CN113846051A - 一种通用型化学成分限定cho细胞传代培养基及其应用 - Google Patents
一种通用型化学成分限定cho细胞传代培养基及其应用 Download PDFInfo
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- CN113846051A CN113846051A CN202111148178.7A CN202111148178A CN113846051A CN 113846051 A CN113846051 A CN 113846051A CN 202111148178 A CN202111148178 A CN 202111148178A CN 113846051 A CN113846051 A CN 113846051A
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Abstract
本发明提供了一种通用型化学成分限定CHO细胞传代培养基,其组分包括氨基酸,维生素,无机盐,微量元素,脂类,糖类和缓冲剂,其制备方法包括按配比分别称量培养基中的各组分并用合适的溶剂溶解,将溶解好的培养基组分混合,调节pH并加水定容,利用氯化钠调节渗透压在280~320mOsm/kg,用0.22μm滤膜过滤。本发明通用型化学成分限定CHO细胞传代培养基其可适用于各种亚型的CHO细胞的种子细胞培养,具有普适性广,使用方便,培养性能突出等优势,在大规模生产前期种子传代与扩增阶段,具有较大的应用前景。
Description
技术领域
本发明涉及无血清细胞培养基技术领域,具体涉及一种通用型化学成分限定CHO细胞传代培养基,及该培养基在细胞传代培养中的应用。
背景技术
蛋白类药物在大规模生产前,需要对种子细胞进行传代扩增培养;待种子细胞扩增到较大的体积,同时增长到合适的密度,方能接种到大规模生物反应器中进行生产。可以说前期种子细胞的传代扩培是生产成功的关键因素之一。
中国仓鼠卵巢细胞(CHO细胞)因其可以悬浮培养,蛋白糖基化修饰与人类似,且背景来源清晰,在蛋白类大分子药物的生产中扮演着越来越重要的角色。目前,市面上超过80%的单克隆抗体药物都由CHO细胞生产而来。
然而,CHO细胞自1957年从中国仓鼠卵巢组织中分离出来后,经过科学家几十年的研究及优化到如今已经衍生出了很多亚型,包括CHO-K1、CHO-K1SV、CHO-DG44、CHO-ZN、CHO-DXB11、CHO-S等。不同类型的CHO细胞其生长和代谢特性都不尽相同,往往培养时对营养成分的需求也不一样。
当前,科研人员针对不同类型CHO细胞的特性,开发出种类繁多的种子培养基,但这些培养基通常均缺乏通用性。要想同时满足CHO细胞诸多亚型的生长代谢具有较大的挑战性,本领域迫切需要开发一种通用型化学成分限定CHO细胞传代培养基。
发明内容
为了解决上述问题,本发明提供了一种通用型化学成分限定CHO细胞传代培养基配方及其在细胞传代培养中的应用。该通用型CHO细胞传代培养基能很好的支持当前工业和科研界常用CHO细胞株的传代扩增培养,具有较高的应用价值。
本发明是采用如下技术方案实现的:
一种通用型化学成分限定CHO细胞传代培养基,其组分包括氨基酸,维生素,无机盐,微量元素,脂类,糖类,缓冲剂;其中,
氨基酸部分包括如下含量范围的成分:
维生素部分包括如下含量范围的成分:
无机盐和微量元素部分包括如下含量范围的成分:
脂类,糖类,缓冲剂部分包括如下含量范围的成分:
作为优选的技术方案,所述通用型化学成分限定CHO细胞传代培养基,包括如下组分:
氨基酸部分包括如下含量范围的成分:
维生素部分包括如下含量范围的成分:
无机盐和微量元素部分包括如下含量范围的成分:
脂类,糖类,缓冲剂部分包括如下含量范围的成分:
本发明还进一步公开了上述通用型化学成分限定CHO细胞传代培养基的制备方法,包括如下步骤:
按配比分别称量培养基中的各组分,其中α-硫辛酸、叶酸、核黄素、生物素采用NaOH水溶液溶解;氯化亚锡、偏钒酸钠采用HCl水溶液溶解;亚油酸采用无水乙醇溶解,其余组分采用超纯水溶解;将溶解好的培养基组分混合,调节pH在7.0-7.4,加水定容,利用氯化钠调节渗透压在280~320mOsm/kg。最后,用0.22μm滤膜过滤,即得所述通用型化学成分限定CHO细胞传代培养基。
