从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞
Summary
Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, α-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.
Abstract
成纤维细胞/肌成纤维细胞（MFS）已获得越来越多的关注他们在发病中的作用及其对肿瘤微环境的两个伤口愈合和推广贡献。虽然目前有很多技术的MF的胃肠道（GI）组织中分离，该协议引入了一种新的从冰冻组织这些间质细胞的分离的元素。冷冻胃肠组织标本不仅允许研究者获得来自世界各地的合作者，生物库，和商业供应商的样品，它也允许新鲜样品的延迟处理。所描述的协议将始终如一地产生特征梭形细胞表达标记CD90，α-SMA和波形对MF型。因为这些细胞来自患者样品衍生，使用原代细胞也赋予密切模仿隶属函数的受益于疾病状态，即癌和炎症性肠疾病。这种技术具有蜂ñ证实胃，小肠和结肠MF原代培养一代。主培养物的MF可以使用在实验繁多在若干通路和它们的纯度评估由两个免疫细胞化学和流式细胞术分析。
Introduction
肌成纤维细胞/成纤维细胞（MFS）表示细胞在胃肠道黏膜丰富的人口。这些基质细胞就位于下方的上皮形成和在肠道内的粘膜固有层的互连网络。微丝不仅负责细胞外基质的沉积，但通过旁分泌调节可影响电解质转运，恢复原状的，屏障功能相邻上皮1,2。此外，隶属函数已被证明在炎症和组织中发挥关键作用重塑3,4。肌成纤维细胞是在肿瘤微环境，在那里他们也被称为癌相关成纤维细胞的一个关键部分，并有助于肿瘤细胞的生长，并作为一个利基癌症干细胞3。新出现的数据表明，微丝也可用作本地抗原呈递细胞。此外，隶属函数功能的固有免疫和适应性免疫反应的重要调节器，机生产线CING多种细胞因子和生长因子5。
在健康个体中，微丝细胞被认为从间充质干细胞分化和表达在其细胞表面上，所述间充质标记物，CD90 3,5，这些细胞也表达vimentin，但阴性上皮细胞和造血细胞标记物的EpCAM和CD45分别。肌成纤维细胞被建议是成纤维细胞的活化形式，并且可以从非活化的成纤维细胞用α-SMA 5的表达来区分。
在过去十年中，多种方法用于从人结肠黏膜肌成纤维细胞的分离已公布-大多是基于最初由Mahida 等人 5-8中记载的方法。虽然个别的研究报道来自各种肠粘膜，对于隶属函数的从胃肠道的多个区域（即，胃，小和升隔离没有通用协议分离程序阿尔赫肠粘膜）已经出版。本文提出的方案已经过测试，并成功地用于所有三个类型的组织上面提到的。 。此外，还没有被报道用于从冷冻胃肠粘膜分离的MF流程。
这里，我们提出一种优化方法，它是基于酶消化，并且同时允许对人体肠道粘膜的MF的培养分离和流式细胞术分析在新鲜消化，单细胞，粘膜制剂。这种技术可靠地得到原代培养与MF的表型。此外，同样的方法可以用来从胃和小肠的组织冷冻样品隔离微丝为好。从新鲜的GI组织成肌纤维细胞的分离先前已描述;然而，冷冻样品的利用带来了许多好处。即，研究人员将能够收集和存储组织免受任何数目在全世界合作者谁具有capabi的lity运送冷冻组织样本。此外，研究人员可能会发现从手术室和/或内窥镜套件和立即丢弃组织的处理的组织为隔离与他们当前的实验时间表冲突的集合。此外，由于手术的不可预测性，可能会出现组织采购非常晚了一天，这将限制左为加工组织的时间。冷冻组织进行后续处理会减轻这些挑战。
最后，这些方法已被成功地用于在结肠，胃和小肠肌纤维母细胞在疾病状态如大肠癌和炎症性肠疾病的隔离。
Protocol
Representative Results
Discussion
虽然该协议是为研究中的应用开发只，在成长为一个潜在的癌症预后标志物和治疗靶点基质细胞至关重要的光，冻结“功能性”生物标本以备后用的能力提供了一个显著优势。这是一个独特的优势，为创作临床biorepository的，可作为额外的工具服务促进个性化医疗10,11的发展，类似于先前针对脂肪间充质干细胞从冷冻的基质血管级分中分离报告的结果，我们进行了未观察到在标记和增殖/免?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Institute of Health (1R01DK103150-01A1, T32 DK007639, R01CA175803, K08CA125209) and the Institute for Translational Sciences at the University of Texas Medical Branch, and a Clinical and Translational Science Award (8UL1TR000071).
