CN112063578B - 适应全悬浮细胞培养的培养基及其制备方法和应用 - Google Patents
适应全悬浮细胞培养的培养基及其制备方法和应用 Download PDFInfo
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Abstract
一种适用于全悬浮培养基的培养基,适用于包括基础代谢物、核苷酸、维生素、无机盐、抗细胞结团剂、剪切力保护剂、流感病毒增殖促进剂和其他化学成分明确的添加物。本发明的培养基,化学组分完全明确,不含有水解物,且不含有任何动物源成分,包括动物源蛋白(如:胰岛素和转铁蛋白)等,质量可控,重复性好,能适用于全悬浮细胞培养,还能将贴壁细胞驯化为单个悬浮细胞,显著提高细胞密度。
Description
技术领域
本发明涉及一种用于生产生物制品的组合物,尤其涉及一种培养细胞的培养基,应用于全悬浮细胞培养和MDCK细胞培养。
背景技术
70多年以来,鸡胚一直是流感疫苗的传统生产基质,然而因其供应不足、安全性、批次差异性等问题,采用动物细胞培养技术来生产流感疫苗近年来得到了飞速的发展。
MDCK(Mardin-Darby canine kidney)细胞是目前公认的最适于扩增甲型和乙型流感病毒的细胞基质之一,生产工艺按照细胞性质可分为贴壁培养工艺和悬浮培养工艺。对于贴壁细胞培养工艺来说,血清的使用会造成培养工艺的复杂性和批次间不稳定性;细胞在微载体上的贴附生长易受到培养基和培养环境的影响,细胞密度也通常受到贴壁面积的制约;需要添加额外的胰酶使细胞脱离微载体,增加了操作复杂性。
因此,通过无血清悬浮培养MDCK细胞来制备流感疫苗越来越得到研究者和疫苗生产企业的青睐。目前,国内仍十分缺乏利于MDCK悬浮细胞高效生长的商业化无血清培养基,少量的现有技术所提供的MDCK无血清培养基中因培养基组分的不合理设计导致细胞结团;且组分中均含有转铁蛋白、胰岛素等动物源成分,价格昂贵。另外大多培养基中含有植物类或动物类水解物,培养基组分并不明确,安全性不佳,不利于疫苗的下游纯化工作以及人用流感疫苗的大规模开发和生产。
基于以上原因,有必要去开发一款化学组分完全明确、配制简单、安全性好、成本低的化学限定培养基,能够快速驯化MDCK贴壁细胞至单个悬浮生长且生长速率高,为基于MDCK细胞全悬浮培养的流感疫苗生产工艺提供良好的基础。
发明内容
本发明的一个目的在于提供一种培养基,其化学组分完全明确,能适用于全悬浮细胞培养。
本发明的另一个目的在于提供一种培养基,其化学组分完全明确,能适用于全悬浮细胞的高密度培养培养。
本发明的再一个目的在于提供一种培养基,利于将贴壁细胞驯化为单个悬浮细胞,提高细胞的培养效率。
本发明的又一个目的在于提供一种培养基,适用于对MDCK细胞实施全悬浮细胞培养,实现细胞的高密度培养。
本发明的第五个目的在于提供一种培养基,适用于采用MDCK细胞的疫苗制取。
一种培养基,包括如下组分:
第一类营养物,用于满足细胞的基础代谢,包括:但不限于
2000~10000mg/L D-葡萄糖、40~600 mg/L丙酮酸钠、5~30mg/L L-丙氨酸、L-精氨酸 100~600 mg/L、5~60 mg/L L-天冬酰胺、5~60 mg/L L-天冬氨酸、5~80 mg/L L-胱氨酸、10~150mg/L L-半胱氨酸、5~60mg/L L-谷氨酸、300~1500mg/L L-谷氨酰胺、5~100mg/L甘氨酸、10~150mg/L L-组氨酸;20~150mg/L L-异亮氨酸、40~250mg/L L-亮氨酸、40~150mg/L L-赖氨酸、10~150mg/L L-甲硫氨酸、10~250mg/L L-苯丙氨酸、10~200mg/L L-脯氨酸、40~150mg/L L-丝氨酸、40~200mg/L L-苏氨酸、10~100mg/L L-色氨酸、10~100 mg/L L-酪氨酸和40~200mg/L L-缬氨酸;
