CN102021139A - 一种中国仓鼠卵巢细胞培养基及其制备方法和应用 - Google Patents
一种中国仓鼠卵巢细胞培养基及其制备方法和应用 Download PDFInfo
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- CN102021139A CN102021139A CN2009101955057A CN200910195505A CN102021139A CN 102021139 A CN102021139 A CN 102021139A CN 2009101955057 A CN2009101955057 A CN 2009101955057A CN 200910195505 A CN200910195505 A CN 200910195505A CN 102021139 A CN102021139 A CN 102021139A
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Abstract
本发明公开了一种中国仓鼠卵巢细胞培养基及其制备方法和应用，所述培养基含有抗坏血酸、盐酸吡多醇、维生素B12、烟酰胺、核黄素、盐酸硫胺素、氯化胆碱、叶酸、泛酸钙、丙酮酸钠、还原型谷胱甘肽、肌醇、生物素、次黄嘌呤、腐胺、硫辛酸、亚油酸、胸腺嘧啶、葡萄糖、HEPES缓冲液、硫酸葡聚糖、Pluronic-F68，以及多种氨基酸及其盐，和多种无机盐。该培养基成本较低，组成中无动物源成分、不含蛋白质且化学成分确定，其通过各组分的调配协同，除满足细胞生长基本要求外，能够有效调节、传递并控制细胞的培养，实现良好的高密度细胞培养，其培养效果与现有培养基培养效果相比至少相当且甚至优于现有培养基。
Description
技术领域
本发明涉及一种中国仓鼠卵巢细胞培养基及其制备方法和应用。
背景技术
中国仓鼠卵巢细胞(Chinese hamster ovary cell，CHO细胞)是目前最广泛使用的哺乳动物细胞系，已被用于生产各种基因工程蛋白产品，例如，组织纤溶酶原激活物(t-PA)、人促红细胞生成素(rHuEPO)、乙肝表面抗原(HBsAg)、人促血小板生成素(rhTPO)、各种重组抗体等。
CHO细胞的培养通常是在添加了胎牛血清或者小牛血清的合成培养基中进行。但是其中添加的血清属于成分不明确的物质，其含有很多蛋白质和其他成分不确定的大分子物质，影响产品的分离纯化，降低产品的收率和纯度。同时来源动物的血清还存在病毒感染的潜在危险。此外，血清的添加将大大增加生产的成本。因此，许多科研工作者致力于开发低血清或者无血清培养基。
美国的Casser(In Vitro Cell Devel Biol 1985，88：588)开发了一种适于CHO细胞生长的无血清培养基，以DMEM/F12为基础培养基，添加了转铁蛋白、白蛋白、胰岛素以及亚硒酸钠等多种成分，成为现在被广泛采用的无血清培养基的基础配方。但是该无血清培养基中含有转铁蛋白、白蛋白、胰岛素等动物来源的蛋白，这些蛋白不仅是潜在的污染源，也大大提高了培养基的成本，加大了分离纯化的难度。所以，科研工作者又开始寻找到了可以替代这些蛋白质的无机盐，研发出无蛋白的培养基。
美国的Leopold Grillberger(美国专利2006/0094104 A1)开发了一种适合于动物细胞培养的无蛋白培养基，该培养基不含任何动物来源的蛋白质，使用动物来源或者植物来源的蛋白水解物代替。这类无蛋白培养基虽然成本已经很低，潜在的污染危险也大大降低，但是其中仍然含有化学成分不确定的成分，仍然一定程度上限制了细胞的生长、增殖、分化调节等的研究。因此，亟待开发一种适合CHO细胞高密度培养，并且培养效果与有血清培养基、无血清培养基、无蛋白质培养基相当或更佳的无动物源成分、无蛋白质、化学成分确定的培养基。
发明内容
