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Monitoring MSCs during expansion in a stirred tank bioreactor
S16
Poster abstracts
effects of FGF2 on BM-MSCs from 17 pediatric patients, cultured in the absence or presence of FGF2 as both heterogeneous bulk populations and colonies derived from single cells isolated since the initial BM mononuclear fraction. FGF2 induced morphologic changes in both bulk and cloned populations with cells showing smaller dimensions, more refractive appearance, reduced adherence to plastic support and reduced adhesion to each other. Both surface and mRNA expression of adhesion molecules such as VCAM1/CD106, ICAM1/CD54 and ALCAM/CD166 were down-regulated in FGF-dependent cultures in comparison to untreated ones, as studied by flow cytometry and quantitative RT-PCR. Moreover, FGF2 induced a significant dose-dependent down-regulation of CD90 expression which was paralleled by significant increase of surface HLA-DR and Class II major histocompatibility complex transactivator (CIITA) mRNA expression, as demonstrated by flow cytometry and RT-PCR, respectively. Despite this effect, FGF2-cultured MSCs were indeed negative for co-stimulatory molecules such as CD40, CD80 and CD86, and along with this, they were unable to stimulate allogeneic CD4+ T cells or allogeneic NK cells. Finally, in terms of differentiation, FGF2-cultured MSCs more easily differentiated towards cartilage than non FGF2-treated cells. These results suggest that FGF2 possess several unexpected activities on BM-MSCs and further studies are needed to understand the molecular pathways involved.
41 CONDITIONAL IMMUNOSUPRESSIVE PROPERTY OF MENSTRUAL BLOOD-DERIVED STEM CELLS IN VITRO P Luz-Crawford1, A Fernandez2, M Torres1, F Alcayaga1, L Salazar1, F Figueroa1, M Khoury1 1 Universidad de Los Andes, miRNA and stem cell laboratory, Santiago, Chile, 2Universidad de Andres Bello, Santiago, Chile Aim: Menstrual blood-derived stem cells (MenSCs) are a recently identified population of stem cells, exhibiting mesenchymal stem cell (MSC)-like properties with self-renewal and multipotency capacities. Furthermore, they showed an isolation, culture and expansion advantages. This study focus at comparatively characterizing MenSCs as opposed to the presently available MSC sources, with regard to immunoregulatory properties and then extend the investigation to unravel new mechanism involved in the observed effect. Methods: Menstrual fluids were collected from healthy donors and characterized according to their immunophenotype and differentiation potential. MenSCs and bone marrow-MSCs (BM-MSCs) were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) labeled with CFSE in the presence of PHA at different cell ratios. In parallel, MenSC and BM-MSCs were cultured in the presence of IFN-g and IL-1b and the production of different factors were assessed. Results: MenSCs inhibit the proliferative response of PBMCs in a dosedependent manner. At 1:10 ratio, they showed a comparable immunosuppression level as BM-MSCs, however, this effect was reversed at a lower ratio, where they stimulated the proliferation of T cells. In contrast, BM-MSCs maintained their immunosuppressive effect at the same ratio. Furthermore, IFN-g and IL-1b-activated MenSCs produced immunosuppressive factors such as IDO and PDL-1. However, the expression of these factors was 3-4 folds lower than BM-MSCs. Conclusion: MenSCs exert immunosuppressive effect on allogeneic PBMCs in a dose-dependent manner. However, in inflammatory conditions, they were unable to match the BM-MSCs for the production of immunomodulatory factors. These findings must be carefully taken into consideration for future clinical applications.
42 MONITORING MSCS DURING EXPANSION IN A STIRRED TANK BIOREACTOR J Murrell, S Punreddy, A Verma, K Mann, D Jing, D Kehoe, N Sunil, K Niss, M Rook EMD Millipore, Bedford, MA Human mesenchymal stromal/stem cells (hMSCs) are an attractive target for clinical study as therapeutic agents. However, multilayer flatbed culture expansion paradigms are cumbersome, time consuming and typically limited in the ability to monitor cell characteristics during the growth process. We described a new expansion paradigm that uses a scalable, single use, stirred tank bioreactor with microcarrier scaffold for hMSC expansion. In addition to ease of handling improvements and lower medium volume requirements, the bioreactor system enables sampling of the hMSCs at any time during the expansion campaign. The cells can then be assessed for a variety of markers that denote desired identity and purity, and for expected cell health status. Beyond the obvious cell viability assessment, apoptosis and cell cycle provide a toolbox of assays for cell health. Changes in the protein markers or cell health status can be used during process development to identify optimal conditions or as a process quality assessment following expansion. In this work, we demonstrate that the combination of cell health assays and protein markers for identity and purity are key assessment tools to support and characterize the expansion of hMSCs in a scalable bioreactor system.
