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Will Not Be Presented
S40
Poster Abstracts
101 WILL NOT BE PRESENTED 102 ANALYSIS OF QUANTUM MOLECULAR RESONANCE EFFECTS ON HUMAN MESENCHYMAL STROMAL CELLS S. Sella1, E. Amati1, V. Adami2, P. Gatto2, G. Pozzato3, G. Astori1 1 Hematology and Cellular Therapy, Advanced Cellular Therapy Laboratory, Vicenza Hospital, Vicenza, Italy, 2High Throughput Screening Core Facility, Center for Integrative Biology, University of Trento, Trento, Italy, 3Telea Electronic Engineering SRL, Sandrigo, Italy Background: Endogenous electric fields play an essential role in cellular functions such as proliferation, migration and gene expression. Quantum Molecular Resonance (QMR) produces waves with a specific form at high frequencies and low intensity through electric fields without increase of temperature. Few is known about QMR mechanism of action on the inter/intracellular processes. This work aims to evaluate as QMR acts on bone marrow derived mesenchymal stromal cells (BM-MSC). Material and Methods: BM-MSC, were treated with QMR Technology (supplied and patented by Telea, Italy) for 10 minutes for 4 consecutive days/week for 2 weeks. Cell morphology, phenotype, viability, proliferation and migration were investigated. QMR effects on BM-MSC were further investigated by microarray after 1 week of stimulation. For the latter, samples were processed according to the “Agilent Gene Expression Analysis”. Data were analysed by using the Limma package (R language). Differentially expressed genes between conditions were selected based on a p-value cut-off of 0.05. Gene enrichment analysis was performed using ToppGene Suite and Ingenuity Pathway Analysis tools. Results: The observations related to morphology and phenotype suggested the maintenance of MSCs identity after 1 and 2 weeks of QMR treatment. Furthermore, no alteration of cellular viability, proliferation and migration were observed between samples and controls. At molecular level, the QMR treatment seemed to slightly affect the cell transcriptome: the identified up-regulated genes were mainly involved in cell tissue and vasculature development while the down-regulated genes were involved in cellular growth, phosphorylation, movement and anchoring. Conclusions: The QMR treatment did not lead to an alteration of cell phenotype maintaining the MSCs identity and growth. The microarray analysis supported the morphological evidence, showing no profound transcriptional differences between conditions. Anyway, the gene enrichment in tissue and vasculature development processes might suggest that QMR could activate angiogenesis. This concept requires more experimental evidences. 103 COST-EFFECTIVE CREATION OF BIOFUNCTIONALISED SCAFFOLDS, TAILORED TO FUNCTION AS STEM CELL NICHES FOR EXPANSION, TRANSPORT AND DELIVERY M. Bilek1, E. Kosobrodova1, E.A. Wakelin1, A. Kondyurin1, G. Yeo1,2, L. Martin1, D. McKenzie1, D.W. Hutmacher3, A.S. Weiss2 1 School of Physics, University of Sydney, Sydney, New South Wales, Australia, 2School of Molecular Biosciences, University of Sydney, Sydney, New South Wales, Australia, 3Institute of Health Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia Scaffold development in tissue engineering has explored the use of natural and synthetic hydrogels as well as thermoplastic polymers such as PCL which allow structures to be printed in 3D using technologies often referred to as additive manufacturing. Combining these platforms shows promise in creating hierarchical structures that mechanically mimic in-vivo tissue environments. Synthetic (eg thermoplastic) scaffold surfaces typically need to be modified to make them biocompatible and to allow functionalisation with biomolecules that communicate with cells via receptors in the cell membranes. Current technologies for functionalisation utilise multiple wet chemistry steps often involving complex protocols that require solvents and potentially toxic reactants making the processes commercially impractical and often unsuitable for regulatory approval. We have developed a dry plasma processing approach to activate complex interconnected 3D polymeric networks that enables subsequent direct covalent immobilisation of bioactive molecules from buffer solution. The bioactive molecules are applied simply and safely by immersion, spotting or painting of the protein/peptide solutions. Since the covalent immobilisation is facilitated
