CN104603263A - 高浓度干细胞的生产方法 - Google Patents
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Abstract
本发明涉及一种用于以高浓度制备干细胞的方法。本发明使得在短时期内以足够临床使用的量培育干细胞成为可能，并且使得相对高效地增加被给药的干细胞有效地到达靶组织并以稳定地方式起作用的能力成为可能，因而能够显著提高使用干细胞的细胞治疗的功效。
Description
技术领域
本发明涉及一种用于制备高浓度干细胞的方法，更特定而言，涉及一种以足以用于临床应用的高收率来培育干细胞的方法。
背景技术
干细胞是指不仅具有自我复制能力还具有分化成至少两种类型的细胞的能力的细胞，且能够划分为全能干细胞、亚全能干细胞(pluripotent stem cell)和多能干细胞(multipotent stem cell)。全能干细胞是具有能够发育成一个完整个体的全能性质的细胞，而这些性质为卵母细胞和精子受精之后直到八细胞期的细胞所拥有。当这些细胞分离并移植到子宫内时，它们可发育成一个完整的个体。亚全能干细胞，即能够发育成外胚层、中胚层和内胚层来源的各种细胞和组织的细胞，来源于位于受精4-5天后生成的胚囊内部的内细胞群。这些细胞被称为“胚胎干细胞”，能够分化成各种其他组织细胞，但不能形成新的活有机体。多能干细胞，即仅能够分化成特异于包含这些细胞的组织和器官的细胞的干细胞，不仅涉及胎儿期、新生儿期和成体期的各种组织和器官的生长和发育，还涉及成体组织的稳态的维持和组织损伤后诱发再生的功能。组织特异性多能细胞统称为“成体干细胞”。
成体干细胞通过从各种人器官中取得细胞并使该细胞发育成干细胞而获得，并且特征在于它们分化成仅特异性的组织。然而，近来，用于使成体干细胞分化成包括肝细胞的各种组织的试验取得巨大成功，这成为了热点。特定而言，在再生医学领域中已经做出努力，所述再生医学通过使用细胞来再生生物组织和器官，并恢复它们由于疾病或事故等导致丧失的功能。在再生医学领域中经常使用的方法包括以下步骤：从患者采集干细胞、血液来源的单核细胞或骨髓来源的单核细胞；通过试管培养诱导细胞的增殖和/或分化；和通过移植将所选择的未分化的(干细胞和/或祖细胞)和/或已分化的细胞引入到患者体内。因此，期望用细胞/组织替代疗法来替代通过药物或外科手术来治疗疾病的现有经典方法，所述细胞/组织替代疗法是用健康细胞、组织或器官来替代受损细胞、组织或器官，因而干细胞的用途将进一步增加。
因此，目前正在研究干细胞的各种功能。特定而言，由于使用间充质干细胞的细胞疗法技术开始受到关注，故已经开发了用于改进自人体分离间充质干细胞以便适合于治疗目的的技术(WO 2006/019357，韩国专利第0795708号和韩国专利第0818214号)。
然而，还没有充分地研究出以足够用于临床应用的量来增殖经分离的干细胞的方法。
因此，本发明者们进行了许多努力来以大的量制备干细胞，并且已经发现当在包含基础培养基和至少两种选自下组的成分的培养基中培养干细胞时，干细胞能够在短时期内以高浓度增殖，所述成分为：N-乙酰基-L-半胱氨酸(NAC)、抗坏血酸、胰岛素或胰岛素样因子、皮质醇、碱性成纤维细胞生长因子(bFGF)、EGF(表皮生长因子)和抗氧化剂，从而完成了本发明。
本背景技术部分揭示的上述信息仅用于增进对本发明背景技术的理解，因而其可包含并未构成已为本领域普通技术人员所知的现有技术的信息。
发明内容
本发明的一个目的在于提供一种在短时期内以高浓度制备干细胞的方法。
