Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4 degrees C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4 degrees C, or UC performed at 37 degrees C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield

Hemokinin-1 Gene Expression Is Upregulated in Trigeminal Ganglia in an Inflammatory Orofacial Pain Model: Potential Role in Peripheral Sensitization

A large percentage of primary sensory neurons in the trigeminal ganglia (TG) contain neuropeptides such as tachykinins or calcitonin gene-related peptide. Neuropeptides released from the central terminals of primary afferents sensitize the secondary nociceptive neurons in the trigeminal nucleus caudalis (TNC), but also activate glial cells contributing to neuroinflammation and consequent sensitization in chronic orofacial pain and migraine. In the present study, we investigated the newest member of the tachykinin family, hemokinin-1 (HK-1) encoded by the Tac4 gene in the trigeminal system. HK-1 had been shown to participate in inflammation and hyperalgesia in various models, but its role has not been investigated in orofacial pain or headache. In the complete Freund's adjuvant (CFA)-induced inflammatory orofacial pain model, we showed that Tac4 expression increased in the TG in response to inflammation. Duration-dependent Tac4 upregulation was associated with the extent of the facial allodynia. Tac4 was detected in both TG neurons and satellite glial cells (SGC) by the ultrasensitive RNAscope in situ hybridization. We also compared gene expression changes of selected neuronal and glial sensitization and neuroinflammation markers between wild-type and Tac4-deficient (Tac4-/-) mice. Expression of the SGC/astrocyte marker in the TG and TNC was significantly lower in intact and saline/CFA-treated Tac4-/- mice. The procedural stress-related increase of the SGC/astrocyte marker was also strongly attenuated in Tac4-/- mice. Analysis of TG samples with a mouse neuroinflammation panel of 770 genes revealed that regulation of microglia and cytotoxic cell-related genes were significantly different in saline-treated Tac4-/- mice compared to their wild-types. It is concluded that HK-1 may participate in neuron-glia interactions both under physiological and inflammatory conditions and mediate pain in the trigeminal system

Identification of Disease- and Headache-Specific Mediators and Pathways in Migraine Using Blood Transcriptomic and Metabolomic Analysis

Recent data suggest that gene expression profiles of peripheral white blood cells can reflect changes in the brain. We aimed to analyze the transcriptome of peripheral blood mononuclear cells (PBMC) and changes of plasma metabolite levels of migraineurs in a self-controlled manner during and between attacks.Twenty-four patients with migraine were recruited and blood samples were collected in a headache-free (interictal) period and during headache (ictal) to investigate disease- and headache-specific alterations. Control samples were collected from 13 age- and sex-matched healthy volunteers. RNA was isolated from PBMCs and single-end 75 bp RNA sequencing was performed using Illumina NextSeq 550 instrument followed by gene-level differential expression analysis. Functional analysis was carried out on information related to the role of genes, such as signaling pathways and biological processes. Plasma metabolomic measurement was performed with the Biocrates MxP Quant 500 Kit.We identified 144 differentially-expressed genes in PBMCs between headache and headache-free samples and 163 between symptom-free patients and controls. Network analysis revealed that enriched pathways included inflammation, cytokine activity and mitochondrial dysfunction in both headache and headache-free samples compared to controls. Plasma lactate, succinate and methionine sulfoxide levels were higher in migraineurs while spermine, spermidine and aconitate were decreased during attacks.It is concluded that enhanced inflammatory and immune cell activity, and oxidative stress can play a role in migraine susceptibility and headache generation

Efficiency of ultracentrifugation (UC) methods under various conditions.

<p>(A) Scheme of serial UC of the supernatant. Exosomal marker CD63 and TSG101 and albumin content of exosomal isolates after serial UC of the supernatant with 1h UC at 4°C (B; n = 3; p>0.05), with 10-fold dilution at 4°C (C, top), with smaller volume of loaded sample at 4°C (C, middle) and with 1h UC at 37°C (C, bottom) as evaluated with Western blot.</p

Efficiency of ultracentrifugation (UC) methods under various conditions.

<p>(A) Scheme of serial UC of the supernatant. Exosomal marker CD63 and TSG101 and albumin content of exosomal isolates after serial UC of the supernatant with 1h UC at 4°C (B; n = 3; p>0.05), with 10-fold dilution at 4°C (C, top), with smaller volume of loaded sample at 4°C (C, middle) and with 1h UC at 37°C (C, bottom) as evaluated with Western blot.</p

Size exclusion chromatography isolation of exosomes from rat blood plasma on a large-scale Sephacryl S-400 column.

<p>(A) Particle concentration and protein content (absorbance at 280 nm) of eluted fractions. (B) Concentration and size distribution of particles in pooled fractions by nanotracking analysis. (C) CD63, TSG101 and albumin content of pooled</p

Efficiency and selectivity of size exclusion chromatography (SEC) on exosome isolation performed with various matrices.

<p>(A) CD63, TSG101 and albumin content of different fractions collected during SEC on Sepharose 2B (top), Sepharose CL-4B (middle) and Sephacryl S-400 (bottom) columns with equal volumes (left column) or equal protein amounts of fractions (right column) loaded for Western blot. (B) Size distribution of particles isolated with various SEC matrices evaluated with dynamic light scattering. Sepharose 2B (top), Sepharose CL-4B (middle) and Sephacryl S-400 (bottom) columns.</p

Exosomes can be isolated with 1h ultracentrifugation method from blood plasma.

<p>(A) Transmission electron microscopy images of exosome isolates from rat and human blood plasma. (B) CD63, TSG101 and albumin content of the rat and human exosomal isolates as evaluated with Western blot. (C) Size distribution of particles isolated from rat and human blood plasma analyzed with dynamic light scattering (averages of n = 3–4).</p

The effect of various ultracentrifugation (UC) duration on the exosomal yield and purity.

<p>(A) Transmission electron microscopy images of rat exosomes isolated with 1, 3, 6 or 14h UC period. (B) Size distribution of rat exosomes based on transmission electron microscopy image analysis (1h UC: n = 2,440; 3h UC: n = 353). (C) Size distribution of particles isolated with different UC duration evaluated with dynamic light scattering (averages of n = 3). (D) Protein concentration of exosome homogenates isolated with different UC duration as assessed with bicinchoninic acid assay (n = 3–7; *, #, &: p<0.05 vs. other three groups). (E) CD63 and albumin content of exosome isolates with different UC length (n = 3; *, #, &: p<0.05 vs. corresponding other three groups; §: p<0.05 vs. 1h).</p