CN114981410A - 降低渗透压的培养基 - Google Patents
降低渗透压的培养基 Download PDFInfo
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- CN114981410A CN114981410A CN202180009157.9A CN202180009157A CN114981410A CN 114981410 A CN114981410 A CN 114981410A CN 202180009157 A CN202180009157 A CN 202180009157A CN 114981410 A CN114981410 A CN 114981410A
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Abstract
本发明提供降低渗透压的培养基，其特征在于，含基础培养基，渗透压调整到150～250mOsm/kg。
Description
【技术领域】
本发明涉及渗透压降低的培养基组合物及使用其的细胞培养方法。
【背景技术】
使用细胞的再生医疗技术作为可治疗至今难以治疗的各种各样的疾病或损伤的手段之一，近年，非常地关注。由于再生医疗需要大量的细胞，意欲推进有效培养细胞的方法的开发。例如，在专利文献1中报告了包括在培养基中将干细胞用ROCK抑制剂处理的干细胞的培养方法。
为了将细胞稳定培养、增殖，定期的的培养基更换或传代等的作业是必须的。由于涉及的作业有重复进行烦琐的过程的必要，作业者的负担大，或还有因作业者的技术的巧拙而对细胞增殖效率等有影响这样的问题。因此，近年，监测培养基的pH或气体浓度这样的培养环境，根据需要进行自动优化这些细胞培养装置(在本说明书中，有时称为“生物反应器”等)的开发或活用。作为涉及的生物反应器的一例，可举出例如，Sartorius公司的“ambr(注册商标)”细胞培养装置。
【现有技术文献】
【专利文献】
专利文献1：特开2008-099662号公报
【发明的概要】
【发明要解决的课题】
由生物反应器的使用，大幅地改善细胞培养中的作业者的负担或起因于作业者的问题。但是一方面，在使用生物反应器时，对于当采用这种条件时可优化细胞增殖的效率，在现时点难说有充分的探讨。
从而，本发明以在使用生物反应器的细胞培养中，提升细胞增殖效率的手段的提供作为目的。
【用于解决课题的手段】
本发明人针对上述课题进行锐意探讨的结果查明，在细胞培养中，培养基的渗透压是给细胞增殖效率影响的要因之一。另外发现，此事实在使用生物反应器的细胞培养中极其重要。即，在细胞培养中，随着时间经过而由细胞的生命活动而培养基的pH降低，但生物反应器检测培养基中的所述pH的降低，将pH调整剂(例、碳酸氢钠)自动添加到培养基中。由此成为培养基的pH维持在中性附近，一方面，随着pH调整剂的添加量增加而培养基的渗透压也升高。进而，本发明人查明，由于当培养基的渗透压超一定水平时开始给细胞的增殖效率带来不良影响，这抑制使用生物反应器的细胞培养的高密度培养。本发明人通过基于涉及的见解推进进一步研究，从而完成本发明。即，本发明如下。
[1]降低渗透压的培养基，其特征在于，含基础培养基，渗透压调整到150～250mOsm/kg。
[2][1]所述的降低渗透压的培养基，其中降低渗透压的培养基的渗透压是170～230mOsm/kg。
[3][1]或[2]所述的降低渗透压的培养基，其中渗透压通过在构成基础培养基的成分之中，使氯化钠的量降低来调整。
[4]细胞的培养方法，其包括以培养基的渗透压成为220～310mOsm/kg的方式用[1]～[3]之任一项所述的降低渗透压的培养基更换使用中的培养基的一部分或全量的工序。
[5][4]所述的方法，其中细胞培养是悬浮培养。
【发明的效果】