为了实现上述目的，本发明提供了一种用于制备高浓度干细胞的方法，该方法包括在培养基中培养干细胞，所述培养基含有基础培养基；和至少两种选自下组的成分：N-乙酰基-L-半胱氨酸(NAC)、抗坏血酸、胰岛素或胰岛素样因子、皮质醇、地塞米松(dexamethasone)、bFGF(碱性成纤维细胞生长因子)、硫酸乙酰肝素、2-巯基乙醇、EGF(表皮生长因子)和抗氧化剂。
通过以下详细描述和所附权利要求书，本发明的其他特征和实施方式将更加明显。
附图说明
图1是示出在每种培养基中持续4天培养获得自20、30、70和80岁成年男性的脂肪来源的干细胞期间的细胞群倍增水平的图表。
具体实施方式
除非另有定义，本文所使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解相同的含义。一般而言，本文使用的命名和实验方法是公知的以及本领域通常采用的那些。
如本文所使用，术语“干细胞”是指不仅具有自我复制能力而且具有分化成至少两种类型的细胞的能力的细胞。“成体干细胞”是指在发育过程之后形成胚胎的各个器官的时期或者在成体时期出现的干细胞。
如本文所使用，术语“间充质干细胞”是指分离自人或哺乳动物的组织的未分化干细胞，其可来源于各种组织。特定而言，间充质干细胞可以是脐带来源的间充质干细胞、脐带血来源的间充质干细胞、骨髓来源的间充质干细胞、脂肪来源的间充质干细胞、肌肉来源的间充质干细胞、神经来源的间充质干细胞、皮肤来源的间充质干细胞、羊膜来源的间充质干细胞和胎盘来源的间充质干细胞。从各个组织分离干细胞的技术是本领域已知的。
如本文所使用，术语“脂肪来源的间充质干细胞”是分离自脂肪组织的未分化的成体干细胞，在本文中也称为“脂肪来源的成体干细胞”、“脂肪干细胞”或“脂肪来源的干细胞”。这些细胞能够通过本领域已知的任何常规方法获得。用于分离脂肪来源的间充质干细胞的方法例如如下。即，脂肪来源的间充质干细胞可以通过以下方法来分离：(在生理盐水中)培养通过脂肪抽吸获得的含脂肪的悬液，然后或者通过胰蛋白酶处理来采集附着到诸如烧瓶等培养容器的干细胞层，或者使用刮刀通过刮擦直接采集悬浮在少量生理盐水中的那些干细胞。
如本文所使用，术语“高浓度干细胞的制备”是指干细胞的数量以高收率增殖。如本文所使用，术语“高浓度的干细胞”也被称为“大量干细胞”。在本发明的实施例中，已证实当在根据本发明的培养基中培养干细胞分离组织时，所获得的干细胞数量显著增加。优选地，证实当经3-5次传代培养时，能够以1×107-5×108个细胞/ml的浓度获得干细胞。
如本文所使用，术语“传代培养”是指通过将细胞转移到新的培养皿并通过将培养基基质用新鲜培养基替换以便长期培养健康细胞而周期性地传代一部分细胞。随着细胞数量在培养皿的有限空间内增加，细胞由于预定时间内营养耗尽或废物积累而自然死亡。因此，传代培养用于增加健康细胞的数量。典型地，1次传代是指通过替换一次基质(培养皿)或细胞群体分离一次的培养物。可以使用本领域已知的任何传代培养方法而没有任何限制，但优选进行机械或酶分离方法。
如本文所使用，术语“机械分离”是指物理或机械地分离细胞聚集物。可以使用本领域已知的任何机械分离方法而没有任何限制，但细胞可优选通过使用刀片、组织切块机、针、移液、EBD(胚状体分配器，embryoid bodydivider)或刮刀分离。根据本发明的优选实施方式，已证实通过使用刀片或组织切块机传代培养干细胞能够实现干细胞的大规模增殖。
如本文所使用，术语“酶分离”是指通过酶处理解离细胞聚集物。可以使用本领域已知的任何酶分离方法而没有任何限制，但可优选通过用包括胶原蛋白酶I、II、III和IV的胶原蛋白酶、Accutase、分散酶或胰蛋白酶处理来解离细胞聚集物，接着传代培养。
