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Feb 2019

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Fibroblast Gap-closure Assay-Microscopy-based in vitro Assay Measuring the Migration of Murine Fibroblasts
基于成纤维细胞间隙封闭显微成像的鼠成纤维细胞的体外迁移分析   

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Abstract

Pulmonary fibrosis is characterized by pathological scaring of the lung. Similar to other fibrotic diseases, scar formation is driven by excessive extracellular matrix deposition by activated, proliferative, and migratory fibroblasts.

Currently, the two most widely used chemotaxis and cell migration assays are the scratch assay and the transmembrane invasion assay. Here we present a gap closure assay that employs commercially available cell lines, equipment and reagents and is time efficient as well as straightforward. The protocol uses an Oris pro cell migration assay 96-well plate with a dissolvable plug in the center of each well to create a cell free area at the time of seeding. Cell repopulation of the empty zone is captured via light microscopy at different time points and quantified with free image analysis software. The clear advantages of this assay in comparison to similar protocols are the use of uncomplicated cell culture methods and the ability to image the experiment throughout.

Keywords: Fibroblast (成纤维细胞), Migration (迁移), Fibrosis (纤维化), Gap closure (间隙闭合), Chemotaxis (趋化作用)

Background

Few treatments for fibrosing diseases exist because of an incompletely understood and complex etiology (Rockey et al., 2015). Current efforts to develop therapies for organ fibrosis have focused on pathologic fibroblasts, also known as myofibroblasts (Blackwell et al., 2014). In addition to secretion of matrix proteins such as collagen, which comprise scar, a hallmark of pathologic fibroblasts is their increased proliferative and migratory capacity (Kendall and Feghali-Bostwick, 2014). A number of studies have shown that innate immune cells interact with and mediate fibroblast activation (Desai et al., 2018). Thus, we investigated the effects of lung macrophages on fibroblasts in a recent study, where we found a novel population of macrophages that localizes to fibrotic scar in murine lung (Aran et al., 2019). These macrophages highly expressed PDGF-AA, a secreted factor known for promoting fibroblast migration and proliferation. Thus, we treated mouse 3T3 embryonic fibroblasts with conditioned media from cultured mouse lung macrophages, with and without PDGF-AA blocking antibody, and measured fibroblast gap closure.

Here we describe the protocol for the assay, which should be useful for other investigators studying paracrine signaling between adjacent cellular lineages. Since the method presented employs an established, adherent cell line, it may be easily adapted to other cell types and treatments in fields where cell migration is an important pathological characteristic, such as cancer or wound healing. Importantly, fibroblasts and the extracellular matrix have been recently recognized as fundamental players in the tumor microenvironment and as such are of the outmost interest in oncology research (Bu et al., 2019).

Compared with other methods for investigating chemotaxis (Justus et al., 2014), such as the scratch assay, our approach does not introduce mechanical stress to the cells, which can potentially activate fibroblasts and obscure results. It also does not require optimization of cell culture as required for the transwell/chamber invasion assay, and data from the same well may be collected at multiple timepoints while cells are visualized in real time. Our assay can be easily scaled up to a high throughput format for drug screening purposes. One limitation of the assay is that it does not address directional cell migration. Also, our protocol measures both migration and proliferation of fibroblast cells; a proliferation assay should be conducted if further distinction is needed.

Materials and Reagents

  1. Oris pro cell migration assay plate, 96-well (Platypus Technologies, catalog number: PROCMA1), stored at room temperature
  2. 3T3 cells (ATCC, catalog number: CRL-1658), stored in liquid nitrogen
  3. DMEM (Corning, catalog number: 10-101-CV), stored at 4 °C
  4. Fetal Bovine Serum (HyClone, catalog number: SH3039603LR), stored at 4 °C
  5. Antibiotic-antimycotic solution (Corning, catalog number: 30-004-CI), stored at 4 °C
  6. Trypsin 0.25% (Corning, catalog number: 25053CL), stored at 4 °C
  7. PDGF-AA antibody (Millipore, catalog number: 07-1436), stored at -20 °C
  8. Cell culture media (see Recipes)
  9. Fully supplemented media (see Recipes)
  10. Serum free media (see Recipes)

Equipment

  1. Microscope
    Zeiss Axio Observer D1 (Carl Zeiss) equipped with a Yocogawa spinning wheel coupled to a photometrics EMCCD camera (Evolve 512 delta)

