我们衷心感谢 Dr. 本. 福勒在 TEM 实验中的技术帮助。

本议定书遵循俄克拉荷马州健康科学中心/机构审查委员会 (IRB #4509) 的指导方针。《议定书》的所有部分都符合《赫尔辛基宣言》的原则。角膜标本取自国家发展和研究所 (第九) 和俄克拉何马州狮子眼银行。
1. 分离原细胞
<ol><li>在收到人类的角膜组织样本后, 将组织转移到21.5 厘米的2培养皿中, 其中含有2毫升的无菌 Dulbecco 的磷酸盐缓冲盐水 (DPBS) (1x)。</li>
	<li>通过定位角膜圆顶面来刮除角膜上皮。为了确保完全去除, 用刀片在45°角上彻底刮掉上皮层。接下来, 翻转角膜圆顶面朝下, 彻底刮掉从基质中的内皮细胞与一个45°角刀片, 以确保完全清除。用 DPBS 冲洗基质组织。</li>
	<li>将基质组织切成小块, 使用10号手术刀片, 用 DPBS 将基质片保持湿润。用无菌镊子和无培养基的 T25 培养瓶中挑选基质片 (每瓶4或5片 2 x 2 毫米的组织)。</li>
	<li>允许外植体粘附在37° c 的烧瓶底部约 30-40 分钟。一旦加入, 慢慢添加鹰的最低基本培养基 (EMEM) 含有10% 胎牛血清 (FBS) 和1% 抗生素/抗的瓶子, 而不干扰外植体。</li>
	<li>轻轻地将烧瓶放在37° c, 5% CO2的孵化器中。让外植体保持不受干扰直到细胞开始分离并在烧瓶中迁移。在大约 2-4 星期以后, 细胞应该到达100% 汇合。在100% 汇合处, 通过去除组织培养介质和用 DPBS 洗涤细胞, 通过细胞。</li>
	<li>将胰蛋白酶添加到细胞中, 然后在37° c 孵育。在大约5分钟后, 查看细胞在光显微镜下, 以确认他们已经分离, 然后通过添加新的媒体停止蛋白水解。</li>
	<li>将细胞悬浮液转化为15毫升的锥形管, 用于离心 1000 x g. 在不干扰颗粒的情况下, 小心去除上清, 并在新鲜 EMEM 10% FBS 1% 抗生素中悬浮细胞。计算细胞和种子大约 1 x 106细胞在 T75 培养烧瓶进一步扩展与新鲜 EMEM 1% FBS 1% 抗生素。</li>
</ol>2. 主要 HCFs 文化和3D 建筑装配
<ol><li>从以前生长在 EMEM 10% FBS 1% 抗生素/抗, 通道和计数 HCFs 的外植体组装3D 结构。</li>
	<li>种子大约 1 x 106细胞/主要 HCFs 在聚碳酸酯膜插入物与0.4 µm 孔从3D 构造, 连同1.5 毫升新鲜 EMEM 10% FBS 1% 抗生素/抗到结构的顶部。将同一介质的另 1.5 mL 添加到每个构造的底部。</li>
	<li>在 24 h 细胞播种之后, 培养细胞与 EMEM 10% FBS 1% 抗生素或抗, 刺激与0.5 毫米 2 O α D-基 l-抗坏血酸 (维生素 C) 通过增加1.5 毫升在顶端和1.5 毫升在每个构造的底部。</li>
	<li>保持培养在37° c, 5% CO2为3星期, 并提供新鲜的媒体每隔一天在整个研究期间的孵化器。不需要进一步的通道。</li>
</ol>3. 细胞分化与共培养
<ol><li>3周后, 在先前组装的3D 结构的顶部添加 8 x 103细胞/SH-SY5Y 人神经母细胞瘤, 其中含有1.5 毫升的 EMEM 含有 10% FBS, 1% 抗生素/抗, 和0.5 毫米 2 O α-d-基-l--抗坏血酸酸 (维生素 C)。<ol>
<li>在细胞分化之前, 允许 SH-SY5Y 神经母细胞瘤细胞在角膜基质干细胞的顶端生长至少24小时。</li>
	</ol>
</li>
	<li>启动 SH-SY5Y 细胞分化的刺激细胞与 EMEM 含有 1% FBS 和1.5 毫升的10µM 维甲酸的顶部的结构。添加1.5 毫升的 EMEM 10% FBS 1% 抗生素/抗0.5 毫米 2 O α-d-基, l-抗坏血酸 (维生素 C) 的底部的结构。请注意, 维甲酸的步骤应该在黑暗中进行。<ol>
<li>继续培养这种分化介质中的细胞5天。</li>
	</ol>
</li>
	<li>当细胞达到分化阶段, 将细胞转换为1.5 毫升无血清培养基, 其中含有 2 nM 脑源性神经营养因子 (BDNF) 和1% 抗生素/抗添加到 EMEM, 增加到该结构的顶部。添加1.5 毫升的 EMEM 10% FBS 1% 抗生素/抗0.5 毫米 2 O α-d-基, l-抗坏血酸 (维生素 C) 的底部的结构。注意, BDNF 的步骤应该在黑暗中进行。</li>
	<li>在处理表 1中所示的任何进一步分析之前, 对单元格进行2天的培养。</li>
</ol>

