CN112608894A - 一种间充质干细胞培养基 - Google Patents
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Abstract
本发明属于生物技术领域,具体公开了一种间充质干细胞培养基。本发明间充质干细胞培养基包括基础培养基和添加剂,所述添加剂包括非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂。本发明间充质干细胞培养基能够为干细胞生长提供细胞生长所需的营养物质,维持细胞生长环境,维持细胞活性,促进干细胞分裂增殖,提高干细胞扩增速度,缩短干细胞培养时间;同时,还能抑制干细胞分化但不影响干细胞的分化潜能;采用本发明的培养基对干细胞进行多次传代次后,干细胞依然能够保持其原有的生物学特性和多向分化潜能。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种间充质干细胞培养基。
背景技术
间充质干细胞(mesenchymal stem cell,MSC)是一种源于中胚层的多能干细胞,能够无限制自我更新,多向分化。在合适的条件下,能在体外或者体内定向分化为脂肪细胞、软骨细胞、肌细胞等等,作为组织修复的种子细胞。在器官移植时使用,可以降低免疫排斥,提高移植的成功率,同时加快手术创口的恢复。间充质干细胞可以在体内或者体外培养扩增,并能够分泌大量的细胞因子,具有免疫调节、炎症抑制等功能,用于治疗免疫相关疾病。而且间充质干细胞基本不具备免疫原性,能够异体移植。总的来说,间充质干细胞具有不菲的临床医用价值。
间充质干细胞广泛存在于骨髓、脐带、胎盘、脂肪等组织和血液中,但是细胞数量极其有限,比如外周血中只有0.1%-0.01%的粒细胞为干细胞,1克脂肪组织仅含有约5×104个脂肪干细胞。为了满足科研和临床治疗的需要,初步分离的干细胞需要经过体外培养增殖,这是干细胞从研究到应用的一个重要阶段。目前大多数采用的是传统方法培养,这种情况下,培养基的组分通常为基础培养基,加上10%-20%的胎牛血清和一定量的抗生素,其中的血清主要功能是提供各种生长因子。然而血清的具体成分至今仍不是十分清楚,且由于其来源和批次的区别,质量和功效也存在差别,为标准化培养带来了困难。使用动物血清时,更无法完全避免来自于动物本身的感染源的污染,血清中包含的动物来源的成分因子也可能引起不良反应,给临床使用带来了风险。
曾经有研究尝试使用自体人源血清或者血小板裂解物来作为生长补充剂,虽然能够满足间充质干细胞生长所需、避免交叉污染,但是因为来源有限而无法推广。随着间充质干细胞需求增大,非常有必要开发一种新的培养基,排除动物血清的使用、减少潜在的风险,同时能够提供间充质干细胞生长所需要营养物质、最大限度的促进间充质干细胞的生长增殖。
自上世纪70年代开始,科学家就开始了无血清培养基的研究。最初,研究人员希望从血清中直接纯化各种营养物质和生长因子,但是因为血清的复杂性和技术手段的限制,这种方法无法满足实际需求。随着技术水平的发展,研究人员能够逐步了解了血清中的各种成分的功能,主要包括:营养物质、生长激素、细胞基质成分和转运蛋白等等,而且能够通过基因工程的手段大量生产这些成分,至此无血清培养基的研究开发开始取得了坚实的发展。目前市场上的比较成熟的产品分别来源于SIGMA,STEM CELL和Thermofisher等公司,但其针对不同来源的间充质干细胞,比如脂肪或者血液,都有不同的成分配方和添加剂。在此本发明充分考虑了间充质干细胞的特殊性后,并结合市场需求,开发了一种能够快速促进不同来源间充质干细胞生长的,替代传统培养基的无血清间充质干细胞培养基。
发明内容
针对现有技术中存在的问题和不足,本发明的目的旨在提供一种间充质干细胞培养基。
为实现发明目的,本发明采用的技术方案如下:
本发明第一方面提供了一种间充质干细胞培养基。所述间充质干细胞培养基由基础培养基和添加剂组成,所述添加剂包括非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂;所述基础培养基为DMEM或DMEM/F12。
根据上述的间充质干细胞培养基,优选地,所述促进细胞膜合成的有机物质成分组成及含量如下:胆固醇 10~15mg/L、卵磷脂8~16mg/L、鞘磷脂 80~100 mg/L、人低密度脂蛋白5~15mg/L、油酸20~25mg/L、乙醇胺 5~10mg/L和腐胺 1~5 mg/L;所述细胞基质成分的组成及含量如下:血清白蛋白 100~500mg/L、纤连蛋白2~5mg/L和层粘连蛋白1~5mg/L。