本发明所要解决的技术问题是克服了现有的中国仓鼠卵巢细胞培养基化学成分不确定、使用动物源组分或者含有蛋白质，从而使培养细胞具有潜在污染威胁，且成本较高，增加分离纯化难度，一定程度限制细胞研究的发展等缺陷，提供了无动物源成分、无蛋白、化学成分确定且培养效果相当甚至优于现有培养基的一种中国仓鼠卵巢细胞培养基及其制备方法和应用。
本发明涉及一种中国仓鼠卵巢细胞培养基，其含有：9.02～141.06mg/L L-色氨酸(L-Tryptophan)、31.29～148.74mg/L L-胱氨酸(L-Cystine)、17.56～159.87mg/L L-半胱氨酸(L-Cystein)、38.4～377.28mg/L L-酪氨酸盐钠盐二水(L-Tyrosine disodium salt dihydrate)、54.47～654mg/L L-异亮氨酸(L-Isoleucine)、59.05～874.32mg/L L-亮氨酸(L-Leucine)、50～420mg/L L-缬氨酸(L-Valine)、80～720mg/L L-精氨酸(L-Arginine)、31～480mg/L L-组氨酸(L-Histidine)、80～852.3mg/L L-赖氨酸(L-Lysine)、10～184.7mg/L L-蛋氨酸(L-Methionine)、20～438.3mg/L L-苯丙氨酸(L-Phenylalanine)、1～235.8mg/L L-谷氨酸(L-Glutamic acid)、70～540mg/L L-天冬酰胺(L-Asparagine)、1～40mg/L甘氨酸(Glycine)、10～620mg/L L-脯氨酸(L-Proline)、0.1～63mg/L L-丙氨酸(L-Alanine)、10～223.6mg/L L-天冬氨酸(L-Apartic acid)、20～661.2mg/L L-丝氨酸(L-Serine)、40～893.4mg/L L-苏氨酸(L-Threonine)、365～1168mg/L L-谷氨酰胺(L-Glutamine)、10～160mg/L无水氯化钙(Calcium Chloride(anhydrous)，CaCl2)、0.001～0.003mg/L五水硫酸铜(Cupric Sulfate，CuSO4·5H2O)、0.05～1mg/L九水硝酸铁(Ferric Nitrate，Fe(NO3)3·9H2O)、0.1～1mg/L七水硫酸亚铁(Ferrous Sulfate，FeSO4·7H2O)、100～400mg/L氯化钾(Patassium Chloride，KCl)、20～60mg/L氯化镁(Magnesium Chloride，MgCl2)、5～200mg/L一水磷酸二氢钠(Sodium Phosphate Monobasic，NaH2PO4·H2O)、50～300mg/L无水磷酸氢二钠(Sodium Phosphate Dibasic(anhydrous)，Na2HPO4)、0.1～2mg/L七水硫酸锌(Zinc Sulfate，ZnSO4·7H2O)、8～50mg/L柠檬酸铁(Ferric Citrate，FeC6H5O7)、0.001～0.05mg/L亚硒酸钠(Sodium Selenite，Na2SeO3)、3000mg/L碳酸氢钠(Sodium Bicarbonate)、2～20mg/L抗坏血酸(Ascorbic Acid)、0.1～4mg/L盐酸吡多醇(Pyridoxine HCl)、0.2～2.72mg/L维生素B12(Vitamin B12)、0.5～6.5mg/L烟酰胺(Niacinamide)、0.05～1.5mg/L核黄素(Riboflavin)、0.1～6.81mg/L盐酸硫胺素(Thiamine HCl)、2.57～26.94mg/L氯化胆碱(Choline Chloride)、0.2～3mg/L叶酸(Folic