43 HUMAN DERIVED ALTERNATIVES TO FETAL CALF SERUM IN CELL CULTURE K Ploederl1,2, K Höller1,2, A Lindenmair3,2, D Theiß1,2, J Kemptner1,2, F Hildner1,2, H Redl3,2, S Hennerbichler-Lugscheider1,2, C Gabriel1,2 1 Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria, 2Austrian Cluster for Tissue Regeneration, Austria, 3Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria Background: Cell expansion for clinical applications is mainly performed in the presence of fetal calf serum (FCS). As FCS is known to cause disease transmission and incorporation of xenogenic proteins during cultivation, alternative supplements for cell culture media are desired. In recent years, human serum (huS) and human platelet lysate (hPL) turned out to be as potential or even better when used instead of FCS. We established production processes for huS and two different types of hPL in the last years. Now their efficacy in application as cell culture supplement should be determined. Methods: Dermal fibroblasts and adipose tissue derived stem cells (ASC) were cultivated in the presence of 10% human serum (huS), 5% hPL in sodium chloride (hPLNaCl) or 5% hPL in plasma (hPL V1.2). hPL V1.2 is prepared from 36 pooled buffy coats upon centrifugation and a freeze-thaw cycle. The preparation of hPL V2 comprises two centrifugation steps and the plasma is replaced by sodium chloride in order to remove plasma proteins. For control purposes, cells were also expanded using 10% FCS. Growth curves and population doublings were determined. Concerning ASC, assays for chondrogenic, osteogenic and adipogenic differentiation were performed. Production of huS and hPL was controlled by residual white and red blood cells and microbiology testing. Additionally, samples for 2D-gel electrophoresis / MALDI-TOF were drawn. Results: Residual cell counts were in the normal range for every product. Results obtained from proteomic analysis will be presented. Growth curves for both cell lines already indicate that different cells have different preferences. Data concerning population doublings and differentiation capacity will be shown. Conclusion: Expansion of dermal fibroblasts and ASC obtained higher population doublings in the presence of hPL and huS. We therefore conclude that huS and hPL can replace FCS in cell culture for clinical applications.
44 INVESTIGATION OF THE ASSESSING METHODS ABOUT MESENCHYMAL STEM CELL TO CANCER CELL INTERACTION S Kim1,2 1 KFDA, Cheongwon-gun, Chuncheongbuk-do, Korea, Republic of, 2College of Life Sciences and Biotechnology, Korea University, Seoul, Korea, Republic of Figure 1. MenSC and BM-MSCs inhibit the proliferation of activated PBMC at 1:10 ratio, however, the effect is lost at a 1:100 ratio, while BMMSCs the immunosuppressive effect is maintained at the same ratio.
Many evidences are elucidating that specific paracrine factors secreted by mesenchymal stem cells (MSCs) affect the characteristics of the cancer cells.
Poster abstracts
effects of FGF2 on BM-MSCs from 17 pediatric patients, cultured in the absence or presence of FGF2 as both heterogeneous bulk populations and colonies derived from single cells isolated since the initial BM mononuclear fraction. FGF2 induced morphologic changes in both bulk and cloned populations with cells showing smaller dimensions, more refractive appearance, reduced adherence to plastic support and reduced adhesion to each other. Both surface and mRNA expression of adhesion molecules such as VCAM1/CD106, ICAM1/CD54 and ALCAM/CD166 were down-regulated in FGF-dependent cultures in comparison to untreated ones, as studied by flow cytometry and quantitative RT-PCR. Moreover, FGF2 induced a significant dose-dependent down-regulation of CD90 expression which was paralleled by significant increase of surface HLA-DR and Class II major histocompatibility complex transactivator (CIITA) mRNA expression, as demonstrated by flow cytometry and RT-PCR, respectively. Despite this effect, FGF2-cultured MSCs were indeed negative for co-stimulatory molecules such as CD40, CD80 and CD86, and along with this, they were unable to stimulate allogeneic CD4+ T cells or allogeneic NK cells. Finally, in terms of differentiation, FGF2-cultured MSCs more easily differentiated towards cartilage than non FGF2-treated cells. These results suggest that FGF2 possess several unexpected activities on BM-MSCs and further studies are needed to understand the molecular pathways involved.
41 CONDITIONAL IMMUNOSUPRESSIVE PROPERTY OF MENSTRUAL BLOOD-DERIVED STEM CELLS IN VITRO P Luz-Crawford1, A Fernandez2, M Torres1, F Alcayaga1, L Salazar1, F Figueroa1, M Khoury1 1 Universidad de Los Andes, miRNA and stem cell laboratory, Santiago, Chile, 2Universidad de Andres Bello, Santiago, Chile Aim: Menstrual blood-derived stem cells (MenSCs) are a recently identified population of stem cells, exhibiting mesenchymal stem cell (MSC)-like properties with self-renewal and multipotency capacities. Furthermore, they showed an isolation, culture and expansion advantages. This study focus at comparatively characterizing MenSCs as opposed to the presently available MSC sources, with regard to immunoregulatory properties and then extend the investigation to unravel new mechanism involved in the observed effect. Methods: Menstrual fluids were collected from healthy donors and characterized according to their immunophenotype and differentiation potential. MenSCs and bone marrow-MSCs (BM-MSCs) were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) labeled with CFSE in the presence of PHA at different cell ratios. In parallel, MenSC and BM-MSCs were cultured in the presence of IFN-g and IL-1b and the production of different factors were assessed. Results: MenSCs inhibit the proliferative response of PBMCs in a dosedependent manner. At 1:10 ratio, they showed a comparable immunosuppression level as BM-MSCs, however, this effect was reversed at a lower ratio, where they stimulated the proliferation of T cells. In contrast, BM-MSCs maintained their immunosuppressive effect at the same ratio. Furthermore, IFN-g and IL-1b-activated MenSCs produced immunosuppressive factors such as IDO and PDL-1. However, the expression of these factors was 3-4 folds lower than BM-MSCs. Conclusion: MenSCs exert immunosuppressive effect on allogeneic PBMCs in a dose-dependent manner. However, in inflammatory conditions, they were unable to match the BM-MSCs for the production of immunomodulatory factors. These findings must be carefully taken into consideration for future clinical applications.