由本发明，可在细胞培养的过程中，为了维持pH而将经时升高的培养基的渗透压维持在适合的范围。从而，当应用本发明时，例如，与使用生物反应器的自动细胞培养中从前进行的实施方式比较，更优选的高密度培养变得可能。
【附图的简单的说明】
【图1】图1是显示实施例1中的渗透压对iPS细胞的增殖的影响的图(n＝1)。
【图2】图2是显示实施例1中的对iPS细胞进行培养的培养基的，day3及day12的时间点的渗透压的图。
【图3】图3是显示实施例2中的渗透压对iPS细胞的增殖的影响的图(n＝1)。
【具体实施方式】
以下，对本发明详细地进行说明。
【定义】
在本说明书中，“悬浮培养”是指在细胞不接附于培养容器的状态下进行的细胞培养方法。在本发明中，悬浮培养可伴随对于液体培养基的自外部的压力或振动、或者，所述液体培养基中的振荡或旋转操作，也可不伴随。
在本说明书中，“基础培养基”是指含有对于细胞的培养必须的碳源、氮源及无机盐等的培养基。
【1.降低渗透压的培养基】
本发明提供含基础培养基的，渗透压调整到150～250mOsm/kg的，降低渗透压的培养基(以下，有时称为“本发明的培养基”)。
本发明的培养基含有基础培养基。在本发明的培养基中可作为其构成成分使用的基础培养基不特别限定，只要对应于培养的细胞种适宜选择即可。基础培养基可由公知的方法调制，也可使用市售品。
作为可含有的基础培养基，可举出例如，Dulbecco改变Eagle培养基(Dulbecco'sModified Eagle's Medium)(DMEM)、HamF12培养基(Ham's Nutrient Mixture F12)、DMEM/F12培养基、McCoy5A培养基(McCoy's 5A Medium)、最小必须培养基(Minimum EssentialMedium)(MEM)、Eagle最小必须培养基(Eagle's Minimum Essential Medium)(EMEM)、α改变型Eagle最小必须培养基(αModified Eagle's Minimum Essential Medium)(αMEM)、Roswell Park记念研究所(Roswell Park Memorial Institute)(RPMI)1640培养基、Iscove改变Dulbecco培养基(Iscove's Modified Dulbecco's Medium)(IMDM)、MCDB131培养基、William培养基E(William's Medium E)、Fisher培养基(Fischer's Medium)等。
另外，特别是，作为在作为干细胞的培养用调制本发明的培养基时使用的基础培养基，可举出STEMPRO(注册商标)hESC SFM培养基(Life Technologies公司)、mTeSR1培养基(STEMCELL Technologies公司)、TeSR2培养基(STEMCELL Technologies公司)、TeSR-E8培养基(STEMCELL Technologies公司)、Essential8培养基(Life Technologies公司)、HEScGRO(商标)Serum-Free Medium for hES cells(Millipore公司)、PluriSTEM(商标)Human ES/iPS Medium(EMD Millipore公司)、NutriStem(注册商标)hESC XF培养基(Biological Industries Israel Beit-Haemek公司)、NutriStem(商标)XF/FF CultureMedium(Stemgent公司)、AF NutriStem(注册商标)hESC XF培养基(BiologicalIndustries Israel Beit-Haemek公司)、S-medium(DSPharmaBiomedical株式会社)、StemFit(注册商标)AK03N培养基(味之素株式会社)、hESF9培养基、hESF-FX培养基、CDM培养基、DEF-CS 500Xeno-Free 3D Spheroid Culture Medium(Cellartis公司)、StemFlex培养基(Thermo Fisher Scientific公司)等。
