CN107075476B - 使用水凝胶诱导干细胞三维成骨分化的方法 - Google Patents
使用水凝胶诱导干细胞三维成骨分化的方法 Download PDFInfo
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Abstract
本发明涉及诱导间充质干细胞成骨分化的方法,更具体地,涉及使用多孔膜和可生物降解的合成生物凝胶培养细胞的短时间成骨分化方法,其中细胞不接触细胞培养容器。与常规的成骨分化方法相比,本发明可以显著缩短成骨分化的诱导期,并且具有细胞在分化后也容易分离的效果。
Description
技术领域
本发明涉及在间充质干细胞的成骨分化诱导中在短时间内在合成生物凝胶上的成骨分化方法。
背景技术
由于组织工程和再生医学的最新发展,能够以与常规方法不同的方式开发能够治疗受损组织和器官的方法,并且通过间充质干细胞的细胞治疗受到最大的关注。干细胞是指可以在未分化状态下无限增殖以及在特定环境和条件下分化以具有特定功能和形状的细胞。干细胞的实例是:源自人胚胎的胚胎干细胞;和成体干细胞,例如不断产生血细胞的骨髓细胞。胚胎干细胞可以分化成构成人体的所有细胞和组织,但是出于伦理原因,其使用受到限制。另一方面,成体干细胞从脐带血或完全生长的成年人的骨髓和血液中提取,使得在体内移植后能够分化成特定组织和器官,并且具有分化的灵活性以转分化成不同于原始细胞特征的其他组织的细胞。成人细胞广泛用于组织工程而没有伦理限制。近年来,通过生长干细胞,然后将干细胞分化为特定细胞,在医疗领域中正在进行各种尝试和早期临床试验,以用于患者的组织或器官的再生和置换。间充质干细胞是发育后存在于身体各种器官或血液中的一种类型的成体干细胞,并且是易于维持且没有伦理问题的细胞来源。目前间充质干细胞是再生医学领域中最显著的干细胞,但是缺点在于与胚胎干细胞相比,间充质干细胞在体外传代培养和分化潜能方面具有限制。
对人和动物的研究已经证实,来自成体干细胞的骨髓来源的干细胞分化成成骨细胞(Friedenstein A.J.et al.,Transplantation.,6:230-247,1968),并且最近的研究已经进行了用于培养从骨髓分离的干细胞以将干细胞分化为成骨细胞的方法,并且使用该方法的临床应用的可能性增加(Ohgushi H.et al.,J.Biomed Mater Res.,48:913-927,1999)。最近,主要研究了从间充质干细胞分化成骨细胞的方法。
另一方面,用于在二维孔板中培养和分化细胞的方法目前最广泛地用于干细胞的分化。然而,最近的论文报道,与三维细胞培养相比,二维(单层)细胞培养降低细胞功能并显着改变形态(Proc.Natl.Acad.Sci.USA,100:1943-1948,2003;Cell,111:923-925,2002;Cancer Cell 2:205-216,2002)。以如上所述不利地影响细胞状态的方式进行的细胞培养和分化导致分化困难并且需要很长时间。为了克服这种细胞培养的缺点,韩国专利第10-0733914号公开了一种三维微细胞培养系统,其特征在于细胞存在于三维凝胶中,但是在干细胞的培养或分化后,凝胶需要溶解以分离存在于凝胶中的细胞,引起严重的细胞损伤。
发明内容
技术问题