by reactive radicals [1] created in the scaffold structures by the plasma activation process, the protein solution pH can be varied to manipulate the orientation of the biological molecules as they arrive at the surface to be covalently coupled. Once covalently linked the preferred orientations are locked in and the functional epitopes exposed can interact with cells inhabiting the structures to provide biological cues. Reference [1] Bilek MM, et al. Proc Natl Acad Sci U S A 2011;108:14405–10. 104 USE OF STANDARDIZED AND CLINICAL GRADE HUMAN PLATELET LYSATE HYDROGEL (HPLG) AS A 3D STRUCTURE FOR THE OPTIMIZATION OF LARGE-SCALE EXPANSION OF HUMAN MESENCHYMAL STEM CELLS T. Bouckenooghe, P. Bertholet, L. Chabrand, S. Viau, B. Delorme Biotherapy, Macopharma, Mouvaux, France The amplification of stem cells, such as human Bone-Marrow Mesenchymal Stem Cells (MSC), in standardized and xeno-free conditions represents a major challenge for the production of safe and reproducible cell therapy products in clinical applications. Recently, human platelet lysate has emerged as a safe and efficient substitute for Fetal Calf Serum (FCS), a traditionally-used animal product showing risks of pathogens transmission and xenogeneic immune reactions. We evaluated the use of a standardized HPLG manufactured under GMP conditions as a 3D-supportive environment for large-scale expansion of MSC. In culture on HPLG, MSC were progressively degrading the gel, releasing the growth factors entrapped (VEGF, PDGF-AB, IGF-1, TGF-b1, bFGF) with specific kinetic profiles. PDGFR blocking experiments with antibodies demonstrated that PDGFs were preferential mitogens (rather than bFGF) used by MSC to proliferate on HPLG. Fluorescently labeled MSC seeded on HPLG surface were shown to migrate inside the gel during culture allowing 3D connections. Cell harvesting from HPLG was quicker and easier than on plastic using a clinical grade enzymatic solution. Comparing MSC’s expansion on plastic with a 10%FCS-bFGF supplemented-medium to a 10% HPLG without any medium supplementation, we demonstrated 1) On HPLG, on 3 consecutive passages, the maintenance of MSC proliferation rate around 90% when a 60% decrease was observed at passage 3 on plastic; 2) no modification for CD13, CD29, CD44, CD45 and CD73 expression; 3) an increase expression of CD90 on HPLG; 4) a similar osteogenic and adipogenic differentiation potential. Interestingly, without any medium change on a 10-days period, we observed that the total number of cells continued to efficiently increase on HPLG, while on plastic, proliferation stopped after D5. Taken together, our data clearly demonstrate that HPLG represents a new, secured and efficient way to amplify MSCs for cell therapy in an animal-free environment. 105 THE USE OF AUTOLOGOUS AND ALLOGENIC FIBRIN GLUES AS SCAFFOLDS FOR MESENCHYMAL STEM CELLS AND PRECHONDROCYTES IN TRAUMATOLOGY AND ORTHOPEDICS N. Enukashvily1,2, A. Ayzenshtadt2,3, V. Bagaeva2, T. Zolina2, L. Aleksandrova2, O. Supilnikova2,3, S. Adilov2 1 Institute of Cytology, St. Petersburg, Russian Federation, 2Cell Cultures Dept, Stem Cell Bank Pokrovsky, Ltd, St. Petersburg, Russian Federation, 3 Cell Technology Lab, Northwest Medical University Named After Mechnikov, St. Petersburg, Russian Federation Mesenchymal stem cells (MSC) and MSC-derived prechondrocytes are an effective tool for the regeneration and maintenance of articular cartilage. The MSCbased strategies include use of MSC both as progenitor cells for cartilage implants, and as producers of bioactive factors in the damaged joint. Delivery of MSC and MSC-derived prechondrocytes into joint might be attained by direct intraarticular implantation of cells either suspended in liquid or immobilized in special constructs—scaffolds. Scaffolds based on fibrin glue (FG) are used because of the glue high biocompatibility, biodegradability, injectability, and ease of handling. Usually FG is made from commercial kits (e.g. Tissucol®, Tisseel®, Greenplast kit®). However FG made from autologous plasma or allogenic cord blood plasma cryoprecypitates can be considered as a better choice in some cases.
