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Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles.

ABSTRACT: Extracellular vesicles (EVs) play a significant role in cell-cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

SUBMITTER: Li J

PROVIDER: S-EPMC4447833 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles.

Li Jinghuan J Lee Yi Y Johansson Henrik J HJ Mäger Imre I Vader Pieter P Nordin Joel Z JZ Wiklander Oscar P B OP Lehtiö Janne J Wood Matthew J A MJ Andaloussi Samir El SE

Journal of extracellular vesicles 20150527

Extracellular vesicles (EVs) play a significant role in cell-cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblasto ...[more]

PMID: 26022510

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Project description:Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-WheelTM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGROTM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between -15.5 ± 1.6 mV and -19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 1011, 3.1 ± 1.3 × 1011, and 4.1 ± 1.7 × 1011 EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.

Project description:The zebrafish (Danio rerio) is a valuable model organism for studying human biology due to its easy genetic manipulation and small size. It is optically transparent and shares genetic similarities with humans, making it ideal for studying developmental processes, diseases, and drug screening via imaging-based approaches. Solid malignant tumors often contain hypoxic areas that stimulate the release of extracellular vesicles (EVs), lipid-bound structures released by cells into the extracellular space, that facilitate short- and long-range intercellular communication and metastatization. Here we investigate the effects of EVs derived from neuroblastoma (NB), a pediatric solid tumor, on metastatic niche formation using the zebrafish as an in vivo model. Intravascular injection in zebrafish embryos allows a non-invasive visualization of EVs dispersion, uptake, and interactions with host cells. To improve repeatability of our results and ease the injection steps, we used an agarose device replica molded from a custom designed micromilled aluminum mold. We first demonstrated that EVs released under hypoxic conditions promote angiogenesis and are more easily internalized by endothelial cells than those purified from normoxic cells. We also showed that injection of with hypoxic EVs increased macrophages mobilization. We then focused on the caudal hematopoietic tissue (CHT) region of the embryo as a potential metastatic site. After hypoxic EVs injection, we highlighted changes in the expression of mmp-9 and cxcl8b genes. Furthermore, we investigated the ability of NB-derived EVs to prime a metastatic niche by a two-step injection of EVs first, followed by NB cells. Interestingly, we found that embryos injected with hypoxic EVs had more proliferating NB cells than those injected with normoxic EVs. Our findings suggest that EVs released by hypoxic NB cells alter the behavior of recipient cells in the zebrafish embryo and promote metastatic outgrowth. In addition, we demonstrated the ability of the zebrafish embryo to be a suitable model for studying the interactions between EVs and recipient cells in the metastatic process.

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Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles.

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Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles.

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