在常规二维细胞培养容器中的成骨分化诱导具有缺点,即只有一部分干细胞与培养基接触,因此,分化所需的营养物、诱导成分和空气的流入不容易,从而使分化更加困难并导致分化周期更长,例如三至五周。在凝胶内培养细胞的常规三维培养方法具有不能分离细胞的缺点。因此,需要开发一种三维细胞培养系统,以促进干细胞成骨分化后的分化细胞的分化和使用。
技术方案
因此,为了增加细胞与培养基的接触表面积以促进成骨分化,同时容易地分离细胞,本发明的一个方面提供一种方法,其中细胞以非接触的方式存在于细胞培养容器内部,并且在所有方向上与培养基接触以促进成骨分化,从而缩短成骨分化的时间。
有益效果
在使用本发明的诱导干细胞的成骨分化的方法进行干细胞的成骨分化的情况下,干细胞可以与具有更宽表面积的培养基接触,从而促进干细胞的成骨分化诱导,因此与常规的成骨分化方法相比,显著缩短了成骨分化诱导的时间;虽然细胞粘附在水凝胶的内外表面,但凝胶相水凝胶在低于细胞培养温度(37℃)的温度下变为溶胶相,因此即使分化后细胞也容易分离。
附图说明
图1示出了根据本发明的用于在三维状态中分化干细胞的方法的示意图。
图2示出了使用根据常规技术的培养皿在二维状态中分化干细胞的方法的示意图。
图3示出了仅使用没有水凝胶的聚合物膜的干细胞三维分化的方法的示意图。
图4证实了通过比较例1的方法(14天:对照1,5天:对照II)、比较例2的方法(泳道3,水凝胶浓度0%)和实施例的方法诱导的骨髓来源的间充质干细胞的成骨分化。
图5证实了通过比较例1的方法、比较例2的方法(泳道2)和实施例的方法诱导的骨髓来源的间充质干细胞的成骨分化。
图6证实了通过比较例2(对照)的方法、比较例2的方法+100ng/ml BMP2或20ng/mlWnt3a和实施例的方法诱导的脐带间充质干细胞的成骨分化程度。
图7证实了通过实施例的方法诱导1天、3天、5天或7天的脐带间充质干细胞的成骨分化程度。
图8示出了通过比较例1的方法、比较例2的方法(没有水凝胶)和实施例的方法,骨髓间充质干细胞(BMMSC)、脂肪来源的间充质干细胞(ADMSC)、脐带间充质干细胞(UCMSC)、胚胎干细胞衍生的间充质干细胞(ESMSC)和牙周膜细胞(PDL)的成骨分化诱导结果。
具体实施方式
最佳实施方式
在下文中,将参考以下实施例详细描述本发明。然而,本发明可以以各种不同的形式实现,因此不限于本文所描述的实施例。
根据本发明的一个方面,提供在细胞培养容器中诱导干细胞成骨分化的方法,其中细胞培养容器中,以非接触方式放置多孔膜,其中多孔膜一个表面包被有水凝胶。
在一个实施方案中,本发明可以诱导干细胞的成骨分化,包括:
以非接触方式将多孔膜置于细胞培养容器内;
在多孔膜的一个表面上施加水凝胶溶液以通过溶胶-凝胶相转变在其上包被水凝胶;
在包被的水凝胶上接种干细胞;和
在成骨分化诱导培养基中培养干细胞。
在一个实施方案中,方法可以进一步包括在干细胞接种后在成骨分化诱导培养基中培养干细胞之前,在成骨分化预处理培养基中培养干细胞10-24小时的步骤。预处理培养基可以具有1.5g/l或更低的葡萄糖浓度。
在一个实施方案中,多孔膜可以以非接触方式与细胞培养容器的底部平行放置。水凝胶在多孔膜上的包被顺序和多孔膜以非接触方式放置在细胞培养容器内部的顺序没有特别限制。在这种情况下,多孔膜可以以非接触方式放置在细胞培养容器的底部上方,然后可以在其上包被水凝胶,或者可以预先将水凝胶包被在多孔膜上,然后包被的多孔膜可以以非接触方式放置在细胞培养容器内。
