CN110564670A - 一种昆虫细胞无血清培养基及其制备工艺 - Google Patents
一种昆虫细胞无血清培养基及其制备工艺 Download PDFInfo
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- CN110564670A CN110564670A CN201910835365.9A CN201910835365A CN110564670A CN 110564670 A CN110564670 A CN 110564670A CN 201910835365 A CN201910835365 A CN 201910835365A CN 110564670 A CN110564670 A CN 110564670A
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- chloride
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- serum
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Abstract
本发明公开了一种昆虫细胞无血清培养基及其制备工艺。该昆虫细胞无血清培养基,包括:11~16g/L氨基酸、5~15g/L碳水化合物、7~12g/L无机盐、0.01~0.2g/L维生素、3~10g/L蛋白水解物、1~5g/L酵母粉以及15~35g/L脂质乳化剂。本发明提供的昆虫细胞无血清培养基的组分明确,维生素含量低,成本较低,制备工艺简单,有利于昆虫细胞大规模培养,可大幅提高昆虫细胞培养效率,具有良好的商业化前景。
Description
技术领域
本发明涉及细胞培养技术领域,特别涉及一种昆虫细胞无血清培养基及其制备工艺。
背景技术
细胞培养是将细胞在体外进行繁殖和培养,是生物学研究领域不可缺少的技术。传统细胞培养基主要是通过在基础培养基中添加适量的血清或者组织提取物配制而成的,因动物血清提取技术的局限,使含有血清的培养基的来源困难且价格格外昂贵,质量不稳定,批间差异大,重复性差,给细胞培养的标准化带来困难。由于传统细胞血清培养基存在诸多问题,促使越来越多的研究人员致力于研发无血清培养基。
近年来,细胞无血清培养技术发展迅速,已报导有多重细胞株系在相应的无血清培养基中生长和增殖获得成功。但是现有的无血清培养基需要添加多种小分子添加物(如重组蛋白)等或高浓度的维生素,使培养基的成本过高,不利于大规模的昆虫细胞培养。
发明内容
本发明目的在于是提供一种昆虫细胞无血清培养基及其制备工艺,以解决上述技术问题。
为了解决上述技术问题,本发明的技术方案为:
第一方面,本发明提供的一种昆虫细胞无血清培养基,包括:11~16g/L氨基酸、5~15g/L碳水化合物、7~12g/L无机盐、0.01~0.2g/L维生素、3~10g/L蛋白水解物、1~5g/L酵母粉以及15~35g/L脂质乳化剂。
优选的,所述氨基酸选自天冬氨酸、谷氨酸、丝氨酸、甘氨酸、蛋氨酸、异亮氨酸、亮氨酸、苯丙氨酸、组氨酸、精氨酸、缬氨酸、赖氨酸、苏氨酸、谷氨酰胺、天冬酰胺、酪氨酸、脯氨酸中的一种或多种。
优选的,所述昆虫细胞无血清培养基包括300~800mg/L天冬氨酸、1000~1800mg/L谷氨酸、350~600mg/L丝氨酸、1000~1500mg/L甘氨酸、100~300mg/L蛋氨酸、200~400mg/L异亮氨酸、250~500mg/L亮氨酸、210~300mg/L苯丙氨酸、300~800mg/L组氨酸、2000~3500mg/L精氨酸、1100~1600mg/L缬氨酸、500~1000mg/L赖氨酸、700~950mg/L苏氨酸、450~600mg/L谷氨酰胺、1000~1500mg/L天冬酰胺、200~400mg/L酪氨酸、100~200mg/L脯氨酸。
