CN108934158B - 细胞冻存用培养基组合物及其应用 - Google Patents
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Abstract
本发明涉及细胞冻存用培养基组合物,以及包括所述培养基组合物和治疗性细胞的药物组合物,所述培养基组合物表现出优异的细胞复苏率、细胞存活率和解冻后的细胞活性。此外,本发明的所述培养基组合物使得冻存的治疗性细胞容易施用给受试对象而无需诸如洗涤、分离等的额外步骤。因此,所述培养基组合物可以作为用以细胞冻存的优良培养基组合物或者作为良好的治疗性组合物。
Description
技术领域
本发明涉及细胞冻存用培养基组合物,其表现出优异的细胞复苏率(cellrecovery rates)、细胞存活率和解冻后的细胞活性,本发明还涉及包括所述培养基组合物和治疗性细胞的药物组合物。
背景技术
随着动物细胞培养方法的发展,这些方法已广泛应用到包括蛋白质药物生产在内的各个领域的生物学研究。在利用动物细胞生产蛋白质药物时,冻存(cryopreservation)主要用作允许长期维持细胞系特性的保藏方法。
通过冻存可以克服细胞培养和维持中的困难,可以防止细菌、支原体等的污染。此外,可以防止细胞系的转化,还可以保证实验和生产的可重现性。
一般来说,动物细胞在冻存过程中会受到细胞损伤,即,细胞暴露于低温会导致细胞器官受损和细胞功能受损。因此,为了保证冻存后的细胞身份,应进行能维持细胞的特性和存活率的冻存方法的开发和验证。
作为动物细胞冻存的一般方法,将10%的二甲基亚砜(DMSO)添加到补充有用于保护细胞膜的血清的细胞培养基中,将细胞逐渐冷却并储存在-196℃下的液氮中。然而,由于血清的问题,在诸如蛋白质生产等的过程中这种方法的使用频率正在降低。此外,虽然血清中的微量元素给细胞的生长施加了影响,但存在难以分析这些要素的问题,且这些要素可以根据生产时间和地点而变化。
此外,大多数血清是来自动物,即使它来自人类,也会受到病毒等的感染。因此,在使用含血清作为直接治疗药物的组合物(细胞治疗剂)中存在安全问题。
发明内容
技术问题
因此,本发明的发明人建立了冻存用培养基组合物,其允许长期冷冻动物细胞并且其不含血清,因此可用作细胞治疗剂用的组合物,且本发明的发明人已完成了本发明。
因此,本发明的一个目的是提供细胞冻存用培养基组合物。
本发明的另一个目的是提供一种药物组合物,其包括上述细胞冻存用培养基组合物。
解决问题的技术方案
根据本发明的一个目的,本发明提供了一种细胞冻存用培养基组合物,基于所述培养基组合物的总体积,所述培养基组合物包括15-55v/v%的白蛋白、20-30v/v%的葡聚糖、2-8v/v%的二甲基亚砜和15-55v/v%的细胞培养基。
根据本发明的另一个目的,本发明提供了一种预防或治疗癌症或感染性疾病的药物组合物,所述药物组合物包括本发明的培养基组合物和作为活性成分的免疫细胞。
根据本发明的又一个目的,本发明提供了一种预防或治疗退行性疾病(degenerative disorder)和免疫相关紊乱的药物组合物,所述药物组合物包括本发明的培养基组合物和作为活性成分的干细胞。
本发明的有益效果
如果细胞是冷冻在本发明的细胞冻存用所述培养基组合物中,那么当细胞被解冻时,细胞的存活率很高,并且还维持了细胞的活性。此外,本发明的所述培养基组合物允许冻存的治疗性细胞施用给受试对象而无需诸如洗涤、分离等的额外步骤。因此,所述培养基组合物可以作为用于细胞冻存的优良培养基组合物或者作为良好的治疗性组合物。
附图说明