第二类营养物,系核苷酸,包括:
1~25mg/L次黄嘌呤、0.1~1mg/L胸苷、1~15mg/L腺苷、1~15mg/L尿苷、1~15mg/L胞苷和1~15mg/L鸟苷;
第三类营养物,系维生素,包括:
0.01~0.20mg/L D-生物素、1~10mg/L叶酸 、1~10mg/L烟酰胺、1~10mg/L吡哆醇、1~10mg/L硫胺素、0.1~1mg/L核黄素、10~50mg/L氯化胆碱、2~10mg/L D-泛酸钙和10~50 mg/L肌醇;
第四类营养物,系无机盐,包括:
10~50mg/L硝酸铁、0.1~1mg/L硫酸亚铁、20~100mg/L硫酸镁、100~500mg/L氯化钾、4000~9000mg/L氯化钠、50~120mg/L磷酸氢二钠、50~120mg/L磷酸二氢钠和15~120mg/L亚硒酸钠;以及
500~2500mg/L剪切力保护剂(如:Pluronic F-68)、20~150mg/L等。
另一种培养基,包括如下组分:
第一类营养物,用于满足细胞的基础代谢,包括:但不限于
5000mg/L D-葡萄糖、200mg/L丙酮酸钠、15~17mg/L L-丙氨酸、285~295mg/L L-精氨酸 、35~40mg/L L-天冬酰胺、35~40mg/L L-天冬氨酸、45~50mg/L L-胱氨酸、75~80mg/L L-半胱氨酸、30~35mg/L L-谷氨酸、840~860mg/L L-谷氨酰胺、45~55mg/L甘氨酸、70~80mg/L L-组氨酸;80~90mg/L L-异亮氨酸、130~140mg/L L-亮氨酸、100~110mg/L L-赖氨酸、80~90mg/L L-甲硫氨酸、100~110mg/L L-苯丙氨酸、90~100mg/L L-脯氨酸、70~80mg/L L-丝氨酸、120~130mg/L L-苏氨酸、50~60mg/L L-色氨酸、55~65mg/L L-酪氨酸和90~100mg/L L-缬氨酸;
第二类营养物,系核苷酸,包括:
10~15mg/L次黄嘌呤、0.3~0.6mg/L胸苷、6~9mg/L腺苷、6~9mg/L尿苷、6~9mg/L胞苷和6~9mg/L鸟苷;
第三类营养物,系维生素,包括:
0.05~0.10mg/L D-生物素、4~8mg/L叶酸、4~8mg/L烟酰胺、4~8mg/L吡哆醇、3~5mg/L硫胺素、0.4~0.6mg/L核黄素、30~4mg/L氯化胆碱、5~8mg/L D-泛酸钙和30~35mg/L肌醇;
第四类营养物,系无机盐,包括:
20~30mg/L硝酸铁、0.3~0.6mg/L硫酸亚铁、30~50mg/L硫酸镁、320~330mg/L氯化钾、6500~7500mg/L氯化钠、65~75mg/L磷酸氢二钠、60~70mg/L磷酸二氢钠和50~60mg/L亚硒酸钠;
1500~2000mg/L剪切力保护剂(如:Pluronic F-68)、50~70mg/L抗细胞结团剂(如:硫酸葡聚糖)等。
本发明的培养基,还加入第五类营养物,包括:
10~40mg/L柠檬酸铁铵、1~10mg/L乙醇胺和0.5~5mg/L谷胱甘肽。
柠檬酸铁铵用于铁的运输,保证细胞对铁的摄取。还原型谷胱甘肽参与三羧酸循环及糖代谢,促进糖、脂肪及蛋白质代谢,且可加速自由基的排泄,使细胞免受损。乙醇胺作为细胞脂类合成(如:磷脂和磷脂酰乙醇胺等)的前体物质加入到细胞培养基中。
另一种第五类营养物,包括:
20~30mg/L柠檬酸铁铵、3.0~5.0mg/L乙醇胺和1.0~2.0mg/L谷胱甘肽。
本发明的培养基还包括第六类营养物,系流感病毒增殖促进剂,包括:
1~10mg/L胆固醇、0.4~4mg/L DL-α-生育酚醋酸酯、0.04~0.6mg/L豆蔻酸、0.04~0.6mg/L棕榈酸、0.04~0.6 mg/L硬脂酸、500~5000mg/L氯化镁、25~250mg/L氯化钙、1~20mg/L二甲基亚砜、0.2~2.0mg/L硫酸锌、5~25mg/L硫酸铜、0.0001~0.001mg/L硫酸锰和0.001~0.005mg/L偏钒酸铵。
