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.

1.聚苯乙烯块聚（N -isopropylacrylamide）通过两步可逆加成断裂链转移（RAFT）自由基聚合合成<ol><li>溶解苯乙烯（153.6毫摩尔），4-氰基-4-（ethylsulfanylthiocarbonyl）sulfanylpentanoic酸（ECT; 0.2毫摩尔）和4,4&#39;-偶氮二（4-氰基戊酸）（ACVA; 0.04毫摩尔）在40ml 1， 4-二恶烷。冻结真空下在液氮中的溶液15-20分钟以除去反应性物质，并逐步在RT解冻。确保该解决方案完全解冻并重复这一冷冻泵解冻循环脱气三次。 </li><li>得到的聚苯乙烯（PST）（分子量:13500），通过聚合一大分子RAFT试剂在70℃的油浴中15小时。 </li><li>沉淀聚苯乙烯大分子RAFT试剂用800毫升乙醚，并在真空干燥。 </li><li>溶解在4ml的1,4-二恶烷的IPAAm单体（4.32毫摩尔），聚苯乙烯大分子RAFT试剂（0.022毫摩尔），和ACVA（0.004毫摩尔）。 </li><li>去掉如在步骤1.1中提到在由冷冻 - 抽吸 - 解冻脱气循环溶液中的氧气。 </li><li>在70℃下进行聚合为在脱气处理后的油浴15小时。获得合成的St-IPAAm分子（MW:32800）中相同的方式作为聚苯乙烯大分子RAFT试剂。 </li></ol> 2.硅烷化修饰的疏水玻璃基板的制备<ol><li>洗涤玻璃基板（24毫米×50毫米）与过量的丙酮和乙醇超声的为5分钟，除去表面的污染物。 </li><li>干燥烘箱中在基片在65℃下30分钟。然后用氧等离子体（400瓦特，3分钟）在RT以激活基片的表面上。 </li><li>浸入基板中含有1％己基三甲过夜在RT silanize基板甲苯。 </li><li>洗硅烷化基板在甲苯和丙酮中浸泡30分钟以除去未反应试剂。 </li><li>在110℃下进行2小时退火衬底以彻底固定在Surface。 </li><li>削减玻璃刀硅烷化衬底25毫米×24毫米，以适应细胞培养皿（碟尺寸:φ35毫米）。 </li></ol> 3.朗缪尔电影和电影调入表面的制备<ol><li>放置朗缪尔膜仪器在机柜防止尘埃积聚。 </li><li>洗朗缪尔槽（尺寸:580毫米×145毫米）中，用蒸馏水和乙醇以除去污染物的障碍。 </li><li>通过用絮的毛巾擦干槽和障碍。然后填充约1.1毫升蒸馏水槽，并设置在槽两侧的障碍。请注意，应在不在下面的步骤溢出从3.5至3.13中加入蒸馏水。 </li><li>热的铂的Wilhelmy板（周长:39.24毫米），用于与一个气体燃烧器监测的表面张力，直到板变红，然后用蒸馏水洗涤以除去污染物。暂停威廉米悬片上附接至电线表面压力测量仪。 </li><li>根据制造商的协议零表面压力测量仪。通过在槽的两侧的障碍压缩上槽的空气-水界面直到接口达到大约50厘米2没有聚合物的任何液滴。 </li><li>抽吸小的污染物，直到表面压力几乎为0达因/厘米。 </li><li>重新定位在两侧的障碍，并加入蒸馏水，以补偿的蒸馏水从步骤3.6的减少。 </li><li>溶解5毫克合成的St-IPAAm分子在5毫升氯仿中显影溶液。 注意:二氯甲烷或甲苯，也可以用作溶剂。 </li><li>轻轻放下圣IPAAm的27微升溶于氯仿到使用微量或微量低谷。 </li><li>等待5分钟后，以允许氯仿完全蒸发，水平移动两个壁垒以压缩的St-IPAAm molecu勒在界面。保持在0.5毫米/秒，直到50达到厘米2目标区域的障碍压缩率。 注:一种快速的压缩率会导致在朗缪尔薄膜缺陷。 </li><li>测量表面压力具有根据制造商的协议压缩过程中附着于表面压力测量仪器的铂的Wilhelmy板（π）-A 米等温线。 </li><li>到达目标区域的大小后，保持表面5分钟，以使圣IPAAm分子放松;该分子不压缩后立即达到平衡。 </li><li>使用传送装置5分钟以有力吸附膜转移的Langmuir膜疏水改性的玻璃基板。固定在装置上平行疏水玻璃基板。将设备连接到对准平台和垂直移动。 </li><li>在一个desiccato传送装置和干燥1天水平提起衬底河</li></ol> 4.培养细胞和朗缪尔电影转印表面优化细胞粘附和支队<ol><li>至在37℃，在5％的组织培养聚苯乙烯（TCPS）用含有10％胎的Dulbecco改良的Eagle培养基（DMEM）制备细胞悬浮液，培养的牛颈动脉内皮细胞（牛主动脉内皮）至三分之一合流的 CO 2和95％空气牛血清（FBS）和100单位/毫升青霉素。 </li><li>达到汇合后，在37℃下在5％CO 2和95％空气治疗的BAECs用3ml的0.25％胰蛋白酶-EDTA的3分钟。 </li><li>通过加入10毫升含有10％FBS的所述DMEM中停用胰蛋白酶-EDTA，并收集细胞悬液至50ml锥形管中。 </li><li>离心，在120×g离心5分钟，吸出上清液。重悬用10ml的DMEM中的细胞。 </li><li>将在紫外线照射下的圣IPAAm表面干净的长椅上消毒5分钟。 </li><li>种子回收CE在圣IPAAm LLS表面以1.0×10 4个细胞/ cm 2通过一次性血细胞计数器计数的浓度，并通过在37℃下装有的培养箱，用5％ 的 CO 2和95％的显微镜观察表面上的细胞空气。 注:装备净化台紫外线消毒的圣IPAAm表面。 </li><li>通过用放大10倍相差显微镜，在37℃大约24.5小时粘附的BAECs的记录时间推移的图像。后BAEC粘附，在20℃下从圣IPAAm表面的BAECs的记录脱离大约3.5小时。 </li></ol>在朗缪尔电影调入表面5.细胞板材加工<ol><li>以相同的方式用于培养牛主动脉内皮第4节中叙述。 </li><li>种子上的St-IPAAm表面共1.0×10 5个细胞/ cm 2的和在5％CO 2在37℃下孵育3天。汇合的BAECs在20℃自发分离。</li>
</ol>

<ol>
	<li>Bae, Y. H., Kwon, I. C., Pai, C. M., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+of+macromolecules+from+electrical+and+chemical+stimuli-responsive+hydrogels.">Controlled release of macromolecules from electrical and chemical stimuli-responsive hydrogels.</a> Makromol. Chem., Macromol. Symp. 70-71 (1), 173-181 (1993).</li><li>Fu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+control+of+free+energy+and+topography+of+nanostructured+surfaces.">Reversible control of free energy and topography of nanostructured surfaces.</a> J. Am. Chem. Soc. 126 (29), 8904-8905 (2004).