人鼻上皮细胞球体的生成与个体化囊性纤维膜电导调节的研究
Summary
这里我们描述了一种生成三维的人鼻上皮细胞的球形培养方法。球体被刺激以驱动囊性纤维化跨膜电导调节剂 (CFTR) 依赖的离子和液体分泌物, 将球体腔内大小的变化量化为 CFTR 功能的代表。
Abstract
囊性纤维化跨膜电导调节剂 (CFTR) 调制药物的引入改变了囊性纤维化 (CF) 的护理, 目前使用的基因型定向治疗模型有若干局限性。首先, 罕见或理解突变组被排除在明确的临床试验。此外, 随着额外的调制药物进入市场, 它将变得难以优化的调制器选择一个单独的主题。这两个问题都是通过使用病人衍生的, 个性化的临床前模型系统的 CFTR 功能和调制。人鼻上皮细胞 (HNEs) 是一种容易获得的呼吸道组织的来源, 这种模式。在此, 我们描述了一个三维球体模型的 CFTR 函数和调制使用初级 HNEs。HNEs 是以微创的方式与受试者隔绝, 在有条件的重新编程条件下扩展, 并播种到球体文化中。在播种2周内, 球体培养产生他球体, 可刺激与 3 ‘, 5 ‘-循环腺苷 (阵营) 产生的激动剂激活 CFTR 功能。球体肿胀被量化为 CFTR 活动的代表。他球体利用微创, 但鼻腔细胞的呼吸道起源, 生成一个可访问的, 个性化的模型, 与反映疾病发病率和死亡率的上皮细胞有关。与空气液界面他培养相比, 球体的成熟度相对快, 降低了整体污染率。在目前的形式, 模型是有限的吞吐量低, 虽然这是抵消了相对容易的组织获取。他球体可以用来可靠地量化和表征 CFTR 活动的个人水平。目前正在进行的一项研究将这种量化与体内药物反应联系起来, 将确定他球体是否是患者对 CFTR 调制的反应的真正的临床前预测因子。
Introduction
囊性纤维化 (CF) 是一种致命的, 常染色体隐性疾病, 影响全球7万多人1。这种寿命缩短的遗传疾病是由囊性纤维化跨膜电导调节蛋白 (CFTR) 的突变引起的2。CFTR 是三磷酸腺苷结合盒系列的成员, 作为离子通道, 允许氯和碳酸氢在多个极化上皮的顶端膜上运动, 包括胃肠道、汗腺、和呼吸树, 其中其他3,4。因此, 功能失调的 CFTR 导致多系统上皮功能障碍, 大多数死亡率来自呼吸系统疾病的 1.在 CF 肺, 失去 CFTR 驱动的气道表面液体 (手语) 调节和粘液释放导致增厚的粘液, 气道梗阻, 慢性感染, 和进步气道重塑和丧失肺功能1,5。
尽管 CFTR 功能障碍的鉴别作为疾病的病因, 在 CF 的治疗传统上侧重于缓解症状 (例如, 胰酶替代疗法, 气道清除疗法)1。这种方法最近被革命性的新疗法, 称为 “CFTR 调制器”, 直接针对功能失调的 CFTR。这种方法已将临床景观从症状管理转移到疾病修改护理, 但带有若干限制6、7、8、9、10。调制器活动是特定的蛋白质缺陷伴随着每个 CFTR 突变和限制选择基因种群11。这种限制是由蛋白质缺陷的异质性所驱动的, 也是罕见突变组临床试验的不切实际。此外, 在研究良好基因型的学科中 (例如, F508del/F508del CFTR, 最常见的 CFTR 突变), 疾病负担和调制器响应的范围有很大的变异性6,7,8 ,9,11。
为了克服这两个问题, 调查人员建议使用个性化模型进行临床前测试12。这个概念利用患者特定的组织生成一个个性化的前体模型系统进行复合测试, 预测体内对治疗的个性化方式的反应。一旦经过验证, 这种模型可以被临床医生用来驱动精确治疗, 不管病人的潜在 CFTR 基因型。
在肺移植时从外植体组织获得的人支气管上皮 (HBE) 细胞建立了这样一种模型的可能性 CF13,14。HBEs 生长在空气液界面 (阿里) 允许功能 CFTR 量化直接通过电生理学测试或间接通过措施的手语稳定13,15。这个模型对于理解 CFTR 生物学是至关重要的, 是 CFTR 调制器16的关键驱动因素。不幸的是, HBE 模型不成立作为一个个性化的模型, 由于侵入性质的采集 (肺移植或支气管刷牙) 和缺乏样本的那些罕见的突变或轻度疾病。反之, 肠道组织, 从直肠或十二指肠活检标本, 可用于肠道电流测量 (ICM) 或肿胀为基础的 organoid 试验研究个性化 CFTR 功能17,18,19. Organoid 化验, 特别是, 是非常敏感的模型 CFTR 活动20,21,22。两种模型均受组织来源 (肠道组织, 而大多数疾病病理学是呼吸道) 和半侵入性采集方法的限制。另外, 一些调查人员研究了人鼻上皮 (他) 细胞, 以模拟 CFTR 恢复23,24,25。HNEs 可以通过刷子或刮除在任何年龄的主题, 并在阿里养殖时, 重述 HBEs25,26,27,28的许多特征的安全收获。他单层培养传统上受鳞状转变限制, 长时间成熟29。此外, 报告的短路电流测量在 HNEs 比 HBEs, 建议一个较小的窗口, 以检测治疗效果25。
鉴于需要一个个性化的, 非侵入性的, 呼吸组织培养模型的 CFTR 功能, 我们试图合并的敏感性, 肿胀基础, organoid 检测与非侵入性和呼吸性质的 HNEs。在这里, 我们描述了我们的方法, 生成3维 “球形” 文化的 HNEs 的个性化 CFTR 研究在肿胀为基础的化验30。他球体极化可靠与上皮尖向球形中心, 或流明。该模型概括了下呼吸道上皮的许多特征, 并且比阿里文化更快地成熟。作为一种功能性检测, 他球体可靠地量化了 CFTR 功能的范围, 以及在研究的突变组 (例如、F508del CFTR) 中的调制。这种基于肿胀的化验方法利用了 CFTR 的离子/盐传输特性, 间接地测量了液体流入球体的量, 因为水跟随顶端的盐流出。以这种方式, 刺激球体与完全功能 CFTR 膨胀强劲, 而那些功能失调的 CFTR 膨胀较少或萎缩。通过对前、1小时刺激后球体的图像分析, 测量腔区面积, 确定百分比变化, 对此进行量化。然后, 可以将这一措施与实验组进行比较, 以特定于病人的方式筛查药物的生物活性。
Protocol
Representative Results
Discussion