另一种第六类营养物,包括:
3~5mg/L胆固醇、1.0~2.0mg/L DL-α-生育酚醋酸酯、0.1~0.3mg/L豆蔻酸、0.3~0.4mg/L棕榈酸、0.2~0.4mg/L硬脂酸、3000~3300mg/L氯化镁、170~180mg/L氯化钙、10~15mg/L二甲基亚砜、1.0~1.3mg/L硫酸锌、15~20mg/L硫酸铜、0.0003~0.0005mg/L硫酸锰和0.001~0.003mg/L偏钒酸铵。
本发明的培养基,能适用于全悬浮细胞培养,利于将贴壁细胞驯化为单个悬浮细胞,提高细胞的培养密度,尤其是应用于采用MDCK细胞的疫苗制取中。
一种制取本发明培养基的方法,包括:
先将各组分混合后磨成干粉,于30℃下溶解,并充分混合20分钟后得到混合液;
然后,调节混合液的pH至6.3~6.7,继续搅拌混匀;
最后,加入NaHCO3粉末(如:3g/L),利于形成在培养基中形成缓冲液体系,即得。
制得的培养基还应过滤除菌和避光保存。
本发明技术方案实现的有益效果:
本发明的培养基化学组分完全明确,不含有水解物,且不含有任何动物源成分,包括动物源蛋白(如:胰岛素和转铁蛋白)等,质量可控,重复性好,能适用于全悬浮细胞培养,还能将贴壁细胞驯化为单个悬浮细胞,显著提高细胞密度。
本发明培养基以小分子物质代替,实现了相同功能,因此安全性高,成本低,利于下游纯化工作。在培养基中加入流感病毒增殖促进剂后,则能适用于对MDCK细胞实施全悬浮细胞培养,以及人用疫苗的开发和大规模制取。
与现有技术相比,本发明的培养基可快速将MDCK贴壁细胞驯化为悬浮细胞,所获细胞呈单个悬浮生长,生长速率高。
与现有技术相比,本发明的培养基配制方法简单快速,大大缩短了配制时间,适用于人用疫苗产品的大规模生产。
附图说明
图1为本发明制取的RS-2培养基驯化8代以后的MDCK细胞活细胞密度和活率结果图;
图2为本发明制取的RS-2培养基驯化8代以后的MDCK细胞形态图。
具体实施方式
以下结合附图详细描述本发明的技术方案。本发明实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围中。
本发明以下实施例采用的MDCK细胞来自华东理工大学赠与,其原始来源为ATCCMDCK CCL-34贴壁细胞株。
本发明以下实施例的培养基,其制取方法如下:
(1)将各原料混合后磨成干粉,然后将其在30℃温度下溶解,并充分混合20分钟后得到混合液;
(2)用NaOH固体调节培养基混合液的pH至6.3~6.7,继续搅拌混合(如:20分钟),料液变澄清;
(3)加入NaHCO3粉末形成培养基缓冲液体系,并定容得到用于MDCK细胞全悬浮培养的化学限定培养基。
本发明以下实施例对细胞进行观察的方法如下:
吸取20μL混合均匀的细胞悬液,轻轻滴于干净的载玻片上,用盖玻片轻压盖住细胞液,于显微镜下合适放大倍数下观察细胞形态。
实施例1
按组分和用量制取用于MDCK全悬浮培养的培养基,并记为RS-1。
基础代谢营养物:D-葡萄糖 3500mg/L;丙酮酸钠92mg/L;L-丙氨酸 10.7mg/L;L-精氨酸207.4mg/L;L-天冬酰胺 24.5mg/L;L-天冬氨酸 23.4mg/L;L-胱氨酸 23.54mg/L;L-半胱氨酸 55.44mg/L;L-谷氨酸 22.44mg/L;L-谷氨酰胺 536mg/L;甘氨酸 19.86mg/L;L-组氨酸 35.48mg/L;L-异亮氨酸 42.13mg/L;L-亮氨酸 75.59mg/L;L-赖氨酸 84.51mg/L;L-甲硫氨酸 42.33mg/L;L-苯丙氨酸 50.87mg/L;L-脯氨酸 46.67mg/L;L-丝氨酸60.06mg/L;L-苏氨酸80.67mg/L;L-色氨酸 33.73mg/L;L-酪氨酸 48.95mg/L;L-缬氨酸53.01mg/L。
