Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

Publisher Copyright: © 2022Background: Extracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge. Objective: To probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells. Methodology: First, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM). Results: A peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield. Conclusions: We demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.Peer reviewe

Proteomics of Deep Cervical Lymph Nodes after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) damages the glymphatic-lymphatic system. We hypothesized that brain injury associated with trauma results in the enrichment of brain-relevant proteins in deep cervical lymph nodes (DCLNs), the end station of meningeal lymphatic vessels, and that some of these proteins will present mechanistic tissue biomarkers for TBI. Proteomes of rat DCLNs were investigated in the left DCLN (ipsilateral to injury) and right DCLN at 6.5 months after severe TBI induced by lateral fluid percussion injury or after sham operation. DCLN proteomes were identified using sequential window acquisition of all theoretical mass spectra. Group comparisons, together with functional protein annotation analyses, were used to identify regulated protein candidates for further validation and pathway analyses. Validation of a selected candidate was assessed using enzyme-linked immunosorbent assay. Analysis comparing post-TBI animals with sham-operated controls revealed 25 upregulated and 16 downregulated proteins in the ipsilateral DCLN and 20 upregulated and 28 downregulated proteins in the contralateral DCLN of post-TBI animals. Protein class and function analyses highlighted the dysregulation of enzymes and binding proteins. Pathway analysis indicated an increase in autophagy. Biomarker analysis suggested that a subgroup of post-TBI animals had an increase in zonula occludens-1 coexpressed with proteins linked to molecular transport and amyloid precursor protein. We propose here that, after TBI, a subgroup of animals exhibit dysregulation of the TBI-relevant protein interactome in DCLNs, and that DCLNs might thus serve as an interesting biomarker source in future studies aiming to elucidate pathological brain functioning.Peer reviewe

Western blot characterization of exosomes isolated from rat plasma and evaluation of the efficiency of a precipitation method in exosome isolation

