Name: bioprintable alginate/gelatin hydrogel 3d in vitro model systems induce cell spheroid formation
Uploaded: 2018-07-02T14:30:00
Description: Watch this Scientific Journal Video about Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation at JoVE.com

陶江感谢中国奖学金委员会 (201403170354) 和麦吉尔工程博士奖 (90025) 为他们提供奖学金。何塞 g. Munguia-洛佩兹感谢 CONACYT (250279, 290936 和 291168) 和 FRQNT (258421) 为他们的奖学金资助。萨尔瓦多 CONACYT 感谢他们的奖学金资助 (751540)。约瑟夫 m. 金塞拉感谢国家科学和工程研究理事会, 加拿大创新基金会, 汤森-Lamarre 家庭基金会, 麦吉尔大学为他们提供资金。我们要感谢艾伦 Ehrlicher 允许我们使用他的流变仪, 丹 Nicolau 允许我们使用他的共焦显微镜, Morag 公园让我们访问荧光标记的细胞线。

1. 材料、水凝胶和细胞培养材料的制备
<ol><li>
材料和溶液制备
	<ol>
<li>洗涤和干250毫升和100毫升玻璃烧杯, 磁性搅拌器, 铲子, 10 毫升墨盒, 25 克圆柱形喷嘴 (长度0.5 英寸和内径250µm)。在121摄氏度/15 min/1 atm 机上热处理, 对材料进行杀菌灭菌. 使物料在无菌条件下使用。 注: 请参考物料表以了解供应商信息。</li>
		<li>重3克海藻酸盐 (3% 瓦特/v) 和7克的明胶 (7% 瓦特/v) (表示?...

