采用Elephant Ear桨叶的生物反应器结构优化和细胞剪切特性研究
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摘要
随着生物技术由实验室研究向实用化和产业化的不断转化,细胞培养作为其重要的手段已成为许多国家争先研究的领域。目前,国内外对于细胞培养的研究不仅仅只停留在实验室规模,而是迈向工业级别的大规模培养,这就对细胞体外培养技术的核心装置生物反应器提出了更高的要求。通过对细胞大规模培养的影响因素进行分析,发现反应器内流场混合的均匀性和搅拌剪切力是制约细胞大规模培养的主要原因,因此把这两个因素作为研究方向,进行一系列的仿真计算,为细胞大规模培养生物反应器的研究设计提供资料和理论依据。
首先,采用CFD数值模拟方法分析一种新型的Elephant Ear桨叶,把它和三种常用的搅拌桨叶在流场混合均匀性和剪切力这两方面进行了仿真对比,得出该桨叶不仅产生的剪切力较小而且流场的混合也比较均匀,采用该桨叶作为大规模生物反应器的搅拌桨叶。
在此基础上,使用FLUENT仿真软件对采用Elephant Ear桨叶的100L生物反应器进行了结构优化,优化了反应器的高径比、反应器罐底半径、桨叶安装高度、挡板数量;同时对Elephant Ear桨叶也进行了仿真分析。
然后,运用有限元分析软件ABAQUS从宏观角度对CHO细胞在流场内的力学特性进行了仿真分析,得到了在剪切力作用下细胞产生的应力和变形规律;分析了培养液压力和剪切力复合作用下的细胞力学特性,认为剪切力是引起细胞变形破坏的主要原因。
最后,把CHO细胞在不同转速下的培养实验和仿真分析结合起来,得出最适合CHO细胞生长的流场剪切力为0.3Pa,对细胞在该剪切力作用下进行了仿真分析,得出细胞在U方向的最大变形位移为8.5μm。
With the continuous transformation of biotechnology from laboratory research to practical and industrial, cell culture technology which is the important means of it has become a rushed field of study in many countries. Currently cell culture studies does not just stop at the laboratory scale, but develops towards the large-scale industrial-level training both at home and abroad, therefore, the bioreactor which is the core device of cell culture technology is required higher. In this paper, based on the analysis of affected factors of large-scale cultivation of cells, we found the main reason for restricting cell mass culture, which is mixed flow reactor uniformity and mixing shear force, therefore, making the two factors as research directions, we carried out a series of simulation calculations and provide information and theoretical basis for the research and design of the large-scale cell culture bioreactor.
Firstly, using CFD simulation to analyze a new type of Elephant Ear impeller, and make a simulation comparison on it and the three commonly used impeller in the mixed flow uniformity and shear force, as a result, it founds that it not only produces less shear force but also produces more uniform flow mixing field, therefore, the impeller was used for the bioreactor.
On this basis, using the simulation software FLUENT to optimize the structure of 100L bioreactor which is use Elephant Ear impeller, optimization of the reactor diameter ratio, bottom of the tank radius, impeller mounting height and the number of baffles; simultaneously, Elephant Ear impeller is analyzed too.
Secondly, using the finite element analysis software ABAQUS simulation analysis the mechanical properties of CHO cells in the flow field from a broad perspective, as a result, obtained the law of cells stress and deformation by Shear force; Analysis of the mechanical properties of cells which is in the medium pressure and shear stress, and get the conclusion that the shear stress is the main reason for deformation and damage of cell.
Finally, we combine the CHO cells cultured experiment under different speed test and simulation analysis, get the result that the most suitable shear stress for the growth of CHO cells in the shear flow field is 0.3Pa, simultaneously, simulate the effect of the shear stress on cells and get the result that the maximum deformation of cell in the U direction is 8.5μm.