可以通过各种途径，例如静脉内给药、动脉内给药或腹膜内给药，将干细胞给药到体内。在这些给药途径当中，优选静脉内给药，因为其能够以方便且安全的方式治疗疾病而无需外科手术操作。为了静脉内给予干细胞以安全地到达靶位点并呈现预期的治疗效果，应当满足各种需求。首先，应当以一定的浓度或较高的浓度给予干细胞，使得干细胞在到达靶位点后呈现预期的治疗效果。因而，大量获得被希望临床应用的干细胞很重要。此外，干细胞应当具有适合血管内给药的尺寸，使得当血管内给药时，这些干细胞既不会降低血液流速也不会形成血栓块。而且，干细胞在血管内给药之前应当不会破碎或聚集，并应当以单个细胞安全到达其靶位点而即使在血管内给药之后也不会破碎或聚集。
鉴于如上所述的若干需求，本发明意在提供临床有效的一定浓度或更高浓度的干细胞。根据常规方法的干细胞培养物，必须进行许多次传代培养来获得干细胞的高收率，因而花费许多人力和时间。特定而言，由于一部分传代培养必需的基质成分非常昂贵，故在经济效率方面是不利的。然而，本发明能够以足够临床应用的高浓度制备干细胞。
一方面，本发明涉及一种制备高浓度干细胞的方法，该方法包括在含有基础培养基和至少两种选自下组的成分的培养基中培养干细胞的步骤：N-乙酰基-L-半胱氨酸(NAC)、抗坏血酸、胰岛素或胰岛素样因子、皮质醇、地塞米松、bFGF(碱性成纤维细胞生长因子)、硫酸乙酰肝素、2-巯基乙醇、EGF(表皮生长因子)和抗氧化剂。
优选地，本发明中使用的干细胞可使用成体干细胞，特别是获得自脂肪组织或诸如毛根鞘囊肿、羊膜等上皮组织的成体干细胞。更优选地，可使用脂肪组织来源的成体干细胞作为所述干细胞。间充质干细胞(MSC)可用作所述干细胞，特别是脂肪组织来源的间充质干细胞可用作所述干细胞。脂肪组织或上皮组织优选源自哺乳动物，更优选源自人。在本发明的实施例中，使用人脂肪组织来源的间充质干细胞(AdMSC)。
本发明中使用的基础培养基是指具有本领域已知的适于干细胞培养的简单组成的典型培养基。通常用于培养干细胞的基础培养基的实例包括MEM(最低必需培养基)、DMEM(Dulbecco Modified Eagle Medium)、RPMI(Roswell Park Memorial Institute Medium)和K-SFM(Keratinocyte Serum FreeMedium)。作为本发明中使用的基础培养基，可以使用任何培养基而没有限制，只要它们在本领域使用。优选地，基础培养基可选自下组：M199/F12(混合物)(GIBCO)、MEM-α培养基(GIBCO)、含低浓度葡萄糖的DMEM培养基(Welgene)、MCDB 131培养基(Welgene)、IMEM培养基(GIBCO)、K-SFM、DMEM/F12培养基、PCM培养基和MSC扩增培养基(Chemicon)。特定而言，其中可优选使用K-SFM。
用于获得经培养的间充质干细胞的基础培养基可补充本领域已知的添加剂，所述添加剂促进处于未分化状态的间充质干细胞的增殖同时抑制其分化。并且，培养基可包含等渗溶液中的中性缓冲液(如磷酸盐和/或高浓度碳酸氢盐)，和蛋白质营养物(例如，血清如FBS、FCS(胎牛血清，fetal calf serum)或马血清、血清替代物、白蛋白或者必需或非必需氨基酸如谷氨酰胺或L-谷氨酰胺)。而且，其可以包含脂类(脂肪酸、胆固醇、血清的HDL或LDL提取物)和存在于大多数这类储存介质中的其他成分(如胰岛素或转铁蛋白、核苷或核苷酸、丙酮酸盐、诸如葡萄糖的糖源、呈任何离子化形式或盐的硒、诸如皮质醇的糖皮质激素和/或诸如β-巯基乙醇的还原剂)。
并且，出于防止细胞彼此粘附、粘附于容器壁或形成太大的团簇的目的，培养基可有利地包含抗凝聚剂，如Invitrogen销售的抗凝聚剂(Cat#0010057AE)。
其中，可以有利地使用一种或多种以下额外的添加剂：
·干细胞因子(SCF，青灰因子)、二聚化c-kit的其他配体或抗体以及同一信号通路的其他激活剂