核苷酸:次黄嘌呤 6.2 mg/L;胸苷 0.22 mg/L;腺苷 3.5 mg/L;尿苷 3.5 mg/L;胞苷 3.5 mg/L;鸟苷 3.5 mg/L。
维生素:D-生物素 0.034 mg/L;叶酸 2.40 mg/L;烟酰胺 1.55 mg/L;吡哆醇1.66mg/L;硫胺素 1.62 mg/L;核黄素 0.24 mg/L;氯化胆碱 13.51 mg/L;D-泛酸钙 2.14 mg/L;肌醇 13 mg/L。
无机盐:硝酸铁 25.64 mg/L;硫酸亚铁 0.456 mg/L;硫酸镁 24.5 mg/L;氯化钾324.4 mg/L;氯化钠 5000 mg/L;磷酸氢二钠 71 mg/L;磷酸二氢钠 62.5 mg/L;亚硒酸钠24.53 mg/L。
剪切力保护剂:1000 mg/L。
抗细胞结团剂:30 mg/L。
流感病毒增殖促进剂:胆固醇 1.59 mg/L;DL-α-生育酚醋酸酯 0.61 mg/L;豆蔻酸 0.1314 mg/L;棕榈酸 0.128 mg/L;硬脂酸 0.135 mg/L;氯化镁 1897 mg/L;氯化钙112.3 mg/L;二甲基亚砜 6.5 mg/L;硫酸锌 0.56 mg/L;硫酸铜 7.33 mg/L;硫酸锰0.000165 mg/L;偏钒酸铵 0.00129 mg/L。
其它添加物: 柠檬酸铁铵 15 mg/L;乙醇胺 3.5 mg/L;谷胱甘肽 0.7 mg/L。
实施例2
按组分和用量制取用于MDCK全悬浮培养的培养基,并记为RS-2。
基础代谢营养物;D-葡萄糖 5000 mg/L;丙酮酸钠200 mg/L;L-丙氨酸 15.7mg/L;L-精氨酸287.4 mg/L;L-天冬酰胺 37.5 mg/L;L-天冬氨酸 39.4mg/L;L-胱氨酸 46.54mg/L;L-半胱氨酸 75.44 mg/L;L-谷氨酸 32.54mg/L;L-谷氨酰胺 856 mg/L;甘氨酸49.86 mg/L;L-组氨酸 77.88mg/L;L-异亮氨酸 86.33 mg/L;L-亮氨酸 132.39 mg/L;L-赖氨酸 102.21 mg/L;L-甲硫氨酸 85.35 mg/L;L-苯丙氨酸 106.62 mg/L;L-脯氨酸 93.77mg/L;L-丝氨酸 75.56mg/L;L-苏氨酸 123.47 mg/L;L-色氨酸 53.33 mg/L;L-酪氨酸58.65mg/L;L-缬氨酸 93.41 mg/L。
核苷酸:次黄嘌呤 11.2 mg/L;胸苷 0.52 mg/L;腺苷 7.5 mg/L;尿苷 7.5 mg/L;胞苷 7.5 mg/L;鸟苷 7.5 mg/L。
维生素:D-生物素 0.084 mg/L;叶酸 5.40 mg/L;烟酰胺 4.65 mg/L;吡哆醇4.56mg/L;硫胺素 3.65 mg/L;核黄素 0.54 mg/L;氯化胆碱 33.51 mg/L;D-泛酸钙 6.44 mg/L;肌醇 32 mg/L。
无机盐:硝酸铁 25.64 mg/L;硫酸亚铁 0.456 mg/L;硫酸镁 40.5 mg/L;氯化钾324.4 mg/L;氯化钠 7000 mg/L;磷酸氢二钠 71 mg/L;磷酸二氢钠 62.5 mg/L;亚硒酸钠54.33 mg/L。
剪切力保护剂:1500 mg/L;
抗细胞结团剂:60 mg/L;
流感病毒增殖促进剂:胆固醇 3.89 mg/L;DL-α-生育酚醋酸酯 1.51 mg/L;豆蔻酸 0.2334 mg/L;棕榈酸 0.2864 mg/L;硬脂酸 0.299 mg/L;氯化镁 3244mg/L;氯化钙178.3 mg/L;二甲基亚砜 12.5 mg/L;硫酸锌 1.12 mg/L;硫酸铜 17.33 mg/L;硫酸锰0.00045 mg/L;偏钒酸铵 0.00199 mg/L;
其它添加物:
柠檬酸铁铵 25 mg/L;乙醇胺 3.5 mg/L;谷胱甘肽 1.5 mg/L。