首先,采用CFD数值模拟方法分析一种新型的Elephant Ear桨叶,把它和三种常用的搅拌桨叶在流场混合均匀性和剪切力这两方面进行了仿真对比,得出该桨叶不仅产生的剪切力较小而且流场的混合也比较均匀,采用该桨叶作为大规模生物反应器的搅拌桨叶。
在此基础上,使用FLUENT仿真软件对采用Elephant Ear桨叶的100L生物反应器进行了结构优化,优化了反应器的高径比、反应器罐底半径、桨叶安装高度、挡板数量;同时对Elephant Ear桨叶也进行了仿真分析。
然后,运用有限元分析软件ABAQUS从宏观角度对CHO细胞在流场内的力学特性进行了仿真分析,得到了在剪切力作用下细胞产生的应力和变形规律;分析了培养液压力和剪切力复合作用下的细胞力学特性,认为剪切力是引起细胞变形破坏的主要原因。
最后,把CHO细胞在不同转速下的培养实验和仿真分析结合起来,得出最适合CHO细胞生长的流场剪切力为0.3Pa,对细胞在该剪切力作用下进行了仿真分析,得出细胞在U方向的最大变形位移为8.5μm。
With the continuous transformation of biotechnology from laboratory research to practical and industrial, cell culture technology which is the important means of it has become a rushed field of study in many countries. Currently cell culture studies does not just stop at the laboratory scale, but develops towards the large-scale industrial-level training both at home and abroad, therefore, the bioreactor which is the core device of cell culture technology is required higher. In this paper, based on the analysis of affected factors of large-scale cultivation of cells, we found the main reason for restricting cell mass culture, which is mixed flow reactor uniformity and mixing shear force, therefore, making the two factors as research directions, we carried out a series of simulation calculations and provide information and theoretical basis for the research and design of the large-scale cell culture bioreactor.
Firstly, using CFD simulation to analyze a new type of Elephant Ear impeller, and make a simulation comparison on it and the three commonly used impeller in the mixed flow uniformity and shear force, as a result, it founds that it not only produces less shear force but also produces more uniform flow mixing field, therefore, the impeller was used for the bioreactor.
On this basis, using the simulation software FLUENT to optimize the structure of 100L bioreactor which is use Elephant Ear impeller, optimization of the reactor diameter ratio, bottom of the tank radius, impeller mounting height and the number of baffles; simultaneously, Elephant Ear impeller is analyzed too.
Secondly, using the finite element analysis software ABAQUS simulation analysis the mechanical properties of CHO cells in the flow field from a broad perspective, as a result, obtained the law of cells stress and deformation by Shear force; Analysis of the mechanical properties of cells which is in the medium pressure and shear stress, and get the conclusion that the shear stress is the main reason for deformation and damage of cell.
Finally, we combine the CHO cells cultured experiment under different speed test and simulation analysis, get the result that the most suitable shear stress for the growth of CHO cells in the shear flow field is 0.3Pa, simultaneously, simulate the effect of the shear stress on cells and get the result that the maximum deformation of cell in the U direction is 8.5μm.
引文
1何辉,樊瑜波.动物细胞培养用生物反应器及其力学环境.生物医学工程学杂志, 2004, 21(3): 498~501
2 Hu WS, Aunins JG. Large-scale mammalian cell culture. Current Opinion in Biotechnology, 1997, 8: 148
3林福玉,陈昭烈,刘红等.大规模动物细胞培养的问题及对策.生物技术通报. 1999, (1): 32~35
4赵佼,谭文松,俞俊棠.动物细胞培养工程的现状与展望.华东理工大学学报. 1997, 4: 131～136
5 L. Hacker, M. D. Jesus. 25 Years of Recombinant Proteins from Reactor-grown Cells. Biotechnology Advances. 2009, 5: 1~5
6 Goutams, Alok B, Satyabrotos. Modeling of the oxygen transfer characteristics of a‘see-saw’bioreactor. Chemical Engineering & Technology. 2001, 24(1): 97~101
7 Zhaolie C, Dirk L, Kai I J. High-density culture of recombinant Chinese hamster overay cells producing prothrombin in protein-free medium. Biotechnology Letters. 2008, 23(10): 767~770
8 Liu CM, Hong L N. Development of a shaking bioreactor system for animal cell cultures. Biochemical Engineering Journal. 2001, 7(2) :121~125
9陈洪章,李佐虎.生物反应器工程.生物工程进展.1998, l(18): 4～8
10张嗣良.生物技术产业化与微生物过程优化.药品评价. 2004, 1: 99~105
11张恂,张伟平,张嗣良.用于生物反应器多尺度问题研究的数据采集系统.华东理工大学学报. 2003, (29): 259~263
12 Zhangsilian, Chuju, Zhang Yingping. A multi-scale study of industrial fermentation professor and their optimization. Advanced Biochemical Engineer. 2004, 87:97~105
13邹寿长,李干祥,杨葆生等.大规模动物细胞培养技术研究进展.生命科学研究. 2001, 6(5): 2
14钱凯先.人体与动物细胞大量培养的进展.生物技术. 1993; 3(6): 1~3
15 H. Miyoko, S. George, C. Joy. Transplantation of Engineered Bone Tissue Using a Rotary Three-dimensional Culture System. In Vitro Cellular & Developmental Biology-Animal. 2007, 42(2): 49～58.
