CN111566204B - 培养细胞的制造方法、脊髓损伤疾病的治疗剂的制造方法 - Google Patents
培养细胞的制造方法、脊髓损伤疾病的治疗剂的制造方法 Download PDFInfo
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Abstract
本发明的课题是提供一种细胞培养方法,其不采用由病毒所引起的基因导入法,而进行安全的GDNF基因表现(mRNA)的增加。本发明的解决手段是一种培养细胞的制造方法,其包含:培养工序,在包含SAG、嘌吗啡胺(Purmorphamine)以及音猬因子(Sonic hedgehog,SHH)蛋白质的任一种或二种以上的无血清培养基中培养人类皮肤来源干细胞;无血清培养基可更包含B‑27添加剂、ROCK抑制剂、EGF、FGF2。
Description
技术领域
本发明是涉及一种细胞的制造方法,所述细胞是培养由人类皮肤组织所得的细胞而得且用于治疗脊髓损伤疾病群,并且,是涉及一种医疗药品的制造方法,所述医疗药品含有通过所述方法而得的细胞或培养培养基或外泌体等的分泌物且治疗脊髓损伤疾病群。
背景技术
脊髓损伤(Spinal Cord Injury)主要是因对脊柱施加强大的外力而损坏脊椎,对脊髓造成损伤的病状。并且,因脊髓瘤及疝气及颈椎脊髓症等内在原因也会发生类似的障碍。脊髓损伤的推测患者数,在日本是10万人。脊髓损伤的大部份原因是由交通事故及从高处落下所导致的外伤。
与末梢神经不同,包含脊髓的中枢神经系统一旦损伤便无法修复/再生。再生被破坏的脊髓、再次恢复功能一事,是全世界的脊损者的愿望,在各式各样的领域中正在进行研究。
目前在刚受伤后,在美国,只要是在刚受伤后48~72小时内,便会进行大量的类固醇药剂的投予。在日本的关西医科大学,仍是对于刚受伤后的患者培养自已的骨髓液并注入脊髓,而日本的庆应大学等的团队,则正进行在因事故等损伤脊髓的78小时内投予肝细胞生长因子(HFG)等的尝试。
在成为慢性期的情形,会进行复健。但是,脊髓损伤的复健无法使已失去的功能恢复。因为只要神经不再生,这就是不可能的。复健的目的在于「要怎么使用残存的功能让ADL(日常生活动作)成为可能」这点。再者,若成为慢性期,则会发生褥疮及尿道感染症等的并发症,照护极为困难。
即使是所述慢性期,目前最被看好的有效治疗法是尝试使用骨髓或神经的干细胞的神经再生。在动物实验中虽被报告有部份效果,但就应用在人体并有助于治疗而言,尚在基础研究的阶段,正期望研究的强力推进。主要正在研究人工多能性干细胞、胚胎干细胞的使用,但应解决的课题仍多。举例而言,在使用人工多能性干细胞(iPS)的情形,虽认为可避免免疫反应,但被指出成为肿瘤的可能性。并且,针对胚胎干细胞,除了免疫反应及肿瘤化的问题,也被指出伦理面的问题。
但是,日本在2010年发表报告,所述报告是在已克服伦理面与免疫排斥的问题而使用由基因导入所制造的iPS细胞对于脊损动物的投予中,在脊髓内会造成肿瘤(非专利文献1)。
2005年到目前,唯一进行临床治疗的是在中国的北京首都医科大学,企图通过注入嗅鞘神经胶质细胞(OEG)而再生脊髓。但是,在由同所大学经过长期的治疗效果的验证中,并未提出让世界上的研究者认同的数据,且残留剧烈疼痛、OEG的取得来源(从堕胎胎儿采取)的问题及异体移植的问题。即使在2014年所发表的移植自体鼻粘膜的日本临床试验中,也未报告有戏剧性效果(可行走案例)。
2010年10月,美国的杰龙公司开始对脊髓损伤的患者4人使用ES细胞的临床试验,但在2011年11月发表撤出。此原因虽不明,但缺乏临床效果、或出现免疫性排斥反应、或在脊髓内发生畸胎瘤的可能性高。
庆应大学的中村雅也准教授人等的团队在京都市召开的日本再生医学学会上,发表在2017年度开始对于脊髓损伤患者的iPS细胞临床研究的计划。对象是从事故起2~4周后、患部的炎症结束且伤口开始愈合前的患者,不适用于慢性期的患者。
如此,虽干细胞治疗受到关注,但已上市的再生医学产品,在美国由干细胞所制造的产品只有1品目,在欧洲联盟作为先进治疗医药品(Advanced Therapy MedicinalProduct)只有1品目。然而,现状是对于脊髓损伤不存在已被承认的再生医学产品。
并且,人类皮肤来源干细胞(Mesenchymal stem cells)在移植后难以诱导成神经细胞,会成为软骨细胞或骨细胞的可能性高,脑来源脑神经干细胞则有治疗效果低等的缺点(非专利文献13)。
另一方面,来自皮肤的前驱细胞被报告在脊损的治疗效果比来自脑的神经干细胞高(非专利文献14)。
再者最近,井上及熊谷人等也报告人类表皮角质细胞也具有对脊髓损伤的治愈效果(非专利文献2)。
然而,目前对于脊髓损伤,尚不存在一种细胞的培养制备法,其特定治疗效果的标记,且所述细胞表现所述标记。
另一方面,作为细胞因子,胶质细胞源性神经营养因子(Glial cell line-derived neurotrophic factor:GDNF)是最明确地对脊髓损伤显示效果的细胞因子(非专利文献4-8)。
于是,欲制造更提高脊髓损伤的治愈效果者的尝试,是对于期待治愈效果的细胞进一步导入GDNF的基因的尝试(非专利文献8-10)。作为导入基因的细胞,使用嗅鞘细胞(Olfactory ensheathing cells)、施万细胞(Schwann cells)等,此细胞本身就具有治愈效果,因此会增大治疗效果。
但是,此方法的缺点是因为使用病毒进行基因导入,所以在移植至生物体时癌化的可能性高。
相反地,GDNF的蛋白本身不稳定且操作处理困难(非专利文献15)。
并且,GDNF也被报告作为脊髓损伤的恢复所需要的内因性因子(非专利文献16)。
再者,GDNF也被报告抑制困扰脊髓损伤患者的异痛症的效果(非专利文献17-18)。
脊髓损伤的治疗大多是针对进入慢性期的患者,对于慢性期也具有效果的治疗法是最被期望的。所幸,GDNF被报告也对慢性期的脊髓损伤有效果(非专利文献19)。
因此,使进行移植的细胞表现GDNF一事,会关系到划时代地提高脊髓损伤的治疗效果(非专利文献20)。
现有技术文献
专利文献
专利文献1:日本特开2012-29684号公报
非专利文献
非专利文献1:Tsuji O,et al.:Therapeutic potential of appropriatelyevaluated self-induced pluripotent stem cells for spinal cord injury.PNAS
2010;107(28),12704-12709
非专利文献2:Inoue H,et al.:Improvement of hind-paralysis followingtraumatic spinal cord injury in rats by grafting normal human keratinocytes:new cell-therapy strategy for nerve regeneration.J Artif Organs.2011;14(4):375-80
非专利文献3:Inoue H,et al.:Improvement of hind-limb paralysisfollowing traumatic spinal cord injury in rats by grafting normal humankeratinocytes:new cell-therapy strategy for nerve regeneration.J ArtifOrgans.2011;14(4):375-80
非专利文献4:Ansorena E,et al.:Injectable alginate hydrogel loadedwith GDNF promotes functional recovery in a hemisection model of spinal cordinjury.Int J Pharm.2013;455(1-2):148-58.