图1a-1d显示了NK细胞在解冻和用IL-2培养后的复苏率(图1a和1c)和存活率(图1b和1d),其中NK细胞被冷冻在根据本发明一实施方式制备的细胞冻存用培养基组合物中。
图2a-2d显示了冷冻在根据本发明一实施方式制备的细胞冻存用培养基组合物中的NK细胞对K562细胞的细胞毒性,其中,图2a和图2b显示了细胞一经解冻后的测量值,图2c和图2d显示了在解冻和用IL-2培养后的测量值。
图3a和图3b显示了HuT78细胞在解冻和培养3天后的细胞数目(图3a)和存活率(图3b),其中,HuT78细胞被冷冻在根据本发明一实施方式制备的细胞冻存用培养基组合物中。
图4a和图4b显示了K562细胞在解冻和培养3天后的细胞数目(图4a)和存活率(图4b),其中,K562细胞被冷冻在根据本发明一实施方式制备的细胞冻存用培养基组合物中。
实施本发明的最佳方式
在下文中,将更具体地描述本发明。
本发明提供了一种细胞冻存用培养基组合物,基于所述培养基组合物的总体积,所述培养基组合物包括15-55v/v%的白蛋白、20-30v/v%的葡聚糖、2-8v/v%的二甲基亚砜和15-55v/v%的细胞培养基。
如本文所用的,术语“冻存”指通过冷冻可以长期稳定地维持细胞。通常地,当培养细胞时,突变以1/10000的比例发生,并且当细胞经过长期的传代培养时,细胞群体可能改变,并变得不同于原始群体。严重的情况下,细胞可能通过传代培养失去其特有的功能。此外,细胞可能在传代培养过程中感染支原体或类似物。由于这样问题的存在,因此需要进行细胞冻存以在细胞失去其内在特性之前冷冻和保存细胞,并在需要时使用它们。
细胞冻存可以通过用冷冻保护剂(cryportectant)处理待冻存的细胞而进行。本发明的培养基组合物包括白蛋白、葡聚糖、二甲基亚砜和细胞培养基,以防止因冷冻保护剂引起的细胞损伤,从而提高在冻存细胞中的安全性和稳定性。
本文所用的术语“冷冻保护剂”指当细胞储存在-80℃至-200℃的超低温下时所用的物质。特别地,该物质可以最大限度地减少冰晶的形成,以及因离子和渗透压的不平衡而造成的细胞损伤,其中,冷冻和解冻过程不可避免地伴随着离子和渗透压的不平衡。
如本文所用的,术语“白蛋白”指具有生物活性的总的人白蛋白或一部分的人白蛋白。白蛋白可用作不含动物成分的替代品(animal-free substitute),其在冻存中相当于或优于血清,并被添加到所述冻存用组合物中以稳定细胞。
白蛋白可以是美国国立卫生研究院的GenBank数据库中登录号为ANC98520.1的蛋白质,且可包括展示出与上述蛋白质具有70%或更高相似性的氨基酸序列,特别地具有80%或更高相似性,更确切地说具有95%或更高相似性,最确切地说具有98%或更高相似性。此外,也可以包括其中部分序列被删除、修饰、替换或添加的氨基酸序列,只要氨基酸序列具有与上述白蛋白相同或对应的生物活性。
基于所述培养基组合物的总体积,本发明的所述培养基组合物中含有的白蛋白的含量可以为15-55v/v%,具体为18-40v/v%,更具体地为20-35v/v%。根据本发明的一个实施方式,基于总的所述培养基组合物,白蛋白的含量可以是20v/v%、35v/v%或50v/v%。这里“v/v%”指每种成分占所述培养基组合物的总体积的体积百分比。
如本文所用的,术语“葡聚糖(dextran)”指一种多糖,它是D-葡萄糖的聚合物。根据重均分子量,葡聚糖可被分类成葡聚糖1、葡聚糖40、葡聚糖70等。在一实施方式中,葡聚糖可以为葡聚糖40。