本发明的细胞培养容器通常指用于细胞培养的皿或孔板,并且细胞培养容器没有特别限制,只要其用于细胞培养并且可以将多孔膜以非接触方式引入容器底部。
在一个实施方案中,细胞在细胞培养容器内非水平培养以诱导干细胞成骨分化的情况下,可以将水凝胶溶液施加在多孔膜的一个表面上,细胞可以粘附到水凝胶,然后将所得膜以非接触方式置于细胞培养容器内部以诱导成骨分化。
在一个实施方案中,干细胞可以在成骨分化诱导培养基中培养3-7天。
在一个实施方案中,培养基在多孔膜和细胞培养容器之间流动,以增加干细胞和培养基之间的接触表面积,从而缩短成骨分化诱导的时间。本发明的多孔膜和水凝胶允许空气和培养基的透过,并且培养基流动以填充多孔膜和细胞培养容器之间的空间(该空间是由多孔膜和细胞培养容器以非接触方式放置而产生的),使得培养基和空气流入细胞的粘附部分中,从而干细胞与具有较宽表面积的培养基和空气接触,从而缩短成骨分化诱导时间。例如,现有的成骨分化方法需要三至五周,但是通过本发明的方法成骨分化在3-7天内发生。
本发明的细胞培养容器通常指用于细胞培养的皿或孔板,并且细胞培养容器没有特别限制,只要其用于细胞培养并且可以以非接触方式在容器中引入多孔膜。
在一个实施方案中,水凝胶溶液(水溶胶)溶胶-凝胶相转变成水凝胶可以在37℃进行1至2小时。
在一个实施方案中,水凝胶可以包括1-40%的水凝胶,更优选1-15%的水凝胶。对于0%水凝胶,干细胞的分化不发生;并且对40%或更多水凝胶,成骨分化的效率不增加,因此40%或更多水凝胶没有意义。然而,水凝胶的浓度不限于此。
在一个实施方案中,1-40%水凝胶可以以200-300μl/cm2包被在多孔膜上,并且通过溶胶-凝胶相变形成的水凝胶的厚度可以为1-4mm。
在一个实施方案中,根据水凝胶的浓度(%),水凝胶在37℃的粘度可以为1.E+00至1.E+06(100至106)mPa·s。在孔径超出该范围的水凝胶中,干细胞的粘附或分化可能困难。本发明的水凝胶在37℃的细胞培养温度下处于凝胶相,因此在凝胶内外培养细胞。本发明的水凝胶在低于细胞培养温度的温度下变为溶胶相,利于细胞分化后的细胞分离。
在一个实施方案中,多孔膜可以具有0.1-8μm的孔径,但是孔径不受限制,只要孔径为培养基和空气可以通过多孔膜但是水凝胶不能通过多孔膜。
在一个实施方案中,干细胞可以是脐带间充质干细胞、脂肪来源的间充质干细胞、胚胎干细胞来源的间充质干细胞、牙周韧带细胞或骨髓来源的间充质干细胞。干细胞的来源没有特别限制,其实例可以是来源于人、猴、猪、马、牛、绵羊、狗、猫、小鼠或兔的细胞。干细胞优选是源自人的干细胞,但不限于此。
如本文所用,术语“多孔膜”或“聚合物膜”是指培养基或空气通过但是水凝胶不能通过的多孔膜、可透过膜或膜型材料。细胞培养基和空气通过的任何多孔结构没有特别限制。
如本文所用,术语“水凝胶”是指这样的材料,包含水作为分散介质的液体通过溶胶-凝胶相转变而固化,从而失去流动性并形成多孔结构。适合于细胞粘附和培养的任何水凝胶没有特别限制,在本发明的一个实施方案中,使用可生物降解的合成生物凝胶。