根据上述的间充质干细胞培养基,优选地,所述细胞生长因子的成分组成及含量如下:类胰岛素生长因子IGF-1 5~10 μg/L、表皮生长因子EGF 5~10 μg/L、肝细胞生长因子HGF 5~15μg/L、肝素结合性表皮生长因子HB-EGF 10~15 μg/L、成纤维细胞生长因子FGF2 40~50 μg/L、成纤维细胞生长因子FGF4 1~5 μg/L、成纤维细胞生长因子FGF19 5~10 μg/L、血管内皮生长因子VEGF 1~10μg/L、转化生长因子TGF-β5~10 μg/L、白血病抑制因子LIF 1~5μg/L、干细胞因子SCF 1~5 μg/L、干细胞生长因子SCGF-α 5~10 μg/L、胰岛素 25~30μg/L、血小板衍生生长因子PDGFAB 40~50 μg/L、血小板衍生生长因子PDGFBB100~150μg/L、凝血酶 10~15 U/L、转铁蛋白0.2%、红细胞生长素EPO 5~10μg/L、单核细胞趋化蛋白MCP-1 5~10μg/L、白介素-2 5~10μg/L、白介素-8 5~10μg/L、粒细胞集落刺激因子G-CSF 5~10μg/L、巨噬细胞集落刺激因子M-CSF 5~10 μg/L、脂质运载蛋白 5~10μg/L。
根据上述的间充质干细胞培养基,优选地,所述抗氧化剂的成分组成及含量为:还原型谷胱甘肽 1~1.5mg/L、β-巯基乙醇0.1mM;所述维生素的成分组成及含量为:维生素A1~10 mg/L、维生素C 5~10mg/L、维生素E 10~15 mg/L;所述微量元素的成分组成及含量为:硫酸铜20~25mg/L、氯化镁15~20 mg/L、硫酸锌 15~20 mg/L、亚硒酸钠1~5 μg/L。
根据上述的间充质干细胞培养基,优选地,所述添加剂还包括含有干细胞外分泌物的溶液,所述含有干细胞外分泌物的溶液的制备方法为:采用权利要求1 所述的间充质干细胞培养基对干细胞进行培养,培养48~72h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
根据上述的间充质干细胞培养基,优选地,所述含有干细胞外分泌物的溶液与基础培养基的体积比为5%~15%。
根据上述的间充质干细胞培养基,优选地,所述pH调节剂为HEPES,所述HEPES与基础培养基的体积比为1%。
根据上述的间充质干细胞培养基,优选地,所述抗生素与基础培养基的体积比为1%~2%。
根据上述的间充质干细胞培养基,优选地,所述基础培养基为DMEM/F12。
根据上述的间充质干细胞培养基,优选地,所述细胞基质成分、细胞生长因子中的蛋白均为人源化的蛋白,通过DNA重组技术表达获得。
根据上述的间充质干细胞培养基,优选地,所述添加成分中的各物质可以是冻干粉或是以基础培养基为溶剂的溶液。
根据上述的间充质干细胞培养基,优选地,所述非必需氨基酸与基础培养基的体积比为1%~2%,所述非必需氨基酸为MEM NEAA非必需氨基酸溶液。
本发明第二方面提供了上述第一方面所述间充质干细胞培养基在体外培养干细胞中的应用。
根据上述的应用,优选地,所述干细胞为间充质干细胞。
根据上述的应用,优选地,所述干细胞包括但不限于脐带间充质干细胞、脂肪干细胞或骨髓干细胞。
本发明第三方面提供了一种利用上述第一方面所述间充质干细胞培养基进行干细胞分离培养方法,包括以下步骤:
(1)将采用缓冲液清洗后的含有干细胞的组织剪碎,向剪碎后的组织中加入含有消化酶的基础培养基,混匀后于37℃震荡消化;
(2)向经步骤(1)处理后的消化产物中加入含FBS的基础培养基,终止消化,然后对消化产物进行低温离心,离心后弃上清,得到沉淀物;
(3)将步骤(2)得到的沉淀物用本发明第一方面所述的间充质干细胞培养基进行重悬,得到细胞悬液,将细胞悬液置于培养容器中于37℃、饱和湿度、CO2体积分数为5%的培养箱内培养2~6h,然后采用缓冲液进行洗涤,洗去未贴壁的细胞,再向培养容器中加入本发明第一方面所述的间充质干细胞培养基进行培养。
根据上述的干细胞分离培养方法,优选地,步骤(1)中所述消化酶为Liberase酶,基础培养基中消化酶的浓度为1mg/ml。
根据上述的干细胞分离培养方法,优选地,步骤(3)中所述缓冲液为HBSS溶液。
根据上述的干细胞分离培养方法,优选地,步骤(1)中所述干细胞为脐带间充质干细胞或脂肪干细胞。
根据上述的干细胞分离培养方法,优选地,步骤(2)中离心处理的温度为4℃,离心处理的转速为1000~2000rpm。
根据上述的干细胞分离培养方法,优选地,所述基础培养基为DMEM 或DMEM/F12。
根据上述的干细胞分离培养方法,优选地,步骤(1)中所述缓冲液为含有抗生素的HBSS溶液,所述缓冲液中抗生素的浓度为1%。
与现有技术相比,本发明取得的积极有益效果为:
(1)本发明间充质干细胞培养基中含有胆固醇、卵磷脂、鞘磷脂、人低密度脂蛋白、油酸、乙醇胺和腐胺等有机物质,该有机物质能给干细胞提供细胞营养,促进细胞膜合成和细胞生长。
