CN1878861B - 分离和培养来自冻存脐带血的间充质干细胞的方法 - Google Patents
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Abstract
本发明涉及使用冻存的脐带血分离和培养最适合用于细胞治疗的间充质干细胞的方法。该方法包括将冻存的脐带血的解冻并向其中加入αMEM(α-极限必需培养基),随后离心收集单核细胞;自获得的单核细胞中分离CD133阳性细胞;并将分离的细胞在α MEM中进行悬浮培养,培养基中含有干细胞因子、GM-CSF(粒细胞巨噬细胞集落刺激因子)、G-CSF(粒细胞集落刺激因子)、IL-3(白细胞介素-3)和IL-6(白细胞介素-6)。
Description
技术领域
本发明涉及利用对细胞治疗而言最为理想的脐带血来分离和培养间充质干细胞(mesenchymal stem cells)的方法。更具体地,其涉及利用冻存于-196℃的脐带血来分离和培养间充质干细胞的可重复性(reproducible)方法。
背景技术
间充质干细胞是指能够分化为例如骨、软骨、脂肪组织、神经和肌肉等的原始细胞,已知骨髓中含有大量的间充质干细胞。实际上,当前由骨髓中分离间充质干细胞,然后用于特定目的的研究或广泛用于多种疾病的临床试验。
尽管可自骨髓中容易地获得间充质干细胞,但在获得骨髓方面存在困难。此外,目前还难以解决与将干细胞植入其他个体时所出现的免疫排斥反应有关的问题。
而相对于骨髓而言,脐带血比较容易获得,同时,如果获得了数量巨大的脐带血单位,则有可能使用与患者的组织相容性基因相同或最为相似的脐带血干细胞,并由此可能解决与免疫排斥有关的问题。不过,相对于骨髓而言,自脐带血获得间充质干细胞比较困难,且因此在研究和临床应用方面均存在困难。
通常,一直主要采用以密度梯度离心方法自生产后24小时内的脐带血中分离并培养间充质干细胞的方法。不过,如果将密度梯度离心方法应用于冻存脐带血,则难以分离细胞,且易于造成细胞丢失,因此使得很难培养脐带血中的那些极少量的间充质干细胞。
作为分离和培养间充质干细胞的常规方法,可参照美国专利5,197,985和5,486,359,其公开了通过自人类骨髓分离间充质干细胞并培养这些细胞以增殖间充质干细胞的方法。也就是说,美国专利5,197,985涉及用于诱导来自人类骨髓的间充质干细胞分化为骨形成细胞的方法,其包括:通过将骨髓样品加到含有刺激间充质细胞生长但不刺激其分化的因子的培养基中,并在培养过程中仅容许间充质干细胞选择性粘附于一种基质表面,由此提供来自人类骨髓的分离的、纯化的、并培养扩增的间充质干细胞;将所述来自人类骨髓的分离的、纯化的、并培养扩增的间充质干细胞加到多孔载体上;并将含有培养扩增的来自人类骨髓的间充质干细胞的多孔载体植入含有使得人类间充质干细胞分化为骨细胞所必需的因子的环境中。在该方法中,所述多孔载体包含羟磷灰石和磷酸三钙(tricalcium phosphate),且其中所述培养基包括添加了胎牛血清(FBS)的BGJb培养基或F-12Nutrient Mixture。此外,美国专利5,486,359公开了能够分化为一种以上组织类型(例如骨、软骨、肌肉或骨髓基质)的细胞的分离的人类间充质干细胞,分离、纯化和培养扩增人类间充质干细胞的方法,以及此类细胞的表征和用途,特别是其研究试剂、诊断和治疗用途。在该专利中,间充质干细胞来自骨髓,并培养于含有胎牛血清的BGJb培养基中。
发明内容
技术问题
因此,本发明是针对培养间充质干细胞中的问题而做出的,本发明的一个目的在于提供一种用于分离和培养间充质干细胞的可重复性方法,其中可自冻存于-196℃的脐带血获得间充质干细胞,而不会在密度梯度离心过程中造成细胞丢失。
技术方案
根据本发明的一个方面,提供了一种用于自冻存的脐带血中分离间充质干细胞并培养这些细胞的方法,所述方法包括以下步骤:
将冻存的脐带血解冻并向其中加入αMEM(α-极限必需培养基),随后通过离心收集单核细胞;
自获得的单核细胞中分离CD133阳性细胞;并
将分离的细胞在αMEM中进行悬浮培养,培养基中含有干细胞因子、GM-CSF(粒细胞巨噬细胞集落刺激因子)、G-CSF(粒细胞集落刺激因子)、IL-3(白细胞介素-3)和IL-6(白细胞介素-6)。
本发明提供了自冻存于-196℃的脐带血单位中分离和培养间充质干细胞的可重复性方法。也就是说,其目的在于通过找出自细胞数相对较少的脐带血中分离和培养间充质干细胞的最佳条件,从而有助于使用脐带血来治疗难治性疾病。
为实现这一目的,通过自冻存的脐带血中获得单核细胞,分离CD133阳性细胞,然后将这些细胞培养于含有干细胞因子、GM-CSF、G-CSF、IL-3和IL-6的αMEM中,由此能够保护原始间充质干细胞,并提高细胞培养的成功率,由此完成了本发明。自含有与单核细胞相混合的红细胞的冻存的脐带血中分离单核细胞十分困难。因此,必须选择并培养被认为含有干细胞的CD133阳性细胞。根据本发明的方法,细胞培养的成功率可高达大约90%。尚未报道可用常规方法自冻存的脐带血或骨髓中培养干细胞。
附图说明
根据下面的详细描述并结合附图,可更加清楚地了解本发明的上述的以及其他的目的、特征和其他优点。