Acid)、0.1～2.5mg/L泛酸钙(D-Ca Pantothenate)、2～150mg/L丙酮酸钠(Sodium Pyruvate)、0.3～2.25mg/L谷胱甘肽(还原型)(Glutathione(reduced))、5～25mg/L肌醇(i-Inositol)、0.0001～0.5mg/L生物素(Biotin)、0.008～8mg/L次黄嘌呤(Na Hypoxanthine)、0.001～9mg/L腐胺(Sodium Putrescine-2HCl)、0.003～0.8mg/L硫辛酸(Lipoic Acid)、0.003～1.2mg/L亚油酸(Linoleic Acid)、0.02～6mg/L胸腺嘧啶(Thymidine)、2150～7000mg/L葡萄糖(D-glucose(Dextrose))、1500～4000mg/L HEPES缓冲液、5～70mg/L硫酸葡聚糖(Dextran Sulfate Sodium Salt)以及500～2000mg/LPluronic-F68(普朗尼克F68)，其余为水(即水补足至1L)。
本发明一较佳实例中的中国仓鼠卵巢细胞培养基，其含有：72mg/L L-色氨酸、50mg/L L-胱氨酸、87.6mg/L L-半胱氨酸、130.2mg/L L-酪氨酸盐钠盐二水、324.6mg/L L-异亮氨酸、376.8mg/L L-亮氨酸、190.6mg/L L-缬氨酸、321.2mg/L L-精氨酸、206.1mg/L L-组氨酸、425.3mg/L L-赖氨酸、84.7mg/L L-蛋氨酸、196.7mg/L L-苯丙氨酸、182.6mg/L L-谷氨酸、213mg/LL-天冬酰胺、13mg/L甘氨酸、210mg/L L-脯氨酸、7mg/L L-丙氨酸、113.4mg/L L-天冬氨酸、326mg/L L-丝氨酸、412mg/L L-苏氨酸、584mg/L L-谷氨酰胺、116mg/L无水氯化钙、0.0015mg/L五水硫酸铜、0.09mg/L九水硝酸铁、0.417mg/L七水硫酸亚铁、311.8mg/L氯化钾、40mg/L氯化镁、62.5mg/L一水磷酸二氢钠、71mg/L无水磷酸氢二钠、1mg/L七水硫酸锌、29.8mg/L柠檬酸铁、0.01mg/L亚硒酸钠、3000mg/L碳酸氢钠、12.5mg/L抗坏血酸、2.6mg/L盐酸吡多醇、1.2mg/L维生素B12、4mg/L烟酰胺、0.68mg/L核黄素、3.65mg/L盐酸硫胺素、19.8mg/L氯化胆碱、1.78mg/L叶酸、1.6mg/L泛酸钙、98mg/L丙酮酸钠、1.25mg/L谷胱甘肽(还原型)、12.6mg/L肌醇、0.01mg/L生物素、4mg/L次黄嘌呤、1.8mg/L腐胺、0.5mg/L硫辛酸、0.08mg/L亚油酸、3.65mg/L胸腺嘧啶、3150mg/L葡萄糖、2000mg/L HEPES缓冲液、35mg/L硫酸葡聚糖以及1200mg/L Pluronic-F68，其余为水(即水补足至1L)。
本发明中，所述的水为本领域常规用水，一般为超纯水。
本发明的中国仓鼠卵巢细胞培养基较佳地还含有：0.5～2.5mg/L二水氯化亚锡(SnCl2·2H2O)、0.02～0.18mg/L矾酸铵(NH4VO3)、0.005～0.03mg/L乙酸钡(Ba(C2H3O2O)2)、0.001～0.04mg/L硝酸银(AgNO3)、0.005～0.03mg/L硅酸钠(Na2SiO3)、0.1～1.0mg/L六水氯化铝(AlCl3·6H2O)、0.001～0.01mg/L八水硫酸镉(CdSO4·8H2O)、0.01～0.3mg/L六水氯化钴(CoCl·6H2O)、0.07～0.5mg/L一水硫酸锰(MnSO4·H2O)、0.01～0.1mg/L四水钼酸铵((NH4)6Mo7O24·4H2O)、0.1～0.9mg/L六水氯化镍(NiCl2·6H2O)、0.00001～0.0003mg/L溴化钾(KBr)、0.001～0.01mg/L氟化钠(NaF)、0.00005～0.00048mg/L碘化钾(KI)、0.001～0.008mg/L花生四烯酸、0.11～0.88mg/L胆固醇、0.035～0.28mg/L DL-醋酸α-生育酚、0.003～0.05mg/L豆蔻酸、0.002～0.04mg/L油酸、0.005～0.04mg/L棕榈酸烯、0.005～0.04mg/L棕榈酸以及0.001～0.09mg/L硬脂酸中的一种或多种。
