（前續(xù)：Optima MAX-XP臺式超速離心機在外泌體分離操作中時間的設(shè)定）

        關(guān)于超速離心機轉(zhuǎn)頭的選擇、離心管容量、RCF設(shè)置及離心時間設(shè)置對外泌體離心分離效能影響的討論，說明外泌體超速離心分離過程中，轉(zhuǎn)頭類型與工作性能，在諸多因素中具有關(guān)鍵基礎(chǔ)性作用。
        而目前細胞外囊泡分離技術(shù)條件下，不同來源生物樣本、研究人員個人經(jīng)驗和儀器使用方法的差異，使得細胞外囊泡功能和表征結(jié)果的解釋變得復雜和不確定。
        為此，2018年11月，國際細胞外囊泡學會（International Society for Extracellular Vesicles, ISEV)）發(fā)布了《細胞外囊泡研究最小信息指南》（Minimal information for studies of extracellular vesicles 2018, MISEV2018），對細胞外囊泡研究實驗的流程、方法和質(zhì)控標準提出了一系列強制性要求，以提高實驗結(jié)果的可重復性。

表10  MISEV2018細胞外囊泡超速離心分離流程細節(jié)信息報告清單（Checklist）

        Checklist中標示為++的要素，屬于Mandatory if applicable（如適用則必須提供），相當于該信息為必須報告事項。

         歸納起來，外泌體超速離心實驗細節(jié)報告要點包括：實驗所用轉(zhuǎn)頭型號及實際工作k因子值、離心管貨號、轉(zhuǎn)速/RCF設(shè)定值、離心溫度、離心時間和轉(zhuǎn)頭加減速設(shè)置。
        為了加深對MISEV2018 Checklist內(nèi)容的理解和印象，我們不煩可參考一下按MISEV018指南要求設(shè)計實施的外泌體分離實例中實驗流程細節(jié)部分匯報內(nèi)容的編寫方法。

1、外泌體（sEVs）離心分離操作細節(jié)信息報告參考范文
實例1 ：腦組織外泌體分離與純化(Nat Protoc; 2022 Nov)
        Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues》一文由美國紐約奧蘭治堡Nathan S. Kline精神病學研究所癡呆癥研究中心E. Levy領(lǐng)導的研究團隊發(fā)表。他們是第一個開發(fā)使用基于蔗糖密度梯度離心方法從腦組織中分離EVs的小組。文中明確宣示他們的腦組織分離外泌體實驗操作流程按MISEV2018指南標準進行的。
        其實驗整體流程如下圖所示，中文翻譯可參考《Optima MAX-XP臺式超速離心機在外泌體分離中應(yīng)用的主要類型》中“2.9.2 腦組織外泌體分離和純化”內(nèi)容。

1）實驗材料部分

       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）外泌體粗提物分離（Crude EVs purification）離心操作

       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內(nèi)容：超速離心機品牌、主機型號/貨號、轉(zhuǎn)頭/型號/貨號/k因子值、離心管類型/容量/貨號、離心溫度設(shè)定、RCF設(shè)定。

3）密度梯度純化環(huán)節(jié)

       除描述了離心機、轉(zhuǎn)頭、離心管和工作條件、加減速設(shè)定外，還按指南規(guī)定，將密度梯度溶液配制方法、梯度也鋪墊順序、離心結(jié)束后各密度餾分采集順序、體積和所用離心管細節(jié)做了詳盡披露。

實例2：內(nèi)耳組織細胞來源的外泌體的分離（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內(nèi)容：RCF設(shè)置、時間設(shè)置、離心溫度、主機型號、轉(zhuǎn)頭型號、離心管貨號。

實例2 前列腺癌細胞系PC-3和VCaP的培養(yǎng)物中外泌體的分離（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內(nèi)容：主機型號、轉(zhuǎn)頭型號/k-factor、RCF、時間設(shè)定、工作溫度。

       正常情況下，參考文獻所提供的操作流程細節(jié)，在樣品類型和體積相同或接近時，讀者根據(jù)實際所用超速離心轉(zhuǎn)頭的工作性能指標，優(yōu)化RCF設(shè)置、離心時間設(shè)定條件，有助于獲得與可驗證的實驗效果。RCF設(shè)置和離心設(shè)置方法，可參考《Optima MAX-XP超速離心機轉(zhuǎn)頭的選擇對外泌體分離效果影響的分析》、《Optima MAX-XP臺式超速離心機在外泌體分離操作中時間的設(shè)定》文中的有關(guān)內(nèi)容。