Software

  1. Zeiss Zen Blue (Carl Zeiss)
  2. Fiji/ImageJ (https://fiji.sc/)
  3. Prism (https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Establish healthy culture of 3T3 cells in a fully supplemented media (10% serum) according to instructions from ATCC.
  2. On the day of the experiment, trypsinize the cells, count and re-suspend in a pre-warmed serum-free media (100 μl per well). Add approximately 5 x 104 cells per well (100% confluent) to a migration assay plate; this step may need optimization depending on cell size and doubling time. Plan to have 3 wells per condition. Let the cells to adhere for 3 h. Note that the plaque in the center of the well dissolves within 30 min; therefore, it is suitable only for rapidly adherent cells. For example, we found that freshly sorted primary mouse lung fibroblasts needed a longer time to adhere, and we failed to obtain consistent results from primary cells.
  3. Using the light microscope (phase contrast for unlabeled cells), verify the cell attachment and the presence of a circular cell-free zone in each well. Exclude the wells that have an excessive amount of cells in a cell-free zone or have uneven distribution of cells in cell growth area (approximately 5% of wells). Take the 0 h timepoint picture of each well, remembering to number or name the wells and images. We recommend using a microscope with a built-in incubator chamber to avoid additional stress to the cells.
  4. Add 100 μl of pre-warmed media with antibodies/drug or conditioned media. Make sure that all of the wells have the same serum conditions. For example, our conditioned media from mouse macrophages contained 10% serum; we therefore used 10% serum media for blocking antibody and all control wells.
  5. Acquire images at 24 h and, if needed, at later timepoints for each well. We found that at 48 h the gaps in majority of wells were completely closed and the most significant difference of the gap closure occurred during the first 24 h. It is possible that shorter timepoints might be necessary for other cell types, or when using conditions with potent mitogens.

Data analysis

We used Zeiss microscope software Zen Blue to stitch the phase-contrast images of the wells acquired at 20x magnification. Fiji (ImageJ) was used for all further image processing and data analysis. As seen in Figure 1, inverting the colors of the pictures greatly improved clarity of the cell-free zone border. We manually selected the cell-free zones using the circular selection tool for 0 h timepoint images and the circular or manually drawn (if the area was not circular) selection tool for 24 h timepoint images. We took individual pictures of wells at time 0 h, because we noticed slight size variability of the cell free zones between the wells. Next, the area of selection was measured by implementing the commands in Fiji: analyze > measure > compute area. We calculated the migration area as a difference between area at time 0 h and 24 h for each well, averaged the area for triplicate wells, and then normalized the data (i.e., we divided all data points from all groups by a mean of the ‘baseline’ group). We used two-sided Wilcoxon rank-sum test as the statistical test.


Figure 1. Representative images and quantification of fibroblast gap closure assay. Pictures of 3T3 fibroblasts were taken at time 0 h and 24 h. Colors in the image were inverted for clarity, the yellow circle indicates the border of the cell free zone at time 0 h and blue circle at time 24 h. Representative data reproduced from Aran et al., 2019; 3T3 fibroblasts were incubated with conditioned media (CM) from lung macrophages sorted by MHCII expression, with and without PDGF-AA blocking antibody (AB). Wilcoxon test two-sided P values are presented. **P < 0.01.

Recipes

  1. Cell culture media
    DMEM
    Fetal Bovine Serum
    Antibiotic-antimycotic solution
    Trypsin
    Prepare cell culture media in sterile conditions
  2. Serum free media
    DMEM
    1% antibiotic-antimycotic
  3. Fully supplemented media
    DMEM
    10% serum
    1% antibiotic-antimycotic

Acknowledgments

This work was supported by a UCSF Marcus Award and a National Institutes of Health grant (HL131560) to Mallar Bhattacharya.
Our assay uses commercially available Oris Pro Migration Assay (https://www.platypustech.com/cell-migration), which has been optimized and modified for this protocol.

Competing interests

The authors declare no competing financial interest.