如本文所用,术语“干细胞”是指具有自我更新和分化能力的未分化细胞。干细胞根据其分化能力包括多能(pluripotent)干细胞、专能(multipotent)干细胞和单能(unipotent)干细胞的亚组。多能干细胞是指具有分化为构成活生物体的所有组织或细胞的能力的细胞,专能干细胞是指具有分化为多种而不是所有组织或细胞的能力的细胞。单能干细胞是指具有分化成特定组织或细胞的能力的细胞。多能干细胞可以包括胚胎干细胞(ES细胞),胚胎生殖细胞(EG细胞),诱导多能干细胞(iPS细胞)等。专能干细胞可以包括成体干细胞,例如间充质干细胞(例如,来自脂肪、骨髓、脐带血或脐带等),造血干细胞(来自骨髓或外周血),神经干细胞,生殖干细胞等。单能干细胞可以包括用于肝细胞的定向干细胞,其通常是具有低自我更新能力的休眠状态,但在某些条件下有力地分化成肝细胞。
根据本发明的一个方面,提供一种用于干细胞分化的装置,其特征在于,包括:
细胞培养容器;和
多孔膜,其被配置为一个表面上附有水凝胶,细胞粘附到所述水凝胶,
其中附有水凝胶的所述多孔膜以非接触方式放置在所述细胞培养容器的内部。
在一个实施方案中,多孔膜和水凝胶可以允许空气和培养基透过。
具体实施例
通过以下实施例更详细地描述本发明的内容。然而,提供以下实施例仅用于说明本发明而不限制本发明的范围。
[实施例]
实施例1:三维状态的干细胞分化方法
为了促进干细胞的分化,进行了三维分化的试验。为此,提供厚度为0.4-1μm的聚合物膜(Corning,USA),以便以非接触方式与细胞培养皿或孔的底部水平。将可生物降解的合成生物凝胶(BASF,德国)溶解于无菌的三级蒸馏水中,以制备具有各种浓度(%)的凝胶。然后,将制备的可生物降解的合成生物凝胶以250μl/cm2包被在聚合物膜上,然后在37℃下固化1小时30分钟,由此制备细胞培养容器。溶胶-凝胶相转变后的生物降解性的合成生物凝胶的厚度为1mm以上,平均为2.5mm。在CEFOgro ADMSC培养基(CB-ADMSC-GM,CEFO,韩国)中的脂肪来源的间充质干细胞、CEFOgro BMMSC培养基(CB-BMMSC-GM,CEFO,韩国)中的骨髓来源的间充质干细胞、CEFOgro ESMSC培养基(CB-ESMSC-GM,CEFO,韩国)中的胚胎干细胞来源的间充质干细胞、CEFOgro UCMSC培养基(CB-UCMSC-GM,CEFO,韩国)中的脐带间充质干细胞、CEFOgro PDL培养基(CB-PDL-GM,CEFO,韩国)中的牙周韧带细胞,在37℃的CO2培养箱中培养3-4天。然后,将各种干细胞接种在生物凝胶上,添加成骨分化前处理培养基(CB-DM-Osteo-PT,CEFO,韩国),在37℃的CO2培养箱中培养18小时。然后,用成骨分化诱导培养基(CB-DM-Osteo,CEFO,韩国)交换培养基,然后在CO2培养箱中在37℃诱导成骨分化5天(图1)。
比较例1:二维状态的干细胞分化方法
通过作为常规干细胞分化方法的二维分化方法分化干细胞。具体地,将细胞接种在12孔细胞培养容器(皿)中,并在预处理培养基(CB-DM-Osteo-PT,CEFO,韩国)中培养,用于诱导成骨分化直至细胞密度达到85-90%。然后,用成骨分化诱导培养基(CB-DM-Osteo,CEFO,韩国)交换培养基,随后进行成骨分化诱导14-21天(图2)。
比较例2:在没有水凝胶的三维状态下的干细胞分化方法
为了研究当实施例1中的聚合物膜上的可生物降解的合成生物凝胶为0%(仅存在聚合物膜)时,干细胞的分化,将干细胞接种在聚合物膜上,然后像实施例1一样培养5天(图3)。
试验例1:骨髓来源间充质干细胞的成骨分化的验证