Poster Abstracts
101 WILL NOT BE PRESENTED 102 ANALYSIS OF QUANTUM MOLECULAR RESONANCE EFFECTS ON HUMAN MESENCHYMAL STROMAL CELLS S. Sella1, E. Amati1, V. Adami2, P. Gatto2, G. Pozzato3, G. Astori1 1 Hematology and Cellular Therapy, Advanced Cellular Therapy Laboratory, Vicenza Hospital, Vicenza, Italy, 2High Throughput Screening Core Facility, Center for Integrative Biology, University of Trento, Trento, Italy, 3Telea Electronic Engineering SRL, Sandrigo, Italy Background: Endogenous electric fields play an essential role in cellular functions such as proliferation, migration and gene expression. Quantum Molecular Resonance (QMR) produces waves with a specific form at high frequencies and low intensity through electric fields without increase of temperature. Few is known about QMR mechanism of action on the inter/intracellular processes. This work aims to evaluate as QMR acts on bone marrow derived mesenchymal stromal cells (BM-MSC). Material and Methods: BM-MSC, were treated with QMR Technology (supplied and patented by Telea, Italy) for 10 minutes for 4 consecutive days/week for 2 weeks. Cell morphology, phenotype, viability, proliferation and migration were investigated. QMR effects on BM-MSC were further investigated by microarray after 1 week of stimulation. For the latter, samples were processed according to the “Agilent Gene Expression Analysis”. Data were analysed by using the Limma package (R language). Differentially expressed genes between conditions were selected based on a p-value cut-off of 0.05. Gene enrichment analysis was performed using ToppGene Suite and Ingenuity Pathway Analysis tools. Results: The observations related to morphology and phenotype suggested the maintenance of MSCs identity after 1 and 2 weeks of QMR treatment. Furthermore, no alteration of cellular viability, proliferation and migration were observed between samples and controls. At molecular level, the QMR treatment seemed to slightly affect the cell transcriptome: the identified up-regulated genes were mainly involved in cell tissue and vasculature development while the down-regulated genes were involved in cellular growth, phosphorylation, movement and anchoring. Conclusions: The QMR treatment did not lead to an alteration of cell phenotype maintaining the MSCs identity and growth. The microarray analysis supported the morphological evidence, showing no profound transcriptional differences between conditions. Anyway, the gene enrichment in tissue and vasculature development processes might suggest that QMR could activate angiogenesis. This concept requires more experimental evidences. 103 COST-EFFECTIVE CREATION OF BIOFUNCTIONALISED SCAFFOLDS, TAILORED TO FUNCTION AS STEM CELL NICHES FOR EXPANSION, TRANSPORT AND DELIVERY M. Bilek1, E. Kosobrodova1, E.A. Wakelin1, A. Kondyurin1, G. Yeo1,2, L. Martin1, D. McKenzie1, D.W. Hutmacher3, A.S. Weiss2 1 School of Physics, University of Sydney, Sydney, New South Wales, Australia, 2School of Molecular Biosciences, University of Sydney, Sydney, New South Wales, Australia, 3Institute of Health Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia Scaffold development in tissue engineering has explored the use of natural and synthetic hydrogels as well as thermoplastic polymers such as PCL which allow structures to be printed in 3D using technologies often referred to as additive manufacturing. Combining these platforms shows promise in creating hierarchical structures that mechanically mimic in-vivo tissue environments. Synthetic (eg thermoplastic) scaffold surfaces typically need to be modified to make them biocompatible and to allow functionalisation with biomolecules that communicate with cells via receptors in the cell membranes. Current technologies for functionalisation utilise multiple wet chemistry steps often involving complex protocols that require solvents and potentially toxic reactants making the processes commercially impractical and often unsuitable for regulatory approval. We have developed a dry plasma processing approach to activate complex interconnected 3D polymeric networks that enables subsequent direct covalent immobilisation of bioactive molecules from buffer solution. The bioactive molecules are applied simply and safely by immersion, spotting or painting of the protein/peptide solutions. Since the covalent immobilisation is facilitated