Eksosomit ovat halkaisijaltaan 40-100 nm kokoisia solunulkoisia vesikkeleitä, joita solut erittävät solunsisäisen multivesikulaarisen rakkulan yhdistyessä solukalvoon. Eksosomit ovat tärkeässä roolissa solujen välisessä viestinnässä, sillä ne kuljettavat monenlaisia biomolekyylejä, kuten useita eri RNA-tyyppejä, lipidejä ja proteiineja. Aivoissa eksosomit liittyvät neurodegeneratiivisiin sairauksiin, kuten Parkisonin tautiin, Alzheimerin tautiin ja aivovammaan. Eksosomit kykenevät läpäisemään veri-aivoesteen, ja tutkijat ovatkin esittäneet paljon kiinnostusta eksosomien mahdollisuuksiin uutena hoitokeinona. Lisäksi eksosomien kuljettamat molekyylit sisältävät tietoa eksosomit erittäneen solun toiminnoista, minkä ansiosta ne voisivat mahdollisesti toimia biomarkkereina eri sairauksille. Eksosomeja voidaan puhdistaa usealla tavalla, esimerkiksi saostamalla, ultrafuugaamalla ja pylväskromatografialla. Puhdistuksen onnistumisen toteamiseksi suositellaan eksosomien karakterisointia ISEV:n (International Society for Extracellular Vesicles) asettamien suuntaviivojen mukaisesti. Tämän tutkimuksen tavoitteet olivat 1) Western blot -menetelmän pystyttäminen rotan plasmanäytteille 2) Western blot menetelmän pystyttäminen eksosomien karakterisointia varten 3) saostusmenetelmän tehokkuuden arviointi eksosomipuhdistuksessa rotan plasmasta. Western blot -menetelmän pystyttämiseen sisältyi protokollan ja karakterisoinnissa käytettävien vasta-aineiden optimointi. Vasta-aineilla tunnistettavat proteiinit olivat Alix (vesikkelimarkkeri), kalneksiini (solukontaminaatiomarkkeri) ja albumiini (plasmaproteiinien kontaminaatiomarkkeri). Saostusmenetelmän tehokkuus arvioitiin usealla menetelmällä (NTA, SDS-PAGE, Western blot). Anti-Alix, anti-kalneksiini ja anti-albumiini vasta-aineiden optimointi onnistui, mutta eksosomimarkkeriproteiini CD63:n ja flotilliini-1:n tunnistavia vasta-aineita ei saatu toimimaan. Anti-CD63 ja anti-flotilliini-1 eivät luultavasti toimineet ollenkaan, sillä niillä ei saatu signaalia edes positiivikontrollista. Tutkimuksessa huomattiin, että sekundaarivasta-aineet reagoivat vahvasti rotan plasmassa esiintyvien immunoglobuliinien kanssa, mikä aiheutti ylimääräisiä vyöhykkeitä Western blotissa. Eksosomien karakterisointi paljasti, että eristystuote sisälsi eksosomeja, mutta myös paljon albumiinia. NTA:n tulokset näyttivät, että suurempien partikkelien osuus kasvoi, kun taas 60 nanometriä pienempien partikkelien (proteiinit ja lipoproteiinit) osuus pieneni selkeästi eristysprosessin aikana. Arvioinnin muut tulokset osoittivat, että saostusmenetelmä onnistui poistamaan noin 60 % plasmaproteiineista, joista albumiini on kaikista yleisin. Tutkimuksessa onnistuttiin pystyttämään Western blot -menetelmä rotan plasmanäytteiden karakterisoinnille. Lisäksi havaittiin, että saostusmenetelmä ennemmin rikasti kuin puhdisti eksosomeja, sillä eksosomien ohella saostui paljon plasman proteiineja. Tulevaisuudessa olisi kannattavaa yrittää parantaa eksosomien eristystä kokeilemalla jotakin toista puhdistusmenetelmää, esimerkiksi pylväskromatografiaa.Exosomes are 40–100 nm membrane-enclosed extracellular vesicles (EVs) secreted outside the cell by the fusion of multivesicular bodies with the plasma membrane. Exosomes play an important role in intercellular signaling by carrying different biomolecules, such as several types of RNA (mRNA, miRNA and other non-coding RNA), lipids and proteins. In the brain, exosomes are involved in neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease and traumatic brain injury (TBI). Exosomes can pass the blood-brain barrier, and there has been a growing interest in the therapeutic potential of exosomes. Also, the contents of exosomes carry information about the ongoing processes in their cell of origin, which makes the exosome cargo, for example miRNAs, potential disease biomarkers. To claim the presence of exosomes in the isolate, they are recommended to be characterized according to the guidelines of International Society for Extracellular Vesicles (ISEV). The aims of this study were 1) to set up a Western blot method for samples from rat plasma 2) to set up a Western blot method for characterization of exosomes according to the ISEV guidelines 3) evaluate the efficiency of the precipitation method in exosome isolation from rat plasma. The set-up of the Western blot method included optimization of the protocol and the antibodies, which were then used in exosome characterization for detection of Alix (EV marker), calnexin (cell contamination marker) and albumin (plasma protein contamination marker). Efficiency of the exosome precipitation was analysed by nanoparticle tracking analysis (NTA), SDS-PAGE and Western blot. It was succeeded to optimize the anti-Alix, anti-calnexin and anti-albumin primary antibodies. However, finding working conditions for antibodies targeting other exosome marker proteins (CD63 and flotillin-1) failed, possibly because of unfunctional antibodies. In addition, the secondary antibodies were observed to strongly cross-react with the immunoglobulins naturally present in rat plasma, which caused unwanted bands in Western blot. Characterization confirmed the presence of exosomes, however, also the vast amount of contaminating plasma proteins in the exosome isolate. NTA results indicated that the share of larger particles increased during the isolation process, whereas the share of <60 nm particles (proteins and lipoproteins) clearly decreased. The results of the evaluation of the precipitation method efficiency by SDS-PAGE and Western blot indicated that the method removed ~60 % of the contaminating plasma proteins, of which albumin is the most abundant. In conclusion, it was succeeded to establish a Western blot characterization method for rat plasma samples. It was also concluded that the precipitation method enriched exosomes rather than purified them, since a lot of plasma proteins were coisolated with exosomes. To improve the isolation of exosomes from plasma in future studies, use of alternative isolation methods i.e. size-exclusion chromatography should be investigated

Plasma Neurofilament Light Chain (NF-L) Is a Prognostic Biomarker for Cortical Damage Evolution but Not for Cognitive Impairment or Epileptogenesis Following Experimental TBI

Plasma neurofilament light chain (NF-L) levels were assessed as a diagnostic biomarker for traumatic brain injury (TBI) and as a prognostic biomarker for somatomotor recovery, cognitive decline, and epileptogenesis. Rats with severe TBI induced by lateral fluid-percussion injury (n = 26, 13 with and 13 without epilepsy) or sham-operation (n = 8) were studied. During a 6-month follow-up, rats underwent magnetic resonance imaging (MRI) (day (D) 2, D7, and D21), composite neuroscore (D2, D6, and D14), Morris-water maze (D35&ndash;D39), and a 1-month-long video-electroencephalogram to detect unprovoked seizures during the 6th month. Plasma NF-L levels were assessed using a single-molecule assay at baseline (i.e., na&iuml;ve animals) and on D2, D9, and D178 after TBI or a sham operation. Plasma NF-L levels were 483-fold higher on D2 (5072.0 &plusmn; 2007.0 pg/mL), 89-fold higher on D9 (930.3 &plusmn; 306.4 pg/mL), and 3-fold higher on D176 32.2 &plusmn; 8.9 pg/mL after TBI compared with baseline (10.5 &plusmn; 2.6 pg/mL; all p &lt; 0.001). Plasma NF-L levels distinguished TBI rats from na&iuml;ve animals at all time-points examined (area under the curve [AUC] 1.0, p &lt; 0.001), and from sham-operated controls on D2 (AUC 1.0, p &lt; 0.001). Plasma NF-L increases on D2 were associated with somatomotor impairment severity (&rho; = &minus;0.480, p &lt; 0.05) and the cortical lesion extent in MRI (&rho; = 0.401, p &lt; 0.05). Plasma NF-L increases on D2 or D9 were associated with the cortical lesion extent in histologic sections at 6 months post-injury (&rho; = 0.437 for D2; &rho; = 0.393 for D9, p &lt; 0.05). Plasma NF-L levels, however, did not predict somatomotor recovery, cognitive decline, or epileptogenesis (p &gt; 0.05). Plasma NF-L levels represent a promising noninvasive translational diagnostic biomarker for acute TBI and a prognostic biomarker for post-injury somatomotor impairment and long-term structural brain damage