(2)本发明间充质干细胞培养基中含有重组人血清白蛋白、重组人纤连蛋白、重组人层粘连蛋白等细胞基质成分,这些细胞基质成分能够在无血清环境中为干细胞提供附着支撑,维持干细胞的生长形态,增强干细胞对体外营养物质的反应,为干细胞提供优良的成长环境。
(3)本发明经过大量的研究筛选到了一些既快速扩增干细胞而又不影响其分化潜能的细胞生长因子,这些因子是天然存在的调节分子,能与细胞表面上的受体结合,来改变细胞的生化活性和生长,以及调节细胞的增殖速率,来影响细胞分化,从而刺激细胞和组织功能。本发明通过各细胞生长因子之间的相互配合、相互作用,能够促进干细胞分裂增殖,提高干细胞扩增速度,缩短干细胞培养时间,同时还能抑制干细胞分化但不影响干细胞的分化潜能,有利于干细胞的快速扩增。
(4)本发明间充质干细胞培养基中还含有干细胞外分泌物,干细胞外分泌物含有特定的蛋白质,多为性质不同的细胞生长因子,免疫调节因子,具有刺激干细胞生长和相对抑制其分化的作用。
(5)本发明间充质干细胞培养基中还含有基础培养基、非必须氨基酸、pH调节剂、抗生素、维生素、微量元素等成分,能够为干细胞生长提供细胞生长所需的基础营养物质,维持细胞生长环境,维持细胞活性。
(6)与现有的血清培养基相比,本发明的间充质干细胞培养基成分确定,不含血清成分,质量可控,能够规模化生产,用其进行干细胞体外培养,干细胞能够正常生长;而且,本发明间充质干细胞培养基有利于干细胞的分裂增殖,极大地提高干细胞扩增速率,缩短干细胞的培养时间;同时,采用本发明的培养基对干细胞进行多次传代次后,干细胞依然能够保持其原有的生物学特性和多向分化潜能。
附图说明
图1为脂肪干细胞在不同培养基中培养的形态图,其中,A为传统血清培养基培养的脂肪干细胞形态照片,B为本发明间充质干细胞培养基培养的脂肪干细胞形态照片;
图2为脂肪干细胞在不同培养基中培养的生长曲线;
图3为脂肪干细胞成脂诱导分化实验的结果;其中,A为活性脂肪干细胞的形态照片,照片显示具有正常生长状态的脂肪干细胞;B为脂肪干细胞经过诱导分化成脂肪细胞的形态照片,照片中的亮点表明含有大量脂肪细胞;C为活性脂肪干细胞经过BioTracker488Green Lipid Droplet Dye 进行染色的染色结果图,脂肪干细胞几乎不显示绿色阳性,紫色为DAPI染色表示细胞核;D为脂肪干细胞经过诱导分化成脂肪细胞经过BioTracker488Green Lipid Droplet Dye 进行染色的染色结果图;经诱导分化的脂肪细胞显示绿色阳性,紫色为DAPI染色表示细胞核。
具体实施方式
以下通过具体的实施例对本发明作进一步详细说明,但并不限制本发明的范围。
实施例1:间充质干细胞培养基的配制
一种间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素和抗氧化剂。
其中,所述基础培养基为DMEM液体培养基,DMEM液体培养基的用量为500ml;所述非必须氨基酸为MEM NEAA非必需氨基酸溶液,MEM NEAA非必需氨基酸溶液与基础培养基的体积比为1%;所述pH调节剂为HEPES, HEPES与基础培养基的体积比为1%;所述抗生素与基础培养基的体积比为1%。
所述促进细胞膜合成的有机物质成分组成及在间充质干细胞培养基中的含量如下:胆固醇 15mg/L、卵磷脂16mg/L、鞘磷脂 100 mg/L、人低密度脂蛋白15mg/L、油酸25mg/L、乙醇胺10mg/L和腐胺 5 mg/L。
所述细胞基质成分的组成及其在间充质干细胞培养基中的含量如下:血清白蛋白100~500mg/L、纤连蛋白2~5mg/L和层粘连蛋白1~5 mg/L。
所述细胞生长因子的成分组成及其在间充质干细胞培养基中的含量如下:类胰岛素生长因子IGF-1 10 μg/L、表皮生长因子EGF 10 μg/L、肝细胞生长因子HGF 15μg/L、肝素结合性表皮生长因子HB-EGF 15 μg/L、成纤维细胞生长因子FGF2 50 μg/L、成纤维细胞生长因子FGF4 5 μg/L、成纤维细胞生长因子FGF19 10 μg/L、血管内皮生长因子VEGF 10μg/L、转化生长因子TGF-β10 μg/L、白血病抑制因子LIF 5μg/L、干细胞因子SCF 5 μg/L、干细胞生长因子SCGF-α 10 μg/L、胰岛素 30μg/L、血小板衍生生长因子PDGFAB 50 μg/L、血小板衍生生长因子PDGFBB 150μg/L、凝血酶 15 U/L、转铁蛋白0.2%、红细胞生长素EPO 10μg/L、单核细胞趋化蛋白MCP-1 10μg/L、白介素-2 10μg/L、白介素-8 10μg/L、粒细胞集落刺激因子G-CSF 10μg/L、巨噬细胞集落刺激因子M-CSF 10 μg/L、脂质运载蛋白 10 μg/L。
