Method Article
Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.
The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells’ native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.
临床相关,模拟白内障手术,这里所描述的体外上皮伤口愈合模型的开发是为了提供调查,在响应于损伤调节修复上皮组织的机制的一种工具。在创建该模型旨在为关键特征包括1)提供条件精密地复制在体内响应于伤人在培养设置,2)缓和调制修复的调控元件,以及3)能力图像的修复过程中,其整体,在实际时间。我们面临的挑战,因此,是在细胞的天然微环境创造一种文化模式,即有可能学习和操作,上皮创面修复。这种创伤修复模型的有效性开辟了新的可能性,标识来自调节修复过程基质蛋白,细胞因子和趋化因子的内源性信号提示。另外,该模型是理想的研究如何一Ñ 上皮能够移动作为集体片重新epithelialize伤口区域2,3,和用于确定在伤口边缘起作用的引导受伤上皮4的集体迁移间充质领袖细胞的谱系。这种模式还提供了一个用以标识疗法,可以有效的促进伤口愈合,防止异常创面修复5的平台。
目前已经有许多可用的创伤修复模式,无论是在文化和体内 ,它提供了当今大多数所谓对伤口修复过程。在动物损伤模型,如角膜6-12和皮肤13-17,有研究的组织的响应伤人的所有修复介质是可以参与的过程中,包括来自所述的上下文中,机会血管和神经系统。但是,也有限制操纵experi体内的精神的条件下,它是目前无法进行体内修复反应的成像研究,随时间连续。与此相反,大多数体外伤口修复培养模型,如划痕,可以很容易地操纵和随后随着时间的推移,但缺乏研究伤口愈合中的体内组织的环境背景。虽然体外模型提供了细胞的微环境加上调节修复的分子调节的过程中的任何时间点的能力范围内不断研究损伤修复过程中随着时间的优势,很少有车型适合这些参数。
这里描述的方法,以产生高度可重复的离体上皮的伤口愈合是再现上皮组织的响应于生理伤人培养物。使用鸡胚镜头作为组织源, 体外 MOCķ白内障手术被执行。该透镜是一种理想的组织用于这些研究,因为它是自包含在一个厚的基底膜胶囊,无血管,没有神经支配,且没有任何相关联的基质18,19。在人类疾病,白内障手术涉及视力丧失,由于透镜的混浊,并且包括除去晶状体纤维细胞块,其包含大量的透镜组成。白内障手术后视力是通过人工眼内透镜的插入恢复。在白内障手术过程中,通过除去纤维细胞,诱导在相邻透镜上皮,这是为了响应通过再上皮晶状体囊的后部区域已被占用的纤维细胞的损伤的反应。在白内障手术中,因为在大多数伤口修复反应,还有,有时会发生的异常纤维化结果向伤口愈合反应,与肌纤维母细胞的出现,这在透镜被称为后验Capsu相关联乐混浊20-22。为了产生白内障手术伤口愈合的模型,一个白内障手术的过程是模仿从鸡胚眼中除去产生生理损伤镜头。显微手术切除晶状体纤维细胞导致的晶状体上皮细胞包围着一个非常一致的圆形伤口面积。该细胞群保持牢固地附着在透镜基底膜胶囊和由外科手术过程中受伤。上皮细胞迁移到内源性基底膜的裸露面积愈合伤口的带动下,在维修过程中被称为领导者细胞1波形丰富的间充质细胞群。用该模型的上皮损伤的响应可容易地可视化并随后随时间在细胞的微环境的上下文中。细胞是易于接触到的表达或活化有望发挥在伤口修复中的作用的分子的修饰。日的一项强大功能是模型是分离和研究在伤口愈合的框架迁移特异性改变的能力。准备大量老年匹配体外伤口愈合培养物用于研究的能力是该模型的另一优点。因此,这个模型系统提供了一个独特的机会,梳理出创面修复机制和试验治疗为他们的伤口愈合过程中的作用。预计在体外模拟白内障手术模式,具有广泛的适用性,为研究损伤修复机制的重要资源。
下面的协议符合托马斯·杰斐逊大学机构动物护理和使用委员会的指导方针,并与ARVO声明为动物的视觉研究使用。
1.安装镜头,并准备于离体培养伤口
2.执行模拟白内障手术
3. Preparin克受伤的镜头体外培养
4.分离中央迁移区(CMZ),在那里再上皮后囊的创面的发生,从原始附件晶状体上皮细胞的区(OAZ),定量分析。
注:细胞开始响应损伤立即移动到CMZ区域。通过在培养一天足够的细胞迁移横跨CMZ分子和生化分析,继CMZ和OAZ通过显微解剖23分离。该协议涉及除去一个翼片(OAZ)中的从胶囊的受伤区域中的时间。
离体模型创建研究伤口愈合过程中的细胞的天然微环境