<ol>
	<li>Cui, X., Hartanto, Y., Zhang, 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+multicellular+spheroids+formation.">Advances in multicellular spheroids formation.</a> Journal of the Royal Society Interface. 14 (127), (2017).</li><li>Yip, D., Cho, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicellular+3D+heterospheroid+model+of+liver+tumor+and+stromal+cells+in+collagen+gel+for+anti-cancer+drug+testing.">A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing.</a> Biochemical and Biophysical Research Communications. 433 (3), 327-332 (2013).</li><li>Breslin, S., O'Driscoll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+using+3D+cell+cultures,+in+addition+to+2D+monolayer+cultures,+when+evaluating+breast+cancer+drug+sensitivity+and+resistance.">The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance.</a> Oncotarget. 7 (29), 45745-45756 (2016).</li><li>Yue, X., Lukowski, J. K., Weaver, E. M., Skube, S. B., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+and+phosphoproteomic+comparison+of+2D+and+3D+colon+cancer+cell+culture+models.">Quantitative proteomic and phosphoproteomic comparison of 2D and 3D colon cancer cell culture models.</a> Journal of Proteome Research. 15 (12), 4265-4276 (2016).</li><li>Priwitaningrum, D. 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=Tumor+stroma-containing+3D+spheroid+arrays:+a+tool+to+study+nanoparticle+penetration.">Tumor stroma-containing 3D spheroid arrays: a tool to study nanoparticle penetration.</a> Journal of Controlled Release. 244 (Pt B), 257-268 (2016).</li><li>Hong, 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=Cellular+behavior+in+micropatterned+hydrogels+by+bioprinting+system+depended+on+the+cell+types+and+cellular+interaction.">Cellular behavior in micropatterned hydrogels by bioprinting system depended on the cell types and cellular interaction.</a> Journal of Bioscience and Bioengineering. 116 (2), 224-230 (2013).</li><li>Dolati, F., 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=In+vitro+evaluation+of+carbon-nanotube-reinforced+bioprintable+vascular+conduits.">In vitro evaluation of carbon-nanotube-reinforced bioprintable vascular conduits.</a> Nanotechnology. 25 (14), 145101 (2014).</li><li>Murphy, S. V., 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=3D+bioprinting+of+tissues+and+organs.">3D bioprinting of tissues and organs.</a> Nature Biotechnology. 32 (8), 773-785 (2014).</li><li>Kang, H. 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=A+3D+bioprinting+system+to+produce+human-scale+tissue+constructs+with+structural+integrity.">A 3D bioprinting system to produce human-scale tissue constructs with structural integrity.</a> Nature Biotechnology. 34 (3), 312-319 (2016).</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 Materials. 11 (9), 768-774 (2012).</li><li>Jia, 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=Direct+3D+bioprinting+of+perfusable+vascular+constructs+using+a+blend+bioink.">Direct 3D bioprinting of perfusable vascular constructs using a blend bioink.</a> Biomaterials. 106, 58-68 (2016).</li><li>Kolesky, D. B., Homan, K. A., Skylar-Scott, M. A., 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=Three-dimensional+bioprinting+of+thick+vascularized+tissues.">Three-dimensional bioprinting of thick vascularized tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (12), 3179-3184 (2016).</li><li>Lee, 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=Design+and+fabrication+of+human+skin+by+three-dimensional+bioprinting.">Design and fabrication of human skin by three-dimensional bioprinting.</a> Tissue Engineering Part C: Methods. 20 (6), 473-484 (2014).</li><li>Jiang, 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=Directing+the+self-assembly+of+tumour+spheroids+by+bioprinting+cellular+heterogeneous+models+within+alginate/gelatin+hydrogels.">Directing the self-assembly of tumour spheroids by bioprinting cellular heterogeneous models within alginate/gelatin hydrogels.</a> Scientific Reports. 7 (1), 4575 (2017).</li><li>Knowlton, S., Onal, S., Yu, C. H., Zhao, J. J., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+for+cancer+research.">Bioprinting for cancer research.</a> Trends in Biotechnology. 33 (9), 504-513 (2015).</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=Printing+and+prototyping+of+tissues+and+scaffolds.">Printing and prototyping of tissues and scaffolds.</a> Science. 338 (6109), 921-926 (2012).</li><li>Nair, 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+cell+viability+during+bioprinting+processes.">Characterization of cell viability during bioprinting processes.</a> Biotechnology Journal. 4 (8), 1168-1177 (2009).</li><li>Costa, E. C., 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=3D+tumor+spheroids:+an+overview+on+the+tools+and+techniques+used+for+their+analysis.">3D tumor spheroids: an overview on the tools and techniques used for their analysis.</a> Biotechnology Advances. 34 (8), 1427-1441 (2016).</li><li>Zhao, 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=Three-dimensional+printing+of+Hela+cells+for+cervical+tumor+model+in+vitro.">Three-dimensional printing of Hela cells for cervical tumor model in vitro.</a> Biofabrication. 6 (3), 035001 (2014).</li><li>Ling, 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=Bioprinting-based+high-throughput+fabrication+of+three-dimensional+MCF-7+human+breast+cancer+cellular+spheroids.">Bioprinting-based high-throughput fabrication of three-dimensional MCF-7 human breast cancer cellular spheroids.</a> Engineering. 1 (2), 269-274 (2015).</li><li>Liang, 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=A+cell-instructive+hydrogel+to+regulate+malignancy+of+3D+tumor+spheroids+with+matrix+rigidity.">A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity.</a> Biomaterials. 32 (35), 9308-9315 (2011).