2、外泌體離心分離乃至多數(shù)其它超速離心實驗細節(jié)信息披露存在問題的普遍性

       而調(diào)研發(fā)現(xiàn)，這份2018年11月就頒布的用于規(guī)范細胞外囊泡實驗過程和實驗報告的權(quán)威指南，至今仍未在科研實驗公開發(fā)文中得到全面有效的遵循。
       在收集的63篇與Optima MAX-XP有關(guān)高分期刊發(fā)文中，只有37篇披露了超速離心機的主機型號、轉(zhuǎn)頭信息。能達到實例1-3中對外泌體分離超速離心流程細節(jié)信息報告詳細程度的屈指可數(shù)。
       就連刊載MISEV2018指南的J Extracell Vesicles期刊，2019 – 2023年刊發(fā)的14篇文章中，大部分也只提供了主機型號、轉(zhuǎn)頭型號、RCF設(shè)置、離心溫度，并未嚴格按Checklist清單要求做到完整詳細的細節(jié)匯報。
       JEV作為專業(yè)研究領(lǐng)域權(quán)威雜志，在MISEV2018頒布施行4年多的今天，編審人員對EV研究報告的審核尚把關(guān)不嚴。那其它綜合性期刊的刊文中對MISEV2018指南的遵循要求普及程度及態(tài)度可想而知。從以下國內(nèi)學者的近期發(fā)表的多篇有關(guān)文章可資佐證（注：作者單位恕不披露）。
       如2023年8月底在線發(fā)表的《M2 exosomes modified by hydrogen sulfide promoted bone regeneration by moesin mediated endocytosis》一文的外泌體分離實驗部分，只提供了自引方法、主機型號信息。追查其方法出處為2018年6月于Nat Commun在線發(fā)表的《Tet1 and Tet2 maintain mesenchymal stem cell homeostasis via demethylation of the P2rX7 promoter》，文中細胞外囊泡分離方法部分只提供了離心機主機型號、100000×g和70 min三個信息。
       類似的還有2023年2月份發(fā)表于Chem Sci的《Drug repurposing screens identify compounds that inhibit α-synuclein oligomers' membrane disruption and block antibody interactions》一文。相關(guān)內(nèi)容不過“centrifuged for 1 h at 40000 rpm using Optima MAX-XP at 25℃”短短一言。
       最離奇的要數(shù)Mol Ther Nucleic Acids上2020年11月26日在線發(fā)表的《Transfer of microRNA-25 by colorectal cancer cell-derived extracellular vesicles facilitates colorectal cancer dEVselopment and metastasis》。其外泌體分離方法是：“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).”做到了無轉(zhuǎn)頭型號、無RCF設(shè)置、無離心時間信息和無方法來源出處。
       其實，超速離心操作流程信息披露不全、不實的現(xiàn)象，并非只存在于外泌體超速離心實驗應(yīng)用中。

       本次調(diào)研收錄的與Optima MAX-XP有關(guān)、涵蓋其它多種研究對象的實驗文獻中，提供了超速離心機主機型號、所用轉(zhuǎn)頭、RCF設(shè)置這三個最基本信息的不過265例。近半數(shù)的文獻中的轉(zhuǎn)頭型號、離心管規(guī)格貨號無從考證。這既是長期以來，對科學出版物中超速離心實驗操作細節(jié)信息報告缺少標準規(guī)范造成的，當然也與超速離心實驗技術(shù)本身的復雜性和超速離心實驗的普及程度也有密切關(guān)系。

3、對外泌體離心分離流程操作細節(jié)要點信息報告的建議

       綜合MISEV2018和前面的討論內(nèi)容，我們認為，在外泌體差速離心法分離流程中技術(shù)細節(jié)的描述中，以下關(guān)鍵信息披露的是關(guān)鍵、必要和有益的：
1）方法的來源出處（便于正本清源，以正視聽）；
2）每個步驟的離心溫度和時間和離心介質(zhì)溶液（便于根據(jù)溶液中樣品組分沉降系數(shù)的變化調(diào)整有效離心時間）；
3）低速離心機主機、水平轉(zhuǎn)頭及吊籃型號、離心管品牌貨號（便于根據(jù)起始樣品材料體積選擇合適的離心管、離心工具）；
4）高速離心機主機、轉(zhuǎn)頭型號和離心管品牌貨號；
5）超速離心機主機、轉(zhuǎn)頭型號和離心管品牌貨號、RCF設(shè)置和轉(zhuǎn)頭實際工作k因子值、加減速設(shè)置（便于選擇不同轉(zhuǎn)頭和調(diào)整實驗設(shè)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）外泌體密度梯度純化流程，梯度液溶度配制、裝樣方法（如適用）；
7）細胞培養(yǎng)基自帶外泌體排空操作方法（如適用）；
8）外泌體分析中陰性對照的設(shè)置；
9) 外泌體熒光標記環(huán)節(jié)中游離熒光染料離心方法（如適用）。

4、對Optima MAX-XP在外泌體超速離心分離中應(yīng)用調(diào)研結(jié)果大總結(jié)

       對臺式超速離心機Optima MAX-XP在外泌體分離應(yīng)用調(diào)研的數(shù)據(jù)表明，Optima MAX-XP用于外泌體沉淀分離、密度梯度純化處理，技術(shù)切實可行。事實上，不僅是外泌體超速分離，它還被廣泛成功地運用于各類蛋白質(zhì)及蛋白復合物、亞細胞組分、納米粒子制劑、脂質(zhì)體和病毒類顆粒等分離制備。Optima MAX-XP臺式超離與大塊頭立式超離Optima L-XP、Optima XPN、Optima XE在科研實驗研究對象，基本重疊。

       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容量范圍的轉(zhuǎn)頭使用頻率較高。具體到外泌體分離操作，最常用是MLA-55、TLA-55和MLA-50 (6×32.4mL)三款角轉(zhuǎn)頭。
       超速離心機在外泌體離心分離以及各類有關(guān)超速離心實驗的正式匯報文件中長期存在、具有普遍性的問題是操作流程關(guān)鍵細節(jié)信息的缺失，不利于對復雜實驗結(jié)果的解釋，還給實驗設(shè)計方法的借鑒造成困擾，降低了學術(shù)成果傳播價值（他引少了嘛）。

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