Emerging technologies for quality control of cell-based, advanced therapy medicinal products.

Advanced therapy medicinal products (ATMP) are complex medicines based on gene therapy, somatic cell therapy, and tissue engineering. These products are rapidly arising as novel and promising therapies for a wide range of different clinical applications. The process for the development of well-established ATMPs is challenging. Many issues must be considered from raw material, manufacturing, safety, and pricing to assure the quality of ATMPs and their implementation as innovative therapeutic tools. Among ATMPs, cell-based ATMPs are drugs altogether. As for standard drugs, technologies for quality control, and non-invasive isolation and production of cell-based ATMPs are then needed to ensure their rapidly expanding applications and ameliorate safety and standardization of cell production. In this review, emerging approaches and technologies for quality control of innovative cell-based ATMPs are described. Among new techniques, microfluid-based systems show advantages related to their miniaturization, easy implementation in analytical process and automation which allow for the standardization of the final product.

Mitigating Oxidative Stress in Perinatal Cells: A Critical Step toward an Optimal Therapeutic Use in Regenerative Medicine.

Oxidative stress (OS) occurs when the production of reactive oxygen species (ROS) is not balanced by the body's antioxidant defense system. OS can profoundly affect cellular health and function. ROS can have a profound negative impact on cells that undergo a predestined and time-regulated process of proliferation or differentiation, such as perinatal stem cells. Due to the large-scale employment of these immunotolerant stem cells in regenerative medicine, it is important to reduce OS to prevent them from losing function and increase their application in the regenerative medicine field. This goal can be achieved through a variety of strategies, such as the use of antioxidants and other compounds that can indirectly modulate the antioxidant defense system by enhancing cellular stress response pathways, including autophagy and mitochondrial function, thereby reducing ROS levels. This review aims to summarize information regarding OS mechanisms in perinatal stem cells and possible strategies for reducing their deleterious effects.

Plumbagin, a Natural Compound with Several Biological Effects and Anti-Inflammatory Properties.

Phytochemicals from various medicinal plants are well known for their antioxidant properties and anti-cancer effects. Many of these bioactive compounds or natural products have demonstrated effects against inflammation, while some showed a role that is only approximately described as anti-inflammatory. In particular, naphthoquinones are naturally-occurring compounds with different pharmacological activities and allow easy scaffold modification for drug design approaches. Among this class of compounds, Plumbagin, a plant-derived product, has shown interesting counteracting effects in many inflammation models. However, scientific knowledge about the beneficial effect of Plumbagin should be comprehensively reported before candidating this natural molecule into a future drug against specific human diseases. In this review, the most relevant mechanisms in which Plumbagin plays a role in the process of inflammation were summarized. Other relevant bioactive effects were reviewed to provide a complete and compact scenario of Plumbagin's potential therapeutic significance.

Non-Permissive Parvovirus B19 Infection: A Reservoir and Questionable Safety Concern in Mesenchymal Stem Cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells with differentiation, immunoregulatory and regenerative properties. Because of these features, they represent an attractive tool for regenerative medicine and cell-based therapy. However, MSCs may act as a reservoir of persistent viruses increasing the risk of failure of MSCs-based therapies and of viral transmission, especially in immunocompromised patients. Parvovirus B19V (B19V) is a common human pathogen that infects bone marrow erythroid progenitor cells, leading to transient or persistent anemia. Characteristics of B19V include the ability to cross the placenta, infecting the fetus, and to persist in several tissues. We thus isolated MSCs from bone marrow (BM-MSCs) and fetal membrane (FM-MSCs) to investigate their permissiveness to B19V infection. The results suggest that both BM- and FM- MSCs can be infected by B19V and, while not able to support viral replication, allow persistence over time in the infected cultures. Future studies are needed to understand the potential role of MSCs in B19V transmission and the conditions that can favor a potential reactivation of the virus.

Characterization of Perinatal Stem Cell Spheroids for the Development of Cell Therapy Strategy.

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton's jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM.

The Medium Obtained from the Culture of Hodgkin Lymphoma Cells Affects the Biophysical Characteristics of a Fibroblast Cell Model.

