Mixture与响应面法结合开发BHK-21细胞无血清悬浮培养基

doi:10.13560/j.cnki.biotech.bull.1985.2015.09.009

生物技术通报 ›› 2015, Vol. 31 ›› Issue (9): 70-78.doi: 10.13560/j.cnki.biotech.bull.1985.2015.09.009

Mixture与响应面法结合开发BHK-21细胞无血清悬浮培养基

汪梁, 刘旭平, 谭文松

(华东理工大学生物反应器工程国家重点实验室，上海 200237)

收稿日期:2015-06-10 出版日期:2015-09-15 发布日期:2015-09-16
作者简介:汪梁，女，硕士研究生，研究方向：动物细胞的大规模培养；E-mail：aaa098503013@163.com
基金资助:
国家自然科学基金项目(21206040，21406066)，国家“863”计划项目(2012AA02A303)，国家重大专项(2013ZX10004003-003-003)，中央高校基本科研业务费(WF1214035)

Optimization of Suspended Serum-free Medium for BHK-21 Cells by Combining Mixture with Response Surface Analysis

Wang Liang, Liu Xuping, Tan Wensong

(State Key Laboratory of Bioreactor Engineering，East China University of Science and Technology，Shanghai 200237)

Received:2015-06-10 Published:2015-09-15 Online:2015-09-16

摘要/Abstract

摘要： 采用Mixture与响应面实验设计相结合的方法开发BHK-21细胞无血清悬浮培养基。在实验室已知配方的6种培养基A-F的基础上，通过Mixture实验筛选出BHK-21细胞无血清培养基的最优组合为A:n>B:n>C:n>D:n>E:n>F=0:n>0:n>11:n>0:n>9:n>0。利用响应面法针对培养基中的几种关键组分进行浓度优化，确定谷氨酰胺、酪氨酸、牛血清白蛋白和钙离子的最优浓度分别为3 mmol/L、2.5 g/L、0 g/L和0 mmol/L。该无血清悬浮培养基能很好地支持BHK细胞悬浮生长，培养时细胞最大活细胞密度可达140.21×10⁵个/mL，比商业培养基提高了1.95倍。采用Mixture试验设计和响应面分析法能够在较短的时间内开发出适合BHK-21细胞生长的无血清悬浮培养基，为采用BHK-21细胞大规模工业化生产口蹄疫疫苗奠定了基础。

关键词: BHK-21细胞, 无血清悬浮培养基, Mixture实验设计, 响应面分析

Abstract: This study is to optimize the suspended serum-free medium for BHK-21 cells by combining Mixture experiment design with response surface analysis(RSA). Based on 6 media A-F of known formulation in the laboratory, the optimal combination of the medium with A：B：C：D：E：F = 0：0：11：0：9：0 was screened by the Mixture test. The contents of several key constituents in the medium were optimized by RSA, and the optimal concentrations of glutamine, tyrosine, bovine serum albumin(BSA)and calcium ion were 3 mmol/L, 2.5 g/L, 0 g/L and 0 mmol/L respectively. The serum-free medium effectively supported the suspending growth of BHK cells. The maximum viable cell density of BHK cells in cell suspension culture reached 140.21×10⁵ cells/ml, which was 1.95 times in basal medium. The suspended serum-free medium for BHK-21 cells was developed by combining Mixture experiment design with RSA, which lays a foundation for the industrial production of foot-and-mouth disease(FMD)vaccine while using BHK-21 cells.

Key words: BHK-21 cells, suspended serum-free medium, Mixture experiment design, response surface analysis

汪梁, 刘旭平, 谭文松. Mixture与响应面法结合开发BHK-21细胞无血清悬浮培养基[J]. 生物技术通报, 2015, 31(9): 70-78.

Wang Liang, Liu Xuping, Tan Wensong. Optimization of Suspended Serum-free Medium for BHK-21 Cells by Combining Mixture with Response Surface Analysis[J]. Biotechnology Bulletin, 2015, 31(9): 70-78.