调查涉及细胞的微环境原生内调节上皮伤口愈合机制,创建了一个临床相关的体外模拟白内障手术模式。这个模型是从晶状体组织提供了许多优点,由于其内在特性创建:1)透镜是一个自包含的器官由粗基底膜称为晶状体囊包围; 2)它是无血管,3)没有神经支配和4)免费相关基质。因此,检查了修复过程仅限于细胞是先天的透镜适当。为了创建这个模型中,透镜从胚胎(E)的第15天的小鸡胚胎( 图1A)除去。一个小切口,在晶状体前囊和其相关联的晶状体上皮细胞,通过该透镜纤维细胞块被移除通过水力洗脱,创建一个生理相关伤人( 图1A)的经典白内障的过程。的晶状体上皮,这是目前作为沿前细胞和晶状体囊的赤道方面围绕纤维细胞块,连同其内源性的波形蛋白富含间充质修复细胞祖细胞1群的连续单层,在留在透镜提取纤维细胞( 图1A)。在除去纤维细胞团,五切口的在晶状体囊的前方面和上皮扁平细胞面朝上,面向平台。此过程使受伤的上皮细胞的各种微观的方法,包括时间推移显微镜( 视频1)响应的成像。在晶状体前囊这些附加切口创建伤员透镜的星形植与由岑除去纤维细胞块创建的圆形伤口之三植( 图1B)。白内障手术后的组织的扁平化,伤员上皮位于星,它被称为原始附件区(OAZ)的点( 图1A,B)。
该从其附着部位的晶状体后囊切除纤维细胞团创建了一个高度重复性的伤口面积的基底膜,用受伤的上皮细胞所包围 ( 图1B中的C)。的透镜赤道上皮的暴露边缘,只是相邻于被附着在纤维细胞,是伤口的前缘。紧接损伤,波形蛋白富含间充质修复细胞亚群被激活,并迁移至上皮1的伤口边缘。的晶状体上皮,与这些间充质修复细胞在它们的前缘,快速移动到内源性b的无细胞区asement膜囊,中央迁移区(CMZ),开始愈合的伤口。晶状体上皮细胞移动的统称,作为片材,进为首的间充质领袖电池1,其延伸突起沿着基底和指导伤口愈合过程的CMZ(F igure 1D,视频1)。伤口愈合的进展,覆盖伤口(67%)1天在培养物( 图1C)的一个显著区域,并且通常内培养3天( 图1B中的C)完成。
一个明显的物理区别可以在OAZ和CMZ区域之间进行早1天,这是界定为这两个区域( 图2A,箭头)之间的折痕或皱纹。这种现象提供了思路由在分离这些区域在伤口愈合过程中的任何点。用细尖镊子,文化可以是微观解剖SEPAR吃了OAZ和CMZ地区,以分析这些不同的区域( 图2B)之间的差异的分子。这是已经被用于标识与伤口修复有关的迁移特异性改变一个功能强大的方法。在此之前,人们发现,有伤口愈合过程5中的增加粘着斑激酶(FAK)的活化在体外伤员培养物。 FAK在细胞迁移26-29行之有效的作用。的能力,以丰富的OAZ与CMZ现在使得能够检查这种增加的FAK的激活是否是特定于迁移特定CMZ区域。对于这项研究中,OAZ和CMZ区域上分离天1 - 3(整个伤口愈合过程)和在FAK的激活(FAK pY397)生物化学变化进行分析。结果表明,增加的FAK活化与迁移特定CMZ区域( 图2C)相关联。该这里描述的体外模型系统提供了在其调查参与协调创伤修复过程中的分子节目的独特和宝贵的机会。
图1.创建一个体外模型,其中,研究细胞的天然微环境中的伤口愈合过程中响应于生理伤人。白内障模拟手术上E15小鸡镜片进行的。晶状体纤维细胞块(白色)是通过在所述前囊由水力洗脱的切口移除。这个过程留下上皮细胞(绿色),该保持牢固附着到晶状体囊和小区裸露基底膜(BM)到其上的细胞会迁移到愈合伤口(A)中 。五切口向上皮变平,并创建一个星形体外崇拜 URE(B)。残留的晶状体上皮细胞,填补了星点被称为原始附件区(OAZ)(A,B)。裸露的BM到其上的细胞将迁移被称为中央迁移区(CMZ)(A,B)。立即响应于损伤,细胞开始移动到CMZ区域上的小区裸露的BM(B)中 。开放性伤口面积进行定量随时间(C)中。伤口愈合通常通过D3中培养(B,C)完成。在(B,C)T0表示伤人的时候,D1-3表示1-3天。内的CMZ,细胞的两个群体可以区分,晶状体上皮细胞和间充质领导细胞定位于伤口边缘,沿着基材(D)延伸的突出部。这个数字是从Menko 等 23重印。目标="_空白">点击此处查看该图的放大版本。
图2.分离OAZ和CMZ区域以识别与伤口愈合相关的迁移特异性改变,通过每日1次,OAZ和CMZ区可以从彼此区分由一个折缝(箭头),它可以用来作为指导以分离这些区域(A)。在此折痕,细尖镊子可以用于夹住OAZ / CMZ线的边缘。文化可以沿着这条线让OAZ和CMZ区(B)的隔离分开。显微解剖的OAZ和CMZ每天,从(D1)的第1天 - 第3天(D3)进行,以确定是否发生在创伤修复的区域中的FAK的激活迁移特异性改变。从各区域的用裂解液Western印迹分析或者FAK激活(pFAK Y39检查7)或总FAK表达(C)。而观察到在总FAK水平的变化不大,在增加的FAK活化与迁移特定CMZ区(C)相关联。
视频1.在体外模拟白内障手术模型创面愈合之后是时间推移显微镜从时间0伤后通过向第3天伤口闭合,从伤口区域的中心观看。
Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryonic lenses requires developing skills in the handling of small tissues that can be acquired with practice, as the embryonic lenses are only about 2mm in diameter. The cataract surgery procedure is performed under the dissecting microscope, and the critical technical steps include making a small incision in the anterior capsule, removing the fiber cell mass by hydro-elution, and making additional cuts in the anterior epithelium that make it possible to flatten the ex vivo post-cataract surgery lens tissue on the substrate to which it is pinned for study in culture. The microsurgical cataract surgery procedure produces a highly reproducible wound area, exposing a circular region of the tissue’s endogenous basement membrane onto which the injured epithelium migrates as it closes the wound. This model has been used to study mechanisms regulating wound repair of epithelia, including how the epithelial cells are able to move collectively into the wound area 30 and the leader cell function of an endogenous vimentin-rich repair cell population of mesenchymal lineage at the wound edge1,23.
This ex vivo wound model lends itself readily to live microscopic observation throughout the repair process including time-lapse imaging, and confocal image analysis following immuno-localization of proteins of interest. Making cuts in the anterior lens capsule so that the capsule with its attached injured epithelium can be flattened on the culture substrate makes such ease of observation possible. Including this critical step is essential for viewing the behavior of the response of the epithelial cells in the original zone of attachment and the movement of the injured epithelium onto the wounded area of the posterior capsule from the time of injury. A great advantage for biochemical or molecular biology analyses is that the cells that are associated with the basement membrane in the original attachment zone (OAZ) can be separated by micro-dissection from those cells migrating across the wounded area of the basement membrane (CMZ) at any time during the wound repair process.