16 F. Meuwly, F. Loviat. Oxygen Supply for CHO Cells Immobilized on a Packed-bed of Fibra-Cell Disks. Biotechnology and Bioengineering. 2006, 93(4): 791～800
17 Zeng B. J. Self-organization and specific expression of genes. First International Conference and Third National Conference on Transgenic Animals, Beijing, Nov.1996, 11: 18~23
18张嗣良,张徇,唐寅等.发展我国大规模细胞培养生物反应器装备制造业.中国生物工程杂志. 2005, 25(7): 1~8
19白力,张淑香,唐寅,郭美锦等.锥底生物反应器的动物细胞培养.华东理工大学学报(自然科学版). 2008, 6(34): 338~341
20李松涛.基于计算流体力学的生物反应器流场模拟及结构优化.哈尔滨工业大学硕士学位论文. 2008, 7: 3
21 Papoutsakis ET. Fluid mechanical damage of animal cells inbioreactors . Trends in Biotechnology. 1991, 9 (12): 427
22福军.计算流体动力学分析. 1.清华大学出版社, 2004, 7: 1~4
23孙庆丰.搅拌釜式生物反应器设计及优化.哈尔滨工业大学硕士学位论文. 2008, 7: 20
24 Luo J.V, Gosman A.D, Issa R.I, Middle J.C, Fitzgerald M.K. Full Flow Field Computation of Mixing in Baffled Stirred Reactors. Trans. I. Chem. E. 1993, 71A: 42~344
25 J.P.Van Doormal, G. G. Raithby. Enhancement of the SIMPLE method for predicting incompressible fluid flows. Numerical Heat Transfer. 1984, 7: 147~163
26 Brucato A.Ciofalo M.Grisafi F.Micale G. Numerical prediction of flow fields in baffled stirred vessels. A comparison of alternative modeling approaches. 1998, 7: 5
27马鑫.搅拌桨叶表面应力的测量及分析.北京化工大学硕士学位论文. 2000, 7: 51~53
28 Hui Zhu, Alvin W. Nienow, Waldek Bujalski, Mark J.H. Simmons. Mixing studies in a model aerated bioreactor equipped with Elephant Ear agitator. Chemical Engineering Research and Design. 2009, 87: 307~317
29 H. Wu, P. K. Patterson. Laser-doppler Measurements of Turbulent-flow Parameters in a Stirred Tank. Chem.Eng.Sci. 1989, 10: 2207~2221
30任杰.搅拌反应器流场与动力性能的模拟及实验研究.郑州大学硕士论文. 2007, 5: 3~5
31樊学军.细胞力学.力学进展. 1995, 5(25): 2
32付志一,焦群英.植物细胞力学模型研究进展.力学进展. 2005, 8(35): 3
33 Benjamin Geiger.Environmental sensing through focal adhesions. Molecular Cell Biology of Nature Reviews. 2009(10): 21~33
34程琴.基于有限元方法的三维细胞模型与力学分析.重庆大学硕士学位论文. 2009, 4: 3~34
35 Kawase Y, Halard B,Moo-Yong M. Liquid-phase mass transfer coefficients in bioreactors. Biotechnol & Bioeng. 1992, 39: 1133~1140
36 Jhanvi H.Macrorheology and adaptive microrheology of endothelial cells subjected to fluid shear stress. Am J Physiol Cell Physiol. 2007, 293: 1568~1575
37李晓宁,樊瑜波.剪切力环境下内皮细胞力学研究的形态学指标.生物医学工程学杂志. 2003, 20(3): 555~558
38商桂敏,施中东等.剪切应力对红豆杉细胞悬浮培养的影响及CFD模拟研究.高枝化学工程学报. 2002, 10(16): 5
39 Leckie F.Scragg A H.Cliff K R Effect of bioreactor design and agitator speed on the growth and alkaloid accumulation by cultures of Catharanthus roseus. Enzyme Microb Technol .1991, 13: 296~305