本发明另一较佳实例中的中国仓鼠卵巢细胞培养基，其还含有：1.5mg/L二水氯化亚锡、0.08mg/L矾酸铵、0.02mg/L乙酸钡、0.01mg/L硝酸银、0.01mg/L硅酸钠、0.7mg/L六水氯化铝、0.003mg/L八水硫酸镉、0.1mg/L六水氯化钴、0.2mg/L一水硫酸锰、0.033mg/L四水钼酸铵、0.45mg/L六水氯化镍、0.00002mg/L溴化钾、0.0058mg/L氟化钠、0.00023mg/L碘化钾、0.0035mg/L花生四烯酸、0.52mg/L胆固醇、0.11mg/L DL-醋酸α-生育酚、0.018mg/L豆蔻酸、0.025mg/L油酸、0.019mg/L棕榈酸烯、0.024mg/L棕榈酸以及0.046mg/L硬脂酸中的一种或多种。
本发明的中国仓鼠卵巢细胞培养基的制备方法可选用本领域常规方法，较佳的包括下列步骤：将上述成分溶于水中简单均匀混合，即可。其中，在均匀混合之后，一般按照本领域常规方式除菌。其中，培养基中涉及含有柠檬酸铁或矾酸铵时，需要将柠檬酸铁或矾酸铵沸水浴加热溶解之后再与其他组分均匀混合；培养基中涉及含有硫辛酸或氯化亚锡时，需要将硫辛酸或氯化亚锡使用无水乙醇进行溶解，之后再与其他组分均匀混合。
本发明还涉及本发明的中国仓鼠卵巢细胞培养基在培养中国仓鼠卵巢细胞中的应用。
本发明中的中国仓鼠卵巢细胞培养基在培养CHO细胞过程中，一般按照本领域常规方法使用即可，较佳地为将CHO细胞接种于本发明的中国仓鼠卵巢细胞培养基中，在常规CHO细胞的培养条件下培养。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明提供了一种中国仓鼠卵巢细胞培养基及其应用。本发明的培养基成本较低，组成中无动物源成分、不含蛋白质且化学成分确定，其通过各组分的调配协同，除满足细胞生长基本要求外，能够有效调节、传递并控制细胞的培养，实现良好的高密度细胞培养，其培养效果与现有的血清培养基、蛋白培养基或蛋白水解物培养基的培养效果相比相当且甚至优于现有培养基。本发明还涉及中国仓鼠卵巢细胞培养基在培养中国仓鼠卵巢细胞中的应用，支持CHO细胞高密度生长，完全可以代替血清培养基用于CHO细胞的培养。
附图说明
图1为实施例1与对比例1的中国仓鼠卵巢细胞培养基分别培养CHO细胞，其细胞密度随时间变化关系图；其中，◆为GIBCO CD CHO AGTTM培养基；■为实施例1的培养基。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。
实施例1
按照下述各成分浓度配制本发明的无动物源成分、无蛋白质、化学成分确定的培养基，将各成分用水溶解，其中，柠檬酸铁需要沸水浴加热溶解之后再与其他物质均匀混合；硫辛酸使用无水乙醇进行溶解之后再均匀混合；均匀混合后使用0.22微米的硝酸纤维素膜进行过滤除菌。
