Summary

伤口划痕试验的优化来探测在小鼠间充质干细胞迁移的可溶性香烟烟雾提取物受损后

Published: December 03, 2015
doi:

Summary

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Abstract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

Introduction

细胞迁移是高度协调和至关重要的许多生理过程,如组织发育,修复和再生,以及病理过程,如癌症转移和动脉硬化1。细胞迁移的理解是特别相关的用于修复受损的组织和治疗的病理状态,包括细胞移植技术和人工组织嫁接2新兴疗法。鉴于间充质干细胞(MSCs)在介导组织修复3,用分析是严格计量的,适应性强,具有成本效益的感兴趣量化这些细胞的迁移能力的作用,目前的利息。重要的是,这样的测定必须是足够敏感的损害后,以检测在细胞迁移能力相对微妙的变化。目前的方法用于定量细胞的迁移,包括刮测定法,反式井迁移测定(Boyden小通道琥珀),微柱阵列,和细胞排除区检测具有一系列在再现性,可定制,量化,和成本效益-1,4,5-限制。此处所描述的优化划痕试验表明健壮的结果,量化的和基于图像的分析能力,成本效益,并适应其它应用。

划痕试验已被用于多种能力来评估不同的实验条件下5细胞迁移和增殖。该检测限嗣继承播种指定的细胞,他们成长,完成或接近汇合,和刮擦产生的单层用消毒针头或吸管尖6。是最常见的,在随机选择的位置7-9比较所述划痕在多个时间点的宽度进行分析。尽管其突出使用,划痕试验通过与重现性和定量的问题混淆。变异代划痕不仅改变细胞的微环境,而且还可以通过破坏该板面和底层的细胞外基质5阻碍细胞的迁移。测定是经常进行了超过7〜12小时,但对于细胞株显示较慢的迁移和更长的实验时间,增殖成为一个混杂变量7,10。最后,通过划痕过程中产生的衰老细胞中可以释放与缩小差距,在单层1所需的细胞外信令干扰因素。优化划痕测定需要创建一个一致的间隙不与表面性质干扰,减少分析时间长度,并防止操作过程中不希望的细胞死亡。止动基础的分析是小区排除区域测定的优化。该测定法利用塞子放置在排除细胞生长的孔的中间,但是允许细胞可围绕中心隔离区电镀。为了评估迁移,挡块被删除,并将所得排除区提供了用于迁移发生的表面。然而,该测定是困难的定制或调整10和对于一些应用,这种技术也可以成本过高。

在对比划痕测定和它们的衍生物,反-井迁移测定(或Boyden小室测定法)通过定量细胞移动从一个腔室的数目,通过微孔滤膜过滤,到含有趋化剂8,11的室评估迁移12。这种技术具有有限的效用为贴壁细胞如间充质干因为以下通过多孔膜的迁移,细胞粘附在膜侧暴露于趋化剂,并且可能很难准确量化。虽然检测是能够研究一些三维的迁移模式,受限制的细 ​​胞类型,这就是它能够准确量化细胞迁移限制其效用10。另一替代划伤试验采用了微柱阵列,它通过一个三维空间使用细胞变形并迁移到数组作为替代的能力测量细胞运动。聚二甲基硅氧烷(PDMS)弹性体固化过精密模具和用臭氧处理和纤连蛋白产生均匀和不可降解的微环境。根据需要,以评估细胞的进入阵列 4的能力的微柱间距也可以变化。模具是通过硅晶片的深反应离子蚀刻创建以创建高宽比阵列 13的负版本。虽然试验是由它的可定制性加强,能够通过细胞迁移的直接可视化模型的三维迁移和分析,难以产生微柱阵列在经济上阻碍了它的广泛使用。

在本协议中所描述的优化划痕试验提供了一个高效,COS叔有效的制造方法,可以使用免费提供的软件进行分析一致划痕。而不是之前和细胞迁移后,整个从头做起简单的宽度测量,该软件能够使用户在迁移前后测定总划伤的地方。这个前进限制了试图确定何处划痕宽度测量应采取的问题,以及是否划痕的宽度是沿着它的长度是均匀的。此外,细胞数,细胞融合和类型和损伤造成对细胞的程度的谨慎的优化,以便进一步优化测定法进行了讨论。

Protocol

注意:对于该研究中,肺间质基质细胞(LR-的MSC)从远端肺组织中分离或者基于它们的细胞表面标记物的表达(CD45 阴性 ,CD31 阴性 ,的Sca-1的高,EPCAM 负)14,15,用植出生长16,或使用酶消化17。粘附LR-的MSCs培养在DMEM高糖和无谷氨酰胺,15%FBS,1×抗生素/抗 ​​真菌剂,和2mM谷氨酰胺(此后完全培养基)并在37℃,5%的CO 2。 LR-MSCs的传代…

Representative Results

这里介绍的划痕试验用鼠肺间质居民基质细胞(LR-MSCS)进行,分离引用在协议中说明。在LR-MSCs的接种500,000个细胞在60毫米组织培养皿的密度,并在48小时生长至90%汇合。为了产生损害,4%CSE(如上所述)中的与细胞一起培养24小时后的初始播种期间,但在划痕试验前。对于每个测定,用200μl移液管尖端部中产生的划痕。最初的图像拍摄已产生的划痕后,任何松散的细胞和碎片已经被冲走完整的?…

Discussion

这里描述的协议提供了一个强大的定量,标准化的方法来执行和分析划痕试验。简单划痕测定法常规使用的在研究的许多不同领域,研究细胞迁移。然而,传统上的划痕试验一直缺乏标准化设置和定量协议,这已导致问题重复性7,10。许多的修改和优化用于提高划痕试验的减少这些问题,包括个人的活细胞成像,标准化塞为基础的测定,和三维微柱测定-4,8,10。但是,这些测定法可?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We are grateful to the other members of the lab for their technical assistance in developing the CSE damage assay, and their support and advice during the development of this assay. In addition, we are grateful to the Holy Cross Fenwick Scholar Committee for their support of Nicholas Cormier, and the Holy Cross College Honors program for their support of Alexander Yeo.