实施例3
按组分和用量制取用于MDCK全悬浮培养的培养基,并记为RS-3。
基础代谢营养物:D-葡萄糖 8500mg/L;丙酮酸钠450mg/L;L-丙氨酸 23.7mg/L;L-精氨酸582.4mg/L;L-天冬酰胺 47.5mg/L;L-天冬氨酸 49.3mg/L;L-胱氨酸 67.24 mg/L;L-半胱氨酸 105.34 mg/L;L-谷氨酸 52.64mg/L;L-谷氨酰胺 1656mg/L;甘氨酸 50.86mg/L;L-组氨酸 139.67mg/L;L-异亮氨酸 137.90mg/L;L-亮氨酸 299.34 mg/L;L-赖氨酸143.21 mg/L;L-甲硫氨酸 110.35 mg/L;L-苯丙氨酸 206.62 mg/L;L-脯氨酸 146.41 mg/L;L-丝氨酸 115.86 mg/L;L-苏氨酸 113.47 mg/L;L-色氨酸 75.32 mg/L;L-酪氨酸78.95 mg/L;L-缬氨酸 143.41 mg/L。
核苷酸:次黄嘌呤 21.2 mg/L;胸苷 0.52 mg/L;腺苷 10.5mg/L;尿苷 10.5 mg/L;胞苷 10.5 mg/L;鸟苷 10.5 mg/L。
维生素:D-生物素 0.144 mg/L;叶酸 7.48 mg/L;烟酰胺 6.66 mg/L;吡哆醇6.86mg/L;硫胺素 6.05 mg/L;核黄素 0.74 mg/L;氯化胆碱 38.51 mg/L;D-泛酸钙 8.44 mg/L;肌醇 32 mg/L。
无机盐:硝酸铁 25.64 mg/L;硫酸亚铁 0.456 mg/L;硫酸镁 70.5 mg/L;氯化钾324.4 mg/L;氯化钠 8000 mg/L;磷酸氢二钠 71 mg/L;磷酸二氢钠 62.5 mg/L;亚硒酸钠74.33 mg/L。
剪切力保护剂:2200 mg/L。
抗细胞结团剂:90 mg/L。
流感病毒增殖促进剂:胆固醇 6.29 mg/L;DL-α-生育酚醋酸酯 2.11mg/L;豆蔻酸0.3334 mg/L;棕榈酸 0.384 mg/L;硬脂酸 0.419 mg/L;氯化镁 4244mg/L;氯化钙248.3mg/L;二甲基亚砜 15.5 mg/L;硫酸锌 1.52 mg/L;硫酸铜 17.33 mg/L;硫酸锰0.00045mg/L;偏钒酸铵 0.00299 mg/L;
其它添加物: 柠檬酸铁铵 40 mg/L;乙醇胺 10.5 mg/L;谷胱甘肽 1.9 mg/L。
实施例4
将实施例1~实施例3获得的培养基RS-1、RS-2和RS-3进行细胞培养验证。采用的细胞为已适应全悬浮培养的MDCK悬浮细胞。以1.0×106个细胞/mL的细胞密度接种细胞至摇瓶中,在37℃、5% CO2条件下进行批培养,每隔24小时进行取样计数。所用对照培养基为商业话无血清培养基Ex-cell。结果如表1所示。
表1批培养不同培养基中的细胞生长情况
时间(h) | RS-1 | RS-2 | RS-3 | Ex-cell |
0 | 1.0 | 1.0 | 1.0 | 1.0 |
24 | 2.13 | 2.34 | 2.03 | 1.73 |
48 | 4.53 | 4.76 | 4.29 | 4.02 |
72 | 8.32 | 9.32 | 8.05 | 5.32 |
96 | 9.46 | 9.89 | 9.01 | 6.21 |
120 | 8.43 | 9.01 | 8.34 | 5.34 |
最大比生长速率(h<sup>-1</sup>) | 0.029 | 0.031 | 0.029 | 0.023 |
与商业化培养基Ex-cell相比,本发明所提供的化学限定培养基RS-1、RS-2和RS-3均能支持MDCK悬浮细胞的高密度生长,最大密度均超过8×106个细胞/mL,高于对照组中Ex-cell中的最大细胞密度6×106个细胞/mL。此外,在对数生长期RS-1、RS-2和RS-3三个培养基中的细胞最大比生长速率约为0.030 h-1,大于对照组中的0.023 h-1。
