Influence of short term storage conditions, concentration methods and excipients on extracellular vesicle recovery and function

Wakker, Simonides Immanuel van de; Oudheusden, J. van; Mol, Emma A.; Roefs, Marieke T.; Zheng, Wenyi; Görgens, André; Andaloussi, Samir El; Sluijter, Joost P.G.; Vader, Pieter

doi:10.1016/j.ejpb.2021.11.012

Cited by 36 publications

(36 citation statements)

References 37 publications

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“…Based on particle concentrations measured, storing EVs at ‐20/80˚C in buffer formulations 3, 4, 5, 10, 13, 14, 16 and 21 seemed to stabilize EVs best. Storing EVs frozen in PBS supplemented with albumin (Buffer 2) showed consistent stabilizing effects compared to PBS alone (Buffer 1), which is in line with previous reports showing increased recovery rates in the presence of albumin (van de Wakker et al., 2021 ; Yamashita et al., 2016 ; Zhang et al., 2020 ). Moreover, adding further additives such as trehalose (Buffer 3), HEPES (Buffer 4) or DMSO (Buffer 5) appeared to further improve EV recovery rates when samples were frozen (Figure 2B/C/E/F ).…”

Section: Resultssupporting

confidence: 87%

“…It has been reported before that EV recovery in context of EV isolation procedures can be reduced through adsorption to tube walls, SEC column surfaces or filters, and it has been shown that albumin pre‐coating of the tubes or filters can prevent this, at least partially (Evtushenko et al., 2021 ; van de Wakker et al., 2021 ; Yamashita et al., 2016 ; Zhang et al., 2020 ). Here, we demonstrate that the usage of albumin‐supplemented PBS as storage buffer or diluent results in clearly improved EV recovery rates compared to using PBS alone.…”

Section: Discussionmentioning

confidence: 99%

“…A few studies in the last years have investigated the impact of storage temperature and time (Cheng et al., 2019 ; Lőrincz et al., 2014 ; Park et al., 2018 ), focused on the role of plastic tubes used for isolation or storage (Evtushenko et al., 2021 ; Resnik et al., 2020 ), or explored the use of cryoprotective additives such as DMSO (Tegegn et al., 2016 ) or trehalose (Bosch et al., 2016 ). More recent comprehensive studies investigated EV stability in context of lyophilisation (Trenkenschuh et al., 2022 ), storage‐related particle loss through vesicle fusion (Gelibter et al., 2022 ), and the short‐term influence of storage conditions and concentration methods on EV recovery and function (van de Wakker et al., 2021 ). Generally, reported findings have sometimes been conflicting, likely related to a focus on storage procedures for specific biofluid samples, to specific storage strategies in terms of EV isolation procedures, or to limited downstream read‐outs only covering partial aspects of EVs such as small RNA quantification or bulk protein content in several studies (Jeyaram & Jay, 2017 ; Kusuma et al., 2018 ; Qin et al., 2020 ; Welch et al., 2017 ; Yuan et al., 2021 ).…”

Section: Introductionmentioning

confidence: 99%

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Identification of storage conditions stabilizing extracellular vesicles preparations

Görgens

Corso

Hagey

et al. 2022

J of Extracellular Vesicle

156

100

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Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS‐HAT). We report that PBS‐HAT buffer facilitates clearly improved short‐term and long‐term EV preservation for samples stored at ‐80°C, stability throughout several freeze‐thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

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Section: Resultssupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of storage conditions stabilizing extracellular vesicles preparations

Görgens

Corso

Hagey

et al. 2022

J of Extracellular Vesicle

156

100

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“…Practically, the CELLine adherent bioreactor flasks have many advantages compared to producing EVs using conventional tissue culture flasks. For example, limiting the EV production to the 15 mL cell culture chamber removes the need for tangential flow and centrifugal concentrators prior to EV isolation which are known to lead to significant EV loss [54][55][56][57]. In addition, cells do not need to be passaged regularly as they naturally turn over during their continuous cultures which adds convenience for holidays and, of current relevance, hindered laboratory access.…”

Section: Discussionmentioning

confidence: 99%

Investigating the Consistency of Extracellular Vesicle Production from Breast Cancer Subtypes Using CELLine Adherent Bioreactors

Hisey

Artuyants

Guo

et al. 2022

Preprint

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Extracellular vesicle (EV) research has grown rapidly in recent years, largely due to the potential use of EVs as liquid biopsy biomarkers or therapeutics. However, in-depth characterisation and validation of EVs produced using conventional in vitro cultures can be challenging due to the large area of cell monolayers and volumes of culture media required. To overcome this obstacle, multiple bioreactor designs have been tested for EV production with varying success, but the consistency of EVs produced over time in these systems has not been reported previously. In this study, we demonstrate that several breast cancer cell lines of different subtypes can be cultured simultaneously in space, resource, and time efficient manner using CELLine AD 1000 systems, allowing the consistent production of vast amounts of EVs for downstream experimentation. We report an improved workflow used for inoculating, maintaining, and monitoring the bioreactors, their EV production, and the characterisation of the EVs produced. Lastly, our proteomic analyses of the EVs produced throughout the lifetime of the bioreactors show that core EV-associated proteins are relatively consistent, with few minor variations over time, and that tracking the production of EVs may be a convenient method for indirectly monitoring the bioreactors' health. These findings will aid future studies requiring the simultaneous production of large amounts of EVs from several cell lines of different subtypes of a disease and other EV biomanufacturing applications.

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“…Some researchers warned that isolated EVs could be adsorb onto tube walls, which would decrease EV concentration. Using Eppendorf Protein LoBind tubes or adding bovine serum albumin or Tween-20 to block the wall can decrease the loss [ 104 , 105 ]. Hermida-Nogueira et al (2020) found that seven days following a pathogen reduction technology (PRT) + Mirasol ® (vitamin B2 with ultraviolet B light), 151 proteins, including EV markers and regulatory proteins released by platelets, were upregulated compared to their levels after two days of storage [ 106 ].…”

Section: Biofluids and Extracellular Vesicles Characterization Under ...mentioning

confidence: 99%

Impact of Storage Conditions on EV Integrity/Surface Markers and Cargos

Sivanantham

Jin

2022

Life

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Extracellular vesicles (EVs) are small biological particles released into biofluids by every cell. Based on their size, they are classified into small EVs (<100 nm or <200 nm) and medium or large EVs (>200 nm). In recent years, EVs have garnered interest for their potential medical applications, including disease diagnosis, cell-based biotherapies, targeted drug delivery systems, and others. Currently, the long-term and short-term storage temperatures for biofluids and EVs are −80 °C and 4 °C, respectively. The storage capacity of EVs can depend on their number, size, function, temperature, duration, and freeze–thaw cycles. While these parameters are increasingly studied, the effects of preservation and storage conditions of EVs on their integrity remain to be understood. Knowledge gaps in these areas may ultimately impede the widespread applicability of EVs. Therefore, this review summarizes the current knowledge on the effect of storage conditions on EVs and their stability and critically explores prospective ways for improving long-term storage conditions to ensure EV stability.

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Influence of short term storage conditions, concentration methods and excipients on extracellular vesicle recovery and function

Cited by 36 publications

References 37 publications

Identification of storage conditions stabilizing extracellular vesicles preparations

Identification of storage conditions stabilizing extracellular vesicles preparations

Investigating the Consistency of Extracellular Vesicle Production from Breast Cancer Subtypes Using CELLine Adherent Bioreactors

Impact of Storage Conditions on EV Integrity/Surface Markers and Cargos

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