CN111343997A - 组织再生用药物和其制造方法 - Google Patents
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Abstract
本发明涉及包含间充质干细胞的细胞药物和其制造方法。更详细而言,涉及包含间充质干细胞的细胞药物,其中,a)前述间充质干细胞通过炎性细胞因子的刺激而表达CX3CL1;和/或b)前述间充质干细胞的90%以上表达EGFR和/或ITGA4;以及包含间充质干细胞的细胞药物的制造方法,其包括:a)向包含间充质干细胞的培养物中添加炎性细胞因子,确认间充质干细胞表达CX3CL1的步骤;和/或b)确认间充质干细胞的90%以上表达EGFR和/或ITGA4的步骤。
Description
技术领域
相关申请
本说明书包括作为本申请的优先权的基础的日本特愿2017-216356号(2017年11月9日提交)的说明书中记载的内容。
技术领域
本发明涉及包含间充质干细胞的细胞药物和其制造方法。更详细而言,涉及在损伤部位的蓄积、免疫调节作用、神经保护作用优异、适合于组织再生药物的包含间充质干细胞的细胞药物和其制造方法。
背景技术
间充质干细胞(Mesenchymal Stem Cell:MSC)中,已知存在脑(实质和血管)的保护作用。脑梗塞后的MSC给药减少梗塞体积、改善行动功能使用实验性梗塞模型得到确认(非专利文献1~3、专利文献1)。还大量实施通过MSC的静脉给药来治疗脑梗塞患者,报告了运动功能、损伤部位的改善(非专利文献4、专利文献2)。此外,针对脊髄损伤患者,也通过MSC的静脉给药,确认了功能恢复、和促进轴索再生、减少损伤部位。
针对MSC的治疗机理,推测有多种作用机制,它们被分类为利用神经营养因子的神经营养和保护作用、血管新生作用(脑血流的恢复)、神经再生这3种。预测神经营养和保护作用经由作为神经营养因子的BDNF(Brain Derived Neurotrophic Factor,脑源性神经营养因子)、GDNF(Glial Derived Neurotrophic Factor,胶细胞源性神经营养因子)等体液因子而得以发挥。
针对对脊髄损伤的神经保护作用,报告了BDNF、NT-3、NGF、PDGF、GDNF等大量神经营养因子、生长因子的参与(非专利文献5),Honmou等人确认了体内(in vivo)的BDNF的神经保护作用(非专利文献3和6)。此外,通过MSC的静脉给药,确认锥体束和锥体外束的轴索再生和出芽(Sprouting)、大脑皮质的皮质脊束神经元的保护,但已知如果将操作基因以使得强制表达BDNF的MSC进行静脉内给药,则这些效果进一步增强(非专利文献7)。
血管新生作用中,可以认为有2种机理,一个为在病源部位蓄积的MSC分泌血管新生因子等而诱导血管新生,另一个为所给药的MSC自身分化为血管内皮而形成新的血管。神经再生作用也可以认为有2种机理,一个为在病源部位蓄积的MSC促进内因性的神经形成,另一个为所给药的MSC自身分化为神经细胞和胶质细胞。
针对MSC的免疫调节作用,报告了通过MSC所分泌的TSG-6、TGF-β1、CX3CL1而调节小胶质细胞,该小胶质细胞从分泌TNF-α、IL-1β、IL-6等炎性细胞因子的细胞毒性的M1型变化为具有细胞保护作用的M2型。(非专利文献8~11)。此外,报告了通过M2型小胶质细胞所分泌的IL-4、IL-13、BDNF、IGF等,抑制了神经细胞和胶质细胞中的炎症,其结果是,抑制了伴随坏死、凋亡的神经继发性损伤(非专利文献8~11)。进一步,报告了所移植的MSC在脊髄的损伤部位处增加IL-4、IL-13的表达,另一方面减少TNF-α、IL-6,由此从具有炎症作用的M1型诱导为具有抗炎症作用的M2型的巨噬细胞,促进了脊髄损伤后的轴索再生、功能恢复(非专利文献12)。
现有技术文献
专利文献
专利文献1:WO2002/000849号
专利文献2:WO2009/002503号
非专利文献
非专利文献1:Iihoshi S.等人,Brain Res.2004;1007:1-9.
非专利文献2:Nomura T.等人,Neuroscience.2005;136:161-169.
非专利文献3:Honma T.等人,Exp.Neurol.2006;199:56-66.
非专利文献4:Honmou O.等人,Brain.2011;134:1790-1807.