<ol>
	<li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. 96 (5), 614-618 (2012).</li><li>R&#246;nkk&#246;, S., Vellonen, K. -. S., J&#228;rvinen, K., Toropainen, E., Urtti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+cell+culture+models+for+drug+toxicity+studies.">Human corneal cell culture models for drug toxicity studies.</a> Drug Delivery and Translational Research. 6 (6), 660-675 (2016).</li><li>Ghezzi, C. E., Rnjak-Kovacina, J., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+tissue+engineering:+recent+advances+and+future+perspectives.">Corneal tissue engineering: recent advances and future perspectives.</a> Tissue Eng Part B Rev. 21 (3), 278-287 (2015).</li><li>Shafaie, S., Hutter, V., Cook, M. T., Brown, M. B., Chau, D. Y. S. <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+Cell+Models+for+Ophthalmic+Drug+Development+Applications.">In Vitro Cell Models for Ophthalmic Drug Development Applications.</a> BioResearch Open Access. 5 (1), 94-108 (2016).</li><li>Shaheen, B. S., Bakir, M., Jain, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+nerves+in+health+and+disease.">Corneal nerves in health and disease.</a> Surv Ophthalmol. 59 (3), 263-285 (2014).</li><li>Beuerman, R. W., Schimmelpfennig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+denervation+of+the+rabbit+cornea+affects+epithelial+properties.">Sensory denervation of the rabbit cornea affects epithelial properties.</a> Exp Neurol. 69 (1), 196-201 (1980).</li><li>Heigle, T. J., Pflugfelder, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+tear+production+in+patients+with+neurotrophic+keratitis.">Aqueous tear production in patients with neurotrophic keratitis.</a> Cornea. 15 (2), 135-138 (1996).</li><li>Wang, 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=In+vitro+3D+corneal+tissue+model+with+epithelium,+stroma,+and+innervation.">In vitro 3D corneal tissue model with epithelium, stroma, and innervation.</a> Biomaterials. 112, 1-9 (2017).</li><li>Ross, R. A., Spengler, B. A., Biedler, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+morphological+and+biochemical+interconversion+of+human+neuroblastoma+cells.">Coordinate morphological and biochemical interconversion of human neuroblastoma cells.</a> J Natl Cancer Inst. 71 (4), 741-747 (1983).</li><li>Griffith, L. G., Naughton, G. <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+-+Current+challenges+and+expanding+opportunities.">Tissue engineering - Current challenges and expanding opportunities.</a> Science. 295 (5557), (2002).</li><li>Guo, X., 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=Morphologic+characterization+of+organized+extracellular+matrix+deposition+by+ascorbic+acid-stimulated+human+corneal+fibroblasts.">Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts.</a> Invest Ophthalmol Vis Sci. 48 (9), 4050-4060 (2007).</li><li>Karamichos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+tissue+engineering:+current+and+future+directions.">Ocular tissue engineering: current and future directions.</a> J Funct Biomater. 6 (1), 77-80 (2015).</li><li>Karamichos, D., Brown, R. A., Mudera, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+stiffness+regulates+cellular+contraction+and+matrix+remodeling+gene+expression.">Collagen stiffness regulates cellular contraction and matrix remodeling gene expression.</a> J Biomed Mater Res A. 83 (3), 887-894 (2007).</li><li>Ruberti, J. W., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prelude+to+corneal+tissue+engineering+-+gaining+control+of+collagen+organization.">Prelude to corneal tissue engineering - gaining control of collagen organization.</a> Prog Retin Eye Res. 27 (5), 549-577 (2008).</li><li>Karamichos, D., Guo, X. Q., Hutcheon, A. E., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+fibrosis:+an+in+vitro+model.">Human corneal fibrosis: an in vitro model.</a> Invest Ophthalmol Vis Sci. 51 (3), 1382-1388 (2010).</li><li>Chen, F. M., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+biomaterials+of+human+origin+for+tissue+engineering.">Advancing biomaterials of human origin for tissue engineering.</a> Prog Polym Sci. 53, 86-168 (2016).