分离来自癌细胞系的低内毒素含量细胞外囊泡
Summary
拟议的方案包括有关如何在从细胞培养上清液中分离细胞外囊泡以及如何正确评估它们期间避免内毒素污染的指南。
Abstract
细胞外囊泡（EV）是细胞在 体外 和 体内释放的膜囊泡的异质群。它们作为生物信息载体的无所不在和重要作用使它们成为有趣的研究对象，需要可靠和重复的方案来分离它们。然而，充分发挥他们的潜力是困难的，因为他们的研究仍然存在许多技术障碍（如适当的收购）。本研究提出了一种基于差异离心从肿瘤细胞系培养上清液中分离小型 EV（根据 MISEV 2018 命名法）的方案。该协议包括有关如何在分离电动汽车期间避免内毒素污染以及如何正确评估它们的指南。电动汽车的内毒素污染会严重阻碍后续实验，甚至掩盖其真正的生物效应。另一方面，被忽视的内毒素的存在可能会导致错误的结论。当提到免疫系统细胞（包括单核细胞）时，这一点尤其重要，因为单核细胞构成了对内毒素残基特别敏感的群体。因此，强烈建议筛查 EV 的内毒素污染，尤其是在处理内毒素敏感细胞（如单核细胞、巨噬细胞、髓源性抑制细胞或树突状细胞）时。
Introduction
根据MISEV 2018命名法，细胞外囊泡（EV）是一个统称，描述了细胞分泌的膜囊泡的各种亚型，这些囊泡在许多生理和病理过程中起着至关重要的作用1，2。此外，电动汽车有望成为各种疾病的新型生物标志物，以及治疗剂和药物输送载体。然而，充分发挥其潜力是困难的，因为与获取它们相关的技术障碍仍然存在许多3.其中一个挑战是分离无内毒素的电动汽车，这在许多情况下被忽视了。最常见的内毒素之一是脂多糖（LPS），它是革兰氏阴性细菌细胞壁的主要成分，由于各种细胞释放大量炎性细胞因子，可引起急性炎症反应4，5。LPS通过与LPS结合蛋白结合诱导反应，然后与骨髓细胞上的CD14 / TLR4 / MD2复合物相互作用。这种相互作用导致MyD88和TRIF依赖性信号通路的激活，进而触发核因子κB（NFkB）。NFkB易位到细胞核启动细胞因子的产生6。单核细胞和巨噬细胞对 LPS 高度敏感，暴露于 LPS 会导致炎症细胞因子和趋化因子（例如 IL-6、IL-12、CXCL8 和 TNF-α）的释放7，8。CD14结构能够结合具有相似亲和力的不同LPS物种，并作为其他toll样受体（TLR）（TLR1，2，3，4，6，7和9）的辅助受体6。关于EV对单核细胞/巨噬细胞影响的研究数量仍在增加9，10，11。特别是从研究单核细胞、其亚群和其他免疫细胞的功能的角度来看，内毒素的存在，甚至它们在EV中的掩盖存在非常重要12。被忽视的电动汽车内毒素污染可能会导致误导性结论并掩盖其真正的生物活性。换句话说，使用单核细胞需要对没有内毒素污染的信心13。内毒素的潜在来源可以是水、商业获得的培养基和血清、培养基成分和添加剂、实验室玻璃器皿和塑料器皿5，14，15。
因此，本研究旨在制定一种分离低内毒素电动汽车的方案。该协议提供了有关如何在EV分离过程中避免内毒素污染而不是从EV中去除内毒素的简单提示。以前，已经提出了许多关于如何从例如纳米医学中使用的工程纳米颗粒中去除内毒素的方案;然而，它们都对电动汽车等生物结构没有用。纳米颗粒的有效去热原可以通过乙醇或乙酸漂洗，在175°C加热3h，γ照射或Triton X-100处理进行;然而，这些程序导致电动汽车的破坏16，17。
所提出的协议是一项开创性研究，专注于避免 EV 中的内毒素杂质，这与之前关于 EV 对单核细胞影响的研究不同9. 将拟议的原则应用于实验室实践可能有助于获得可靠的研究结果，这在考虑电动汽车作为临床治疗剂的潜在用途时至关重要12。
Protocol
1. 超速离心管的制备 使用无菌一次性试管。如果无法做到这一点，请在使用无菌巴斯德移液管或其他一次性涂抹器用洗涤剂清洗后重复使用超速离心管。请记住，超速离心管应专用于一种类型的离心材料（细胞培养上清液/血清/血浆）和物种（人/小鼠/等）。 洗涤剂洗涤后，用去离子的无LPS水冲洗超速离心管3次。注意：请勿使用劣质水。 干燥超速离心管，?…
Representative Results
该方案的先决条件或强制性步骤是从试剂中排除可能的内毒素污染。所有使用的试剂，如FBS、DMEM、RPMI、PBS，甚至超速离心管，都必须不含内毒素（<0.005 EU/mL）。维持无内毒素污染并不容易，因为例如，用于细胞培养的常规/标准血清可能是其丰富的来源（0.364 EU/mL;见 表1）。 尽管该协议旨在分离内毒素含量低的EV，但表征分离的EV非常重要。在这项研究中，从两种…
Discussion
在过去几年中，正确分离电动汽车的方法变得越来越重要，使其能够进一步进行可靠的分析，例如，在获得可靠的组学和功能数据的背景下24。根据以前的研究经验，似乎不仅隔离方法的类型，而且该过程期间的其他条件都可能很重要。使用耗尽电动汽车的FBS被广泛认为是必需品25，26;然而，对电动汽车内毒素污染的监测往往被忽视。<…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了波兰国家科学中心的支持，批准号为2019/33/B/NZ5/00647。我们要感谢雅盖隆大学医学院分子医学微生物学系的Tomasz Gosiewski教授和Agnieszka Krawczyk教授在检测电动汽车中细菌DNA方面的宝贵帮助。
Materials
| Alix (3A9) Mouse mAb | Cell Signaling Technology | 2171 | |