其中NaOH水溶液和HCl水溶液可采用本领域常规的可适用的浓度,例如NaOH水溶液可采用0.4mol/L的NaOH水溶液,HCl水溶液可采用6mol/L的HCl水溶液。
本发明还进一步公开了上述通用型化学成分限定CHO细胞传代培养基在CHO细胞传代培养中的应用,其可适用于各种亚型的CHO细胞的种子细胞培养。可适用的CHO细胞包括CHO-K1、CHO-K1SV、CHO-DG44、CHO-ZN、CHO-DXB11、CHO-S等。
与现有技术相比,本发明提供的通用型化学成分限定CHO细胞传代培养基具有如下有益效果:
CHO细胞的种类繁多,其生长代谢特性不一,通常使用的种子传代培养基也各不相同。本发明的种子传代培养基是通过分析不同类型的CHO细胞代谢组学和营养消耗情况,优化培养基中的组分而来的,具有普适性广,使用方便,培养性能突出等优势。
以下结合附图及实施例进一步说明本发明。
附图说明
图1为实施例1-6使用本发明培养基Media C培养不同CHO细胞的活细胞密度随时间变化图;
图2为实施例1-6使用本发明培养基Media C培养不同CHO细胞的细胞活率随时间变化图;
图3为实施例1-6使用本发明培养基Media C培养不同CHO细胞的细胞直径随时间变化图;
图7为对比例7-12使用B-9培养基培养不同CHO细胞的活细胞密度随时间变化图;
图8为对比例7-12使用B-9培养基培养不同CHO细胞的细胞活率随时间变化图;
图9为对比例7-12使用B-9培养基培养不同CHO细胞的细胞直径随时间变化图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明的优选实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明中的试剂或仪器未注明生产厂商的,均为常规商业化试剂或仪器。
实施例中,本发明通用型化学成分限定CHO细胞传代培养基(以下简称“Media C”)的成份及含量如下:
氨基酸部分:
成份 | 含量mg/L | 成份 | 含量mg/L |
L-甲硫氨酸 | 130 | L-丝氨酸 | 720 |
L-苯丙氨酸 | 280 | L-苏氨酸 | 500 |
L-天冬酰胺 | 1200 | L-缬氨酸 | 480 |
L-羟脯氨酸 | 220 | L-异亮氨酸 | 410 |
L-组氨酸盐酸盐 | 270 | L-谷氨酸 | 350 |
L-脯氨酸 | 700 | L-精氨酸 | 380 |
L-赖氨酸盐酸盐 | 620 | L-半胱氨酸盐酸盐 | 180 |
L-亮氨酸 | 650 | L-酪氨酸 | 360 |
L-天冬氨酸 | 180 | L-胱氨酸 | 170 |
L-谷胱甘肽 | 2 |
维生素部分:
成份 | 含量mg/L | 成份 | 含量mg/L |
α-硫辛酸 | 2 | 烟酰胺 | 3 |
氰钴胺素 | 1.1 | 硫胺盐酸盐 | 3.2 |
生物素 | 2.2 | 核黄素 | 0.36 |
叶酸 | 4 | 对氨基苯甲酸 | 1.3 |
维生素C | 22 | 吡哆醇 | 4 |
泛酸钙 | 3 | 氯化胆碱 | 70 |
肌醇 | 80 |
无机盐和微量元素部分:
脂类,糖类,缓冲剂部分:
成份 | 含量mg/L | 成份 | 含量mg/L |
亚油酸 | 0.08 | P188 | 2000 |
丙酮酸钠 | 150 | HEPES | 1500 |
葡萄糖 | 6000 |
配制Media C的方法如下:
(1)使用浓度为0.4mol/L的NaOH溶解α-硫辛酸,叶酸,核黄素,生物素配置成母液A;
(2)使用6mol/L HCl溶解氯化亚锡,偏钒酸钠配置成母液B;
(3)使用无水乙醇溶解亚油酸配置成母液C;
(4)使用超纯水溶解剩余培养基组分;
(5)将母液A,B,C与步骤(4)中的液体混合,搅拌10min;
(6)调节pH在7.0-7.4,加水定容,搅拌10min;
(7)使用氯化钠调节渗透压在280~320mOsm/kg;