Materials
| MEM, 1X | Corning | MT-10-010-CV | |
| Hanks' Balanced Salt Solution | Sigma-Aldrich | H6648 | |
| Sodium pyruvate | Sigma-Aldrich | S8636 | |
| Antibiotic-Antimycotic, 100X | Gibco | 15240-062 | |
| Ciprofloxacin HCl | Corning | 61-277-RF | |
| L-Glutamine, 100x | Corning | 25-005-CI | |
| MEM Nonessential Amino Acids | Corning | 25-025-CI | |
| Dimethyl Sulfoxide (DMSO) Hybri-Max | Sigma-Aldrich | D2650 | |
| DL-Dithiothreitol solution (DTT) | Sigma-Aldrich | 646563 | |
| 0.5M EDTA, pH 8.0 | Cellgro | 46-034-CI | |
| DNAse | Worthington | LS002139 | |
| Collagenase from Clostridium histolyticum, Type I | Sigma-Aldrich | C1639 | |
| Collagenase from Clostridium histolyticum, Type II | Sigma-Aldrich | C1764 | |
| Collagenase from Clostridium histolyticum, Type IV | Sigma-Aldrich | C5138 | |
| Accumax cell dissociation solution | Sigma-Aldrich | A7089 | |
| gentleMACS C Tubes | Miltenyi Biotec | 130-093-237 | |
| gentleMACS Dissociator | Miltenyi Biotec | 130-093-235 | |
| Countess Automated Cell Counter | Invitrogen | C10227 | |
| Cell Strainer (70µm) | Corning | 352350 | |
| PE Mouse Anti-Human Vimentin | BD Biosciences | 562337 | Clone RV202 |
| FITC Monoclonal Anti-Actin, α-Smooth Muscle | Sigma-Aldrich | F3777 | Clone 1A4 |
| APC Mouse Anti-Human CD90 | BD Biosciences | 559869 | Clone 5E10 |
Referências
- Yamaguchi, H., et al. Stromal Fibroblasts Mediate Extracellular Matrix Remodeling and Invasion of Scirrhous Gastric Carcinoma Cells. PLoS ONE. 9, e85485 (2014).
- Mullan, N., Hughes, K. R., Mahida, Y. R. Primary Human Colonic Myofibroblasts Are Resistant to Clostridium difficile Toxin A-Induced, but Not Toxin B-Induced, Cell Death. Infect Immun. 79, 1623-1630 (2011).
- Powell, D. W., Pinchuk, I. V., Saada, J. I., Chen, X., Mifflin, R. C. Mesenchymal Cells of the Intestinal Lamina Propria. Annu Rev Physiol. Annu Rev Physiol . 73, 213-237 (2011).
- Latella, G., Sferra, R., Speca, S., Vetuschi, A., Gaudio, E. Can we prevent, reduce or reverse intestinal fibrosis in IBD?. Eur Rev Med Pharmacol Sci. 17, 1283-1304 (2013).
- Saada, J. I., et al. Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa. J. Immunol. 177, 5968-5979 (2006).
- Mahida, Y. R., et al. Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2. Am J Physiol. 273, G1341-G1348 (1997).
- Roncoroni, L., et al. Isolation and culture of fibroblasts from endoscopic duodenal biopsies of celiac patients. J Transl Med. 7 (40), (2009).
- Pinchuk, I. V., et al. Stromal Cells Induce Th17 during Helicobacter pylori Infection and in the Gastric Tumor Microenvironment. PLoS ONE. 8, e53798 (2013).
- Pinchuk, I. V., et al. Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease. J. Immunol. 178, 8097-8106 (2007).
- Paulsson, J., Micke, P. Prognostic relevance of cancer-associated fibroblasts in human cancer. Sem Cancer Biol. 25, 61-68 (2014).
- Valcz, G., Sipos, F., Tulassay, Z., Molnar, B., Yagi, Y. Importance of carcinoma-associated fibroblast-derived proteins in clinical oncology. J. Clin. Pathol. 67, 1026-1031 (2014).
- Minonzio, G., et al. Frozen adipose-derived mesenchymal stem cells maintain high capability to grow and differentiate. Cryobiology. 69, 211-216 (2014).
- Gargus, M., Niu, C., Shaker, A. Isolation of Myofibroblasts from Mouse and Human Esophagus. J. Vis. Exp. (95), e52215 (2015).
- Khalil, H., Nie, W., Edwards, R. A., Yoo, J. Isolation of primary myofibroblasts from mouse and human colon tissue. J. Vis. Exp. (80), e50611 (2013).
- Schiller, J. H., Bittner, G. Loss of the Tumorigenic Phenotype with in Vitro, but not in Vivo, Passaging of a Novel Series of Human Bronchial Epithelial Cell Lines: Possible Role of an α5/β1-Integrin-Fibronectin Interaction. Cancer Res. 55, 6215-6221 (1995).
- Augello, A., Kurth, T. B., De Bari, C. Mesenchymal stem cells: a perspective from in vitro cultures to in vivo migration and niches. Eur Cell Mater. 20, 121-133 (2010).
- Meller, D., Pires, R. T. F., Tseng, S. C. G. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol. 86, 463-471 (2002).
- Kuhlmann, I. The prophylactic use of antibiotics in cell culture. Cytotechnology. 19, 95-105 (1995).
- Morris, K. T., Nofchissey, R. A., Pinchuk, I. V., Beswick, E. J. Chronic Macrophage Migration Inhibitory Factor Exposure Induces Mesenchymal Epithelial Transition and Promotes Gastric and Colon Cancers. PLoS ONE. 9, e98656 (2014).