·针对其他酪氨酸激酶相关受体的配体，如针对血小板来源的生长因子(PDGF)的受体、巨噬细胞集落刺激因子、Flt-3配体和血管内皮细胞生长因子(VEGF)
·升高环状AMP水平的因子，如毛喉素(forskolin)
·诱导gp130的因子，如：LIF或制瘤素-M(Oncostatin-M)
·造血生长因子，如促血小板生成素(TPO)
·转化生长因子，如TGFβ1
·神经素营养因子(neurotrophin)，如CNTF
·抗生素，如庆大霉素、青霉素或链霉素。
本发明中使用的培养基除了基础培养基之外，还可包含至少两种选自下组的成分：N-乙酰基-L-半胱氨酸(NAC)、胰岛素或胰岛素样因子、皮质醇、碱性成纤维细胞生长因子(bFGF)和抗氧化剂。
本发明中使用的培养基可包含基础培养基和至少两种选自下组的成分：N-乙酰基-L-半胱氨酸(NAC)、抗坏血酸、胰岛素或胰岛素样因子、皮质醇、地塞米松、bFGF(碱性成纤维细胞生长因子)、硫酸乙酰肝素、2-巯基乙醇、EGF(表皮生长因子)和抗氧化剂。
具体而言，培养基可包含胰岛素样因子作为胰岛素替代物，所述胰岛素样因子通过增强葡萄糖代谢和蛋白质代谢而起到促进细胞生长的作用。特定而言，优选使用重组IGF-1(胰岛素样生长因子-1)。胰岛素样因子的优选含量是10-50ng/ml。如果胰岛素样因子的含量小于10ng/ml，将发生细胞凋亡，而如果含量高于50ng/ml，则将增加细胞毒性和培养基成本。
培养基可包含能够诱导各种类型的细胞体内增殖的碱性成纤维细胞生长因子(bFGF)。优选地，使用重组bFGF蛋白。bFGF优选的含量为1-100ng/ml。
本发明中可使用的抗氧化剂实例包括硒、抗坏血酸、维生素E、儿茶酚、番茄红素、β-胡萝卜素、辅酶Q-10、EPA(二十碳五烯酸)、DHA(二十碳六烯酸)等等。优选地，可以使用硒。在本发明的实施例中，使用硒作为抗氧化剂。培养基中的硒的含量优选为0.5-10ng/ml。如果硒的含量小于0.5ng/ml，则培养基将对氧毒性敏感，而如果含量高于10ng/ml，则将造成严重的细胞毒性。
本发明中使用的培养基可另外包含选自FBS(胎牛血清)、钙和EGF的成分。表皮生长因子(EGF)能够诱导体内各种类型的细胞增殖，且优选使用重组EGF蛋白。表皮生长因子的优选含量为10-50ng/ml。如果培养基中表皮生长因子的含量少于10ng/ml，将不具有特定的效果，而如果含量高于50ng/ml，则将对细胞有毒性。
优选地，当传代培养3-5次时，在根据本发明的培养基中培养的干细胞以1×107-5×108个细胞/ml的浓度增殖。此外，由于在通过传代培养的干细胞的数量增加期间不发生细胞的功能/形态畸变，故根据本发明获得的干细胞能够有效地应用于临床试验。
在本发明的实施例中，在本发明的培养基中培养脂肪来源的间充质干细胞。能够以以下方式获得脂肪来源的间充质干细胞。首先，通过脂肪抽吸或类似方法从腹部获得人脂肪组织，并用PBS洗涤，之后将组织细切并使用包含胶原蛋白酶的DMEM培养基降解。将经降解的组织用PBS洗涤并以1000rpm离心5分钟。去除上清液，并用PBS洗涤留在底部的沉淀，然后以1000rpm离心5分钟。将得到的细胞通过100目的过滤器过滤以去除碎片，然后用PBS洗涤。将细胞在DMEM培养基(10％FBS、2mM NAC、0.2mM抗坏血酸)中培养过夜，然后用PBS将没有附着到培养容器底部的细胞洗涤出去，将细胞进行传代培养，同时以2天为间隔将培养基用含有NAC、抗坏血酸、钙、rEGF、胰岛素、Bfgf、皮质醇和硒的K-SFM培养基替换，从而获得脂肪来源的间充质干细胞。除了该方法，也可以使用本领域已知的方法来获得间充质干细胞。
下面，将参考实施例进一步详细描述本发明。对本领域普通技术人员而言显然这些实施例仅是说明目的而不构成对本发明范围的限制。
实施例1：人脂肪组织来源的间充质干细胞的分离