</li><li>Nykanen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+behavior+and+temperature-responsive+molecular+filters+based+on+self-assembly+of+polystyrene-block-poly(N-isopropylacrylamide)-block-polystyrene.">Phase behavior and temperature-responsive molecular filters based on self-assembly of polystyrene-block-poly(N-isopropylacrylamide)-block-polystyrene.</a> Macromolecules. 40 (16), 5827-5834 (2007).</li><li>Sun, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+switching+between+superhydrophilicity+and+superhydrophobicity.">Reversible switching between superhydrophilicity and superhydrophobicity.</a> Angew. Chem. Int. Ed. Engl. 43 (3), 357-360 (2004).</li><li>Shuler, R. L., Zisman, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Study+of+the+behavior+of+Polyoxyethylene+at+the+air-water+interface+by+wave+damping+and+other+methods.">A Study of the behavior of Polyoxyethylene at the air-water interface by wave damping and other methods.</a> J. Phys. Chem. 74 (7), 1523-1534 (1970).</li><li>Kawaguchi, M., Sauer, B. B., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+monolayer+dynamics+at+the+air/water+interface+by+surface+light+scattering.">Polymeric monolayer dynamics at the air/water interface by surface light scattering.</a> Macromolecules. 22 (4), 1735-1743 (1989).</li><li>Saito, W., Kawaguchi, M., Kato, T., Imae, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+solvent+and+temperature+effects+on+interfacial+properties+of+Poly+(N-isopropylacrylamide)+films+at+the+air-water+interface.">Spreading solvent and temperature effects on interfacial properties of Poly (N-isopropylacrylamide) films at the air-water interface.</a> Langmuir. 7463 (11), 5947-5950 (1996).</li><li>Jheng, K. T., Hsu, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+effect+of+PMMA+on+its+miscibility+with+PS-b-PEO+at+the+air/water+interface.">Molecular weight effect of PMMA on its miscibility with PS-b-PEO at the air/water interface.</a> J. App. Polym. Sci. 125 (3), 1986-1992 (2012).</li><li>Biesalski, M. A., Knaebel, A., Tu, R., Tirrell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+on+a+polymerized+peptide-amphiphile+monolayer.">Cell adhesion on a polymerized peptide-amphiphile monolayer.</a> Biomaterials. 27 (8), 1259-1269 (2006).</li><li>Da Silva, A. M. P. S. G., Lopes, S. I. C., Brogueira, P., Prazeres, T. J. V., Beija, M., Martinho, J. M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-responsiveness+of+poly(N,N-diethylacrylamide)+polymers+at+the+air-water+interface:+The+effect+of+a+hydrophobic+block.">Thermo-responsiveness of poly(N,N-diethylacrylamide) polymers at the air-water interface: The effect of a hydrophobic block.</a> Journal of colloid interface sci. 327 (1), 129-137 (2008).</li><li>Wang, S. Q., Zhu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+method+to+prepare+smooth+and+homogeneous+polymer+brush+surfaces+of+varied+brush+thickness+and+grafting+density.">Facile method to prepare smooth and homogeneous polymer brush surfaces of varied brush thickness and grafting density.</a> Langmuir. 25 (23), 13448-13455 (2009).</li><li>Estillore, N. C., Park, J. Y., Advincula, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langmuir&#8722;Schaefer+(LS)+macroinitiator+film+control+on+the+grafting+of+a+thermosensitive+polymer+brush+via+surface+initiated-ATRP.">Langmuir&#8722;Schaefer (LS) macroinitiator film control on the grafting of a thermosensitive polymer brush via surface initiated-ATRP.