在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡
1Microbiological Analysis Team, Group for Biometrology,Korea Research Institute of Standards and Science (KRISS), 2College of Pharmacy,Chungnam National University (CNU), 3Stem Cell Research Center,Korea Research Institute of Bioscience and Biotechnology (KRIBB), 4Convergent Research Center for Emerging Virus Infection,Korea Research Institute of Chemical Technology (KRICT), 5Department of Bio-Analysis Science,University of Science & Technology (UST)
Summary
这里介绍了了解 乳酸杆菌 无细胞超高分子（LCFS）的抗癌作用的方法。结肠直肠癌细胞系显示在3D培养物中用LCFS治疗时细胞死亡。生成球体的过程可以根据脚手架进行优化，并且所呈现的分析方法可用于评估所涉及的信号通路。
Abstract
这份手稿描述了一个协议，评估癌细胞死亡的三维（3D）球形多细胞类型的癌细胞使用超分子从 乳酸杆菌发酵 细胞培养， 被认为是益生菌培养物。使用 3D 培养物来测试 乳酸杆菌 无细胞超高分子 （LCFS） 比在 2D 单层测试更好，特别是因为 L. 发酵 可以在肠道内产生抗癌效果。 L. 发酵 超高分子被鉴定在3D培养条件下对几种结肠直肠癌（CRC）细胞具有增加的抗增殖作用。有趣的是，这些效应与培养模型密切相关，显示了 L.发酵 诱导癌细胞死亡的显著能力。稳定的球体来自不同的CRC（结肠直肠癌细胞），使用下面提出的协议。生成 3D 球体的这种协议节省时间且具有成本效益。该系统的开发是为了轻松研究LCFS在多种CRC球体中的抗癌作用。不出所料，在实验中用LCFS治疗的CRC球体强烈诱导细胞死亡，并表达由qRT-PCR、西侧印迹和FACS分析分析的特定凋亡分子标记。因此，该方法对于探索细胞生存能力和评估抗癌药物的疗效具有重要的价值。
Introduction
益生菌是肠道中最有利的微生物，可改善免疫平衡和宿主能量代谢1。乳酸杆菌和双歧杆菌的益生菌是肠2、3中同类中最先进的。先前的研究表明，乳酸杆菌对包括结肠直肠癌在内的几种癌症有抑制和反促进作用。此外，益生菌可预防炎症性肠病、克罗恩病和溃疡性结肠炎5、6。然而，大多数益生菌研究都是在生长在固体表面的二维（2D）单层体中进行的。
人工培养系统缺乏环境特征，这对癌细胞来说是不自然的。为了克服这一局限性，开发了3维（3D）文化系统7、8。3D中的癌细胞在基本生物机制方面表现出改善，如细胞生存能力、增殖、形态、细胞细胞通信、药物敏感性和体内相关性9、10。此外，球体由多细胞类型的结肠直肠癌制成，并依赖于细胞-细胞相互作用和细胞外基质（ECM）11。我们先前的研究已经报告说，使用乳酸杆菌发酵产生的益生菌无细胞超纳坦（CFS）对结肠直肠癌（CRC）细胞12的3D培养物具有抗癌作用。我们建议CFS是测试3D球体12的益生菌效应的合适替代策略。
在这里，我们提出了一种方法，可以容纳多细胞类型的3D结肠直肠癌，用于分析益生菌无细胞超高分子（CFS）对几个3D结肠直肠癌模仿系统的治疗效果。这种方法为体外分析相关的益生菌和抗癌效果提供了手段。
Protocol
1. 细菌细胞培养和 无乳酸杆菌 无细胞超高分子（LCFS）的制备
注：步骤 1.2 – 1.9 在厌氧室中执行。
- 准备一个含有 L-西斯坦的人造黄油板和肉汤，并通过自动包装消毒。
- 在H2 厌氧室中预孵育MRS agar板，维持在37°C，含20ppm氧气。
- 解冻乳酸杆菌库存，并接种与细菌培养的阿加板（图1A（i）） 。 <l…
Representative Results
我们描述了从不同的结肠直肠癌细胞系中获取球状体的程序。需要补充甲基纤维素才能产生球状体。我们还介绍了LCFS制备方法，并提出了一个模型来研究益生菌与结肠直肠癌之间的相关性。球形形成和LCFS制备协议在图1A，B中示意图说明。如图2A所示，甲基纤维素浓度为0.6%，将癌细胞转化为紧凑的球体。这一结果表明，球状体可以通过使用…
Discussion
组织微环境，包括相邻细胞和细胞外基质（ECM），是组织生成的基础，在控制细胞生长和组织发育方面至关重要。然而，2D培养物有几个缺点，如细胞相互作用的中断，以及细胞形态的改变，细胞外环境，以及第14分区的方法。3D细胞培养系统经过严格研究，以更好地在体内繁殖效果，并已被证明是更精确的体外癌症检测系统15，16。<sup class="xre…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了”化学和辐射测量标准的制定”、授予编号为KRISS-202020-0003以及”生物材料和医学融合测量标准和技术的制定”的支持，赠款编号为KRISS-2020-GP2020-0004项目，由韩国标准与科学研究所资助。这项研究还得到了韩国科学和信息通信技术部（MSIT）、韩国国家研究基金会（NRF-2019M3A9F3065868）、卫生福利部（MOHW）、韩国卫生产业发展研究所（KHIDI、HI20C0558）、贸易、工业和能源部（MOTIE）和韩国工业技术评估研究所（KEIT，20009350）的支持。ORCID ID （何敏宇： 0000-0002-5951-2137：杜金康： 0000-0002-5924-9674：西尔·金： 0000-0003-3465-7118：李俊恩： 0000-0002-2495-1439：李吉娜： 0000-0002-3661-3701）。我们感谢张伍公园对实验的协助。