实施例5
用实施例2中的化学限定培养基RS-2驯化MDCK贴壁细胞为无血清悬浮培养,具体操作如下:
(1)当在含10%胎牛血清的DMEM中培养的贴壁MDCK细胞生长至汇合度80%时,弃去原有培养基并用PBS润洗细胞表面两遍后,加入适量胰酶,使其正好覆盖所有MDCK贴壁细胞,消化15分钟。
(2)至细胞变圆后加入少量血清终止消化,并加适量所述化学限定培养基重悬并1000 rpm离心5 min后弃上清。将剩余的细胞团用所述化学限定培养基RS-2重悬至细胞密度约1.0×106个细胞/mL,并转移到摇瓶中,在转速130rpm、温度37℃、5%CO2的条件下置于培养箱中培养。
(3)每隔48 h取样计数,并用所述化学限定培养基将细胞密度稀释至约1.0×106个细胞/mL,进行传代,直至获得适应所述化学限定培养基生长的悬浮MDCK细胞,结果如图1和图2所示。
由图1可知,细胞生长稳定,每次传代至1.0×106个细胞/mL后48小时内可生长至5~6×106个细胞/mL。经计算,该MDCK细胞的比生长速率在0.030h-1左右。此外,细胞活率高,传代过程中均高于96.5%。
由图2所示,在RS-2培养基中驯化8代以后的MDCK细胞呈单个分散,细胞大小均一,细胞饱满且透亮。
Claims (1)
1.一种适用于全悬浮细胞培养的培养基在将MDCK贴壁细胞驯化为单个悬浮细胞中的应用,其特征在于所述培养基由下列组成:
第一类营养物,用于满足细胞的基础代谢,为:
5000mg/L D-葡萄糖、200mg/L丙酮酸钠、15.7mg/L L-丙氨酸、287.4mg/L L-精氨酸、37.5mg/L L-天冬酰胺、39.4mg/L L-天冬氨酸、46.54mg/L L-胱氨酸、75.44mg/LL-半胱氨酸、32.54mg/L L-谷氨酸、856mg/L L-谷氨酰胺、49.86mg/L甘氨酸、77.88mg/L L-组氨酸;86.33mg/L L-异亮氨酸、132.39mg/L L-亮氨酸、102.21mg/L L-赖氨酸、85.35mg/L L-甲硫氨酸、106.62mg/L L-苯丙氨酸、93.77mg/L L-脯氨酸、75.56mg/L L-丝氨酸、123.47mg/LL-苏氨酸、53.33mg/L L-色氨酸、58.65mg/L L-酪氨酸和93.41mg/L L-缬氨酸;
第二类营养物,系核苷酸,为:
11.2mg/L次黄嘌呤、0.52mg/L胸苷、7.5mg/L腺苷、7.5mg/L尿苷、7.5mg/L胞苷和7.5mg/L鸟苷;
第三类营养物,系维生素,为:
0.084mg/L D-生物素、5.40mg/L叶酸、4.65mg/L烟酰胺、4.56mg/L吡哆醇、3.65mg/L硫胺素、0.54mg/L核黄素、33.51mg/L氯化胆碱、6.44mg/L D-泛酸钙和32mg/L肌醇;
第四类营养物,系无机盐,为:
25.64mg/L硝酸铁、0.456mg/L硫酸亚铁、40.5mg/L硫酸镁、324.4mg/L氯化钾、7000mg/L氯化钠、71mg/L磷酸氢二钠、62.5mg/L磷酸二氢钠和54.33mg/L亚硒酸钠;
1500mg/L剪切力保护剂和60mg/L抗细胞结团剂;
第五类营养物,为25mg/L柠檬酸铁铵、3.5mg/L乙醇胺和1.5mg/L谷胱甘肽;
流感病毒增殖促进剂,为3.89mg/L胆固醇、1.51mg/L DL-α-生育酚醋酸酯、0.2334mg/L豆蔻酸、0.2864mg/L棕榈酸、0.299mg/L硬脂酸、3244mg/L氯化镁、178.3mg/L氯化钙、12.5mg/L二甲基亚砜、1.12mg/L硫酸锌、17.33mg/L硫酸铜、0.00045mg/L硫酸锰和0.00199mg/L偏钒酸铵。
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