Identification of storage conditions stabilizing extracellular vesicles preparations

Abstract

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.

Keywords: diluent; exosomes; extracellular vesicles; liposomes; preservation; stability; storage; storage buffer; vesicles.

FIGURE 1

EVs appear unstable when stored in PBS. (A) HEK293T EVs were isolated by UC (A‐E) or UF/SEC (F), and aliquots were stored in PBS at +4˚C, ‐20˚C or ‐80˚C and analysed at different time points. (B) Particle concentration and diameter estimated by NTA measurement of samples stored at +4˚C in PBS up to 8 days and estimated bulk protein amount and RNA quantifications from same sample volumes. Measurements were done daily up to 8 days (n = 2). Values are expressed relative to fresh samples. (C) Particle concentration and diameter estimated by NTA measurement of samples stored at +4˚C, ‐20˚C or ‐80˚C for 1 week in PBS. Estimated bulk protein amount and RNA quantifications from same sample volumes (n = 2). (D) NTA and protein concentration measurements from EVs stored at ‐20˚C in PBS for up to 26 weeks (n = 3; RNA data not available). (E) Results from EVs stored at ‐80˚C in PBS for up to 26 weeks (n = 6). (F) Results from TFF/SEC‐purified EVs stored at ‐20˚C or ‑80˚C in PBS for 1 week, 4 weeks or 12 weeks (n = 3). Statistical significance was assessed by one‐way ANOVA followed by Dunnet's posthoc tests. All p‐values are referring to fresh values (*:P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001; mean±SD; d: days; wks: weeks; n.d.: not detected))

FIGURE 2

Sample concentrations measured by NTA after EV storage in different candidate buffers. (A) EVs were enriched from MSC or HEK293T:CD63eGFP conditioned medium by TFF/UF, diluted 10x in different buffer formulations and stored at +4˚C, ‐20˚C or ‐80˚C. NTA particle counts were measured as surrogate read‐out for EV stability freshly and after 5 and 15 weeks of storage. NTA‐based concentrations are provided for HEK293T:CD63eGFP EVs stored at ‐20˚C (B) and ‐80˚C (C). Most HEK293T:CD63eGFP EV aliquots stored at +4˚C showed signs of contamination and were excluded from the analysis (not shown). (D‐F) Particle concentrations for MSC EVs stored at +4˚C (D), ‐20˚C (E) and ‐80˚C (F)

FIGURE 3

Stage 3 candidate buffer formulation overview and experimental outline. (A) Candidate storage buffer formulations, including PBS and Buffers 2, 3, 4, 5, and 10 from Stage 2 as well as two new variations (PBS‐HA and NaCl‐HA) and the commercial buffer ‘Exocap Storage Booster’ (ExoCap). Additives were added at the same concentrations as for Stage 2 (HSA 0.2 %; Trehalose 25 mM; HEPES 25 mM; DMSO 1%). (B) Summary of experimental outline for Stage 3

FIGURE 4

Sample concentrations measured by NTA after EV storage in different candidate Stage 3 buffers. (A) Engineered EVs were isolated from MSC:TNFR1 or HEK293T:CD63mNG conditioned medium by TFF/UF and subsequent BE‐SEC, diluted 10x in nine different buffer formulations and stored at +4˚C, ‐20˚C or ‐80˚C. (B) NTA particle concentrations measured freshly (dotted line) and after 6, 12 and 20 weeks of storage

FIGURE 5

Using fluorescence‐tagged EVs to evaluate stability in various buffers. (A) HEK293T:CD63mNG engineered fluorescent EVs were stored in respective buffers for 6, 12, and 20 weeks at +4˚C, ‐20˚C or ‐80˚C. Equal volumes from aliquots were subjected to bulk fluorescence measurements (B; n = 2; mean±SD) or cellular uptake assays (C; dotted line indicates fold change values obtained for fresh samples)

FIGURE 6

Inhibitory functionality of engineered EVs displaying TNF‐α binding decoy receptors. (A) MSC:TNFR1 engineered decoy EVs were stored in respective buffers for 6, 12, and 20 weeks at +4˚C, ‐20˚C or ‐80˚C. (B) Equal volumes from aliquots were evaluated for TNF‐α decoy in a reporter cell assay responsive to TNF‐α induced NF‐κB activation. Data were normalized to cells treated with TNF‐α only (no EVs; 5 ng/ml; n = 2; mean±SD)

FIGURE 7

Analysis of EVs stored in selected buffers by EM, WB and multiplex bead‐based flow cytometry. (A) MSC:TNFR1 engineered decoy EVs or HEK293T:CD63mNG derived EVs were stored in respective buffers 6–20 weeks at +4˚C, ‐20˚C or ‐80˚C before analysis. (B) Representative EM images of MSC:TNFR1 EVs after storage for 20 weeks. (C) Western blot analysis (anti‐Syntenin) of equal volumes of HEK293T:CD63mNG EVs post storage for 20 weeks. Entire blot provided in Figure S3. Other EV‐related proteins were tested but below detection limit for all samples. (D) Multiplex bead‐based flow cytometry analysis of EVs stored for 6–12 weeks. A mixture of APC‐labelled anti‐CD9, CD63 and CD81 antibodies was used for detection. Background subtracted APC MFIs are shown. Equal volumes of 60 μl EV suspension from each aliquot were taken as assay input