非专利文献5:Hervey等人,2015,Brain Resarch 1619:36-71
非专利文献6:Osaka等人,2010,Brain Research 1343:226-235
非专利文献7:Sasaki等人,2009,Journal of Neuroscience 29(47):14932-14941
非专利文献8:Giunti等人,2012,Stem Cells 30,2044-53,
非专利文献9:Yoo等人,2013,Neurobiology of Disease 58,249-257,
非专利文献10:Liu等人,2014,Journal of Neuroinflammation 11,135,
非专利文献11:Noh等人,2016,Stem Cells Translatoinal Medicine 5,1-12
非专利文献12:Nakajima等人,2012,Journal of Neurotrauma 29,1614-25
发明内容
发明要解决的课题
本发明的课题在于,提供在损伤部位的蓄积、免疫调节作用(炎症调节作用)、神经保护作用等治疗效果优异的包含MSC的细胞药物和其制造方法。
解决课题的手段
发明人等为了解决上述课题,针对用于评价MSC的功能的指标进行研究。并且,制备为细胞药物用途的MSC被发现通过细胞因子刺激而表达CX3CL1,EGFR和/或ITGA4的表达为90%以上,能够以这些为指标来评价MSC的免疫调节能力(炎症调节能力)、在损伤部位的蓄积能力。
本发明基于上述见解而完成,包括以下的(1)~(11)。
(1)包含间充质干细胞的细胞药物的制造方法,其包括:
a)向包含间充质干细胞的培养物中添加炎性细胞因子,确认间充质干细胞表达CX3CL1的步骤;和/或
b)确认间充质干细胞的90%以上表达EGFR和/或ITGA4的步骤。
(2)根据(1)所述的方法,其进一步包括:确认包含间充质干细胞的培养物中的选自BDNF、VEGF、和HGF中任一种以上的分泌能力的步骤;前述方法优选包括确认BDNF和/或VEGF的分泌能力的步骤,更优选包括确认BDNF的分泌能力的步骤。应予说明,BDNF、VEGF、HGF的分泌可以观察来自未刺激的细胞的分泌,也可以观察来自炎性细胞因子刺激后的细胞的分泌。
(3)根据(1)或(2)所述的方法,其中,炎性细胞因子为选自TNF-α、INFγ、IL-1、IL-6、IL-8、IL-12、和IL-18中的1种以上。
(4)根据(1)或(2)所述的方法,其中,炎性细胞因子包含TNF-α、INFγ、和IL-6;炎性细胞因子优选为TNF-α、INFγ、和IL-6的混合物。
(5)包含间充质干细胞的细胞药物,其中
a)前述间充质干细胞通过炎性细胞因子的刺激而表达CX3CL1;和/或
b)前述间充质干细胞的90%以上表达EGFR和/或ITGA4。
(6)根据(5)所述的细胞药物,其中,前述间充质干细胞具有选自BDNF、VEGF、和HGF中任一种以上的分泌能力。前述间充质干细胞优选具有BDNF和/或VEGF的分泌能力,更优选具有BDNF的分泌能力。
(7)根据(5)或(6)所述的细胞药物,其中,炎性细胞因子为选自TNF-α、INFγ、IL-1、IL-6、IL-8、IL-12、和IL-18中的1种以上。
(8)根据(5)或(6)所述的细胞药物,其中,炎性细胞因子包含TNF-α、INFγ、和IL-6。
(9)根据(5)或(6)所述的细胞药物,其中,因TNF-α、INFγ、和IL-6的混合物的刺激而导致的CX3CL1表达量比因TNF-α、INFγ、和IL-6各自单独的刺激而导致的CX3CL1表达量的总和更多。
(10)评价包含间充质干细胞的细胞药物的免疫调节能力的方法,其包括:用炎性细胞因子刺激间充质干细胞,测定CX3CL1的表达的步骤。所使用的“炎性细胞因子”优选为选自TNF-α、INFγ、IL-1、IL-6、IL-8、IL-12、和IL-18中的1种以上,更优选包含TNF-α、INFγ、和IL-6,进一步优选为TNF-α、INFγ、和IL-6的混合物。
(11)评价包含间充质干细胞的细胞药物在损伤部位的蓄积能力的方法,其包括:评价前述间充质干细胞中的EGFR和/或ITGA4的表达是否为90%以上的步骤。
发明的效果
根据本发明,能够简便地评价MSC的免疫调节作用(炎症调节作用)、在损伤部位的蓄积、神经保护作用,能够提供功能高的包含MSC的细胞药物。
附图说明
图1示出通过实时RT-PCR法测定MSC样品(KN-011、KY-14、KA-17,3个批次)中的(A)TSG-6、(B)CX3CL1、和(C)TGF-β1的基因表达(相对表达比)的结果。图从左起为无添加(对照)、添加TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)。
图2示出通过ELISA测定MSC样品(KN-011、KY-14、KA-17,3个批次)中的(A)TSG-6、(B)CX3CL1、和(C)TGF-β1的表达量(pg/1.0x104个细胞)的结果。图从左起为无添加(初始)、添加TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)。
图3-1示出通过实时RT-PCR法测定MSC样品(KN-011、KY-14、KA-17,3个批次)中的(A)VEGF、(B)HGF、(C)NGF、(D)GDNF的基因表达(相对表达比)的结果。图从左起为无添加(对照)、添加TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)。