用PBS洗涤由脂肪抽吸从腹部组织分离的脂肪组织并细切，之后于37℃下在补充有1型胶原蛋白酶(1mg/ml)的DMEM培养基中消化所述组织2小时。用PBS洗涤经胶原蛋白酶处理的组织并以1000rpm离心5分钟。去除上清液，并用PBS洗涤沉淀，然后以1000rpm离心5分钟。将得到的细胞通过100目的过滤器过滤以去除碎片，之后用PBS洗涤细胞，并在包含10％FBS、2mM NAC(N-乙酰基-L-半胱氨酸)和0.2mM抗坏血酸的DMEM培养基中培养过夜。
然后，用PBS洗涤去除未附着的细胞，用包含5％FBS、2mM NAC、0.2mM抗坏血酸、0.09mM钙、5ng/ml rEGF、5μg/ml胰岛素、10ng bFGF和74ng/ml皮质醇的K-SFM(角质细胞无血清培养基)以2天为间隔替换培养基同时将留下的细胞培养4代，从而分离脂肪来源的间充质干细胞。
实施例2：培养基成分对干细胞增殖能力的效果研究
制备包含全部如下成分的培养基(即，培养基1)：FBS、N-乙酰基-L-半胱氨酸(NAC)、抗坏血酸、胰岛素、皮质醇、bFGF(碱性成纤维细胞生长因子)、EGF(表皮生长因子)和硒，上述成分是添加到实施例1中使用的K-SFM培养基中的活性成分，并制备不含至少一种上述活性成分的培养基(即，培养基2-9)。培养基组成如下：
培养基1(M1)：K-SFM培养基+FBS+NAC+抗坏血酸+胰岛素+皮质醇+bFGF+EGF+硒
培养基2(M2)：K-SFM培养基+NAC+抗坏血酸+胰岛素+皮质醇+bFGF+EGF+硒(从培养基1的成分中排除FBS)
培养基3(M3)：K-SFM培养基+FBS+NAC+抗坏血酸+胰岛素+皮质醇+EGF+硒(从培养基1的成分中排除bFGF)
培养基4(M4)：K-SFM培养基+FBS+NAC+抗坏血酸+皮质醇+bFGF+EGF+硒(从培养基1的成分中排除胰岛素)
培养基5(M5)：K-SFM培养基+FBS+NAC+抗坏血酸+胰岛素+bFGF+EGF+硒(从培养基1的成分中排除皮质醇)
培养基6(M6)：K-SFM培养基+FBS+NAC+抗坏血酸+胰岛素+皮质醇+bFGF+硒(从培养基1的成分中排除EGF)
培养基7(M7)：K-SFM培养基+FBS+NAC+胰岛素+皮质醇+bFGF+EGF+硒(从培养基1的成分中排除抗坏血酸)
培养基8(M8)：K-SFM培养基+FBS+抗坏血酸+胰岛素+皮质醇+bFGF+EGF+硒(从培养基1的成分中排除NAC)
培养基9(M9)：K-SFM培养基+FBS+NAC+抗坏血酸+胰岛素+皮质醇+bFGF+EGF+(从培养基1的成分中排除硒)
培养基10(M10)：K-SFM培养基+FBS+NAC+抗坏血酸+胰岛素+皮质醇+硒(从培养基1的成分中排除bFGF和EGF)。
在培养基中培养脂肪来源的干细胞。使用自20、30、70和80岁成年男性获得的脂肪来源的干细胞作为脂肪干细胞。在每种培养基中培养传代4次后，将获得的脂肪来源的间充质干细胞用胰蛋白酶处理，然后用共焦显微镜测量细胞数量。从下表1可见，列出了当将1×105个脂肪来源的间充质干细胞以3次传代接种到培养基中并培育4天时，基于培养基和天数获得的细胞数量。结果，根据本发明的干细胞培养方法，能够证实当培养干细胞4天时，依赖于培养基的成分能够以2.5×106个细胞/ml的最大浓度制备干细胞(见表1)。此外，尽管细胞群体倍增水平(CPDL)根据年龄稍有差异，但培养基9表现出最高的细胞群体倍增水平(CPDL)。在20岁和30年龄的成年男性中，能够证实当将干细胞以1×105个细胞/ml的浓度接种到培养基中并接种4天时，细胞数量增加13-25倍。在70岁和80年龄的成年男性的情况下，同样能够证实细胞数量增加7-15倍(见图1)。此外，未证实发生突变的细胞(未显示)。
