CN114214287A - 换液培养基及其应用 - Google Patents
换液培养基及其应用 Download PDFInfo
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Abstract
本发明涉及一种换液培养基及其应用,属于病毒载体包装技术领域。该换液培养基包括基础培养基和形态维持剂,所述形态维持剂由以下成分组成:体积百分含量为3‑10%的血清替代物,1±0.1g/L的胰岛素、0.55±0.1g/L的转铁蛋白、0.00067±0.0001wt%的亚硒酸钠、0.20±0.05g/L的乙醇胺、1±0.02mmol/L的丙酮酸钠,25±5μmol/L的磷酸氯喹。本发明通过将上述形态维持剂加入到基础培养基中,从而通过营养物质的补充维持细胞状态的良好,配合少量血清替代物(血清替代物),可以获得比使用10%胎牛血清包装的更高滴度的慢病毒。
Description
技术领域
本发明涉及病毒载体包装技术领域,特别是涉及一种换液培养基及其应用。
背景技术
慢病毒载体是一种对分裂细胞和非分裂细胞均具有感染能力的基因治疗载体,常用于嵌合抗原受体-T细胞治疗(Chimeric Antigen Receptor T-Cell Immunotherapy,CAR-T)。
当前,慢病毒的生产主要分为两种工艺系统,一种采用1-40层的细胞工厂的贴壁系统,另外一种为293T细胞经过无血清驯化后利用发酵培养技术的悬浮系统,其中慢病毒贴壁工艺往往采用低血清驯化细胞后进行慢病毒包装,存在驯化周期较长,在包装过程中仍旧存在较低血清的问题。其次胎牛血清存在很大的批间差异,对慢病毒的稳定性造成一定的影响。同时胎牛血清主要来源于牛,使用胎牛血清进行包装的慢病毒需去除BSA的残留,才可满足临床需求。总之,在贴壁系统中,细胞需经过低血清驯化,实验周期较长,对下游慢病毒BSA的清除产生压力,并且胎牛血清(FBS)存在批间差,这将直接影响各批次慢病毒产品的稳定性。
针对293T贴壁细胞,很多企业研发并生产了一系列的血清替代产品,可以与胎牛血清结合使用来降低胎牛血清的使用量,但是所得慢病毒包装的滴度较低,并且用于免疫细胞治疗的人源的血清替代成品的造价昂贵。
用于悬浮系统的293T细胞必须经过一系列的悬浮驯化后方可使用,293T细胞需在无血清培养基或者低血清培养基中进行驯化适应后才可以进行慢病毒包装,常见的无血清培养基有HyCell TransFX,FreeStyle 293,SFM4TranFx-293等,但培养基成本较高。且所获得的的慢病毒滴度大多在106左右;近年来,通过工艺的优化,也逐渐出现病毒滴度在108的培养工艺。但是单次生产慢病毒的成本高,细胞驯化的周期长,不适合GMP级别的慢病毒需求量较少的临床项目。
因而,目前仍缺乏一种能够适用于无血清贴壁293T细胞生产慢病毒的培养基,以及获得高滴度,低BSA残留的生产工艺。
发明内容
基于此,有必要针对上述问题,提供一种换液培养基,采用该换液培养基进行无血清贴壁病毒载体包装,可获得低BSA残留,高滴度特点的慢病毒载体。
一种换液培养基,包括基础培养基和形态维持剂,所述形态维持剂由以下成分组成:
体积百分含量为3-10%的血清替代物,1±0.1g/L的胰岛素、0.55±0.1g/L的转铁蛋白、0.00067±0.0001wt%的亚硒酸钠、0.20±0.05g/L的乙醇胺、1±0.02mmol/L的丙酮酸钠,25±5μmol/L的磷酸氯喹。
本发明人在长期实践的基础上进行调研和研究后认为,如细胞培养环境缺少血清中的各种粘附贴壁因子如纤粘连蛋白、层粘连蛋白、胶原、玻表粘连蛋白等,细胞将无法达到贴壁状态,而在常规病毒载体包装培养过程中,由于宿主细胞处于产毒状态,细胞转态较差,更难以达到贴壁状态,因而给血清降低或者血清去除带来了难度。
在此基础上,发明人通过将上述形态维持剂加入到基础培养基中,从而通过上述有效成分的配合,能够补充细胞所需成分,维持细胞状态的良好,再配合少量血清替代物,即可获得比使用10%胎牛血清包装的更高滴度的病毒载体。