参考文献

[1] 谢庆阁. 口蹄疫[M]. 北京：中国农业出版社, 2004：175-176.
[2] 刘旭平, 范里, 朱明龙, 等. MOI对 HEK293细胞感染病毒后的生长代谢和腺病毒扩增效率的影响[J]. 高校化学工程学报, 2010, 24(4)：638-644.
[3] 李晓璐, 范里, 赵亮, 等. 基于单纯型设计和部件搜索方法的CHO细胞无血清培养基的高通量优化[J]. 高校化学工程学报, 2014, 28(4)：777-783.
[4] Bolwell C, Brown AL, Barnett PV, et al. Host cell selection of antigenic variants of foot-and-mouth disease virus[J]. J Gen Virol, 1989, 70(Pt 1)：45-57.
[5] Amadori M, Volpe G, Defilippi P, et al. Phenotypic features of BHK-21 cells used for production of foot-and-mouth disease vaccine[J]. Biologicals, 1997, 25(1)：65-73.
[6] Duygu AT, Ayse N, Murat E, et al. Cultivation and comparison of BHK-21 anchorage semi-dependent cell line in different production systems[J]. Turkish Journal of Biochemistry-Turk J Biochem, 2012, 37(3)：280-286.
[7] Cruz HJ, Freitas CM, Alves PM, et al. Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells[J]. Enzyme and Microbial Technology, 2000, 27(1-2)：43-52.
[8] Rourou S, van der Ark A, van der Velden T, et al. Development of an animal-component free medium for vero cells culture[J]. Biotechnol Prog, 2009, 25(6)：1752-1761.
[9] Lee YY, Yap MG, Hu WS, et al. Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production[J]. Biotechnol Prog, 2003, 19(2)：501-509.
[10] Vohra A, Satyanarayana T. Statistical optimization of the medium components by response surface methodology to enhance phytase production by Pichia anomala[J]. Process Biochem, 2002, 37(9)：999-1004.
[11] 俞锦锋, 陈慧萍, 缑向楠, 等. CHO DG44细胞无血清培养基关键组分的优化与替代[J]. 南昌大学学报：工科版, 2013, 35(2)：125-130.
[12] Lars E, Mats B, Anders L. Optimization of a chondrogenic medium through the use of factorial design of experiments[J]. BioResearch Open Access, 2012, 1(6)：306-313.
[13] Sung HK, Gyun ML. Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments[J]. Appl Microbiol Biotechnol, 2009, 83(4)：639-648.
[14] Yasuhiko I, Yoshinobu K, Ikuya N, et al. Effects of L-glutamine deprivation on growth of HVJ(Sendai Virus)in BHK Cells[J]. Journal of Virology, 1974, 13(3)：557-566.
[15] 张淑香, 李东晓, 朱明龙, 等. 谷氨酰胺限制对杂交瘤细胞生长, 代谢和单抗生产的影响[J]. 高校化学工程学报, 2008, 22(1)：77-82.
[16] Lea PJ, Robinson SA, Stewart GR. The enzymology and metabolism of glutamine, glutamate, and asparagine[J]. The Biochemistry of Plants, 1990, 16(10)：121-159.
[17] Peshwa MV, Kyung YS, McClure DB, et al. Cultivation of mammalian cells as aggregates in bioreactors：effect of calcium concentration on spatial distribution of viability[J]. Biotechnol Bioeng, 1993, 41(2)：179-187.
[18] 赵亮, 朱明龙, 张旭, 等. 钙离子对293细胞结团和生长的影响[J]. 生物工程学报, 2008, 21(3)：482-485.
[19] Nesbitt WS, Giuliano S, Kulkarni S, et al. Intercellular calcium communication regulates platelet aggregation and thrombus growth[J]. J Cell Biol, 2003, 160(7)：1151-1161.
[20] Haiming Z, Srinivasan D. Effects of calcium and magnesium Ions on aggregation of whey protein isolate and its effect on foaming properties[J]. J Agric Food Chem, 1994, 42(4)：856-862.

Mixture与响应面法结合开发BHK-21细胞无血清悬浮培养基

Optimization of Suspended Serum-free Medium for BHK-21 Cells by Combining Mixture with Response Surface Analysis

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 14

编辑推荐

Metrics

本文评价

[1]	张美君, 吴庆, 尹翠, 王妮, 马晓庆, 马晓霞, 曹云娥. 尖镰孢黄瓜专化型枯萎病菌拮抗菌的筛选、鉴定及培养条件优化[J]. 生物技术通报, 2020, 36(9): 125-136.
[2]	杨胜男, 刘娜, 宋东辉. 菌藻混合体系去除Cr（VI）的条件优化及Cr（VI）还原酶活性的测定[J]. 生物技术通报, 2019, 35(9): 83-92.
[3]	雒晓芳, 陈丽华, 夏苗苗, 许淑娟, 熊梅. 苏云金芽孢杆菌对PAHs的降解研究[J]. 生物技术通报, 2019, 35(9): 125-133.
[4]	吕翻洋, 许泽华, 毛晓洁, 孙建光. 咪唑乙烟酸降解菌的分离鉴定及降解条件优化[J]. 生物技术通报, 2017, 33(10): 163-168.
[5]	樊星,王淑婷,李春艳. 一株高效铁锰氧化细菌P1的分离鉴定及氧化条件优化[J]. 生物技术通报, 2016, 32(4): 175-183.
[6]	张谦,贾佳,林智,杨晓锋,郭宏涛,王剑英,Carol Sze Ki Lin. 产脂肪酶黑曲霉摇瓶发酵条件优化研究[J]. 生物技术通报, 2015, 31(12): 227-233.
[7]	朱海峰, 吴丹, 吴敬. 毕赤酵母GS115中N-乙酰转移酶在大肠杆菌中的克隆表达与性质研究[J]. 生物技术通报, 2015, 31(11): 179-185.
[8]	柴秀娟,李曹龙,孔德真,崔郑龙,王爱英. 产纤维素酶菌株的筛选、鉴定及产酶条件的优化[J]. 生物技术通报, 2014, 0(9): 164-170.
[9]	刘倩, 齐计英, 韩静, 万阅 ,郑士彬. 响应面法优化回流提取紫薯花青素工艺[J]. 生物技术通报, 2014, 0(12): 97-104.
[10]	张学林, 唐湘华, 李俊俊, 宋拓, 慕跃林, 许波, 杨云娟, 黄遵锡. 响应面法优化小桐子油制备生物柴油工艺的研究[J]. 生物技术通报, 2013, 0(6): 183-187.
[11]	赵超, 曾峰, 黄一帆, 刘斌. Box-Behnken响应面设计法优化黑木耳菌质多糖提取工艺[J]. 生物技术通报, 2013, 0(6): 188-193.
[12]	史馨怡, 刘丹, 陈黎黎, 陈欣. 逆境对红酵母发酵生产类胡萝卜素的促进作用[J]. 生物技术通报, 2013, 0(1): 191-197.
[13]	韦杰;段永忠;唐湘华;李俊俊;黄遵锡;. 响应面分析法对甘蔗渣中木聚糖提取条件的优化[J]. , 2009, 0(S1): 291-296.
[14]	杜少平;梁淑娃;杨冠东;蓝碧锋;彭中健;. 响应面法优化两歧双歧杆菌发酵培养基[J]. , 2008, 0(S1): 431-434.