</li><li>Priyadarsini, S., Sarker-Nag, A., Rowsey, T. G., Ma, J. X., Karamichos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+3D+In+Vitro+Model+to+Accelerate+the+Development+of+Human+Therapies+against+Corneal+Diabetes.">Establishment of a 3D In Vitro Model to Accelerate the Development of Human Therapies against Corneal Diabetes.</a> PLoS One. 11 (12), e0168845 (2016).</li><li>Karamichos, D., Hjortdal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratoconus:+tissue+engineering+and+biomaterials.">Keratoconus: tissue engineering and biomaterials.</a> J Funct Biomater. 5 (3), 111-134 (2014).</li><li>Wilson, S. L., Yang, Y., El Haj, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Stromal+Cell+Plasticity:+In+Vitro+Regulation+of+Cell+Phenotype+Through+Cell-Cell+Interactions+in+a+Three-Dimensional+Model.">Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model.</a> Tissue Engineering Part A. 20 (1-2), 225-238 (2014).</li><li>Proulx, 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=Reconstruction+of+a+human+cornea+by+the+self-assembly+approach+of+tissue+engineering+using+the+three+native+cell+types.">Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.</a> Molecular Vision. 16 (234-236), 2192-2201 (2010).</li><li>Gonzalez-Andrades, 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=Establishment+of+a+novel+in+vitro+model+of+stratified+epithelial+wound+healing+with+barrier+function.">Establishment of a novel in vitro model of stratified epithelial wound healing with barrier function.</a> Sci Rep. 6, 19395 (2016).</li><li>Hopkins, A. M., DeSimone, E., Chwalek, K., Kaplan, D. L. <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+modeling+of+the+central+nervous+system.">3D in vitro modeling of the central nervous system.</a> Prog Neurobiol. 125, 1-25 (2015).</li><li>Schulz, 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=Natural+Corneal+Cell-Based+Microenvironment+as+Prerequisite+for+Balanced+3D+Corneal+Epithelial+Morphogenesis:+A+Promising+Animal+Experiment-Abandoning+Tool+in+Ophthalmology.">Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology.</a> Tissue Engineering Part C-Methods. 20 (4), 297-307 (2014).</li><li>Gao, J., Wang, Y., Zhao, X., Chen, P., Xie, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204-5p-Mediated+Regulation+of+SIRT1+Contributes+to+the+Delay+of+Epithelial+Cell+Cycle+Traversal+in+Diabetic+Corneas.">MicroRNA-204-5p-Mediated Regulation of SIRT1 Contributes to the Delay of Epithelial Cell Cycle Traversal in Diabetic Corneas.</a> Invest Ophthalmol Vis Sci. 56 (3), 1493-1504 (2015).</li><li>Koulikovska, 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=Enhanced+regeneration+of+corneal+tissue+via+a+bioengineered+collagen+construct+implanted+by+a+nondisruptive+surgical+technique.">Enhanced regeneration of corneal tissue via a bioengineered collagen construct implanted by a nondisruptive surgical technique.</a> Tissue Eng Part A. 21 (5-6), 1116-1130 (2015).</li><li>Ljubimov, A. V., Saghizadeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+corneal+wound+healing.">Progress in corneal wound healing.</a> Progress in retinal and eye research. 49, 17-45 (2015).</li><li>Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+and+wound+healing.">Extracellular matrix and wound healing.</a> Curr Opin Ophthalmol. 12 (4), 237-241 (2001).</li><li>Karamichos, D., Hutcheon, A. E., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta3+regulates+assembly+of+a+non-fibrotic+matrix+in+a+3D+corneal+model.">Transforming growth factor-beta3 regulates assembly of a non-fibrotic matrix in a 3D corneal model.</a> J Tissue Eng Regen Med. 5 (8), e228-e238 (2011).</li><li>Karamichos, D., Lakshman, N., Petroll, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+for+assessing+fibroblast+migration+in+3-D+collagen+matrices.">An experimental model for assessing fibroblast migration in 3-D collagen matrices.</a> Cell Motil Cytoskeleton. 66 (1), 1-9 (2009).</li><li>Karamichos, 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=Novel+in+Vitro+Model+for+Keratoconus+Disease.">Novel in Vitro Model for Keratoconus Disease.</a> J Funct Biomater. 3 (4), 760-775 (2012).</li></ol>