| 1250ul Filter Universal Pipette Tips, Clear, Polypropylene, Non-Pyrogenic | GoogLab Scientific | GBFT1250-R-NS | |
| BD FACSCanto II Flow Cytometr | BD Biosciences | ||
| CBA Human Th1/Th2 Cytokine Kit II | BD Biosciences | 551809 | |
| CD9 (D8O1A) Rabbit mAb | Cell Signaling Technology | 13174 | |
| ChemiDoc Imaging System | Bio-Rad Laboratories, Inc. | 17001401 | |
| DMEM (Dulbecco’s Modified Eagle’s Medium) | Corning | 10-013-CV | |
| ELX800NB, Universal Microplate Reader | BIO-TEK INSTRUMENTS, INC | ||
| Fetal Bovine Serum | Gibco | 16000044 | |
| Fetal Bovine Serum South America Ultra Low Endotoxin | Biowest | S1860-500 | |
| Gentamicin, 50 mg/mL | PAN – Biotech | P06-13100 | |
| Goat anti-Mouse IgG- HRP | Santa Cruz Biotechnology | sc-2004 | |
| Goat anti-Rabbit IgG- HRP | Santa Cruz Biotechnology | sc-2005 | |
| Immun-Blot PVDF Membrane | Bio-Rad Laboratories, Inc. | 1620177 | |
| LPS from Salmonella abortus equi S-form (TLRGRADE) | Enzo Life Sciences, Inc. | ALX-581-009-L002 | |
| Mini Trans-Blot Electrophoretic Transfer Cell | Bio-Rad Laboratories, Inc. | 1703930 | |
| Nanoparticle Tracking Analysis | Malvern Instruments Ltd | ||
| NuPAGE LDS Sample Buffer (4X) | Invitrogen | NP0007 | |
| NuPAGE Sample Reducing Agent (10x) | Invitrogen | NP0004 | |
| Parafilm | Sigma Aldrich | P7793 | transparent film |
| Perfect 100-1000 bp DNA Ladder | EURx | E3141-01 | |
| PierceTM Chromogenic Endotoxin Quant Kit | Thermo Scientific | A39552 | |
| PP Oak Ridge Tube with sealing caps | Thermo Scientific | 3929, 03613 | |
| RPMI 1640 | RPMI-1640 (Gibco) | 11875093 | |
| SimpliAmp Thermal Cycler | Applied Biosystem | A24811 | |
| Sorvall wX+ ULTRA SERIES Centrifuge with T-1270 rotor | Thermo Scientific | 75000100 | |
| Sub-Cell GT Horizontal Electrophoresis System | Bio-Rad Laboratories, Inc. | 1704401 | |
| SuperSignal West Pico PLUS Chemiluminescent Substrate | Thermo Scientific | 34577 | |
| SW480 cell line | American Type Culture Collection(ATCC) | ||
| SW480 cell line | American Type Culture Collection (ATCC) | ||
| Syringe filter 0.22 um | TPP | 99722 | |
| Trans-Blot SD Semi-Dry Transfer Cell | Bio-Rad Laboratories, Inc. | 1703940 | Transfer machine |
| Transfer pipette, 3.5 mL | SARSTEDT | 86.1171.001 |
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