by reactive radicals [1] created in the scaffold structures by the plasma activation process, the protein solution pH can be varied to manipulate the orientation of the biological molecules as they arrive at the surface to be covalently coupled. Once covalently linked the preferred orientations are locked in and the functional epitopes exposed can interact with cells inhabiting the structures to provide biological cues. Reference [1] Bilek MM, et al. Proc Natl Acad Sci U S A 2011;108:14405–10. 104 USE OF STANDARDIZED AND CLINICAL GRADE HUMAN PLATELET LYSATE HYDROGEL (HPLG) AS A 3D STRUCTURE FOR THE OPTIMIZATION OF LARGE-SCALE EXPANSION OF HUMAN MESENCHYMAL STEM CELLS T. Bouckenooghe, P. Bertholet, L. Chabrand, S. Viau, B. Delorme Biotherapy, Macopharma, Mouvaux, France The amplification of stem cells, such as human Bone-Marrow Mesenchymal Stem Cells (MSC), in standardized and xeno-free conditions represents a major challenge for the production of safe and reproducible cell therapy products in clinical applications. Recently, human platelet lysate has emerged as a safe and efficient substitute for Fetal Calf Serum (FCS), a traditionally-used animal product showing risks of pathogens transmission and xenogeneic immune reactions. We evaluated the use of a standardized HPLG manufactured under GMP conditions as a 3D-supportive environment for large-scale expansion of MSC. In culture on HPLG, MSC were progressively degrading the gel, releasing the growth factors entrapped (VEGF, PDGF-AB, IGF-1, TGF-b1, bFGF) with specific kinetic profiles. PDGFR blocking experiments with antibodies demonstrated that PDGFs were preferential mitogens (rather than bFGF) used by MSC to proliferate on HPLG. Fluorescently labeled MSC seeded on HPLG surface were shown to migrate inside the gel during culture allowing 3D connections. Cell harvesting from HPLG was quicker and easier than on plastic using a clinical grade enzymatic solution. Comparing MSC’s expansion on plastic with a 10%FCS-bFGF supplemented-medium to a 10% HPLG without any medium supplementation, we demonstrated 1) On HPLG, on 3 consecutive passages, the maintenance of MSC proliferation rate around 90% when a 60% decrease was observed at passage 3 on plastic; 2) no modification for CD13, CD29, CD44, CD45 and CD73 expression; 3) an increase expression of CD90 on HPLG; 4) a similar osteogenic and adipogenic differentiation potential. Interestingly, without any medium change on a 10-days period, we observed that the total number of cells continued to efficiently increase on HPLG, while on plastic, proliferation stopped after D5. Taken together, our data clearly demonstrate that HPLG represents a new, secured and efficient way to amplify MSCs for cell therapy in an animal-free environment. 105 THE USE OF AUTOLOGOUS AND ALLOGENIC FIBRIN GLUES AS SCAFFOLDS FOR MESENCHYMAL STEM CELLS AND PRECHONDROCYTES IN TRAUMATOLOGY AND ORTHOPEDICS N. Enukashvily1,2, A. Ayzenshtadt2,3, V. Bagaeva2, T. Zolina2, L. Aleksandrova2, O. Supilnikova2,3, S. Adilov2 1 Institute of Cytology, St. Petersburg, Russian Federation, 2Cell Cultures Dept, Stem Cell Bank Pokrovsky, Ltd, St. Petersburg, Russian Federation, 3 Cell Technology Lab, Northwest Medical University Named After Mechnikov, St. Petersburg, Russian Federation Mesenchymal stem cells (MSC) and MSC-derived prechondrocytes are an effective tool for the regeneration and maintenance of articular cartilage. The MSCbased strategies include use of MSC both as progenitor cells for cartilage implants, and as producers of bioactive factors in the damaged joint. Delivery of MSC and MSC-derived prechondrocytes into joint might be attained by direct intraarticular implantation of cells either suspended in liquid or immobilized in special constructs—scaffolds. Scaffolds based on fibrin glue (FG) are used because of the glue high biocompatibility, biodegradability, injectability, and ease of handling. Usually FG is made from commercial kits (e.g. Tissucol®, Tisseel®, Greenplast kit®). However FG made from autologous plasma or allogenic cord blood plasma cryoprecypitates can be considered as a better choice in some cases.