Materials

DMEM (high glucose, no added glutamine) Life Technologies 31053-036
Fetal Bovine Serum, Thermo Scientific HyClone Fisher Scientific SH3091002
Antibiotic/antimycotic, Thermo Scientific HyClone  Fisher Scientific SV3007901
Glutamine, 200mM, Thermo Scientific HyClone Fisher Scientific SH3003401
TypeLE cell dissociation reagent Life Technologies 12563-01
Dulbecco's Phosphate Buffered Saline, Thermo Scientific HyClone  Fisher Scientific SH3002802
Falcon standard 60mm tissue culture dishes Fisher Scientific 08-772B
Fisherbrand Sterile 200ul pipet tips Fisher Scientific 02-707-502
Inverted light microscope with camera attachment Variable
ImageJ software  http://imagej.nih.gov/ij/
MRI_Wound_Healting plugin http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool
Statistical analysis software Microsoft Excel

References

  1. Gough, W., Hulkower, K. I., Lynch, R., et al. A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay. J. Biomol. Screen. 16 (2), 155-163 (2011).
  2. Ridley, A. J., Schwartz, M. A., Burridge, K., et al. Cell migration: integrating signals from front to back. Science. 302 (5651), 1704-1709 (2013).
  3. Sharma, R. R., Pollock, K., Hubel, A., McKenna, D. Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices. Transfusion. 54 (5), 1418-1437 (2014).
  4. Booth-Gauthier, E. A., Du, V., Ghibaudo, M., et al. Hutchinson-Gilford progeria syndrome alters nuclear shape and reduces cell motility in three dimensional model substrates. Integr. Biol. (Camb). 5 (3), 569-577 (2013).
  5. Keese, C. R., Wegener, J., Walker, S. R., Giaever, I. Electrical wound-healing assay for cells in vitro. Proc. Natl. Acad. Sci. U.S.A. 101 (6), 1554-1559 (2004).
  6. Pinco, K. A., He, W., Yang, J. T. Alpha4beta1 Integrin Regulates Lamellipodia Protrusion Via a Focal Complex/focal Adhesion-Independent Mechanism. Mol. Biol. Cell. 13 (9), 3203-3217 (2002).
  7. Fronza, M., Heinzmann, B., Hamburger, M., Laufer, S., Merfort, I. Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts. J. Ethnopharmacol. 126 (3), 463-467 (2009).
  8. Justus, C. R., Leffler, N., Ruiz-Echevarria, M., Yang, L. V. In vitro cell migration and invasion assays. J. Vis. Exp. (88), (2014).
  9. Walter, M. N., Wright, K. T., Fuller, H. R., MacNeil, S., Johnson, W. E. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays. Exp. Cell Res. 316 (7), 1271-1281 (2010).
  10. Goetsch, K. P., Niesler, C. U. Optimization of the scratch assay for in vitro skeletal muscle wound healing analysis. Anal. Biochem. 411 (1), 158-160 (2011).
  11. Chung, C. A., Chen, C. Y. The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber. J. Theor. Biol. 261 (4), 610-625 (2009).
  12. Fabbri, M., Bianchi, E., Fumagalli, L., Pardi, R. Regulation of lymphocyte traffic by adhesion molecules. Inflamm. Res. 48 (5), 239-246 (1999).
  13. Saez, A., Ghibaudo, M., Buguin, A., Silberzan, P., Ladoux, B. Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates. Proc. Natl. Acad. Sci. U.S.A. 104 (20), 8281-8286 (2007).
  14. Paxson, J. A., Gruntman, A. M., Davis, A. M., et al. Age Dependence of Lung Mesenchymal Stromal Cell Dynamics Following Pneumonectomy. Stem Cells Dev. 22 (24), 3214-3225 (2013).
  15. McQualter, J. L., Brouard, N., Williams, B., et al. Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction. Stem Cells. 27 (3), 623-633 (2009).
  16. Hoffman, A. M., Paxson, J. A., Mazan, M. R., et al. Lung-Derived Mesenchymal Stromal Cell Post-Transplantation Survival, Persistence, Paracrine Expression, and Repair of Elastase-Injured Lung. Stem Cells Dev. 20 (10), 1779-1792 (2011).
  17. Beane, O. S., Fonseca, V. C., Cooper, L. L., Koren, G., Darling, E. M. Impact of aging on the regenerative properties of bone marrow-, muscle-, and adipose-derived mesenchymal stem/stromal cells. PLoS One. 9 (12), e115963 (2014).
  18. Chen, L., Ge, Q., Tjin, G., et al. Effects of cigarette smoke extract on human airway smooth muscle cells in COPD. Eur. Respir. J. 44 (3), 634-646 (2014).
  19. Itahana, K., Campisi, J., Dimri, G. P. Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay. Methods Mol. Biol. 371, 21-31 (2007).

Play Video

Cite This Article
Cormier, N., Yeo, A., Fiorentino, E., Paxson, J. Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract. J. Vis. Exp. (106), e53414, doi:10.3791/53414 (2015).

View Video