优选的,所述维生素选自维生素B1、维生素B6、维生素B12、泛酸、生物素、烟酸、核黄素、叶酸、氯化胆碱中的一种或多种。
优选的,所述昆虫细胞无血清培养基包括0.1~0.3mg/L维生素B1、0.02~0.1mg/L维生素B6、0.05~0.1mg/L维生素B12、0.05~0.2mg/L泛酸、0.05~0.2mg/L生物素、0.1~0.2mg/L烟酸、0.01~0.1mg/L核黄素、0.1~0.2mg/L叶酸、0.05~0.2g/L氯化胆碱。
优选的,所述无机盐选自钾盐、钙盐、钠盐、磷酸盐、铜盐、锰盐、锌盐、铁盐、钼酸盐、钴盐、镍盐、钡盐中的一种或多种。
进一步的,所述钾盐为氯化钾。
进一步的,所述钙盐选自氯化钙、硫酸钙中的任意一种。
进一步的,所述钠盐选自碳酸氢钠、氯化钠、磷酸二氢钠中的一种或多种。
进一步的,所述磷酸盐选自磷酸二氢钠、磷酸二氢钾中的一种或多种。
进一步的,所述镁盐为硫酸镁。
进一步的,所述铜盐为氯化铜。
进一步的,所述锰盐为氯化锰。
进一步的,所述锌盐为氯化锌。
进一步的,所述铁盐为硫酸亚铁。
进一步的,钼酸盐为钼酸铵。
进一步的,所述钴盐为氯化钴。
进一步的,所述镍盐为氯化镍。
进一步的,所述钡盐为氯化钡。
优选的,所述昆虫细胞无血清培养基包括0.5~3g/L氯化钙、0.5~3g/L氯化钠、0.5~3g/L磷酸二氢钠、0.5~3g/L氯化钾、0.1~0.3g/L氯化铜、0.05~0.2g/L氯化锰、0.05~0.1g/L氯化锌、0.3~3g/L硫酸镁、0.3~0.8g/L硫酸亚铁、0.05~0.1g/L钼酸铵、0.01~0.05g/L氯化钴、0.01~0.05g/L氯化镍。
第二方面,本发明提供的一种如第一方面所述的昆虫细胞无血清培养基的制备工艺,包括如下步骤:
(1)将无机盐溶解于水中,得到无机盐溶液;
(2)将维生素溶解于水中,得到维生素溶液;
(3)将氨基酸溶于水中,得到氨基酸溶液;
(4)将碳水化合物、蛋白水解物、脂质乳化剂加入水中,搅拌均匀后,再加入无机盐溶液和的维生素溶液、氨基酸溶液,搅拌均匀后,经定容得到昆虫细胞无血清培养基。
与现有技术相比,本发明的有益效果在于:
本发明提供的昆虫细胞无血清培养基的组分明确,维生素含量低,成本较低,制备工艺简单,有利于昆虫细胞大规模培养,可大幅提高昆虫细胞培养效率,具有良好的商业化前景。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
实施例1
本实施例提供的昆虫细胞无血清培养基的制备工艺,包括如下步骤:
(1)将表1中各无机盐成分加入20mL去离子水中溶解,得到无机盐溶液;
(2)将表2中各维生素成分加入20mL去离子水中溶解,得到维生素溶液;
(3)将表3中各氨基酸成分加入20mL去离子水中溶解,得到氨基酸溶液;
(4)将所得的无机盐溶液、维生素溶液、氨基酸溶液加入到盛放有800mL去离子水的烧杯中,搅拌均匀后,将如表4所示的碳水化合物、蛋白水解物、酵母粉、脂质乳化剂依次加入烧杯中,搅拌均匀,定容至1000mL即可得到昆虫细胞无血清培养基。
表1昆虫细胞无血清培养基中各无机盐成分及其含量
序号 | 无机盐成分 | 含量(g/L) | 序号 | 无机盐成分 | 含量(g/L) |
1 | 氯化钙 | 2.72 | 7 | 氯化锌 | 0.08 |
2 | 氯化钠 | 1.82 | 8 | 硫酸镁 | 2.54 |
3 | 磷酸二氢钠 | 1.68 | 9 | 硫酸亚铁 | 0.5 |
4 | 氯化钾 | 1.8 | 10 | 钼酸铵 | 0.07 |
5 | 氯化铜 | 0.12 | 11 | 氯化钴 | 0.04 |
6 | 氯化锰 | 0.15 | 12 | 氯化镍 | 0.03 |
表2昆虫细胞无血清培养基中各维生素成分及其含量