References

  1. Aran, D., Looney, A. P., Liu, L., Wu, E., Fong, V., Hsu, A., Chak, S., Naikawadi, R. P., Wolters, P. J., Abate, A. R., Butte, A. J. and Bhattacharya, M. (2019). Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat Immunol 20(2): 163-172.
  2. Blackwell, T. S., Tager, A. M., Borok, Z., Moore, B. B., Schwartz, D. A., Anstrom, K. J., Bar-Joseph, Z., Bitterman, P., Blackburn, M. R., Bradford, W., Brown, K. K., Chapman, H. A., Collard, H. R., Cosgrove, G. P., Deterding, R., Doyle, R., Flaherty, K. R., Garcia, C. K., Hagood, J. S., Henke, C. A., Herzog, E., Hogaboam, C. M., Horowitz, J. C., King, T. E., Jr., Loyd, J. E., Lawson, W. E., Marsh, C. B., Noble, P. W., Noth, I., Sheppard, D., Olsson, J., Ortiz, L. A., O'Riordan, T. G., Oury, T. D., Raghu, G., Roman, J., Sime, P. J., Sisson, T. H., Tschumperlin, D., Violette, S. M., Weaver, T. E., Wells, R. G., White, E. S., Kaminski, N., Martinez, F. J., Wynn, T. A., Thannickal, V. J. and Eu, J. P. (2014). Future directions in idiopathic pulmonary fibrosis research. An NHLBI workshop report. Am J Respir Crit Care Med 189(2): 214-222.
  3. Bu, L., Baba, H., Yoshida, N., Miyake, K., Yasuda, T., Uchihara, T., Tan, P. and Ishimoto, T. (2019). Biological heterogeneity and versatility of cancer-associated fibroblasts in the tumor microenvironment. Oncogene 38(25): 4887-4901.
  4. Desai, O., Winkler, J., Minasyan, M. and Herzog, E. L. (2018). The role of immune and inflammatory cells in idiopathic pulmonary fibrosis. Front Med (Lausanne) 5: 43.
  5. Justus, C. R., Leffler, N., Ruiz-Echevarria, M. and Yang, L. V. (2014). In vitro cell migration and invasion assays. J Vis Exp (88). doi: 10.3791/51046.
  6. Kendall, R. T. and Feghali-Bostwick, C. A. (2014). Fibroblasts in fibrosis: novel roles and mediators. Front Pharmacol 5: 123.
  7. Rockey, D. C., Bell, P. D. and Hill, J. A. (2015). Fibrosis--a common pathway to organ injury and failure. N Engl J Med 373(1): 96.

简介

肺纤维化的特征在于肺的病理性瘢痕。与其他纤维化疾病类似,瘢痕形成由活化的,增殖的和迁移的成纤维细胞过度的细胞外基质沉积驱动。
目前,两种最广泛使用的趋化性和细胞迁移测定是划痕测定和跨膜侵袭测定。在这里,我们提出了一种间隙闭合测定法,该测定法使用商业上可获得的细胞系,设备和试剂,并且具有时间效率和直接性。该方案使用Oris pro细胞迁移测定96孔板,在每个孔的中心具有可溶解的塞子,以在接种时产生无细胞区域。在不同时间点通过光学显微镜捕获空区的细胞再增殖,并用免费图像分析软件定量。与类似方案相比,该测定的明显优点是使用无复杂的细胞培养方法和整个实验成像的能力。
【背景】由于不完全了解和复杂的病因,很少有针对纤维化疾病的治疗方法(Rockey et al。,2015)。目前开发用于器官纤维化的疗法的努力集中于病理性成纤维细胞,也称为肌成纤维细胞(Blackwell 等人,2014)。除了包含瘢痕的基质蛋白如胶原蛋白的分泌外,病理性成纤维细胞的标志是它们增加的增殖和迁移能力(Kendall和Feghali-Bostwick,2014)。许多研究表明,先天免疫细胞与成纤维细胞活化相互作用并介导成纤维细胞活化(Desai et al。,2018)。因此,我们在最近的一项研究中研究了肺巨噬细胞对成纤维细胞的影响,我们发现了一种新的巨噬细胞群,它定位于小鼠肺纤维化瘢痕(Aran et al。,2019)。这些巨噬细胞高度表达PDGF-AA,这是一种已知用于促进成纤维细胞迁移和增殖的分泌因子。因此,我们用来自培养的小鼠肺巨噬细胞的条件培养基处理小鼠3T3胚胎成纤维细胞,使用和不使用PDGF-AA阻断抗体,并测量成纤维细胞间隙闭合。
在这里,我们描述了该测定的方案,这对于研究相邻细胞谱系之间的旁分泌信号传导的其他研究者应该是有用的。由于所提出的方法采用已建立的贴壁细胞系,因此可以容易地适应细胞迁移是重要病理特征的领域中的其他细胞类型和治疗,例如癌症或伤口愈合。重要的是,成纤维细胞和细胞外基质最近被认为是肿瘤微环境中的基本参与者,因此对肿瘤学研究最为关注(Bu et al。,2019)。
与用于研究趋化性的其他方法(Justus et al。,2014)相比,例如划痕试验,我们的方法不会向细胞引入机械应力,这可能潜在地激活成纤维细胞并模糊结果。它也不需要优化细胞培养物,如transwell /室入侵测定所需,并且可以在多个时间点收集来自相同孔的数据,同时实时观察细胞。我们的分析可以轻松扩展到高通量格式,用于药物筛选目的。该测定的一个限制是它不能解决定向细胞迁移问题。此外,我们的协议测量成纤维细胞的迁移和增殖;如果需要进一步区分,应进行增殖试验。

