- Email: [email protected]
A mixed lymphocyte reaction as a functional assay for extracellular vesicles of different origins
Poster Abstract Presentations
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5 days in the absence or presence of EVs. Thereafter, cell morphologies are documented and cells are phenotypically characterized by flow cytometry. By analyzing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by therapeutically active MSC-EVs. For example we observed that in the presence of active MSC-EVs more CD14+ (monocytes) and CD56+ (natural killer cells) are recovered from the MLR than in corresponding control samples. In contrast, in the presence of active MSC-EVs contents of CD4+ and CD8+ T cells got slightly decreased. Focusing on T cells, we learned that active MSC-EVs reduced the content of CD4 and CD8 T cells expressing T cell activation markers like CD54 and CD25. Currently, we compare the immunomodulatory capabilities of EVs of different cell types. Furthermore, we proceed in optimizing the marker panel to distinguish immune cell subtypes such as the different types of CD4+ cell types (T 1, T 2, T 17 and T).
195 MSC-EVS PROTECT MICE FROM GRAFT-VERSUS-HOST DISEASE PATHOLOGY IN A PREPARATION DEPENDENT MANNER R. Madel1, V. B€ orger2, M. Bremer2, P. Horn2, H.A. Baba3, S. Brandau4, J. Buer1, B. Giebel5 & C. Kirschning1 1 Institute of Medical Microbiology, Essen, Germany, 2Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, 3Institute of Pathology, Essen, Germany, 4Department of Otorhinolaryngology, Essen, Germany, 5Institute for Transfusion Medicine, University Duisburg-Essen, Essen, NRW, Germany Background & Aim: Extracellular vesicles (EVs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (MSCs) can suppress acute inflammatory cues in a variety of different diseases, including Graft-versus-Host Disease (GvHD) and ischemic stroke, and promote regeneration of affected tissues. Following a successful clinical treatment attempt of a steroid refractory GvHD patient, we intend to optimize MSCEV production strategies for further clinical applications. Methods, Results & Conclusion: As we observed functional differences of independent MSC-EV preparations in vitro, we aimed to set up an in vivo GvHD model for the more advanced functional testing of different MSC-EV preparations. To this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (IIR). GvHD was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine T cells. If not treated otherwise, myeloablated mice developed severe GvHD symptoms. The GvHD symptoms were effectively suppressed if MSC-EV preparations, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay, were applied at 3 consecutive days. MSC-EV preparations lacking in vitro immune modulating activities within the MLR assay, however, hardly improved the symptoms of the GvHD mice. Thus, our results demonstrate that not all MSC-EV preparations harvested from adult bone marrow-derived MSCs are functional equivalent. Thus, successful transplantation of MSCEVs into the clinics requires a platform allowing identification of MSC-EV preparations with sufficient therapeutic, most probably immune modulating activities. 196 A MIXED LYMPHOCYTE REACTION AS A FUNCTIONAL ASSAY FOR EXTRACELLULAR VESICLES OF DIFFERENT ORIGINS M. Bremer, V. B€ orger, P. Horn & B. Giebel Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany Background & Aim: Extracellular vesicles (EVs), such as exosomes and microvesicles, are shed by all cell types and found in all body fluids. EVs transmit specific information from their cells of origin to specific target cells and are key factors in a novel form of intercellular communication. Depending on their origin, EVs can modulate immune responses and either act proinflammatory (e.g. mature DC-EVs) or anti-inflammatory (e.g. mesenchymal stem cell (MSC) and many tumor cell-derived EVs). Methods, Results & Conclusion: Aiming to analyze immune-modulating properties of EVs from different sources, in vitro, we established a novel form of a mixed lymphocyte reaction (MLR) assay. Here, human peripheral bloodderived mononuclear cells (MNCs) were pooled from up to 12 different healthy donors warranting high cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed MNCs are cultured for
197 TANGENTIAL FLOW FILTRATION, A POTENTIAL METHOD FOR THE SCALED PREPARATION OF EXTRACELLULAR VESICLES V. B€ orger1, R. Dittrich1, S. Staubach1, S. Zumegen1, P. Horn1 & B. Giebel2 1 Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, 2Institute for Transfusion Medicine, University Duisburg-Essen, Essen, NRW, Germany Background & Aim: Human mesenchymal stem/stromal cells (MSCs) have been applied in more than 900 NIH-registered clinical trials with different outcomes. In a proportion of studies pro-regenerative and/or immunomodulatory therapeutic effects were observed. Against the initial assumption, preclinical data demonstrated that if functional - MSCs act in a paracrine rather than in a cellular manner. Efforts to search for the active paracrine components ended in the identification of extracellular vesicle (EVs). Indeed, in several preclinical models EVs harvested from MSC conditioned media exerted comparable therapeutic effects than the