非专利文献5:Tang XQ,et al.:Adenovirus-mediated delivery of GDNFameliorates corticospinal neuronal atrophy and motor function deficits inrats with spinal cord injury.Neuroreport.2004;15(3):425-9.
非专利文献6:Guzen FP,et al.:Glial cell line-derived neurotrophicfactor added to a sciatic nerve fragment grafted in a spinal cord gapameliorates motor impairments in rats and increases local axonalgrowth.Restor Neurol Neurosci.2009;27(1):1-16.
非专利文献7:Kao CH,et al.:Exogenous administration of glial cellline-derived neurotrophic factor improves recovery after spinal cord injury.
Resuscitation.2008;77(3):395-400.
非专利文献8:Koelsch A,et al.:Transgene-mediated GDNF expressionenhances synaptic connectivity and GABA transmission to improve functionaloutcome after spinal cord contusion.J Neurochem.2010;113(1):143-52.
非专利文献9:Cao L,et al.:Olfactory ensheathing cells geneticallymodified to secrete GDNF to promote spinal cord repair.Brain.2004Mar;127(Pt3):535-49.
非专利文献10:Deng LX,et al.:A novel growth-promoting pathway formedby GDNF-overexpressing Schwann cells promotes propriospinal axonalregeneration,synapse formation,and partial recovery of function after spinalcord injury.J Neurosci.2013Mar 27;33(13):5655-67.
非专利文献11:Stephen J.A.Daviees,et al.:Transplantaion of specifichuman astrocytes promotes functional recovery after spinal cord injury.PLoSONE 2011;6(3):e17328
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发明内容
发明所欲解决的课题
本发明的目的是提供一种细胞培养方法,其不采用由病毒所引起的基因导入法,而进行安全的GDNF基因表现(mRNA)的增加。
本发明的目的在于提供一种细胞培养方法,其也表现神经干细胞的标记等各种标记。
本发明的目的在于提供一种脊髓损伤疾病的治疗剂的制造方法。
解决课题的技术方案
本发明基本上是基于由以下实施例而来的见解:在包含SAG、嘌吗啡胺(Purmorphamine)以及音猬因子(Sonic hedgehog,SHH)蛋白质的任一种或二种以上的无血清培养基中培养人类皮肤来源干细胞,借此可使胶质细胞源性神经营养因子(GDNF)mRNA高表现(例如,通常培养的10倍以上的表现)。
本发明的第一态样是涉及一种培养细胞的制造方法,所述培养细胞包含胶质细胞源性神经营养因子(GDNF)mRNA。此方法包含:培养工序,其在包含SAG、嘌吗啡胺(Purmorphamine)以及音猬因子(Sonic hedgehog,SHH)蛋白质的任一种或二种以上的无血清培养基中培养人类皮肤来源干细胞。此方法的胶质细胞源性神经营养因子(GDNF)mRNA会高表现。
人类皮肤来源干细胞优选为源自患者本人的皮肤细胞。尤其,优选为采取来自在采取时对组织的损伤轻微且恢复快的皮肤组织的细胞,而获得培养细胞。干细胞的培养方法因为是公知,所以基本上只要遵循本说明书记载的方法与公知的方法(例如日本特许第5409359号公报或日本特许第6041270号公报所记载的方法)培养细胞即可。人类皮肤来源干细胞的制造方法也被记载于上述公报且为公知的技术。
SAG是以CAS编号364590-63-6为人所知,且化学物质名称是N-甲基-N’-(3-吡啶基苄基)-N’-(3-氯苯并[b]噻吩-2-羰基)-1,4-二氨基环己烷(N-Methyl-N’-(3-pyridinylbenzyl)-N’-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane)的化合物。
