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  2. 產品課堂

    雷霆收罷江海凝——Optima MAX-XP臺式超速離心機在外泌體分離操作細節匯報中應注意的問題

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

            關于超速離心機轉頭的選擇、離心管容量、RCF設置及離心時間設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,轉頭類型與工作性能,在諸多因素中具有關鍵基礎性作用。
            而目前細胞外囊泡分離技術條件下,不同來源生物樣本、研究人員個人經驗和儀器使用方法的差異,使得細胞外囊泡功能和表征結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、方法和質控標準提出了一系列強制性要求,以提高實驗結果的可重復性。

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

    方法

    報告要點

    重要性

    標準差速離心分離法

    reference number of   tube(s)  離心管貨號

    ++

    rotor(s) 轉頭型號

    ++

    time+ speed+ rotor,   volume/density of centrifugation conditions

    每個離心步驟轉頭、轉速、樣品容量

    ++

    temperature 離心溫度設置

    ++

    brake settings 轉頭減速制動檔位設置

    ++




    密度梯度二次分離法

    nature of matrix 密度梯度介質成份

    ++

    method of   generating gradient 生成梯度的方法

    ++

    reference (and   size) of tubes 離心管貨號和管尺寸

    ++

    bottom-up (sample   at bottom, high density) or top-bottom (sample on top, low density)

    溶液垂直方向上密度梯度的分布和樣品層的位置

    ++

    centrifugation   speed and time (with brake specified)

    離心速度和時間及減速檔位設置

    ++

    method and volume   of fraction recovery餾分回收的方法和體積

    ++

            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報告要點包括:實驗所用轉頭型號及實際工作k因子值、離心管貨號、轉速/RCF設定值、離心溫度、離心時間和轉頭加減速設置。
            為了加深對MISEV2018 Checklist內容的理解和印象,我們不煩可參考一下按MISEV018指南要求設計實施的外泌體分離實例中實驗流程細節部分匯報內容的編寫方法。

    1、外泌體(sEVs)離心分離操作細節信息報告參考范文
    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領導的研究團隊發表。他們是第一個開發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用的主要類型
    中“2.9.2 腦組織外泌體分離和純化”內容。

    Optima MAX-XP臺式超速離心機用于腦組織EVs分離實驗流程圖.jpg

    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內容:超速離心機品牌、主機型號/貨號、轉頭/型號/貨號/k因子值、離心管類型/容量/貨號、離心溫度設定、RCF設定。

    3)密度梯度純化環節

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

     

    實例2:內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內容:RCF設置、時間設置、離心溫度、主機型號、轉頭型號、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內容:主機型號、轉頭型號/k-factor、RCF、時間設定、工作溫度。

           正常情況下,參考文獻所提供的操作流程細節,在樣品類型和體積相同或接近時,讀者根據實際所用超速離心轉頭的工作性能指標,優化RCF設置、離心時間設定條件,有助于獲得與可驗證的實驗效果。RCF設置和離心設置方法,可參考《Optima MAX-XP超速離心機轉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定》文中的有關內容。


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

           而調研發現,這份2018年11月就頒布的用于規范細胞外囊泡實驗過程和實驗報告的權威指南,至今仍未在科研實驗公開發文中得到全面有效的遵循。
           在收集的63篇與Optima MAX-XP有關高分期刊發文中,只有37篇披露了超速離心機的主機型號、轉頭信息。能達到實例1-3中對外泌體分離超速離心流程細節信息報告詳細程度的屈指可數。
           就連刊載MISEV2018指南的J Extracell Vesicles期刊,2019 – 2023年刊發的14篇文章中,大部分也只提供了主機型號、轉頭型號、RCF設置、離心溫度,并未嚴格按Checklist清單要求做到完整詳細的細節匯報。
           JEV作為專業研究領域權威雜志,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度可想而知。從以下國內學者的近期發表的多篇有關文章可資佐證(注:作者單位恕不披露)。
           如2023年8月底在線發表的《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。相關內容不過“centrifuged for 1 h at 40000 rpm using Optima MAX-XP at 25℃”短短一言。
           最離奇的要數Mol Ther Nucleic Acids上2020年11月26日在線發表的《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).”做到了無轉頭型號、無RCF設置、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、不實的現象,并非只存在于外泌體超速離心實驗應用中。

           本次調研收錄的與Optima MAX-XP有關、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、所用轉頭、RCF設置這三個最基本信息的不過265例。近半數的文獻中的轉頭型號、離心管規格貨號無從考證。這既是長期以來,對科學出版物中超速離心實驗操作細節信息報告缺少標準規范造成的,當然也與超速離心實驗技術本身的復雜性和超速離心實驗的普及程度也有密切關系。


    3、對外泌體離心分離流程操作細節要點信息報告的建議

           綜合MISEV2018和前面的討論內容,我們認為,在外泌體差速離心法分離流程中技術細節的描述中,以下關鍵信息披露的是關鍵、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溶液(便于根據溶液中樣品組分沉降系數的變化調整有效離心時間);
    3)低速離心機主機、水平轉頭及吊籃型號、離心管品牌貨號(便于根據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、轉頭型號和離心管品牌貨號;
    5)超速離心機主機、轉頭型號和離心管品牌貨號、RCF設置和轉頭實際工作k因子值、加減速設置(便于選擇不同轉頭和調整實驗設置)。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置;
    9) 外泌體熒光標記環節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用調研結果大總結

           對臺式超速離心機Optima MAX-XP在外泌體分離應用調研的數據表明,Optima MAX-XP用于外泌體沉淀分離、密度梯度純化處理,技術切實可行。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及蛋白復合物、亞細胞組分、納米粒子制劑、脂質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)三款角轉頭。
           超速離心機在外泌體離心分離以及各類有關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鍵細節信息的缺失,不利于對復雜實驗結果的解釋,還給實驗設計方法的借鑒造成困擾,降低了學術成果傳播價值(他引少了嘛)。

     

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