基于所述培养基组合物的总体积,本发明的所述培养基组合物中含有的葡聚糖的含量可以为20-30v/v%,具体地为23-27v/v%。根据本发明的一实施方式,所述葡聚糖的含量可以是总的所述培养基组合物的25v/v%。
如本文所用的,“二甲基亚砜(DMSO)”用作细胞膜渗透性冻存剂。它可渗透细胞膜以取代细胞内水,并抑制冻结过程中冰晶的形成。
基于所述培养基组合物的总体积,本发明的所述培养基组合物中含有的二甲基亚砜的含量可以是2-8v/v%,具体地为4-6v/v%。根据本发明的一实施方式,所述二甲基亚砜的含量可以是总的所述培养基组合物的5v/v%。
如本文所用的,术语“细胞培养基”指用于细胞体外生长和增殖的混合物,含有糖、氨基酸、各种营养物、无机物质等细胞生长和增殖的必需元素。可以对所述细胞培养基进行优化以用于特定细胞培养,例如,用于维持细胞生长的基本培养基、所制备的用以促进由细胞生产蛋白质的细胞培养生产培养基、通过将营养物质浓缩到高浓度而制备的浓缩培养基。
上述细胞培养基可以含有选自下组中的至少一种成分:L-丙氨酸、L-精氨酸盐酸盐、L-半胱氨酸盐酸盐、L-谷氨酰胺、L-组氨酸盐酸盐、L-赖氨酸盐酸盐、L-蛋氨酸、L-脯氨酸、L-丝氨酸、L-苏氨酸、L-缬氨酸、L-天冬酰胺一水合物、L-天冬氨酸、L-半胱氨酸·2HCl、L-谷氨酸、L-异亮氨酸、L-亮氨酸、L-苯丙氨酸、L-色氨酸、L-酪氨酸二水合物、内消旋肌醇、盐酸硫胺素、烟酰胺、吡哆醇盐酸盐、生物素、D-泛酸钙、叶酸、核黄素、维生素B12、氯化钠(NaCl)、碳酸氢钠(NaHCO3)、氯化钾(KCl)、氯化钙(CaCl2)、一水合磷酸氢钠(NaH2PO4-H2O)、五水合硫酸铜(CuSO4-5H2O)、七水硫酸铁(FeSO4-7H2O)、氯化镁(无水)、硫酸镁(MgSO4)磷酸氢二钠(Na2HPO4)、七水硫酸锌((ZnSO4-7H2O)、D-葡萄糖(右旋糖)、丙酮酸钠、次黄嘌呤钠(hypoxanthin Na)、亚麻酸、硫辛酸、腐胺·2HCl和胸苷(thymidine)。
本发明的所述细胞培养基可以通过人工制造来制备,或者使用市售的细胞培养基。市售的细胞培养基包括DMEM(Dulbecco改良的Eagle培养基)、MEM(最小必需培养基)、BME(Eagle基本培养基)、RPMI1640、F-10、F-12、α-MEM(α-最小必需培养基)、G-MEM(Glasgow最小必需培养基)、cellgro SCGM、X-VIVO、AIM-V等。
基于所述培养基组合物的总体积,所述细胞培养基的含量可以是15-55v/v%,具体地为30-52v/v%。根据本发明的一实施方式,基于100v/v%的总的培养基组合物,所述细胞培养基的含量可以是20v/v%、35v/v%或50v/v%。
可以用本发明的冻存用培养基组合物进行保存的细胞可以是动物细胞,但是也可以能被本发明的培养基组合物稳定地冻存的任何细胞。这些细胞可以来自人、猴、猪、马、牛、羊、狗、猫、老鼠、兔子等,具体地可以是来自动物的免疫细胞、肿瘤细胞、脐血细胞、干细胞、上皮细胞、原代细胞等。
此外,本发明的冻存用培养基组合物还可以包括缓冲剂、等渗剂或凋亡抑制剂。该缓冲剂可以是选自柠檬酸盐、磷酸盐、琥珀酸盐、酒石酸盐、富马酸盐、葡萄糖酸盐、草酸酯、乳酸酯、乙酸盐、组氨酸和tris(三羟基氨基甲烷)中的任何一种。另外,等渗剂可以选自氯化钠、氯化钾、硼酸、硼酸钠、甘露醇、甘油、丙二醇、聚乙二醇、麦芽糖、蔗糖、赤藓糖醇、阿拉伯糖醇、木糖醇、山梨糖醇、海藻糖和葡萄糖中的一种。凋亡抑制剂可以选自Rho相关激酶(ROCK)抑制剂、过氧化氢酶和zVAD-fmk中的一种。
