Name: printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture
Uploaded: 2013-07-10T14:00:00
Description: Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

我们感谢德博拉·STUDER的帮助与bioprinter。 这项工作是由欧盟第七框架计划（FP7/2007-2013）根据赠款协议Ñ°NMP4-SL-2009-229292。

1。下游的泊洛沙姆407的解决方案如果可用，泊洛沙姆溶液进行准备在寒冷的房间（4℃）。如果没有可用的，一个玻璃瓶放置在烧杯中，用冰冷的水填充。在较高的温度下，将泊洛沙姆凝胶点以上，因此无法正确地溶解。 <ol><li>加入60毫升冰冷的PBS溶液倒入玻璃瓶中，并用磁力搅拌器剧烈搅拌。 </li><li>称取24.5克泊洛沙姆，并将其添加少量的冷PBS。等待，直到泊洛沙姆部分溶...

<ol>
	<li>Fedorovich, N. E., 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=Evaluation+of+photocrosslinked+Lutrol+hydrogel+for+tissue+printing+applications.">Evaluation of photocrosslinked Lutrol hydrogel for tissue printing applications.</a> Biomacromolecules. 10, 1689-1696 (2009).</li><li>Lee, K. B., Park, S. J., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+nanoarrays+generated+by+Dip-Pen+Nanolithography.">Protein nanoarrays generated by Dip-Pen Nanolithography.</a> Abstr Pap Am Chem S. 223, C94 (2002).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annual review of biomedical engineering. 3, 335-373 (2001).</li><li>Mironov, V., Reis, N., Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+bioprinting:+a+beginning.">Review: bioprinting: a beginning.</a> Tissue engineering. 12, 631-634 (2006).</li><li>Odde, D. J., Renn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-guided+direct+writing+of+living+cells.">Laser-guided direct writing of living cells.</a> Biotechnology and bioengineering. 67, 312-318 (2000).</li><li>Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting:+inkjet+printing+proteins+and+hybrid+cell-containing+materials+and+structures.">Bioprinting: inkjet printing proteins and hybrid cell-containing materials and structures.</a> J Mater Chem. 18, 5717-5721 (1039).</li><li>Therriault, D., White, S. R., Lewis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaotic+mixing+in+three-dimensional+microvascular+networks+fabricated+by+direct-write+assembly.">Chaotic mixing in three-dimensional microvascular networks fabricated by direct-write assembly.</a> Nature. 2, 265-271 (2003).</li><li>Engelhardt, 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=Fabrication+of+2D+protein+microstructures+and+3D+polymer-protein+hybrid+microstructures+by+two-photon+polymerization.">Fabrication of 2D protein microstructures and 3D polymer-protein hybrid microstructures by two-photon polymerization.</a> Biofabrication. 3, 025003 (2011).</li><li>Mironov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+technology+to+produce+living+tissue.">Printing technology to produce living tissue.</a> Expert opinion on biological therapy. 3, 701-704 (2003).</li><li>Mironov, V., Kasyanov, V., Drake, C., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+promises+and+challenges.">Organ printing: promises and challenges.</a> Regenerative medicine. 3, 93-103 (2008).</li><li>Mironov, V., Kasyanov, V., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+from+bioprinter+to+organ+biofabrication+line.">Organ printing: from bioprinter to organ biofabrication line.</a> Current opinion in biotechnology. 22, 667-673 (2011).</li><li>Fedorovich, N. E., De Wijn, J. R., Verbout, A. J., Alblas, J., Dhert, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fiber+deposition+of+cell-laden,+viable,+patterned+constructs+for+bone+tissue+printing.">Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing.</a> Tissue engineering. Part A. 14, 127-133 (2008).</li><li>Dhariwala, B., Hunt, E., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+of+tissue-engineering+constructs,+using+photopolymerizable+hydrogels+and+stereolithography.">Rapid prototyping of tissue-engineering constructs, using photopolymerizable hydrogels and stereolithography.</a> Tissue engineering. 10, 1316-1322 (2004).</li><li>Cook, C., Wang, T., Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet+Printing+of+Enzymes+for+Glucose+Biosensors.">Inkjet Printing of Enzymes for Glucose Biosensors.</a> Mater Res Soc Symp P. 1191, 103-109 (2009).</li><li>Cui, X., Gao, G., Qiu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+myotube+formation+using+bioprinting+technology+for+biosensor+applications.">Accelerated myotube formation using bioprinting technology for biosensor applications.