图3-2示出通过实时RT-PCR法测定MSC样品(KN-011、KY-14、KA-17,3个批次)中的(E)PDGF-A、(F)PDGF-B、(G)PIGF、和(H)BDNF的基因表达(相对表达比)的结果。图从左起为无添加(对照)、添加TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)。
图4-1示出通过ELISA测定MSC样品(KN-011、KY-14、KA-17,3个批次)中的(A)proBDNF、(B)maturePDNF、(C)NGF、和(D)GDNF的表达量(pg/1.0x104个细胞)的结果。图从左起为无添加(初始)、添加TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)。
图4-2示出通过ELISA测定MSC样品(KN-011、KY-14、KA-17,3个批次)中的(E)VEGF、(F)PIGF、(G)HGF、和(H)PDGF-AB的表达量(pg/1.0x104个细胞)的结果。图从左起为无添加(初始)、添加TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)。
图5-1示出MSC样品(KN-011、KY-14、KA-17,3个批次)中的趋化因子受体(CCR1、CCR2、CCR3、CCR4、CCR5、CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CX3CR1)、生长因子受体(PDGFRa、PDGFRb、FGF-R2、EGFR、HGFR、NGFR、IGF1R、VEGFR1、VEGFR2、Tie-2)的利用流式细胞术的表达分析结果。
图5-2示出MSC样品(KN-011、KY-14、KA-17,3个批次)中的粘附因子(NCAD、HCAM(CD44)、NCAM、ALCAM、ITGAV、ITGA4、ITGB1、ITGB4、VCAM1、ICAM2)的利用流式细胞术的表达分析结果。
图6示出因趋化因子、生长因子的刺激而导致的MSC样品(KN-011、KY-14、KA-17,3个批次)的迁移测定的结果(相对于未刺激培养的相对表达比,平均值±SD,*1.5倍改变v.s初始)。
图7-1示出通过实时RT-PCR法测定MSC样品(KN-011、KY-14、KA-17,3个批次)中的粘附因子((A)ITGB1、(B)ITGA4)和浸润相关蛋白质((C)MMP1)的基因表达(相对表达比)的结果。图从左起为无添加(对照)、添加TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)。
图7-2示出通过实时RT-PCR法测定MSC样品(KN-011、KY-14、KA-17,3个批次)中的浸润相关蛋白质((D)MMP2、(E)TIMP1、(F)TIMP2)的基因表达(相对表达比)的结果。图从左起为无添加(对照)、添加TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)。
具体实施方式
1.包含间充质干细胞的细胞药物
本发明的细胞药物包含间充质干细胞,其特征在于,a)前述间充质干细胞通过细胞因子的刺激而表达CX3CL1;和/或b)前述间充质干细胞的90%以上表达EGFR和/或ITGA4。
[间充质干细胞]
本发明中使用的“间充质干细胞”是指在间充质组织的间质细胞中微量存在的具有多向分化能力和自我复制能力的干细胞,已知不仅具有分化为骨细胞、软骨细胞、脂肪细胞等结缔组织细胞的分化能力,还具有分化为神经细胞、心肌细胞的分化能力。
针对MSC的治疗机理,推测和提出了多种作用机制。例如,已知MSC通过在损伤部位蓄积,能够实现有效的组织再生。此外,MSC被报告具有细胞死亡的抑制作用、炎症调节作用,经由神经营养因子的分泌而具有神经保护作用(前述)。
[CX3CL1的表达]
本发明中使用的MSC的特征在于,通过炎性细胞因子的刺激而表达CX3CL1。
CX3CL1在CXXXC基序的趋化因子中也被称为不规则趋化因子(fractalkine),在活化血管内皮细胞、神经细胞、树突状细胞和肠上皮细胞中表达,通过炎症系细胞因子的刺激而诱导表达。趋化因子对中性粒细胞、单核细胞、淋巴细胞等白细胞具有趋化活性,被称为在炎症反应中发挥重要功能的一组分子量10kDa前后的碱性生理活性肽。趋化因子根据结构上的特征被分类为CXC、CC、C、CX3C的4个亚家族,对这些趋化因子亚家族,确定被分类为CXCR、CCR、XCR、CX3CR的7次跨膜三聚体G蛋白偶联受体(GPCR)家族。在生物体内,CX3CL1采取膜结合型和分泌型的2种形态,不仅作为趋化因子,还作为不依赖整合素而示出细胞粘附能力的细胞粘附分子而发挥功能。CX3CL1的表达已知参与类风湿性关节炎、动脉硬化等各种各样的疾病。
针对细胞死亡的抑制效果、炎症调节作用,已知利用在损伤部位的MSC的小胶质细胞和巨噬细胞的Modulation(调节)作用相关,发明人等通过实时RT-PCR和ELISA分析,发现对炎性细胞因子刺激应答的CX3CL1的特征性表达作为MSC的炎症调节作用(免疫调节作用)的指标是有用的。表达CX3CL1的MSC调节小胶质细胞/巨噬细胞,从具有炎症作用的M1型诱导为抗炎症性的M2型,由此期待发挥免疫调节(炎症调节)作用。
作为所使用的“炎性细胞因子”,可以举出例如IL-1、IL-6、IL-8、IL-12、IL-18等白细胞介素类、TNF-α、IFN-γ。其中,优选为IL-6、TNF-α、IFN-γ,更优选使用IL-6、TNF-α、和IFN-γ的混合物。
