生物技术通报 ›› 2020, Vol. 36 ›› Issue (10): 62-71.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0343
收稿日期:
2020-03-27
出版日期:
2020-10-26
发布日期:
2020-11-02
作者简介:
陈敏,女,博士,研究方向:动物细胞培养和组织工程;E-mail:CHEN Min1(), LIU Xu-ping1, ZHAO Liang2
Received:
2020-03-27
Published:
2020-10-26
Online:
2020-11-02
摘要:
优化无血清培养基,以实现BHK-21悬浮细胞的超高密度生长和口蹄疫病毒高效扩增。依据BHK-21细胞的代谢特点和动力学分析识别无血清培养基中的关键营养成分,优化组分浓度,对比优化前后细胞的生长和产毒能力,并在生物反应器中验证。优化后的无血清培养基支持批培养模式下最高活细胞密度达到1.78×107cells/mL,代谢副产物积累的情况得到改善;口蹄疫病毒TCID50与含低比例血清的培养基相比具有相似水平,约为7.25 lgTCID50/0.1mL;146s含量与低血清培养基相比提高50.7%,达到15.6 μg/mL。以细胞的生长需求和代谢规律为基础,优化营养成分和降低代谢副产物积累能有效解除“细胞密度效应”,实现超高细胞密度接毒和高产口蹄疫病毒。
陈敏, 刘旭平, 赵亮. 支持高密度BHK-21细胞培养和高产FMD病毒的无血清培养基开发与优化[J]. 生物技术通报, 2020, 36(10): 62-71.
CHEN Min, LIU Xu-ping, ZHAO Liang. Development and Optimization of Serum-Free Medium for High-density Culture of Suspended BHK-21 Cells and High-yield of FMD Virus[J]. Biotechnology Bulletin, 2020, 36(10): 62-71.
添加 物质 | 接种后第3天 剩余比例/% | 0-3 d比消耗速率 mmol/(109 cells?day) | 推荐增加的 浓度比例/% |
---|---|---|---|
Asp | 6.95 | 0.12 | 32.92 |
69.17 | |||
Asn | 11.48 | 0.20 | 26.46 |
60.94 | |||
Gln | 14.09 | 0.17 | 22.73 |
56.20 |
表1 氨基酸浓度优化的理论添加量
添加 物质 | 接种后第3天 剩余比例/% | 0-3 d比消耗速率 mmol/(109 cells?day) | 推荐增加的 浓度比例/% |
---|---|---|---|
Asp | 6.95 | 0.12 | 32.92 |
69.17 | |||
Asn | 11.48 | 0.20 | 26.46 |
60.94 | |||
Gln | 14.09 | 0.17 | 22.73 |
56.20 |
实验组 | Asp浓度 | Asn浓度 | Gln浓度 |
---|---|---|---|
S1-SFM-a | + | + | + |
S1-SFM-b | + | - | + |
S1-SFM-c | + | + | - |
S1-SFM-d | + | - | - |
S1-SFM-e | - | + | + |
S1-SFM-f | - | - | + |
S1-SFM-g | - | + | - |
S1-SFM-h | - | - | - |
S1-SFM-i | 0 | 0 | 0 |
表2 氨基酸浓度优化的实验设计
实验组 | Asp浓度 | Asn浓度 | Gln浓度 |
---|---|---|---|
S1-SFM-a | + | + | + |
S1-SFM-b | + | - | + |
S1-SFM-c | + | + | - |
S1-SFM-d | + | - | - |
S1-SFM-e | - | + | + |
S1-SFM-f | - | - | + |
S1-SFM-g | - | + | - |
S1-SFM-h | - | - | - |
S1-SFM-i | 0 | 0 | 0 |
实验组 | 铜离子浓度/(mmol?L-1) | 锌离子浓度/(mmol?L-1) |
---|---|---|
S2-SFM-a | 0.04 | 0.2 |
S2-SFM-b | 0.04 | 0.02 |
S2-SFM-c | 0.04 | 0.002 |
S2-SFM-d | 0.004 | 0.2 |
S2-SFM-e | 0.004 | 0.02 |
S2-SFM-f | 0.004 | 0.002 |
S2-SFM-g | 0.0004 | 0.2 |
S2-SFM-h | 0.0004 | 0.02 |
S2-SFM-i | 0.0004 | 0.002 |
S2-SFM-j | 0 | 0 |
表3 铜锌离子浓度实验设计表
实验组 | 铜离子浓度/(mmol?L-1) | 锌离子浓度/(mmol?L-1) |
---|---|---|
S2-SFM-a | 0.04 | 0.2 |
S2-SFM-b | 0.04 | 0.02 |
S2-SFM-c | 0.04 | 0.002 |
S2-SFM-d | 0.004 | 0.2 |
S2-SFM-e | 0.004 | 0.02 |
S2-SFM-f | 0.004 | 0.002 |
S2-SFM-g | 0.0004 | 0.2 |
S2-SFM-h | 0.0004 | 0.02 |
S2-SFM-i | 0.0004 | 0.002 |
S2-SFM-j | 0 | 0 |
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