另外，本发明的培养基也可除了基础培养基之外，进一步添加对于细胞增殖优选的成分。作为涉及的成分，可举出例如，葡萄糖、果糖、蔗糖、麦芽糖等的糖；天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸等的氨基酸；白蛋白、转铁蛋白等的蛋白质；甘氨酰甘氨酰甘氨酸、大豆肽等的肽；血清；胆碱、维生素A、维生素B组(硫胺素、核黄素、吡哆素、氰基钴胺、生物素、叶酸、泛酸、烟酰胺等)、维生素C、维生素E等的维生素；油酸、花生四烯酸、亚油酸等的脂肪酸；胆甾醇等的脂质；氯化钠、氯化钾、氯化钙、硫酸镁、磷酸二氢钠等的无机盐；锌、铜、硒等的微量元素；N,N-双(2-羟基乙基)-2-氨基乙磺酸(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid(BES))、4-(2-羟基乙基)-1-哌嗪乙磺酸(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid(HEPES))、N-[三(羟基甲基)甲基]甘氨酸(N-[tris(hydroxymethyl)methyl]glycine(Tricine))等的缓冲剂；两性霉素B、卡那霉素、庆大霉素、链霉素、青霉素等的抗生物质；I型胶原、II型胶原、纤连蛋白、层粘连蛋白、聚-L-赖氨酸、聚-D-赖氨酸等的细胞接附因子及细胞外基质成分；白细胞介素、成纤维细胞生长因子(FGF)、肝细胞生长因子(HGF)、转化生长因子(TGF)-α、转化生长因子(TGF)-β、血管内皮生长因子(VEGF)、激活素A等的细胞因子及生长因子；地塞米松、氢化可的松、雌二醇、黄体酮、胰高血糖素、胰岛素等的激素等，可对应于培养的细胞的种类选择使用适合的成分。
在优选的一实施方式中，也可将D-葡萄糖及5种氨基酸(色氨酸、丝氨酸、半胱氨酸(或者胱氨酸)、甲硫氨酸、精氨酸)追加添加到本发明的培养基中。这些成分的添加量只要参考以下的“2.细胞的培养方法”中的对应记载，根据培养的目的或各种培养条件(例、细胞密度、培养基更换的频度)等适宜设定即可。
另外，在将本发明的培养基适用于干细胞时，在优选的一实施方式中，也可将bFGF和/或胆碱追加添加到本发明的培养基中。
本发明的培养基的特征在于，与通常的培养基比较而渗透压降低。本发明的培养基的渗透压的上限可通常为250mOsm/kg、优选为240mOsm/kg、更优选为235mOsm/kg、再优选为230mOsm/kg、特别优选为220mOsm/kg。另外，渗透压的下限可通常为150mOsm/kg、优选为160mOsm/kg、更优选为165mOsm/kg、再优选为170mOsm/kg、特别优选为180mOsm/kg。在一实施方式中，本发明的培养基的渗透压可设为通常150～250mOsm/kg、优选为160～240mOsm/kg、更优选为165～235mOsm/kg、再优选为170～230mOsm/kg、特别优选为180～220mOsm/kg。
为了使培养基的渗透压降低，可举出例如，使贡献于渗透压的升高的培养基成分的量减少的方法。例如，只要通过使基础培养基中的氯化钠等的成分的量适量减少，使渗透压降低至期望的水平即可。或者，即使通过将基础培养基用适量的水稀释，也可容易地调整渗透压。再者，培养基的渗透压的测定只要使用公知的方法即可。