</li><li>Szot, C. S., Buchanan, C. F., Freeman, J. W., Rylander, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+in+vitro+bioengineered+tumors+based+on+collagen+I+hydrogels.">3D in vitro bioengineered tumors based on collagen I hydrogels.</a> Biomaterials. 32 (31), 7905-7912 (2011).</li><li>Carey, S. P., Kraning-Rush, C. M., Williams, R. M., Reinhart-King, 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=Biophysical+control+of+invasive+tumor+cell+behavior+by+extracellular+matrix+microarchitecture.">Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture.</a> Biomaterials. 33 (16), 4157-4165 (2012).</li><li>Hospodiuk, M., Dey, M., Sosnoski, D., Ozbolat, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioink:+a+comprehensive+review+on+bioprintable+materials.">The bioink: a comprehensive review on bioprintable materials.</a> Biotechnology Advances. 35 (2), 217-239 (2017).</li><li>Caliari, S. R., Burdick, 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=A+practical+guide+to+hydrogels+for+cell+culture.">A practical guide to hydrogels for cell culture.</a> Nature Methods. 13 (5), 405-414 (2016).</li><li>Bhutani, U., Laha, A., Mitra, K., Majumdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+alginate+and+gelatin+hydrogels:+viscosity+effect+on+hydrophobic+drug+release.">Sodium alginate and gelatin hydrogels: viscosity effect on hydrophobic drug release.</a> Materials Letters. 164, 76-79 (2016).</li><li>Biswal, D., 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=Effect+of+mechanical+and+electrical+behavior+of+gelatin+hydrogels+on+drug+release+and+cell+proliferation.">Effect of mechanical and electrical behavior of gelatin hydrogels on drug release and cell proliferation.</a> Journal of the Mechanical Behavior of Biomedical Materials. 53, 174-186 (2016).</li><li>Rowley, J. A., Madlambayan, G., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+hydrogels+as+synthetic+extracellular+matrix+materials.">Alginate hydrogels as synthetic extracellular matrix materials.</a> Biomaterials. 20 (1), 45-53 (1999).</li><li>Djabourov, M., Leblond, J., Papon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelation+of+aqueous+gelatin+solutions.+I.+Structural+investigation.">Gelation of aqueous gelatin solutions. I. Structural investigation.</a> Journal de Physique (France). 49 (2), 319-332 (1988).</li><li>Djabourov, M., Leblond, J., Papon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelation+of+aqueous+gelatin+solutions.+II.+Rheology+of+the+sol-gel+transition.">Gelation of aqueous gelatin solutions. II. Rheology of the sol-gel transition.</a> Journal de Physique (France). 49 (2), 333-343 (1988).</li><li>Coussot, 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> Rheometry of Pastes, Suspensions, and Granular Materials: Applications in Industry and Environment. , (2005).</li><li>Ouyang, L., Yao, R., Zhao, Y., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+bioink+properties+on+printability+and+cell+viability+for+3D+bioplotting+of+embryonic+stem+cells.">Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.</a> Biofabrication. 8 (3), 035020 (2016).</li><li>Michon, C., Cuvelier, G., Launay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+dependence+of+the+critical+viscoelastic+properties+of+gelatin+at+the+gel+point.">Concentration dependence of the critical viscoelastic properties of gelatin at the gel point.</a> Rheologica Acta Rheologica Acta: An International Journal of Rheology. 32 (1), 94-103 (1993).</li><li>Mouser, V. H., 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=Yield+stress+determines+bioprintability+of+hydrogels+based+on+gelatin-methacryloyl+and+gellan+gum+for+cartilage+bioprinting.">Yield stress determines bioprintability of hydrogels based on gelatin-methacryloyl and gellan gum for cartilage bioprinting.</a> Biofabrication. 8 (3), 035003 (2016).</li><li>Benbow, J. J., Oxley, E. W., Bridgwater, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extrusion+mechanics+of+pastes-the+influence+of+paste+formulation+on+extrusion+parameters.">The extrusion mechanics of pastes-the influence of paste formulation on extrusion parameters.</a> Chemical Engineering Science. 42 (9), 2151-2162 (1987).</li><li>Bingham, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluidity and plasticity. , (1922).</li><li>Horrobin, D. J., Nedderman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+entry+pressure+drops+in+paste+extrusion.">Die entry pressure drops in paste extrusion.</a> Chemical Engineering Science. 53 (18), 3215-3225 (1998).</li><li>Soman, P., 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=Cancer+cell+migration+within+3D+layer-by-layer+microfabricated+photocrosslinked+PEG+scaffolds+with+tunable+stiffness.">Cancer cell migration within 3D layer-by-layer microfabricated photocrosslinked PEG scaffolds with tunable stiffness.</a> Biomaterials. 33 (29), 7064-7070 (2012).</li><li>Asghar, 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=Engineering+cancer+microenvironments+for+in+vitro+3-D+tumor+models.">Engineering cancer microenvironments for in vitro 3-D tumor models.</a> Materials Today. 18 (10), 539-553 (2015).</li><li>Lin, R. Z., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+three-dimensional+multicellular+spheroid+culture+for+biomedical+research.">Recent advances in three-dimensional multicellular spheroid culture for biomedical research.</a> Biotechnology Journal. 3 (9-10), 1172-1184 (2008).</li><li>Akasov, R., 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=Formation+of+multicellular+tumor+spheroids+induced+by+cyclic+RGD-peptides+and+use+for+anticancer+drug+testing+in+vitro.">Formation of multicellular tumor spheroids induced by cyclic RGD-peptides and use for anticancer drug testing in vitro.</a> International Journal of Pharmaceutics. 506 (1-2), 148-157 (2016).</li></ol>