如同国际公开WO2014-084085号小册子所公开,SAG是使音猬因子(Shh)活化的蛋白质。SAG1.1的化学物质名称是N-甲基-N’-(3-(4-苯甲腈)-4-甲氧基苄基)-N-(3-氯苯并[b]噻吩-2-羰基)-1,4-二氨基环己烷(N-Methyl-N-(3-(4-benzonitrile)-4-methoxybenzyl)-N’-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane))。关于SAG,详细内容在文献Sinha S,ChenJK.Nat Chem Biol.2006Jan;2(1):29-30.以及Chen JK,Taipale J,Young KE,Maiti T,Beachy PA.Proc Natl Acad Sci U S A.2002Oct 29;99(22):14071-6.。同样地,关于SAG1.1,详细内容在文献Chen,W.,Ren,X.R.,Nelson,C.D.,Barak,L.S.,Chen,J.K.,Beachy,P.A.,de Sauvage,F.&Lefkowitz,R.J.(2004)Science 306,(5705)2257-2260。
嘌吗啡胺(Purmorphamine)的通用名称是(9-环己基-N-[4-(4-吗啉基)苯基]-2-(1-萘氧基)-9H-嘌呤-6-胺))((9-Cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine))。嘌吗啡胺是也被称为Purmorphamine的平滑激动剂,被记载于日本特许6210881号公报、以及日本特许5620821号公报(在培养基中添加2uM)。
音猬因子(Sonic hedgehog,SHH)蛋白质是公知的蛋白质,例如在日本特许4900587号公报中,在无血清培养基中添加有200~400ng/ml的SHH蛋白质。
无血清培养基
本发明的培养基是无血清培养基,可适当包含公知的培养基中的成分。并且,例如在日本特许第4385076号公报以及日本特开2012-157263号公报中公开一种用于无血清培养动物细胞的培养基。如此,也可在本发明的培养基中适当地添加公知的文献所记载的要素。
本发明的培养基可适当使用作为基本培养基而公知的培养基。此种基本培养基的例子是MEM培养基、杜尔贝可MEM(注册商标)培养基、MCDB153、哈姆F12(注册商标)培养基、麦考伊5A(注册商标)培养基、199培养基厄尔溶液(注册商标)、RPMI1640(注册商标)培养基、F―10哈姆培养基、MEM-α培养基、DMEM/F12培养基、MCDB131、MCDB153以及MCDB201。
培养基的pH是在被5%浓度CO2环境进行平衡化之际被调整为pH6.8-7.8的范围,更优选为pH7.2(±0.1)。可使用缓冲材(例如,碳酸氢钠)、盐酸以及氢氧化钠等pH调整剂,适当调整基本培养基的酸性度。
本发明的培养基可适当包含各式各样的低分子化合物。例如,本发明的培养基优选为包含抗氧化剂。抗氧化剂的例子是选自由褪黑激素(Melatonin)、n-乙酰-L-半胱氨酸、还原型谷胱甘肽以及抗坏血酸所组成的群组的一或二个以上的成分。本发明的培养基也可适当包含磷脂质(例如,磷脂酰丝氨酸、磷脂酰乙醇胺以及磷脂酰胆碱等)及脂肪酸(例如,亚油酸、油酸、亚麻酸、花生四烯酸、肉豆蔻酸、棕榈酰酸、棕榈酸以及硬脂酸)。可添加至本发明的培养基。其他成分的例子是转铁蛋白、硒酸盐、葡萄糖、D-生物素、D-泛酸钙、氯化胆碱、叶酸、肌醇、烟酰胺、p-氨基安息香酸、盐酸吡哆醛、盐酸吡哆醇、核黄素、盐酸硫胺、维生素B12、丙酮酸钠、胸苷、次黄嘌呤、亚硒酸钠、硫酸链霉素、青霉素G钾盐以及酚红等。
上述培养细胞的制造方法优选为无血清培养基更包含B-27添加剂。若无血清培养基包含B-27添加剂,则如同实施例中所示,巢蛋白mRNA会高表现。巢蛋白基因是神经干细胞的标记。例如像在日本特许6185907号公报以及日本特许6137626号公报中被添加至培养基般,B-27添加剂是公知的要素。
上述培养细胞的制造方法优选为无血清培养基更包含ROCK抑制剂。若无血清培养基包含ROCK抑制剂,则会维持干细胞的稳定性。ROCK抑制剂被定义为抑制Rho-激酶(ROCK)的激酶活性的物质,可列举例如,Y-27632(4-[(1R)-1-氨基乙基]-N-吡啶-4-基环已烷-1-甲酰胺)或其二盐酸盐(例如参照Ishizaki et al.,Mol.Pharmacol.57,976-983(2000);Narumiya et al.,Methods Enzymol.325,273-284(2000))、Fasudil/HA1077(1-(5-异喹啉磺酰基)高哌嗪)或其二盐酸盐(例如参照Uenata et al.,Nature 389:990-994(1997))、H-1152((S)-(+)-2-甲基-1-[(4-甲基-5-异喹啉)磺酰基]-六氢-1H-1,4-二氮杂卓)或其二盐酸盐(例如参照Sasaki et al.,Pharmacol.Ther.93:225-232(2002))、Wf-536((+)-(R)-4-(1-氨基乙基)-N-(4-吡啶基)苯甲酰胺一盐酸盐)(例如参照Nakajima et al.,Cancer Chemother.Pharmacol.52(4):319-324(2003))以及其等的衍生物、以及对于ROCK的反义核酸、RNA干涉诱导性核酸(例如,siRNA)、显性负性突变体、以及其等的表达载体。并且,作为ROCK抑制剂也已知其他低分子化合物,因此在本发明中也可使用此种化合物或其等的衍生物(例如参照美国专利申请公开第20050209261号、同第20050192304号、同第20040014755号、同第20040002508号、同第20040002507号、同第20030125344号、同第20030087919号、以及国际公开第2003/062227号、同第2003/059913号、同第2003/062225号、同第2002/076976号、同第2004/039796号)。在本发明中,能使用至少一种的ROCK抑制剂。ROCK抑制剂一般能在100nM~50μM的范围被使用。