The neoplastic Hodgkin-Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) represent only 1-10% of cells and are surrounded by an inflammatory microenvironment. The HL cytokine network is a key point for the proliferation of HRS cells and for the maintenance of an advantageous microenvironment for HRS survival. In the tumor microenvironment (TME), the fibroblasts are involved in crosstalk with HRS cells. The aim of this work was to study the effect of lymphoma cell conditioned medium on a fibroblast cell population and evaluate modifications of cell morphology and proliferation. Hodgkin lymphoma-derived medium was used to obtain a population of "conditioned" fibroblasts (WS-1 COND). Differences in biophysical parameters were detected by the innovative device Celector®. Fibroblast-HL cells interactions were reproduced in 3D co-culture spheroids. WS-1 COND showed a different cellular morphology with an enlarged cytoplasm and enhanced metabolism. Area and diameter cell values obtained by Celector® measurement were increased. Co-culture spheroids created with WS-1 COND showed a tighter aggregation than those with non-conditioned WS-1. The presence of soluble factors derived from HRS cells in the conditioned medium was adequate for the proliferation of fibroblasts and conditioned fibroblasts in a 3D HL model allowed to develop a representative model of the in vivo TME.

Perinatal Stem Cell Therapy to Treat Type 1 Diabetes Mellitus: A Never-Say-Die Story of Differentiation and Immunomodulation.

Human term placenta and other postpartum-derived biological tissues are promising sources of perinatal cells with unique stem cell properties. Among the massive current research on stem cells, one medical focus on easily available stem cells is to exploit them in the design of immunotherapy protocols, in particular for the treatment of chronic non-curable human diseases. Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells and perinatal cells can be harnessed both to generate insulin-producing cells for beta cell replenishment and to regulate autoimmune mechanisms via immunomodulation capacity. In this study, the strong points of cells derived from amniotic epithelial cells and from umbilical cord matrix are outlined and their potential for supporting cell therapy development. From a basic research and expert stem cell point of view, the aim of this review is to summarize information regarding the regenerative medicine field, as well as describe the state of the art on possible cell therapy approaches for diabetes.

Quality Control Platform for the Standardization of a Regenerative Medicine Product.

Adipose tissue is an attractive source of stem cells due to its wide availability. They contribute to the stromal vascular fraction (SVF), which is composed of pre-adipocytes, tissue-progenitors, and pericytes, among others. Because its direct use in medical applications is increasing worldwide, new quality control systems are required. We investigated the ability of the Non-Equilibrium Earth Gravity Assisted Dynamic Fractionation (NEEGA-DF) method to analyze and separate cells based solely on their physical characteristics, resulting in a fingerprint of the biological sample. Adipose tissue was enzymatically digested, and the SVF was analyzed by NEEGA-DF. Based on the fractogram (the UV signal of eluting cells versus time of analysis) the collection time was set to sort alive cells. The collected cells (F-SVF) were analyzed for their phenotype, immunomodulation ability, and differentiation potential. The SVF profile showed reproducibility, and the alive cells were collected. The F-SVF showed intact adhesion phenotype, proliferation, and differentiation potential. The methodology allowed enrichment of the mesenchymal component with a higher expression of mesenchymal markers and depletion of debris, RBCs, and an extracellular matrix still present in the digestive product. Moreover, cells eluting in the last minutes showed higher circularity and lower area, proving the principles of enrichment of a more homogenous cell population with better characteristics. We proved the NEEGA-DF method is a "gentle" cell sorter that purifies primary cells obtained by enzymatic digestion and does not alter any stem cell function.

A Tailored Lipid Supplement Restored Membrane Fatty Acid Composition and Ameliorates In Vitro Biological Features of Human Amniotic Epithelial Cells.