MSCs themselves. Methods, Results & Conclusion: EVs released by virtually all cell types represent a heterogenous population of vesicles of different origin, including exosomes (70-150 nm), microvesicles (100-1000 nm), and apoptotic bodies (> 500 nm). Classically, they are prepared by differential centrifugation. Due to volume restricitions during ultracentrifugation only smaller amounts of EV containing liquids can be processed with this method. To efficiently translate MSC-EVs into the clinics, they need to be produced in a scaled manner. Previously, we established and successfully used a polyehtylen glycol (PEG) precipitation procedure allowing to process up to 10 l conditioned medium. Intending to further scale the MSC-EV production, optimally in a closed system, we have started to optimize tangential flow filtration (TFF) protocols, in principal being scalable up to several 1000 litres. At first, we compared different TFF devices and selected the holofiber-based TFF system. Although EVs can efficiently be prepared from serum-free conditioned media, the processing of serum or human platelet lysate (hPL) containing conditioned media remains challenging. The high protein content of serum and hPL supplemented media quickly results in protein aggregate formation and clogging of the pores of the TFF system, which negatively affect the EV purification process. Currently, by changing flow rates and and re-addition of buffers, we optimize the TFF protocols to enable us obtaining MSC-EV preparations with comparable purities than with the PEG method. 198 FREE FLOW ELECTROPHORESIS ALLOWS PREPARATION OF EXTRACELLULAR VESICLES WITH HIGH PURITY S. Staubach2,1, C. Reiter1, G. Weber1 & B. Giebel2 1 FFE Service GmbH, Feldkirchen, Germany, 2Institute for Transfusion Medicine, University Duisburg-Essen, Essen, NRW, Germany Background & Aim: Currently, it remains a challenge to prepare extracellular vesicles (EVs) to high purity. Neither fractionation by density nor by size alone is sufficient to separate EVs from all contaminants e.g. high and low density lipoprotein (HDL/LDL) and adherent proteins. For now, a time consuming combination of two methods (density and size separation) is required to enrich EVs to high purity at the expense of time and low recovery. A fast and efficient alternative is represented by Free Flow Electrophoresis (FFE). It is a well-established (semi-) preparative method to separate analytes with inherent difference of charge density and/or difference of pI-value. Methods, Results & Conclusion: A Free Flow Interval Zone Electrophoresis (FF-IZE) method has been developed, for the purification and isolation of EVs as well DNA and RNA from human plasma samples, using a set of
S57
5 days in the absence or presence of EVs. Thereafter, cell morphologies are documented and cells are phenotypically characterized by flow cytometry. By analyzing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by therapeutically active MSC-EVs. For example we observed that in the presence of active MSC-EVs more CD14+ (monocytes) and CD56+ (natural killer cells) are recovered from the MLR than in corresponding control samples. In contrast, in the presence of active MSC-EVs contents of CD4+ and CD8+ T cells got slightly decreased. Focusing on T cells, we learned that active MSC-EVs reduced the content of CD4 and CD8 T cells expressing T cell activation markers like CD54 and CD25. Currently, we compare the immunomodulatory capabilities of EVs of different cell types. Furthermore, we proceed in optimizing the marker panel to distinguish immune cell subtypes such as the different types of CD4+ cell types (T 1, T 2, T 17 and T).
195 MSC-EVS PROTECT MICE FROM GRAFT-VERSUS-HOST DISEASE PATHOLOGY IN A PREPARATION DEPENDENT MANNER R. Madel1, V. B€ orger2, M. Bremer2, P. Horn2, H.A. Baba3, S. Brandau4, J. Buer1, B. Giebel5 & C. Kirschning1 1 Institute of Medical Microbiology, Essen, Germany, 2Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, 3Institute of Pathology, Essen, Germany, 4Department of Otorhinolaryngology, Essen, Germany, 5Institute for Transfusion Medicine, University Duisburg-Essen, Essen, NRW, Germany Background & Aim: Extracellular vesicles (EVs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (MSCs) can suppress acute inflammatory cues in a variety of different diseases, including Graft-versus-Host Disease (GvHD) and ischemic stroke, and promote regeneration of affected tissues. Following a successful clinical treatment attempt of a steroid refractory GvHD patient, we intend to optimize MSCEV production strategies for further clinical applications. Methods, Results & Conclusion: As we observed functional differences of independent MSC-EV preparations in vitro, we aimed to set up an in vivo GvHD model for the more advanced functional testing of different MSC-EV preparations. To this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (IIR). GvHD was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine T cells. If not treated otherwise, myeloablated mice developed severe GvHD symptoms. The GvHD symptoms were effectively suppressed if MSC-EV preparations, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay, were applied at 3 consecutive days. MSC-EV preparations lacking in vitro immune modulating activities within the MLR assay, however, hardly improved the symptoms of the GvHD mice. Thus, our results demonstrate that not all MSC-EV preparations harvested from adult bone marrow-derived MSCs are functional equivalent. Thus, successful transplantation of MSCEVs into the clinics requires a platform allowing identification of MSC-EV preparations with sufficient therapeutic, most probably immune modulating activities. 