上述培养细胞的制造方法优选为无血清培养基更包含EGF。若无血清培养基包含EGF,则维持神经干细胞,巢蛋白的mRNA会高表现。
EGF是选自表皮生长因子(EGF:epidermal growth factor)家族的生长因子。例如,在日本专利6191694号公报中,相对于培养基,EGF的含量的最终浓度是0.5~200ng/mL。
GFAP
GFAP基因是被认为适于对脊损进行移植的成体神经干细胞(adult neural stemcell)或星状细胞(astrocytes)的标记(非专利文献1、20、22)。
上述培养细胞的制造方法优选为无血清培养基更包含FGF2。若无血清培养基包含ROCK抑制剂,则如同实施例中所示,EGF的mRNA会高表现,再者CD73蛋白质会表现。FGF(纤维母细胞生长因子)2也被表示为FGF-2。例如,在日本特许5856029号公报中,FGF2是以0.1~50ng/mL,更优选为1~10ng/mL,最优选为5ng/mL的浓度被添加至培养基。CD73(细胞外-5’-核苷酸酶:Ecto-5’-核苷酸酶)缓和脊损患者特有的疼痛的可能性高。
上述培养细胞的制造方法优选为无血清培养基的钙离子浓度是0.03mM以上且0.12mM以下。通过将钙离子浓度维持在此范围中,可抑制干细胞的分化。
此外,上述优选态样可适当组合。上述的SAG、嘌吗啡胺、音猬因子(SHH)蛋白质、B-27添加剂、ROCK抑制剂、EGF、FGF2等添加物,只要考虑其等的物质性质而将适当量添加至培养基即可。此等物质公知为被添加至培养基的物质,因此只要基于公知的资料适当调整添加量即可。例如,只要分别在培养基中以成为0.1ng/mL以上且20μg/mL以下(或0.2ng/mL以上且10μg/mL以下)的方式进行添加即可。只要因应此等的纯化程度及需要量而适当调整并添加即可。
上述培养细胞的制造方法优选为人类皮肤来源干细胞是将所采取的人类皮肤以分散酶处理、胰蛋白酶处理以及胶原酶处理的顺序进行处理而得的细胞。分散酶处理、胰蛋白酶处理以及胶原酶处理皆是酶处理。酶处理条件能是:在被缓冲成生理学能容许的pH例如约6~8,优选为约7.2~7.6的等张盐溶液;例如是胰蛋白酶的情形是在PBS或Hanks平衡盐溶液中,例如是分散酶、胶原酶的情形则是在例如MEM培养基中;在约4~40℃,优选为约4~39℃;用于分解结缔组织的充分时间例如是约1~1000分钟,优选为5~720分钟;为此的充分浓度例如约0.0001~5%w/v,优选为约0.001%~0.5%w/v。
本发明的第二态样是也提供脊髓损伤疾病的治疗剂的制造方法。脊髓是贯穿脊椎内部的中枢神经,能使用于修复此神经的是脊髓损伤疾病的治疗剂。此方法是使用上述方法制造培养细胞。其后,制造脊髓损伤疾病的治疗剂(本发明的剂),所述治疗剂是将培养细胞、培养工序中所得的培养基、以及培养工序中所得的分泌物(外泌体等)的任一种或二种以上作为有效成分。分泌物(外泌体等)可来自培养上清液。并且,培养基可仅使用培养上清液。
本发明的剂只要利用发明所属技术领域的普通技术人员公知的方法制造即可。本发明的剂可被制造作为口服制剂以及非口服制剂,但优选为非口服制剂。此种非口服制剂可为液剂(水性液剂、非水性液剂、悬浮性液剂、乳浊性液剂等),也可为固形剂(粉末填充制剂、冷冻干燥制剂等)。并且,本发明的剂可为缓释制剂。作为将活细胞作为剂的情形的剂型,优选为液剂,作为将细胞的一部分成分或死细胞整体作为剂的情形的剂型,可选择液剂或固形剂。
本发明的剂的主成分是所培养的人类皮肤来源干细胞、培养工序中所得的培养基、以及来自培养细胞的分泌物(外泌体等)的任一种或二种以上。无论如何,例如作为细胞的数量的指标,只要设计有效量即可。最终的剂中也可去除干细胞。并且,分泌物可被包含在培养上清液中。每一次的投予量与投予次数、投予的频率会依据投予的对象、年龄、症状等而变化。一般而言,1日的人类皮肤来源干细胞剂的投予量的例子,作为静脉内注射1投予单位,是1×105细胞以上1×109细胞以下,优选为1×106细胞以上5×108细胞以下,更优选为1×107细胞以上2×108细胞以下。或,每1kg体重的人类皮肤来源干细胞的1日投予量的例子是1×104细胞以上1×108细胞以下,优选为1×105细胞以上5×107细胞以下,更优选为1×106细胞以上2×107细胞以下。本发明的剂可每3日投予1次且合计投予8次,也可持续一周连日投予。并且,也可在组合各式各样的频率的投予期间使用。在1次便确认到症状改善的显着效果例中,可仅投予首次便结束。如此,本发明的剂能依据疾病及症状的背景而多种多样地使用,再者,在各投予中的移植细胞数在上述范围中能任意地设定。
本发明也提供上述本发明的人类皮肤来源干细胞的使用,其用于制造脊髓损伤的治疗剂。
本发明也提供脊髓损伤的治疗方法。各种疾病的治疗方法包含以下工序:将有效量的如上述般制造的本发明的剂投予至罹患脊髓损伤的患者。
发明效果
本发明可提供一种细胞培养方法,其不采用由病毒所引起的基因导入法,而进行安全的GDNF基因表现(mRNA)的增加。
本发明可提供一种细胞培养方法,其也表现神经干细胞的标记等各种标记。
本发明可提供一种脊髓损伤疾病的治疗剂的制造方法。