本发明的中国仓鼠卵巢细胞培养基含有：72mg/L L-色氨酸、50mg/L L-胱氨酸、87.6mg/L L-半胱氨酸、130.2mg/L L-酪氨酸盐钠盐二水、324.6mg/LL-异亮氨酸、376.8mg/L L-亮氨酸、190.6mg/L L-缬氨酸、321.2mg/L L-精氨酸、206.1mg/L L-组氨酸、425.3mg/L L-赖氨酸、84.7mg/L L-蛋氨酸、196.7mg/L L-苯丙氨酸、182.6mg/L L-谷氨酸、213mg/L L-天冬酰胺、13mg/L甘氨酸、210mg/L L-脯氨酸、7mg/L L-丙氨酸、113.4mg/L L-天冬氨酸、326mg/L L-丝氨酸、412mg/L L-苏氨酸、584mg/L L-谷氨酰胺、116mg/L无水氯化钙、0.0015mg/L五水硫酸铜、0.09mg/L九水硝酸铁、0.417mg/L七水硫酸亚铁、311.8mg/L氯化钾、40mg/L氯化镁、62.5mg/L一水磷酸二氢钠、71mg/L无水磷酸氢二钠、1mg/L七水硫酸锌、29.8mg/L柠檬酸铁、0.01mg/L亚硒酸钠、3000mg/L碳酸氢钠、12.5mg/L抗坏血酸、2.6mg/L盐酸吡多醇、1.2mg/L维生素B12、4mg/L烟酰胺、0.68mg/L核黄素、3.65mg/L盐酸硫胺素、19.8mg/L氯化胆碱、1.78mg/L叶酸、1.6mg/L泛酸钙、98mg/L丙酮酸钠、1.25mg/L谷胱甘肽(还原型)、12.6mg/L肌醇、0.01mg/L生物素、4mg/L次黄嘌呤、1.8mg/L腐胺、0.5mg/L硫辛酸、0.08mg/L亚油酸、3.65mg/L胸腺嘧啶、3150mg/L葡萄糖、2000mg/L HEPES缓冲液、35mg/L硫酸葡聚糖以及1200mg/L Pluronic-F68。
在制得的该培养基中接入CHO细胞(CHO种子细胞为该培养基驯化细胞，来源上海富莼科芯生物技术公司)，接种密度为5×105细胞/ml，进行悬浮培养，培养条件为：37℃，5％CO2(v/v)，湿度大于95％的环境进行培养，细胞的最大密度达到7.3×106cells/ml。
其中，CHO种子细胞按下述方法进行驯化培养：将贴壁生长的CHO细胞用0.25％(w/v)胰酶进行消化，用含血清培养基(DMEM+10％Hyclone小牛血清，购于上海世仪生物科技公司)进行重悬后离心，倾倒上清，将细胞重悬于前述配制的本发明培养基中驯化适应培养，CHO细胞生长方式可由贴壁生长转化为悬浮生长，由此可进一步使CHO细胞在本培养基中扩增生长。
实施例2
在实施例1中培养基含有的成分基础上，再添加1.5mg/L二水氯化亚锡、0.08mg/L矾酸铵、0.02mg/L乙酸钡、0.01mg/L硝酸银、0.01mg/L硅酸钠、0.7mg/L六水氯化铝、0.003mg/L八水硫酸镉、0.1mg/L六水氯化钴、0.2mg/L一水硫酸锰、0.033mg/L四水钼酸铵、0.45mg/L六水氯化镍、0.00002mg/L溴化钾、0.0058mg/L氟化钠、0.00023mg/L碘化钾、0.0035mg/L花生四烯酸、0.52mg/L胆固醇、0.11mg/L DL-醋酸α-生育酚、0.018mg/L豆蔻酸、0.025mg/L油酸、0.019mg/L棕榈酸烯、0.024mg/L棕榈酸以及0.046mg/L硬脂酸。其中，柠檬酸铁和矾酸铵需要沸水浴加热溶解之后再与其他物质均匀混合；硫辛酸和氯化亚锡使用无水乙醇进行溶解，之后再均匀混合，然后使用0.22微米的硝酸纤维素膜进行过滤除菌。
在制得的该培养基中接入实施例1中CHO细胞，接种密度为5×105cells/ml，进行悬浮培养，培养条件为：37℃，5％CO2(v/v)，湿度大于95％的环境进行培养，细胞的最大细胞密度为8.0×106cells/ml。
对比例1
对比例1所用的培养基为GIBCO CD CHO AGTTM培养基(上海源智生化科技有限公司，此培养基属于化学成分确定的培养基)，在该培养基中接入CHO细胞(种子细胞为该培养基驯化细胞)，接种密度为5×105cells/ml，进行悬浮培养，培养条件为：37℃，5％CO2，湿度大于95％的环境进行培养，细胞最大密度为4.5×106cells/ml。