所述抗氧化剂的成分组成及其在间充质干细胞培养基中的其在间充质干细胞培养基中的含量为:还原型谷胱甘肽 1.5mg/L、β-巯基乙醇0.1mM。
所述维生素的成分组成及其在间充质干细胞培养基中的含量为:维生素A 10 mg/L、维生素C 10mg/L、维生素E 15 mg/L;所述微量元素的成分组成及含量为:硫酸铜25mg/L、氯化镁20 mg/L、硫酸锌 20 mg/L、亚硒酸钠5 μg/L。
实施例2:间充质干细胞培养基的配制
一种间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素和抗氧化剂。
其中,所述基础培养基为DMEM液体培养基,DMEM液体培养基的用量为500ml;所述非必须氨基酸为MEM NEAA非必需氨基酸溶液,MEM NEAA非必需氨基酸溶液与基础培养基的体积比为1%;所述pH调节剂为HEPES, HEPES与基础培养基的体积比为1%;所述抗生素与基础培养基的体积比为1%。
所述促进细胞膜合成的有机物质成分组成及在间充质干细胞培养基中的含量如下:胆固醇 10mg/L、卵磷脂8mg/L、鞘磷脂 80mg/L、人低密度脂蛋白5mg/L、油酸20mg/L、乙醇胺 5mg/L和腐胺 1mg/L。
所述细胞基质成分的组成及其在间充质干细胞培养基中的含量如下:血清白蛋白100mg/L、纤连蛋白2mg/L和层粘连蛋白1mg/L。
所述细胞生长因子的成分组成及其在间充质干细胞培养基中的含量如下:类胰岛素生长因子IGF-1 5μg/L、表皮生长因子EGF 5μg/L、肝细胞生长因子HGF 5μg/L、肝素结合性表皮生长因子HB-EGF 10μg/L、成纤维细胞生长因子FGF2 40μg/L、成纤维细胞生长因子FGF4 1μg/L、成纤维细胞生长因子FGF19 5μg/L、血管内皮生长因子VEGF 1μg/L、转化生长因子TGF-β5μg/L、白血病抑制因子LIF 1μg/L、干细胞因子SCF 1μg/L、干细胞生长因子SCGF-α 5μg/L、胰岛素 25μg/L、血小板衍生生长因子PDGFAB 40μg/L、血小板衍生生长因子PDGFBB 100μg/L、凝血酶 10U/L、转铁蛋白0.2%、红细胞生长素EPO 5μg/L、单核细胞趋化蛋白MCP-1 5μg/L、白介素-2 5μg/L、白介素-8 5μg/L、粒细胞集落刺激因子G-CSF 5μg/L、巨噬细胞集落刺激因子M-CSF 5μg/L、脂质运载蛋白 5μg/L。
所述抗氧化剂的成分组成及其在间充质干细胞培养基中的其在间充质干细胞培养基中的含量为:还原型谷胱甘肽 1mg/L、β-巯基乙醇0.1mM。
所述维生素的成分组成及其在间充质干细胞培养基中的含量为:维生素A 1mg/L、维生素C 5mg/L、维生素E 10mg/L;所述微量元素的成分组成及含量为:硫酸铜20mg/L、氯化镁15mg/L、硫酸锌 15mg/L、亚硒酸钠1μg/L。
实施例3:间充质干细胞培养基的配制
一种间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素和抗氧化剂。
其中,所述基础培养基为DMEM液体培养基,DMEM液体培养基的用量为500ml;所述非必须氨基酸为MEM NEAA非必需氨基酸溶液,MEM NEAA非必需氨基酸溶液与基础培养基的体积比为1%;所述pH调节剂为HEPES, HEPES与基础培养基的体积比为1%;所述抗生素与基础培养基的体积比为2%。
所述促进细胞膜合成的有机物质成分组成及在间充质干细胞培养基中的含量如下:胆固醇 12mg/L、卵磷脂10mg/L、鞘磷脂 90 mg/L、人低密度脂蛋白10mg/L、油酸23mg/L、乙醇胺 8mg/L和腐胺 3mg/L。
所述细胞基质成分的组成及其在间充质干细胞培养基中的含量如下:血清白蛋白300mg/L、纤连蛋白4mg/L和层粘连蛋白3 mg/L。
所述细胞生长因子的成分组成及其在间充质干细胞培养基中的含量如下:类胰岛素生长因子IGF-1 8 μg/L、表皮生长因子EGF 8μg/L、肝细胞生长因子HGF 10μg/L、肝素结合性表皮生长因子HB-EGF 12 μg/L、成纤维细胞生长因子FGF2 45 μg/L、成纤维细胞生长因子FGF4 3μg/L、成纤维细胞生长因子FGF19 8μg/L、血管内皮生长因子VEGF 5μg/L、转化生长因子TGF-β8μg/L、白血病抑制因子LIF 4μg/L、干细胞因子SCF 4μg/L、干细胞生长因子SCGF-α 8μg/L、胰岛素 28μg/L、血小板衍生生长因子PDGFAB 45μg/L、血小板衍生生长因子PDGFBB 125μg/L、凝血酶 12U/L、转铁蛋白0.2%、红细胞生长素EPO 8μg/L、单核细胞趋化蛋白MCP-1 8μg/L、白介素-2 8μg/L、白介素-8 8μg/L、粒细胞集落刺激因子G-CSF 8μg/L、巨噬细胞集落刺激因子M-CSF 8μg/L、脂质运载蛋白 8 μg/L。
所述抗氧化剂的成分组成及其在间充质干细胞培养基中的其在间充质干细胞培养基中的含量为:还原型谷胱甘肽 1.2mg/L、β-巯基乙醇0.1mM。
所述维生素的成分组成及其在间充质干细胞培养基中的含量为:维生素A 5 mg/L、维生素C 8mg/L、维生素E 12mg/L;所述微量元素的成分组成及含量为:硫酸铜22mg/L、氯化镁18mg/L、硫酸锌 18mg/L、亚硒酸钠3μg/L。
实施例4:间充质干细胞培养基的配制
一种间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素和抗氧化剂。
其中,所述基础培养基为DMEM液体培养基,DMEM液体培养基的用量为500ml;所述非必须氨基酸为MEM NEAA非必需氨基酸溶液,MEM NEAA非必需氨基酸溶液与基础培养基的体积比为1%;所述pH调节剂为HEPES, HEPES与基础培养基的体积比为1%;所述抗生素与基础培养基的体积比为1%。
所述促进细胞膜合成的有机物质成分组成及在间充质干细胞培养基中的含量如下:胆固醇 10mg/L、卵磷脂10mg/L、鞘磷脂 100 mg/L、人低密度脂蛋白10mg/L、油酸20mg/L、乙醇胺 10mg/L和腐胺 5 mg/L。
所述细胞基质成分的组成及其在间充质干细胞培养基中的含量如下:血清白蛋白100mg/L、纤连蛋白5mg/L和层粘连蛋白5 mg/L。
所述细胞生长因子的成分组成及其在间充质干细胞培养基中的含量如下:类胰岛素生长因子IGF-1 10 μg/L、表皮生长因子EGF 10 μg/L、肝细胞生长因子HGF 10μg/L、肝素结合性表皮生长因子HB-EGF 10 μg/L、成纤维细胞生长因子FGF2 50 μg/L、成纤维细胞生长因子FGF4 5 μg/L、成纤维细胞生长因子FGF19 5μg/L、血管内皮生长因子VEGF 10μg/L、转化生长因子TGF-β5μg/L、白血病抑制因子LIF 5μg/L、干细胞因子SCF 5 μg/L、干细胞生长因子SCGF-α 10 μg/L、胰岛素 30μg/L、血小板衍生生长因子PDGFAB 50 μg/L、血小板衍生生长因子PDGFBB 100μg/L、凝血酶 10 U/L、转铁蛋白0.2%、红细胞生长素EPO 5μg/L、单核细胞趋化蛋白MCP-1 5μg/L、白介素-2 5μg/L、白介素-8 5μg/L、粒细胞集落刺激因子G-CSF 5μg/L、巨噬细胞集落刺激因子M-CSF 5μg/L、脂质运载蛋白 5μg/L。
所述抗氧化剂的成分组成及其在间充质干细胞培养基中的其在间充质干细胞培养基中的含量为:还原型谷胱甘肽 1mg/L、β-巯基乙醇0.1mM。
所述维生素的成分组成及其在间充质干细胞培养基中的含量为:维生素A 10 mg/L、维生素C 5mg/L、维生素E 10mg/L;所述微量元素的成分组成及含量为:硫酸铜20mg/L、氯化镁20 mg/L、硫酸锌 20 mg/L、亚硒酸钠5 μg/L。
实施例5:间充质干细胞培养基的配制
一种间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素和抗氧化剂。
其中,所述基础培养基为DMEM液体培养基,DMEM液体培养基的用量为500ml;所述非必须氨基酸为MEM NEAA非必需氨基酸溶液,MEM NEAA非必需氨基酸溶液与基础培养基的体积比为2%;所述pH调节剂为HEPES, HEPES与基础培养基的体积比为1%;所述抗生素与基础培养基的体积比为2%。
所述促进细胞膜合成的有机物质成分组成及在间充质干细胞培养基中的含量如下:胆固醇 10mg/L、卵磷脂10mg/L、鞘磷脂 80 mg/L、人低密度脂蛋白10mg/L、油酸20mg/L、乙醇胺10mg/L和腐胺 5 mg/L。
所述细胞基质成分的组成及其在间充质干细胞培养基中的含量如下:血清白蛋白100mg/L、纤连蛋白2mg/L和层粘连蛋白1 mg/L。
所述细胞生长因子的成分组成及其在间充质干细胞培养基中的含量如下:类胰岛素生长因子IGF-1 5 μg/L、表皮生长因子EGF 5μg/L、肝细胞生长因子HGF 5μg/L、肝素结合性表皮生长因子HB-EGF 10μg/L、成纤维细胞生长因子FGF2 50 μg/L、成纤维细胞生长因子FGF4 5 μg/L、成纤维细胞生长因子FGF19 10 μg/L、血管内皮生长因子VEGF 10μg/L、转化生长因子TGF-β5μg/L、白血病抑制因子LIF 1μg/L、干细胞因子SCF 1μg/L、干细胞生长因子SCGF-α 5μg/L、胰岛素 30μg/L、血小板衍生生长因子PDGFAB 40μg/L、血小板衍生生长因子PDGFBB 100μg/L、凝血酶 10U/L、转铁蛋白0.2%、红细胞生长素EPO 5μg/L、单核细胞趋化蛋白MCP-1 5μg/L、白介素-2 5μg/L、白介素-8 5μg/L、粒细胞集落刺激因子G-CSF 5μg/L、巨噬细胞集落刺激因子M-CSF 5μg/L、脂质运载蛋白 5μg/L。
