cleaning and reusing polycarbonate ultracentrifuge tubes for sustainable extracellular vesicle isolation

מיחזור יעיל של צינורות אולטרה-צנטריפוגות פוליקרבונט לפרוטאומיקה

Extracellular vesicles (EVs) are lipid-bilayer-delimited particles released from cells that carry biologically active cargo, such as protein, and participate in various biological processes, including cell-cell communication and the formation of biologic mineralization1. These particles are found in all body fluids and tissues, and their biological activities and uses are a rapidly evolving field of scientific research. Isolation and validation of these nanoparticles present various challenges due to their small size and bio-similarity to other particles, such as liposomes and protein aggregates. The most recent International Society of Extracellular Vesicles guidelines, Minimal information for studies of extracellular vesicles 2018 (MISEV2018), are the recognized standard for EV scientific research2.
Various methods, often in tandem, must be used for EV isolation depending on the upstream source and the downstream applications. As of 2015, the most common primary isolation method for EVs was differential ultracentrifugation (UC)2. In principle, initial lower speed centrifugation separates&#160;larger or denser unwanted components, such as cells and cell debris, leaving EVs in the supernatant. Subsequently, UC uses very high centrifugal force to pellet out and thus separate and purify EVs from other particles that are smaller or less dense but which may also contain dense non-EV particles. Most protocols will often use, at one step or another, UC to isolate EVs from a fluid3. Further, UC is used in other methods of EV isolation, such as density gradient ultracentrifugation (DGUC), which uses mediums such as OptiprepTM iodixanol and centrifugal force to separate EVs according to their buoyant density4. Other methods of EV isolation exist3.
Considering the rapidly evolving understanding of the biological processes dictated by EVs and their potential as biomarkers and information regarding pathophysiology, discovery-based analyses such as proteomics have gained traction5,6,7,8. EVs are small, and, depending on the source, have a low yield of protein (&#60;1 &#956;g) when compared to whole tissue or cell lysate. Isolation of EVs for proteomics analysis requires special considerations, such as the removal of non-EV protein contaminants from upstream liquid or tissue, consideration of EV protein degradation during the isolation process, and the utilization of methods that create protein solutions that are chemically compatible with peptide preparation and mass spectrometry analyses.
Research laboratory consumables are often plastic and disposable in nature. These single-use materials contribute to the global plastic pollution crisis and consumable costs. Specialized polycarbonate and polystyrene UC tubes are designed to withstand the high centrifugal forces required to pellet EVs in laboratory applications. While it is possible to sterilize and disinfect UC tubes for re-use, proteomic analysis, especially those of low protein yields such as EVs, requires special attention. Not only is sufficient removal of residual protein from the previous use paramount, chemical compatibility with mass-spectroscopy and plastic-derived polymer contamination must also be considered.
Here we present a cleaning protocol of polycarbonate tubes using detergents suitable for mass-spectrometry and perform experiments to validate its successful removal of residual protein below limits of detection and lack of detectable polymer contaminants. To validate the cleaning protocol for EV proteomic applications using both UC and DGUC purposes, we obtained tubes from isolations of human vasculature tissue EVs with a combined UC and DGUC protocol. Tubes were cleaned using the protocol described, and the experimental process was repeated without samples comparing a blank never-used UC tube and a cleaned UC tube. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the cost associated with such experiments.

מחבר זרקור: שימוש חוזר יעיל בצינורות פוליקרבונט לבידוד שלפוחית חוץ תאית

שימוש חוזר בצינור אולטרה-צנטריפוגה פוליקרבונט באנליזות פרוטאומיות של שלפוחיות חוץ-תאיות

We study cardiovascular diseases, specifically the role of extracellular vesicles in the pathological calcification of cardiovascular tissues, such as valves and blood vessels. Researchers in our group have recently defined methods to extract tissue and trapped extracellular vesicles from fibro calcific cardiovascular tissue to determine their proteomic profile. Our group has demonstrated that these extracellular vesicles contain important protein cargo for initiating calcification in disease tissues.The specialized tubes used in the ultracentrifugation step of extracellular vesicle isolation can be cleaned and sterilized for reuse. However, proteomic analysis requires special consideration as any contamination could greatly affect downstream results. Here we validate a protocol for reusing consumables, specifically polycarbonate ultracentrifuge tubes for extracellular vesicle isolation that is suitable for downstream low input proteomics applications.Our protocol is simple and effective, and it will allow researchers to reduce both the consumable costs and the waste typically associated with these isolation protocols.

ניקוי ושימוש חוזר בצינורות אולטרה-צנטריפוגות פוליקרבונט לבידוד שלפוחית חוץ-תאי בר-קיימא

כדי להתחיל, לאסוף צינורות פוליקרבונט בשימוש ולהשליך את המדגם הקודם ביסודיות. כדי למנוע התייבשות של הדגימה לצד הצינור, מיד לטבול את צינורות ultracentrifuge במי ברז, ולאחר מכן לדגור את הצינור 0.1% נתרן דודציל סולפט או SDS במשך 10 דקות. דקנט את רוב, אך לא את כל פתרון SDS מהצינור.ובאמצעות צמר גפן לנגב ביסודיות את האזור של הצינור שבו היה גלולת שלפוחית חוץ-תאית. כדי לשטוף, מלא את הצינור במי ברז חמים ורוקן לחלוטין. לאחר שלוש שטיפות, באמצעות בקבוק תרסיס, יש לרסס מים נטולי מינרלים לתוך הצינור ולאחר מכן 70% אתנול.מניחים את הצינור הפוך לייבוש מלא ומעבירים לצנצנת נקייה לשימוש חוזר.

cleaning-reusing-polycarbonate-ultracentrifuge-tubes-for-sustainable

Research

JoVE Journal

Biology

Cleaning and Reusing Polycarbonate Ultracentrifuge Tubes for Sustainable Extracellular Vesicle Isolation

94 Views.  Brigham and Women’s Hospital, Harvard Medical School. כדי להתחיל, לאסוף צינורות פוליקרבונט בשימוש ולהשליך את המדגם הקודם ביסודיות. כדי למנוע התייבשות של הדגימה לצד הצינור, מיד לטבול את צינורות ultracentrifuge במי ברז, ולאחר מכן לדגור את הצינור 0.1% נתרן דודציל סולפט או SDS במשך 10 דקות. דקנט את רוב, אך לא את כל פתרון SDS מהצינור.ובאמצעות ?...

Video: ניקוי ושימוש חוזר בצינורות אולטרה-צנטריפוגות פוליקרבונט לבידוד שלפוחית חוץ-תאי בר-קיימא

Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins.
This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples.
Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (&#8804;20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

A detailed protocol is provided for cleaning and re-using polycarbonate ultracentrifuge tubes to perform extracellular vesicle isolation suitable for proteomics experiments.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ...