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Increasing yield of msc-evs in scalable xeno-free manufacturing
S58
Poster Abstract Presentations
buffer media of different pH-values ranging from pH 8.0 to pH 4.8. Upon processing supernatants of mesenchymal stem/stromal cells (MSCs) cultured in the presence of 10% human platelet lysate, EVs are recovered in a small number of FFE fractions lacking most proteins of the conditioned media. Currently, we characterize the identified fractions in more detail. Prepared EVs are quantified by Nano Particle Tracking Analysis (NTA) and imaging flow cytometry. Furthermore, the presence of several EV markers and the absence of contaminants are analyzed by Western Blot and mass spectrometry-based proteomics. We conclude FFE is a feasible and quick method to highly purify EVs in an accurate manner. 199 INCREASING YIELD OF MSC-EVS IN SCALABLE XENO-FREE MANUFACTURING K. Adlerz, M. Trempel, J.A. Rowley & T. Ahsan RoosterBio, Inc., Frederick, Maryland, United States Background & Aim: Extracellular vesicles (EVs) are increasingly being investigated as both drug delivery vehicles and as a cell-free therapy. EVs from mesenchymal stem/stromal cells (MSC-EVs), for example, have been shown to recapitulate many of the therapeutic effects of the cells themselves. A critical barrier in the development of MSC-EVs as a commercial therapy, however, is generating the large amount of EVs that will be required per dose. In a recent survey, the majority of those working with EVs were working with less than 100mL of sample (ISEV 2016). In this study, two strategies were investigated to maximize EV yield. First, timing parameters for culturing human bonemarrow derived MSCs and collecting MSC-EVs were evaluated to increase EV yield. Second, conditioning the cells was investigated by adding an EV activator during the EV collection period. Methods, Results & Conclusion: MSCs were first cultured in an expansion media (RoosterNourishTM, RoosterBio) in a fed-batch system. The cultures were then switched to a collection media (RoosterCollectTM-EV, RoosterBio) with or without an EV activator (EV BoostTM, RoosterBio). After two days, the conditioned media was harvested. The EVs in the collected conditioned media were analyzed for particle concentration and particle size distributions by nanoparticle tracking analysis, protein expression, RNA expression, and wound healing capability. Particle concentration (particles per L of media) was used as a measure of EV yield. Particle concentration increased with increased culture time in expansion media, with confluent cultures generating the most EVs. The EV activator at a low concentration increased EV yield about 2-fold compared to without the activator after 24hrs. The EV yield from the low EV activator culture at 24hrs was still about 45% greater than the yield of the culture without the activator after 48hrs. After 6hrs of collection, the high EV activator concentration increased EV yield to about the same level as without the activator after 48hrs of collection. Optimizing protocols for EV yield will become increasingly important as MSC-EVs move towards the clinic and lot sizes of clinically-relevant doses are required. Pre-conditioning of cells or conditioning cells during EV collection offers one possibility to further increase EV yield and decrease process time.
their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. It was recently found that the number of exosomes released from 2 million MSCs in 48 hours is equivalent to a single dose for a rodent (Phinney, Pittenger 2017). Hence, most indications would require an MSC production lot size that is hardly achievable in 2D planar cell culture; larger scalable bioreactor systems will be crucial to generate enough EVs for clinical doses. Therefore, this study compared the yield and characteristics of MSC-derived EVs generated in typical 2D culture with those from scalable 3D bioreactor systems. Methods, Results & Conclusion: Human bone marrow-derived MSCs were cultured on microcarriers in 3L and 15L bioreactors. After initial cell inoculation, a bioreactor feed was added on Day 3, and cultures were switched to an EV collection media on Day 4, then cultured for 3 additional days. Samples of the conditioned media were analyzed for particle size and concentration. The collected MSC-EVs were evaluated for protein and RNA expression and wound healing capability. MSC-EVs were compared between bioreactor scales and with a 2D control cultured in a 10-layer cell stack. Higher cell densities were achieved in 3D compared to 2D culture, with similar cell densities in the 3L and 15L bioreactors. The conditioned media was analyzed after the 3-day EV collection period. Particle size distributions were similar across culture systems. The particle concentration in the conditioned media (particles/L media) was similar between the 3L and 15L bioreactors, with the 3D cultures about 3x higher than 2D culture. When particle concentration was normalized to cell number, the productivity (particles/cell) was comparable in the two bioreactor cultures, with 3D cultures greater than 2D. Optimizing EV yield will become increasingly important as EVs are used in the clinic. We have developed a scalable process for MSC-EV generation in a bioreactor. The comparison between 2D and bioreactor cultures demonstrates that a greater EV yield can be accomplished in 3D culture. We intend to continue to scale this process up to production scale (80L) bioreactors, which is critical to generating lot sizes for clinically relevant doses of MSC-EVs.
