稳定肝细胞表型采用优化合成表面
Summary
本文将重点发展聚合物涂层表面进行长期，干细胞的培养稳定来源的人肝细胞。
Abstract
Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.
Introduction
生物材料已经被广泛用于多能干细胞1的维持和分化。同时使这些生物基质通常包含未定义了大量元器件。基质胶是一种常用的基质干细胞的培养和分化。不幸的是，它的可变成分影响细胞功能和表型。虽然各种各样的替代方案中，更明确的生物基质已用于2-7，其动物来源或可扩展性差，使它们不适合的候选用于工业制造。因此合成的替代品，具有固定的成分，性能可靠的鉴定，是干细胞研究的主要目标。
在克服不确定的细胞培养基质的限制的尝试，化学和生物学的交叉学科的合作已经确定了合成材料，以支持细胞表型的能力。合成器客位基板具有可扩展性，成本效益和可制造成复杂的三维结构，模仿体内环境。由于这些特性的合成底物已被广泛使用，以支持和驱动许多细胞类型8-10的分化。
先进的高通量检测提供了便利合成材料的快速筛选，从大型图书馆，并提供新的材料，具有灵活的特性，在生物医药研究和开发11-13广泛的应用。利用高通量，聚合物微阵列筛选技术，我们迅速确定了一个简单的聚氨酯（PU134），适用于维护人类干细胞衍生的肝细胞。该聚合物被认为是优于动物源性底物对于肝细胞的分化和功能14-16。我们随后优化的涂覆条件，地形和灭菌过程来访问效果在稳定肝细胞的功能和寿命聚合物的性能。这有针对了解肝细胞生物学细胞建模和再生医学应用基础显著影响。
此处所描述的技术代表了如何由合成聚合物的表面可以进行优化，以保持细胞的表型的实例。我们认为，这种技术提供了高效的无血清肝细胞分化方案的组合有可能提供一种可伸缩的生产肝细胞在体外模型和再生医学中使用。
Protocol
1，合成PHNGAD（聚[1,6-hexanodiol /新戊二醇/二（乙二醇）-alt – 己二酸]二醇）的 方案1：PHNAGD合成 PHNAGD的合成的示意图。 PHNAGD制备的1,6-Hexanodiol的反应中，二甘醇，neoppentyl乙二醇和己二酸。 PHNAGD，聚[1,6-hexanodiol /新戊二醇/二（乙二醇）-alt – 己二酸]二醇。 适用于热处理的单体1,6…
Representative Results
聚合物溶剂影响聚合物涂覆的表面的形貌 聚氨酯134溶解在氯仿中，无论是单独使用或与甲苯或四氢呋喃或二氯甲烷中，并在载玻片上旋涂一层的不同制剂的组合。扫描电子显微镜（SEM）和原子力显微镜（AFM）进行了表征的聚合物涂层的物理性能（ 图1）。使用甲苯或氯仿中得到的涂层不是均匀的，这表明一个不均匀的涂层与PU134沉淀（ 图1A）。…
Discussion
许多用于产生从干细胞的肝细胞的现有方法依赖于动物来源的未定义的矩阵。这些基材可以是昂贵的，充满变数，影响细胞的功能和稳定性，较显著障碍中的应用。因此，我们进行了一个屏幕的量支持的干细胞衍生的肝细胞培养的合成材料。我们已经确定了，一个简单的聚氨酯（PU134），通过聚合PHNGAD，MDI和一个扩展，在结合强大的肝细胞分化的方式形成，稳定肝细胞的表型，当与基质胶15</su…
Declarações
The authors have nothing to disclose.
Acknowledgements
大昌行，MB和FK是由EPSRC按照有关基金的支持。 BL-V和DS分别支持MRC博士助学金。 KC是由来自英国的再生医学平台提供资金支持。
Materials
| Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips | |||
| Shaker | Edmun Bühler | KS-15 | |
| Irradiator | CIS Biointernational | IBL 637 | |
| Spin coater | Specialty Coating System | P-6708 | |
| Scanning Electron Microscope | Philips | XL30CPSEM | |
| Atomic Force Microscope | DimensionV Nanoscope, VEECO | ||
| p4-GLO CYP3A4 | Promega | V8902 | |
| UV bulb | ESCO | ||
| NanoScope analysis software | VEECO | version 1.20 | |
| Fluorescence microscope | Olympus | TH45200 | Use Volocity 4 Software |
| Tissue culture plates | Corning, UK | 3527 | |
| glass slides | Scientific Laboratory Supplies | MIC3308 | |
| Diethylene glycol | Sigma–Aldrich | 93171 | |
| 1,6-hexanediol | Sigma–Aldrich | 240117 | |
| Neopentyl glycol | Sigma–Aldrich | 408255 | |
| Adipic acid | Sigma–Aldrich | 9582 | |
| anhydrous N,N-Dimethylformamide | Sigma–Aldrich | 227056 | |
| Diethyl ether | Sigma–Aldrich | 676845 | |
| titanium (IV) butoxide | Sigma–Aldrich | 244112 | |
| 1,4-butanediol | Sigma–Aldrich | 493732 | |
| Vacuum oven | Thermoscientific | ||
| 4,4’-Methylenebis(phenyl isocyanate) | Sigma–Aldrich | 101688 | |
| Tetrahydrofurane | Sigma–Aldrich | 401757 | |
| Sputter coater | Bal-Tec SCD 050 | ||
| Inmunostaining | |||
| Phosphate buffer saline (-MgCl2, -CaCl2) | Gibco | 10010031 | Store at room temperature |
| PBST, PBS made up with 0.1% TWEEN 20 | Scientific Laboratory Supplies Ltd | EC607 | |
| Methanol | Scientific Laboratory Supplies Ltd | CHE5010 | |
| Bovine Serum Albumin | Sigma-Aldrich, UK | A7906 | |
| MOWIOL 488 DAPI | Calbiochem | 475904 | Made up in Tris HCL and glycerol as per manufacturers instructions |
| Cell culture and Functional assay | |||
| CYP3A activity pGLO kit | Promega | V8902 | |
| Hepatozyme | Gibco | 17705021 | |
| TryLE express | Life Technologies | 12604013 |
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Citar este artigo
Lucendo-Villarin, B., Cameron, K., Szkolnicka, D., Travers, P., Khan, F., Walton, J. G., Iredale, J., Bradley, M., Hay, D. C. Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces. J. Vis. Exp. (91), e51723, doi:10.3791/51723 (2014).