在本发明的方法中,可利用无血清/无饲养层细胞培养法,从在人类组织中特别容易采取的皮肤获得脊髓损伤的治疗用细胞。在本发明的方法中,发现将在脊髓损伤的治疗中最必要的GDNF的表现量提升10倍的方法,与此同时,确认不进行基因导入而获得对脊髓损伤治癒效果高的细胞,所述细胞会使神经干细胞的标记的巢蛋白表现、使对脊髓损伤的治疗有效的GFAP表现、更使防止因脊髓损伤所引起的痛觉过敏的CD73也表现。由本发明的方法所得的细胞被确认在大鼠脊髓损伤模式具有治愈效果。并且,此细胞在致癌性试验中是阴性。再者,通过利用腰椎穿刺法一个月2次投予此细胞,获得以下结果:因2年以上的脊髓损伤(Th3)而无法行走的47岁患者在投予1个月后变得能行走。另一方面,在由2014年的TRI研讨会所发表的日本的嗅粘膜移植中,对于4例的35岁以下的下肢完全麻痹(Th4~7),连1例的能行走例都没有。
附图说明
图1是取代显示GDNF mRNA的表现量增加效果(实验例2)的图片的图表。
图2是取代显示巢蛋白mRNA的表现(实验例3)的图片的图表。
图3是取代显示GFAP mRNA的表现(实验例4)的图片的图表。
图4是取代显示CD73的表现(CD表现量变曲线)(实验例5)的图片的图表。
图5是取代显示在大鼠脊髓损伤模式的效果(BBB评分)(实验例6)的图片的图表。
图6是取代显示致癌性评价的图片的图表。
具体实施方式
以下,针对用于实施本发明的方式进行说明。本发明不受限于以下说明的方式,也包含发明所属技术领域的普通技术人员由以下方式在显而易知的范围进行适当修正的方式。
本发明提供一种方法,其利用以下工序获得对脊髓损伤效果高的细胞,并提供直接投予的细胞:
从人类皮肤采取皮肤,并将组织进行除菌的组织采取工序;
细胞采取播种工序;
细胞增殖工序;
继代工序;
分化诱导工序;
移植/加工工序;
或提供一种提供细胞的方法,所述细胞是用于与其他基剂结合的医疗设备。再者,提供一种方法,其投予由以上工序所得的培养后培养基成分。
以下,针对各工序,举例说明其详细内容。以下的说明是举例,本发明不受限于以下的例子,也包含在对于发明所属技术领域的普通技术人员而言是显而易知的范围内适当变化条件的方式。此外,u有时意指微米。
(组织采取工序)
由本工序所采取的皮肤期望是颈部以上的部位,再者,由采取后的美容上的观点而言,最期望是耳廓后部的皮肤。不问皮肤的性别及年龄,能到70多岁为止。以下工序全部以无菌操作进行。采取组织的除菌是利用已通过0.1um孔径的除菌过滤器去除芽孢的70%乙醇水进行。
(细胞采取播种工序)
将已裁切的组织在含10uM Y-27632的分散酶溶液(PBS(-))中于4℃浸渍1晚。将此组织再在含10uM Y-27632的胰蛋白酶溶液(PBS(-))中于37℃浸渍20分钟。再者,将此组织在胶原酶溶液(DMEM)中浸渍,于37℃,以搅拌器搅拌1~2小时,消化皮肤。将此搅拌后的悬浮液通过100um网格而去除残渣。将此液利用PBS(-)进行3次离心、清洗而去除酶,获得细胞。将此细胞置入胰蛋白酶抑制剂中静置10分钟。进行离心而从此细胞去除抑制剂后,悬浮在含10uM Y-27632的培养基中,并播种至培养皿。
(细胞增殖工序)
在播种6日为止前不进行培养基交换,仅添加培养基。其后的培养基交换是将在条件培养基中增殖浮游的细胞进行离心并回收,将悬浮在新培养基中的细胞返回培养系统而进行培养基交换。
(継代工序)
将细胞进行30分钟以上的10uM Y-27632处理,利用EDTA与TrypLEx Express从培养皿回收细胞。接下来置入胰蛋白酶抑制剂中静置10分钟。进行离心,从细胞去除抑制剂后,悬浮在含10uM Y-27632的培养基中,播种到原来的4~5倍面积的培养皿中。在継代第1代培养5个的100mm2培养皿,若达到汇合,则将其中3个用于移植,或在进行移植前冷冻保存。将剩余2个进一步继代培养成6个100mm2培养皿,若达到汇合则移往因子添加诱导工序。
(因子添加诱导工序)
因子添加诱导工序是在移植前第3日对需要的细胞进行药物处理且添加必要因子并进行培养的工序。
通过本工序,能获得高表现GDNFmRNA的细胞,例如表现通常培养的10倍以上的细胞。并且,通过本工序,能获得CD13是阴性的细胞(CD13(-))。并且,通过本工序,能获得CD34是阴性的细胞(CD34
(-))。并且,通过本工序,能获得CD45是阴性的细胞(CD45
(-))。并且,通过本工序,能获得CD90是阴性的细胞(CD90
(-))。并且,通过本工序,能获得CD73是阳性的细胞(CD73
(+))。培养细胞之中,CD73是阳性的细胞(CD73(+))的比例(细胞数)的例子可为20%以上且100%以下,也可为40%以上且100%以下,也可为60%以上且99%以下,也可为80%以上且99%以下。结束因子添加诱导工序的细胞优选为在3日后用于移植,或在移植前进行冷冻保存。
(移植/加工工序)
将经过各过程而达到治疗需要数量的细胞进行30分钟以上的10uM Y-27632处理,利用EDTA与TrypLEx Express从培养皿回收细胞。接下来置入胰蛋白酶抑制剂中静置10分钟。进行离心,从细胞去除抑制剂后,在利用脊髓穿刺进行移植的情形,以107cell/ml浓度悬浮在临床用输液中而使用。此细胞提供与其他基剂或细胞的加工。
以下,使用实施例具体地说明本发明。本发明也包含基于以下实施例而发明所属技术领域的普通技术人员可使用公知的技术进行适当调整的范围。
实施例1
(来自人类皮肤的皮肤干细胞的采取/培养)
(组织的采取)
使用56岁女性在拉皮美容整形手术时从脸部切除废弃的约2cm2的皮肤组织的全层部分。以下工序全部以无菌操作进行。将此采取组织在已利用0.1um(微米)孔径(poresize)的除菌过滤器除菌的70%乙醇水中浸渍30秒钟,再立刻利用磷酸缓冲食盐水(Phosphate buffer saline)(PBS(-))清洗3次,清洗掉乙醇。从此组织留下毛根存在的地方并利用铗尽可能地去除皮下脂肪以及真皮结缔组织,接下来利用手术刀裁切成1mm2左右的大小。
(细胞采取播种工序1)