(8)最后,用0.22μm滤膜过滤,即得Media C培养基。
实施例1
从液氮罐中取一支CHO-K1细胞冻存管,在37℃水浴中震荡溶解,加入到含有10mL新鲜Media C培养基的15mL离心管中,混匀。随后,在200×g下离心5min,弃去上清液。用10mL Media C培养基重悬细胞,计数细胞密度;继续添加培养基将细胞密度调整到3×105cells/mL接种到125mL摇瓶中进行传代培养。每两天取样,用细胞计数仪检测活细胞密度、细胞活率和细胞直径,再次以同样的密度传代。连续传代1个月后结束培养。
活细胞密度、活率及直径分别见图1、2和3,细胞生长数据见表1。使用Media C连续传代培养1个月,CHO-K1细胞株活细胞密度维持在1.99×106cells/mL左右,活率在98.70%左右,细胞直径在14.17μm左右。细胞的倍增时间在17.67h左右。
实施例2
从液氮罐中取一支CHO-K1SV细胞冻存管,在37℃水浴中震荡溶解,加入到含有10mL新鲜Media C培养基的15mL离心管中,混匀。随后,在200×g下离心5min,弃去上清液。用10mL Media C培养基重悬细胞,计数细胞密度;继续添加培养基将细胞密度调整到5×105cells/mL接种到125mL摇瓶中进行传代培养。每两天取样,用细胞计数仪检测活细胞密度、细胞活率和细胞直径,再次以同样的密度传代。连续传代1个月后结束培养。
活细胞密度、活率及直径分别见图1、2和3,细胞生长数据见表1。使用Media C连续传代培养1个月,CHO-K1SV细胞株活细胞密度维持在2.44×106cells/mL左右,活率在99.43%左右,细胞直径在14.45μm左右。细胞的倍增时间在21.13h左右。
实施例3
从液氮罐中取一支CHO-DG44细胞冻存管,在37℃水浴中震荡溶解,加入到含有10mL新鲜Media C培养基的15mL离心管中,混匀。随后,在200×g下离心5min,弃去上清液。用10mL Media C培养基重悬细胞,计数细胞密度;继续添加培养基将细胞密度调整到4×105cells/mL接种到125mL摇瓶中进行传代培养。每两天取样,用细胞计数仪检测活细胞密度、细胞活率和细胞直径,再次以同样的密度传代。连续传代1个月后结束培养。
活细胞密度、活率及直径分别见图1、2和3,细胞生长数据见表1。使用Media C连续传代培养1个月,CHO-DG44细胞株活细胞密度维持在2.36×106cells/mL左右,活率在96.39%左右,细胞直径在14.68μm左右。细胞的倍增时间在18.8h左右。
实施例4
从液氮罐中取一支CHO-ZN细胞冻存管,在37℃水浴中震荡溶解,加入到含有10mL新鲜Media C培养基的15mL离心管中,混匀。随后,在200×g下离心5min,弃去上清液。用10mL Media C培养基重悬细胞,计数细胞密度;继续添加培养基将细胞密度调整到5×105cells/mL接种到125mL摇瓶中进行传代培养。每两天取样,用细胞计数仪检测活细胞密度、细胞活率和细胞直径,再次以同样的密度传代。连续传代1个月后结束培养。
活细胞密度、活率及直径分别见图1、2和3,细胞生长数据见表1。使用Media C连续传代培养1个月,CHO-ZN细胞株活细胞密度维持在1.48×106cells/mL左右,活率在97.94%左右,细胞直径在14.18μm左右。细胞的倍增时间在31.13h左右。
实施例5
从液氮罐中取一支CHO-DXB11细胞冻存管,在37℃水浴中震荡溶解,加入到含有10mL新鲜Media C培养基的15mL离心管中,混匀。随后,在200×g下离心5min,弃去上清液。用10mL Media C培养基重悬细胞,计数细胞密度;继续添加培养基将细胞密度调整到3×105cells/mL接种到125mL摇瓶中进行传代培养。每两天取样,用细胞计数仪检测活细胞密度、细胞活率和细胞直径,再次以同样的密度传代。连续传代1个月后结束培养。
活细胞密度、活率及直径分别见图1、2和3,细胞生长数据见表1。使用Media C连续传代培养1个月,CHO-DXB11细胞株活细胞密度维持在1.72×106cells/mL左右,活率在99.27%左右,细胞直径在14.58μm左右。细胞的倍增时间在19.08h左右。
