雷霆收罢江海凝——Optima MAX-XP台式超速离心机在外泌体分离操作细节汇报中应注意的问题

（前续：Optima MAX-XP台式超速离心机在外泌体分离操作中时间的设定）

        关于超速离心机转头的选择、离心管容量、RCF设置及离心时间设置对外泌体离心分离效能影响的讨论，说明外泌体超速离心分离过程中，转头类型与工作性能，在诸多因素中具有关键基础性作用。
        而目前细胞外囊泡分离技术条件下，不同来源生物样本、研究人员个人经验和仪器使用方法的差异，使得细胞外囊泡功能和表征结果的解释变得复杂和不确定。
        为此，2018年11月，国际细胞外囊泡学会（International Society for Extracellular Vesicles, ISEV)）发布了《细胞外囊泡研究最小信息指南》（Minimal information for studies of extracellular vesicles 2018, MISEV2018），对细胞外囊泡研究实验的流程、方法和质控标准提出了一系列强制性要求，以提高实验结果的可重复性。

表10  MISEV2018细胞外囊泡超速离心分离流程细节信息报告清单（Checklist）

        Checklist中标示为++的要素，属于Mandatory if applicable（如适用则必须提供），相当于该信息为必须报告事项。

         归纳起来，外泌体超速离心实验细节报告要点包括：实验所用转头型号及实际工作k因子值、离心管货号、转速/RCF设定值、离心温度、离心时间和转头加减速设置。
        为了加深对MISEV2018 Checklist内容的理解和印象，我们不烦可参考一下按MISEV018指南要求设计实施的外泌体分离实例中实验流程细节部分汇报内容的编写方法。

1、外泌体（sEVs）离心分离操作细节信息报告参考范文
实例1 ：脑组织外泌体分离与纯化(Nat Protoc; 2022 Nov)
        Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues》一文由美国纽约奥兰治堡Nathan S. Kline精神病学研究所痴呆症研究中心E. Levy领导的研究团队发表。他们是第一个开发使用基于蔗糖密度梯度离心方法从脑组织中分离EVs的小组。文中明确宣示他们的脑组织分离外泌体实验操作流程按MISEV2018指南标准进行的。
        其实验整体流程如下图所示，中文翻译可参考《Optima MAX-XP台式超速离心机在外泌体分离中应用的主要类型》中“2.9.2 脑组织外泌体分离和纯化”内容。

1）实验材料部分

       Single edge razor blades (Stanley, cat. no. 11–515)

       40 μm cell strainers (Fisherbrand, cat. no. 22–363-547)

       70 mL ultracentrifugation polycarbonate bottles (Beckman Coulter, cat. no. 355622)

       Type 45Ti, titanium fixed-angle rotor (Beckman Coulter, cat. no. 339160)

       Optima XE-90 floor-type ultracentrifuge (Beckman Coulter, cat. no. A94471)

       Allegra X-30R tabletop-type refrigerated centrifuge (Beckman Coulter, cat. no. B06320)

       14 mL open-top, thin wall, ultra-clear tubes (Beckman Coulter, cat. no. 344060)

       6.5 mL open-top, thick wall, polycarbonate tubes (Beckman Coulter, cat. no. 355647)

       MLA-80 fixed angle rotor (Beckman Coulter, cat. no. 367096) or Type 70.1Ti, titanium fixed-angle rotor (Beckman Coulter, cat. no. 342184)

       SW 40Ti, titanium swinging-bucket rotor (Beckman Coulter, cat. no. 331301)

       Optima XE-90 floor-type ultracentrifuge (Beckman Coulter, cat. no. A94471)

       Optima MAX-XP tabletop-type ultracentrifuge (Beckman Coulter, cat. no. 393315)

2）外泌体粗提物分离（Crude EVs purification）离心操作

       Transfer the supernatant into a 70 mL ultracentrifugation polycarbonate bottle. Add ice-cold PBS to bring the total volume up to 50 mL.

       Centrifuge at 10,000×g for 30 min at 4℃. If using a Type 45Ti rotor, this corresponds to 11,000 rpm (k-factor: 2,218).

       Centrifuge at 10,000×g for 30 min at 4℃. If using a Type 45Ti rotor, this corresponds to 11,000 rpm (k-factor: 2,218).

