An rCHO cell line expressing recombinant human prourokinase （pro-UK） at the level of 5μg/ 10^6cells/d was cultivated on Cytopore cellulose porous microcarriers in a 7.5L Biostat CT stirred tank reactor. A periodic ...An rCHO cell line expressing recombinant human prourokinase （pro-UK） at the level of 5μg/ 10^6cells/d was cultivated on Cytopore cellulose porous microcarriers in a 7.5L Biostat CT stirred tank reactor. A periodic pressure oscillation of 0.04 MPa and 0.04 Hz was adopted to introduce a physical stimulus on the rCHO cells and to improve mass transfer characteristic between cells and medium in the process of porous microcarrier CHO cell culture. Compared to constant pressure culture, the oscillation culture didn＇t influence specific cell growth rate significantly, but could enhance the pro-UK specific production by 10% - 40%, and reduce production of lactate by 10% - 30%. In the perfusion culture of recombinant CHO cell with serum-free medium for 67 days, cell density could reach 2.64×10^7/ml, the maximal prourokinase concentration in harvested supernatant was about 118mg/L, a total of 21.1 grams of prourokinase was produced in 313 liters of supernatant. In conclusion, the perfusion cell culture with periodic pressure oscillation can enhance the production of recombinant protein and increase the reactor specific productivity.展开更多
Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcar...Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml. the cell density could reach 4 to 5 × 106 cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method. i. e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system. we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0. 5 to 3working volumes. Prourokinase was stably secreted.展开更多
CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system.We successfully cultivated CD-1 cells to a very high density (over1×107cells/ml). Prourokinase was stably s...CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system.We successfully cultivated CD-1 cells to a very high density (over1×107cells/ml). Prourokinase was stably secreted at about 180 IU/ 1e6 cells/24 h. Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers, then reatthch and proliferate on fresh microcarriers. This makes it very easy to scale up preduction. The microcarriers could be reused several times without affecting adhesion. proliferation and prourokinase secretion. With CMPECC membrane radial flow chromatography and MPG chromatography, the prourokinase in conditioned medium could be purified to a specific activity of 1×105 IU/mg of protein. The purification factor was about 600 fold, and approxiamately 90 % of the biological activity was recovered.展开更多
The fabrication of functional microcarriers capable of achieving in vivo-like three-dimensional cell culture is important for many tissue engineering applications. Here,inspired by the structure of Buddha beads, which...The fabrication of functional microcarriers capable of achieving in vivo-like three-dimensional cell culture is important for many tissue engineering applications. Here,inspired by the structure of Buddha beads, which are generally composed of moveable beads strung on a rope, we present novel cell microcarriers with controllable macropores and heterogeneous microstructures by using a capillary array microfluidic technology. Microfibers with a string of moveable and releasable microcarriers could be achieved by an immediate gelation reaction of sodium alginate spinning and subsequent polymerization of cell-dispersed gelatin methacrylate emulsification. The sizes of the microcarriers and their inner macropores could be well tailored by adjusting the flow rates of the microfluidic phases; this was of great importance in guaranteeing a sufficient supply of nutrients during cell culture. In addition, by infusing multiple cell-dispersed pregel solutions into the capillaries, the microcarriers with spatially heterogeneous cell encapsulations for mimicking physiological structures and functions could also be achieved.展开更多
Microcarriers have a demonstrated value for biomedical applications,in particular for drug delivery and three-dimensional cell culture.Attempts to develop this technique tend to focus on the fabrication of functional ...Microcarriers have a demonstrated value for biomedical applications,in particular for drug delivery and three-dimensional cell culture.Attempts to develop this technique tend to focus on the fabrication of functional microparticles by using convenient methods with innovative but accessible materials.Inspired by the process of boiling eggs in everyday life,which causes the solidification of egg proteins,we present a new microfluidic‘‘cooking"approach for the generation of egg-derived microcarriers for cell culture and drug delivery.As the egg emulsion droplets are formed with exquisite precision during the microfluidic emulsification,the resultant egg microcarriers present highly monodisperse and uniform morphologies at the size range of hundred microns to one millimeter.Benefiting from the excellent biocompatibility of the egg protein components,the obtained microcarriers showed good performances of cell adherence and growth.In addition,after a freezing treatment,the egg microcarriers were shown to have interconnected porous structures throughout their whole sphere,could absorb and load different kinds of drugs or other active molecules,and work as microcarrier-based delivery systems.These features point to the potential value of the microfluidic egg microcarriers in biomedicine.展开更多
Cell microcarriers have emerged as a powerful cell culture platform in biomedical areas, but their functions are usually limited to simply capturing and proliferating cells,because of the simplicity of their component...Cell microcarriers have emerged as a powerful cell culture platform in biomedical areas, but their functions are usually limited to simply capturing and proliferating cells,because of the simplicity of their components. Thus, in this study, we developed a new near-infrared（NIR） light-responsive graphene oxide（GO） hydrogel microcarrier system for controllable cell culture. The microcarriers were generated by using capillary microfluidics to emulsify the GO dispersed poly（N-isopropylacrylamide）（pNIPAM） and gelatin methacrylate（GelMA） pre-gel solution. The composite GO hydrogel microcarriers exhibited photothermally responsive cell capture, as well as the capacity for proliferation and release due to the NIR absorption of GO, the thermally responsive shape transition of pNIPAM, and the high biocompatibility of Gel MA. It was found that the NIR-responsive GO hydrogel microcarriers could prevent the cultured cells from being attacked by the immune system and promote the formation of tumor models in immunocompetent mice, which is desired for tumor and drug research. These features make the NIR-responsive GO hydrogel microcarriers excellent functional materials for different biomedical applications.展开更多
文摘An rCHO cell line expressing recombinant human prourokinase （pro-UK） at the level of 5μg/ 10^6cells/d was cultivated on Cytopore cellulose porous microcarriers in a 7.5L Biostat CT stirred tank reactor. A periodic pressure oscillation of 0.04 MPa and 0.04 Hz was adopted to introduce a physical stimulus on the rCHO cells and to improve mass transfer characteristic between cells and medium in the process of porous microcarrier CHO cell culture. Compared to constant pressure culture, the oscillation culture didn＇t influence specific cell growth rate significantly, but could enhance the pro-UK specific production by 10% - 40%, and reduce production of lactate by 10% - 30%. In the perfusion culture of recombinant CHO cell with serum-free medium for 67 days, cell density could reach 2.64×10^7/ml, the maximal prourokinase concentration in harvested supernatant was about 118mg/L, a total of 21.1 grams of prourokinase was produced in 313 liters of supernatant. In conclusion, the perfusion cell culture with periodic pressure oscillation can enhance the production of recombinant protein and increase the reactor specific productivity.