本发明还提供了一种细胞冻存方法,包括冷冻细胞的步骤,其中,向细胞中添加冻存用培养基组合物。
可以通过常规的方法对向其中添加有冻存用培养基组合物的细胞进行冷冻,例如玻璃化冷冻(vitrification freezing)、慢速冷冻(slow freezing)等。玻璃化冷冻是一种冷冻细胞的方法,它是通过使用诸如控速冷冻装置(controlled rate freezing,CRF)的设备以每分钟恒定的速率来控制温度。当温度达到-80℃时,将冷冻的细胞储存在氮气罐中。同时,慢速冷冻是一种冷冻细胞的方法,它通过将装有含细胞的冻存用培养基组合物的小瓶(vial)放入含有异丙醇的容器中冷冻,然后在-70℃或更低温度的超低温冰箱中以恒定的速率降温12-24小时。之后,将冷冻的细胞储存在液氮罐中。
根据本发明的一实施方式,所述细胞冻存方法可以是玻璃化冷冻法。所述玻璃化冷冻是采用CRF进行,通过:1)在室温下将细胞冷却至4℃;2)以-1℃/分钟的速率冷却至-8℃;3)以-25℃/分钟的速率冷却至-65℃;4)以15℃/分钟的速率升温至-14℃;5)以-1℃/分钟的速率冷却至-45℃;6)以-10℃/分钟的速率冷却至-90℃。
以上细胞冻存方法可以通过小瓶内含有适当浓度的细胞来进行。细胞的浓度可以为1 x 104至1 x 108细胞/ml,具体地可以为1 x 105至1 x 107细胞/ml小瓶,但该浓度可以根据细胞的类型而改变。
本发明提供一种预防或治疗癌症或感染性疾病的药物组合物,包括本发明的培养基组合物及作为活性成分的免疫细胞。
所述培养基组合物与上述细胞冻存用培养基组合物相同。然而,当用作药物组合物时,可以包含的细胞培养基优选为不含诸如血清、酚红或β-巯基乙醇的有毒物质的培养基。因此,本发明的所述药物组合物不具有人体毒性,因此可在解冻后直接给受试对象服用。
如本文所用的,术语“免疫细胞”指参与人体免疫反应的细胞,包括自然杀伤细胞、T细胞、B细胞和树突状细胞等。具体地,所述免疫细胞可以是天然杀伤细胞。
上述癌症可以包括:膀胱癌、乳腺癌、胃癌、肺癌、卵巢癌、甲状腺癌、宫颈癌、中枢神经系统癌、胶质瘤、肝癌、皮肤癌、胰腺癌、胃癌、结肠癌、直肠癌、食道癌、肾癌、肺癌、上皮癌、血癌、前列腺癌、软组织肉瘤、多发性硬化等。
如本文所用的,术语“感染性疾病”指由病毒或病原体感染引起的病理状态,包括通过呼吸道、血液、皮肤接触等可能传播或感染的所有疾病。这类感染性疾病的例子包括但不限于乙型肝炎和丙型肝炎、人乳头瘤病毒(HPV,human papilloma virus)感染、巨细胞病毒感染、病毒性呼吸道疾病、流感等。
本发明还提供了一种预防或治疗退行性疾病的药物组合物,包括本发明的培养基组合物和作为活性成分的干细胞。
所述培养基组合物与上述细胞冻存用培养基组合物相同。然而,当用作药物组合物时,可以包含的细胞培养基优选为不含诸如血清、酚红或β-巯基乙醇的有毒物质的培养基。因此,本发明的所述药物组合物不具有人体毒性,因此可在解冻后直接给受试对象服用。
如本文所用的,术语“干细胞”指具有多能性的细胞,能够分化成各种细胞。这样的干细胞可以包括骨髓干细胞、间充质干细胞、人神经干细胞、口腔粘膜固有层干细胞等。具体地,所述干细胞可以为间充质干细胞。
如本文所用的,术语“退行性疾病”指因组织的不可逆定量丧失而组织丧失原有功能的病理状态。这样的退行性障碍包括但不限于颅神经障碍、缺血性疾病、皮肤损伤、骨骼障碍和退行性关节炎等。
本发明还提供了一种预防或治疗免疫紊乱的药物组合物,包括本发明的培养基组合物和作为活性成分的间充质干细胞。