序号 | 维生素成分 | 含量(mg/L) | 序号 | 氨基酸成分 | 含量(mg/L) |
1 | 维生素B<sub>1</sub> | 0.15 | 6 | 烟酸 | 0.12 |
2 | 维生素B<sub>6</sub> | 0.06 | 7 | 核黄素 | 0.06 |
3 | 维生素B<sub>12</sub> | 0.08 | 8 | 叶酸 | 0.16 |
4 | 泛酸 | 0.1 | 9 | 氯化胆碱 | 140 |
5 | 生物素 | 0.1 | —— | —— | —— |
表3昆虫细胞无血清培养基中各氨基酸成分及其含量
表4昆虫细胞无血清培养基中其它成分及其含量
序号 | 其它成分 | 含量(g/L) |
1 | 蛋白水解物 | 8 |
2 | 酵母粉 | 4 |
3 | 脂质乳化剂 | 32 |
为了进一步说明本发明的有益效果,取本实施例的培养基放入容量为1L的摇瓶中悬浮培养Sf9细胞,根据培养效果可知,本实施例的培养基培养的细胞,最大活细胞密度不低于1.4×107cells/mL。
实施例2
本实施例提供的昆虫细胞无血清培养基的制备工艺,包括如下步骤:
(1)将表5中各无机盐成分加入20mL去离子水中溶解,得到无机盐溶液;
(2)将表6中各维生素成分加入20mL去离子水中溶解,得到维生素溶液;
(3)将表7中各氨基酸成分加入20mL去离子水中溶解,得到氨基酸溶液;
(4)将所得的无机盐溶液、维生素溶液、氨基酸溶液加入到盛放有800mL去离子水的烧杯中,搅拌均匀后,将如表8所示的碳水化合物、蛋白水解物、酵母粉、脂质乳化剂依次加入烧杯中,搅拌均匀,定容至1000mL即可得到昆虫细胞无血清培养基。
表5昆虫细胞无血清培养基中各无机盐成分及其含量
表6昆虫细胞无血清培养基中各维生素成分及其含量
序号 | 维生素成分 | 含量(mg/L) | 序号 | 氨基酸成分 | 含量(mg/L) |
1 | 维生素B<sub>1</sub> | 0.1 | 6 | 烟酸 | 0.2 |
2 | 维生素B<sub>6</sub> | 0.02 | 7 | 核黄素 | 0.1 |
3 | 维生素B<sub>12</sub> | 0.05 | 8 | 叶酸 | 0.1 |
4 | 泛酸 | 0.2 | 9 | 氯化胆碱 | 50 |
5 | 生物素 | 0.2 | —— | —— | —— |
表7昆虫细胞无血清培养基中各氨基酸成分及其含量
序号 | 氨基酸成分 | 含量(mg/L) | 序号 | 氨基酸成分 | 含量(mg/L) |
1 | 天冬氨酸 | 800 | 10 | 精氨酸 | 3500 |
2 | 谷氨酸 | 1800 | 11 | 缬氨酸 | 1600 |
3 | 丝氨酸 | 600 | 12 | 赖氨酸 | 1000 |
4 | 甘氨酸 | 1500 | 13 | 苏氨酸 | 950 |
5 | 蛋氨酸 | 100 | 14 | 谷氨酰胺 | 450 |
6 | 异亮氨酸 | 200 | 15 | 天冬酰胺 | 1000 |
7 | 亮氨酸 | 250 | 16 | 酪氨酸 | 200 |
8 | 苯丙氨酸 | 300 | 17 | 脯氨酸 | 200 |
9 | 组氨酸 | 800 | —— | —— | —— |
表8昆虫细胞无血清培养基中其它成分及其含量
序号 | 其它成分 | 含量(g/L) |
1 | 蛋白水解物 | 3 |
2 | 酵母粉 | 5 |
3 | 脂质乳化剂 | 35 |
为了进一步说明本发明的有益效果,本实施例的培养基放入容量为1L的摇瓶中悬浮培养Sf9细胞,根据培养效果可知,本实施例的培养基培养的细胞,最大活细胞密度约为1.23×107cells/mL。
实施例3
本实施例提供的昆虫细胞无血清培养基的制备工艺,包括如下步骤:
(1)将表9中各无机盐成分加入20mL去离子水中溶解,得到无机盐溶液;