实施例6
从液氮罐中取一支CHO-S细胞冻存管,在37℃水浴中震荡溶解,加入到含有10mL新鲜Media C培养基的15mL离心管中,混匀。随后,在200×g下离心5min,弃去上清液。用10mLMedia C培养基重悬细胞,计数细胞密度;继续添加培养基将细胞密度调整到3×105cells/mL接种到125mL摇瓶中进行传代培养。每两天取样,用细胞计数仪检测活细胞密度、细胞活率和细胞直径,再次以同样的密度传代。连续传代1个月后结束培养。
活细胞密度、活率及直径分别见图1、2和3,细胞生长数据见表1。
表1实施例1-6活细胞密度、活率及直径检测指标
使用Media C连续传代培养1个月,CHO-S细胞株活细胞密度维持在2.31×106cells/mL左右,活率在99.21%左右,细胞直径在14.96μm左右。细胞的倍增时间在16.36h左右。
对比例1-6
选取Merck的CD CHO Fusion(货号:14365C)为对照组1,分别采用和实施例1-6相同的传代培养操作连续传代培养CHO-K1、CHO-K1SV、CHO-DG44、CHO-ZN、CHO-DXB11、CHO-S细胞,得到对应的对比例1-6。细胞的活细胞密度、活率及直径分别见图4、5和6,细胞生长数据见表2。
表2对比例1-6活细胞密度、活率及直径检测指标
使用CD CHO Fusion连续传代培养1个月,可以发现对于CHO-K1、CHO-K1SV、CHO-ZN、CHO-DXB11、CHO-S细胞而言,细胞的生长状况与Media C很接近。但在CHO-DG44细胞上,使用CD CHO Fusion后细胞的密度偏低(降低43.22%),倍增时间也相对较长(增长36.06%)。
对比例7-12
选取申请人在先申请专利CN111676184A中公开的B-9培养基为对照组2,分别采用和实施例1-6相同的传代培养操作连续传代培养CHO-K1、CHO-K1SV、CHO-DG44、CHO-ZN、CHO-DXB11、CHO-S细胞,得到对应的对比例7-12。细胞的活细胞密度、活率及直径分别见图7、8和9,细胞生长数据见表3。
表3对比例7-12活细胞密度、活率及直径检测指标
使用B-9培养基连续传代培养1个月,可以发现对于CHO-K1、CHO-K1SV、CHO-DG44、CHO-ZN、CHO-DXB11、CHO-S细胞的密度和倍增速率都要低于Media C。且在CHO-ZN细胞上,B-9培养基表现最差,倍增时间最长。
综合以上实验结果显示,本发明通用型化学成分限定CHO细胞传代培养基能支持当前工业和科研界多种CHO细胞的生长代谢,满足大规模生产前期种子传代与扩增阶段的需求,具有较大的应用前景。且与商业对照培养基(对照组1)相比,在CHO-DG44细胞上具有较大的优势;与对照组2培养基相比,活细胞密度和倍增速率均具有明显优势,显示出本发明具有更为优异的通用性。
本领域的技术人员应理解,上述描述及附图中所示的本发明的实施例只作为举例而并不限制本发明。本发明的目的已经完整并有效地实现。本发明的功能及结构原理已在实施例中展示和说明,在没有背离所述原理下,本发明的实施方式可以有任何变形或修改。
Claims (5)
3.权利要求1或2所述通用型化学成分限定CHO细胞传代培养基的制备方法,其特征在于,包括如下步骤:按配比分别称量培养基中的各组分,其中α-硫辛酸、叶酸、核黄素、生物素采用NaOH水溶液溶解;氯化亚锡、偏钒酸钠采用HCl水溶液溶解;亚油酸采用无水乙醇溶解,其余组分采用超纯水溶解;将溶解好的培养基组分混合,调节pH在7.0-7.4,加水定容,利用氯化钠调节渗透压在280~320mOsm/kg,最后用0.22μm滤膜过滤,即得所述通用型化学成分限定CHO细胞传代培养基。
4.权利要求1或2所述通用型化学成分限定CHO细胞传代培养基在CHO细胞传代培养中的应用。
5.如权利要求4所述的应用,其特征在于,所述CHO细胞包括CHO-K1、CHO-K1SV、CHO-DG44、CHO-ZN、CHO-DXB11、CHO-S。
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