Cell culture conditions influence several biological and biochemical features of stem cells (SCs), including the membrane lipid profile, thus limiting the use of SCs for cell therapy approaches. The present study aims to investigate whether the in vitro culture may alter the membrane fatty acid signature of human Amniotic Epithelial Cells (hAECs). The analysis of the membrane fatty acid composition of hAECs cultured in basal medium showed a loss in polyunsaturated fatty acids (PUFA), in particular in omega-6 (ω-6) content, compared to freshly isolated hAECs. The addition to the basal culture medium of a chemically defined and animal-free tailored lipid supplement, namely Refeed®, partially restored the membrane fatty acid signature of hAECs. Although the amelioration of the membrane composition did not prolong hAECs culture lifespan, Refeed® influenced cell morphology, counteracted the onset of senescence, and increased the migratory capacity as well as the ability of hAECs to inhibit Peripheral Blood Mononuclear Cell (PBMC) proliferation. This study provides new information on hAEC features during culture passages and demonstrates that the maintenance of the membrane fatty acid signature preserved higher cell quality during in vitro expansion, suggesting the use of lipid supplementation for SC expansion in cell-based therapies.

Effective Label-Free Sorting of Multipotent Mesenchymal Stem Cells from Clinical Bone Marrow Samples.

Mesenchymal stem cells (MSC) make up less than 1% of the bone marrow (BM). Several methods are used for their isolation such as gradient separation or centrifugation, but these methodologies are not direct and, thus, plastic adherence outgrowth or magnetic/fluorescent-activated sorting is required. To overcome this limitation, we investigated the use of a new separative technology to isolate MSCs from BM; it label-free separates cells based solely on their physical characteristics, preserving their native physical properties, and allows real-time visualization of cells. BM obtained from patients operated for osteochondral defects was directly concentrated in the operatory room and then analyzed using the new technology. Based on cell live-imaging and the sample profile, it was possible to highlight three fractions (F1, F2, F3), and the collected cells were evaluated in terms of their morphology, phenotype, CFU-F, and differentiation potential. Multipotent MSCs were found in F1: higher CFU-F activity and differentiation potential towards mesenchymal lineages compared to the other fractions. In addition, the technology depletes dead cells, removing unwanted red blood cells and non-progenitor stromal cells from the biological sample. This new technology provides an effective method to separate MSCs from fresh BM, maintaining their native characteristics and avoiding cell manipulation. This allows selective cell identification with a potential impact on regenerative medicine approaches in the orthopedic field and clinical applications.

Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome.

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

A New Predictive Technology for Perinatal Stem Cell Isolation Suited for Cell Therapy Approaches.

The use of stem cells for regenerative applications and immunomodulatory effect is increasing. Amniotic epithelial cells (AECs) possess embryonic-like proliferation ability and multipotent differentiation potential. Despite the simple isolation procedure, inter-individual variability and different isolation steps can cause differences in isolation yield and cell proliferation ability, compromising reproducibility observations among centers and further applications. We investigated the use of a new technology as a diagnostic tool for quality control on stem cell isolation. The instrument label-free separates cells based on their physical characteristics and, thanks to a micro-camera, generates a live fractogram, the fingerprint of the sample. Eight amniotic membranes were processed by trypsin enzymatic treatment and immediately analysed. Two types of profile were generated: a monomodal and a bimodal curve. The first one represented the unsuccessful isolation with all recovered cell not attaching to the plate; while for the second type, the isolation process was successful, but we discovered that only cells in the second peak were alive and resulted adherent. We optimized a Quality Control (QC) method to define the success of AEC isolation using the fractogram generated. This predictive outcome is an interesting tool for laboratories and cell banks that isolate and cryopreserve fetal annex stem cells for research and future clinical applications.

Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations.

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

A Reliable Flow-Based Method for the Accurate Measure of Mass Density, Size and Weight of Live 3D Tumor Spheroids.

Gathering precise information on mass density, size and weight of cells or cell aggregates, is crucial for applications in many biomedical fields with a specific focus on cancer research. Although few technical solutions have been presented for single-cell analysis, literature does not cover this aspect for 3D models such as spheroids. Since the research interest on such samples is notably rising, here we describe a flow-apparatus, and the associated physical method and operative protocol for the accurate measurements of mass density, size and weight. The technique is based on the detection of the terminal velocity of a free-falling sample into a specifically conceived analysis flow-channel. Moreover, in order to demonstrate the accuracy and precision of the presented flow-device, analyses were initially carried out on standardized polystyrene beads. Finally, to display the application of the proposed system for biological samples, mass density, size and weight of live SW620 tumor spheroids were analyzed. The combined measurements of such parameters can represent a step toward a deeper understanding of 3D culture models.