该协议描述了病人衍生的鼻细胞球形培养的产生, 能够产生一个个性化的, 特定的 CFTR 功能模型。在这个过程中有几个关键步骤, 应该密切关注, 以避免困难。首先是一个很好的样本采集从病人的鼻子。一个好的样本应该有 > 5万细胞, 有限的粘液/碎片, 并准备在4小时内处理 (虽然成功也很容易实现与隔夜航运冰)。练习用刮匙 (或刷子来获取样品是必要的, 这有助于通过将早期样品采集集中到一个或两…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了囊性纤维化基础疗法, 格兰特号 CLANCY14XX0, 并通过囊性纤维化基金会的支持, CLANCY15R0 号。作者希望感谢克里斯蒂娜. 雷在病人招募和监管监督方面的协助。作者还要感谢他的工作组, 该组织在囊性纤维化基金会的支持下, 帮助培养他文化能力: 普雷斯顿 Bratcher, 卡尔文棉花, 玛蒂娜 Gentzsch, 伊丽莎白 Joseloff, 迈克尔 Myerburg, 戴夫尼科尔斯,斯科特 Randell, 史蒂夫罗, g. 所罗门, 凯瑟琳. Tuggle。
Materials
| 1.5 mL Eppendorf Tube | USA Scientific | 4036-3204 | |
| 150 mL Filter Flask | Midsci | TP99150 | To filter Media |
| 15 mL Conical Tube | Midsci | TP91015 | |
| 1 L Filter Flask | Midsci | TP99950 | To filter Media |
| 35 mm Glass-Bottom Dish | MatTek Corporation | P35G-0-20-C | Optional |
| 3-Isobutyl-1-Methylxanthine (IBMX) | Fisher Scientific | AC228420010 | Prepare a 100 mM stock solution of 22.0 mg in 1 mL of DMSO |
| 50 mL Conical Tube | Midsci | TP91050 | |
| Accutase | Innovative Cell Technologies, Inc. | AT-104 | Cell detachment solution |
| Adenine | Sigma-Aldrich | A2786-25G | See Table 1 |
| Amphotericin B | Sigma-Aldrich | A9528-100MG | See Table 1 |
| Bovine Brain Extract (9mg/mL) | Lonza | CC-4098 | See Table 2 |
| Ceftazidime hydrate | Sigma-Aldrich | C3809-1G | See Table 1 |
| Cell Scrapers 20 cm | Midsci | TP99010 | |
| CFTR Inh172 | Tocris Bioscience | 3430 | Prepare a 10 mM stock solution of 4.0 mg in 1 mL of DMSO |
| Cholera Toxin B (From Vibrio cholerae) | Sigma-Aldrich | C8052-.5MG | See Table 1 |
| CYB-1 | Medical Packaging Corporation | CYB-1 | Cytology brush |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D5879-500ML | |
| Dulbecco's Modified Eagle Media (DMEM)/F12 Hepes | Life Technologies | 11330-057 | Base Medium; See Tables 1 and 2 |
| Epidermal Growth Factor (Recombinant Human Protein, Animal-Origin Free) | Thermo Fisher Scientific | PHG6045 | See Table 1 |
| Epinephrine | Sigma-Aldrich | E4250-1G | See Table 2 |
| Ethanol | Fisher Scientific | 2701 | |
| Ethanolamine | Sigma-Aldrich | E0135-500ML | 16.6 mM solution; See Table 2 |
| Ethylenediaminetetraacetic Acid (EDTA) | TCI America | E0084 | |
| Fetal Bovine Serum (high performance FBS) | Invitrogen | 10082147 | See Table 1 |
| Forskolin | Sigma-Aldrich | F6886-50 | Prepare a 10 mM stock solution of 4.1 mg in 1 mL of DMSO |
| Growth Factor-Reduced Matrigel | Corning, Inc. | 356231 | Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, LDEV-Free, 10 mL. |
| Hemacytometer | Hausser Scientific | 1483 | |
| Human Collagen Solution, Type I (VitroCol; 3 mg/mL) | Advanced BioMatrix | 5007-A | Collagen solution |
| HyClone (aka FetalClone II) | GE Healthcare | SH30066.03HI | See Table 2 |
| Hydrocortisone | StemCell Technologies | 07904 | See Tables 1 and 2 |
