倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台
Summary
This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.
Abstract
维持肝细胞功能的体外，用于测试异生素“的细胞毒性，研究病毒感染和开发针对肝脏的药物为目的的能力，需要在哪些小区得到适当的生物化学和机械线索的平台。最近肝组织工程系统已采用合成的或天然的水凝胶组成的三维（3D）支架，由于其高保水性和其提供由该细胞所需要的机械性刺激的能力。出现了在倒胶态晶体（ICC）的脚手架越来越大的兴趣，最近的发展，其允许高空间组织，同型和异型细胞相互作用，以及细胞外基质（ECM）的相互作用。在此，我们描述了一个协议使用聚（乙二醇）二丙烯酸酯（PEGDA），颗粒沥滤方法来制造IC卡支架。简要地说，一个晶格是由微颗粒制成，在这之后的预聚合物：R染料溶液加入，适当地聚合，然后颗粒被除去，或者浸出，使用有机溶剂（ 如，四氢呋喃）。晶格结果与控制的孔尺寸和interconnectivities允许媒体更容易地达到电池的高度多孔支架的溶解。这种独特的结构允许所述细胞附着的能力来涂覆PEGDA IC卡支架与蛋白质还示出对电池性能有显着影响的孔之间以及容易的通信，和高的表面积。我们分析了支架的形态，以及肝癌细胞（的Huh-7.5）行为存活率方面和功能探索ICC结构和ECM涂料的效果。总体而言，本文提供了在组织工程中广泛应用，尤其是肝脏组织工程支架出现了详细的协议。
Introduction
肝脏是一个高度血管器官与多种功能，包括血液解毒，异生素的代谢，以及生产的血清蛋白。肝组织具有复杂的三维（3D）微结构，其包含多种细胞类型，胆管，正弦，和不同的生物基质的组成和不同的氧浓度的区域。鉴于这种精细结构中，它已经难于创建体外 1适当肝模型。然而，对于在体外模型官能托管人肝细胞用于测试药物毒性2平台和学习与肝3相关联的疾病的需求上升。
当前肝组织工程平台已经通过分离，或者集中于几个，肝脏的参数，单元4即共培养，所述透明的生化组成简化了肝脏的复杂性升微环境5，流动动力学6,7和生物基质8的结构。在生物基质的结构可分为参数，如支架材料，细胞外基质（ECM）蛋白的组合物，基质硬度以及支架的设计和结构。已经有使用合成水凝胶，特别是聚（乙二醇）（PEG）水凝胶9的组织工程研究的上升，由于能够调整水凝胶的机械性能，生物活性，和降解速率。对于肝脏相关的研究，生物相容性水凝胶应用于肝病3的病毒感染研究。作为肝细胞平台设计，许多研究已经利用肝细胞三明治培养10,11和电池封装的水凝胶12,13内，以提供3D环境和细胞ECM和细胞-细胞相互作用这是必不可少的体内微环境模仿。豪版本，这些平台不具有高度的控制和空间组织的，通过支架14导致非均匀的性质。
倒晶胶体（ICC）14脚手架是，首先在21世纪初引进的细胞培养具有高度组织性的三维支架。所述支架的独特结构可以归因于使用的胶态晶体，可变直径的胶体粒子的有序晶格的简单的制造过程。简要地说，以总结的过程中，颗粒被整齐排列并利用热，以形成晶格退火。这种晶格的浸出，通过一种有机溶剂，在六边形填充球形腔室15具有高表面积的聚合水凝胶的结果。这种高度有序的支架已与合成和天然材料先前已经制成，包括但不限于聚（丙烯酰胺）16-21，聚（乳酸-共-乙醇酸）15,22-30，聚（乙二醇）31,32，聚（2-羟乙基甲基丙烯酸酯）21,33-35，和脱乙酰壳多糖36-39。由非污染材料ICC支架往往空腔14,23,40内促进细胞球体。多种细胞类型已经显示成功增殖，此配置中的分化和功能，包括软骨细胞41，骨髓基质细胞42，和干细胞43,44。关于肝细胞，研究已经与由钠2的SiO 3和聚（丙烯酰胺）的IC卡的支架，但不PEG进行。用简单的生物结合策略（ 即 ，通过EDC / NHS胺偶联），ECM蛋白缀合的PEG基的支架可被制造，这可以证明更多细胞的结合位点是一个更象环境体内和增强肝功能。
在这份手稿和相关的视频，我们详细介绍了ICC支架的制造使用聚（乙二醇）二丙烯酸酯（PEGDA）水凝胶和聚苯乙烯微球的晶格，用于肝癌优化（的Huh-7.5）培养。我们证明在脚手架拓扑和电池性能方面的一般非粘着裸PEGDA ICC支架和胶原涂层PEGDA ICC脚手架之间的差异。细胞活力和功能测定定性和定量评估的Huh-7.5细胞的行为。
Protocol
1. ICC脚手架制作（图1） 制备聚苯乙烯（PS）棱（直径= 6毫米;珠8-13层）。 为了准备模具，从0.2毫升煮沸型离心管切的提示关闭在40微升的水平。附着切割管的顶端与防水胶24×60毫米2显微镜盖玻璃滑动。 放包含在水悬浮液中进20ml小瓶在PS球（直径= 140微米），小心地吸取出来的水悬浮液，并加入18毫升70％的乙醇溶液到小瓶。把球体溶液放入超声波浴松开凝集球体。重?…
Representative Results
对于IC卡支架的结构表征和各IC卡支架状态的功效的在培养的肝细胞比较代表结果示和说明。在这些结果中使用的IC卡的支架条件是0微克/毫升（裸），20微克/毫升（胶原20），200微克/毫升（胶原200），和400微克/毫升（胶原400）和初始胶原涂层咦-7.5细胞种植数量为1×10 6。 ICC孔互连和支架的拓扑结构表征。 <p class="jov…