将已裁切的组织在含10uM Y-27632的已预先冷却的分散酶(Dispase)溶液(25units/ml of PBS(-))5ml中,于4℃浸渍1晩。
(细胞采取播种工序2)
在将此进行离心去除上清液后,在含10uM Y-27632的TrypLE Express溶液(5x稀释PBS(-))中,于37℃浸渍20分钟。
(细胞采取播种工序3)
在将此组织进行离心去除上清液后,浸渍在20ml的胶原酶溶液(GIBCO:type I)(1400units/ml DMEM培养基)中,于37℃利用搅拌器搅拌1.5小时。
(细胞采取播种工序4)
将搅拌后的悬浮液通过100um网格而去除未消化的残渣。将此液利用PBS(-)进行3次离心/清洗而去除酶,获得细胞。
(细胞采取播种工序5)
将此细胞置入大豆胰蛋白酶抑制剂(Soy bean Trypsin Inhibitor)(0.25%/PBS(-))5ml中静置10分钟。
(细胞采取播种工序6)
进行离心,从细胞去除抑制剂后,悬浮在添加10uM Y-27632的培养培养基中,并播种至培养皿。因初始组织宽广(1~2cm2),故播种至1个6孔盘(CellBind:COSTAR)的全部孔。
(培养基制备工序)
培养培养基是如同以下般制备。亦即,在基本培养基的MCDB153培养基(Sigma:Stemline Keratinocyte Basal Medium)中,添加胰岛素(10mg/L)、全转铁蛋白(5.5mg/L)、硒(6.7ug/L)、乙醇胺(2mg/L)、维生素C(L-抗坏血酸2-磷酸酯半镁盐)(50ug/L)、KGF(10ug/L)、脂质(脂肪酸混合物:花生四烯酸(20ug/L)、胆固醇(2.2mg/L)、DL-a-生育酚-醋酸酯(700ug/L)、亚油酸(100ug/L)、亚麻酸(100ug/L)、肉豆蔻酸(100ug/L)、油酸(100ug/L)、棕榈油酸(100ug/L)、棕榈酸(100ug/L)、硬脂酸(100ug/L)、吐温80
(22mg/L)、普朗尼克F-68(1000mg/L)、EGF(20ug/L)、FGF(10ug/L)、Y-27632(1uM)、1%B-27添加剂XenoFree CTS(美国英杰生命技术有限公司)而制备。
(细胞增殖工序1)
原代培养是在培养7日前不进行培养基交换而是以1/3量的比例一边添加培养基一边进行驯化培养。其后一边每2日进行培养基交换一边进行培养。在此培养基交换中,将旧培养基进行离心,舍弃其2/3量,将残留的1/3量的培养基与已利用离心而沉淀的浮游细胞进行混合,加入2/3量的新培养基再加回培养皿。在到达100%汇合前需要10日。
(継代工序)
在所培养的细胞的培养基中以10uM浓度加入Y-27632,在37℃静置30分钟。接下来去除全部的培养基,以2ml/孔添加0.05%EDTA/PBS(-),在37℃静置10分钟。再以0.5ml/孔添加已利用PBS(-)5倍稀释的TrypLEx Express,在37℃静置5分钟。接下来利用滴管吸取而从孔剥离细胞。在此,添加2.5ml/孔的胰蛋白酶抑制剂(0.05%/PBS(-)),将所回收的细胞进行离心。去除上清液,置入2.5ml的胰蛋白酶抑制剂(0.25%/PBS(-)),将细胞进行悬浮,静置10分钟。进行离心,从细胞去除抑制剂后,悬浮在含10uM Y-27632的培养基中,播种至原本培养皿的5倍面积的六孔盘(CellBind:COSTAR)5个。在1周后,因到达100%汇合,所以作为以下实施例的实验材料。
实施例2
(GDNF(Glial cell-derived neurotrophic factor)mRNA的表现量增加法)
有可能用于发现使细胞的GDNF(Glial cell-derived neurotrophic factor)mRNA表现增加的化合物或细胞因子的方法已探讨以下六个方法。将丙戊酸(Valproicacid,VA)、血小板诱导生长因子(Platelete-derived growth factor,PDGF)、SAG(或者嘌吗啡胺或rh-音猬因子(PeproTech))、骨形态发生蛋白4(Bone morphogenic protein 4,BMP4)此等6个利用以下的组合添加至实施例1已汇合的7孔中。(1)控制组(无添加)(2)VA(1uM)(3)VA(1uM)+PDGF(10ng/ml)(4)VA(1uM)+SAG(0.3uM)(5)PDGF(10ng/ml)(6)SAG(0.3uM)(7)MBP-4(10ng/ml):在添加后72小时后,利用TRIzol法从各孔提取总RNA。使用PCR用cDNA合成试剂盒「ReverTra Ace(注册商标)qPCR RT Kit」,对于ReverTra Ace(注册商标)qPCR RT Kit的10μl反应系统,以0.5ug分别添加各总RNA,进行各cDNA合成。
在此等cDNA样品中的GDNF mRNA的表现比较,使用人类GDNF实时定量PCR引物试剂盒(Roche 5326257),利用实时定量PCR法,进行使用美国应用生物系统公司的ABI PRISM(注册商标)7700的嵌入剂鉴定法。qRT-PCR反应是使用东洋纺的SYBR(注册商标)GreenRealtime PCR Master Mix-Plus-而制备反应液。
反应液的制备
将反应液置入96孔盘的各孔而进行PCR反应。
PCR循环是利用3步骤法,将退火60℃/伸长72℃设为条件,将(1)~(2)进行40循环。在最后的步骤进行熔化曲线(Melting Curve)分析,确认未见到引物二聚体的形成。
95℃60秒钟
(1)↓
(2)95℃15秒钟
(3)60℃15秒钟
(4)72℃45秒钟(数据收集)
