Dissecting the dynamics of SARS-CoV-2 reinfections in blood donors with pauci- or asymptomatic COVID-19 disease course at initial infection.

BACKGROUND: Understanding the dynamics of SARS-CoV-2 reinfections is crucial for public health policy, vaccine development, and long-term disease management. However, data on reinfections in the general population remains scarce. OBJECTIVES: This study aimed to investigate SARS-CoV-2 antibody dynamics among Austrian blood donors, representing healthy adults, over two years following primary infection and to evaluate the reinfection risk. METHODS: 117,895 blood donations were analysed for SARS-CoV-2 total anti-N levels from June 2020 to December 2023. We examined anti-N and anti-S antibody dynamics and in vitro functionality in 230 study participants at five defined times during 24 months, assessing associations with demographics, vaccination status, and reinfection awareness. RESULTS: The seroprevalence of SARS-CoV-2 infection-derived anti-N antibodies increased over time, reaching 90% by February 2023 and remaining at that level since then. According to serological screenings, we found an 88% reinfection rate, which is in contrast to participants' reports indicating a reinfection rate of 59%. Our data further reveal that about 26% of reinfections went completely unnoticed. Antibody dynamics were independent of age, sex, and ABO blood group. Interestingly, individuals with multiple reinfections reported symptoms more frequently during their primary infection. Our results further show that vaccination modestly affected reinfection risk and disease course. CONCLUSION: SARS-CoV-2 reinfections were uncommon until the end of 2021 but became common with the advent of Omicron. This study highlights the underestimation of reinfection rates in healthy adults and underscores the need for continued surveillance, which is an important support for public health policies and intervention strategies.

Collection efficiency of mononuclear cells in offline extracorporeal photopheresis: can processing time be shortened?

BACKGROUND: Extracorporeal photopheresis (ECP) is a well-established but lengthy and burdensome cell-based therapy for various diseases such as cutaneous T-cell lymphoma, graft-versus-host disease and organ rejection after transplantation. The number of mononuclear cells (MNCs) that needs to be collected to obtain a clinical response to ECP is still under debate. The purpose of this retrospective study was to determine the number of lymphocytes, monocytes and neutrophils in mononuclear cell products (MCP) by flow cytometry and the collection efficiency in the offline ECP setting. MATERIALS AND METHODS: We collected data from 10 different patients undergoing 162 ECP procedures using the Spectra Optia device for MNC collection. White blood cell (WBC) count of MCP was determined using a hematology analyzer. MNCs were analyzed for CD45 and CD14 expression by flow cytometry to exactly determine the collected lymphocyte and monocyte fractions. RESULTS: Collected MCP showed high cell yields with 55.3×106/kg MNCs and 41.1×106/kg lymphocytes. MCP were characterized by high MNC (81.3%) and low neutrophils (18.7%) percentage. Mean collection efficiency for WBCs and for MNCs was 23.9% and 62.0%, respectively. The MNC fraction showed a moderate to high correlation between peripheral blood cell count of patients and MCP count. DISCUSSION: This study is one of a few reports showing the monocyte-to-lymphocyte relation in MCP for ECP determined by flow cytometry. In comparison to historical data from inline ECP, the offline ECP processing one total blood volume results in considerably higher cell yields. For this reason, and to reduce the burden on patients, we propose that the offline ECP processing time can be substantially reduced.

Epstein-Barr virus reactivation is not causative for post-COVID-19-syndrome in individuals with asymptomatic or mild SARS-CoV-2 disease course.