</a> Macromolecules. 43 (16), 6588-6598 (2010).</li><li>Seo, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanowire+and+mesh+conformations+of+diblock+copolymer+blends+at+the+air/water+interface.">Nanowire and mesh conformations of diblock copolymer blends at the air/water interface.</a> Nano Lett. 4 (3), 483-486 (2004).</li><li>Lu, Q., Bazuin, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent-assisted+formation+of+nanostrand+networks+from+supramolecular+diblock+copolymer/surfactant+complexes+at+the+air/water+interface.">Solvent-assisted formation of nanostrand networks from supramolecular diblock copolymer/surfactant complexes at the air/water interface.</a> Nano lett. 5 (7), 1309-1314 (2005).</li><li>Nagano, S., Matsushita, Y., Ohnuma, Y., Shinma, S., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+a+highly+ordered+dot+array+of+surface+micelles+of+a+block+copolymer+via+liquid+crystal-hybridized+self-assembly.">Formation of a highly ordered dot array of surface micelles of a block copolymer via liquid crystal-hybridized self-assembly.</a> Langmuir. 22 (12), 5233-5236 (2006).</li><li>Perepichka, I. I., Borozenko, K., Badia, A., Bazuin, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+order+transition+in+nanodot-forming+diblock.">Pressure-induced order transition in nanodot-forming diblock.</a> J. Am. Chem. Soc. 133 (493), 19702-19705 (2011).</li><li>Wang, X. L., Ma, X. Y., Zang, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+behavior+of+polystyrene-b-poly(acrylicacid)+at+the+air-water+interface.">Aggregation behavior of polystyrene-b-poly(acrylicacid) at the air-water interface.</a> Soft Matter. 9 (2), 443-453 (2013).</li><li>Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sheet+engineering.">Cell sheet engineering.</a> Mater. Today. 7 (5), 42-47 (2004).</li><li>Rollason, G., Daviest, J. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+report+on+cell+culture+on+a+thermally+reversible+copolymer.">Preliminary report on cell culture on a thermally reversible copolymer.</a> Biomaterials. 14 (2), 153-155 (1993).</li><li>Park, Y. S., Ito, Y., Imanishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeation+control+through+porous+membranes+immobilized+with+thermosensitive+polymer.">Permeation control through porous membranes immobilized with thermosensitive polymer.</a> Langmuir. 14 (4), 910-914 (1998).</li><li>Kwon, O. H., Kikuchi, A., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+cell+sheet+recovery+by+co-grafting+of+PEG+with+PIPAAm+onto+porous+cell+culture+membranes.">Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes.</a> Biomaterials. 24 (7), 1223-1232 (2003).</li><li>Kikuchi, A., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+designs+of+biomedical+materials:+applications+of+cell+sheet+engineering+to+functional+regenerative+tissues+and+organs.">Nanostructured designs of biomedical materials: applications of cell sheet engineering to functional regenerative tissues and organs.</a> J. Control. Release. 101 (1-3), 69-84 (2005).