Materials
10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl |
Biorad | 4561036 | Pkg of 10 |
| Applied Biosystems MicroAmp Optical Adhesive Film | Thermo Fisher Scientific | 4311971 | 100 covers |
| 10x transfer buffer | Intron | IBS-BT031A | 1 L |
| 10X Tris-Glycine (W/SDS) | Intron | IBS-BT014 | 1 L |
| Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case | Corning | SCT-200-C | 500 Tubes/Pack, 10 Packs/Case |
| BD Difco Bacto Agar | BD | 214010 | 500 g |
| BD Difco Lactobacilli MRS Broth | BD | DF0881-17-5 | 500 g |
| CellTiter-Glo 3D Cell viability assay | Promega | G9681 | 100μl/assay in 96-well plates |
| Complete Protease Inhibitor Cocktail | Sigma-Aldrich | 11697498001 | vial of 20 tablets |
| Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1 | Corning | 21-040-CV | 500 mL |
| EMD Millipore Immobilon-P PVDF Transfer Membranes | fisher Scientific | IPVH00010 | 26.5cm x 3.75m roll; Pore Size: 0.45um |
| Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap | Corning | 352235 | 25/Pack, 500/Case |
| Fetal Bovine Serum, certified, US origin | Thermo Fisher Scientific | 16000044 | 500 mL |
| iScript cDNA Synthesis Kit, 25 x 20 µl rxns #1708890 | Biorad | 1708890 | 25 x 20 µL rxns |
| iTaq Universal SYBR Green Supermix | Biorad | 1725121 | 5 x 1 mL |
| Lactobacillus fermentum | Korean Collection for Type Cultures | KCTC 3112 | |
| L-Cysteine hydrochloride monohydrate | Sigma-Aldrich | C6852-25G | 25 g |
| Methyl Cellulose (3500-5600mPa·s, 2% in Water at 20°C) | TCI | M0185 | 500 g |
| MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL | Applied Biosystems | 4346906 | 20 plates |
| Millex-GS Syringe Filter Unit, 0.22 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilized | Millipore | SLGS033SB | 250 |
| PE Annexin V Apoptosis Detection Kit with 7-AAD | Biolegend | 640934 | 100 tests |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | 100 mL |
| Propidium Iodide | Introgen | P1304MP | 100 mg |
| RIPA Lysis and Extraction Buffer | Thermo Fisher Scientific | 89901 | 250 mL |
| RNeasy Mini Kit (250) | Qiagen | 74106 | 250 |
| RPMI-1640 | Gibco | 11875-119 | 500 mL |
| Trypsin-EDTA (0.25%), phenol red | Thermo Fisher Scientific | 25200056 | 100 mL |
| Name of Materials/Equipment/Software | Company | Catalog Number | Comments/Description |
| anti – p-IκBα (B-9) | Santa cruze | sc-8404 | 200 µg/mL |