将通过比较例1的方法诱导14天或5天经历成骨分化的骨髓来源间充质干细胞、通过比较例2的方法(0%生物降解性合成生物凝胶)诱导5天经历成骨分化的骨髓来源干细胞以及通过上述实施例的方法(5%、10%或15%可生物降解的合成生物凝胶)诱导的经历成骨分化的骨髓来源干细胞,通过相差显微镜视觉观察成骨分化。此外,为了通过茜素红染色(Alizarin red staining)研究成骨细胞分化的细胞,将细胞用PBS洗涤两次,用70%乙醇在室温下固定10分钟,然后用三级蒸馏水洗涤两次。然后,用茜素红染色试剂盒(CB-SK-Osteo)的Sol I处理细胞,随后在室温下反应30分钟。然后,用Sol II将细胞干净清洗3次,并使用倒置显微镜(LEICA,德国)进行图像分析。此外,为了使结果数字化,在图像合成后用Sol III处理细胞,进行30分钟的反应,使得染色的试剂完全溶解。然后,取100μL溶解的溶液,置于96孔板中,在550nm测量吸光度。结果,通过比较例1的方法诱导成骨分化5天,以及通过比较例2的方法诱导成骨分化5天,不能获得成骨分化;但是通过实施例的方法诱导成骨分化5天,诱导了足够的成骨分化。特别地,可以看出,对于通过上述实施例的方法的成骨分化,成骨分化有利地在所有1至30%的水凝胶中发生,并且最佳成骨分化显示为10%的浓度(图4)。
试验例2:脂肪来源间充质干细胞的成骨分化的验证
将通过比较例1的方法诱导14天的经历成骨分化的脂肪来源间充质干细胞、通过比较例2的方法(0%生物降解性合成生物凝胶)诱导5天的经历成骨分化的骨髓来源干细胞、以及通过上述实施例的方法(5%、10%或15%可生物降解合成生物凝胶)诱导的经历成骨分化的骨髓来源干细胞,通过相差显微镜视觉观察成骨分化。此外,为了通过茜素红染色研究成骨细胞分化的细胞,将细胞用PBS洗涤两次,用70%乙醇在室温下固定10分钟,然后用三级蒸馏水洗涤两次。然后,用茜素红染色试剂盒(CB-SK-Osteo)的Sol I处理细胞,随后在室温下反应30分钟。然后,用Sol II将细胞干净清洗3次,并使用倒置显微镜(LEICA,德国)进行图像分析。结果,可以看出,通过比较例2的方法诱导成骨分化5天时,从未发生成骨分化,但是通过实施例的方法诱导成骨分化5天时,成骨分化在所有1至30%可生物降解的合成水凝胶中有利地获得,并且在5-10%的浓度下显示最佳的成骨分化(图5)。
试验例3:脐带间充质干细胞的成骨分化的验证
将通过比较例2的方法诱导5天的经历成骨分化的脐带间充质干细胞、通过比较例2的方法(用100ng/ml骨形态发生蛋白2(BMP 2,peprotech,以色列)或20ng/ml Wnt3a(peprotech,以色列)处理,然后进行成骨分化诱导5天)接种的脐带间充质干细胞、以及通过上述实施例的方法(10%可生物降解的合成生物凝胶)进行成骨分化诱导的脐带间充质干细胞,通过相差显微镜视觉观察成骨分化。此外,为了通过茜素红染色研究成骨细胞分化的细胞,将细胞用PBS洗涤两次,用70%乙醇在室温下固定10分钟,然后用三级蒸馏水洗涤两次。然后,用茜素红染色试剂盒(CB-SK-Osteo)的Sol I处理细胞,随后在室温下反应30分钟。然后,用Sol I将细胞干净清洗3次,并使用倒置显微镜(LEICA,德国)进行图像分析。此外,为了使结果数字化,在图像合成后用Sol III处理细胞,进行30分钟的反应,使得染色的试剂完全溶解。然后,取100μL溶解的溶液,置于96孔板中,在550nm测量吸光度。结果,使用本发明实施例的方法的脐带间质干细胞的成骨效率非常好,而不是比较例2的方法诱导5天并且有已知促进成骨分化的BMP和Wnt3a处理(图6)。