40 Scragg A H. Allan EJ, Leckie F. Effect of shear on the viability of plant cell suspensions . Enzyme Microb Technol. 1988, 10: 361~367
41 Hooker B S, Lee J M, An G. Cultivation of plant cells in a stirred vessel: Effect of impeller design. Biotechnol Bioeng. 1990, 35: 296~304
42 Meijer J J, ten Hoopen H J G,van Gameren Y M,Luyben K Ch A M, Libbenga K R. Effects of hydrodynamic stress on the growth of Plant cells in batch and continuous culture. Enzyme and Microb Technol. 1994, 16: 467~477
43 WangerF, volgelmann H. PIant Tissue Culture and its Biotechnological Application. Berlin:Springer-verlag. 1977, 245~255
44 Brucato A,Ciofalo M.Grisafi F, Micale G. Numerical predication of flow fields in baffeld stirred vessels. A companson of alternative modeling approaches. Chem Eng Sci. 1998, 53: 3653~3684
45谭文松,戴干策,陈宏志.生物反应器中杂交瘤细胞的物理破损.华东理工大学学报, 1996, 22 (3): 276~280
46吴金辉,张西正等.用于组织工程化培养生物反应器的研究进展.国外医学生物医学工程分册. 2003, (26): 2
2 Hu WS, Aunins JG. Large-scale mammalian cell culture. Current Opinion in Biotechnology, 1997, 8: 148
3林福玉,陈昭烈,刘红等.大规模动物细胞培养的问题及对策.生物技术通报. 1999, (1): 32~35
4赵佼,谭文松,俞俊棠.动物细胞培养工程的现状与展望.华东理工大学学报. 1997, 4: 131～136
5 L. Hacker, M. D. Jesus. 25 Years of Recombinant Proteins from Reactor-grown Cells. Biotechnology Advances. 2009, 5: 1~5
6 Goutams, Alok B, Satyabrotos. Modeling of the oxygen transfer characteristics of a‘see-saw’bioreactor. Chemical Engineering & Technology. 2001, 24(1): 97~101
7 Zhaolie C, Dirk L, Kai I J. High-density culture of recombinant Chinese hamster overay cells producing prothrombin in protein-free medium. Biotechnology Letters. 2008, 23(10): 767~770
8 Liu CM, Hong L N. Development of a shaking bioreactor system for animal cell cultures. Biochemical Engineering Journal. 2001, 7(2) :121~125
9陈洪章,李佐虎.生物反应器工程.生物工程进展.1998, l(18): 4～8
10张嗣良.生物技术产业化与微生物过程优化.药品评价. 2004, 1: 99~105
11张恂,张伟平,张嗣良.用于生物反应器多尺度问题研究的数据采集系统.华东理工大学学报. 2003, (29): 259~263
12 Zhangsilian, Chuju, Zhang Yingping. A multi-scale study of industrial fermentation professor and their optimization. Advanced Biochemical Engineer. 2004, 87:97~105
13邹寿长,李干祥,杨葆生等.大规模动物细胞培养技术研究进展.生命科学研究. 2001, 6(5): 2
14钱凯先.人体与动物细胞大量培养的进展.生物技术. 1993; 3(6): 1~3
15 H. Miyoko, S. George, C. Joy. Transplantation of Engineered Bone Tissue Using a Rotary Three-dimensional Culture System. In Vitro Cellular & Developmental Biology-Animal. 2007, 42(2): 49～58.