</li><li>Fukumori, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+ultra-thin+temperature-responsive+polymer+layer+and+its+polymer+thickness+dependency+on+cell+attachment/detachment+properties.">Characterization of ultra-thin temperature-responsive polymer layer and its polymer thickness dependency on cell attachment/detachment properties.</a> Macromol. biosci. 10 (10), 1117-1129 (2010).</li><li>Takahashi, H., Nakayama, M., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+chain+length+and+graft+density+of+thermoresponsive+polymer+brushes+for+optimizing+cell+sheet+harvest.">Controlled chain length and graft density of thermoresponsive polymer brushes for optimizing cell sheet harvest.</a> Biomacromolecules. 11 (8), 1991-1999 (2010).</li><li>Nakayama, M., Yamada, N., Kumashiro, Y., Kanazawa, H., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+poly(N-isopropylacrylamide)-based+block+copolymer+coating+for+optimizing+cell+sheet+fabrication.">Thermoresponsive poly(N-isopropylacrylamide)-based block copolymer coating for optimizing cell sheet fabrication.</a> Macromol. Biosci. 12 (6), 751-760 (2012).</li><li>Sakuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+adhesion+and+detachment+on+Langmuir-Schaefer+surface+composed+of+dodecyl-terminated+thermo-responsive+polymers.">Control of cell adhesion and detachment on Langmuir-Schaefer surface composed of dodecyl-terminated thermo-responsive polymers.</a> J. Biomater. Sci., Polym. Ed. 25 (5), 431-443 (2014).</li><li>Sakuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+nanostructured+surfaces+generated+by+the+Langmuir-Schaefer+method+are+suitable+for+cell+sheet+fabrication.">Thermoresponsive nanostructured surfaces generated by the Langmuir-Schaefer method are suitable for cell sheet fabrication.</a> Biomacromolecules. 15 (11), 4160-4167 (2014).</li><li>Moad, G., Chong, Y. K., Postma, A., Rizzardo, E., Thang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+RAFT+polymerization:+the+synthesis+of+polymers+with+defined+end-groups.">Advances in RAFT polymerization: the synthesis of polymers with defined end-groups.</a> Polymer. 46 (19), 8458-8468 (2005).</li><li>Nishida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Reconstruction+with+Tissue-Engineered+Cell+Sheets+Composed+of+Autologous+Oral+Mucosal+Epithelium.">Corneal Reconstruction with Tissue-Engineered Cell Sheets Composed of Autologous Oral Mucosal Epithelium.</a> N. Engl. J. Med. 351 (12), 1187-1196 (2004).</li><li>Ohki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+esophageal+stricture+after+endoscopic+submucosal+dissection+using+tissue-engineered+cell+sheets.">Prevention of esophageal stricture after endoscopic submucosal dissection using tissue-engineered cell sheets.</a> Gastroenterology. 143 (3), 582-588 (2012).</li><li>Sawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineered+myoblast+sheets+improved+cardiac+function+sufficiently+to+discontinue+LVAS+in+a+patient+with+DCM:+report+of+a+case.">Tissue engineered myoblast sheets improved cardiac function sufficiently to discontinue LVAS in a patient with DCM: report of a case.</a> Surg. Today. 42 (2), 181-184 (2012).</li></ol>

All authors contributed equally to writing the manuscript and have approved the final version. The authors declare that they have no competing financial interests.