作者声明没有竞争的金融利益。

有几项研究的重点是开发各种动物模型, 可以帮助人们更好地了解角膜疾病以及发现治疗方法。然而, 这些研究对人类的重要价值还没有得到证实。到目前为止, 由于其显著的临床意义, 各种类型的体外模型已经得到了发展和广泛的研究。我们以前建立的 3D在体外模型是一个新的系统, 大大有助于医学科学进步的视觉研究和显示完美的能力, 综述在体内事件在体外<sup class="x...

在人体内, 角膜是最浓密的支配组织。神经是负责的各种感觉, 如触摸, 疼痛, 温度, 也有一个重要的作用, 伤口愈合, 眨眼反射, 撕裂生产, 并分泌5,6,7。在角膜中, 基质神经树干从角膜缘丛中产生, 并放射状地进入周围的角膜基质。基质神经组织是平行的胶原片, 他们进一步分支成更小的束簇, 因为他们走向表面基质5,8。神经纤维进一步穿透上皮层, 从而在角膜上皮和基质中广泛分布。因此, 神经支配在角膜的健康和患病状态中起着至关重要的作用。在这个协议中, 我们揭示了一个新的 3D体外模型的进展, 这是它的第一种模仿体内间质神经相互作用。这项研究使用了 SH-SY5Y 细胞系, 因为它是研究神经元生长的最成熟、最有特征的线之一。SH-SY5Y 细胞线已被描述产生两个基体附着物 (S 型) 和 neuroblastic (N 型) 细胞, 可以接受转分化9。因此, 即使这一细胞线是从三个连续的 N 型细胞克隆的选择, 它也包含了少量的 s-型细胞能够通过使用维甲酸和脑衍生的分化成神经元神经营养因子9。这提供了一个工具, 可能会导致更好地了解与糖尿病视网膜病变 (DM) 和其他眼部疾病相关的角膜并发症。由于与眼部疾病患者获得和培养神经元有关的困难, 这一3D 的体外模型在研究神经元相互作用和与角膜基质的信号传递方面具有重要的意义。
患病的情况往往会影响到身体的各种组织, 在很大的规模, 导致生活质量受损。眼部营养是常见的并发症, 往往与系统性疾病, 导致视力丧失, 甚至永久视力丧失。综合研究往往是必要的, 以更好地了解疾病的情况, 以及在基础细胞水平的影响。为了研究这些疾病的影响, 在组织工程应用的帮助下, 开发了各种体内和体外模型。角膜组织工程应用已在科学的各个领域获得了极大的兴趣10,11,12,13,14, 但仍然有主要的限制实际应用, 包括角膜移植排斥, 感染, 和疤痕10,11,12,13,14。有几项研究已经成功地开发和建立了各种体外模型3、15、16、17、18、 19,20,21,22,23,24,25,26。3D体外模型是最有希望和最具科学意义的。3D 模型可以更好地反映在纤维化和伤口愈合过程中至关重要的体内细胞和生理事件15、27、28、29。这些体外模型在寻找治疗不同疾病 (包括角膜并发症) 的新治疗方法方面起到了不可或缺的作用。尽管神经支配在角膜功能中起着至关重要的作用, 但在角膜组织工程结构中, 几乎没有为促进外周神经元增殖而做出努力2,3。然而, 建议的 3D体外细胞构造模拟目标组织, 以达到所需的组织功能。
虽然糖尿病角膜是一个明显的应用, 在这里所描述的模型, 由于神经元缺陷, 有几个其他的角膜疾病, 可以受益于人类的体外模型, 包括圆锥和福斯营养。我们的3D 模型从这一前景中产生, 并提出了一个体外的角膜组织的表征, 以评估新药的交付和新的眼科药物的安全性。