FIGURE 8

RNA stability is severely affected when EVs are stored in PBS. (A) RNA was analysed from HEK293T:CD63mNG derived EVs stored for 20 weeks. No data for fresh samples available. (B) Representative Agilent Bioanalyzer small RNA profiles and quantification of small RNA (5‐200 bp, white) and miRNA (10‐40, grey) concentrations of selected EV samples stored under different conditions

FIGURE 9

NTA analysis of EV samples stored in different candidate buffers after 2 years. (A) Measured particle concentration in MSC‐TNFR EV aliquots. (B) Measured particle concentration in HEK293T:CD63mNG EV aliquots. (C) Particle diameters measured for MSC:TNFR1 EV aliquots. (D) Particle diameters measured for HEK293T:CD63mNG EV aliquots. All data is presented without normalization to freshly measured samples due to expected instrument performance variation after 2 years. Size distribution graphs are available in Figures S7 and S8

FIGURE 10

Using fluorescence‐tagged EVs to evaluate stability in various buffers after 2 years. (A) HEK293T:CD63mNG engineered fluorescent EVs were stored in respective buffers for 2 years at +4˚C, ‐20˚C or ‐80˚C. Equal volumes from aliquots were subjected to bulk fluorescence measurements (B; n = 2; mean±SD) or cellular uptake assays (C)

FIGURE 11

Analysis of EVs stored for 2 years in selected buffers by multiplex bead‐based flow cytometry. Multiplex bead‐based flow cytometry analysis of EVs stored for 6–12 weeks. A mixture of APC‐labelled anti‐CD9, CD63 and CD81 antibodies was used for detection. Background subtracted APC MFIs are shown for selected positive surface markers. Equal volumes of 60 μl EV suspension from each aliquot were taken as assay input. Full profiles are available as Figure S9

FIGURE 12

Analysis of HEK293T:CD63mNG EV RNA after 2 years of storage under various conditions. (A) Representative Agilent Bioanalyzer small RNA profiles following storage under different conditions. (B) Quantification of small RNA (white) and miRNA (grey) concentrations after storage under different storage conditions. (C) Representative Agilent Bioanalyzer HS DNA profiles of cDNA produced from RNA stored under different conditions. (D) The ratio of cDNA concentration 50–600 bp to that 0.6 to 10 kb from the cDNA profiles such as those presented in C was used to calculate mRNA stability under the different storage conditions (n = 3, mean±SD; *:P < 0.05). (E) Quantification of full‐length cDNA from C (n.s.: P > 0.05). No values for fresh samples available. Statistical significance was assessed by one‐way ANOVA followed by Dunnet's posthoc tests

FIGURE 13

EV quantification by high resolution imaging flow cytometry (IFCM) after 2 years of storage. (A) EV samples after 2 years of storage in respective candidate buffers were subjected to IFCM analysis. Equal volumes from each aliquot were used accordingly. (B) IFCM analysis of fluorescently tagged HEK293T:CD63mNG EVs. Samples were diluted 500 fold in PBS‐HAT before measurement. (C) 25 μl of MSC:TNFR1 EVs were incubated over‐night with 8 nM of APC‐labelled anti‐CD9, anti‐CD63 and anti‐CD81 antibodies, respectively. Samples were diluted 1000 fold in PBS‐HAT before data acquisition. Controls, gating strategy and dotplots for all samples are available as Figure S10. Further details provided in Figure Tables S1 and S2. AF: autofluorescence

FIGURE 14

Evaluation of the impact of diluent, tube type and freeze/thaw cycles on EV recovery. (A) Fluorescently tagged HEK293‐Freestyle EVs (HEK293FS:CD63mNG) were diluted 200,000‐fold in either PBS, PBS‐A, or PBS‐HAT buffer and 10 replicates were acquired by IFCM for 180 s directly afterwards, respectively. Measured concentrations and fluorescence intensities are given in bar graphs, with replicate one measured first and replicate 10 measured last, respectively. (B) IFCM‐based quantification of recovery of HEK293FS:CD63mNG EVs following storage of samples diluted 100,000 fold in PBS or PBS‐HAT and stored for 7 days at +4°C in different tubes (n = 3). (C) HEK293FS:CD63mNG EVs measured freshly and after 1–5 freeze/thaw cycles by IFCM (n = 3). All data is expressed as mean±SD (n.s.: P > 0.05; ***: P < 0.0001; statistical significance was assessed by one‐way ANOVA followed by Tukey's posthoc tests.). AF: autofluorescence. Further details and supplemental data provided in Figures S11‐S13, and Tables S1 and S2