并且,同时进行GAPDHmRNA的检测,将GDNFmRNA的表现量补正作为GDNFmRNA/GAPDHmRNA。其结果,仅(6)SAG获得(1)控制组的10倍以上的GDNFmRNA表现。即使以嘌吗啡胺(1.5uM)、rh-音猬因子(10ng/ml)取代(6)的SAG,也同样地获得接近10倍的GDNFmRNA表现。(图-1)GAPDH的引物序列是利用以下序列而合成。
上游引物:ATCTTCTTTTGCGTCGCC(序列编号1)
下游引物:GATGACAAGCTTCCCGTTC(序列编号2)
表现链长:250bp
图1是显示取代实施例2中的GDNF mRNA的表现量测定结果的图片的图表。
实施例3
(巢蛋白mRNA的由神经分化诱导所引起的表现变化)
在实施例1中到达汇合的3孔之中的2孔中,利用神经前驱细胞的分化鉴定用试剂盒Human/Mouse/Rat Neural Progenitor Cell Functional Identification Kit(R&DSystems SC082)的分化用培养基(medium for NPC differentiation)培养7日。在另一个孔中,利用发明的维持培养基培养相同日。在第7日,与实施例2同样地提取总RNA后,制备cDNA。使用此cDNA,利用RT-PCR,将GAPDH mRNA作为基底状态,测量神经干细胞的标记亦即巢蛋白mRNA的表现比较。巢蛋白的引物是上游CGTTGGAACAGAGGTTGGAG(序列编号3)与下游TCCTGAAAGCTGAGGGAAG(序列编号4),表现链长是262bp。GAPDH的引物是使用与实施例2同样的引物。
将其结果显示于图2。
图2是取代显示实施例3中的巢蛋白mRNA的表现量的图片的图表。如图2所示,若改变成分化培养基,则巢蛋白的表现量(巢蛋白mRNA/GAPDH mRNA)会减少成分化前的1/7左右。此结果显示,发明人的增殖培养基会使神经干细胞维持,分化培养基会使神经干细胞减少。
实施例4
(GFAP mRNA的由神经分化诱导所引起的表现变化)
在实施例1中到达汇合的3孔之中的2孔中,利用神经前驱细胞的分化鉴定用试剂盒Human/Mouse/Rat Neural Progenitor Cell Functional Identification Kit(R&DSystems SC082)的分化用培养基(medium for NPC differentiation)培养7日。在另一个孔中,利用发明的维持培养基培养相同日。在第7日,与实施例2同样地提取总RNA后,制备cDNA。使用此cDNA,利用RT-PCR,将GAPDH mRNA作为基底状态,测量星状细胞或成体神经干细胞的标记亦即GFAP mRNA的表现比较。GFAP的引物是上游:ACATCGAGATCGCCACCTAC(序列编号5)与下游:ACATCACATCCTTGTGCTCC(序列编号6),表现链长是219bp。GAPDH的引物是使用与实施例2同样的引物。将其结果显示于图3。
图3是取代显示实施例4中的GFAP mRNA的表现量的图片的图表。如图4所示,若改变成分化培养基,则GFAP的表现量(GFAP mRNA/GAPDH mRNA)仅稍较分化前增加。此结果显示,发明者的增殖培养基会使神经干细胞维持,也使其中的GFAP的表现维持。
实施例5
(利用流式细胞术的CD抗原表现探讨)
使用胰蛋白酶-EDTA溶液,从培养皿剥离/单离已被培养成为汇合的细胞。接下来,利用3%BSA/PBS(-)溶液并放置30分钟,借此进行封闭,防止非特异性吸附。再者,利用4%多聚甲醛固定10分钟后,在0.5%曲拉通X-100/PBS(-)中、在4℃培育5分钟。将此细胞利用抗CD73的单克隆抗体CD73-PE(BD 561014)在4℃过夜培育。将此细胞利用清洗液清洗5次而去除未结合的抗体,流式细胞术是使用SONY EC800分析抗原的表现。将其结果显示于图4。
图4是取代显示实施例5中的CD73的表现(CD表现量变曲线)的图片的图表。如图4所示,因CD13、CD34、CD45是阴性,所以表示此细胞是非血液系统细胞。并且,因CD90也是阴性,所以也表示不是人类皮肤来源间充质干细胞。并且,CD73强烈表现一事,是此细胞最明确的CD的特征。
实施例6
(在大鼠脊髓损伤模式中的利用BBB评分法的效果探讨)
使用酶ACCUTASE(GIBCO A11105),从培养皿剥离利用SAG处理等而不使GDNF增加的培养细胞。将此剥离细胞1x105个悬浮在200ul的培养液中,在1周前利用锤对脊髓造成损伤,且注入移植至已投予免疫抑制剂的大鼠的脊髓损伤部位。对于对照的大鼠,进行仅注入同量的培养培养基的假手术。细胞移植组是将5只,对照组是将4只大鼠用于实验。脊髓损伤的治疗效果是利用录影装置观察动物的举动并利用进行点数化的BBB评分法而测定。将结果显示于图5。图中,横轴表示细胞移植后的日数,纵轴表示BBB评分。实线表示移植上述细胞的结果,虚线表示对照的结果,*表示利用学生T检验的显着性检验的结果P<0.05,**表示P<0.01,***表示P<0.001。从投予后3日与对照组进行比较,细胞移植组显示在BBB评分有2点差距的统计上有意义的效果(P<0.05)。若移植后经过35日,与对照组进行比较,细胞移植组显示在BBB评分有4点差距的统计上有意义的效果(P<0.001)。在骨髓基质细胞移植的报告(非专利文献23)中,在达到有意义差异为止前会耗时接近2周。并且,在由iPS所诱导的神经干细胞移植(非专利文献1)中,在达到有意义差异为止前需要3周。相较于此等,这次的结果戏剧性地快速,从3日便见到效果。而且,在骨髓基质细胞移植中,虽然经过4周有意义差异也是P<0.05,但本发明在同时期是更优异的P<0.001。此结果显示,由本发明所得的细胞对于脊髓损伤的治疗非常有效。
实施例7
(致肿瘤性的评价)