具体的,上述形态维持剂中,丙酮酸钠的添加有利于重组蛋白的表达,提高RNA逆转录,也有实验证实它可以有效地促进HIV-1的LTR和CMV启动子的作用;磷酸氯喹在细胞内具有显著提高吞饮泡和溶酶体的pH值,阻止外源质粒DNA的过快降解的作用;胰岛素可促进葡萄糖和氨基酸的摄取、脂肪形成、细胞内运输以及蛋白和核酸的合成,从而可通过改善293T细胞的生长状态来提高慢病毒滴度;转铁蛋白是一种铁载体,也有助于降低氧自由基和过氧化物的毒性水平,维持细胞的生长和增殖。硒(以亚硒酸钠提供)是谷胱甘肽过氧化物酶和其他蛋白的一种辅因子,在培养基中用作抗氧化剂;乙醇胺是磷酸甘油酯的前体,对质膜和细胞器的结构至关重要。上述物质的共同使用,能够较好的维持细胞形态,以获得更高滴度的病毒载体。
在其中一个实施例中,所述血清替代物的体积百分含量为5-7%。
在其中一个实施例中,所述血清替代物包括:胆固醇,白蛋白,球蛋白,生长因子和葡萄糖。上述血清替代物中,加入胆固醇可以通过稳定慢病毒的脂质双分子层,保证慢病毒的稳定性,白蛋白对细胞的渗透压具有维持作用,球蛋白可以促进细胞的贴壁与粘附,生长因子有利于细胞的增殖,葡萄糖为细胞的生长提供营养基础,上述成分共同作用,有利于无血清贴壁细胞的病毒包装过程。
在其中一个实施例中,所述基础培养基为DMEM培养基。
本发明还公开了上述换液培养基用于无血清贴壁细胞病毒载体包装中的应用。
在其中一个实施例中,所述病毒载体为慢病毒。
在其中一个实施例中,所述细胞为HEK293T细胞。
本发明还公开了一种无血清贴壁细胞病毒包装的方法,包括以下步骤:
细胞复苏培养:将HEK293T细胞接种至培养瓶中,进行复苏培养;
包装培养:将培养基更换为包装培养基;
转染培养:以质粒转染上述复苏培养后细胞,并将培养液更换为上述的换液培养基进行培养。
上述无血清贴壁细胞病毒包装的方法,无需对HEK293T细胞进行低血清驯化,大大缩短了实验周期,具有操作简便的优势。可以理解的,上述包装培养基采用本领域常规包装培养基即可,如含5%FBS,5%血清替代物,1mmol/L的丙酮酸钠,25μmol/L的磷酸氯喹的包装培养基。
在其中一个实施例中,所述细胞复苏培养步骤中,HEK293T细胞不经过低血清驯化,直接进行复苏培养,待细胞密度达到90±5%,即可进行转染培养步骤。
在其中一个实施例中,所述转染培养步骤中,采用磷酸钙法进行质粒转染,在质粒转染之后的4-6h更换为所述换液培养基进行培养。在质粒转染4-6h更换培养基,可减轻病毒原液中FBS对下游除杂工艺的压力,降低由血清造成的慢病毒载体批间差异,通过添加产毒增强物质生产高低度的慢病毒载体,同时通过补充细胞营养物质,缓解慢病毒出芽对宿主细胞的损伤。
与现有技术相比,本发明具有以下有益效果:
本发明的一种换液培养基,通过向基础培养基中加入形态维持剂,补充维持细胞状态的良好,配合少量血清替代产品,可以获得比使用10%胎牛血清包装的更高滴度的慢病毒。
并且,在包装过程中,不需要添加血清,只需要加入少量血清替代物,促进细胞生长的一些补充物质即可。
即便是比较大的表达载体(如本发明中9kbp以上表达载体),此套工艺亦可在转染后的40-48h收获转导滴度在107左右的慢病毒载体。
附图说明
图1为慢病毒包装表达载体图谱。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
以下实施例所用试剂,如非特别说明,均为市售可得,一下实施例所用方法,如非特别说明,均为常规方法可实现。
实施例1
一种无血清贴壁293T细胞慢病毒包装的方法,其包括以下步骤:
S1:HEK293T细胞(ATCC,货号:11268)不需要进行低血清驯化,经过复苏培养两代后进行慢病毒包装,包装前一日,将2.2×107细胞接种至装有10%FBS+DMEM的T175培养瓶中,次日细胞密度须达到90%;
S2:取出待包装细胞,并更换为包装培养基(基本培养基为:DMEM,并加入5%FBS,5%血清替代物);
S3:采用磷酸钙法进行质粒转染,表达质粒大小在9000kb左右,带有GFP荧光标签,该质粒表达示意如图1所示。;