可适用本发明的培养基的细胞种不特别限定。作为涉及的细胞种，含例如，精子或卵子等的生殖细胞、构成生物体的体细胞、干细胞(多能性干细胞等)、前体细胞、从生物体分离的癌细胞、从生物体分离而获得永生化能而在体外稳定维持的细胞(细胞株)、从生物体分离而人为形成基因改变的细胞、从生物体分离而人为更换核的细胞等。作为构成生物体的体细胞的例，包括但不限于：成纤维细胞、骨髓细胞、B淋巴细胞、T淋巴细胞、嗜中性粒细胞、红细胞、血小板、巨噬细胞、单核细胞、骨细胞、周皮细胞、树突细胞、角质化细胞、脂肪细胞、间充质细胞、上皮细胞、表皮细胞、内皮细胞、血管内皮细胞、肝实质细胞、软骨细胞、卵丘细胞、神经系细胞、神经胶质细胞、神经元、少突胶质细胞、微神经胶质、星状胶细胞、心脏细胞、食道细胞、肌肉细胞(例如，平滑肌细胞或骨骼肌细胞)、胰腺β细胞、黑色素细胞、造血前体细胞(例如，来源于脐带血的CD34阳性细胞)、及单核细胞等。所述体细胞含从例如皮肤、肾脏、脾脏、肾上腺、肝脏、肺、卵巢、胰腺、子宫、胃、结肠、小肠、大肠、膀胱、前列腺、精巢、胸腺、肌肉、结缔组织、骨、软骨、血管组织、血液(含脐带血)、骨髓、心脏、眼、脑或神经组织等的任意的组织采集的细胞。
干细胞是指兼备复制自身的能力和分化为其他多个系统的细胞的能力的细胞，作为其例，包括但不限于：胚胎干细胞(ES细胞)、胚性肿瘤细胞、胚性生殖干细胞、人工多能性干细胞(iPS细胞)、神经干细胞、造血干细胞、间充质干细胞、肝干细胞、胰腺干细胞、肌干细胞、生殖干细胞、肠干细胞、癌干细胞、毛包干细胞等。
细胞株是指由生物体外的人为的操作获得无限的增殖能的细胞。细胞株是指由生物体外的人为的操作获得无限的增殖能的细胞，作为其例，包括但不限于：CHO(中国仓鼠卵巢细胞株)、HCT116、Huh7、HEK293(人胎儿肾细胞)、HeLa(人子宫癌细胞株)、HepG2(人肝癌细胞株)、UT7/TPO(人白血病细胞株)、MDCK、MDBK、BHK、C-33A、HT-29、AE-1、3D9、Ns0/1、Jurkat、NIH3T3、PC12、S2、Sf9、Sf21、High Five(注册商标)、Vero等。
在优选的一实施方式中，细胞是干细胞，更优选为iPS细胞。
本发明的培养基也可在液体的状态下提供，另外，也可设为在比使用时的浓度浓缩的状态或冷冻干燥的粉末等的固体的状态下调制，在使用时用水等的溶剂稀释，或者溶解或分散于水等的溶剂而使用的实施方式。
【2.细胞的培养方法】
本发明另外提供包括以培养基的渗透压成为220～310mOsm/kg的方式用本发明的培养基更换使用中的培养基的一部分或全量的工序的，细胞的培养方法(以下，有时称为“本发明的方法”)。
细胞培养中的培养基更换的频度一般综合考虑细胞密度、培养方法(接附培养/悬浮培养)、培养的细胞种、培养基的组成、培养条件(温度、气体浓度)、更换的培养基的量(全量/一部分的量)、培养基的成本、作业者的生活方式等的多种条件来确定，通常是每2～3天1次、1天1次、或者1天多次(例、2次)程度，在本发明的方法中，也可由这样的频度进行培养基更换。
一方面，也可着眼于将培养基的渗透压维持在对于细胞增殖优选的范围的本发明的一侧面，将渗透压超一定水平(例如，310m Osm/kg)的时间点设定为培养基更换的时机。
作为可适宜地应用本发明的方法的一实施方式，可举出使用生物反应器等的高密度培养。具体而言，在使用生物反应器等进行高密度培养时，起因于多数的细胞的生命活动的培养基的pH的降低可在比较短期间发生。生物反应器检测所述pH的降低，为了将pH维持在中性附近而向培养基自动添加pH调整剂(例、碳酸氢钠)。因此，培养基的渗透压变得经时升高。如以下的实施例中所示，当培养基的渗透压超一定水平时，细胞增殖效率降低。从而，通过以培养基的渗透压作为基准，适时用本发明的培养基更换渗透压升高的培养基的一部分的量或全量，将培养基的渗透压维持在适宜于细胞增殖的范围(即，220～310(优选为240～280)mOsmol/kg)。作为结果，可以比从前可使用生物反应器达成的效率更优选的效率达成细胞增殖。当考虑这样的事实时，本发明的方法，在一侧面，也可另称为使用生物反应器的高密度培养法的改良方法。