所述抗氧化剂的成分组成及其在间充质干细胞培养基中的其在间充质干细胞培养基中的含量为:还原型谷胱甘肽 1mg/L、β-巯基乙醇0.1mM。
所述维生素的成分组成及其在间充质干细胞培养基中的含量为:维生素A 10 mg/L、维生素C 10mg/L、维生素E 15 mg/L;所述微量元素的成分组成及含量为:硫酸铜20mg/L、氯化镁20 mg/L、硫酸锌 20 mg/L、亚硒酸钠1μg/L。
实施例6:间充质干细胞培养基的配制
实施例6的内容与实施例1基本相同,其不同之处在于:所述间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂和含有干细胞外分泌物的溶液;所述含有干细胞外分泌物的溶液与基础培养基的体积比为10%。
所述含有干细胞外分泌物的溶液的制备方法为:采用实施例1所述的间充质干细胞培养基对干细胞进行培养,培养48h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
实施例7:间充质干细胞培养基的配制
实施例7的内容与实施例1基本相同,其不同之处在于:所述间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂和含有干细胞外分泌物的溶液;所述含有干细胞外分泌物的溶液与基础培养基的体积比为5%。
所述含有干细胞外分泌物的溶液的制备方法为:采用实施例1所述的间充质干细胞培养基对干细胞进行培养,培养48h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
实施例8:间充质干细胞培养基的配制
实施例8的内容与实施例1基本相同,其不同之处在于:所述间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂和含有干细胞外分泌物的溶液;所述含有干细胞外分泌物的溶液与基础培养基的体积比为15%。
所述含有干细胞外分泌物的溶液的制备方法为:采用实施例1所述的间充质干细胞培养基对干细胞进行培养,培养48h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
实施例9:间充质干细胞培养基的配制
实施例9的内容与实施例1基本相同,其不同之处在于:所述基础培养基为DMEM/F12培养基。
实施例10:间充质干细胞培养基的配制
实施例10的内容与实施例6基本相同,其不同之处在于:所述基础培养基为DMEM/F12培养基。
实施例11:间充质干细胞培养基的配制
实施例11的内容与实施例2基本相同,其不同之处在于:所述间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂和含有干细胞外分泌物的溶液;所述含有干细胞外分泌物的溶液与基础培养基的体积比为10%。
所述含有干细胞外分泌物的溶液的制备方法为:采用实施例2所述的间充质干细胞培养基对干细胞进行培养,培养48h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
实施例12:间充质干细胞培养基的配制
实施例12的内容与实施例3基本相同,其不同之处在于:所述间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂和含有干细胞外分泌物的溶液;所述含有干细胞外分泌物的溶液与基础培养基的体积比为10%。
所述含有干细胞外分泌物的溶液的制备方法为:采用实施例3所述的间充质干细胞培养基对干细胞进行培养,培养48h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
实施例13:间充质干细胞培养基的配制
实施例13的内容与实施例4基本相同,其不同之处在于:所述间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂和含有干细胞外分泌物的溶液;所述含有干细胞外分泌物的溶液与基础培养基的体积比为10%。
所述含有干细胞外分泌物的溶液的制备方法为:采用实施例4所述的间充质干细胞培养基对干细胞进行培养,培养48h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
实施例14:间充质干细胞培养基的配制