S4:质粒转染4-6h更换无血清换液培养基,换液培养基包括基础培养基和形态维持剂,其中基础培养基为DMEM培养基(gibco,货号C11995),形态维持剂的成分包括:10%(Vol)血清替代物,1g/L胰岛素、0.55g/L转铁蛋白、0.00067%亚硒酸钠、0.20g/L乙醇胺,1mmol/L的丙酮酸钠,25μmol/L的磷酸氯喹;对照组换液培养基含5%FBS,5%血清替代物。
上述血清替代物包括:胆固醇,白蛋白,球蛋白,生长因子和葡萄糖(西宝生物,货号C08001)。
S5:转染48-72h后,收获细胞培养上清10μl,转导HEK 293T细胞,293T细胞数为5×105/孔。
S6:流式上机检测。
实施例2
一种无血清贴壁293T细胞慢病毒包装的方法,其包括以下步骤:
S1:HEK293T细胞不需要进行低血清驯化,经过复苏培养两代后进行慢病毒包装,包装前一日,将2.2×107细胞接种至装有10%FBS+DMEM的T175培养瓶中,次日细胞密度须达到90%;
S2:取出待包装细胞,并更换包装培养基(基本培养基为:DMEM,并加入5%FBS,5%血清替代物);
S3:采用磷酸钙法进行质粒转染,表达质粒大小在9000kb左右;
S4:质粒转染4-6H更换无血清换液培养基,换液培养基包括基础培养基和形态维持剂,其中基础培养基为DMEM培养基(gibco,货号C11995),形态维持剂的成分包括:9%血清替代物,1g/L胰岛素、0.55g/L转铁蛋白、0.00067%亚硒酸钠、0.20g/L乙醇胺,1mmol/L的丙酮酸钠,25μmol/L的磷酸氯喹。对照组换液培养基含5%FBS,5%血清替代物;
上述血清替代物包括:胆固醇,白蛋白,球蛋白,生长因子和葡萄糖(西宝生物,货号C08001)。
S5:转染48-72h后,收获细胞培养上清10μl,转导HEK 293T细胞,293T细胞数为5×105/孔。
S6:流式上机检测。
实施例3
一种无血清贴壁293T细胞慢病毒包装的方法,其包括以下步骤:
S1:HEK293T细胞不需要进行低血清驯化,经过复苏培养两代后进行慢病毒包装,包装前一日,将2.2×107细胞接种至装有10%FBS+DMEM的T175培养瓶中,次日细胞密度须达到90%;
S2:取出待包装细胞,并更换包装培养基(基本培养基为:DMEM,并加入5%FBS,5%血清替代物);
S3:采用磷酸钙法进行质粒转染,表达质粒大小在9000kb左右;
S4:质粒转染4-6H更换无血清换液培养基,换液培养基包括基础培养基和形态维持剂,其中基础培养基为DMEM培养基(gibco,货号C11995),形态维持剂的成分包括:8%血清替代物,1g/L胰岛素、0.55g/L转铁蛋白、0.00067%亚硒酸钠、0.20g/L乙醇胺,1mmol/L的丙酮酸钠,25μmol/L的磷酸氯喹。对照组换液培养基含5%FBS,5%血清替代物;
上述血清替代物包括:胆固醇,白蛋白,球蛋白,生长因子和葡萄糖(西宝生物,货号C08001)。
S5:转染48-72h后,收获细胞培养上清10μl,转导HEK 293T细胞,293T细胞数为5×105/孔。
S6:流式上机检测。
实施例4
一种无血清贴壁293T细胞慢病毒包装的方法,其包括以下步骤:
S1:HEK293T细胞不需要进行低血清驯化,经过复苏培养两代后进行慢病毒包装,包装前一日,将2.2×107细胞接种至装有10%FBS+DMEM的T175培养瓶中,次日细胞密度须达到90%;
S2:取出待包装细胞,并更换包装培养基(基本培养基为:DMEM,并加入5%FBS,5%血清替代物);
S3:采用磷酸钙法进行质粒转染,表达质粒大小在9000kb左右;
S4:质粒转染4-6H更换无血清换液培养基,换液培养基包括基础培养基和形态维持剂,其中基础培养基为DMEM培养基(gibco,货号C11995),形态维持剂的成分包括:7%血清替代物,1g/L胰岛素、0.55g/L转铁蛋白、0.00067%亚硒酸钠、0.20g/L乙醇胺,1mmol/L的丙酮酸钠,25μmol/L的磷酸氯喹。对照组换液培养基含5%FBS,5%血清替代物;