(2)将表10中各维生素成分加入20mL去离子水中溶解,得到维生素溶液;
(3)将表11中各氨基酸成分加入20mL去离子水中溶解,得到氨基酸溶液;
(4)将所得的无机盐溶液、维生素溶液、氨基酸溶液加入到盛放有800mL去离子水的烧杯中,搅拌均匀后,将如表12所示的碳水化合物、蛋白水解物、酵母粉、脂质乳化剂依次加入烧杯中,搅拌均匀,定容至1000mL即可得到昆虫细胞无血清培养基。
表9昆虫细胞无血清培养基中各无机盐成分及其含量
序号 | 无机盐成分 | 含量(g/L) | 序号 | 无机盐成分 | 含量(g/L) |
1 | 氯化钙 | 0.5 | 7 | 氯化锌 | 0.05 |
2 | 氯化钠 | 3.0 | 8 | 硫酸镁 | 0.3 |
3 | 磷酸二氢钠 | 0.5 | 9 | 硫酸亚铁 | 0.3 |
4 | 氯化钾 | 3.0 | 10 | 钼酸铵 | 0.05 |
5 | 氯化铜 | 0.1 | 11 | 氯化钴 | 0.01 |
6 | 氯化锰 | 0.05 | 12 | 氯化镍 | 0.01 |
表10昆虫细胞无血清培养基中各维生素成分及其含量
序号 | 维生素成分 | 含量(mg/L) | 序号 | 氨基酸成分 | 含量(mg/L) |
1 | 维生素B<sub>1</sub> | 0.3 | 6 | 烟酸 | 0.1 |
2 | 维生素B<sub>6</sub> | 0.1 | 7 | 核黄素 | 0.01 |
3 | 维生素B<sub>12</sub> | 0.2 | 8 | 叶酸 | 0.2 |
4 | 泛酸 | 0.05 | 9 | 氯化胆碱 | 200 |
5 | 生物素 | 0.05 | —— | —— | —— |
表11昆虫细胞无血清培养基中各氨基酸成分及其含量
序号 | 氨基酸成分 | 含量(mg/L) | 序号 | 氨基酸成分 | 含量(mg/L) |
1 | 天冬氨酸 | 300 | 10 | 精氨酸 | 2000 |
2 | 谷氨酸 | 1000 | 11 | 缬氨酸 | 1100 |
3 | 丝氨酸 | 350 | 12 | 赖氨酸 | 500 |
4 | 甘氨酸 | 1000 | 13 | 苏氨酸 | 700 |
5 | 蛋氨酸 | 300 | 14 | 谷氨酰胺 | 600 |
6 | 异亮氨酸 | 400 | 15 | 天冬酰胺 | 1500 |
7 | 亮氨酸 | 500 | 16 | 酪氨酸 | 400 |
8 | 苯丙氨酸 | 210 | 17 | 脯氨酸 | 100 |
9 | 组氨酸 | 300 | —— | —— | —— |
表12昆虫细胞无血清培养基中其它成分及其含量
序号 | 其它成分 | 含量(g/L) |
1 | 蛋白水解物 | 10 |
2 | 酵母粉 | 1 |
3 | 脂质乳化剂 | 15 |
为了进一步说明本发明的有益效果,本实施例的培养基放入容量为1L的摇瓶中悬浮培养Sf9细胞,根据培养效果可知,本实施例的培养基培养的细胞,最大活细胞密度不低于1.18×107cells/mL。
对比例1
为了进一步说明本发明的有益效果,按照实施例1的步骤配制成培养基,本对比例与实施例1的区别仅在于:本对比例未添加氯化胆碱、叶酸和核黄素。
为了进一步说明本发明的有益效果,分别取本对比例的培养基放入容量为1L的摇瓶中悬浮培养Sf9细胞,根据培养效果可知,而本对比例的培养基培养的细胞,最大活细胞密度分别约为1.1×107cells/mL。
对比例2
为了进一步说明本发明的有益效果,按照实施例1的步骤配制成培养基,本对比例与实施例1的区别仅在于:本对比例未添加氯化镍、氯化钴、氯化锰。
为了进一步说明本发明的有益效果,分别取本对比例的培养基放入容量为1L的摇瓶中悬浮培养Sf9细胞,根据培养效果可知,而本对比例的培养基培养的细胞,最大活细胞密度分别约为1.17×107cells/mL。