关键字:成纤维细胞, 迁移, 纤维化, 间隙闭合, 趋化作用

材料和试剂

  1. Oris pro细胞迁移测定板,96孔(Platypus Technologies,目录号:PROCMA1),在室温下储存
  2. 3T3细胞(ATCC,目录号:CRL-1658),储存在液氮中
  3. DMEM(Corning,目录号:10-101-CV),储存在4℃
  4. 胎牛血清(HyClone,目录号:SH3039603LR),储存在4℃
  5. 抗生素 - 抗真菌溶液(Corning,目录号:30-004-CI),储存在4℃
  6. 胰蛋白酶0.25%(Corning,目录号:25053CL),储存在4°C
  7. PDGF-AA抗体(Millipore,目录号:07-1436),储存在-20℃
  8. 细胞培养基(见食谱)
  9. 完全补充的媒体(见食谱)
  10. 无血清培养基(见食谱)

设备

  1. 显微镜
    Zeiss Axio Observer D1(卡尔蔡司)配备Yocogawa旋转轮,耦合光度计EMCCD相机(Evolve 512 delta)

软件

  1. 蔡司禅蓝(卡尔蔡司)
  2. 斐济/ ImageJ( https://fiji.sc/ )
  3. 棱镜( https://www.graphpad.com/scientific-software/prism/ )

程序

  1. 根据ATCC的说明,在完全补充的培养基(10%血清)中建立3T3细胞的健康培养物。
  2. 在实验当天,胰蛋白酶消化细胞,计数并重新悬浮在预热的无血清培养基中(每孔100μl)。每孔加入约5×10 4个细胞(100%汇合)至迁移测定板;此步骤可能需要根据单元大小和倍增时间进行优化。计划每个条件有3口井。让细胞粘附3小时。注意,孔中心的斑块在30分钟内溶解;因此,它仅适用于快速粘附的细胞。例如,我们发现新分选的原代小鼠肺成纤维细胞需要更长的粘附时间,并且我们未能从原代细胞获得一致的结果。
  3. 使用光学显微镜(未标记细胞的相差),验证细胞附着和每个孔中是否存在圆形无细胞区。排除在无细胞区域中具有过量细胞的孔或在细胞生长区域中具有不均匀的细胞分布(约5%的孔)。获取每个井的0小时时间点图片,记住对井和图像进行编号或命名。我们建议使用带有内置培养箱的显微镜,以避免对细胞造成额外的压力。
  4. 加入100μl预热的培养基和抗体/药物或条件培养基。确保所有孔具有相同的血清条件。例如,我们来自小鼠巨噬细胞的条件培养基含有10%的血清;因此,我们使用10%血清培养基来阻断抗体和所有对照孔。
  5. 在24小时获取图像,如果需要,在每个孔的稍后时间点获取图像。我们发现在48小时时,大多数井中的间隙完全闭合,并且间隙闭合的最显着差异发生在最初的24小时内。其他细胞类型可能需要更短的时间点,或者使用具有强效促分裂原的条件时可能需要更短的时间点。

数据分析

我们使用Zeiss显微镜软件Zen Blue来缝合以20倍放大率获得的孔的相衬图像。斐济(ImageJ)用于所有进一步的图像处理和数据分析。如图1所示,反转图像的颜色大大提高了无细胞区边界的清晰度。我们使用圆形选择工具手动选择无细胞区域0小时时间点图像和圆形或手动绘制(如果区域不是圆形)选择工具24小时时间点图像。我们在0小时时拍摄了井的单独照片,因为我们注意到孔之间无细胞区的尺寸变化很小。接下来,通过在斐济实施命令来测量选择区域:分析&gt;测量&gt;计算区域。我们将迁移区域计算为每个井在0 h和24 h时间区域之间的差异,平均一式三份井的面积,然后对数据进行归一化(即,我们将所有数据点都划分为所有数据点按“基线”组的平均值分组。我们使用双侧Wilcoxon秩和检验作为统计检验。