文摘Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml. the cell density could reach 4 to 5 × 106 cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method. i. e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system. we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0. 5 to 3working volumes. Prourokinase was stably secreted.
文摘CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system.We successfully cultivated CD-1 cells to a very high density (over1×107cells/ml). Prourokinase was stably secreted at about 180 IU/ 1e6 cells/24 h. Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers, then reatthch and proliferate on fresh microcarriers. This makes it very easy to scale up preduction. The microcarriers could be reused several times without affecting adhesion. proliferation and prourokinase secretion. With CMPECC membrane radial flow chromatography and MPG chromatography, the prourokinase in conditioned medium could be purified to a specific activity of 1×105 IU/mg of protein. The purification factor was about 600 fold, and approxiamately 90 % of the biological activity was recovered.
基金supported by the National Natural Science Foundation of China(21473029 and 51522302)the NSAF Foundation of China(U1530260)+3 种基金the Natural Science Foundation of Jiangsu(BK20140028)the Program for New Century Excellent Talents in Universitythe Scientific Research Foundation of Southeast Universitythe Scientific Research Foundation of Graduate School of Southeast University
文摘The fabrication of functional microcarriers capable of achieving in vivo-like three-dimensional cell culture is important for many tissue engineering applications. Here,inspired by the structure of Buddha beads, which are generally composed of moveable beads strung on a rope, we present novel cell microcarriers with controllable macropores and heterogeneous microstructures by using a capillary array microfluidic technology. Microfibers with a string of moveable and releasable microcarriers could be achieved by an immediate gelation reaction of sodium alginate spinning and subsequent polymerization of cell-dispersed gelatin methacrylate emulsification. The sizes of the microcarriers and their inner macropores could be well tailored by adjusting the flow rates of the microfluidic phases; this was of great importance in guaranteeing a sufficient supply of nutrients during cell culture. In addition, by infusing multiple cell-dispersed pregel solutions into the capillaries, the microcarriers with spatially heterogeneous cell encapsulations for mimicking physiological structures and functions could also be achieved.
基金supported by the National Natural Science Foundation of China (21473029, 51522302)the NSAF Foundation of China (U1530260)+2 种基金the Natural Science Foundation of Jiangsu Province (BK20140028)the Program for New Century Excellent Talents in Universitythe Scientific Research Foundation of Southeast University
文摘Microcarriers have a demonstrated value for biomedical applications,in particular for drug delivery and three-dimensional cell culture.Attempts to develop this technique tend to focus on the fabrication of functional microparticles by using convenient methods with innovative but accessible materials.Inspired by the process of boiling eggs in everyday life,which causes the solidification of egg proteins,we present a new microfluidic‘‘cooking"approach for the generation of egg-derived microcarriers for cell culture and drug delivery.As the egg emulsion droplets are formed with exquisite precision during the microfluidic emulsification,the resultant egg microcarriers present highly monodisperse and uniform morphologies at the size range of hundred microns to one millimeter.Benefiting from the excellent biocompatibility of the egg protein components,the obtained microcarriers showed good performances of cell adherence and growth.In addition,after a freezing treatment,the egg microcarriers were shown to have interconnected porous structures throughout their whole sphere,could absorb and load different kinds of drugs or other active molecules,and work as microcarrier-based delivery systems.These features point to the potential value of the microfluidic egg microcarriers in biomedicine.
基金supported by the National Natural Science Foundation of China (21473029 and 51522302)the NSAF Foundation of China (U1530260)+3 种基金the Scientific Research Foundation of Southeast Universitythe Scientific Research Foundation of Graduate School of Southeast Universitythe Postgraduate Research & Practice Innovation Program of Jiangsu Provincethe Fundamental Research Funds for the Central Universities
文摘Cell microcarriers have emerged as a powerful cell culture platform in biomedical areas, but their functions are usually limited to simply capturing and proliferating cells,because of the simplicity of their components. Thus, in this study, we developed a new near-infrared（NIR） light-responsive graphene oxide（GO） hydrogel microcarrier system for controllable cell culture. The microcarriers were generated by using capillary microfluidics to emulsify the GO dispersed poly（N-isopropylacrylamide）（pNIPAM） and gelatin methacrylate（GelMA） pre-gel solution. The composite GO hydrogel microcarriers exhibited photothermally responsive cell capture, as well as the capacity for proliferation and release due to the NIR absorption of GO, the thermally responsive shape transition of pNIPAM, and the high biocompatibility of Gel MA. It was found that the NIR-responsive GO hydrogel microcarriers could prevent the cultured cells from being attacked by the immune system and promote the formation of tumor models in immunocompetent mice, which is desired for tumor and drug research. These features make the NIR-responsive GO hydrogel microcarriers excellent functional materials for different biomedical applications.