Characterization of the Tissue and Stromal Cell Components of Micro-Superficial Enhanced Fluid Fat Injection (Micro-SEFFI) for Facial Aging Treatment.

BACKGROUND: New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. OBJECTIVES: To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. METHODS: Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector,® an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. RESULTS: Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates, and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages. CONCLUSIONS: Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing.

Early Developmental Zebrafish Embryo Extract to Modulate Senescence in Multisource Human Mesenchymal Stem Cells.

Stem cells undergo senescence both in vivo, contributing to the progressive decline in self-healing mechanisms, and in vitro during prolonged expansion. Here, we show that an early developmental zebrafish embryo extract (ZF1) could act as a modulator of senescence in human mesenchymal stem cells (hMSCs) isolated from both adult tissues, including adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and a perinatal tissue such as the Wharton's Jelly (hWJ-MSCs). In all the investigated hMSCs, ZF1 decreased senescence-associated ß-galactosidase (SA ß-gal) activity and enhanced the transcription of TERT, encoding the catalytic telomerase core. In addition, it was associated, only in hASCs, with a transcriptional induction of BMI1, a pleiotropic repressor of senescence. In hBM-MSCs, hDP-MSCs, and hWJ-MSCs, TERT over-expression was concomitant with a down-regulation of two repressors of TERT, TP53 (p53), and CDKN1A (p21). Furthermore, ZF1 increased the natural ability of hASCs to perform adipogenesis. These results indicate the chance of using ZF1 to modulate stem cell senescence in a source-related manner, to be potentially used as a tool to affect stem cell senescence in vitro. In addition, its anti-senescence action could also set the basis for future in vivo approaches promoting tissue rejuvenation bypassing stem cell transplantation.

Parathyroid Tissue Cryopreservation: Does the Storage Time Affect Viability and Functionality?

Parathyroidectomy is a standard practice to treat recurrent or persistent hyperparathyroidism. However, this can lead to the onset of hypoparathyroidism, treatable with the autotransplantation of parathyroid tissue (PT). Tissue can be transplanted immediately after parathyroidectomy or cryopreserved and transplanted only in case of necessity. Since 2011, the Cord Blood Bank and Cardiovascular Tissue Bank of Emilia-Romagna has been storing PT for potential autologous transplantation. To date, there are highly variable data about the viability and function of PT after thawing. However, it is not clear if the PT quality is affected by different cryopreservation protocols and/or by the storage time. The aim of this study was to assess the ex vivo function and viability of the PTs of ten patients stored in the Bank. Tissue morphology was evaluated before and after cryopreservation through histological investigations. PT function was analyzed by assessing the ability of cryopreserved PT to synthesize and secrete parathyroid hormone (PTH) in response to different calcium concentrations. Moreover, viability and function were also investigated on tissue-isolated cells in culture. These data show that tested tissues appear to be viable and able to produce PTH even after 5 years of storage, and the histological architecture is well preserved.

Exploring the roles of MSCs in infections: focus on bacterial diseases.

Despite human healthcare advances, some microorganisms continuously react evolving new survival strategies, choosing between a commensal fitness and a pathogenic attitude. Many opportunistic microbes are becoming an increasing cause of clinically evident infections while several renowned infectious diseases sustain a considerable number of deaths. Besides the primary and extensively investigated role of immune cells, other cell types are involved in the microbe-host interaction during infection. Interestingly, mesenchymal stem cells (MSCs), the current leading players in cell therapy approaches, have been suggested to contribute to tackling pathogens and modulating the host immune response. In this context, this review critically explores MSCs' role in E. coli, S. aureus, and polymicrobial infections. Summarizing from various studies, in vitro and in vivo results support the mechanistic involvement of MSCs and their derivatives in fighting infection and in contributing to microbial spreading. Our work outlines the double face of MSCs during infection, disease, and sepsis, highlighting potential pitfalls in MSC-based therapy due to the MSCs' susceptibility to pathogens' weapons. We also identify potential targets to improve infection treatments, and propose the potential applications of MSCs for vaccine research.