如果污染 (生物或化学) 发生在过程中的任何一点上, 就会危及细胞的结构。通常, 生物污染是在三天的文化后, 作为一种颜色变化的文化媒体或 bioprinted 结构。因此, 灭菌 (物理和化学消毒) 是所有细胞相关过程的关键步骤。值得注意的是, 热处理明胶改变了其凝胶性质, 这使得它在我们进行的试验中更慢。因此, 我们通过紫外线照射对海藻酸盐和明胶的能量进行杀菌。由于 UV 光的穿透能力非?...

虽然2D 细胞培养在癌症研究中得到广泛应用, 但由于细胞以单层格式生长, 营养和氧气浓度均匀, 所以存在局限性。这些文化缺乏在原生肿瘤微环境中存在的重要细胞细胞和细胞基质相互作用。因此, 这些模型不太概括的生理条件, 导致异常细胞行为, 包括非自然形态, 不规则的受体组织, 膜极化, 和异常的基因表达, 除其他情况1,2,3,4。另一方面, 3D 细胞培养, 细胞被扩展在一个体积空间作为聚合体, 球体, 或 organoids, 提供了一种替代技术, 以创造更准确的体内环境, 以研究基本细胞生物学和生理学。3D 细胞培养模型还可以鼓励细胞 ECM 相互作用, 这是本机1,4,5的重要生理特性。新兴的3D 生物打印技术为构建模仿异类的模型提供了可能性。
3D 生物打印是从快速原型, 并使制造的3D 显微组织, 可以模仿一些复杂的活体标本6,7。目前的生物打印方法包括喷墨、挤出和激光辅助印刷8。其中, 挤出方法可以在不同的初始位置精确定位各种不同类型的材料, 从而在印制矩阵中控制异质性。因此, 它是制造多类型细胞或矩阵的异质体模型的最佳方法。挤压生物打印已成功地用于建立耳形支架9, 血管结构10,11,12, 皮肤组织13, 导致高印刷保真度和细胞可行性。该技术还具有多种材料选择, 有能力储存与已知密度的细胞的材料, 高重现性14,15,16,17.天然和合成水凝胶经常被用作3D 生物打印的 bioinks, 因为它们的生物相容性, 生物活性, 和他们的亲水性网络, 可以工程结构类似 ECM7,18 ,19,20,21,22,23。水凝胶也很有利, 因为它们可以包括细胞的粘接部位、结构元素、养分和气体的渗透性, 以及促进细胞发育的适当机械特性24。例如, 胶原水凝胶提供整合素锚固点, 细胞可以用来连接到基质。凝胶, 变性胶原蛋白, 保留类似的细胞黏附部位。相比之下, 海藻酸盐是 bioinert, 但通过形成 crosslinks 与价离子25,26,27,28提供机械完整性。
在这项工作中, 我们开发了复合水凝胶作为 bioink, 由海藻酸盐和明胶组成, 与原生肿瘤基质的显微结构相似。乳腺癌细胞和成纤维细胞被嵌入在水凝胶中,通过挤压的 bioprinter 印刷, 以创建模拟体内微环境的3D 模型。设计的3D 环境允许癌细胞形成多细胞肿瘤球体 (MCTS), 具有很高的生存能力, 长时间的细胞培养 (> 30 天)。该协议展示了合成复合水凝胶的方法, 表征了材料的显微组织和适印性, 生物打印细胞异构模型, 观察 MCTS 的形成。这些方法可以应用于其他 bioinks 挤压生物打印, 以及不同的设计的异构组织模型的潜在应用, 在药物筛选, 细胞迁移检测, 和研究, 重点在基本细胞生理功能。