Discussion
组织工程支架的快速发展提供一切必要的再生，维持或器官替代的应用程序修复组织，研究疾病，药物开发的物理和生化线索，和许多其他57。在肝组织工程，原代人肝细胞迅速失去其代谢功能，一旦从主体分离，从而为工程支架非常需要和显影平台维持肝功能。 体外肝细胞培养平台目前已经利用不同的生物材料。在这方面的研究已集中于模仿体内肝微环境的各种特征，如EC…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者希望从国家研究基金会奖学金（NRF -NRFF2011-01）与竞争研究计划（NRF-CRP10-2012-07）承认支持。
Materials
| 0.2 mL PCR tube | Axygen Scientific | PCR-02D-C | Boil-proof |
| Gorilla Glue | Gorilla Glue, Inc. | Depends on vendor. This was purchased from a local store. | |
| Glass slides | VWR | 631-1575 | Dimensions: 24×60 mm |
| Polystyrene spheres | Fisher Scientific | TSS#4314A | Diameter = 140 um; 3×10^4 particles per milliliter and 1.4% size distribution |
| Ethanol | Merck | 1.00983.1011 | absolute for analysis EMSURE; Dilute to 70% with Milli-Q water |
| Ultrasonic Bath | Elma | S10H | Equiment |
| Furnace | Nabertherm | N7/H | Equipment |
| 200 µL pipette tip | Axygen Scientific | T-210-Y-R-S | |
| Rocking shaker | VWR | 444-0142 | |
| Polyethylene Glycol (PEG) | Merck | 1.09727.0100 | Mw= 4kDa; acrylation of PEG monomers and purification of the resulting precipitate produces a PEGDA macromer with Mw = 4.6kDa |
| Centrifuge | Beckman Coulter | 392932 | Equipment |
| Acrylate-Poly (Ethylene Glycol) – Succinimidyl Valerate | Laysan Bio | ACRL-PEG-SVA-3400-1g | Mw = 3.4 kDa |
| 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone | Sigma Aldrich | 410896 | |
| Vortex | VWR | 58816-123 | Equipment |
| Microcentrifuge | Eppendorf | 5404 000.413 | |
| Paraffin Film | Parafilm M | #PM996 | Kept at 9" with allows intensity of 10.84 mW/cm^2 |
| Bluewave 200 UV spotlight | Blaze Technology | 120008, 122300 | |
| Tetrahydrofuran (THF) | Merck | 107025 | |
| Orbital shaker | Heidolph | 543-123120-00-5 | From rat |
| Collagen Type I | Sigma Aldrich | C3867-1VL | 1X, w/o CaCl & MgCl; Ph = 7.2 |
| Phosphate Buffered Saline (PBS) | Gibco | 20012-027 | 16% W/V AQ. 10x10ml |
| Paraformaldehyde | VWR | 43368.9M | Equipment |
| Freezone 4.5 freeze drier | Labconco | 7750020 | Equipment |
| Sputter coater | Jeol Ltd. | JFC-1600 | Equipment |
| Scanning Electron Microscope | Jeol Ltd. | JSM 5310 | |