Microfractures and hydrogel scaffolds in the treatment of osteochondral knee defects: A clinical and histological evaluation.

BACKGROUND: Osteochondral knee defects (OCD) are often symptomatic, causing pain and functional impairment even in young and active patients. Regenerative surgical options, aiming to stimulate natural cartilage healing, have been recently used as a first line treatment. In this study, a new hydrogel is investigated in its capacity to regenerate the ultra-structural quality of hyaline cartilage when combined with a classical microfracture technique. MATERIAL AND METHODS: Forty-six patients, affected by grade III and IV knee chondropathies, were consecutively treated between 2013 and 2015 with microfractures followed by application of a modern hydrogel in the lesion site. All patients underwent clinical evaluation (WOMAC) pre-operatively, at 6,12 and at 24 months postoperatively: the results were compared with a subsequent, consecutive, matched, control group of 23 patients treated with microfractures alone. In a parallel and separate in-vitro histological study, adipose derived mesenchymal stem cells (ADMSCs) were encapsulated in the hydrogel scaffold, induced to differentiation into chondrocytes, and observed for a 3 weeks period. RESULTS: The initial WOMAC score of 58.6 ±â€¯11.0 in the study group was reduced by 88% at 6 months (7.1 ±â€¯9.2) and 95% at 24 months (2.9 ±â€¯5.9). The "in-vitro" study revealed a histological characterization typical of hyaline cartilage in study group. Separate biopsies performed at 12 months post-op in the study group also revealed type 2 collagen and hyaline-like cartilage in the regenerated tissue. CONCLUSION: Our study demonstrated high patient satisfaction rates after microfractures combined with a modern hydrogel scaffold; histologic evaluation supported the hypothesis of creating an enhanced chondrogenic environment. Microfracture "augmentation" using modern acellular biomaterials, like hydrogels, might improve the clinical outcomes of this classical bone marrow stimulating procedure.

Restoring In Vivo-Like Membrane Lipidomics Promotes Exosome Trophic Behavior from Human Placental Mesenchymal Stromal/Stem Cells.

Human mesenchymal stem cells (hMSCs) are an effective tool in regenerative medicine notably for their intrinsic plentiful paracrine activity rather than differentiating properties. The hMSC secretome includes a wide spectrum of regulatory and trophic factors, encompassing several naked molecules as well as different kinds of extracellular vesicles (EVs). Among EVs, exosomes represent an intriguing population, able to shuttle proteins, transcription factors, and genetic materials, with a relevant role in cell-to-cell communication, modulating biological responses in recipient cells. In this context, the extracellular milieu can greatly impact the paracrine activity of stem cells, modifying their metabolism, and the dynamics of vesicle secretion. In the present study, we investigated the effects elicited on exosome patterning by tailored, ad hoc formulated lipid supplementation (Refeed®) in MSCs derived from human fetal membranes (hFM-MSCs). Wound healing experiments revealed that stem cell exposure to exosomes obtained from Refeed®-supplemented hFM-MSCs increased their migratory capability, although the amount of exosomes released after Refeed® supplementation was lower than that yielded from non-supplemented cells. We found that such a decrease was mainly due to a different rate of exosomal exocytosis rather than to an effect of the lipid supplement on the endocytic pathway. Endoplasmic reticulum homeostasis was modified by supplementation, through the upregulation of PKR-like ER kinase (PERK) and inositol-requiring enzyme 1α (IRE1α). Increased expression of these proteins did not lead to stress-induced, unfolded protein response (UPR)-mediated apoptosis, nor did it affect phosphorylation of p38 kinase, suggesting that PERK and IRE1α overexpression was due to augmented metabolic activities mediated by optimization of a cellular feeding network afforded through lipid supplementation. In summary, these results demonstrate how tailored lipid supplementation can successfully modify the paracrine features in hFM-MSCs, impacting both intracellular vesicle trafficking and secreted exosome number and function.