| Insulin, human recombinant, zinc solution | Life Technologies | 12585014 | 4 mg/mL solution; see Table 2 |
| IVF 4-Well Dish, Non-treated | NUNC (via Fisher Scientific) | 12566350 | 4-well plate for spheroids; similar well size to a 24-well plate |
| MEF-CF1-IRR | Globalstem | GSC-6001G | Irradiated murine embryonic fibroblasts |
| Metamorph 7.7 | Molecular Devices | Analysis Software; https://www.moleculardevices.com/systems/metamorph-research-imaging/metamorph-microscopy-automation-and-image-analysis-software for a quote | |
| Olympus IX51 Inverted Microscope | Olympus Corporation | Discontinued | Imaging Microscope. Replacment: Olympus IX53, https://www.olympus-lifescience.com/pt/microscopes/inverted/ix53/ for a quote |
| Pen Strep | Life Technologies | 15140122 | See Table 2 |
| Phsophoryletheanolamine | Sigma-Aldrich | P0503-5G | See Table 2 |
| Retinoic Acid | Sigma-Aldrich | R2625-50MG | See Table 2 |
| Rhinoprobe | Arlington Scientific, Inc. | 96-0905 | Nasal curette |
| Slidebook 5.5 | 3i, Intelligent Imaging Innovations | Discontinued | Imaging Software. Replacement: Slidebook 6, https://www.intelligent-imaging.com/slidebook for a quote |
| Sterile Phosphate Buffered Saline (PBS) | Thermo Fisher Scientific | 20012050 | |
| Sterile Water | Sigma-Aldrich | W3500-6X500ML | |
| Tissue Culture Dish 100 | Techno Plastic Products | 93100 | Tissue culture dish for expansion |
| Tobramycin | Sigma-Aldrich | T4014-100MG | See Table 1 |
| Transferrin (Human Transferrin 0.5 mL) | Lonza | CC-4205 | See Table 2 |
| Triiodothryonine | Sigma-Aldrich | T6397-1G | 3,3′,5-Triiodo-L-thyronine sodium salt [T3]; See Table 2 |
| Trypsin from Porcine Pancreas | Sigma-Aldrich | T4799-10G | |
| Ultroser-G | Crescent Chemical (via Fisher Scientific) | NC0393024 | 20 mL lypophilized powder; See Table 2 |
| Vancomycin hydrochloride from Streptomyces orientalis | Sigma-Aldrich | V2002-5G | See Table 1 |
| VX770 | Selleck Chemicals | S1144 | Prepare a 1 mM stock solution of 0.4 mg in 1 mL of DMSO |
| VX809 | Selleck Chemicals | S1565 | Purchase or prepare a 10 mM stock solution of 4.5 mg in 1 mL of DMSO |
| Y-27632 Dihydrochloride ROCK inhibitor | Enzo LifeSciences | ALX-270-333-M025 | See Table 1 |
References
- Rowe, S. M., Miller, S., Sorscher, E. J. Cystic fibrosis. N Engl J Med. 352 (19), 1992-2001 (2005).
- Rommens, J. M., et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science. 245 (4922), 1059-1065 (1989).
- Anderson, M. P., et al. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell. 67 (4), 775-784 (1991).
- Boucher, R. C. Human airway ion transport. Part one. Am J Respir Crit Care Med. 150 (1), 271-281 (1994).