       Checklist内容：超速离心机品牌、主机型号/货号、转头/型号/货号/k因子值、离心管类型/容量/货号、离心温度设定、RCF设定。

3）密度梯度纯化环节

       除描述了离心机、转头、离心管和工作条件、加减速设定外，还按指南规定，将密度梯度溶液配制方法、梯度也铺垫顺序、离心结束后各密度馏分采集顺序、体积和所用离心管细节做了详尽披露。

实例2：内耳组织细胞来源的外泌体的分离（J Clin Invest/2020 May 1）

       Exosomes were isolated from utricle-conditioned medium using an abbrEVsiated version of a prEVsiously described protocol (29). Cells and large cellular debris were removed by centrifugation (300×g, 10 minutes, 4℃), followed by sedimentation of large vesicles and additional cellular debris (10000×g, 30 minutes, 4℃). Exosomes were pelleted by subjecting the supernatant from the second spin to a high-speed centrifugation step (100000×g, 70 minutes, 4℃) in polycarbonate tubes (349622, Beckman Coulter) using a TLA-100.3 rotor and an Optima MAX-XP ultracentrifuge (Beckman Coulter). The exosome pellet was resuspended in PBS or culture medium by trituration with a micropipette.

Checklist内容：RCF设置、时间设置、离心温度、主机型号、转头型号、离心管货号。

实例2 前列腺癌细胞系PC-3和VCaP的培养物中外泌体的分离（J Extracell Vesicles;2019 Apr 4）

       A flow chart of the isolation and analyses is presented in Figure 1. EVs were isolated from the conditioned media using differential ultracentrifugation as prEVsiously described [5] with slight modifications.

       In brief, the conditioned medium (180 ml from conventional cell cultures and 10 ml from bioreactors) was first centrifuged to remove cell debris and apoptotic bodies at 2500×g for 30 min. The supernatant was then centrifuged at 20000×g for 60 min for the 20K EVs pellet, and the final supernatant was ultracentrifuged at 110000×g for 2 h in +4℃ to obtain the 110K EVs pellet using Optima LE-80K ultracentrifuge with rotor Ti 50.2, k-factor 143.3 (Beckman Coulter). For the metabolomics analysis, media were incubated in the absence of cells at 37℃, and after 3 days ultracentrifuged at 110000×g for 2 h, as a recommended control [18]. The collected EVs were washed with 500μl of PBS and re-pelleted by ultracentrifugation at 100000×g, +4℃ for 2 h using Optima MAX-XP (Beckman Coulter) ultracentrifuge with rotor TLA-55, k-factor 81.3 (Beckman Coulter). The 20K, 110K pellets and media controls were then resuspended in 50μl of Dulbecco’s phosphate buffered saline (DPBS) (Gibco, Life Technologies), and stored at 80℃ for further analysis.

Checklist内容：主机型号、转头型号/k-factor、RCF、时间设定、工作温度。

       正常情况下，参考文献所提供的操作流程细节，在样品类型和体积相同或接近时，读者根据实际所用超速离心转头的工作性能指标，优化RCF设置、离心时间设定条件，有助于获得与可验证的实验效果。RCF设置和离心设置方法，可参考《Optima MAX-XP超速离心机转头的选择对外泌体分离效果影响的分析》、《Optima MAX-XP台式超速离心机在外泌体分离操作中时间的设定》文中的有关内容。

2、外泌体离心分离乃至多数其它超速离心实验细节信息披露存在问题的普遍性

       而调研发现，这份2018年11月就颁布的用于规范细胞外囊泡实验过程和实验报告的权威指南，至今仍未在科研实验公开发文中得到全面有效的遵循。
       在收集的63篇与Optima MAX-XP有关高分期刊发文中，只有37篇披露了超速离心机的主机型号、转头信息。能达到实例1-3中对外泌体分离超速离心流程细节信息报告详细程度的屈指可数。
       就连刊载MISEV2018指南的J Extracell Vesicles期刊，2019 – 2023年刊发的14篇文章中，大部分也只提供了主机型号、转头型号、RCF设置、离心温度，并未严格按Checklist清单要求做到完整详细的细节汇报。
       JEV作为专业研究领域权威杂志，在MISEV2018颁布施行4年多的今天，编审人员对EV研究报告的审核尚把关不严。那其它综合性期刊的刊文中对MISEV2018指南的遵循要求普及程度及态度可想而知。从以下国内学者的近期发表的多篇有关文章可资佐证（注：作者单位恕不披露）。
       如2023年8月底在线发表的《M2 exosomes modified by hydrogen sulfide promoted bone regeneration by moesin mediated endocytosis》一文的外泌体分离实验部分，只提供了自引方法、主机型号信息。追查其方法出处为2018年6月于Nat Commun在线发表的《Tet1 and Tet2 maintain mesenchymal stem cell homeostasis via demethylation of the P2rX7 promoter》，文中细胞外囊泡分离方法部分只提供了离心机主机型号、100000×g和70 min三个信息。
       类似的还有2023年2月份发表于Chem Sci的《Drug repurposing screens identify compounds that inhibit α-synuclein oligomers' membrane disruption and block antibody interactions》一文。相关内容不过“centrifuged for 1 h at 40000 rpm using Optima MAX-XP at 25℃”短短一言。
       最离奇的要数Mol Ther Nucleic Acids上2020年11月26日在线发表的《Transfer of microRNA-25 by colorectal cancer cell-derived extracellular vesicles facilitates colorectal cancer dEVselopment and metastasis》。其外泌体分离方法是：“After 48 h, conditioned medium (CM) was collected and filtered through a 0.22 μm filter (Merck Millipore, Billerica, MA, USA). EVs in CM were separated by ultracentrifugation using the Optima Max-XP instrument (Beckman Coulter, CA, USA).”做到了无转头型号、无RCF设置、无离心时间信息和无方法来源出处。
       其实，超速离心操作流程信息披露不全、不实的现象，并非只存在于外泌体超速离心实验应用中。