本文所用的术语“免疫紊乱”指因过度的或不希望的免疫反应而导致组织受损的任何病理状态。因此,术语“免疫紊乱”与“多动免疫障碍(hyperactive immune disorder)”具有相同的含义,且“预防或治疗免疫紊乱的组合物”与“免疫反应抑制剂(immunosuppressive agent)”具有相同的含义。
这样的免疫紊乱包括但不限于,移植物抗宿主病、移植排斥反应、慢性炎症性疾病、炎性疼痛、神经性疼痛、慢性阻塞性肺病(COPD)和自身免疫性疾病。
本文所用的术语“自身免疫性疾病”指当免疫细胞攻击自身而不区分自身与外来物质时发生的病理状态。自身免疫性疾病包括但不限于,类风湿性关节炎、桥本氏甲状腺炎(Hashimoto's thyroiditis)、毒性弥漫性甲状腺肿(Grave's病)、多发性硬化、硬皮病、重症肌无力、I型糖尿病、变态反应性脑脊髓炎、肾小球肾炎(glomerulonephritis)、白癜风、贝克氏病(Bechet's disease)、克罗恩病(Crohn's disease)、强直性脊柱炎、血小板减少性紫癜、寻常型天疱疮、自身免疫性贫血(autoimmune anemia)、肾上腺脑白质营养不良(ALD)和系统性红斑狼疮(SLE)。
本发明的药物组合物还可以包括除上述活性成分之外的药学上可接受的添加剂。
此外,所述药物组合物可以配制成适用于通过药剂学领域的常规方法施用给病人身体的单位剂型。适合这种用途的制剂包括作为肠胃外给药制剂的溶液或悬浮液,或者作为外用制剂的软膏。当配制本发明的药物组合物时,可以一起使用填料、填充剂、粘合剂、润湿剂、崩解剂、稀释剂(如表面活性剂)、赋形剂等。
相对于总的药物组合物,所述药物组合物的剂量可以为每1公斤体重约1μg-50mg,且诸如免疫细胞和干细胞的治疗性细胞的成人剂量可以为1-108/天、10-105/天、102-103/天。上述施用可以每日一次或每日分剂量进行数次。然而,可以考虑患者的体重、年龄和性别以及疾病的严重程度和给药途径等因素来确定给药的剂量和次数。
本发明的实施例
下文将参照以下实施例、比较例和实验例来详细描述本发明。然而,以下实施例、比较例和实验例旨在说明本发明而不是限制本发明的范围。
实施例1-3制备冻存用培养基组合物
为制备冻存用培养基组合物,将5ml的DMSO、25ml的葡聚糖40、50ml的白蛋白注射液、20ml的作为细胞培养基的RPMI1640进行混合(实施例1)。在实施例2和3中,也采用与实施例1相同的方法但以下表1所示的量来制备冻存用培养基组合物,其含有二甲基亚砜(DMSO,Bioniche Pharma,美国)、葡聚糖40(大韩制药株式会社(Dai Han Pharm.Co.,Ltd))、白蛋白注射液(韩国绿十字公司)和RPMI1640(美国Gibco公司)。
对比例1-6制备冻存用培养基组合物
作为上述实施例的比较例,将5ml的DMSO和50ml的白蛋白注射液进行混合以制备冻存用培养基组合物(对比例1)。在对比例2-6中,也采用与实施例1相同的方法但以下表1所示的量来制备冻存用培养基组合物,其含有二甲基亚砜(DMSO,Bioniche Pharma,美国)、葡聚糖40(大韩制药株式会社)、白蛋白注射液(韩国绿十字公司)和RPMI1640(美国Gibco公司)。
表1
实验例1冷冻和解冻后的NK细胞的评价
1.1NK细胞的冷冻和解冻
将NK细胞冷冻在实施例1-3和对比例1-6中制备的冻存用培养基组合物中,然后进行解冻以评估冻存用培养基组合物。