196 A MIXED LYMPHOCYTE REACTION AS A FUNCTIONAL ASSAY FOR EXTRACELLULAR VESICLES OF DIFFERENT ORIGINS M. Bremer, V. B€ orger, P. Horn & B. Giebel Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany Background & Aim: Extracellular vesicles (EVs), such as exosomes and microvesicles, are shed by all cell types and found in all body fluids. EVs transmit specific information from their cells of origin to specific target cells and are key factors in a novel form of intercellular communication. Depending on their origin, EVs can modulate immune responses and either act proinflammatory (e.g. mature DC-EVs) or anti-inflammatory (e.g. mesenchymal stem cell (MSC) and many tumor cell-derived EVs). Methods, Results & Conclusion: Aiming to analyze immune-modulating properties of EVs from different sources, in vitro, we established a novel form of a mixed lymphocyte reaction (MLR) assay. Here, human peripheral bloodderived mononuclear cells (MNCs) were pooled from up to 12 different healthy donors warranting high cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed MNCs are cultured for
197 TANGENTIAL FLOW FILTRATION, A POTENTIAL METHOD FOR THE SCALED PREPARATION OF EXTRACELLULAR VESICLES V. B€ orger1, R. Dittrich1, S. Staubach1, S. Zumegen1, P. Horn1 & B. Giebel2 1 Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, 2Institute for Transfusion Medicine, University Duisburg-Essen, Essen, NRW, Germany Background & Aim: Human mesenchymal stem/stromal cells (MSCs) have been applied in more than 900 NIH-registered clinical trials with different outcomes. In a proportion of studies pro-regenerative and/or immunomodulatory therapeutic effects were observed. Against the initial assumption, preclinical data demonstrated that if functional - MSCs act in a paracrine rather than in a cellular manner. Efforts to search for the active paracrine components ended in the identification of extracellular vesicle (EVs). Indeed, in several preclinical models EVs harvested from MSC conditioned media exerted comparable therapeutic effects than the MSCs themselves. Methods, Results & Conclusion: EVs released by virtually all cell types represent a heterogenous population of vesicles of different origin, including exosomes (70-150 nm), microvesicles (100-1000 nm), and apoptotic bodies (> 500 nm). Classically, they are prepared by differential centrifugation. Due to volume restricitions during ultracentrifugation only smaller amounts of EV containing liquids can be processed with this method. To efficiently translate MSC-EVs into the clinics, they need to be produced in a scaled manner. Previously, we established and successfully used a polyehtylen glycol (PEG) precipitation procedure allowing to process up to 10 l conditioned medium. Intending to further scale the MSC-EV production, optimally in a closed system, we have started to optimize tangential flow filtration (TFF) protocols, in principal being scalable up to several 1000 litres. At first, we compared different TFF devices and selected the holofiber-based TFF system. Although EVs can efficiently be prepared from serum-free conditioned media, the processing of serum or human platelet lysate (hPL) containing conditioned media remains challenging. The high protein content of serum and hPL supplemented media quickly results in protein aggregate formation and clogging of the pores of the TFF system, which negatively affect the EV purification process. Currently, by changing flow rates and and re-addition of buffers, we optimize the TFF protocols to enable us obtaining MSC-EV preparations with comparable purities than with the PEG method. 198 FREE FLOW ELECTROPHORESIS ALLOWS PREPARATION OF EXTRACELLULAR VESICLES WITH HIGH PURITY S. Staubach2,1, C. Reiter1, G. Weber1 & B. Giebel2 1 FFE Service GmbH, Feldkirchen, Germany, 2Institute for Transfusion Medicine, University Duisburg-Essen, Essen, NRW, Germany Background & Aim: Currently, it remains a challenge to prepare extracellular vesicles (EVs) to high purity. Neither fractionation by density nor by size alone is sufficient to separate EVs from all contaminants e.g. high and low density lipoprotein (HDL/LDL) and adherent proteins. For now, a time consuming combination of two methods (density and size separation) is required to enrich EVs to high purity at the expense of time and low recovery. A fast and efficient alternative is represented by Free Flow Electrophoresis (FFE). It is a well-established (semi-) preparative method to separate analytes with inherent difference of charge density and/or difference of pI-value. Methods, Results & Conclusion: A Free Flow Interval Zone Electrophoresis (FF-IZE) method has been developed, for the purification and isolation of EVs as well DNA and RNA from human plasma samples, using a set of