实施例14的内容与实施例5基本相同,其不同之处在于:所述间充质干细胞培养基,由基础培养基和添加剂组成,所述基础培养基为DMEM培养基,所述添加剂为非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂和含有干细胞外分泌物的溶液;所述含有干细胞外分泌物的溶液与基础培养基的体积比为10%。
所述含有干细胞外分泌物的溶液的制备方法为:采用实施例5所述的间充质干细胞培养基对干细胞进行培养,培养48h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
实施例15:本发明间充质干细胞培养基对脂肪干细胞培养、增殖、诱导分化的研究
为了研究本发明间充质干细胞培养基对干细胞培养、增殖、诱导分化的影响,以本发明实施例6制备的间充质干细胞培养基为例,采用本发明实施例6制备的间充质干细胞培养基对脂肪干细胞进行培养。具体实验操作及结果如下。
、脂肪干细胞分离培养、传代:
(1)获取脂肪组织,用预冷的含有1%抗生素的HBSS溶液(Hank's平衡盐溶液)清洗,去除血污,将脂肪组织在冰上进行剥离,去除不需要的组织。
(2)将经剥离处理后的脂肪组织剪碎,加入含有1mg/ml Liberase酶的DMEM/F12培养基,混匀后于37℃水浴震荡消化30-45min。
(3)将经步骤(1)处理后的消化产物中加入等体积的含10%FBS的DMEM/F12培养基终止消化;然后将消化后的产物转移至离心管中,冰上放置10min,于4℃ 1000~2000rpm离心5-10min,离心后弃上清,得到沉淀物。
(4)将步骤(3)得到的沉淀物用实施例6制备的间充质干细胞进行重悬,得到细胞悬液,将细胞悬液置于培养容器中于37℃、饱和湿度、CO2体积分数为5%的培养箱内培养4h;然后采用HBSS溶液进行洗涤,洗去未贴壁的细胞,再向培养容器中加入实施例6制备的间充质干细胞培养基进行培养。
(5)细胞培养24h后,采用HBSS溶液进行洗涤,洗涤后加入实施例6制备的间充质干细胞培养基进行培养。
(6)当培养容器中细胞汇合度达到80%时,用HBSS溶液进行洗涤,再用0.25%胰蛋白酶溶液进行消化处理,收集脱落的细胞,将脱落细胞用实施例6制备的间充质干细胞培养基进行重悬,得到细胞悬液,将细胞悬液按1:3的比例接种进行传代培养。
脂肪干细胞扩增到第3代后,收集细胞,进行形态学分析。
同时,为了与本发明间充质干细胞培养基进行对比,本发明还采用传统血清培养基(含10%FBS的DMEM/F12培养基)培养分离培养脂肪干细胞(采用传统血清培养基分离培养脂肪干细胞的方法与本发明的方法基本相同,其不同之处在于:上述步骤(4)、(5)(6)中采用的培养基均为传统血清培养基)。采用传统血清培养基、本发明间充质干细胞培养的脂肪干细胞的形态学分析结果如图1所示。
由图1可知,采用本发明间充质干细胞培养的脂肪干细胞的细胞形态与传统血清培养基一致。由此说明,本发明间充质干细胞培养基能够用于干细胞的培养和传代。
、脂肪干细胞的增殖:
为了分析本发明间充质干细胞培养基对脂肪干细胞增殖能力的影响,本实施例采用四种培养基分别对第三代脂肪干细胞进行培养。四种培养基分别为:传统血清培养基(含10%FBS的DMEM/F12培养基)、低浓度(LOW)间充质干细胞培养基(按本发明实施例6间充质干细胞培养基所有添加成份配成100浓缩液,然后将100倍的浓缩液进行500倍稀释)、中浓度(Medium)间充质干细胞培养基(按本发明实施例6的间充质干细胞培养基的所有添加成份配成100浓缩液,然后将100倍的浓缩液进行100倍稀释)和高浓度(High)间充质干细胞培养基(按本发明实施例6的间充质干细胞培养基的所有添加成份配成100浓缩液,然后将100倍的浓缩液进行50倍稀释)。
具体的培养方法为:选取生长良好的第三代脂肪干细胞,以5×10-3/孔接种于96孔板中,每孔加入100μl培养基于37℃、饱和湿度、CO2体积分数为5%的培养箱内进行培养;每24小时随机选取一组细胞(包含3个重复孔),用promega的celltiter-Glo测定细胞活性读值。连续测定5天,绘制细胞生长曲线。
脂肪干细胞的生长曲线测定结果如图2所示。由图2可知,本方案培养基相比传统培养基比较,能够促进脂肪干细胞的生长速度,其中高浓度和中浓度配方的培养基比低浓度配方的培养基在96小时对数生长期内快30%、 比传统培养基快约一倍。
、脂肪干细胞的成脂诱导分化:
为了证明本方案所培养的干细胞具有分化潜能,选取生长良好的第三代脂肪干细胞,按照thermofisher公司的 StemPro™ Adipogenesis Differentiation Kit 说明书进行成脂诱导分化操作,并对分化后的脂肪进行BioTracker488 Green Lipid Droplet Dye染色。