42 MONITORING MSCS DURING EXPANSION IN A STIRRED TANK BIOREACTOR J Murrell, S Punreddy, A Verma, K Mann, D Jing, D Kehoe, N Sunil, K Niss, M Rook EMD Millipore, Bedford, MA Human mesenchymal stromal/stem cells (hMSCs) are an attractive target for clinical study as therapeutic agents. However, multilayer flatbed culture expansion paradigms are cumbersome, time consuming and typically limited in the ability to monitor cell characteristics during the growth process. We described a new expansion paradigm that uses a scalable, single use, stirred tank bioreactor with microcarrier scaffold for hMSC expansion. In addition to ease of handling improvements and lower medium volume requirements, the bioreactor system enables sampling of the hMSCs at any time during the expansion campaign. The cells can then be assessed for a variety of markers that denote desired identity and purity, and for expected cell health status. Beyond the obvious cell viability assessment, apoptosis and cell cycle provide a toolbox of assays for cell health. Changes in the protein markers or cell health status can be used during process development to identify optimal conditions or as a process quality assessment following expansion. In this work, we demonstrate that the combination of cell health assays and protein markers for identity and purity are key assessment tools to support and characterize the expansion of hMSCs in a scalable bioreactor system.
43 HUMAN DERIVED ALTERNATIVES TO FETAL CALF SERUM IN CELL CULTURE K Ploederl1,2, K Höller1,2, A Lindenmair3,2, D Theiß1,2, J Kemptner1,2, F Hildner1,2, H Redl3,2, S Hennerbichler-Lugscheider1,2, C Gabriel1,2 1 Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria, 2Austrian Cluster for Tissue Regeneration, Austria, 3Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria Background: Cell expansion for clinical applications is mainly performed in the presence of fetal calf serum (FCS). As FCS is known to cause disease transmission and incorporation of xenogenic proteins during cultivation, alternative supplements for cell culture media are desired. In recent years, human serum (huS) and human platelet lysate (hPL) turned out to be as potential or even better when used instead of FCS. We established production processes for huS and two different types of hPL in the last years. Now their efficacy in application as cell culture supplement should be determined. Methods: Dermal fibroblasts and adipose tissue derived stem cells (ASC) were cultivated in the presence of 10% human serum (huS), 5% hPL in sodium chloride (hPLNaCl) or 5% hPL in plasma (hPL V1.2). hPL V1.2 is prepared from 36 pooled buffy coats upon centrifugation and a freeze-thaw cycle. The preparation of hPL V2 comprises two centrifugation steps and the plasma is replaced by sodium chloride in order to remove plasma proteins. For control purposes, cells were also expanded using 10% FCS. Growth curves and population doublings were determined. Concerning ASC, assays for chondrogenic, osteogenic and adipogenic differentiation were performed. Production of huS and hPL was controlled by residual white and red blood cells and microbiology testing. Additionally, samples for 2D-gel electrophoresis / MALDI-TOF were drawn. Results: Residual cell counts were in the normal range for every product. Results obtained from proteomic analysis will be presented. Growth curves for both cell lines already indicate that different cells have different preferences. Data concerning population doublings and differentiation capacity will be shown. Conclusion: Expansion of dermal fibroblasts and ASC obtained higher population doublings in the presence of hPL and huS. We therefore conclude that huS and hPL can replace FCS in cell culture for clinical applications.
44 INVESTIGATION OF THE ASSESSING METHODS ABOUT MESENCHYMAL STEM CELL TO CANCER CELL INTERACTION S Kim1,2 1 KFDA, Cheongwon-gun, Chuncheongbuk-do, Korea, Republic of, 2College of Life Sciences and Biotechnology, Korea University, Seoul, Korea, Republic of Figure 1. MenSC and BM-MSCs inhibit the proliferation of activated PBMC at 1:10 ratio, however, the effect is lost at a 1:100 ratio, while BMMSCs the immunosuppressive effect is maintained at the same ratio.
Many evidences are elucidating that specific paracrine factors secreted by mesenchymal stem cells (MSCs) affect the characteristics of the cancer cells.