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的控制。 应采取措施，以防止空气 - 水界面的污染的圣IPAAm表面的制备过程中，以避免在纳米结构的缺陷。下探聚合物分子到界面上，contaminan之前TS应该吸气，直到表面压力达到约0达因/厘米。污染往往积累围绕槽和威廉米悬片的边缘。因为威廉米悬板具有在其表面上的一些污染，它被退火。当使用纸的Wilhelmy板，在界面处的抽吸步骤应至少重复两次。该πA M等温线也取决于污染的在空气-水界面的存在。我们建议的等温线的曲线膜的制造之前得到一次以上。因为疏水性修饰的玻璃基板的污染，也可能发生，衬底应与新鲜空气或氮气吹入以去除污染。 始终如一地制造的Langmuir膜转印表面（圣IPAAm表面），聚合物的疏水性链段是重要的，因为朗缪尔膜与疏水性修饰的玻璃I之间的疏水相互作用<html> S中的反应的驱动力。在这项研究中，由于嵌段共聚物由聚苯乙烯，这是强疏水性的，并在室温下亲水PIPAAm的，被转印膜稳定地，即使在水或细胞培养条件附着到基底上。这表明，疏水性链段中制造一个健壮的St-IPAAm表面发挥重要的作用。 为了控制上的温度响应表面的细胞粘附和脱离，两者的分子组成和密度的精确控制是重要的。在此朗缪尔-谢弗方法中，通过下降分子的给定量和空气-水界面的目标区域合成的聚合物的每个分子的面积（A M）可以通过压缩进行控制。滴加圣IPAAm分子的A M可以在理论上由下列公式计算: D / 53465 / 53465eq1.jpg"/&gt; 其中A是接口，M W为面积 是圣IPAAms的分子量，c是氯仿溶液的浓度，N A是阿伏伽德罗数，V是聚合物溶液的体积减少。纳米海岛结构加工后的St-IPAAm面观察，因为两亲聚合物形成空气-水界面自组织结构，因为在以往的研究描述了13-17。纳米结构的尺寸和量通过A M和St-IPAAms的分子量控制。 细胞粘附和脱离上通过各种方法，包括电子束照射，表面引发RAFT聚合和旋涂19-27制造PIPAAm涂层表面进行评价。因为密度，分子量，和PIPAAm结构影响CEL升粘附和脱离，用于制造PIPAAm涂覆表面精确的技术是重要的。在此朗缪尔 - 谢弗方法中，如上所述密度，分子量和表面的纳米结构可以控制。圣-IPAAm表面上的细胞粘附和脱离者急剧各A M和St-IPAAm分子组合物的影响，和圣IPAAm的一些条件面为细胞粘附和脱离可以被进一步优化。这些结果表明，该方法可以控制的St-IPAAm表面和细胞之间的相互作用。 为再现地恢复完整的细胞片，两亲性聚合物的设计是最重要的。当仅亲水性聚合物是由朗缪尔 - 谢弗方法的表面上制成，涂覆聚合物容易被蒸馏水或培养基冲洗掉。这个结果表明，弱疏水聚合物不应被用于在此甲基再现地回收细胞片OD因为聚合物改性，表面是在水环境不稳定。在我们的方法中，两亲聚合物的亲水性链段使表面上健壮制造不受水溶液冲洗掉。另外，由于在朗缪尔膜在这项研究中平行转移到玻璃基板上，观察到的表面上的纳米级的海岛结构。虽然朗缪尔膜的上基底侧的结构是公知的是，从不同的顶侧，该结构固定由一个平行转移。 细胞疗法和再生医学一直专注于医治病人谁不响应其他治疗的可能性。我们的实验室已使用具有在组织工程应用潜力上制造温度响应培养表面细胞片工程开发的原始的，独特的自由支架组织工程方法。与细胞片几个临床试验是alreaDY正在进行和已经证明成功的结果为多种类型的组织29-31。虽然牛主动脉内皮被用作细胞来源以制造细胞片层，其它细胞来源，包括干细胞，可用于通过优化的米和组合的St-IPAAm分子的分子的组合物来制造上的圣IPAAm表面片。这种非传统的技术将不仅有助于容易生产纳米结构表面，而且还用于在再生医学使用基本细胞培养物的策略。

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<html>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"&gt;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。

<table border="0" cellpadding="0" cellspacing="0" width="777">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">N-isopropylacrylamide</td>
			<td style="width:193px;">Kohjin</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">No catalog number</td>
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			<td height="21" style="height:21px;width:299px;">Azobis(4-cyanovaleric acid)</td>
			<td style="width:193px;">Wako Pure Chemicals</td>
			<td style="width:119px;">016-19332</td>
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			<td height="21" style="height:21px;width:299px;">Styrene</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:119px;">S4972</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1,3,5-trioxane</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:119px;">T81108</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1,4-Dioxane</td>
			<td style="width:193px;">Wako Pure Chemicals</td>
			<td style="width:119px;">045-24491</td>
