CAUTION, only handle in chemical fume hood. |
| Dimethylsulfoxide | Sigma Aldrich | 276855 | CAUTION, only handle in chemical fume hood. |
| Propargylamine | Sigma Aldrich | P50900 | CAUTION, only handle in chemical fume hood. |
| 200 Proof Ethanol | University of Wisconsin Material Distribution Services | 2292 | CAUTION, only handle in chemical fume hood. |
| Azide-PEG3-Biotin | ClickChemistryTools | AZ104-100 | Solubilized in DMF |
| Copper(II) Sulfate | Sigma Aldrich | C1297 | CAUTION, limit exposure with surgical mask. |
| Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) | Sigma Aldrich | 678937 | |
| L-Ascorbic Acid | Sigma Aldrich | A7506 | |
| Phosphate Buffer Saline | Invitrogen | 14190144 | |
| Donkey Serum | Sigma Aldrich | D9663 | Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (e.g. BSA) are available and serve this purpose. |
| 12-Well Polystyrene Plate | Thermo Scientifit – NUNC | 07-200-81 | Plates can be purchased form a number of suppliers with varying dimensions. |
| DBCO-PEG4-Biotin | Clickchemistytools | A105P4-10 | Solubilized in DMF |
| Streptavidin, Alexa Fluor 488 Conjugate | Life Technologies | S-11223 | Solubilized in PBS |
| Streptavidin, Alexa Fluor 546 conjugate | Life Technologies | S-11225 | Solubilized in PBS |
| Nikon A1-R Confocal Microscope | Nikon | Nikon Eclipse Ti, A1R | An epifluorescent microscope is sufficient to image functionalized micropatterned substrates. |
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