</a> Biotechnol Lett. , 1-7 (2012).</li><li>Wang, T. M., Cook, C., Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+a+Glucose+Biosensor+by+Piezoelectric+Inkjet+Printing.">Fabrication of a Glucose Biosensor by Piezoelectric Inkjet Printing.</a> , 82-85 (2009).</li><li>Shim, J. H., Lee, J. S., Kim, J. Y., Cho, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+of+a+mechanically+enhanced+three-dimensional+dual+cell-laden+construct+for+osteochondral+tissue+engineering+using+a+multi-head+tissue/organ+building+system.">Bioprinting of a mechanically enhanced three-dimensional dual cell-laden construct for osteochondral tissue engineering using a multi-head tissue/organ building system.</a> J. Micromech. Microeng. 22, (2012).</li><li>Malmsten, M., Lindman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembly+in+Aqueous+Block+Copolymer+Solutions.">Self-Assembly in Aqueous Block Copolymer Solutions.</a> Macromolecules. 25, 5440-5445 (1021).</li><li>Cebotari, 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=Clinical+application+of+tissue+engineered+human+heart+valves+using+autologous+progenitor+cells.">Clinical application of tissue engineered human heart valves using autologous progenitor cells.</a> Circulation. 114, I132-I137 (2006).</li><li>Matsumura, G., Hibino, N., Ikada, Y., Kurosawa, H., Shin'oka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+application+of+tissue+engineered+vascular+autografts:+clinical+experience.">Successful application of tissue engineered vascular autografts: clinical experience.</a> Biomaterials. 24, 2303-2308 (2003).</li><li>Kropp, B. P., Zwischenberger, J. B. <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+autologous+bladders:+new+possibilities+for+cystoplasty.">Tissue-engineered autologous bladders: new possibilities for cystoplasty.</a> Nature clinical practice. Urology. 3, 588-589 (2006).</li><li>Oberpenning, F., Meng, J., Yoo, J. J., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+reconstitution+of+a+functional+mammalian+urinary+bladder+by+tissue+engineering.">De novo reconstitution of a functional mammalian urinary bladder by tissue engineering.</a> Nature. 17, 149-155 (1999).</li><li>Wood, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+skin.">Tissue engineering of skin.</a> Clinics in plastic surgery. 39, 21-32 (2012).</li><li>Groeber, F., Holeiter, M., Hampel, M., Hinderer, S., Schenke-Layland, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+tissue+engineering--in+vivo+and+in+vitro+applications.">Skin tissue engineering--in vivo and in vitro applications.</a> Clinics in plastic surgery. 39, 33-58 (2012).</li><li>Bannasch, H., Momeni, A., Knam, F., Stark, G. B., Fohn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+skin+substitutes.">Tissue engineering of skin substitutes.</a> Panminerva medica. 47, 53-60 (2005).</li><li>Jakab, K., Neagu, A., Mironov, V., Forgacs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+fiction+or+science.">Organ printing: fiction or science.</a> Biorheology. 41, 371-375 (2004).</li><li>Boland, T., Mironov, V., Gutowska, A., Roth, E. A., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+organ+printing+2:+fusion+of+cell+aggregates+in+three-dimensional+gels.">Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels.</a> The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology. 272, 497-502 (2003).</li><li>Mironov, V., 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=Organ+printing:+tissue+spheroids+as+building+blocks.">Organ printing: tissue spheroids as building blocks.</a> Biomaterials. 30, 2164-2174 (2009).</li><li>Raimondi, M. 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=Two-photon+laser+polymerization:+from+fundamentals+to+biomedical+application+in+tissue+engineering+and+regenerative+medicine.">Two-photon laser polymerization: from fundamentals to biomedical application in tissue engineering and regenerative medicine.</a> Journal of applied biomaterials. 10, 56-66 (2012).</li><li>Miller, J. 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=Rapid+casting+of+patterned+vascular+networks+for+perfusable+engineered+three-dimensional+tissues.">Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues.</a> Nature. 11, 768-774 (2012).