| Anti-mouse primary antibodies against Collagen type I | Abcam | ab6308 | |
| Anti-mouse secondary antibody conjugated with Alexa Fluor 488 | Life Technologies | A21121 | |
| Plate, Tissue Culture 24 Well, Flat Bottom (Nunclon) | Bio-Rev PTE LTD | 3820-024 | |
| Dulbecco's Modified Eagle's Medium(DMEM) 2.5 g/L Glucose w/ L-Gln |
Lonza | 12-604F | |
| Fetal Bovine Serum (FBS) | Gibco | A15-151 | |
| Penicillin-Streptomycin (P/S) | Life Tchnologies | 15140-122 E | |
| APC49‐Huh ‐7.5 Cell Line | Apath | ||
| 100 mm Corning non-treated culture dishes | Sigma Aldrich | CLS430591 | |
| 0.25% Trypsin-EDTA | Gibco | 25200-056 | Equipment; 37°C, 5% Humidity |
| Forma Steri-Cycle CO2 Incubators | Thermofisher Scientific | 371 | |
| Hausser Bright-Line Phase Hemacytometer | Thermofisher Scientific | 02-671-6 | |
| Live/Dead Viability/Cytotoxicity Kit 'for mammalian cells | Life Technologies | L3224 | |
| CCK-8 Assay | Dojindo Laboratories | CK04-11 | Monosodium-salt reagent (MSR) |
| Infinite 200 PRO microplate reader | Tecan | ||
| Albumin Human ELISA kit | Abcam | ab108788 | |
| Triton X-100 | Bio-Rad | #1610407 | |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A2153-50G | |
| Anti-mouse primary antibodies (against CYP3A4, albumin) | Santa Cruz Biotechnology | sc-53850; sc-271605 | |
| DAPI | Life Technologies | D3571 | |
| Alexa Fluor 555 labelled Phalloidin | Life Technologies | A34055 | |
| Trizol | Life Technologies | 15596-026 | |
| Chloroform | VWR | 22706.326 | |
| Isopropanol | Fisher Scientific | 67-63-0 | |
| DPEC water | Thermofisher Scientific | AM9916 | |
| Nanodrop 2000c Spectrophotometer | Thermofisher Scientific | ND-2000 | |
| iScript Reverse Transcription Supermix | Bio-Rad Laboratories | 1708840 | |
| SYBR select Master Mix for CFX | Life Technology | 4472937 | |
| Primers (to be chosen) | |||
| CFX96 Real-Time System, C-1000 Touch Thermal Cycler | Bio Rad Laboratories | SOFT-CFX-31-PATCH |
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Cite This Article
Shirahama, H., Kumar, S. K., Jeon, W., Kim, M. H., Lee, J. H., Ng, S. S., Tabaei, S. R., Cho, N. Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering. J. Vis. Exp. (114), e54331, doi:10.3791/54331 (2016).