如果根据炎性细胞因子的刺激,MSC表达CX3CL1,则该MSC可以期待炎症调节作用(免疫调节作用)优异。
特别地,在使用TNF-α、INFγ、和IL-6进行刺激的情况下,可以将因TNF-α、INFγ、和IL-6的混合物的刺激而导致的CX3CL1表达量比因TNF-α、INFγ、和IL-6各自单独的刺激而导致的CX3CL1表达量的总和更多设为功能性(炎症调节作用优异)MSC的特征性指标。
[EGFR和/或ITGA4的表达]
本发明中使用的MSC的特征在于,90%以上表达EGFR(Epidermal Growth FactorReceptor,表皮生长因子受体)和/或ITGA4(Integrin subunit Alpha4,整合素亚基α4)。
EGFR是酪氨酸激酶型受体的1种,作为配体,除了与表皮生长因子(EGF)之外,还与TGF-α、双调节蛋白等结合。以EGFR为首的受体型酪氨酸激酶将因细胞外的生长因子而导致的刺激传递给细胞内,将该刺激通过信号转导而传递给核。其结果是,核内的转录活性提高,改变蛋白质合成、细胞的功能、结构。EGFR已知在体内的各种各样的细胞的增殖、器官的发育和形成中示出重要功能。
整合素(integrin)是细胞表面蛋白质的1种,是主要参与对细胞外基质的细胞粘附、来自细胞外基质的信息转导的细胞粘附分子。整合素分子是α链与β链以1:1缔合的异源二聚体,α链中被报告有至少18种,ITGA4是其中一种。ITGB1、ITGA4被报告对于对血管内皮的粘附是重要的,与迁移的细胞在损伤部位的蓄积相关(James等人,2007;Brigitte等人,2006))。
在损伤部位的蓄积涉及MSC的迁移能力。发明人等针对与迁移性相关的趋化因子和生长因子,发现进行利用流式细胞术的受体分析和迁移测定,EGFR和/或ITGA4的表达作为MSC的迁移能力、在损伤部位的蓄积能力的指标是有用的。
在外伤患者中的组织的损伤部位,已知EGF、NGF等生长因子等分泌增加。此外,在创伤治愈过程中由血液中的血小板确认EGF、bFGF、IL-6、IL-8的释放(Ono等人,1995,Burns21,352-355;Zhuang等人,2013,Asian Pacific Journal of Tropical Medicine,383-386;Werner等人,2003,Physiol Rev 83,835-870)。然而,针对MSC中的EGFR、ITGA4的表达与在损伤部位的蓄积之间的关系,尚无报告。
如果MSC的EGFR和/或ITGA4的表达为90%以上,则该MSC可以期待在损伤部位的蓄积能力优异。
[BDNF的表达]
本发明中使用的MSC除了CX3CL1的表达、EGFR和/或ITGA4的表达之外,还优选分泌选自BDNF、VEGF、和HGF中的1种以上的营养因子。其中,重要的是分泌BDNF和/或VEGF,特别重要的是分泌BDNF。
BDNF(Brain-derived Neurotrophic Factor,脑源性神经营养因子)是与靶细胞表面上的特异性受体TrkB结合,调节神经细胞的生长的体液蛋白。BDNF作用于中枢神经系统、外周神经系统的部分神经元,促进其维持和生长,促进向新的神经元、突触的分化。在脑中,已知BDNF在海马、大脑皮质、大脑基底核被活化,对长期记忆是重要的,但对视网膜、运动神经元、肾脏、唾液腺、前列腺也发挥作用。
VEGF(Vascular Endothelial Growth Factor,血管内皮生长因子)是从垂体细胞的培养液中分离得到的对血管内皮细胞发挥特异性作用的增殖因子。VEGF具有以血管内皮细胞的增殖为首的血管新生过程的促进、血管透过性的亢进作用,因此被推定与血管新生发挥重要功能的各种疾病和症状(癌、糖尿病性视网膜症、类风湿性关节炎、创伤治愈过程)相关。
HGF(Hepatocyte Growth Factor,肝细胞生长因子)是作为强烈促进原代培养肝细胞的增殖的因子而被提纯出的细胞因子,是促进肝再生的重要因子。HGF经由靶细胞中表达的c-Met受体而发挥生物活性,不仅对肝细胞,而且对各种各样的细胞促进细胞增殖促进、细胞运动促进、抗凋亡(细胞死亡)、形态形成诱导、血管新生。
神经保护作用中,可以认为有MSC所分泌的营养因子等的参与。发明人等通过实时RP-PCR和ELISA,研究MSC所分泌的神经营养因子的表达,确认了BDNF、VEGF、HGF的表达、其中BDNF和/或VEGF的表达、特别是BDNF的表达作为MSC的神经保护作用的指标是有用的。
如果MSC具有BDNF、VEGF和/或HGF分泌能力,则该MSC可以期待具有损伤区域的修复和再生能力、神经保护作用优异。MSC即使未刺激也分泌BDNF、VEGF和/或HGF,但分泌能力的确认可以评价来自未刺激的细胞的分泌,也可以评价来自炎性细胞因子刺激后的细胞的分泌。
如后述实施例所示那样,本发明中使用的MSC作为与炎症调节作用(免疫调节作用)相关的因子,除了CX3CL1之外还表达TGF-β1。此外,作为与迁移能力相关的因子,除了EGFR和/或ITGA4之外,还确认了NCAM、ALCAM、ITGAV、ITGB1的表达。
[表达分析]
本发明中,上述CX3CL1、EGFR、ITGA4、BDNF、VEGF、HGF的表达可以通过本领域中公知的方法而容易地测定。例如,基因水平下的表达分析可以利用实时PCR(实时RT-PCR)、微阵列、Northern印迹法等。此外,蛋白质水平下的表达分析可以利用ELISA、流式细胞术(FCM)、蛋白质芯片等。