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			<td height="21" style="height:21px;">PBS</td>
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			<td style="width:119px;">11482-15</td>
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			<td height="21" style="height:21px;width:299px;">Streptomycin</td>
			<td style="width:193px;">GIBCO BRL</td>
			<td style="width:119px;">15140-163</td>
			<td></td>
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			<td height="21" style="height:21px;width:299px;">Penicillin</td>
			<td style="width:193px;">GIBCO BRL</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
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			<td height="21" style="height:21px;width:299px;">Trypsin-EDTA</td>
			<td style="width:193px;">Sigma</td>
			<td style="width:119px;">T4174</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FBS</td>
			<td style="width:193px;">Japan Bioserum</td>
			<td style="width:119px;">JBS-11501</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:299px;">BAECs</td>
			<td style="width:193px;">Health Science Reserch Resources Bank</td>
			<td style="width:119px;">JCRB0099</td>
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			<td height="21" style="height:21px;width:299px;">Cover Glasses</td>
			<td style="width:193px;">Matsunami Glass Industry</td>
			<td style="width:119px;">C024501</td>
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			<td height="21" style="height:21px;width:299px;">AFM NanoScope V</td>
			<td style="width:193px;">Veeco</td>
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			<td></td>
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			<td height="24" style="height:24px;width:299px;">1H NMR INOVA 400</td>
			<td style="width:193px;">Varian, Palo Alto</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:299px;">ATR/FT-IR NICOLET 6700</td>
			<td style="width:193px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">GPC HLC-8320GPC</td>
			<td style="width:193px;">Tosoh</td>
			<td style="width:119px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">TSKgel Super AW2500, AW3000, AW4000</td>
			<td style="width:193px;">Tosoh</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:299px;">Langmuir-Blodgett Deposition Troughs&#160;</td>
			<td style="width:193px;">KSV Instruments</td>
			<td style="width:119px;">KN 2002</td>
			<td>KSV NIWA Midium trough</td>
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			<td height="21" style="height:21px;width:299px;">Nikon ECLIPSE TE2000-U</td>
			<td style="width:193px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of thermoresponsive nanostructured surfaces for tissue engineering

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.