Histograms, normalized to concentration, show EVs maintained similar size profiles across culture systems. The productivity of MSCs, measured as particles/cell, was higher in bioreactor systems than in 2D culture.
201 ACUTE ANAPHYLAXIS STUDY OF MESENCHYMAL STEM CELL-DERIVED EXOSOMES IN GUINEA PIGS G. Yoo1, A. Lee1, S. Park1, S. Han1, J. Kim1, S. Lee1, S. Na1, M. Yoo1, D. Kim1, Y. Cho2 & K. Moon1 1 Korea Institute of Toxicology, Daejeon, the Republic of Korea, 2Hanyang University ERICA, Ansan, the Republic of Korea
A Parameters for MSC expansion time and EV collection time were investigated in two donors. B The addition of an EV activator to the collection media increased EV productivity, similar particle level to 48hrs without EV activator was achieved at 24 hours with low EV activator and 6hrs with high EV activator. 200 COMPARISON OF MSC-EVS MANUFATURED IN 2D VERSUS SCALABLE 3D BIOREACTOR SYSTEMS K. Adlerz, M. Trempel, D. Wang, R.D. Kirian, J.A. Rowley & T. Ahsan RoosterBio, Inc., Frederick, Maryland, United States Background & Aim: There have been over 800 registered clinical trials using mesenchymal stem/stromal cells (MSCs) for therapeutic applications. Due to
Background & Aim: Mesenchymal stem cells (MSC) therapy in global regenerative medicine market has grown rapidly in the last decade. However, there are still limitations of MSC-based therapy, such as limited survival of stem cells and delivery approach to clinics. Recently, exosomes of MSC are proposed to be potential alternative to MSC therapy. MSC-derived exosomes are known to have similar therapeutic effects as its parental cells, while equipped with convenience of long term storage and easier laboratory manipulation. On the other hand, toxicological assessments of MSC-derived exosomes remain scarce. Methods, Results & Conclusion: Here, we used mesenchymal stem cell derived exosomes of defined quality to assess acute anaphylactic response in guinea pigs. For sensitization, guinea pigs were subcutaneously administered with low dosage (1 £ 108 particles/head) or high dosage (1 £ 109 particles/head) of MSC-derived exosome for thrice in the period of two weeks. Two weeks after the last exosome administration, animals were challenged by intravenous injection of the exosomes with high dosage (1 £ 109 particles/head) and then their anaphylactic responses were scored. We found
Poster Abstract Presentations
buffer media of different pH-values ranging from pH 8.0 to pH 4.8. Upon processing supernatants of mesenchymal stem/stromal cells (MSCs) cultured in the presence of 10% human platelet lysate, EVs are recovered in a small number of FFE fractions lacking most proteins of the conditioned media. Currently, we characterize the identified fractions in more detail. Prepared EVs are quantified by Nano Particle Tracking Analysis (NTA) and imaging flow cytometry. Furthermore, the presence of several EV markers and the absence of contaminants are analyzed by Western Blot and mass spectrometry-based proteomics. We conclude FFE is a feasible and quick method to highly purify EVs in an accurate manner. 199 INCREASING YIELD OF MSC-EVS IN SCALABLE XENO-FREE MANUFACTURING K. Adlerz, M. Trempel, J.A. Rowley & T. Ahsan RoosterBio, Inc., Frederick, Maryland, United States Background & Aim: Extracellular vesicles (EVs) are increasingly being investigated as both drug delivery vehicles and as a cell-free therapy. EVs from mesenchymal stem/stromal cells (MSC-EVs), for example, have been shown to recapitulate many of the therapeutic effects of the cells themselves. A critical barrier in the development of MSC-EVs as a commercial therapy, however, is generating the large amount of EVs that will be required per dose. In a recent survey, the majority of those working with EVs were working with less than 100mL of sample (ISEV 2016). In this study, two strategies were investigated to maximize EV yield. First, timing parameters for culturing human bonemarrow derived MSCs and collecting MSC-EVs were evaluated to increase EV yield. Second, conditioning the cells was investigated by adding an EV activator during the EV collection period. Methods, Results & Conclusion: MSCs were first cultured in an expansion media (RoosterNourishTM, RoosterBio) in a fed-batch system. The cultures were then switched to a collection media (RoosterCollectTM-EV, RoosterBio) with or without an EV activator (EV BoostTM, RoosterBio). After two days, the conditioned media was harvested. The EVs in the collected conditioned media were analyzed for particle concentration and particle size distributions by nanoparticle tracking analysis, protein expression, RNA expression, and wound healing capability. Particle concentration (particles per L of media) was used as a measure of EV yield. Particle concentration increased with increased culture time in expansion media, with confluent cultures generating the most EVs. The EV activator at a low concentration increased EV yield about 2-fold compared to without the activator after 24hrs. The EV yield from the low EV activator culture at 24hrs was still about 45% greater than the yield of the culture without the activator after 48hrs. After 6hrs of collection, the high EV activator concentration increased EV yield to about the same level as without the activator after 48hrs of collection. Optimizing protocols for EV yield will become increasingly important as MSC-EVs move towards the clinic and lot sizes of clinically-relevant doses are required. Pre-conditioning of cells or conditioning cells during EV collection offers one possibility to further increase EV yield and decrease process time.