特别地,蛋白质水平下的表达在EGFR、ITGA4等细胞表面蛋白质的情况下,从简便性和敏感性的观点出发,优选使用流式细胞术(FCM),在CX3CL1、BDNF、VEGF、HGF等分泌蛋白质的情况下,从简便性和敏感性的观点出发,优选使用微珠测定法。
[MSC的制备]
作为本发明中使用的MSC的来源,有骨髄、外周血、脐带血、胎儿胚胎、脑等,本发明中,优选为人骨髄或血液来源的MSC(骨髄间充质干细胞),特别优选为人骨髄MSC。
细胞可以为由ES细胞、人工多能性干细胞(iPS细胞等)分化诱导得到的细胞,也可以为确立细胞系的细胞,也可以为从生物体分离并增殖得到的细胞。细胞可以为异体细胞来源或自体细胞来源,优选为自体细胞来源(来源于患者自身的细胞)的MSC。
本发明中使用的MSC优选选自CD73、CD90、和CD105中的至少1种以上的表达为90%以上,和/或CD34或CD45的表达为5%以下。更优选地,本发明中使用的MSC的特征在于,选自CD73、CD90、和CD105中的至少2种以上的表达为90%以上,和/或CD34和CD45的表达为5%以下。进一步优选地,本发明中使用的MSC的特征在于,CD73、CD90、和CD105的表达为90%以上,且CD34或CD45的表达为5%以下。
此外,本发明中使用的MSC优选为作为分化标志物的CD24阴性、且维持未分化状态的细胞。维持未分化状态的MSC具有增殖率和生物体内导入后的生存率高的特征。像这样未分化的MSC的取得方法也被开发出来,其详情记载于WO2009/034708号。
适合于本发明的细胞药物的功能性MSC可以通过例如下述方式制备:将从骨髄液等中在不与抗凝固剂(肝素等)发生实质性接触的条件下分离得到的细胞,使用包含人血清(优选为自体血清)、且不含或者以极低浓度包含抗凝固剂(肝素等)的培养基进行增殖,从而制备。应予说明,“不含或者以极低浓度包含抗凝固剂”是指作为抗凝固剂不含有效量的抗凝固剂。具体而言,如果为例如肝素、其衍生物,则通常作为抗凝固剂的有效量为约20-40U/mL左右,但在前述方法中,通过预先将用于试样采集的采血管中添加的量设为最小限度,从生物体采集的试样中的量低于5U/mL、优选低于2U/mL、进一步优选低于0.2U/mL,在培养细胞时在培养基中存在的量相对于培养基的容积低于0.5U/mL、优选低于0.2U/mL、进一步优选低于0.02U/mL(参照WO2009/034708号)。
培养基中的细胞的密度对细胞的性质和分化的方向性造成影响。在MSC的情况下,如果培养基中的细胞密度大于8,500个/cm2,则细胞的性质发生变化,因此优选最大在8,500个/cm2以下的条件下进行传代培养,更优选为在达到5,500个/cm2以上的时点进行传代培养。
前述方法中,由于使用含人血清的培养基,因此考虑到血清供体的负担,期望培养基交换尽可能次数少,例如进行至少每周1次、更优选为每周1~2次的培养基交换。
培养反复进行传代培养直至细胞的总数达到108个以上。所需的细胞数可以根据使用目的而变化,例如用于脑梗塞的治疗的移植所需的MSC的数量可以认为为107个以上。根据前述方法,可以以12天左右得到107个MSC。
增殖的MSC根据需要,可以至使用为止通过冷冻保存等手段(例如在-152℃的深冷库中)保存。冷冻保存中,使用包含血清(优选为人血清、更优选为自体血清)、葡聚糖、DMSO的培养基(RPMI等哺乳动物细胞用的培养基)作为冷冻保存液。例如,可以在包含进行了通常的过滤灭菌的RPMI20.5mL、和从患者采集的自体血清20.5mL、葡聚糖5mL、DMSO 5mL的冷冻保存液中悬浮细胞,在-150℃下进行冷冻保存。例如,作为DMSO,可以使用ニプロ株式会社制的クライオザーブ,葡聚糖使用大塚制药制的低分子葡聚糖L注射液,但不限于此。
[细胞药物(细胞制剂)]
本发明的细胞药物中包含的MSC的细胞数越多越优选,但考虑到对对象的给药时期、培养所需要的时间,实用的是示出效果的最小量。因此,本发明的优选的方式中,MSC的细胞数为107个以上、优选为5x107个以上、更优选为108个以上、进一步优选为5x108个以上。给药次数不限于1次,可以给药2次以上。
本发明的细胞药物优选为肠胃外给药制剂,更优选为肠胃外全身给药制剂,特别是静脉内给药制剂。作为适合于肠胃外给药的剂型,可以举出溶液性注射剂、混悬性注射剂、乳液性注射剂、即用制备型注射剂等注射剂、植入片等。肠胃外给药用制剂是水性或非水性的等渗性无菌溶液或悬浮液的形态,例如适当组合药理学上可接受的载体或介质,具体而言灭菌水、生理食盐水、培养基(特别地,RPMI等哺乳动物细胞的培养中使用的培养基)、PBS等生理缓冲液、植物油、乳化剂、悬浮剂、表面活性剂、稳定剂、赋形剂、溶媒、防腐剂、结合剂等,制剂化为适当的单位给药形态。
作为注射用的水溶液,可以举出例如生理食盐水、培养基、PBS等生理缓冲液、包含葡萄糖、其他助剂的等渗液、例如D-山梨糖醇、D-甘露糖、D-甘露醇、氯化钠等,可以与适当的溶解助剂、例如醇、具体而言为乙醇、多元醇、丙二醇、聚乙二醇、非离子性表面活性剂、例如聚山梨酯80、HCO-50等组合使用。
本发明的细胞药物通过组织再生用药物、特别是损伤部位(病变部分)中的突触形成和可塑性促进效果,对认知功能障碍、慢性期的脑梗塞、慢性期的脊髄损伤、神经改性疾病、精神疾病、高级功能障碍等治疗是有用的。
2.包含间充质干细胞的细胞药物的制造方法
本发明还提供包含间充质干细胞的细胞药物的制造方法。本发明的细胞药物的制造方法的特征在于,包括:a)向包含间充质干细胞的培养物中添加细胞因子,确认间充质干细胞表达CX3CL1的步骤;和/或b)确认间充质干细胞表达EGFR和/或ITGA4的步骤。