首先,从人外周血(韩国首尔国立大学医院)分离出单核细胞。采用CliniMACS系统从分离的单核细胞中取出CD3阳性细胞,并将其用作种子细胞。同时,将以2000cGy的γ射线照射的单核细胞作为NK细胞培养的支持细胞。将种子细胞和支持细胞以0.5 x 106至1 x106细胞/ml的浓度培养在SCGM培养基(德国CellGenix公司)中,该培养基添加有500IU/ml的IL-2、10μg/ml的抗-CD3抗体OKT3和1v/v%的血清。在37℃和5%CO2的条件下进行培养14-21天。
收集培养后的NK细胞,并在4℃和1500rpm的条件下离心10分钟。将所得球团(pellet)用RPMI 1640培养基洗涤,计算细胞数。将细胞以1x108细胞/试管的浓度分配到15ml的管中,然后在上述相同条件下离心以得到球团。将得到的球团冷冻在实施例1-3和对比例1-6中制备的冻存用培养基组合物中。
在下面表2所示的条件下,将细胞放置于控速冷冻装置中,并将冷冻的细胞储存在LN2蒸气罐中一段时间以进行细胞冷冻。收集冷冻后的细胞,并在37℃恒温浴中快速解冻。将细胞悬浮在相当于10倍细胞体积的含10%胎牛血清的RPMI1640培养基中,然后在4℃和1200rpm的条件下离心10分钟,获得细胞。
表2
0步 | 末端温度+4℃ |
1步 | 末端温度-8℃;斜率-1℃/min |
2步 | 末端温度-65℃;斜率-25℃/min |
3步 | 末端温度-14℃;斜率+15℃/min |
4步 | 末端温度-45℃;斜率-1℃/min |
5步 | 末端温度-90℃;斜率-10℃/min |
1.2 NK细胞的培养
将获得的NK细胞重悬于含500IU/ml的IL-2和10%胎牛血清的RPMI1640培养基中,以得到2 x 106细胞/ml的浓度。将重悬后的细胞(10ml)分散在T75烧瓶中,在培养的第3天,加入等体积的培养基,并培养7天。
1.3检测活细胞数目和存活率
根据制造商手册,使用ADAM细胞计数器Accustain试剂盒(DigialBio)检测培养后的NK细胞的活细胞的数目和存活率。
首先,对实验例1.2中培养的细胞进行计数并用PBS稀释至浓度为1 x 106细胞/ml。将稀释后的细胞以14μl/孔的浓度分配到圆底96孔板的两个孔中。将可测量细胞总数的14μl的T溶液加入到两个孔中的其中一个,并与稀释后的细胞混合,然后收集14μl的混合物并放入计数器芯片的T板中。此外,将可测量死细胞数的14μl的N溶液加入到两个孔中的另一个孔中,并与稀释后的细胞混合,然后收集14μl的混合物并放入计数器芯片的N板中。将芯片插入到ADAM计数器中,测量活细胞的数目,结果如图1a-1d所示。
在将冷冻的细胞解冻及然后用IL-2培养之后的复苏率(如图1a和1c所示)显示成在解冻和用IL-2培养后的活细胞的数目的百分比(如下表3和4所示),这是基于冷冻时瓶内细胞的数目来算。
表3
平均值 | 标准偏差 | |
对比例1 | 36 | 2 |
对比例2 | 60 | 5.8 |
实施例1 | 72 | 18.1 |
实施例2 | 82 | 6.2 |
实施例3 | 85 | 27.6 |
表4
同时,在将冰冻的细胞解冻及然后用IL-2培养之后的复苏率(如图1b和1d所示)显示成基于细胞总数目的活细胞数目的百分比,如下表5和表6所示。
表5
平均值 | 标准偏差 | |
对比例1 | 42 | 23 |
对比例2 | 67 | 13 |
实施例1 | 76 | 6 |
实施例2 | 80 | 11 |
实施例3 | 79 | 7 |
表6
平均值 | 标准偏差 | |
实施例3 | 72 | 6 |
对比例3 | 68 | 15 |