| anti-BclxL (H-5) | Santa cruze | sc-8392 | 200 µg/mL |
| anti-PARP 1 (C2-10) | Santa cruze | sc-53643 | 50 µl ascites |
| anti-β-actin (C4) | Santa cruze | sc-47778 | 200 µg/mL |
| BD FACSVerse | BD Biosciences | San Diego, CA, USA | |
| Synergy HTX Multi-Mode Microplate Reader | BioT | S1LFA | |
| CO2 incubator | Thermo fisher | HERAcell 150i | |
| Conical tube 15 ml | SPL | 50015 | |
| Conical tube 50 ml | SPL | 50050 | |
| Corning Costar Ultra-Low Attachment Multiple Well Plate | Sigma-Aldrich | CLS7007 | |
| Corning Costar Ultra-Low Attachment Multiple Well Plate | Sigma-Aldrich | CLS3471 | |
| Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile | Costar | 4870 | |
| Countess Cell Counting Chamber Slides | Thermofisher | C10228 | |
| Countess II FL Automated Cell Counter | invitrogen | AMQAF1000 | |
| EnSpire Multimode Reader | Perkin Elmer | Enspire 2300 | |
| Eppendorf Research Plus Multi Channel Pipette, 8-channel | Eppendorf | 3122000051 | |
| FlowJo software | TreeStar | Ashland, OR, USA | |
| Goat Anti-Mouse IgG (H+L) | Jackson immunoresearch | 115-035-062 | 1.5 mL |
| Goat Anti-Rabbit IgG (H+L) | Jackson immunoresearch | 111-035-144 | 2.0 mL |
| GraphPad Prism 5 | GraphPad Software | Inc., San Diego, CA, USA | |
| ImageJ | NIH | ||
| ImageQuant LAS 4000 mini | Fujifilm | Tokyo, Japan | |
| Incubated shaker | Lab companion | SIF-6000R | |
| Multi Gauge Ver. 3.0, | Fujifilm | Tokyo, Japan | |
| Optical density (OD)LAMBDA UV/Vis Spectrophotometers | Perkin Elmer | Waltham, MA, USA | |
| Phase-contrast microscope | Olympus | Tokyo, Japan | |
| SPL microcentrifuge tube 1.5mL | SPL | 60015 | |
| SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile | SPL | 21012 | |
| StepOnePlus Real-Time PCR system | Thermo Fisher Scientific | Waltham, MA, USA | |
| Vibra-Cell Ultrasonic Liquid Processors | SONICS-vibra cell | VC 505 | 500 Watt ultrasonic processor |
| Vinyl Anaerobic Chamber | COY LAB PRODUCTS |
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Tags
Keywords:
Cell DeathCell-free SupernatantProbioticsThree-dimensional SpheroidColorectal CancerLactobacillusAnti-cancer EffectIn Vivo MimicryMRS AgarAnaerobic ChamberMRS BrothL-cysteinePBSShaking IncubatorOD620CentrifugationRPMI 1640Fetal Bovine Serum0.22 Micron StrainerColorectal Cancer Cell LinesMonolayersTrypsin-EDTAGrowth Medium
Cite This Article
Lee, J., Lee, J., Kim, S., Kang, D., Yoo, H. M. Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells. J. Vis. Exp. (160), e61285, doi:10.3791/61285 (2020).