上述血清替代物包括:胆固醇,白蛋白,球蛋白,生长因子和葡萄糖(西宝生物,货号C08001)。
S5:转染48-72h后,收获细胞培养上清10μl,转导HEK 293T细胞,293T细胞数为5×105/孔。
S6:流式上机检测。
实施例5
一种无血清贴壁293T细胞慢病毒包装的方法,其包括以下步骤:
S1:HEK293T细胞不需要进行低血清驯化,经过复苏培养两代后进行慢病毒包装,包装前一日,将2.2×107细胞接种至装有10%FBS+DMEM的T175培养瓶中,次日细胞密度须达到90%;
S2:取出待包装细胞,并更换包装培养基(基本培养基为:DMEM,并加入5%FBS,5%血清替代物);
S3:采用磷酸钙法进行质粒转染,表达质粒大小在9000kb左右;
S4:质粒转染4-6H更换无血清换液培养基,换液培养基包括基础培养基和形态维持剂,其中基础培养基为DMEM培养基(gibco,货号C11995),形态维持剂的成分包括:6%血清替代物,1g/L胰岛素、0.55g/L转铁蛋白、0.00067%亚硒酸钠、0.20g/L乙醇胺,1mmol/L的丙酮酸钠,25μmol/L的磷酸氯喹。对照组换液培养基含5%FBS,5%血清替代物;
上述血清替代物包括:胆固醇,白蛋白,球蛋白,生长因子和葡萄糖(西宝生物,货号C08001)。
S5:转染48-72h后,收获细胞培养上清10μl,转导HEK 293T细胞,293T细胞数为5×105/孔。
S6:流式上机检测。
实施例6
一种无血清贴壁293T细胞慢病毒包装的方法,其包括以下步骤:
S1:HEK293T细胞不需要进行低血清驯化,经过复苏培养两代后进行慢病毒包装,包装前一日,将2.2×107细胞接种至装有10%FBS+DMEM的T175培养瓶中,次日细胞密度须达到90%;
S2:取出待包装细胞,并更换包装培养基(基本培养基为:DMEM,并加入5%FBS,5%血清替代物);
S3:采用磷酸钙法进行质粒转染,表达质粒大小在9000kb左右;
S4:质粒转染4-6H更换无血清换液培养基,换液培养基包括基础培养基和形态维持剂,其中基础培养基为DMEM培养基(gibco,货号C11995),形态维持剂的成分包括:5%血清替代物,1g/L胰岛素、0.55g/L转铁蛋白、0.00067%亚硒酸钠、0.20g/L乙醇胺,1mmol/L的丙酮酸钠,25μmol/L的磷酸氯喹。对照组换液培养基含5%FBS,5%血清替代物;
上述血清替代物包括:胆固醇,白蛋白,球蛋白,生长因子和葡萄糖(西宝生物,货号C08001)。
S5:转染48-72h后,收获细胞培养上清10μl,转导HEK 293T细胞,293T细胞数为5×105/孔。
S6:流式上机检测。
实施例7-9,依次将血替成分降低至2%,其包装步骤与实施6一致。
对比例1
一种无血清贴壁293T细胞慢病毒包装的方法,与实施例6的方法基本相同,区别仅在于:换液培养基中不添加磷酸氯喹及丙酮酸钠。
对比例2
一种无血清贴壁293T细胞慢病毒包装的方法,与实施例6的方法基本相同,区别仅在于:换液培养基不添加1g/L的胰岛素、0.55g/L的转铁蛋白、0.00067%的亚硒酸钠、0.20g/L的乙醇胺。
实验例
一、常规培养降血清实验。
采用常规方法进行T175小量慢病毒包装,所用换液培养基为为常规培养基,即仅包括基础培养基,不添加本发明所述的形态维持剂,但加入适当的血清血替,逐渐降低血清加入量,加入量见下表。
表1.血清降低后病细胞生长状态
实验组 | 细胞代数 | 传代密度 | 两天后细胞数量 | 包装代数 | 细胞铺板密度 |
10%血清 | P7-8 | 1×10<sup>7</sup> | 4-5×10<sup>7</sup> | P8 | 2×10<sup>7</sup> |
5%血清 | P7-8 | 1×10<sup>7</sup> | 4×10<sup>7</sup> | P8 | 2.5×10<sup>7</sup> |