their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. It was recently found that the number of exosomes released from 2 million MSCs in 48 hours is equivalent to a single dose for a rodent (Phinney, Pittenger 2017). Hence, most indications would require an MSC production lot size that is hardly achievable in 2D planar cell culture; larger scalable bioreactor systems will be crucial to generate enough EVs for clinical doses. Therefore, this study compared the yield and characteristics of MSC-derived EVs generated in typical 2D culture with those from scalable 3D bioreactor systems. Methods, Results & Conclusion: Human bone marrow-derived MSCs were cultured on microcarriers in 3L and 15L bioreactors. After initial cell inoculation, a bioreactor feed was added on Day 3, and cultures were switched to an EV collection media on Day 4, then cultured for 3 additional days. Samples of the conditioned media were analyzed for particle size and concentration. The collected MSC-EVs were evaluated for protein and RNA expression and wound healing capability. MSC-EVs were compared between bioreactor scales and with a 2D control cultured in a 10-layer cell stack. Higher cell densities were achieved in 3D compared to 2D culture, with similar cell densities in the 3L and 15L bioreactors. The conditioned media was analyzed after the 3-day EV collection period. Particle size distributions were similar across culture systems. The particle concentration in the conditioned media (particles/L media) was similar between the 3L and 15L bioreactors, with the 3D cultures about 3x higher than 2D culture. When particle concentration was normalized to cell number, the productivity (particles/cell) was comparable in the two bioreactor cultures, with 3D cultures greater than 2D. Optimizing EV yield will become increasingly important as EVs are used in the clinic. We have developed a scalable process for MSC-EV generation in a bioreactor. The comparison between 2D and bioreactor cultures demonstrates that a greater EV yield can be accomplished in 3D culture. We intend to continue to scale this process up to production scale (80L) bioreactors, which is critical to generating lot sizes for clinically relevant doses of MSC-EVs.
Histograms, normalized to concentration, show EVs maintained similar size profiles across culture systems. The productivity of MSCs, measured as particles/cell, was higher in bioreactor systems than in 2D culture.
201 ACUTE ANAPHYLAXIS STUDY OF MESENCHYMAL STEM CELL-DERIVED EXOSOMES IN GUINEA PIGS G. Yoo1, A. Lee1, S. Park1, S. Han1, J. Kim1, S. Lee1, S. Na1, M. Yoo1, D. Kim1, Y. Cho2 & K. Moon1 1 Korea Institute of Toxicology, Daejeon, the Republic of Korea, 2Hanyang University ERICA, Ansan, the Republic of Korea
A Parameters for MSC expansion time and EV collection time were investigated in two donors. B The addition of an EV activator to the collection media increased EV productivity, similar particle level to 48hrs without EV activator was achieved at 24 hours with low EV activator and 6hrs with high EV activator. 200 COMPARISON OF MSC-EVS MANUFATURED IN 2D VERSUS SCALABLE 3D BIOREACTOR SYSTEMS K. Adlerz, M. Trempel, D. Wang, R.D. Kirian, J.A. Rowley & T. Ahsan RoosterBio, Inc., Frederick, Maryland, United States Background & Aim: There have been over 800 registered clinical trials using mesenchymal stem/stromal cells (MSCs) for therapeutic applications. Due to
Background & Aim: Mesenchymal stem cells (MSC) therapy in global regenerative medicine market has grown rapidly in the last decade. However, there are still limitations of MSC-based therapy, such as limited survival of stem cells and delivery approach to clinics. Recently, exosomes of MSC are proposed to be potential alternative to MSC therapy. MSC-derived exosomes are known to have similar therapeutic effects as its parental cells, while equipped with convenience of long term storage and easier laboratory manipulation. On the other hand, toxicological assessments of MSC-derived exosomes remain scarce. Methods, Results & Conclusion: Here, we used mesenchymal stem cell derived exosomes of defined quality to assess acute anaphylactic response in guinea pigs. For sensitization, guinea pigs were subcutaneously administered with low dosage (1 £ 108 particles/head) or high dosage (1 £ 109 particles/head) of MSC-derived exosome for thrice in the period of two weeks. Two weeks after the last exosome administration, animals were challenged by intravenous injection of the exosomes with high dosage (1 £ 109 particles/head) and then their anaphylactic responses were scored. We found