</li><li>Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S., Dubruel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+trends+and+limitations+in+hydrogel-rapid+prototyping+for+tissue+engineering.">A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering.</a> Biomaterials. 33, 6020-6041 (2012).</li><li>Murphy, S. V., Skardal, A., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hydrogels+for+bio-printing+applications.">Evaluation of hydrogels for bio-printing applications.</a> Journal of biomedical materials research. Part A. 101, 272-284 (2013).</li><li>He, J., Li, D., Liu, Y., Gong, H., Lu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+fabrication+of+microstructured+chitosan-gelatin+scaffolds+using+rapid+prototyping.">Indirect fabrication of microstructured chitosan-gelatin scaffolds using rapid prototyping.</a> Virtual and Physical Prototyping. 3, 159-166 (2008).</li><li>Sachlos, E., Reis, N., Ainsley, C., Derby, B., Czernuszka, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+collagen+scaffolds+with+predefined+internal+morphology+made+by+solid+freeform+fabrication.">Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication.</a> Biomaterials. 24, 1487-1497 (2003).</li><li>Lee, W., 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=On-demand+three-dimensional+freeform+fabrication+of+multi-layered+hydrogel+scaffold+with+fluidic+channels.">On-demand three-dimensional freeform fabrication of multi-layered hydrogel scaffold with fluidic channels.</a> Biotechnology and bioengineering. 105, 1178-1186 (2010).</li><li>Turturro, M., Christenson, M., Larson, J., Papavasiliou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+(MMP)+sensitive+PEG+diacrylate+(PEGDA)+hydrogels+with+spatial+variations+in+matrix+properties+direct+vascular+cell+invasion.">Matrix metalloproteinase (MMP) sensitive PEG diacrylate (PEGDA) hydrogels with spatial variations in matrix properties direct vascular cell invasion.</a> J. Tissue. 6, 302-302 (2012).</li><li>Butterworth, A., Garcia, M. D. L., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photopolymerized+poly(ethylene)+glycol+diacrylate+(PEGDA)+microfluidic+devices.">Photopolymerized poly(ethylene) glycol diacrylate (PEGDA) microfluidic devices.</a> Roy. Soc. Ch. , 4-6 (2005).</li><li>Shachar, M., Tsur-Gang, O., Dvir, T., Leor, J., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+immobilized+RGD+peptide+in+alginate+scaffolds+on+cardiac+tissue+engineering.">The effect of immobilized RGD peptide in alginate scaffolds on cardiac tissue engineering.</a> Acta biomaterialia. 7, 152-162 (2011).</li><li>Jeon, O., Bouhadir, K. H., Mansour, J. M., Alsberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocrosslinked+alginate+hydrogels+with+tunable+biodegradation+rates+and+mechanical+properties.">Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties.</a> Biomaterials. 30, 2724-2734 (2009).</li><li>Mauck, R. L., 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=Functional+tissue+engineering+of+articular+cartilage+through+dynamic+loading+of+chondrocyte-seeded+agarose+gels.">Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.</a> J. Biomech. Eng-T Asme. 122, 252-260 (2000).</li><li>D'Arrigo, G., 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=Hyaluronic+acid+methacrylate+derivatives+and+calcium+alginate+interpenetrated+hydrogel+networks+for+biomedical+applications:+physico-chemical+characterization+and+protein+release.">Hyaluronic acid methacrylate derivatives and calcium alginate interpenetrated hydrogel networks for biomedical applications: physico-chemical characterization and protein release.</a> Colloid Polym. Sci. 290, 1575-1582 (2012).</li><li>Pescosolido, L., 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=Hyaluronic+Acid+and+Dextran-Based+Semi-IPN+Hydrogels+as+Biomaterials+for+Bioprinting.">Hyaluronic Acid and Dextran-Based Semi-IPN Hydrogels as Biomaterials for Bioprinting.</a> Biomacromolecules. 12, 1831-1838 (2011).</li><li>Guo, 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=Hydrogels+of+collagen/chondroitin+sulfate/hyaluronan+interpenetrating+polymer+network+for+cartilage+tissue+engineering.">Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering.</a> J. Mater. Sci-Mater. M. 23, 2267-2279 (2012).</li></ol>