对比例4 | 66 | 11 |
对比例5 | 66 | 9 |
对比例6 | 65 | 11 |
如图1a-1d及表3-6所示,当使用实施例1-3的冻存用培养基组合物培养7天后,细胞复苏率和存活率高达70%以上。同时,含有DMSO和白蛋白注射液(对比例1),含有DMSO、葡聚糖40及白蛋白注射液(对比例2),含有DMSO、葡聚糖40和小于15V/V%的白蛋白注射液和RPMI1640(对比例3-6)的冻存用培养基组合物导致了低复苏率和存活率。
1.4 NK细胞活性的评价
为了检测冷冻和解冻在实施例1-3及对比例1-6中的冷冻培养基中的NK细胞的细胞活性,采用NK细胞处理肿瘤细胞,并检测肿瘤细胞的杀伤能力。其中,对在实验例1.1中一经解冻后获得的NK细胞和实验例1.2中在培养后获得的NK细胞的活性分别进行了评价。
首先,在37℃和5%CO2的条件下,将肿瘤细胞K562细胞(ATCC,美国)培养在含10%胎牛血清的RPMI1640培养基中。用胰蛋白酶消化培养后的细胞,并悬浮于该培养基中至浓度为1 x 106细胞/ml。然后加入1mM的钙黄绿素AM(calcein AM,美国生命技术公司),得到30μM的最终浓度。在37℃和5%CO2的条件下培养细胞1小时,用荧光标记肿瘤细胞。
将标记的肿瘤细胞用10ml的该培养基洗涤,并以1 x 105细胞/ml的浓度重悬,然后以100μl/孔的浓度分散到96-孔圆底板中。混入在实验例1.1中一经解冻后获得的细胞和在实验例1.2中培养后获得的细胞,以使NK细胞(效应细胞,E)和K562细胞(靶细胞,T)的比例分别为10∶1、3∶1和0.3∶1,然后以100μL/孔加入到板中。其中,为了自发的释放,在肿瘤细胞中加入等量的培养基,为了最大的释放,在肿瘤细胞中加入等量的2%Triton X-100。
将混合物在37℃和5%CO2的条件下反应4小时,然后在4℃下以2000rpm离心3分钟。之后,将100μl的所获得的上清液转移到一个黑色孔板,并采用荧光检测器在385nm/450nm的条件下测量。利用由此获得的荧光测量值,由以下式(1)计算NK细胞对肿瘤细胞的溶解率(%),并如图2a-2d所示,其也在下表7-10中示出。自然杀伤细胞供体的数量在表7中为6,而自然杀伤细胞供体的数量在表8中为3。
[式1]
表7
一经解冻 | 对比例1 | 对比例2 | 实施例1 | 实施例2 | 实施例3 |
E10 | 44 | 60 | 59 | 65 | 76 |
E3 | 22 | 32 | 32 | 42 | 52 |
E1 | 8 | 13 | 12 | 17 | 24 |
E0.3 | 2 | 4 | 9 | 7 | 8 |
表8
表9
培养后 | 对比例1 | 对比例2 | 实施例1 | 实施例2 | 实施例3 |
E10 | 84 | 87 | 86 | 86 | 88 |
E3 | 68 | 82 | 77 | 76 | 77 |
E1 | 45 | 61 | 52 | 46 | 45 |
E0.3 | 17 | 28 | 21 | 19 | 18 |
表10
培养后 | 实施例3 | 对比例3 | 对比例4 | 对比例5 | 对比例6 |
E30 | 84 | 81 | 84 | 82 | 80 |
E10 | 83 | 82 | 82 | 77 | 73 |
E3 | 76 | 71 | 67 | 65 | 56 |
E1 | 50 | 41 | 35 | 34 | 29 |
E0.3 | 22 | 14 | 13 | 14 | 10 |
