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Antonios G. Mikos - One of the best experts on this subject based on the ideXlab platform.
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flow Perfusion Culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics
Annals of Biomedical Engineering, 2005Co-Authors: Heidi L. Holtorf, John A. Jansen, Tiffany L Sheffield, Catherine G Ambrose, Antonios G. MikosAbstract:Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow Perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, Cultured for up to 16 days in static or flow Perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow Perfusion Culture whereas they were located only along the scaffold perimeter in static Culture. At all Culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically Cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the Culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow Perfusion bioreactor for bone tissue engineering applications.
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Flow Perfusion Culture induces the osteoblastic differentiation of marrow stromal cell‐scaffold constructs in the absence of dexamethasone
Journal of Biomedical Materials Research Part A, 2005Co-Authors: Heidi L. Holtorf, John A. Jansen, Antonios G. MikosAbstract:Flow Perfusion Culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static Culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow Perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow Perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were Cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs Cultured under flow Perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell Culture medium under flow Perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow Perfusion Culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow Perfusion Culture are used.
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Flow Perfusion Culture induces the osteoblastic differentiation of marrow stromal cell-scaffold constructs in the absence of dexamethasone
Journal of Biomedical Materials Research - Part A, 2005Co-Authors: Heidi L. Holtorf, John A. Jansen, Antonios G. MikosAbstract:Flow Perfusion Culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static Culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow Perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow Perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were Cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs Cultured under flow Perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell Culture medium under flow Perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow Perfusion Culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow Perfusion Culture are used.
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flow Perfusion Culture of marrow stromal osteoblasts in titanium fiber mesh
Journal of Biomedical Materials Research Part A, 2003Co-Authors: Juliette Van Den Dolder, John A. Jansen, Gregory N Bancroft, Vassilios I Sikavitsas, Paul H M Spauwen, Antonios G. MikosAbstract:The objective of this study was to evaluate the effect of two cell Culture techniques, static and flow Perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material. Thereafter, the constructs were Cultured under static conditions or in a flow Perfusion system for 4, 8, and 16 days. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity. Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections. Results showed an increase in DNA from day 4 to day 8 for the flow Perfusion system. At day 8, a significant enhancement in DNA content was observed for flow Perfusion Culture compared with static Culture conditions, but similar cell numbers were found for each Culture system at 16 days. Calcium measurements showed a large increase in calcium content of the meshes subjected to flow Perfusion at day 16. The SEM examination revealed that the 16-day samples subjected to flow Perfusion Culture were completely covered with layers of cells and mineralized matrix. In addition, this matrix extended deep into the scaffolds. In contrast, meshes Cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes. Evaluation of the light microscopy sections confirmed the SEM observations. On the basis of our results, we conclude that a flow Perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh.
Jean-françois P. Hamel - One of the best experts on this subject based on the ideXlab platform.
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Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG2a Monoclonal Antibody in a Wave Bioreactor‐Perfusion Culture System
Biotechnology Progress, 2008Co-Authors: Ya‐jie Tang, Ryo Ohashi, Jean-françois P. HamelAbstract:A novel wave bioreactor-Perfusion Culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based Perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the Culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based Perfusion controller was designed to balance the medium renewal rate and the harvest rate during Perfusion Culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch Culture. A control strategy for Perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during Perfusion Culture, and the optimal control strategy was increasing Perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing Perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and Perfusion Culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in Perfusion Culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch Culture. Relatively fast cell growth and the Perfusion Culture approach warrant that high biomass and mAb productivity may be obtained in such a novel Perfusion Culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell Culture. The fundamental information obtained in this study may be useful for Perfusion Culture of hybridoma cells on a large scale.
