Name: corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea
Uploaded: 2018-01-24T13:30:00.000+00:00
Duration: 7 min 35 s
Description: 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

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>

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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&amp;rsquo;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&amp;rsquo;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-&amp;mu;m pores</td><td>Corning Costar</td><td>3412</td><td></td></tr><tr><td>2-O-&amp;alpha;-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 de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

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

8.8K 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

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

科研

发育生物学

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

生物工程

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

生物材料膜上人与羊角膜内皮细胞层的生长

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

医学

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

使用分裂的角质按钮的孔膜内皮器官培养模型

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

角膜上皮机械和化学损伤的离体和体内动物模型

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

一种由工程成纤维细胞生长因子1刺激的Descemet剥离的人类角膜器官培养模型，仅具有加速愈合

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

用于轴突再生研究的角膜感觉纤维损伤方案

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

作者聚焦：通过创新模型推进角膜神经支配研究 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

角膜组织工程: 人角膜间质神经相互作用的体外模型

摘要

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在体外和体内模型来研究角膜内皮-间充质转换

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

生物材料膜上人与羊角膜内皮细胞层的生长

创面愈合研究的前活体角膜器官培养模型

使用分裂的角质按钮的孔膜内皮器官培养模型

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

角膜上皮机械和化学损伤的离体和体内动物模型

一种由工程成纤维细胞生长因子1刺激的Descemet剥离的人类角膜器官培养模型，仅具有加速愈合

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

角膜组织工程: 人角膜间质神经相互作用的体外模型

摘要

探索更多视频

此视频中的章节

在体外和体内模型来研究角膜内皮-间充质转换

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

生物材料膜上人与羊角膜内皮细胞层的生长

创面愈合研究的前活体角膜器官培养模型

使用分裂的角质按钮的孔膜内皮器官培养模型

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

角膜上皮机械和化学损伤的离体和体内动物模型

一种由工程成纤维细胞生长因子1刺激的Descemet剥离的人类角膜器官培养模型，仅具有加速愈合

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生