在本发明的方法中，由本发明的培养基的更换可更换使用中的培养基的全量，也可更换一部分(例如，相对于使用中的培养基的全量，10％、20％、30％、40％、50％、60％、70％、80％、或者90％等)。对于更换的培养基量，只要是培养基更换后的渗透压可在适宜于细胞培养的范围(即，220～310(优选为240～280)mOsmol/kg)，就不特别限定。但是，当更换培养基的全量时，也可有从培养环境的急剧的变化给细胞应激的情况。从而，在一实施方式中，由本发明的培养基更换的培养基量可为培养基的一部分。具体而言，可为相对于使用中的培养基的全量，10％～90％、可优选为相对于使用中的培养基的全量，50％～90％、可再优选为相对于使用中的培养基的全量，70％～90％。
在优选的一实施方式中，也可将D-葡萄糖及5种氨基酸(色氨酸、丝氨酸、半胱氨酸(或者胱氨酸)、甲硫氨酸、精氨酸)追加添加到本发明的培养基中。
葡萄糖(或者其盐)可以换算为葡萄糖的浓度而成为通常0.1g/L/天～900g/L/天、优选为1g/L/天～200g/L/天、更优选为1g/L/天～20g/L/天的方式添加到本发明的培养基中。
另外，5种氨基酸(色氨酸、丝氨酸、半胱氨酸(胱氨酸)、甲硫氨酸及精氨酸)可以色氨酸的浓度(换算为游离体的色氨酸的浓度)成为通常0.1mg/L/天～11000mg/L/天、优选为1mg/L/天～1000mg/L/天、更优选为1mg/L/天～100mg/L/天、丝氨酸的浓度(换算为游离体的丝氨酸的浓度)成为通常0.1mg/L/天～425000mg/L/天、优选为1mg/L/天～1000mg/L/天、更优选为1mg/L/天～100mg/L/天、半胱氨酸或胱氨酸的浓度(换算为游离体的半胱氨酸的浓度)成为通常0.1mg/L/天～280000mg/L/天、优选为1mg/L/天～1000mg/L/天、更优选为1mg/L/天～100mg/L/天、甲硫氨酸的浓度(换算为游离体的甲硫氨酸的浓度)成为通常0.1mg/L/天～55000mg/L/天、优选为1mg/L/天～1000mg/L/天、更优选为1mg/L/天～100mg/L/天、精氨酸的浓度(换算为游离体的精氨酸的浓度)成为通常0.1mg/L/天～150000mg/L/天、优选为1mg/L/天～2000mg/L/天、更优选为1mg/L/天～200mg/L/天的方式添加到本发明的培养基中。
另外，在将本发明的方法适用于干细胞时，在优选的一实施方式中，也可将bFGF和/或胆碱追加添加到本发明的培养基中。
在本发明的方法中，培养条件不特别限定，只要对应于细胞种、细胞密度、培养方法(接附培养/悬浮培养等)等选择公知的方法即可。例如，培养温度可通常为25℃～39℃、优选为33℃～39℃。作为二氧化碳浓度，可通常为4体积％～10体积％、优选为4体积％～6体积％。作为氧浓度，可通常为1体积％～25体积％、优选为4体积％～20体积％。
在以下的实施例中对本发明更具体地进行说明，但本发明不受这些例的任何限定。
【实施例】
在以下的实施例中，评价由降低渗透压的培养基的人工多能性干细胞(iPS细胞)的增殖效果。作为iPS细胞，使用自iPSAcademia日本公司购入的1210B2株。另外，作为iPS细胞用培养基，使用市售的StemFit AK03N(味之素公司)。
【实施例1：由使用悬浮培养系的降低渗透压的培养基的iPS细胞的增殖促进效果】