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a Perfusion Culture system using a stirred ceramic membrane reactor for hyperproduction of igg2a monoclonal antibody by hybridoma cells
Biotechnology Progress, 2008Co-Authors: Haodi Dong, Ryo Ohashi, Yajie Tang, Jean-françois P. HamelAbstract:A novel Perfusion Culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred-tank reactor during the Perfusion Culture. Furthermore, the significance of the control strategy of Perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (deltaD, vvd per day, vvdd) of Perfusion rate, the maximal viable cell density of 3.5 x 10(7) cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower deltaD's, the cell growth became limited. Under nutrient-limited condition, the specific cell growth rate (mu) was regulated by deltaD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 +/- 0.02 pg/cell x h in all runs. During the cell growth-limited phase, qmAb was regulated by deltaD within the range of 0.25-0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell x h was obtained during the growth-limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L x day and 340 mg/L x medium, respectively. This study demonstrated that this novel Perfusion Culture system for suspension mammalian cells can support high cell density and efficient mAb production and that deltaD is an important control parameter to regulate and achieve high mAb production.
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Perfusion Culture of hybridoma cells for hyperproduction of igg2a monoclonal antibody in a wave bioreactor Perfusion Culture system
Biotechnology Progress, 2007Co-Authors: Ryo Ohashi, Yajie Tang, Jean-françois P. HamelAbstract:A novel wave bioreactor-Perfusion Culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based Perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the Culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based Perfusion controller was designed to balance the medium renewal rate and the harvest rate during Perfusion Culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch Culture. A control strategy for Perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during Perfusion Culture, and the optimal control strategy was increasing Perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing Perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and Perfusion Culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in Perfusion Culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch Culture. Relatively fast cell growth and the Perfusion Culture approach warrant that high biomass and mAb productivity may be obtained in such a novel Perfusion Culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell Culture. The fundamental information obtained in this study may be useful for Perfusion Culture of hybridoma cells on a large scale.
Yajie Tang - One of the best experts on this subject based on the ideXlab platform.
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a Perfusion Culture system using a stirred ceramic membrane reactor for hyperproduction of igg2a monoclonal antibody by hybridoma cells
Biotechnology Progress, 2008Co-Authors: Haodi Dong, Ryo Ohashi, Yajie Tang, Jean-françois P. HamelAbstract:A novel Perfusion Culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred-tank reactor during the Perfusion Culture. Furthermore, the significance of the control strategy of Perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (deltaD, vvd per day, vvdd) of Perfusion rate, the maximal viable cell density of 3.5 x 10(7) cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower deltaD's, the cell growth became limited. Under nutrient-limited condition, the specific cell growth rate (mu) was regulated by deltaD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 +/- 0.02 pg/cell x h in all runs. During the cell growth-limited phase, qmAb was regulated by deltaD within the range of 0.25-0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell x h was obtained during the growth-limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L x day and 340 mg/L x medium, respectively. This study demonstrated that this novel Perfusion Culture system for suspension mammalian cells can support high cell density and efficient mAb production and that deltaD is an important control parameter to regulate and achieve high mAb production.
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Perfusion Culture of hybridoma cells for hyperproduction of igg2a monoclonal antibody in a wave bioreactor Perfusion Culture system
Biotechnology Progress, 2007Co-Authors: Ryo Ohashi, Yajie Tang, Jean-françois P. HamelAbstract:A novel wave bioreactor-Perfusion Culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based Perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the Culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based Perfusion controller was designed to balance the medium renewal rate and the harvest rate during Perfusion Culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch Culture. A control strategy for Perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during Perfusion Culture, and the optimal control strategy was increasing Perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing Perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and Perfusion Culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in Perfusion Culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch Culture. Relatively fast cell growth and the Perfusion Culture approach warrant that high biomass and mAb productivity may be obtained in such a novel Perfusion Culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell Culture. The fundamental information obtained in this study may be useful for Perfusion Culture of hybridoma cells on a large scale.
Michiyuki Tokashiki - One of the best experts on this subject based on the ideXlab platform.