其中，CHO种子细胞按下述方法进行驯化培养：将贴壁生长的CHO细胞用0.25％(w/v)胰酶进行消化，用含血清培养基(DMEM+10％Hyclone小牛血清，购于上海世仪生物科技公司)进行重悬后离心，倾倒上清，将细胞重悬于GIBCO CD CHO AGTTM培养基中驯化适应培养，CHO细胞生长方式可由贴壁生长转化为悬浮生长，由此可进一步使CHO细胞在该培养基中扩增生长。
在此，将CHO细胞接种于对比例1与实施例1中的培养基培养过程中，细胞密度随时间的变化情况记录于图1。由图1的对比发现，在实施例1的无动物源，无蛋白质，化学成分确定的培养基中培养CHO细胞，细胞的生长优于同样条件培养的市售商业化培养基，完全可以用于大规模培养，且成本较低，远低于商业化化学成份确定的培养基价格，适于在工业化生产中应用。
Claims (7)
1.一种中国仓鼠卵巢细胞培养基，其特征在于：所述的中国仓鼠卵巢细胞培养基含有如下组分：9.02～141.06mg/L L-色氨酸、31.29～148.74mg/L L-胱氨酸、17.56～159.87mg/L L-半胱氨酸、38.4～377.28mg/L L-酪氨酸盐钠盐二水、54.47～654mg/L L-异亮氨酸、59.05～874.32mg/L L-亮氨酸、50～420mg/L L-缬氨酸、80～720mg/L L-精氨酸、31～480mg/L L-组氨酸、80～852.3mg/L L-赖氨酸、10～184.7mg/L L-蛋氨酸、20～438.3mg/L L-苯丙氨酸、1～235.8mg/L L-谷氨酸、70～540mg/L L-天冬酰胺、1～40mg/L甘氨酸、10～620mg/L L-脯氨酸、0.1～63mg/L L-丙氨酸、10～223.6mg/L L-天冬氨酸、20～661.2mg/L L-丝氨酸、40～893.4mg/L L-苏氨酸、365～1168mg/L L-谷氨酰胺、10～160mg/L无水氯化钙、0.001～0.003mg/L五水硫酸铜、0.05～1mg/L九水硝酸铁、0.1～1mg/L七水硫酸亚铁、100～400mg/L氯化钾、20～60mg/L氯化镁、5～200mg/L一水磷酸二氢钠、50～300mg/L无水磷酸氢二钠、0.1～2mg/L七水硫酸锌、8～50mg/L柠檬酸铁、0.001～0.05mg/L亚硒酸钠、3000mg/L碳酸氢钠、2～20mg/L抗坏血酸、0.1～4mg/L盐酸吡多醇、0.2～2.72mg/L维生素B12、0.5～6.5mg/L烟酰胺、0.05～1.5mg/L核黄素、0.1～6.81mg/L盐酸硫胺素、2.57～26.94mg/L氯化胆碱、0.2～3mg/L叶酸、0.1～2.5mg/L泛酸钙、2～150mg/L丙酮酸钠、0.3～2.25mg/L还原型谷胱甘肽、5～25mg/L肌醇、0.0001～0.5mg/L生物素、0.008～8mg/L次黄嘌呤、0.001～9mg/L腐胺、0.003～0.8mg/L硫辛酸、0.003～1.2mg/L亚油酸、0.02～6mg/L胸腺嘧啶、2150～7000mg/L葡萄糖、1500～4000mg/L HEPES缓冲液、5～70mg/L硫酸葡聚糖以及500～2000mg/L Pluronic-F68，其余为水。
2.如权利要求1所述的中国仓鼠卵巢细胞培养基，其特征在于：所述的中国仓鼠卵巢细胞培养基含有如下组分：72mg/L L-色氨酸、50mg/L L-胱氨酸、87.6mg/L L-半胱氨酸、130.2mg/L L-酪氨酸盐钠盐二水、324.6mg/LL-异亮氨酸、376.8mg/L L-亮氨酸、190.6mg/L L-缬氨酸、321.2mg/L L-精氨酸、206.1mg/L L-组氨酸、425.3mg/L L-赖氨酸、84.7mg/L-蛋氨酸、196.7mg/L-苯丙氨酸、182.6mg/L L-谷氨酸、213mg/L L-天冬酰胺、13mg/L甘氨酸、210mg/L L-脯氨酸、7mg/L L-丙氨酸、113.4mg/L