温敏纳米结构表面的组织工程准备
Summary
Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.
Abstract
Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.
Introduction
纳米结构表面最近吸引了大量关注，因为它们的各种潜在应用，包括图案化，细胞培养，清洗，和表面交换。例如，通过荷叶和其它响应表面的纳米结构启发超疏水表面是能够反应以外部刺激1-4。
朗缪尔膜是最广泛研究的聚合物涂层中的一个。朗缪尔膜通过滴两性分子到空气-水界面5-8形成。该膜然后可通过物理或化学吸附被转移到一个固体表面上，并且可以使用垂直和水平转移方法9-12被控制在固体表面上的分子的构象。朗缪尔膜的密度可以通过压缩的空气 – 水界面精确地调节。最近，这种方法也被证明有效的用于制造纳米尺度的海岛structur利用两亲性嵌段共聚物上课。纳米结构被假定为是由疏水性链段的芯和亲水性链段13-17的壳的。此外，在表面上的纳米结构的数量是通过控制在界面上的嵌段共聚物的每个分子的面积（A M）调节。
我们专注于一个原始，独特的无支架组织工程方法，细胞片工程，使用温度响应培养表面。所开发的技术已经应用到再生疗法为各种器官18。温度响应培养表面是由接枝聚（N- -isopropylacrylamide）（PIPAAm），温度响应分子到表面19-27制成。 PIPAAm及其共聚物表现出的低临界溶液温度（LCST）在水性介质中在接近32℃的温度。培养表面还表现出一个温度响应alternati关于疏水性和亲水性之间。在37℃时，PIPAAm接枝的表面变得疏水的，细胞容易附着和增殖的表面上，以及对现有的组织培养聚苯乙烯。当温度降低至20℃时，表面变得亲水，和细胞表面的自发分离。因此，表面上培养的汇合细胞可被收获作为通过改变温度完整片。这些细胞粘附和剥离性能也由朗缪尔膜包衣对实验室演示26,27制成的表面显示。聚苯乙烯（P（ST））和PIPAAm（ST-IPAAm）构成的嵌段共聚物的朗缪尔膜制成。与特定的A M的朗缪尔膜可以水平地转移到疏水改性的玻璃基板。此外，对细胞粘附和脱离由响应于温度的准备好的表面进行评价。
_content“>这里，我们描述协议热 – 反应的两亲嵌段共聚物的玻璃基板上构成的纳米结构的Langmuir膜的制造。我们的方法可以用于在表面科学的各个领域的有机纳米膜提供有效的制造技术，并可以有利于更在有效控制细胞粘附和从表面自发支队。Protocol
Representative Results
Discussion
温度响应性表面由朗缪尔 – 谢弗方法制造，并为细胞粘附/拆卸和细胞片恢复表面性能进行了优化。当使用表面的制造这种方法，几个步骤是关键的。圣-IPAAm分子的分子的组合物具有在表面结构和有很大影响的表面的稳定性，并且通过扩展，对细胞粘附和脱离。特别是，圣IPAAm分子应具有窄的分子量分布。在我们的方法中，两次与不同PIPAAm链长的St-IPAAm分子通过RAFT聚合合成，使分子量和分子量分布?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This study was financially supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Program’s Project for Developing Innovation Systems “Cell Sheet Tissue Engineering Center (CSTEC)” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
Materials
| N-isopropylacrylamide | Kohjin | No catalog number | |
| Azobis(4-cyanovaleric acid) | Wako Pure Chemicals | 016-19332 | |
| Styrene | Sigma-Aldrich | S4972 | |
| 1,3,5-trioxane | Sigma-Aldrich | T81108 | |
| 1,4-Dioxane | Wako Pure Chemicals | 045-24491 | |
| DMEM | Sigma | D6429 | |
| PBS | Nakarai | 11482-15 | |
| Streptomycin | GIBCO BRL | 15140-163 | |
| Penicillin | GIBCO BRL | 15140-122 | |
| Trypsin-EDTA | Sigma | T4174 | |
| FBS | Japan Bioserum | JBS-11501 | |
| BAECs | Health Science Reserch Resources Bank | JCRB0099 | |
| Cover Glasses | Matsunami Glass Industry | C024501 | |
| AFM NanoScope V | Veeco | ||
| 1H NMR INOVA 400 | Varian, Palo Alto | ||
| ATR/FT-IR NICOLET 6700 | Thermo Scientific | ||
| GPC HLC-8320GPC | Tosoh | ||
| TSKgel Super AW2500, AW3000, AW4000 | Tosoh | ||
| Langmuir-Blodgett Deposition Troughs | KSV Instruments | KN 2002 | KSV NIWA Midium trough |
| Nikon ECLIPSE TE2000-U | Nikon |
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