作为所使用的“炎性细胞因子”,可以举出TNF-α、INFγ、IL-1、IL-6、IL-8、IL-12、IL-18,其中优选包含TNF-α、INFγ、和IL-6,更优选使用TNF-α、INFγ、和IL-6的混合物。
发明的细胞药物的制造方法可以进一步包括:确认(细胞因子未添加)培养物中的选自BDNF、VEGF、和HGF中任一种以上的存在的步骤。特别地,重要的是确认BDNF和/或VEGF的存在,最重要的是确认BDNF的存在。
如前所述,如果通过添加炎性细胞因子而使MSC表达CX3CL1,则该MSC可以期待炎症调节作用(免疫调节作用)优异,如果MSC的90%以上表达EGFR和/或ITGA4,则该MSC可以期待在损伤部位的蓄积能力优异。此外,如果在培养基中存在BDNF、VEGF、和HGF等营养因子中任一者,则可以期待包含神经保护作用高的MSC,其中BDNF和/或VEGF的存在、特别是BDNF的存在可以是神经保护作用高的MSC的重要指标。MSC即使未刺激也分泌BDNF、VEGF和/或HGF,但分泌能力的确认可以评价来自未刺激的细胞的分泌,也可以评价来自炎性细胞因子刺激后的细胞的分泌。
上述CX3CL1、EGFR、ITGA4、BDNF、VEGF、HGF的表达与基因水平相比,优选将蛋白质水平下的表达设为指标,可以通过前项记载的方法测定。特别地,在EGFR、ITGA4等细胞表面蛋白质的情况下,从简便性和敏感性的观点出发,优选使用流式细胞术(FCM),在CX3CL1、BDNF、VEGF、HGF等分泌蛋白质的情况下,从简便性和敏感性的观点出发,优选使用微珠测定法。
本发明的细胞药物的制造方法中使用的MSC的制备如前项所述那样,按照WO2009/034708号的记载,通过下述方式制备:将从骨髄液等中在不与抗凝固剂(肝素等)发生实质性接触的条件下分离得到的细胞,使用包含人血清(优选为自体血清)、且不含或者以极低浓度包含抗凝固剂(肝素等)的培养基进行增殖,从而制备。应予说明,“不含或者以极低浓度包含抗凝固剂”是指作为抗凝固剂不含有效量的抗凝固剂。具体而言,如果为例如肝素、其衍生物,则通常作为抗凝固剂的有效量为约20-40U/mL左右,但在前述方法中,通过预先将用于试样采集的采血管中添加的量设为最小限度,从生物体采集的试样中的量低于5U/mL、优选低于2U/mL、进一步优选低于0.2U/mL,在培养细胞时在培养基中存在的量相对于培养基的容积低于0.5U/mL、优选低于0.2U/mL、进一步优选低于0.02U/mL。
3.评价包含间充质干细胞的细胞药物的免疫调节能力的方法
本发明还提供评价包含间充质干细胞的细胞药物的免疫调节能力的方法。前述评价方法包括:用炎性细胞因子刺激间充质干细胞,测定CX3CL1的表达的步骤。所使用的“炎性细胞因子”、CX3CL1的表达的测定方法如1和2中记载那样。
如果用细胞因子刺激后的MSC表达CX3CL1,则包含该MSC的细胞药物能够评价为免疫调节能力高。特别地,在使用TNF-α、INFγ、和IL-6进行刺激的情况下,因TNF-α、INFγ、和IL-6的混合物的刺激而导致的CX3CL1表达量比因TNF-α、INFγ、和IL-6各自单独的刺激而导致的CX3CL1表达量的总和更多的情况下,能够评价为免疫调节能力高。
4.评价包含间充质干细胞的细胞药物在损伤部位的蓄积能力的方法
还提供评价包含间充质干细胞的细胞药物在损伤部位的蓄积能力的方法。前述评价方法包括:确认间充质干细胞表达90%以上EGFR和/或ITGA4的步骤。
如果MSC的90%以上表达EGFR和/或ITGA4,则包含该MSC的细胞药物能够评价为在损伤部位的蓄积能力高。
实施例
以下,通过实施例具体说明本发明,但本发明不限于这些实施例。
实施例1:免疫调节作用
针对MSC的细胞死亡的抑制效果、免疫调节作用,已知利用在损伤部位的MSC的小胶质细胞和巨噬细胞的Modulation(调节)作用相关。因此,为了研究包含MSC的细胞药物中的免疫调节能力,作为相关的因子,通过实时RT-PCR法和ELISA法分析TSG-6、CX3CL1、TGF-β1的表达。
1.实验方法,评价项目
1.1细胞培养
MSC样品使用临床试验用(STRO1)的不同的3个批次的样品(KN-011,KY-14,KA-17)。将前述MSC样品悬浮在培养液14mL(10%人血清,1%青霉素-链霉素,1%L-谷氨酰胺)中,在150mm培养皿中以0.7-1.0×106个细胞/培养皿的密度接种。在温度37℃、5%CO2的条件下培养,确认80%左右的汇合后,进行传代作业,以5.0×105个细胞/培养皿的密度接种。接着,进行传代培养,在第4次传代中在100mm培养皿以3.0×105个细胞/培养皿的密度接种。以下的全部实验体系中,使用传代数4次的细胞。
1.2利用炎性细胞因子的刺激培养上清液回收和总RNA提取
进行第4次传代的24小时后,交换为添加通常的培养液(10%FBS,1%青霉素-链霉素,1%L-谷氨酰胺)、和炎性细胞因子(TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/ml)、TNF-α/IFN-γ/IL-6(各50ng/ml))的10mL的培养液(5个条件,n=3)。上述炎性细胞因子在脊髄损伤部位被分泌,可以认为引起各种各样的细胞毒性。交换48小时后,回收培养上清液,进行离心处理(2280g,20分钟)。其后,在1.5ml管中分别注入各200μl,在-80℃冰箱中保存。上清液回收后,用胰蛋白酶处理,从培养皿中剥离细胞,进行细胞计数。细胞计数后,使用RNA提取试剂盒(QIAGEN),提取总RNA。由总RNA合成cDNA,将它们作为模板,进行实时RT-PCR。