如图2a和图2b及表7-8所示,发现当白蛋白的含量10%以下或70%以上时,相比于实施例,一经解冻的冷冻细胞对肿瘤的细胞毒性较低。一般而言,已知当激活的免疫细胞被冷冻时,细胞的功能会因细胞活性的降低而减少。然而,发现解冻的细胞能基于冻存培养基的组成来维持不同程度的活性。特别地,发现在实施例3中,当细胞在冷冻后解冻时能维持最高的NK细胞活性。
同时,冷冻的细胞在培养一段时间后对肿瘤细胞的毒性检测结果如上述图2c、2d及表9-10所示,这表明所有的实施例和比较例显示出相似的细胞毒性。可以预期该结果是由于培养过程中NK细胞活性的恢复,添加到培养基中的IL-2可以减少NK细胞的活性。
实验例2解冻后肿瘤细胞的评价
检查实施例1-3和比较例1-6中制备的冻存用培养基组合物是否也可用于冷冻肿瘤细胞。
首先,在37℃和5%CO2的条件下,分别在含有10%胎牛血清的RPMI1640培养基及含有20%胎牛血清的RPMI1640培养基中培养人T淋巴瘤细胞系HuT78(ATCC,美国)和人白血病细胞K562(ATCC,美国)。回收培养后的细胞并悬浮于各培养基中。然后,将HuT78细胞和K562细胞放置在小瓶中,并分别以1 x 107细胞/ml和4 x 106细胞/ml的浓度冷冻。细胞冷冻是通过与上述实验例1.1中描述的相同的方法进行。将冷冻后的细胞重悬于各培养基中,对细胞进行传代培养以获得1 x 106细胞/ml的浓度,并在37℃和5%CO2的条件下培养4天。
用与实验例1.3相同的方法测定培养后的细胞的活细胞数和存活率。HuT78细胞的活细胞数和存活率如图3a和3b所示,而K562细胞的活细胞数和存活率如图4a和4b所示。
结果发现,如图3a-4b所示,在实施例1-3的冻存用培养基组合物中和在对比例2的冻存用培养基组合物中的肿瘤细胞HuT78和K562细胞的活细胞数和存活率类似。同时,对比例1的冻存用培养基组合物中显示出极低的活细胞数和存活率。
Claims (3)
1.一种自然杀伤细胞冻存用培养基组合物,其特征在于,基于所述培养基组合物的总体积,所述培养基组合物包括18-40v/v%的20%白蛋白溶液、23-27v/v%的10%葡聚糖溶液、4-6v/v%的二甲基亚砜和30-52v/v%的细胞培养基。
2.如权利要求1所述的培养基组合物,其特征在于,所述培养基组合物是直接施用给具有所述自然杀伤细胞的受试对象。
3.一种预防或治疗癌症或感染性疾病的药物组合物,其特征在于,所述药物组合物包括如权利要求1-2任一项所述的培养基组合物和作为活性成分的自然杀伤细胞。
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WO2017135631A1 (ko) | 2017-08-10 |
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JP2019504643A (ja) | 2019-02-21 |
EP3412149A4 (en) | 2019-09-18 |
KR20180085796A (ko) | 2018-07-27 |
CN108934158A (zh) | 2018-12-04 |
CA3013187C (en) | 2023-02-28 |
KR102175256B1 (ko) | 2020-11-06 |
AU2017215955A1 (en) | 2018-08-23 |
CA3013187A1 (en) | 2017-08-10 |
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