聚苯乙烯和聚（N- -isopropylacrylamide）（圣IPAAms）具有特定分子量的构成的嵌段共聚物通过RAFT自由基聚合来合成。 ECT被作为Moad 等人 28所述制备作为链转移剂。不同PIPAAm链长的二圣IPAAm分子合成，并且所得到的嵌段聚合物进行表征通过1 H核磁共振（NMR）和凝胶渗透色谱（GPC）。圣IPAAms的分子量分别为32800和67900，具有窄分子量分布（1.31和1.50）。聚苯乙烯大分子RAFT试剂和PIPAAm的单体转化率被发现是17.4％和85.0％以上。合成的圣IPAAms被命名为圣IPAAm170和圣IPAAm480分别。 每个分子不同区域（A M），朗缪尔电影以是捏造滴加溶解在氯仿溶液（ 图1A）的St-IPAAm分子空气-水界面。滴加圣IPAAm分子之后，空气-水界面上的表面压力固定为0达因/厘米，不断增加的屏障（ 图1B）在压缩过程中关闭的接口。在形成的空气-水界面上的Langmuir膜的任何缺陷可以从该πA M等温线来推断。所述制备的St-IPAAm朗缪尔膜转印到疏水改性的玻璃基板（圣IPAAm面）（ 图1C）。膜转印表面用原子力显微镜（AFM）进行评价。 图2示出了圣IPAAm170和St-IPAAm480表面的原子力显微镜地形图（1×1微米）。观察的St-IPAAm表面上的纳米结构，而这种结构分别在裸疏水基材上很少观察到。纳米结构的尺寸和形状的强烈dependenT于A M和圣IPAAm组成。 AFM地形图像证实，朗缪尔膜可以均匀地转印到疏水性修饰玻璃基板和，表面形貌可以由分子组成和A M进行控制。 圣-IPAAm朗缪尔膜的稳定性是由衰减全反射评估叶变换红外分光镜（ATR / FT-IR）。 PIPAAms的疏水玻璃基板上的量可以通过从千厘米峰强度比-1从玻璃（的Si-O）1650 -1从PIPAAm（C = O）衍生衍生获得的校准线来估计。校准线，从一系列已知量的疏水性玻璃PIPAAm铸造的计算。被发现的St-IPAAm170（10纳米2 /分子）和St-IPAAm480在衬底上（40纳米2 /分子）的PIPAAm的量为0.87微克/ cm 2，并且分别0.63微克/厘米2。 PIPAAm的表面上，用蒸馏水洗涤后的量几乎相同的表面上，而不洗涤。这些结果表明，所制作的圣IPAAm表面的水分条件是稳定的。 接下来，我们检查了附着力和圣IPAAm表面上的牛颈动脉内皮细胞（牛主动脉内皮）的脱离。在40纳米2 /分子的圣IPAAm480表面上粘附和分离的BAECs的延时拍摄显示在动画图1。粘附的BAECs在37℃来自圣IPAAm170和圣IPAAm480降低后迅速分离温度至20℃。上的St-IPAAm170和St-IPAAm480粘附细胞在37℃的数目为0.6×10 4个细胞/ cm 2和0.9×10 4个细胞/ cm 2，分别表示贴壁细胞的数量S为被圣IPAAm表面的组成进行调制。细胞片层也可通过分离来自这些的St-IPAAm表面汇合培养的牛主动脉内皮回收。将回收的细胞片可视化为二维单元结构（ 图3）。后在圣IPAAm170和St-IPAAm480培养3天达到汇合的细胞表面，在37℃，细胞片减少37℃的温度至20℃后迅速恢复。分子量和St-IPAAms对细胞片恢复A M的效果概括在表1中 。细胞片的生存能力通过台盼蓝染色的BAECs片的分离之后进行评价。用胰蛋白酶-EDTA在37ºC在玻璃基板上处理的细胞用作对照。上的St-IPAAm170，圣IPAAm480和疏水玻璃基板作为控制死细胞比率计算为约11.0％，7.7％，和11.7％之间。这些结果表明，策LL存活率几乎不受温度降低的影响。 <img alt="图1" src="/files/ftp_upload/53465/53465fig1.jpg" /> 图1:一个温敏的Langmuir膜转印表面的制备 （A）中的聚苯乙烯- 嵌段 -聚（N- -isopropylacrylamide）（圣IPAAm）的氯仿溶液轻轻滴到空气-水界面。压缩过程中测得的表面压力，以检测在朗缪尔膜的任何缺陷。 （B）的两个障碍被用来压缩所述接口上的圣IPAAm分子直至50厘米达到2目标区域。 （℃）压缩之后，经疏水改性的覆盖玻璃基片水平放置在接口上与对准平台5分钟。将基板水平地提升并干燥1天。