PURPOSE: Post-COVID-19-Syndrome (PCS) frequently occurs after an infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, the understanding of causative mechanisms is still limited. Aim of this study was to determine the PCS rate among SARS-CoV-2 seropositive blood donors as representatives of supposedly healthy adults, who had experienced an asymptomatic or mild COVID-19 disease course, and to examine whether Epstein-Barr virus (EBV) is reactivated in individuals reporting PCS. METHODS: The PCS rate was determined using questionnaires that included questions about infection and persistent symptoms. Pre-pandemic blood samples and samples collected at regular, pre-defined times after a SARS-CoV-2 infection were analysed for neopterin, a marker for antiviral immune responses, by an enzyme-linked immunosorbent assay (ELISA). Additionally, we determined the rate of SARS-CoV-2 anti-N total antibodies using an electrochemiluminescence immunoassay (ECLIA). Furthermore, quantitative real-time polymerase chain reaction (qPCR) to detect EBV DNA and ECLIA screening for EBV viral capsid-antigen (VCA) IgM, IgG and EBV nuclear antigen 1 (EBNA) IgG were performed. RESULTS: Our data reveal that 18% of all infections result in PCS, with symptoms lasting for up to one year. In individuals reporting PCS, no elevated levels of neopterin were detected, indicating no persisting pro-inflammatory, antiviral immune response. SARS-CoV-2 antibody levels were declining in all participants in comparable manner over time, pointing to a successful virus clearance. In individuals with PCS, no EBV DNA could be detected. Furthermore, no differences in EBV specific antibody levels could be shown in PCS groups compared to non-PCS groups. CONCLUSION: Our data suggest that PCS in per se healthy, immunocompetent adults cannot be ascribed to a reactivation of EBV.

Transcription Factors STAT3 and MYC Are Key Players of Human Platelet Lysate-Induced Cell Proliferation.

Human platelet lysate (HPL) is an efficient alternative for animal serum supplements, significantly enhancing stromal cell proliferation. However, the molecular mechanism behind this growth-promoting effect remains elusive. The aim of this study was to investigate the effect of HPL on cell cycle gene expression in different human stromal cells and to identify the main key players that mediate HPL's growth-enhancing effect. RT-qPCR and an antibody array revealed significant upregulation of cell cycle genes in stromal cells cultured in HPL. As HPL is rich in growth factors that are ligands of tyrosine kinase receptor (TKR) pathways, we used TKR inhibitors and could significantly reduce cell proliferation. Genome profiling, RT-qPCR and Western blotting revealed an enhanced expression of the transcription factors signal transducer and activator of transcription 3 (STAT3) and MYC, both known TKR downstream effectors and stimulators of cell proliferation, in response to HPL. In addition, specifically blocking STAT3 resulted in reduced cell proliferation and expression of cell cycle genes. Our data indicate that HPL-enhanced cell proliferation can, at least in part, be explained by the TKR-enhanced expression of STAT3 and MYC, which in turn induce the expression of genes being involved in the promotion and control of the cell cycle.

SARS-CoV-2 IgG Levels Allow Predicting the Optimal Time Span of Convalescent Plasma Donor Suitability.

Convalescent plasma (CP) has been in use for the treatment of numerous infectious diseases for more than a century, recently also for coronavirus disease 2019 (COVID-19). A major challenge for this treatment is identifying suitable donors with sufficient levels of functional antibodies and to determine the optimal time span for CP donation. In this retrospective study, we analyzed 189 CP donations of 66 donors regarding anti-SARS-CoV-2 anti-S IgG antibody levels. We found a significant correlation between the semi-quantitative SARS-CoV-2 IgG ratio values and in vitro antibody functionality. A time-to-event analysis allowed us to predict the optimal time span of COVID-19 CP donor suitability. We found that high IgG ratio values, which significantly correlate with high in vitro antibody functionality, were suitable for CP donation for a median of 134 days after the first CP donation. Donors with lower IgG ratios were suitable for a median of 53 days. Our data support plasma collection centers to determine optimal points in time for CP donation by means of widely used semi-quantitative laboratory IgG ratio values.

Sample Buffer Containing Guanidine-Hydrochloride Combines Biological Safety and RNA Preservation for SARS-CoV-2 Molecular Diagnostics.

The COVID-19 pandemic has elicited the need to analyse and store large amounts of infectious samples for laboratory diagnostics. Therefore, there has been a demand for sample storage buffers that effectively inactivate infectious viral particles while simultaneously preserving the viral RNA. Here, we present a storage buffer containing guanidine-hydrochloride that fulfils both requirements. Its ability to preserve RNA stability was confirmed by RT-qPCR, and virus-inactivating properties were tested by tissue culture infectious dose assay. Our data revealed that RNA from samples diluted in this storage buffer was efficiently preserved. Spiking samples with RNase A resulted in RNAse concentrations up to 100 ng/mL being efficiently inhibited, whereas spiking samples with infectious SARS-CoV-2 particles demonstrated rapid virus inactivation. In addition, our buffer demonstrated good compatibility with several commercially available RNA extraction platforms. The presented guanidine-hydrochloride-based storage buffer efficiently inactivates infectious SARS-CoV-2 particles and supports viral RNA stability, leading to a reduced infection risk during sample analysis and an increased period for follow-up analysis, such as sequencing for virus variants. Because the presented buffer is uncomplicated to manufacture and compatible with a variety of commercially available test systems, its application can support and improve SARS-CoV-2 laboratory diagnostics worldwide.