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High Density Culture of Hybridoma Cells using a Perfusion Culture Apparatus with Multi-settling Zones
Production of Biologicals from Animal Cells in Culture, 2013Co-Authors: Michiyuki Tokashiki, Takami AraiAbstract:ABSTRACT Mouse-human hybridoma X32 cells were cultivated using a Perfusion Culture apparatus provided with three settling zones where the cells were separated from the Culture medium by gravitational settling, as oxygen was supplied using a perfluorocarbon. Viable cell density reached about 2 × 10 cells·ml–1 at 2.0 vol·vol–1·d–1 specific Perfusion rate, and the monoclonal antibody was continuously produced. This viable cell density was significantly higher than in the Perfusion Culture of a gravitational settling type provided with one settling zone, as oxygen was directly sparged (about 1 × 107 cell·ml–1).
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Large-scale Perfusion Culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones
Applied Microbiology and Biotechnology, 1996Co-Authors: Hiroyuki Takamatsu, K. Hamamoto, K. Ishimura, S. Yokoyama, Michiyuki TokashikiAbstract:A high-cell-density Perfusion Culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from Culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml Culture medium min^−1 at 100 g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this Culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 10^6 cells ml^−1 for 25 days. This centrifuge Culture shows promise as a large-scale Perfusion Culture process.
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Perfusion Culture apparatus for suspended mammalian cells
Cytotechnology, 1993Co-Authors: Michiyuki Tokashiki, Hiroyuki TakamatsuAbstract:A variety of processes have been proposed for mammalian cell Culture in the commercial production of useful substances (e.g., monoclonal antibodies, therapeutic and diagnostic proteins). Among them, the Perfusion Culture of suspended non-immobilized cells is the most advantageous. Perfusion Culture can be classified by the separation process of suspended cells from the Culture mixture into three types, namely filtration, gravitational settling and centrifugation. From a commercial point of view, the present situation and technical problems of suspended-cell Perfusion Culture will be reviewed based on the three types, The recent development of Perfusion Culture has been carried out mainly on the filtration separation process, but the centrifugation process seems to have a promising future because of operation stability and scale-up feasibility. The reasons will be explained in details.
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Transferrin recycling Perfusion Culture of hybridoma cells
Cytotechnology, 1992Co-Authors: Yoshiharu Takazawa, Michiyuki TokashikiAbstract:A Perfusion Culture of hybridoma cells in serum-free medium recycling transferrin was carried out, which greatly reduced the level of transferrin that was needed. The Culture was maintained even without supplying transferrin for nine days. IgG concentration reached 1.1 mg ml^−1 in a month of recycling and its ratio to the total protein was 45.8%. The affinity of the antibody did not decrease and no degradation was observed after long recycling period. The cell density under recycling condition was 2≈3 times higher than that without recycling. It was indicated that there was autocrine growth promoting activity in the Culture supernatant.
Heidi L. Holtorf - One of the best experts on this subject based on the ideXlab platform.
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flow Perfusion Culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics
Annals of Biomedical Engineering, 2005Co-Authors: Heidi L. Holtorf, John A. Jansen, Tiffany L Sheffield, Catherine G Ambrose, Antonios G. MikosAbstract:Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow Perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, Cultured for up to 16 days in static or flow Perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow Perfusion Culture whereas they were located only along the scaffold perimeter in static Culture. At all Culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically Cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the Culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow Perfusion bioreactor for bone tissue engineering applications.
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Flow Perfusion Culture induces the osteoblastic differentiation of marrow stromal cell‐scaffold constructs in the absence of dexamethasone
Journal of Biomedical Materials Research Part A, 2005Co-Authors: Heidi L. Holtorf, John A. Jansen, Antonios G. MikosAbstract:Flow Perfusion Culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static Culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow Perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow Perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were Cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs Cultured under flow Perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell Culture medium under flow Perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow Perfusion Culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow Perfusion Culture are used.
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Flow Perfusion Culture induces the osteoblastic differentiation of marrow stromal cell-scaffold constructs in the absence of dexamethasone
Journal of Biomedical Materials Research - Part A, 2005Co-Authors: Heidi L. Holtorf, John A. Jansen, Antonios G. MikosAbstract:Flow Perfusion Culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static Culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow Perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow Perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were Cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs Cultured under flow Perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell Culture medium under flow Perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow Perfusion Culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow Perfusion Culture are used.