这个数字已经从萨克出版了略作修改UMA 等人 27 <img alt="图2" src="/files/ftp_upload/53465/53465fig2.jpg" /> 图 2: 朗缪尔电影传送面（ST-IPAAm面）的原子力显微镜（AFM）地形图像（1×1微米）左面板:ST-IPAAm170与10纳米2 /分子的A M纳米结构表面。右面板:ST-IPAAm480与40纳米2 /分子的A M纳米结构表面。在用磷酸盐掺杂的硅悬臂用的3 N / m的弹簧常数和70-90千赫共振频率轻敲模式得到的AFM图像。 <img alt="图3" src="/files/ftp_upload/53465/53465fig3.jpg" /> 图 3: 圣IPAAm480朗缪尔膜转印表面上的回收细胞片和40nm的A M的宏观图像 <s高达&gt; 2 /分子。牛颈动脉内皮细胞（牛主动脉内皮）接种于圣IPAAm480表面，并在37℃孵育3天。达到汇合后，牛主动脉内皮自发脱离在20℃，细胞片回收后大约30分钟。插图图像示出了由相差显微镜（10X）观察到的表面上的confluently培养牛主动脉内皮。 <img alt="电影1" src="/files/ftp_upload/53465/53465movie1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53465/movie1.mp4">动画图1: <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53465/movie1.mp4">（</a></a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53465/movie1.mp4">右键点击下载）</a>牛主动脉内皮的粘附和支队与圣IPAAm480和40纳米2 /分子的上午朗缪尔膜转印表面上时间推移照片。该图像是在2分钟间隔收集，收集的图像被提供作为电影在960x速度。该在电影中比例尺为50微米。 <table fo:keep-together.within-page="1"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td colspan="3">圣IPAAm170 </td><td colspan="3">圣IPAAm480 </td></tr><tr height="23"><td> 3 [纳米2 /分子] </td><td> 10纳米2 /分子] </td><td> 40纳米2 /分子] </td><td> 3 [纳米2 /分子] </td><td> 10纳米2 /分子] </td><td> 40纳米2 /分子] </td></tr><tr><td>附着力弱</td><td>好</td><td>很难恢复</td><td>附着力弱</td><td>附着力弱</td><td>好</td></tr></tbody></table> 表1: 用各种密度和PIPAAm链长朗缪尔膜转印表面的牛颈动脉内皮细胞（BAEC）片恢复 "好"代表完好策LL表恢复。 "几乎恢复"是指该细胞可以是confluently培养和培养的细胞，即使在温度降低后显示出从表面很少或没有脱离。 "弱粘附"是指细胞不培养，在37℃至铺满3天。

Watch this Scientific Journal Video about Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering at JoVE.com

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

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.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

preparation-thermoresponsive-nanostructured-surfaces-for-tissue

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.2K 次观看.  Tokyo University of Science. 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.

视频: Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...