在附图中,图1至图6所示的照片(X100)分别是根据本发明的方法培养来自冻存的脐带血的间充质干细胞5、7、10、14、20和25天后的结果。
最佳实施方式
现结合以下实施例来更加详细地描述本发明的用于自冻存的脐带血中分离和培养间充质干细胞的方法。提供这些实施例的目的仅在于示例性地说明本发明,不能将其理解为是对本发明的范围和实质的限制。
实施例
实施例1:分离和培养间充质干细胞
将冻存于-196℃的脐带血单位在37℃水浴中解冻。为了自脐带血分离单核细胞,将脐带血以2倍体积的αMEM(α-极限必需培养基,JeilBiotech Services,Korea)进行稀释,并以300xg在室温离心10min。收集分离的血膜层(buffy coat layer),再以2倍体积的αMEM进行稀释,覆盖在Ficoll-Hypaque上,并以300xg在室温离心30min。
Ficoll-Hypaque是Ficoll(蔗糖聚合物)和Hypaque(sodium ditrizoate)的聚合物,其被广泛用于自血液中分离单核细胞。Ficoll-Hypaque的比重为1.077g/ml,其比单核细胞重但比红细胞轻,因此可以根据单核细胞与红细胞之间的不同比重而将两者分离。也就是说,如果将血液置于Ficoll-Hypaque上并离心,单核细胞会聚集在Ficoll-Hypaque上。
将通过密度梯度离心方法所获得的单核细胞以没有混合添加物的αMEM洗涤2次。
使用分离试剂盒(Miltenyi Bioteck,Germany),按照如下方法自获得的单核细胞中选择CD133阳性细胞:在单核细胞中加入100μl的封闭试剂以去除非特异性结合,然后与100μl的CD133/微珠混合。将混合物在4℃培养30分钟。在培养物中加入10倍体积的PBS(D-磷酸缓冲盐,JeilBiotech Services,Korea),300xg离心10分钟,然后弃掉PBS以获得粘附于管子上的细胞。以500μl的PBS重悬细胞。以3ml的PBS洗涤分离试剂盒的柱子,然后将重悬的细胞加到柱上并在柱上保持15分钟。将柱子以PBS漂洗4次,自试剂盒中取下,然后置于管子中,加入PBS,随后以活塞进行冲洗以选择阳性细胞。
随后,向含有抗生素(1000U/ml青霉素G,1000μg/ml硫酸链霉素,Gibco-BRL)、抗真菌剂(0.25μg/ml两性霉素B)和2mM的谷氨酰胺(Sigma)的αMEM中加入20%胎牛血清(FBS,Jeil Biotech Services,Korea)、干细胞因子(50ng/ml)、GM-CSF(粒细胞巨噬细胞集落刺激因子:10ng/ml)、G-CSF(粒细胞集落刺激因子:10ng/ml)、IL-3(白细胞介素-3:10ng/ml)和IL-6(白细胞介素-6:10ng/ml),并将细胞以1×106/cm2的浓度重悬。
培养5天后,自培养细胞组中去除悬浮的细胞。获得粘附的细胞后,将其在含有20%胎牛血清和抗生素的αMEM中培养25天,每隔2天更换全部培养基,不进行洗涤。
图1至图6所示的照片(X 100)分别是根据本发明的方法将来自冻存的脐带血的间充质干细胞培养5、7、10、14、20和25天后所观察到的结果。如图所示,在培养分离自脐带血的单核细胞时,培养后5天,观察到粘附于瓶底并在瓶底生长的细胞,在第7天,细胞形成集落并生长成具有不同形状的细胞。培养后10天,单核细胞分化为纺锤状细胞,而在培养后25天,这些纺锤状细胞通过细胞分裂和扩增成为彻底的间充质干细胞,由此完成了培养。
实施例2:培养的间充质干细胞的细胞表面抗原的表征
为了确定上述分离和培养的纺锤状间充质干细胞的细胞表面抗原的特征,通过FACS对细胞表面抗原进行分析。结果显示于表1。通过将发光的免疫抗原指示剂附着在细胞表面,将FACS(荧光激活的细胞分选;一种流式细胞仪)用于分析细胞的特征,或可根据所需的目的分离具有特定抗原指示剂的分离的细胞。
表1
指示剂 | 反应 |
CD14 | - |
CD34 | - |
CD45 | - |
SH2 | + |
指示剂 | 反应 |
SH3 | + |
C29 | + |
CD44 | + |
CD90 | + |
CD166 | (+) |
表1证实,对于根据本发明所分离和培养的干细胞,CD34、CD45和CD14(造血干细胞的特征性指示剂)呈阴性反应,SH2、SH3、CD29和CD44(间充质干细胞的特征性指示剂)呈阳性反应,而CD166呈弱阳性反应。对这一事实的解释为,其说明根据本发明所分离并培养的细胞是间充质干细胞。
实施例3:比较间充质干细胞培养的成功率
分别根据常规的方法和本发明的方法培养50单位的脐带血,并比较两者的细胞培养的成功率。结果显示于下面的表2。
具体而言,在常规方法中,脐带血被用于干细胞培养,其中对所用的脐带血的量以及自分娩至收集的时间均没有设定标准。此外,常规方法的培养基中没有添加细胞生长因子,除此之外,采用的细胞的分离过程与本发明的方法相似。
表2
常规方法 | 本发明的方法 | |
所得的间充质干细胞单位的数量 | 0 | 49 |
培养成功率(%) | 0 | 98 |
从以上表2可以看出,通过对间充质干细胞培养的成功率进行比较发现,常规方法的成功率为0%,而本发明具有98%的高成功率。