<table>
	<tbody>
		<tr>
			<td>Healthy corneal tissue</td>
			<td>NDRI</td>
			<td></td>
			<td>Samples from donors with no ocular trauma or systemic disease</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Solution (1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td>Fischer Scientific</td>
			<td>13-812-42</td>
			<td>Fisherbrand Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr>
			<td>Single edge razor blades</td>
			<td>Personna</td>
			<td>270100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical scalpel blades No.10</td>
			<td>Feather Surgical Blade</td>
			<td>2976#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#8217;s Minimum Essential Medium</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td>10% FBS is required for media preparation</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco by Life Technologies</td>
			<td>15240-062</td>
			<td>1% Antibiotic-Antimycotic is required for media preparation</td>
		</tr>
		<tr>
			<td>0.05% Trypsin EDTA(1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane inserts with 0.4-&#956;m pores</td>
			<td>Corning Costar</td>
			<td>3412</td>
			<td></td>
		</tr>
		<tr>
			<td>2-O-&#945;-Dglucopyranosyl-L-ascorbic acid (Vitamin C)</td>
			<td>Sigma-Aldrich</td>
			<td>SMB00390-14</td>
			<td>A concentration of 0.5 mM should be used for the study</td>
		</tr>
		<tr>
			<td>Wax block</td>
			<td>VWR</td>
			<td>50-949-027</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-SY5Y Neuroblastoma cells</td>
			<td>ATCC</td>
			<td>SHSY5YATCC CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3014-10UG</td>
			<td>Final concentration of 10uM needs to be used</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-100MG</td>
			<td>Final concentration of 2nM needs to be used</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide(DMSO)</td>
			<td>VWR-Alfa Aesar</td>
			<td>67-68-5</td>
			<td>Ultra Pure Grade-Sterile DMSO to be used</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T25)</td>
			<td>Fisher Scientific</td>
			<td>12-565-351</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T75)</td>
			<td>Fisher Scientific</td>
			<td>12-565-349</td>
			<td></td>
		</tr>
	</tbody>
</table>

corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea

组织工程学得到了大量的承认, 由于对人类角膜置换的高需求, 估计全世界有1000万人患有角膜视力损失1。为了满足对生存的人眼角膜的需求, 三维 (3D) 组织工程已取得重大进展2,3,4。这些角膜模型范围从简单单层系统到多层模型, 导致3D 全厚度角膜等效物2。
然而, 使用3D 组织工程角膜的环境中的体外疾病模型研究迄今缺乏相似的多层3D 角膜组织结构, 功能, 和网络的不同类型的细胞 (即, 神经,上皮, 基质, 和内皮)2,3。此外, 对体外角膜组织模型的需求也有所增加, 试图减少对药物产品的动物试验。因此, 更复杂的模型需要更好地匹配系统, 以人体生理需求, 和发展一个模型, 这是更相关的病人人口是绝对必要的。鉴于角膜中的多种细胞类型受疾病和营养的影响, 如圆锥、糖尿病角膜和福斯, 该模型包括一个由健康捐献者和神经元组成的人角膜成纤维细胞 (HCFs) 的3D 共培养模型。SH-SY5Y 细胞系。这让我们第一次研究人类角膜组织内两种细胞之间的相互作用。我们认为, 这种模式可能会解剖的基础机制与神经间相互作用的角膜疾病, 表现出神经损害。该3D 模型反映了角膜组织的基本解剖和生理特性在体内, 可以作为一种工具, 用于研究角膜缺损以及在动物试验前筛选各种药物的功效。

图 1是工作 3D体外模型的一步一步代表图像。第一步, 细胞与人类角膜隔绝。然后, 他们生长在聚碳酸酯膜和刺激与维生素 C 组装一个自分泌的3D 矩阵。这个3D 构造系统诱导了多层细胞在体内样基质基质的合成。此后, 神经母细胞瘤在基质细胞的顶端被播种, 然后启动神经细胞分化。这导致了角膜基质细胞和分化神经元细胞在 3D体外...

Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

本议定书描述了一个新的三维的体外模型, 其中角膜基质细胞和分化的神经细胞被培养在一起, 以协助检查和理解两种细胞的相互作用。

角膜组织工程: 人角膜间质神经相互作用的体外模型

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

corneal-tissue-engineering-an-vitro-model-stromal-nerve-interactions

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

8.7K 次观看.  University of Oklahoma Health Sciences Center. 本议定书描述了一个新的三维的体外模型, 其中角膜基质细胞和分化的神经细胞被培养在一起, 以协助检查和理解两种细胞的相互作用。

视频: Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

在SH-SY5Y人神经母细胞瘤分化

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

小儿食管上皮细胞重编程的条件在组织工程和疾病使用调查

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...