<table border="0" cellpadding="0" cellspacing="0" width="925">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Sodium alginate</td>
			<td style="width:213px;">FMC BioPolymer</td>
			<td style="width:173px;">CAS-No: 9005-38-3</td>
			<td style="width:167px;">Protanal LF 10/60 FT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Gelatin</td>
			<td style="width:213px;">Sigma-Aldrich</td>
			<td style="width:173px;">G9391</td>
			<td>Type B gelatin from bovine skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Dubelcco&#39;s phosphate buffered saline (DPBS 1X)</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:173px;">LS14190136</td>
			<td>1&#215;, w/o calcium, w/o magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Magnetic hotplate</td>
			<td style="width:213px;">Corning&#160;</td>
			<td style="width:173px;">N/A</td>
			<td>Stirrer/hot plate model PC-420</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">50 mL centrifuge tubes</td>
			<td style="width:213px;">Corning</td>
			<td style="width:173px;">352098</td>
			<td>Falcon&#174; 50mL High Clarity PP Centrifuge Tube, Conical Bottom, Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Centrifuge</td>
			<td style="width:213px;">GMI</td>
			<td style="width:173px;">N/A</td>
			<td>Sorvall RT6000D, GMI, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride anhydrous</td>
			<td style="width:213px;">Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">MilliQ water</td>
			<td style="width:213px;">Millipore</td>
			<td style="width:173px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Millipore 0.22 &#181;m filters</td>
			<td style="width:213px;">Millipore</td>
			<td style="width:173px;">SLGS033SB</td>
			<td>Millex-GS Syringe Filter Unit, 0.22&#160;&#181;m, mixed cellulose esters, 33&#160;mm, ethylene oxide sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Oscillation rheometer MCR 302</td>
			<td style="width:213px;">Anton Paar</td>
			<td style="width:173px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Rheometer measuring tool CP25</td>
			<td style="width:213px;">Anton Paar</td>
			<td style="width:173px;">79038</td>
			<td>Conical plate geometry for rheometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">RheoCompass</td>
			<td style="width:213px;">Anton Paar</td>
			<td style="width:173px;">N/A</td>
			<td>Software controlling rheometer MCR 302</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Scanning electron microscope</td>
			<td style="width:213px;">Hitachi</td>
			<td style="width:173px;">N/A</td>
			<td>SEM, Hitachi SU-3500 Variable Pressure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Paraformaldehyde, 96%, extra pure</td>
			<td style="width:213px;">Acros Organics</td>
			<td style="width:173px;">416785000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Dulbecco modified eagle medium (DMEM)</td>
			<td style="width:213px;">Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic/Antimycotic solution (100X) stabilized</td>
			<td style="width:213px;">Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Wisent Bioproducts</td>
			<td style="width:173px;">080-150</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:372px;">Cell culture T-75 flasks</td>
			<td style="width:213px;">Sigma-Aldrich</td>
			<td style="width:173px;">CLS430641</td>
			<td>75 cm2 TC-Treated surface treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D bioprinter BioScaffolder 3.1</td>
			<td style="width:213px;">GeSiM</td>
			<td style="width:173px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeSim software</td>
			<td style="width:213px;">GeSiM</td>
			<td style="width:173px;">N/A</td>
			<td>Software controlling BioScaffolder 3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cc cartridge UV resist</td>
			<td>EFD Nordson</td>
			<td>7012126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">End cap</td>
			<td>EFD Nordson</td>
			<td>7014472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tip cap</td>
			<td>EFD Nordson</td>
			<td>7014469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piston&#160;</td>
			<td>EFD Nordson</td>
			<td>7012182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless nozzle G25</td>
			<td>EFD Nordson</td>
			<td>7018345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Water bath</td>
			<td style="width:213px;">VWR</td>
			<td style="width:173px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Agarose</td>
			<td style="width:213px;">Sigma-Aldrich</td>
			<td style="width:173px;">A9539</td>
			<td>Bioreagent, for molecular biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Costar 6-well plates&#160;</td>
			<td style="width:213px;">Corning</td>
			<td style="width:173px;">3516</td>
			<td>TC-Treated Multiple Well Plates, Individually Wrapped, Sterile&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Confocal spinning disk inverted microscope</td>
			<td style="width:213px;">Olympus Life Science</td>
			<td style="width:173px;">N/A</td>
			<td>Olympus IX83</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">MTS assay kit</td>
			<td style="width:213px;">Promega</td>
			<td style="width:173px;">G3582</td>
			<td>CellTiter 96&#174; AQueous&#160;One Solution Cell Proliferation Assay&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Live/Dead viability cytotoxicity kit</td>
			<td>Molecular Probes,ThermoFisher Scientific</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin 0.25/EDTA 1X</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Corning 96-well plate</td>
			<td style="width:213px;">Corning</td>
			<td style="width:173px;">3595</td>
			<td>Clear Flat Bottom Polystyrene TC-Treated Microplate, Individually Wrapped, with Low Evaporation Lid, Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Autoclave Tuttnauer</td>
			<td style="width:213px;">Heidolph Brinkmann</td>
			<td style="width:173px;">N/A</td>
			<td>Heidolph Tuttnauer 2540E Autoclave Sterilizer Electronic Model with 4 Stainless Steel Trays, 23L Capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Trypan blue</td>
			<td style="width:213px;">Invitrogen&#160;</td>
			<td style="width:173px;">T10282</td>
			<td>0.4% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Ethanol</td>
			<td style="width:213px;">Commercial Alcohols</td>
			<td style="width:173px;">P016EA95</td>
			<td>Greenfield Speciality Alcohols</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:372px;">CO2 Incubator</td>
			<td style="width:213px;">Panasonic</td>
			<td style="width:173px;">N/A</td>
			<td>MCO 19AIC-PA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Lyophilizer&#160;</td>
			<td style="width:213px;">SP Scientific</td>
			<td style="width:173px;">N/A</td>
			<td>Virtis Sentry 2.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">SolidWorks</td>
			<td style="width:213px;">Dassault Systems</td>
			<td style="width:173px;">N/A</td>
			<td>A CAD software used to build demostrative propeller-like model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">MATLAB</td>
			<td style="width:213px;">The MathWorks</td>
			<td style="width:173px;">N/A</td>
			<td>A programming software used to generate G-code for BioScaffolder 3.1</td>
		</tr>
	</tbody>
</table>