       本次调研收录的与Optima MAX-XP有关、涵盖其它多种研究对象的实验文献中，提供了超速离心机主机型号、所用转头、RCF设置这三个最基本信息的不过265例。近半数的文献中的转头型号、离心管规格货号无从考证。这既是长期以来，对科学出版物中超速离心实验操作细节信息报告缺少标准规范造成的，当然也与超速离心实验技术本身的复杂性和超速离心实验的普及程度也有密切关系。

3、对外泌体离心分离流程操作细节要点信息报告的建议

       综合MISEV2018和前面的讨论内容，我们认为，在外泌体差速离心法分离流程中技术细节的描述中，以下关键信息披露的是关键、必要和有益的：
1）方法的来源出处（便于正本清源，以正视听）；
2）每个步骤的离心温度和时间和离心介质溶液（便于根据溶液中样品组分沉降系数的变化调整有效离心时间）；
3）低速离心机主机、水平转头及吊篮型号、离心管品牌货号（便于根据起始样品材料体积选择合适的离心管、离心工具）；
4）高速离心机主机、转头型号和离心管品牌货号；
5）超速离心机主机、转头型号和离心管品牌货号、RCF设置和转头实际工作k因子值、加减速设置（便于选择不同转头和调整实验设置）。如：Optima MAX-XP ultracentrifuge (Beckman Coulter, P/N 393315)；MLA-80 fixed angle rotor (Beckman Coulter, P/N 367096)；6.5 mL open-top thick wall polycarbonate tubes (Beckman Coulter, P/N 355647)；Centrifuge at 100,000×g for 90 min (Accel:4/Decel: 9) at 4℃

6）外泌体密度梯度纯化流程，梯度液溶度配制、装样方法（如适用）；
7）细胞培养基自带外泌体排空操作方法（如适用）；
8）外泌体分析中阴性对照的设置；
9) 外泌体荧光标记环节中游离荧光染料离心方法（如适用）。

4、对Optima MAX-XP在外泌体超速离心分离中应用调研结果大总结

       对台式超速离心机Optima MAX-XP在外泌体分离应用调研的数据表明，Optima MAX-XP用于外泌体沉淀分离、密度梯度纯化处理，技术切实可行。事实上，不仅是外泌体超速分离，它还被广泛成功地运用于各类蛋白质及蛋白复合物、亚细胞组分、纳米粒子制剂、脂质体和病毒类颗粒等分离制备。Optima MAX-XP台式超离与大块头立式超离Optima L-XP、Optima XPN、Optima XE在科研实验研究对象，基本重叠。

       就Optima MAX-XP而言， TLA-55（12×1.5mL）、TLS-55（4×2.2mL）、MLS-50（4×5.0mL）、TLA-100.3（6×3.5mL）、TLA-120.2（12×2.0mL）、MLA-80 8×8.0mL）和MLA-55（8×13.5mL）等1.5 – 13.5mL容量范围的转头使用频率较高。具体到外泌体分离操作，最常用是MLA-55、TLA-55和MLA-50 (6×32.4mL)三款角转头。
       超速离心机在外泌体离心分离以及各类有关超速离心实验的正式汇报文件中长期存在、具有普遍性的问题是操作流程关键细节信息的缺失，不利于对复杂实验结果的解释，还给实验设计方法的借鉴造成困扰，降低了学术成果传播价值（他引少了嘛）。

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