这里，我们提出，在第一次，使用牺牲模，可在冷水中迅速溶出，由于约20℃的泊洛沙姆凝胶 - 溶胶转变温敏聚合物快速创建生物大分子结构不能有足够的分辨率打印速度的全过程，使泊洛沙姆有趣。这里描述的技术可以用于图案形成的水凝胶，水凝胶或在另一个先前已报告的其他材料35的微流体通道的创建。泊洛沙姆作为牺牲模具的优点是，它可以被印在任意的几何形状为固体层由层构?...

组织工程方法已经在过去几年取得了很大的进步，对于人体组织和器官的再生19,20。然而，到现在为止，已组织工程的焦点往往是有限的组织，具有简单的结构，或小的尺寸，如膀胱21,22或皮肤23-25。人的身体，但是，包含许多复杂的三维的组织，细胞和细胞外基质被布置在空间定义的方式。为了制造这些组织中，这种技术是必需的，可以放置在指定位置上的一个三维的结构内的细胞和细胞外基质的脚手架。生物印刷有潜力成为这样一种技术，制造复杂的三维组织的愿景，可以实现10,11,26-28。 生物印刷被定义为“材料转让过程使用的图案和组装生物相对埃文特材料-分子，细胞，组织，和可生物降解的生物材料-规定的组织来完成一个或多个生物功能“，它包含了几种不同的技术，工作在不同的分辨率和长度尺度，从亚微米分辨率的两个光子聚合29分辨率为150微米至420微米挤压印刷1,12,30。不是单一材料或材料组合将满足要求每个方法31。挤出印刷，关键参数是粘度和凝胶时间32，高粘度和快速凝胶化是可取的。 3D打印是一种技术，它允许创建复杂的几何形状30,33,34牺牲模具很容易的创建。这个过程是基于模具的建设，利用快速原型技术，如挤压bioprinter。使用创建的牺牲模具难以印刷由于其低的粘度和凝胶时间慢的材料，这些材料以形成复杂的结构。这里介绍的方法涉及到创建一个牺牲模具的材料组成的，在低温下迅速溶解，并可以准确地被挤压。的嵌段共聚物聚（乙二醇）99-聚（丙二醇）67-聚（乙二醇）99（也被称为的Pluronic F127或泊洛沙姆407）满足这些要求。它已经被用于在挤压印刷1中的修改后的版本，但据我们所知，从未被使用未修改的版本，由于它的不稳定性，在液体环境中的打印。泊洛沙姆407还示出了逆热响应行为18 即它从凝胶在冷却时的溶胶。最重要的是，它可以打印任意弯曲成复杂的结构具有非常高的保真度。这允许创建一个结构化的水凝胶从低粘度的材料，在这种情况下慢胶凝琼脂糖，该溶液通过移液到印刷的牺牲模具。具有高的保真度和快速洗脱从铸造的结构化的水凝胶，使得它快速和灵活的方法来创建不同的几何形状的模具，无需使用的平版印刷方法，因为它往往是必需的掩模或邮票打印的牺牲模具的组合。铸造后的结构化的水凝胶，可以进一步与另一种材料是不适合用于挤出印刷由于其低粘度填充。这是在我们的情况下，一个低粘度海藻酸钠丙烯酸甲酯的解决方案。在这里，我们提出的方法反向温敏凝胶图案牺牲模具使用支柱数组的例子。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Poloxamer (Pluronic F127)</td>
 <td>Sigma</td>
 <td>P2443</td>
 <td ></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=10010015" target="_blank">10010-015</a></td>
 <td ></td>
 </tr>
 <tr>
 <td>CAD software</td>
 <td>regenHU</td>
 <td>BioCAD</td>
 <td ></td>
 </tr>
 <tr>
 <td>Alginate methacrylate</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
 <td>Invitrogen</td>
 <td>F13191</td>
 <td ></td>
 </tr>
 <tr>
 <td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Lonza</td>
 <td>50004</td>
 <td ></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Bioprinter</td>
 <td>regenHU</td>
 <td>Biofactory</td>
 <td ></td>
 </tr>
 <tr>
 <td>Valve</td>
 <td>regenHU</td>
 <td>300 &mu;m Nozzel Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>regenHU</td>
 <td>150 &mu;m Inner Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Zeiss Axioobserver with ApoTome</td>
 <td>Zeiss</td>
 <td></td>
 <td ></td>
 </tr>
 <tr>
 <td>UV Light Source</td>
 <td>UVP</td>
 <td>Blak-Ray B-100AP High Intensity UV Lamp</td>
 <td>100 W</td>
 </tr>
 </tbody>
</table>

printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture

生物印刷是一项新兴技术，在快速原型制造行业有它的起源。不同的印刷工艺可分为接触生物印刷1-4（挤出，蘸笔，软光刻技术），非接触式生物印刷5-7（激光的正向转 ​​移，喷墨沉积）和基于激光的技术，例如双光子光聚合8。它可用于许多应用，如组织工程9-13，14-16生物传感器的微细加工作为一种工具，回答基本的生物学问题，如影响共培养不同的细胞类型17。与常见的光刻或软光刻的方法不同的是，挤出生物印刷具有的优点在于，它不要求一个单独的掩模或印戳。使用CAD软件，可以快速地改变结构的设计和调整，根据运营商的要求。这使得生物印刷更灵活，比基于光刻的做法。 在这里，我们展示了印刷的牺牲模具来产生多材料的三维结构内的水凝胶，例如，使用一个数组支柱。这些支柱可以代表一个血管网或管内神经导向导管的空心结构。所选择的材料的牺牲模具是泊洛沙姆407，温度敏感型聚合物具有优异的印刷性能，这是液体在4℃，固体其胶凝温度以上〜20℃时为24.5％w / v的溶液18。这个属性允许基于泊洛沙姆牺牲模具洗脱需求在缓慢溶解的固体材料，尤其是狭窄的几何形状具有优势。泊洛沙姆印在显微镜载玻片创建牺牲模具。琼脂糖移液到模具中并冷却，直至凝胶化。泊洛沙姆在冰冷的水洗脱后，在琼脂糖模具的空隙被填充与藻酸盐丙烯酸甲酯藻IKED FITC标记的纤维蛋白原。填充空隙，然后交与UV落射荧光显微镜成像和结构。

代表性的结果表明的倒模技术（ 图2中所示）将创建结构化的第二材料，可填充有凝胶。在每次印刷过程中的开头的印刷参数首先优化。分步进行调整参数将导致在描绘在图3和图4单行打印时打印的多层构造。如果层厚度（后一个印刷层的针阀升程）过低时，人们会发现，针触摸前述层。如果针太高，出现波纹的表面上印刷的构造，将出现。此可以看出，在<st...

Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

àbioprinter被用于创建图案的水凝胶的基础上牺牲的模具。泊洛沙姆模具与第二水凝胶回填，然后洗脱，留下空隙充满了三分之一的水凝胶。此方法使用快速的洗脱和良好的印刷泊洛沙姆产生复杂的生物大分子结构。

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Research

JoVE Journal

Immunology and Infection

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

科研

免疫与感染

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

类器官的模型传染病：人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

巨型囊泡内单细胞层的细菌细胞培养

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

反向转录循环介导的等温放大（RT-LAMP）测定，用于对罗非鱼湖病毒进行特定和快速检测

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...