16 F. Meuwly, F. Loviat. Oxygen Supply for CHO Cells Immobilized on a Packed-bed of Fibra-Cell Disks. Biotechnology and Bioengineering. 2006, 93(4): 791～800
17 Zeng B. J. Self-organization and specific expression of genes. First International Conference and Third National Conference on Transgenic Animals, Beijing, Nov.1996, 11: 18~23
18张嗣良,张徇,唐寅等.发展我国大规模细胞培养生物反应器装备制造业.中国生物工程杂志. 2005, 25(7): 1~8
19白力,张淑香,唐寅,郭美锦等.锥底生物反应器的动物细胞培养.华东理工大学学报(自然科学版). 2008, 6(34): 338~341
20李松涛.基于计算流体力学的生物反应器流场模拟及结构优化.哈尔滨工业大学硕士学位论文. 2008, 7: 3
21 Papoutsakis ET. Fluid mechanical damage of animal cells inbioreactors . Trends in Biotechnology. 1991, 9 (12): 427
22福军.计算流体动力学分析. 1.清华大学出版社, 2004, 7: 1~4
23孙庆丰.搅拌釜式生物反应器设计及优化.哈尔滨工业大学硕士学位论文. 2008, 7: 20
24 Luo J.V, Gosman A.D, Issa R.I, Middle J.C, Fitzgerald M.K. Full Flow Field Computation of Mixing in Baffled Stirred Reactors. Trans. I. Chem. E. 1993, 71A: 42~344
25 J.P.Van Doormal, G. G. Raithby. Enhancement of the SIMPLE method for predicting incompressible fluid flows. Numerical Heat Transfer. 1984, 7: 147~163
26 Brucato A.Ciofalo M.Grisafi F.Micale G. Numerical prediction of flow fields in baffled stirred vessels. A comparison of alternative modeling approaches. 1998, 7: 5
27马鑫.搅拌桨叶表面应力的测量及分析.北京化工大学硕士学位论文. 2000, 7: 51~53
28 Hui Zhu, Alvin W. Nienow, Waldek Bujalski, Mark J.H. Simmons. Mixing studies in a model aerated bioreactor equipped with Elephant Ear agitator. Chemical Engineering Research and Design. 2009, 87: 307~317
29 H. Wu, P. K. Patterson. Laser-doppler Measurements of Turbulent-flow Parameters in a Stirred Tank. Chem.Eng.Sci. 1989, 10: 2207~2221
30任杰.搅拌反应器流场与动力性能的模拟及实验研究.郑州大学硕士论文. 2007, 5: 3~5
31樊学军.细胞力学.力学进展. 1995, 5(25): 2
32付志一,焦群英.植物细胞力学模型研究进展.力学进展. 2005, 8(35): 3
33 Benjamin Geiger.Environmental sensing through focal adhesions. Molecular Cell Biology of Nature Reviews. 2009(10): 21~33
34程琴.基于有限元方法的三维细胞模型与力学分析.重庆大学硕士学位论文. 2009, 4: 3~34
35 Kawase Y, Halard B,Moo-Yong M. Liquid-phase mass transfer coefficients in bioreactors. Biotechnol & Bioeng. 1992, 39: 1133~1140
36 Jhanvi H.Macrorheology and adaptive microrheology of endothelial cells subjected to fluid shear stress. Am J Physiol Cell Physiol. 2007, 293: 1568~1575
37李晓宁,樊瑜波.剪切力环境下内皮细胞力学研究的形态学指标.生物医学工程学杂志. 2003, 20(3): 555~558
38商桂敏,施中东等.剪切应力对红豆杉细胞悬浮培养的影响及CFD模拟研究.高枝化学工程学报. 2002, 10(16): 5
39 Leckie F.Scragg A H.Cliff K R Effect of bioreactor design and agitator speed on the growth and alkaloid accumulation by cultures of Catharanthus roseus. Enzyme Microb Technol .1991, 13: 296~305
40 Scragg A H. Allan EJ, Leckie F. Effect of shear on the viability of plant cell suspensions . Enzyme Microb Technol. 1988, 10: 361~367
41 Hooker B S, Lee J M, An G. Cultivation of plant cells in a stirred vessel: Effect of impeller design. Biotechnol Bioeng. 1990, 35: 296~304
42 Meijer J J, ten Hoopen H J G,van Gameren Y M,Luyben K Ch A M, Libbenga K R. Effects of hydrodynamic stress on the growth of Plant cells in batch and continuous culture. Enzyme and Microb Technol. 1994, 16: 467~477
43 WangerF, volgelmann H. PIant Tissue Culture and its Biotechnological Application. Berlin:Springer-verlag. 1977, 245~255
44 Brucato A,Ciofalo M.Grisafi F, Micale G. Numerical predication of flow fields in baffeld stirred vessels. A companson of alternative modeling approaches. Chem Eng Sci. 1998, 53: 3653~3684
45谭文松,戴干策,陈宏志.生物反应器中杂交瘤细胞的物理破损.华东理工大学学报, 1996, 22 (3): 276~280
46吴金辉,张西正等.用于组织工程化培养生物反应器的研究进展.国外医学生物医学工程分册. 2003, (26): 2