表1
[表1]
用于高浓度干细胞制备的培养基组成的研究
工业实用性
根据本发明，由于即使培养3-5次传代也能够以足够临床应用的量来制备干细胞，所以能够显著增加血管内给予干细胞对细胞治疗的效果。
尽管本发明已经参考具体特征详细地进行了描述，但对本领域技术人员显然的是，该描述仅仅是优选的实施方式，而不限制本发明的范围。因此，本发明的实质范围将由所附权利要求书及其等同方式所限定。
Claims (6)
1.一种以1×107-5×108个细胞/ml的高浓度制备干细胞的方法，所述方法包括在培养基中培养干细胞，所述培养基包含基础培养基；和至少两种选自下组的成分：N-乙酰基-L-半胱氨酸(NAC)、抗坏血酸、胰岛素或胰岛素样因子、皮质醇、地塞米松、bFGF(碱性成纤维细胞生长因子)、硫酸乙酰肝素、2-巯基乙醇、EGF(表皮生长因子)和抗氧化剂。
2.权利要求1所述的方法，其中所述基础培养基选自下组：M199/F12(混合物)(GIBCO)、MEM-α培养基(GIBCO)、含低浓度葡萄糖的DMEM培养基(Welgene)、MCDB 131培养基(Welgene)、IMEM培养基(GIBCO)、K-SFM、DMEM/F12培养基、PCM培养基和MSC扩增培养基(Chemicon)。
3.权利要求1所述的方法，其中所述抗氧化剂选自下组：硒、抗坏血酸、维生素E、儿茶酚、番茄红素、β-胡萝卜素、辅酶Q-10、EPA(二十碳五烯酸)和DHA(二十碳六烯酸)。
4.权利要求3所述的方法，其中所述抗氧化剂是硒。
5.权利要求1所述的方法，其中所述培养基另外包含选自FBS(胎牛血清)、钙和EGF的成分。
6.权利要求1所述的方法，其中所述干细胞是脂肪组织来源的间充质干细胞。
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- 2013-05-23 US US14/405,320 patent/US20150118748A1/en not_active Abandoned
- 2013-05-23 WO PCT/KR2013/004517 patent/WO2014003319A1/ko active Application Filing
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Also Published As
| Publication number | Publication date |
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| EP2865749A4 (en) | 2015-12-30 |
| KR20140000945A (ko) | 2014-01-06 |
| JP2015526067A (ja) | 2015-09-10 |
| US20150118748A1 (en) | 2015-04-30 |
| BR112014031874A2 (pt) | 2017-08-01 |
| MX2014014337A (es) | 2015-02-20 |
| HK1206781A1 (zh) | 2016-01-15 |
| EP2865749A1 (en) | 2015-04-29 |
| WO2014003319A1 (ko) | 2014-01-03 |
| KR101523040B1 (ko) | 2015-05-26 |
| AU2013281596A1 (en) | 2014-12-04 |
| ZA201408915B (en) | 2015-11-25 |
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