bioprintable alginate/gelatin hydrogel 3d in vitro model systems induce cell spheroid formation

使用常规的二维 (2D) 细胞培养法, 生长永生化癌细胞株不概括本机肿瘤微环境的细胞、生物化学和生物物理异质性。这些挑战可以克服, 通过使用生物打印技术建立异构的三维 (3D) 肿瘤模型, 即不同类型的细胞嵌入。海藻酸盐和明胶是生物打印中使用的两种最常见的生物材料, 其生物相容性、仿生学和力学性能。通过结合这两种聚合物, 我们取得了 bioprintable 复合水凝胶与原生肿瘤基质的显微结构相似。通过流变学研究了复合水凝胶的适印性, 得到了最佳的印刷窗口。将乳腺癌细胞和成纤维细胞嵌入水凝胶中, 并印成3D 模型, 模拟体内微环境。bioprinted 异构模型为长期细胞培养 (> 30 天) 提供了很高的生存能力, 并促进了乳腺癌细胞的自组装成多细胞肿瘤球体 (MCTS)。我们观察了肿瘤相关成纤维细胞 (咖啡馆) 与 MCTS 在该模型中的迁移和相互作用。利用 bioprinted 细胞培养平台作为共培养系统, 为研究肿瘤发生对基质成分的依赖性提供了独特的工具。该技术具有吞吐量高、成本低、重现性高的特点, 也为常规细胞单层培养和动物肿瘤模型的研究提供了一种可替代的模型。

温度扫描显示 A3G7 前驱体在25°c 和37°c 上的明显差异。前体是液体在37°c 和有一个复杂的粘度 1938.1, 84.0 mPa x s, 这是验证由一个更大的 g "超过 g"。随着温度的降低, 前驱体经过物理凝胶, 由于明胶分子自发物理纠缠成三螺旋形成29,30。g ' 和 g ' 增加和汇合在30.6 °c, 表明溶胶-凝胶转折。模数继续增加, 温度降低, 达到 468.5 34.2 pa g ' ?...

Watch this Scientific Journal Video about Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation at JoVE.com

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

我们开发了一种异种乳癌模型, 由永生化的肿瘤和成纤维细胞组成的 bioprintable 海藻酸盐/明胶 bioink。该模型概括体内肿瘤微环境, 促进多细胞肿瘤球体的形成, 对肿瘤的发生机制有深入的认识。

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Research

JoVE Journal

Bioengineering

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

科研

生物工程

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

模拟卵巢癌多细胞球体行为动态三维微型腹膜

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

三维印刷多孔纤维素纳米复合水凝胶脚手架

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

在三维生物打印中使用多层水凝胶生物油墨，实现均匀细胞分布

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

通过交替粘性 - 惯性力喷射进行 体外 水凝胶微载体的3D打印

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...