试验例4:脐带间充质干细胞的成骨分化期间的变化的验证
通过茜素红染色对上述实施例的方法(10%可生物降解的合成生物凝胶)进行成骨分化1、3、5或7天的脐带间充质干细胞进行图像分析。此外,为了数字化结果,在550nm测量吸光度。结果,从成骨分化诱导的第1天开始诱导成骨分化,并且随着成骨分化的时期变长,成骨分化变强。特别地,已知在常规二维方法中比其他类型的间充质干细胞经历较少成骨分化的脐带间充质干细胞,在本发明实施例的三维成骨分化方法中显示有利的成骨分化,与其他类型的间充质干细胞一样(图7)。
试验例5:各种类型的间充质干细胞的成骨分化的验证
使骨髓来源间充质干细胞、脂肪来源间充质干细胞、脐带间充质干细胞、胚胎干细胞来源间充质干细胞和牙周韧带细胞,通过比较例1的方法诱导成骨分化14天、通过比较例2的方法(0%可生物降解的合成生物凝胶)诱导成骨分化5天、或上述实施例的方法(10%可生物降解的合成生物凝胶)诱导成骨分化5天,然后通过茜素红染色研究每种类型的间充质干细胞的成骨分化程度。
结果,通过比较例1的方法和比较例2的方法诱导成骨分化5天,不能诱导间充质干细胞的成骨分化,但是无论间充质干细胞的来源如何,当成骨分化在可生物降解的合成生物凝胶上被三维诱导5天,都有利地诱导成骨分化(图8)。
如上所述,通过常规的二维成骨分化方法证实间充质干细胞的成骨分化需要约2-5周,但当使用本发明的三维成骨分化方法时,成骨分化在3-7天内发生,本发明的方法将聚合物膜以非接触方式置于细胞培养容器上方,在其上包被水凝胶,在其上接种间充质干细胞,在成骨分化预处理培养基中培养间充质干细胞,使用成骨分化诱导培养基处理间充质干细胞。
Claims (4)
1.一种用于在3-7天内从干细胞诱导成骨分化的方法,其特征在于,所述方法包括:
将多孔膜以非接触方式置于细胞培养容器内;
在所述多孔膜的一个表面上施加包括5-15%水凝胶的水凝胶溶液以通过溶胶-凝胶相转变在其上包被水凝胶;
在包被的水凝胶上接种干细胞;和
在成骨分化诱导培养基中培养所述干细胞,
其中所述水凝胶在37℃的细胞培养温度下处于凝胶相,并且所述水凝胶在37℃的粘度为1.E+00至1.E+06(100至106)mPa·s,
其中所述水凝胶在低于所述细胞培养温度的温度下处于溶胶相,
其中通过所述溶胶-凝胶相转变产生的所述水凝胶具有1mm至4mm的厚度,
其中所述多孔膜具有0.1-8μm的孔径,
其中所述多孔膜和所述水凝胶允许空气和培养基透过。
2.根据权利要求1所述的方法,其特征在于,所述培养基在所述多孔膜和所述细胞培养容器之间流动,以增加所述干细胞和所述培养基之间的接触表面积,从而缩短成骨分化诱导的时间。
3.根据权利要求1所述的方法,其特征在于,所述干细胞是脐带间充质干细胞、脂肪来源的间充质干细胞、牙周韧带细胞或骨髓来源的间充质干细胞。
4.根据权利要求1所述的方法,其特征在于,所述溶胶-凝胶相转变在37℃下进行1至2小时。
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