L-天冬氨酸、326mg/L L-丝氨酸、412mg/L L-苏氨酸、584mg/L L-谷氨酰胺、116mg/L无水氯化钙、0.0015mg/L五水硫酸铜、0.09mg/L九水硝酸铁、0.417mg/L七水硫酸亚铁、311.8mg/L氯化钾、40mg/L氯化镁、62.5mg/L一水磷酸二氢钠、71mg/L无水磷酸氢二钠、1mg/L七水硫酸锌、29.8mg/L柠檬酸铁、0.01mg/L亚硒酸钠、3000mg/L碳酸氢钠、12.5mg/L抗坏血酸、2.6mg/L盐酸吡多醇、1.2mg/L维生素B12、4mg/L烟酰胺、0.68mg/L核黄素、3.65mg/L盐酸硫胺素、19.8mg/L氯化胆碱、1.78mg/L叶酸、1.6mg/L泛酸钙、98mg/L丙酮酸钠、1.25mg/L还原型谷胱甘肽、12.6mg/L肌醇、0.01mg/L生物素、4mg/L次黄嘌呤、1.8mg/L腐胺、0.5mg/L硫辛酸、0.08mg/L亚油酸、3.65mg/L胸腺嘧啶、3150mg/L葡萄糖、2000mg/L HEPES缓冲液、35mg/L硫酸葡聚糖以及1200mg/L Pluronic-F68，其余为水。
3.如权利要求1所述的中国仓鼠卵巢细胞培养基，其特征在于：所述的中国仓鼠卵巢细胞培养基还含有如下组分中的一种或多种：0.5～2.5mg/L二水氯化亚锡、0.02～0.18mg/L矾酸铵、0.005～0.03mg/L乙酸钡、0.001～0.04mg/L硝酸银、0.005～0.03mg/L硅酸钠、0.1～1.0mg/L六水氯化铝、0.001～0.01mg/L八水硫酸镉、0.01～0.3mg/L六水氯化钴、0.07～0.5mg/L一水硫酸锰、0.01～0.1mg/L四水钼酸铵、0.1～0.9mg/L六水氯化镍、0.00001～0.0003mg/L溴化钾、0.001～0.01mg/L氟化钠、0.00005～0.00048mg/L碘化钾、0.001～0.008mg/L花生四烯酸、0.11～0.88mg/L胆固醇、0.035～0.28mg/L DL-醋酸α-生育酚、0.003～0.05mg/L豆蔻酸、0.002～0.04mg/L油酸、0.005～0.04mg/L棕榈酸烯、0.005～0.04mg/L棕榈酸以及0.001～0.09mg/L硬脂酸。
4.如权利要求3所述的中国仓鼠卵巢细胞培养基，其特征在于：所述的中国仓鼠卵巢细胞培养基还含有如下组分中的一种或多种：1.5mg/L二水氯化亚锡、0.08mg/L矾酸铵、0.02mg/L乙酸钡、0.01mg/L硝酸银、0.01mg/L硅酸钠、0.7mg/L六水氯化铝、0.003mg/L八水硫酸镉、0.1mg/L六水氯化钴、0.2mg/L一水硫酸锰、0.033mg/L四水钼酸铵、0.45mg/L六水氯化镍、0.00002mg/L溴化钾、0.0058mg/L氟化钠、0.00023mg/L碘化钾、0.0035mg/L花生四烯酸、0.52mg/L胆固醇、0.11mg/L DL-醋酸α-生育酚、0.018mg/L豆蔻酸、0.025mg/L油酸、0.019mg/L棕榈酸烯、0.024mg/L棕榈酸以及0.046mg/L硬脂酸。
5.如权利要求1或2所述的中国仓鼠卵巢细胞培养基的制备方法，其特征在于：其包括下列步骤：(1)将柠檬酸铁沸水浴加热溶解，将硫辛酸使用无水乙醇进行溶解，将其余组分用水溶解；(2)将步骤(1)的各组分均匀混合，然后除菌。
6.如权利要求3或4所述的中国仓鼠卵巢细胞培养基的制备方法，其特征在于：其包括下列步骤：(1)将柠檬酸铁和矾酸铵沸水浴加热溶解，将硫辛酸和氯化亚锡使用无水乙醇进行溶解，将其余组分用水溶解；(2)将步骤(1)的各组分均匀混合，然后除菌。
7.如权利要求1～4任一项所述的中国仓鼠卵巢细胞培养基在培养中国仓鼠卵巢细胞中的应用。
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