2.结果的评价和判定基准
2.1利用实时RT-PCR法的基因表达
由从细胞提取的总RNA,合成cDNA。将合成的cDNA作为模板,使用TSG-6、CX3CL1、TGF-β1的各因子的Taqman探针,进行PCR反应。由各Target和内部标准的Ct值,通过ΔΔCt法添加通常的培养液和炎性细胞因子,对培养的细胞的基因表达量进行比较定量。
试验样本记作无添加(初始)、TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)添加,从培养开始起48小时后,回收mRNA和培养上清液,进行实时RT-PCR。
2.2利用ELISA法的分泌蛋白质定量
将1.1中回收、保存的培养上清液作为样品,对培养上清液中的TSG-6、CX3CL1、TGF-β1进行定量。试验样本记作无添加(初始)、TNF-α(50ng/ml)、IFN-γ(50ng/ml)、IL-6(50ng/mL)和TNF-α/IFN-γ/IL-6(均为50ng/ml)添加,从培养开始起48小时后,回收mRNA和培养上清液,进行实时RT-PCR。
3.结果
3.1实时RT-PCR(图1)
图示出与对照的相对表达比,表示出Ct值。所有批次中,均确认TSG-6、CX3CL1、TGF-β1的基因表达,特别地TSG-6和CX3CL1的表达通过混合添加TNF-α/IFN-γ/IL-6而显著增加。根据本结果,暗示了MSC参与对小胶质细胞和巨噬细胞的Modulation(调节)作用,此外TSG-6、CX3CL1、TGF-β1有助于该作用。
3.2利用ELISA法的分泌蛋白质定量(图2)
TSG-6和TGF-β1在所有批次中均未确认到因细胞因子刺激而导致的表达量的变化,但CX3CL1在未刺激、用细胞因子单独的刺激中几乎未确认到表达,与此相对地,确认到通过混合添加TNF-α/IFN-γ/IL-6而显著表达。此外,RT-PCR中,也得到因TNF-α/IFN-γ/IL-6混合刺激而导致的表达比因各自单独刺激而导致的表达的总和更多的结果。
4.考察
CX3CL1在ELISA中几乎未确认到表达,但通过TNF-α/IFN-γ/IL-6的混合刺激而确认到表达。此外,因TNF-α/IFN-γ/IL-6混合刺激而导致的表达比因各自单独刺激而导致的表达的总和更多这一CX3CL1的表达特征在MSC所分泌的其他免疫调节能力相关因子(TSG-6、TGF-β1)中未被发现,因此,可以推测CX3CL1作为评价MSC的免疫调节能力的指标是有用的。
实施例2:神经保护作用
MSC的神经保护作用中,可以认为有多个营养因子等的参与。分析MSC所分泌的营养因子(VEGF、HGF、NGF、GDNF、PDGF-A、PDGF-A、PlGF、BDNF)的表达。
1.实验方法,评价项目
细胞培养和总RNA的制备通过在实施例1中记载的方法进行。
2.结果的评价和判定基准
与实施例1相同。
3.结果
3.1实时RT-PCR(图3)
图示出与对照的相对表达比,表示出Ct值。在未添加炎性细胞因子的培养液中培养的MSC中,能够确认作为神经营养因子的BDNF、NGF、GDNF、与血管新生相关的VEGF、PDGF-A、PlGF、与损伤组织的修复和再生相关的HGF的表达。TNF-α/IFN-γ/IL-6的混合刺激中,在NGF的表达中确认增加倾向。
3.2利用ELISA法的分泌蛋白质定量(图4)
在未添加炎性细胞因子的培养液中培养的MSC中,确认作为神经营养因子的mature-BDNF和作为其前体的proBDNF、与血管新生相关的VEGF的分泌。此外,HGF、PlGF在3样本中的2样本中得到确认。另一方面,NGF、GDNF、PDGF-AB无法确认分泌。TNF-α/IFN-γ/IL-6的混合刺激中,在VEGF和HGF的分泌量中确认增加倾向。
4.考察
mRNA水平中,虽然确认全部营养因子的表达,但在蛋白质水平下能够在全样品中确认分泌的仅为BDNF、VEGF。PlGF和HGF中,仅在3个批次中的2个批次中能够确认。
针对对脊髄损伤的神经保护作用,报告了BDNF、NT-3、NGF、PDGF、GDNF等大量神经营养因子、生长因子的参与,Honmou等人的体内分析中,确认利用BDNF的神经保护作用(前述Nomura等人,2005;Osaka等人,2010)。此外,已知如果将操作基因以使得强制表达BDNF的BDNF-MSC进行静脉内给药,则这些效果进一步增强(前述Sasaki等人,2009)。
本次结果也与上述报告一致,可以认为BDNF的分泌作为MSC的神经保护作用的评价指标是特别重要的。针对VEGF、HGF,除了BDNF之外,还认为对MSC的功能评价也是有用的。
实施例3:MSC的迁移能力
为了评价MSC在损伤部位的蓄积,通过FCM法、迁移测定和实时RT-PCR法,分析MSC的体外(in vitro)迁移能力。
1.实验方法,评价项目
细胞培养和总RNA的制备通过实施例1中记载的方法进行。
2.结果的评价和判定基准
2.1流式细胞术(FCM)法
首先,作为与迁移性相关的趋化因子和生长因子的分析,使用FCM法,各自分析下述受体的表达。
<迁移性所涉及的受体>
趋化因子受体:
CCR1、CCR2、CCR3、CCR4、CCR5、CXCR1、CXCR2、CXCR3、CXCR4、
CXCR5、CXCR6、CXCR7、CX3CR1
生长因子受体:
VEGFR1、VEGFR2、PDGFRβ、EGFR、IGF-1R、FGF-R2、HGFR、Tie-2
<粘附因子>
ICAM2、VCAM、ALCAM、HCAM(CD44)、ITGAV、ITGA4、ITGB1
2.2迁移测定
接着,将下述所示的趋化因子、生长因子添加到培养基中,使用迁移测定法研究MSC的迁移能力。
<趋化因子和生长因子>
VEGF、EGF、HGF、IGF-1、PDGF-AB、bFGF、ANGPT-1
MCP-1(CCL2)、MIP-1α(CCL3)、RANTES(CCL5)、Eotaxin-1(CCL11)、MDC(CCL22)、
Eotaxin-2(CCL24)、CRO-α(CXCL1)、SDF-1(CXCL12)、Fractalkine(CX3CL1)
迁移测定使用FluoroBlok(コーニング公司)来实施。
1)向孔板中添加迁移因子,放置插入物;
2)在插入物上部添加细胞悬浮液;
3)18小时后,对迁移细胞数,用CalceinAM(同仁化学研究所)计数染色细胞。
结果以将未添加趋化因子、生长因子时的迁移细胞数记作1.0的相对迁移比来评价。
相对迁移比=细胞数(添加迁移因子)/细胞数(未添加迁移因子)
2.3实时RT-PCR
按照实施例1,针对细胞对血管内皮的粘附、对组织的浸润所涉及的因子,通过实时RT-PCR法分析基于有无炎性细胞因子刺激的基因表达。
<粘附因子>
ITGB1、ITGA4
<浸润相关蛋白质>
MMP1、MMP2、TIMP1、TIMP2
3.结果
3.1利用FCM法的MSC的趋化因子受体、生长因子受体、粘附因子的分析(表1和图5)
针对趋化因子受体,尽管在部分细胞中确认CCR5、CXCR3等的表达,但并非在全部细胞中共通表达。另一方面,针对生长因子的受体,确认EGFR、HGFR、NGFR、Tie2的表达。针对粘附因子,确认到据信参与迁移的MSC对血管内皮细胞的粘附的NCAD、CD44、NCAM、ALCAM、ITGA4、ITGB1的表达。
[表1]
利用FCM的趋化因子受体、生长因子受体的表达分析
-:完全不表达
+/-:极少表达
+:表达
3.2迁移测定(图6)
通过EGF、PDGF-AB、βFGF、ANGPT-1、MCP-1(CCL2)、MIP-1α(CCL3),确认促进迁移,特别地,EGF、MCP-1(CCL2)中倾向显著。
3.3实时RT-PCR(图7)
确认表达与作为粘附因子的ITGB1、ITGA4、浸润相关的MMP1、MMP2、TIMP1、TIMP2。此外,确认用炎性细胞因子(TNF-α/IFN-γ/IL-6)刺激的MSC的MMP1的表达大幅增加。ITGB1、ITGA4被报告对血管内皮的粘附是重要的,与迁移的细胞在损伤部位的蓄积(前述James等人,2007)。此外,已知MMP、TIMP家族将细胞的基底膜融解,迁移的细胞对损伤部位浸润(Caroline等人,2008;Mariusz等人,2012)。根据这些报告与上述结果,暗示包含MSC的药物具有与对血管内皮的粘附和对组织的浸润相关的特性。
4.考察
根据利用FCM的受体的分析和迁移测定的结果,确认了作为MSC的迁移能力的指标,EGFR的表达是特别重要的。此外,根据FCM分析和实时RT-PCR分析的结果,确认了作为MSC的迁移能力的指标,ITGA4的表达是重要的。
工业实用性
根据本发明,能够适当测定包含间充质干细胞的细胞药物的功能,能够提供适合于组织再生的包含间充质干细胞的细胞药物。
本说明书中引用的全部出版物、专利和专利申请直接以参考的方式并入本说明书中。
Claims (11)
1.包含间充质干细胞的细胞药物的制造方法,其包括:
a)向包含间充质干细胞的培养物中添加炎性细胞因子,确认间充质干细胞表达CX3CL1的步骤;和/或
b)确认间充质干细胞的90%以上表达EGFR和/或ITGA4的步骤。
2.根据权利要求1所述的方法,其进一步包括:确认包含间充质干细胞的培养物中的选自BDNF、VEGF、和HGF中任一种以上的分泌能力的步骤。
3.根据权利要求1或2所述的方法,其中,炎性细胞因子为选自TNF-α、INFγ、IL-1、IL-6、IL-8、IL-12、和IL-18中的1种以上。
4.根据权利要求1或2所述的方法,其中,炎性细胞因子包含TNF-α、INFγ、和IL-6。
5.包含间充质干细胞的细胞药物,其中
a)前述间充质干细胞通过炎性细胞因子的刺激而表达CX3CL1;和/或
b)前述间充质干细胞的90%以上表达EGFR和/或ITGA4。
6.根据权利要求5所述的细胞药物,其中,前述间充质干细胞具有选自BDNF、VEGF、和HGF中的1种以上的分泌能力。
7.根据权利要求5或6所述的细胞药物,其中,炎性细胞因子为选自TNF-α、INFγ、IL-1、IL-6、IL-8、IL-12、和IL-18中的1种以上。
8.根据权利要求5或6所述的细胞药物,其中,炎性细胞因子包含TNF-α、INFγ、和IL-6。
9.根据权利要求5或6所述的细胞药物,其中,因TNF-α、INFγ、和IL-6的混合物的刺激而导致的CX3CL1表达量比因TNF-α、INFγ、和IL-6各自单独的刺激而导致的CX3CL1表达量的总和更多。
10.评价包含间充质干细胞的细胞药物的免疫调节能力的方法,其包括:用炎性细胞因子刺激间充质干细胞,测定CX3CL1的表达的步骤。
11.评价包含间充质干细胞的细胞药物在损伤部位的蓄积能力的方法,其包括:评价前述间充质干细胞中的EGFR和/或ITGA4的表达是否为90%以上的步骤。
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