Persistence of Naturally Acquired and Functional SARS-CoV-2 Antibodies in Blood Donors One Year after Infection.

The developmental course of antibodies produced after a SARS-CoV-2 infection has been insufficiently investigated so far. Therefore, the aim of this study was to investigate the dynamics of SARS-CoV-2 antibody levels against the viral nucleocapsid- and spike-protein among Austrian blood donors as a representative group of a supposedly healthy population within the first year after a SARS-CoV-2 infection. The impact of age, sex, vaccination status, AB0-blood group and awareness about the infection was evaluated. Our study shows that the level of anti-N antibodies is declining, while anti-S antibody levels remain stable. Antibodies detected were functional in vitro. Age, sex and blood group do not influence antibody dynamics. However, blood group AB shows significantly lower antibody levels and in vitro functionality compared to other blood groups. Our data reveal that one out of five individuals was not aware of a previous SARS-CoV-2 infection and that the disease course neither affects the level of antibody production nor the in vitro functionality. We also found that 14% of participants show persisting COVID-19-related symptoms for up to nine months. Our results provide valuable insights into the dynamics of the immune response after a SARS-CoV-2 infection in a representative cohort of adult blood donors in Central Europe.

Heparin and Derivatives for Advanced Cell Therapies.

Heparin and its derivatives are saving thousands of human lives annually, by successfully preventing and treating thromboembolic events. Although the mode of action during anticoagulation is well studied, their influence on cell behavior is not fully understood as is the risk of bleeding and other side effects. New applications in regenerative medicine have evolved supporting production of cell-based therapeutics or as a substrate for creating functionalized matrices in biotechnology. The currently resurgent interest in heparins is related to the expected combined anti-inflammatory, anti-thrombotic and anti-viral action against COVID-19. Based on a concise summary of key biochemical and clinical data, this review summarizes the impact for manufacturing and application of cell therapeutics and highlights the need for discriminating the different heparins.

Human Platelet Lysate for Good Manufacturing Practice-Compliant Cell Production.

Numerous cell-based therapeutics are currently being tested in clinical trials. Human platelet lysate (HPL) is a valuable alternative to fetal bovine serum as a cell culture medium supplement for a variety of different cell types. HPL as a raw material permits animal serum-free cell propagation with highly efficient stimulation of cell proliferation, enabling humanized manufacturing of cell therapeutics within a reasonable timeframe. Providers of HPL have to consider dedicated quality issues regarding identity, purity, potency, traceability and safety. Release criteria have to be defined, characterizing the suitability of HPL batches for the support of a specific cell culture. Fresh or expired platelet concentrates from healthy blood donors are the starting material for HPL preparation, according to regulatory requirements. Pooling of individual platelet lysate units into one HPL batch can balance donor variation with regard to essential platelet-derived growth factors and cytokines. The increasingly applied pathogen reduction technologies will further increase HPL safety. In this review article, aspects and regulatory requirements of whole blood donation and details of human platelet lysate manufacturing are presented. International guidelines for raw materials are discussed, and defined quality controls, as well as release criteria for safe and GMP-compliant HPL production, are summarized.

First-in-human intracochlear application of human stromal cell-derived extracellular vesicles.

Extracellular vesicles (EVs) derived from the secretome of human mesenchymal stromal cells (MSC) contain numerous factors that are known to exert anti-inflammatory effects. MSC-EVs may serve as promising cell-based therapeutics for the inner ear to attenuate inflammation-based side effects from cochlear implantation which represents an unmet clinical need. In an individual treatment performed on a 'named patient basis', we intraoperatively applied allogeneic umbilical cord-derived MSC-EVs (UC-MSC-EVs) produced according to good manufacturing practice. A 55-year-old patient suffering from Menière's disease was treated with intracochlear delivery of EVs prior to the insertion of a cochlear implant. This first-in-human use of UC-MSC-EVs demonstrates the feasibility of this novel adjuvant therapeutic approach. The safety and efficacy of intracochlear EV-application to attenuate side effects of cochlea implants have to be determined in controlled clinical trials.