Extracellular Vesicles as Diagnostics and Therapeutics for Structural Epilepsies

Extracellular vesicles (EVs) are small vesicles involved in intercellular communication. Data is emerging that EVs and their cargo have potential as diagnostic biomarkers and treatments for brain diseases, including traumatic brain injury and epilepsy. Here, we summarize the current knowledge regarding changes in EV numbers and cargo in status epilepticus (SE) and traumatic brain injury (TBI), which are clinically significant etiologies for acquired epileptogenesis in animals and humans. We also review encouraging data, which suggests that EVs secreted by stem cells may serve as recovery-enhancing treatments for SE and TBI. Using Gene Set Enrichment Analysis, we show that brain EV-related transcripts are positively enriched in rodent models of epileptogenesis and epilepsy, and altered in response to anti-seizure drugs. These data suggest that EVs show promise as biomarkers, treatments and drug targets for epilepsy. In parallel to gathering conceptual knowledge, analytics platforms for the isolation and analysis of EV contents need to be further developed

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

Publisher Copyright: © 2022Background: Extracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge. Objective: To probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells. Methodology: First, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM). Results: A peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield. Conclusions: We demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.Peer reviewe

Plasma miR-9-3p and miR-136-3p as Potential Novel Diagnostic Biomarkers for Experimental and Human Mild Traumatic Brain Injury

Noninvasive, affordable circulating biomarkers for difficult-to-diagnose mild traumatic brain injury (mTBI) are an unmet medical need. Although blood microRNA (miRNA) levels are reportedly altered after traumatic brain injury (TBI), their diagnostic potential for mTBI remains inconclusive. We hypothesized that acutely altered plasma miRNAs could serve as diagnostic biomarkers both in the lateral fluid percussion injury (FPI) model and clinical mTBI. We performed plasma small RNA-sequencing from adult male Sprague–Dawley rats (n = 31) at 2 days post-TBI, followed by polymerase chain reaction (PCR)-based validation of selected candidates. miR-9a-3p, miR-136-3p, and miR-434-3p were identified as the most promising candidates at 2 days after lateral FPI. Digital droplet PCR (ddPCR) revealed 4.2-, 2.8-, and 4.6-fold elevations in miR-9a-3p, miR-136-3p, and miR-434-3p levels (p n = 7/15) and miR-136-3p (n = 5/15) levels higher than one standard deviation above the control mean at <2 days postinjury. In sTBI patients, plasma miR-9-3p levels were 6.5- and 9.2-fold in comparison to the mTBI and control groups, respectively. Thus, plasma miR-9-3p and miR-136-3p were identified as promising biomarker candidates for mTBI requiring further evaluation in a larger patient population

Plasma miR-9-3p and miR-136-3p as Potential Novel Diagnostic Biomarkers for Experimental and Human Mild Traumatic Brain Injury

Noninvasive, affordable circulating biomarkers for difficult-to-diagnose mild traumatic brain injury (mTBI) are an unmet medical need. Although blood microRNA (miRNA) levels are reportedly altered after traumatic brain injury (TBI), their diagnostic potential for mTBI remains inconclusive. We hypothesized that acutely altered plasma miRNAs could serve as diagnostic biomarkers both in the lateral fluid percussion injury (FPI) model and clinical mTBI. We performed plasma small RNA-sequencing from adult male Sprague–Dawley rats (n = 31) at 2 days post-TBI, followed by polymerase chain reaction (PCR)-based validation of selected candidates. miR-9a-3p, miR-136-3p, and miR-434-3p were identified as the most promising candidates at 2 days after lateral FPI. Digital droplet PCR (ddPCR) revealed 4.2-, 2.8-, and 4.6-fold elevations in miR-9a-3p, miR-136-3p, and miR-434-3p levels (p &lt; 0.01 for all), respectively, distinguishing rats with mTBI from naïve rats with 100% sensitivity and specificity. DdPCR further identified a subpopulation of mTBI patients with plasma miR-9-3p (n = 7/15) and miR-136-3p (n = 5/15) levels higher than one standard deviation above the control mean at &lt;2 days postinjury. In sTBI patients, plasma miR-9-3p levels were 6.5- and 9.2-fold in comparison to the mTBI and control groups, respectively. Thus, plasma miR-9-3p and miR-136-3p were identified as promising biomarker candidates for mTBI requiring further evaluation in a larger patient population