以上对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (9)
1.一种昆虫细胞无血清培养基,其特征在于,包括:11~16g/L氨基酸、5~15g/L碳水化合物、7~12g/L无机盐、0.01~0.2g/L维生素、3~10g/L蛋白水解物、1~5g/L酵母粉以及15~35g/L脂质乳化剂。
2.根据权利要求1所述的昆虫细胞无血清培养基,其特征在于,所述氨基酸选自天冬氨酸、谷氨酸、丝氨酸、甘氨酸、蛋氨酸、异亮氨酸、亮氨酸、苯丙氨酸、组氨酸、精氨酸、缬氨酸、赖氨酸、苏氨酸、谷氨酰胺、天冬酰胺、酪氨酸、脯氨酸中的一种或多种。
3.根据权利要求1所述的昆虫细胞无血清培养基,其特征在于,所述昆虫细胞无血清培养基包括300~800mg/L天冬氨酸、1000~1800mg/L谷氨酸、350~600mg/L丝氨酸、1000~1500mg/L甘氨酸、100~300mg/L蛋氨酸、200~400mg/L异亮氨酸、250~500mg/L亮氨酸、210~300mg/L苯丙氨酸、300~800mg/L组氨酸、2000~3500mg/L精氨酸、1100~1600mg/L缬氨酸、500~1000mg/L赖氨酸、700~950mg/L苏氨酸、450~600mg/L谷氨酰胺、1000~1500mg/L天冬酰胺、200~400mg/L酪氨酸、100~200mg/L脯氨酸。
4.根据权利要求1所述的昆虫细胞无血清培养基,其特征在于,所述维生素选自维生素B1、维生素B6、维生素B12、泛酸、生物素、烟酸、核黄素、叶酸、氯化胆碱中的一种或多种。
5.根据权利要求1所述的昆虫细胞无血清培养基,其特征在于,所述昆虫细胞无血清培养基包括0.1~0.3mg/L维生素B1、0.02~0.1mg/L维生素B6、0.05~0.1mg/L维生素B12、0.05~0.2mg/L泛酸、0.05~0.2mg/L生物素、0.1~0.2mg/L烟酸、0.01~0.1mg/L核黄素、0.1~0.2mg/L叶酸、0.05~0.2g/L氯化胆碱。
6.根据权利要求1所述的昆虫细胞无血清培养基,其特征在于,所述无机盐选自钾盐、钙盐、钠盐、磷酸盐、铜盐、锰盐、锌盐、铁盐、钼酸盐、钴盐、镍盐、钡盐中的一种或多种。
7.根据权利要求6所述的昆虫细胞无血清培养基,其特征在于,所述钾盐为氯化钾;所述钙盐选自氯化钙、硫酸钙中的任意一种;所述钠盐选自碳酸氢钠、氯化钠、磷酸二氢钠中的一种或多种;所述磷酸盐选自磷酸二氢钠、磷酸二氢钾中的一种或多种;所述镁盐为硫酸镁;所述铜盐为氯化铜;所述锰盐为氯化锰;所述锌盐为氯化锌;所述铁盐为硫酸亚铁;钼酸盐为钼酸铵;所述钴盐为氯化钴;所述镍盐为氯化镍;所述钡盐为氯化钡。
8.根据权利要求1所述的昆虫细胞无血清培养基,其特征在于,所述昆虫细胞无血清培养基包括0.5~3g/L氯化钙、0.5~3g/L氯化钠、0.5~3g/L磷酸二氢钠、0.5~3g/L氯化钾、0.1~0.3g/L氯化铜、0.05~0.2g/L氯化锰、0.05~0.1g/L氯化锌、0.3~3g/L硫酸镁、0.3~0.8g/L硫酸亚铁、0.05~0.1g/L钼酸铵、0.01~0.05g/L氯化钴、0.01~0.05g/L氯化镍。
9.一种如权利要求1~8中任一项所述的昆虫细胞无血清培养基的制备工艺,其特征在于,包括如下步骤:
(1)将无机盐溶解于水中,得到无机盐溶液;
(2)将维生素溶解于水中,得到维生素溶液;
(3)将氨基酸溶于水中,得到氨基酸溶液;
(4)将碳水化合物、蛋白水解物、脂质乳化剂加入水中,搅拌均匀后,再加入无机盐溶液和的维生素溶液、氨基酸溶液,搅拌均匀后,经定容得到昆虫细胞无血清培养基。
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