使用iPS细胞用30mL一次性生物反应器(ABLE公司、型号BWV-S03A)，将iPS细胞1210B2株以6×105个细胞/mL的细胞密度接种到StemFit AK03N+10μM Y-27632(Wako公司、034-24024)中，在CO2温育器内在37℃、CO2浓度＝5％、搅拌速度＝120rpm的条件下搅拌培养。在接种第2天，将7成的培养基用StemFit AK03N更换。用注射用水(大塚制药公司)稀释StemFit AK03N的A液，制作调制到220mOsmol/kg的StemFit AK03N。另外，用注射用水稀释StemFit AK03N的A液，制作用NaCl(和光纯药公司)添加调制到260mOsmol/kg的StemFitAK03N。在接种第3天，将细胞悬浮液10mL再悬浮于新鲜的220mOsmol/kg StemFit AK03N或260mOsmol/kg StemFit AK03N，移到生物反应器的ambr15(sartorius：001-0881)，持续在37℃、pH＝7.2、溶解氧浓度＝20％、搅拌速度＝300rpm的条件下搅拌培养。将7成的培养基用220mOsmol/kg StemFit AK03N或260mOsmol/kg StemFit AK03N进行1天1次更换，再者向两组添加40mg/L/天Trp(味之素公司)、40mg/L/天Ser(味之素公司)、40mg/L/天Cys(日本蛋白公司)、40mg/L/天Met(味之素公司)、160mg/L/天Arg(味之素公司)、4g/L/天D-葡萄糖(NACALAI TESQUE公司：16806-25)。在培养第12天，使用生死细胞自动分析仪的Vi-CELLTMXR(Beckman Coulter公司)测定活细胞数。另外，使用冰点降低法渗透压计的OSMOMAT auto(Gonotec公司)测定培养第12天的培养上清的渗透压。
以1次重复验证渗透压对iPS细胞的增殖的影响的结果示于图1，另外其时的渗透压示于图2。通过使用将渗透压调整到220mOsmol/kg的培养基，调整培养中的渗透压，得到了显示细胞增殖促进效果的结果。
【实施例2：由使用悬浮培养系的降低渗透压的培养基的iPS细胞的增殖促进效果】
调整StemFit AK03N A液的NaCl，制作渗透压成为220、241、267、307、或者352mOsm/kg的StemFit AK03N。使用iPS细胞用30mL一次性生物反应器(ABLE：BWV-S03A)，将iPS细胞1210B2株以2.6×105个细胞/mL的细胞密度接种到各渗透压的StemFit AK03N+10μM Y-27632(Wako：034-24024)中，在CO2温育器内在37℃、CO2浓度＝5％、搅拌速度＝120rpm的条件下搅拌培养。接种第2天往后、1天1次将7成的培养基用各渗透压的StemFit AK03N更换。在培养第4天或第5天，使用生死细胞自动分析仪的Vi-CELLTM XR(Beckman Coulter公司)测定活细胞数。
以1次重复验证渗透压对iPS细胞的增殖的影响的结果示于图3。通过调整培养基的渗透压，得到了显示细胞增殖促进效果的结果。
【产业上的利用可能性】
由本发明，可用非常地廉价并且简便的方法实现与使用生物反应器的细胞培养中从前进行的实施方式比较更优选的高密度培养。
本申请以在日本申请的特愿2020-003960(申请日：2020年1月14日)作为基础，其内容全部包含在本说明书中。
Claims (5)
1.降低渗透压的培养基，其特征在于，
含基础培养基，
渗透压调整到150～250mOsm/kg。
2.权利要求1所述的降低渗透压的培养基，其中降低渗透压的培养基的渗透压是170～230mOsm/kg。
3.权利要求1或2所述的降低渗透压的培养基，其中通过降低构成基础培养基的成分中的氯化钠的量来调节渗透压。
4.细胞的培养方法，其包括：以使培养基的渗透压成为220～310mOsm/kg的方式用权利要求1～3之任一项所述的降低渗透压的培养基更换使用中的培养基的一部分或全量的工序。
5.权利要求4所述的方法，其中细胞培养是悬浮培养。
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