The mock cataract surgery wound repair culture model was established using lenses from E15 chick embryos. Alternatively, wound repair cultures can be successfully prepared using this protocol from lenses removed at other stages of chick embryonic development, and from lenses isolated from both adult mice and rats. While studies to date have focused on wound repair in the chick embryo lens model, in part because of the ease in obtaining large numbers of age-matched tissue for experimental analysis, there is no reason that would prevent this protocol from being used with lenses isolated from any species. However, it is widely recognized in the literature that wound repair in the embryo often occurs more quickly and with less scarring than in the adult 31-35. The wound repair cultures presented here are typically grown in the absence of serum to maintain conditions close to the in vivo microenvironment, suggesting that the factors that promote wound healing are provided by components of that environment including ones produced by the cells themselves. However, wound repair in this culture model also occurs effectively in defined media conditions, and can be modulated by including specific pharmaceutical inhibitors in the culture media 5,23,30, or knocking down expression of proteins through an siRNA approach 23. Note that inclusion of serum in the culture media induces movement of the cells from the cut edges on the outside of the flattened explant onto the tissue culture substrate, while in serum-free medium the cells remain associated with the capsule and migrate only onto the wounded area of the basement membrane of the lens capsule. The approach of adding serum to the media can be an advantage if one desires to examine the behavior of the wounded epithelium as it moves onto different substrates. To that end, there are other options that can be taken to alter the environment for wound repair that includes use of alternate tissue culture substrates on which to pin the tissue following the mock cataract surgery, such as substrates with defined rigidities as can be provided by collagen or acrylamide gels. While the ex vivo mock cataract surgery cultures can be facilely pinned to tissue culture plastic, collagen gels, or acrylamide gels, affixing the cultures to glass substrates has proven difficult. An important adaptation of this wound repair model to consider is its usefulness for re-injury studies. In this approach a scrape wound is inflicted on the healed lens epithelium after wound repair is completed, which removes an area of the lens epithelium from its underlying basement membrane capsule. This re-injury can be performed in either the original zone of attachment of the epithelium to the lens capsule or in the central migration zone where the epithelium has healed the initial wound area.
A great advantage to this wound-repair model is that the lens is an avascular tissue, not innervated without an associated stromal compartment, thus creating an ideal reductionist model of wound repair. As such, this model allows for examination of the native wound repair process under conditions in which only cells and microenvironment that are intrinsic to this epithelial tissue can modulate the repair process. In order to examine the role of cells that could be recruited following injury from neighboring vasculature or stromal compartments, it would be necessary to add such cells to the culture environment.
In previous culture wound models, the major limitation to investigating how an epithelial tissue responds to wounding is that most are limited to examining cells, often cell lines, plated on tissue culture plastic. Popular models using this approach, such as the scratch wound assay, have provided significant insight into how cells migrate to fill an area of the culture dish from where the cells have been removed. However, in these scratch wound studies the movement of cells onto the exposed area of the culture substrate occurs in the absence of many of the dynamic wound response signals that regulate wound healing in vivo. Therefore, the most important advantage of the mock cataract surgery culture model is that wound repair can be studied ex vivo within the cells’ native microenvironment and the endogenous signals originating from the underlying matrix, growth factors, cytokines and repair cell subpopulations. As this new wound repair model makes it possible to examine and manipulate the response of an epithelium to wounding in its native microenvironment, it is likely to have many applications, and holds great promise as an ideal system for revealing the molecular-mediators of wound-repair.
The authors declare that they have no competing financial interests.
This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).
Name | Company | Catalog Number | Comments |
Sodium Chloride (NaCl) | Fisher Scientific | S271-3 | Use at 140 mM in TD Buffer |
Potassium Chloride (KCl) | Fisher Scientific | P217-500 | Use at 5 mM in TD Buffer |
Sodium Phosphate (Na2HPO4) | Sigma | S0876 | Use at 0.7 mM in TD Buffer |
D-glucose (Dextrose) | Fisher Scientific | D16-500 | Use at 0.5 mM in TD Buffer |
Tris Base | Fisher Scientific | BP152-1 | Use at 8.25 mM in TD Buffer |
Hydrochloric acid | Fisher Scientific | A144-500 | Use to pH TD buffer to 7.4 |
Media 199 | GIBCO | 11150-059 | |
L-glutamine | Corning/CellGro | 25-005-CI | Use at 1% in Media199 |
Penicillin/streptomycin | Corning/CellGro | 30-002-CI | Use at 1% in Media199 |
100 mm petri dishes | Fisher Scientific | FB0875711Z | |
Stericup Filter Unit | Millipore | SCGPU01RE | Use to filter sterilize Media |
Dumont #5 forceps (need 2) | Fine Science Tools | 11251-20 | |
35 mm Cell Culture Dish | Corning | 430165 | |
27 G 1 ml SlipTip with precision glide needle | BD | 309623 | |
Fine Scissors | Fine Science Tools | 14058-11 | |
Standard Forceps | Fine Science Tools | 91100-12 | |
Other Items Needed: General dissection instruments, fertile white leghorn chicken eggs, check egg incubator (humidified, 37.7°C), laminar flow hood, binocular stereovision dissecting microscope |
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