5%血清+5%血替 | P7-8 | 1×10<sup>7</sup> | 4.2×10<sup>7</sup> | P8 | 2.3×10<sup>7</sup> |
2%血清+8%血替 | P7-8 | 1×10<sup>7</sup> | 3.7×10<sup>7</sup> | P8 | 3×10<sup>7</sup> |
2%血清+10%血替 | P7-8 | 1×10<sup>7</sup> | 3.85×10<sup>7</sup> | P8 | 3×10<sup>7</sup> |
表2血清降低后病毒包装滴度测试
而实验中发现,随着胎牛血清(FBS)的降低,细胞生长速度缓慢,慢病毒滴度呈现下降趋势明显;
二、本发明换液培养基降血清实验。
上述实施例1-9中的方案,采用T175进行小量慢病毒包装,可以理解的,除使用T175外还可以采用细胞工厂进行大量包装;形成的磷酸盐-质粒沉淀平均分成三份加入3瓶T175中,其中两瓶设立为实验组,一瓶设立对照组。其中,细胞培养时将血清浓度降低至小于5%时,细胞生长缓慢,慢病毒包装产率下降,详细数据见下表3。
表3.无血清换液培养基包装测试结果
三、与对比例进行比较
分别按照对比例1-2和实施例6的方法,采用T175进行小量慢病毒包装,所得结果如下表4所示。
表4、对比例数据
注:“/”表示该换液培养基中无缺失成分,为实施例6的重复试验。
从上述实验结果可以看出,在使用本发明的换液培养基后,实施1-7的实验组均比对照组(含低血清)产毒滴度高,尽管血清替代物含量的逐步降低,但慢病毒的包装滴度稳定。当血替含量小于4%含量(实施例7)时,慢病毒包装滴度逐渐降低,但其滴度仍旧高于对照组,详细数据见表3。使用本发明的无血清贴壁慢病毒生产工艺,仅需维持血替浓度在4%以上,即可以获得滴度大于1×107的慢病毒载体。
而无论是将其中用于维持形态维持的何种成分省略,均导致所得慢病毒载体滴度下降。
通过上述实验数据我们可以看出,本发明的慢病毒无血清贴壁细胞生产工艺,具有低BSA残留,滴度高的特点,并且针对本套工艺可以使用细胞工厂进行大量包装。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种换液培养基,其特征在于,包括基础培养基和形态维持剂,所述形态维持剂由以下成分组成:
体积百分含量为3-10%的血清替代物,1±0.1g/L的胰岛素、0.55±0.1g/L的转铁蛋白、0.00067±0.0001wt%的亚硒酸钠、0.20±0.05g/L的乙醇胺、1±0.02mmol/L的丙酮酸钠,25±5μmol/L的磷酸氯喹。
2.根据权利要求1所述的换液培养基,其特征在于,所述血清替代物的体积百分含量为5-7%。
3.根据权利要求1所述的换液培养基,其特征在于,所述血清替代物包括:胆固醇,白蛋白,球蛋白,生长因子和葡萄糖。
4.根据权利要求1所述的换液培养基,其特征在于,所述基础培养基为DMEM培养基。
5.权利要求1-4任一项所述的换液培养基用于无血清贴壁细胞病毒载体包装中的应用。
6.根据权利要求5所述的应用,其特征在于,所述病毒载体为慢病毒。
7.根据权利要求5所述的应用,其特征在于,所述细胞为HEK293T细胞。
8.一种无血清贴壁细胞病毒包装的方法,其特征在于,包括以下步骤:
细胞复苏培养:将HEK293T细胞接种至培养瓶中,进行复苏培养;
包装培养:将培养基更换为包装培养基;
转染培养:以质粒转染上述复苏培养后细胞,并将培养液更换为权利要求1-4任一项所述的换液培养基进行培养。
9.根据权利要求8所述的无血清贴壁细胞病毒包装的方法,其特征在于,所述细胞复苏培养步骤中,HEK293T细胞不经过低血清驯化,直接进行复苏培养,待细胞密度达到90±5%,即可进行后续步骤。
10.根据权利要求8所述的无血清贴壁细胞病毒包装的方法,其特征在于,所述转染培养步骤中,采用磷酸钙法进行质粒转染,在质粒转染之后的4-6h更换为所述换液培养基进行培养。
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