工业实用性
如上所述,根据本发明,可以自细胞数相对缺乏的脐带血中有效地分离并培养间充质干细胞,因此,可将原本无用而被废弃的脐带血作为细胞治疗的一种重要的途径,用于治疗各种难治性疾病。
尽管已经示例性地公开了本发明的优选的实施方式,但本领域普通技术人员应该理解,在不脱离如所附权利要求书所述的本发明的范围和实质的情况下,可以存在各种更改、添加和替换。
Claims (3)
1.用于自冻存的脐带血中分离和培养间充质干细胞的方法,所述方法包括以下步骤:
将冻存的脐带血解冻并向其中加入αMEM(α-极限必需培养基),随后离心收集单核细胞;
自获得的单核细胞中分离CD133阳性细胞;并
将分离的细胞在αMEM中进行悬浮培养,所述αMEM中添加了干细胞因子、GM-CSF(粒细胞巨噬细胞集落刺激因子)、G-CSF(粒细胞集落刺激因子)、IL-3(白细胞介素-3)和IL-6(白细胞介素-6)。
2.权利要求1的方法,其中将所述脐带血与2倍体积的αMEM混合,覆盖在Ficoll-Hypaque上,然后进行离心以收集单核细胞。
3.权利要求1的方法,其中所述用于培养单核细胞的αMEM还添加了抗生素、抗真菌剂、谷氨酰胺和胎牛血清。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2003-0079363 | 2003-11-11 | ||
KR10-2003-0079363A KR100534215B1 (ko) | 2003-11-11 | 2003-11-11 | 냉동 보관된 제대혈로부터 중간엽 줄기세포의 분리 및 배양 방법 |
KR1020030079363 | 2003-11-11 | ||
PCT/KR2004/002716 WO2005045011A1 (en) | 2003-11-11 | 2004-10-25 | Method of isolating and culturing mesenchymal stem cell derived from cryopreserved umbilical cord blood |
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EP (1) | EP1687415B1 (zh) |
KR (1) | KR100534215B1 (zh) |
CN (1) | CN1878861B (zh) |
AT (1) | ATE465243T1 (zh) |
DE (1) | DE602004026764D1 (zh) |
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JP2004531256A (ja) | 2001-04-24 | 2004-10-14 | バクシュ,ドロレス | 前駆細胞集団、その増殖、及びそれからの非造血細胞種及び組織の成長 |
US7807458B2 (en) | 2003-01-30 | 2010-10-05 | The United States Of America As Represented By The Secretary Of The Department Of Veterans Affairs | Multilineage-inducible cells and uses thereof |
CN100377165C (zh) * | 2003-04-24 | 2008-03-26 | 皇家飞利浦电子股份有限公司 | 无创式左心室的容积测定 |
DK1725656T3 (da) * | 2004-03-05 | 2014-01-13 | John E Davies | Serumfri suspensionskultursystem til mesenchymale progenitorceller |
KR100679950B1 (ko) * | 2005-05-18 | 2007-02-08 | 한훈 | 제대혈 유래 줄기세포와 만니톨을 이용한근위축성측삭경화증 환자의 세포치료용 조성물 |
AU2006202318A1 (en) * | 2005-06-02 | 2006-12-21 | Wing-Yee Chan | The preparation of multipotent stem cells and the use thereof |
AU2007209870A1 (en) | 2006-01-30 | 2007-08-09 | University Of Virginia Patent Foundation | Methods of preparing and characterizing mesenchymal stem cell aggregates and uses thereof |
WO2008016196A1 (en) * | 2006-08-02 | 2008-02-07 | Intromedic. Co., Ltd. | An endoscope and a method for moving it |