动物以及试验法
在实验中,使用NOD/Shi-scid、IL-2Rγnull(注册商标)系统的小鼠(6周龄,♀,公共财団法人实验动物中央研究所)。在1周的驯化期间后,使用泛用的分组系统(株式会社VISIONS),尽可能地使体重的平均值相同地分成3组。在各组的小鼠的右侧腹部,将利用与实施例1相同的方法所培养的细胞(设为细胞A)、作为阴性对照细胞的培养液、作为阳性对照细胞的HeLa S3(株式会社DS Pharma Biomedical)分别通过注射器(Myjector(注册商标),针规格:27G)进行移植(皆是0.2mL/个体)。将各组设为细胞A组(10个体)、培养液组(10个体)、HeLa S3组(5个体)。此外,细胞A是将利用与实施例1相同方法所培养的细胞准备3系统,分别以1x107cells/mL悬浮在α-MEM培养基中,以不包含空气的方式分注至2ml的CryoTubes。在分注的约3小时后,去除α-MEM培养基,再次悬浮在培养培养基液中,将3系统每次等量细胞数地进行混合,制备5x107 cells/mL的悬浮液,在移植前保存在4℃,在制备开始后2小时内进行移植。此外,将之前已测量的3系统进行混合的检体的移植后的存活率是75%。
各组的小鼠是依循「公益财团法人实验动物中央研究所/关于本所中的实验动物的饲育管理的作业基准」,从移植起饲育13周,观察一般状态,每周进行一次体重测量及结节的观察/尺寸测量。在观察结节之际,利用指头触诊被试验物质移植部位,在有硬触感的情形设为确认到结节的形成,利用游标卡尺测量长径(L)与短径(W)。结节体积(V)是使用「裸小鼠与抗癌剂评价」(野口达次、樱井钦夫、稻叶实编着,蟹书房,1991年6月)的肿瘤体积简易计算式,利用计算式V=LW2/2进行计算(单位是长径与短径是mm,体积是mm3)。此外,HeLaS3组的个体因为在观察期间中确认到结节重量(将比重设为1,由体积计算)超过体重的1/10,所以在第5周~第8周的时间点进行人道的终点,结束观察并处以安乐死。
在处以安乐死之际,小鼠在异氟烷麻醉下通过全放血使其安乐死,观察胸腔内、腹腔内各脏器。并且,采取包含皮下组织的移植部位皮肤,利用10%中性缓冲福尔马林液固定。福尔马林固定标本是利用通常方法在石蜡包埋后进行薄切,并进行HE染色、抗HLA(Human Leucocyte Antigen,人类白细胞抗原)免疫染色,利用光学显微镜观察。
结果
将其结果显示于图6。图6是取代显示致癌性评价的图片的图表。在HeLa S3组中,在移植后3周确认到在100%(5/5例)的个体中形成结节,但在培养液组以及细胞A组中,直到观察结束为止未确认到结节的形成。在HeLa S3组的全部例中形成结节。此结节因表示连续的体积增加且与低分化型癌的组织影像一起显示抗HLA反应,所以判断是源自HeLa S3细胞的肿瘤。在细胞A组中,通过病理组织检查,在6/10例中确认到显示抗HLA反应阳性的纤维增殖,判断移植细胞残存。但是,经过全观察期间,在全部例未确认到结节的形成,在病理组织检查中的HE染色影像中,未确认到会被认为是过度增生变化或肿瘤性变化的结果。此结果表示在本试验的条件下细胞A不显示致肿瘤性。
实施例8
(在人类脊髓损伤患者的效果探讨:GDNF的效果)
从各个自体皮肤培养自体细胞,使用酶ACCUTASE(GIBCO A11105)从培养皿剥离已使用SAG等不使GDNF增加的培养细胞。在事前利用腰椎穿刺采取1ml的脊髓液后,将悬浮在1ml的注射用生理食盐液中的剥离细胞1x107个自体移植到穿刺轨迹。此病例1~7的脊损患者7位的年龄是32~51岁,男性6位,女性1位。投予次数是2~3次,投予间隔是1~3个月。从损伤起至投予为止的期间是3~23年,C4~7是颈髓损伤且四肢麻痹。副作用是投予日会头痛、发烧。7位中的3位无治疗效果。其他4位确认治疗效果,有手臂的流汗、皮肤感觉恢复、尿的储存改善、握力提升、指头的动作改善等自觉症状。仅病例8进行细胞的SAG处理,移植已使GDNF mRNA增加的细胞。在1个月内投予2次与上述相同的细胞数。病例8的患者是47岁男性,胸髓Th3损伤,从损伤起至投予为止的期间是2年半,且下肢麻痹。副作用是投予时会头痛。治疗效果在投予1个月后出现,变得在辅助下能站立行走。相较于病例1~7,病例8是下肢麻痹和轻症,且在投予前仅短短2年半,但效果是戏剧性的且恢复时间也短。
产业上的可利用性
本发明能利用在医药产业。
序列表自由文本
序列编号1引物
序列编号2引物
序列编号3引物
序列编号4引物
序列编号5引物
序列编号6引物
Claims (7)
1.一种培养细胞的制造方法,其特征在于,包含:培养工序,其在包含SAG的无血清培养基中培养人类皮肤来源干细胞,且所述培养细胞包含胶质细胞源性神经营养因子(GDNF)mRNA以及CD73蛋白质。
2.根据权利要求1所述的方法,其特征在于,所述无血清培养基更包含嘌吗啡胺(Purmorphamine)以及音猬因子(Sonic hedgehog,SHH)蛋白质的任一方或两方。
3.根据权利要求1所述的方法,其特征在于,所述无血清培养基更包含B-27添加剂。
4.根据权利要求1所述的方法,其特征在于,所述无血清培养基更包含ROCK抑制剂。
5.根据权利要求1所述的方法,其特征在于,所述无血清培养基更包含EGF或FGF2。
6.根据权利要求1所述的方法,其特征在于,所述无血清培养基的钙离子浓度是0.03mM以上且0.12mM以下。
7.根据权利要求1所述的方法,其特征在于,所述人类皮肤来源干细胞是将所采取的人类皮肤以分散酶处理、胰蛋白酶处理以及胶原酶处理的顺序进行而得的细胞。
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