Seroprevalence of anti-SARS-CoV-2 total antibody is higher in younger Austrian blood donors.

PURPOSE: Frequently the infection with coronavirus 2 (SARS-CoV-2) can be asymptomatic or provoke only mild symptoms. These cases often remain unnoticed, so it is difficult to estimate the actual numbers of infections. Aim of this study was to determine the seroprevalence of anti-SARS-CoV-2 total antibody in Austrian blood donors. METHODS: 20,228 blood donors aged between 18 and 72 years resident in four Austrian federal states were screened for anti-SARS-CoV-2 total antibody between 5th of June and 4th of December 2020. To evaluate the impact of sex, age, AB0-blood group and donation period on the anti-SARS-CoV-2 seroprevalence, multiple logistic regression was done. RESULTS: Our data reveal an anti-SARS-CoV-2 seroprevalence of 2.5% overall, significantly depending on the time point of blood donation: after the first Austrian lockdown the seroprevalence was lower compared to the following months, when the rate was constantly rising. While younger blood donors showed significantly higher seroprevalence, no differences were found concerning sex or AB0 blood group. CONCLUSION: Broad testing strategies are required to better determine the number of SARS-CoV-2 infections. Screening blood donors as a representative group for the adult population could be a valid tool to determine the number of recorded and unrecorded cases of SARS-CoV-2 infection.

Extracellular vesicles from human multipotent stromal cells protect against hearing loss after noise trauma in vivo.

The lack of approved anti-inflammatory and neuroprotective therapies in otology has been acknowledged in the last decades and recent approaches are heralding a new era in the field. Extracellular vesicles (EVs) derived from human multipotent (mesenchymal) stromal cells (MSC) can be enriched in vesicular secretome fractions, which have been shown to exert effects (eg, neuroprotection and immunomodulation) of their parental cells. Hence, MSC-derived EVs may serve as novel drug candidates for several inner ear diseases. Here, we provide first evidence of a strong neuroprotective potential of human stromal cell-derived EVs on inner ear physiology. In vitro, MSC-EV preparations exerted immunomodulatory activity on T cells and microglial cells. Moreover, local application of MSC-EVs to the inner ear significantly attenuated hearing loss and protected auditory hair cells from noise-induced trauma in vivo. Thus, EVs derived from the vesicular secretome of human MSC may represent a next-generation biological drug that can exert protective therapeutic effects in a complex and nonregenerating organ like the inner ear.

Heparin Differentially Impacts Gene Expression of Stromal Cells from Various Tissues.

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.

Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate.

BACKGROUND: Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology. METHODS: Stromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed. RESULTS: Biochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na+ were elevated in pHPL preparations, K+ and Fe3+ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions. CONCLUSION: All pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state.

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.

Selection of Tissue Factor-Deficient Cell Transplants as a Novel Strategy for Improving Hemocompatibility of Human Bone Marrow Stromal Cells.

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation. Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC. Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells. Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.

An In Vitro Potency Assay for Monitoring the Immunomodulatory Potential of Stromal Cell-Derived Extracellular Vesicles.

The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs) is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs) exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.

Manufacturing of Human Extracellular Vesicle-Based Therapeutics for Clinical Use.

Extracellular vesicles (EVs) derived from stem and progenitor cells may have therapeutic effects comparable to their parental cells and are considered promising agents for the treatment of a variety of diseases. To this end, strategies must be designed to successfully translate EV research and to develop safe and efficacious therapies, whilst taking into account the applicable regulations. Here, we discuss the requirements for manufacturing, safety, and efficacy testing of EVs along their path from the laboratory to the patient. Development of EV-therapeutics is influenced by the source cell types and the target diseases. In this article, we express our view based on our experience in manufacturing biological therapeutics for routine use or clinical testing, and focus on strategies for advancing mesenchymal stromal cell (MSC)-derived EV-based therapies. We also discuss the rationale for testing MSC-EVs in selected diseases with an unmet clinical need such as critical size bone defects, epidermolysis bullosa and spinal cord injury. While the scientific community, pharmaceutical companies and clinicians are at the point of entering into clinical trials for testing the therapeutic potential of various EV-based products, the identification of the mode of action underlying the suggested potency in each therapeutic approach remains a major challenge to the translational path.

Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.

BACKGROUND: Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin. METHODS: We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages. RESULTS: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure. CONCLUSIONS: Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.