- Ehre, C., Ridley, C., Thornton, D. J. Cystic fibrosis: an inherited disease affecting mucin-producing organs. Int J Biochem Cell Biol. 52, 136-145 (2014).
- Accurso, F. J., et al. Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. N Engl J Med. 363 (21), 1991-2003 (2010).
- De Boeck, K., et al. Efficacy and safety of ivacaftor in patients with cystic fibrosis and a non-G551D gating mutation. J Cyst Fibros. 13 (6), 674-680 (2014).
- Moss, R. B., et al. Efficacy and safety of ivacaftor in patients with cystic fibrosis who have an Arg117His-CFTR mutation: a double-blind, randomised controlled trial. Lancet Respir Med. 3 (7), 524-533 (2015).
- Wainwright, C. E., et al. Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR. N Engl J Med. , (2015).
- Brewington, J. J., McPhail, G. L., Clancy, J. P. Lumacaftor alone and combined with ivacaftor: preclinical and clinical trial experience of F508del CFTR correction. Expert Rev Respir Med. 10 (1), 5-17 (2016).
- . . CFTR2 Database. , (2017).
- Mou, H., Brazauskas, K., Rajagopal, J. Personalized medicine for cystic fibrosis: establishing human model systems. Pediatr Pulmonol. 50 Suppl 40, S14-S23 (2015).
- Randell, S. H., Fulcher, M. L., O’Neal, W., Olsen, J. C. Primary epithelial cell models for cystic fibrosis research. Methods Mol Biol. 742, 285-310 (2011).
- Whitcutt, M. J., Adler, K. B., Wu, R. A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells. In Vitro Cell Dev Biol. 24 (5), 420-428 (1988).
- Worthington, E. N., Tarran, R. Methods for ASL measurements and mucus transport rates in cell cultures. Methods Mol Biol. 742, 77-92 (2011).
- Van Goor, F., et al. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U S A. 108 (46), 18843-18848 (2011).
- Hug, M. J., Tummler, B. Intestinal current measurements to diagnose cystic fibrosis. J Cyst Fibros. 3 Suppl 2, 157-158 (2004).
- van Barneveld, A., Stanke, F., Ballmann, M., Naim, H. Y., Tummler, B. Ex vivo biochemical analysis of CFTR in human rectal biopsies. Biochim Biophys Acta. 1762 (4), 393-397 (2006).
- Dekkers, J. F., et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med. 19 (7), 939-945 (2013).
- Dekkers, J. F., et al. Optimal correction of distinct CFTR folding mutants in rectal cystic fibrosis organoids. Eur Respir J. 48 (2), 451-458 (2016).
- Dekkers, R., et al. A bioassay using intestinal organoids to measure CFTR modulators in human plasma. J Cyst Fibros. 14 (2), 178-181 (2015).
- Zomer-van Ommen, D. D., et al. Limited premature termination codon suppression by read-through agents in cystic fibrosis intestinal organoids. J Cyst Fibros. , (2015).
- Bridges, M. A., Walker, D. C., Davidson, A. G. Cystic fibrosis and control nasal epithelial cells harvested by a brushing procedure. In Vitro Cell Dev Biol. 27a (9), 684-686 (1991).
- Di Lullo, A. M., et al. An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis. Acta Otorhinolaryngol Ital. 37 (3), 207-213 (2017).
- Pranke, I. M., et al. Correction of CFTR function in nasal epithelial cells from cystic fibrosis patients predicts improvement of respiratory function by CFTR modulators. Sci Rep. 7 (1), 7375 (2017).
- Devalia, J. L., Davies, R. J. Human nasal and bronchial epithelial cells in culture: an overview of their characteristics and function. Allergy Proc. 12 (2), 71-79 (1991).
- Devalia, J. L., Sapsford, R. J., Wells, C. W., Richman, P., Davies, R. J. Culture and comparison of human bronchial and nasal epithelial cells in vitro. Respir Med. 84 (4), 303-312 (1990).
- Thavagnanam, S., et al. Nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies. PLoS One. 9 (1), e85802 (2014).
- Jorissen, M., Van der Schueren, B., Van den Berghe, H., Cassiman, J. J. The preservation and regeneration of cilia on human nasal epithelial cells cultured in vitro. Arch Otorhinolaryngol. 246 (5), 308-314 (1989).
- Brewington, J. J., et al. Detection of CFTR function and modulation in primary human nasal cell spheroids. J Cyst Fibros. , (2017).
- Sun, H., et al. Tgf-beta downregulation of distinct chloride channels in cystic fibrosis-affected epithelia. PLoS One. 9 (9), e106842 (2014).
- Boucher, R. C. Evidence for airway surface dehydration as the initiating event in CF airway disease. J Intern Med. 261 (1), 5-16 (2007).