图1.成纤维细胞间隙闭合测定的代表性图像和定量。在0小时和24小时时拍摄3T3成纤维细胞的图片。为清楚起见,图像中的颜色被反转,黄色圆圈表示0小时时无细胞区域的边界和24小时时的蓝色圆圈。代表性数据来自Aran et al。,2019;将3T3成纤维细胞与来自肺巨噬细胞的条件培养基(CM)一起温育,所述条件培养基通过MHCII表达分选,有和没有PDGF-AA阻断抗体(AB)。给出了Wilcoxon检验双侧P值。 ** P &lt; 0.01。

食谱

  1. 细胞培养基
    DMEM
    胎牛血清
    抗生素 - 抗真菌药物解决方案
    胰蛋白酶
    在无菌条件下制备细胞培养基
  2. 无血清培养基
    DMEM
    1%抗生素 - 抗真菌药
  3. 完全补充的媒体
    DMEM
    10%血清
    1%抗生素 - 抗真菌剂

致谢

这项工作得到了加州大学旧金山分校马库斯奖和国家卫生研究院拨款(HL131560)给Mallar Bhattacharya的支持。
我们的化验使用市售的Oris Pro迁移试验( https://www.platypustech.com/cell-migration),已针对此协议进行了优化和修改。

利益争夺

作者声明没有竞争性的经济利益。

参考

  1. Aran,D.,Looney,AP,Liu,L.,Wu,E.,Fong,V.,Hsu,A.,Chak,S.,Naikawadi,RP,Wolters,PJ,Abate,AR,Butte,AJ and Bhattacharya,M。(2019年)。 基于参考的肺单细胞测序分析显示过渡性促纤维化巨噬细胞。 Nat Immunol 20(2):163-172。
  2. Blackwell,TS,Tager,AM,Borok,Z.,Moore,BB,Schwartz,DA,Anstrom,KJ,Bar-Joseph,Z.,Bitterman,P.,Blackburn,MR,Bradford,W.,Brown,KK, Chapman,HA,Collard,HR,Cosgrove,GP,Deterding,R.,Doyle,R.,Flaherty,KR,Garcia,CK,Hagood,JS,Henke,CA,Herzog,E.,Hogaboam,CM,Horowitz,JC ,King,TE,Jr.,Loyd,JE,Lawson,WE,Marsh,CB,Noble,PW,Noth,I.,Sheppard,D.,Olsson,J.,Ortiz,LA,O'Riordan,TG,Oury ,TD,Raghu,G.,Roman,J.,Sime,PJ,Sisson,TH,Tschumperlin,D.,Violette,SM,Weaver,TE,Wells,RG,White,ES,Kaminski,N.,Martinez,FJ ,Wynn,TA,Thannickal,VJ和Eu,JP(2014)。 特发性肺纤维化研究的未来发展方向。 NHLBI研讨会报告。 Am J Respir Crit Care Med 189(2):214-222。
  3. Bu,L.,Baba,H.,Yoshida,N.,Miyake,K.,Yasuda,T.,Uchihara,T.,Tan,P。和Ishimoto,T。(2019)。 癌症相关成纤维细胞在肿瘤微环境中的生物异质性和多功能性。 癌基因&nbsp; 38(25):4887-4901。
  4. Desai,O.,Winkler,J.,Minasyan,M。和Herzog,E。L.(2018)。 免疫和炎症细胞在特发性肺纤维化中的作用。 Front Med (洛桑) 5:43。
  5. Justus,C.R.,Leffler,N.,Ruiz-Echevarria,M。和Yang,L。V.(2014)。 体外细胞迁移和侵袭分析。 J Vis Exp&nbsp; (88)。 doi:10.3791 / 51046。
  6. Kendall,R。T.和Feghali-Bostwick,C。A.(2014)。 纤维化成纤维细胞:新型角色和介质。 Front Pharmacol 5:123。
  7. Rockey,D.C.,Bell,P。D. and Hill,J.A。(2015)。 纤维化 - 器官损伤和衰竭的常见途径。 N Engl J Med 373(1):96。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Looney, A. P. and Bhattacharya, M. (2019). Fibroblast Gap-closure Assay-Microscopy-based in vitro Assay Measuring the Migration of Murine Fibroblasts. Bio-protocol 9(16): e3333. DOI: 10.21769/BioProtoc.3333.
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