AU2007320031B2 (en) | 2006-10-06 | 2013-05-02 | University Of Virginia Patent Foundation | Methods and compositions useful for diabetic wound healing |
EP2488187A4 (en) * | 2009-10-13 | 2014-03-26 | Allocure Inc | ASSAY FOR PREDICTING THE THERAPEUTIC EFFECT OF MECHANICAL CYCLES AND METHOD OF USE THEREOF |
EA029577B1 (ru) | 2011-07-06 | 2018-04-30 | Селл Терапи Лимитед | Клетки-предшественники мезодермальной линии |
US8940294B2 (en) | 2012-03-02 | 2015-01-27 | Tissuetech, Inc. | Methods of isolating and culturing stem cells |
AR093662A1 (es) * | 2012-11-30 | 2015-06-17 | Pharmacosmos As | Agentes crioprotectores, composiciones crioprotectoras y criopreservadas, sus usos y metodos de criopreservacion |
CN114469876B (zh) * | 2022-01-18 | 2023-11-21 | 济南万泉生物技术有限公司 | 一种用于皮肤组织损伤的细胞因子冻干粉的制备方法 |
CN115109748A (zh) * | 2022-07-29 | 2022-09-27 | 四川新生命干细胞科技股份有限公司 | 解冻后脐带血造血干细胞活率和集落形成能力的检测方法 |
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US5197985A (en) | 1990-11-16 | 1993-03-30 | Caplan Arnold I | Method for enhancing the implantation and differentiation of marrow-derived mesenchymal cells |
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KR100560340B1 (ko) | 2003-11-11 | 2006-03-14 | 한훈 | 제대혈로부터 중간엽 줄기세포의 분리 및 배양 방법 |
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- 2004-10-25 CN CN2004800330364A patent/CN1878861B/zh not_active Expired - Fee Related
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US6335195B1 (en) * | 1997-01-28 | 2002-01-01 | Maret Corporation | Method for promoting hematopoietic and mesenchymal cell proliferation and differentiation |
CN1330142A (zh) * | 2001-07-09 | 2002-01-09 | 中国人民解放军军事医学科学院野战输血研究所 | 人骨髓和脐带血间充质干细胞体外分离扩增和向神经细胞定向诱导的方法 |
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Publication number | Publication date |
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KR100534215B1 (ko) | 2005-12-08 |
US20070105221A1 (en) | 2007-05-10 |
EP1687415A4 (en) | 2007-10-17 |
US7582477B2 (en) | 2009-09-01 |
KR20050045326A (ko) | 2005-05-17 |
ATE465243T1 (de) | 2010-05-15 |
CN1878861A (zh) | 2006-12-13 |
EP1687415A1 (en) | 2006-08-09 |
DE602004026764D1 (de) | 2010-06-02 |
WO2005045011A1 (en) | 2005-05-19 |
EP1687415B1 (en) | 2010-04-21 |
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