成脂诱导分化实验的结果如图3所示。图3中, A为活性脂肪干细胞的形态照片,照片显示具有正常生长状态的脂肪干细胞; B为脂肪干细胞经过诱导分化成脂肪细胞的形态照片,照片中的亮点表明含有大量脂肪细胞;C为活性脂肪干细胞经过BioTracker488Green Lipid Droplet Dye 进行染色的染色结果图,从该染色结果图可以看出脂肪干细胞几乎不显示绿色阳性;D为脂肪干细胞经过诱导分化成脂肪细胞经过BioTracker488 GreenLipid Droplet Dye 进行染色的染色结果图;由该染色结果图可以看出经诱导分化的脂肪细胞显示绿色阳性,表明脂肪干细胞经诱导分化成脂肪细胞。
由图3可知,本发明的间充质干细胞培养基不影响脂肪干细胞的分化潜能,采用本发明间充质干细胞培养的脂肪干细胞,经成脂诱导分化能够分化成脂肪细胞,具有分化潜能。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (10)
1.一种间充质干细胞培养基,其特征在于,由基础培养基和添加剂组成,所述添加剂包括非必需氨基酸、pH调节剂、抗生素、促进细胞膜合成的有机物质成分、细胞基质成分、细胞生长因子、维生素、微量元素、抗氧化剂;所述基础培养基为DMEM或DMEM/F12。
2.根据权利要求1所述的间充质干细胞培养基,其特征在于,所述促进细胞膜合成的有机物质成分组成及含量如下:胆固醇 10~15mg/L、卵磷脂8~16mg/L、鞘磷脂 80~100 mg/L、人低密度脂蛋白5~15mg/L、油酸20~25mg/L、乙醇胺 5~10mg/L和腐胺 1~5 mg/L;所述细胞基质成分的组成及含量如下:血清白蛋白 100~500mg/L、纤连蛋白2~5mg/L和层粘连蛋白1~5 mg/L。
3.根据权利要求2所述的间充质干细胞培养基,其特征在于,所述细胞生长因子的成分组成及含量如下:类胰岛素生长因子IGF-1 5~10 μg/L、表皮生长因子EGF 5~10 μg/L、肝细胞生长因子HGF 5~15μg/L、肝素结合性表皮生长因子HB-EGF 10~15 μg/L、成纤维细胞生长因子FGF2 40~50 μg/L、成纤维细胞生长因子FGF4 1~5 μg/L、成纤维细胞生长因子FGF19 5~10 μg/L、血管内皮生长因子VEGF 1~10μg/L、转化生长因子TGF-β5~10 μg/L、白血病抑制因子LIF 1~5μg/L、干细胞因子SCF 1~5 μg/L、干细胞生长因子SCGF-α 5~10μg/L、胰岛素 25~30μg/L、血小板衍生生长因子PDGFAB 40~50 μg/L、血小板衍生生长因子PDGFBB 100~150μg/L、凝血酶 10~15 U/L、转铁蛋白0.2%、红细胞生长素EPO 5~10μg/L、单核细胞趋化蛋白MCP-1 5~10μg/L、白介素-2 5~10μg/L、白介素-8 5~10μg/L、粒细胞集落刺激因子G-CSF 5~10μg/L、巨噬细胞集落刺激因子M-CSF 5~10 μg/L、脂质运载蛋白 5~10 μg/L。
4.根据权利要求3所述的间充质干细胞培养基,其特征在于,所述抗氧化剂的成分组成及含量为:还原型谷胱甘肽 1~1.5mg/L、β-巯基乙醇0.1mM;所述维生素的成分组成及含量为:维生素A 1~10 mg/L、维生素C 5~10mg/L、维生素E 10~15 mg/L;所述微量元素的成分组成及含量为:硫酸铜20~25mg/L、氯化镁15~20 mg/L、硫酸锌 15~20 mg/L、亚硒酸钠1~5 μg/L。
5.根据权利要求1所述的间充质干细胞培养基,其特征在于,所述添加剂还包括含有干细胞外分泌物的溶液,所述含有干细胞外分泌物的溶液的制备方法为:采用权利要求1 所述的间充质干细胞培养基对干细胞进行培养,培养48~72h后,离心,取上清液,所述上清液为含有干细胞外分泌物的溶液。
6.根据权利要求1所述的间充质干细胞培养基,其特征在于,所述pH调节剂为HEPES,所述HEPES与基础培养基的体积比为1%;所述抗生素与基础培养基的体积比为1%~2%;所述非必需氨基酸与基础培养基的体积比为1%~2%,所述非必需氨基酸为MEM NEAA非必需氨基酸溶液。
7.权利要求1~6任一所述的间充质干细胞培养基在体外培养干细胞中的应用。
8.根据权利要求7所述的应用,其特征在于,所述为脐带间充质干细胞、脂肪干细胞或骨髓干细胞。
9.一种干细胞分离培养方法,其特征在于,包括以下步骤:
(1)将采用缓冲液清洗后的含有干细胞的组织剪碎,向剪碎后的组织中加入含有消化酶的基础培养基,混匀后于37℃震荡消化;
(2)向经步骤(1)处理后的消化产物中加入含FBS的基础培养基,终止消化,然后对消化产物进行低温离心,离心后弃上清,得到沉淀物;
(3)将步骤(2)得到的沉淀物用权利要求1~6任一所述的间充质干细胞培养基进行重悬,得到细胞悬液,将细胞悬液置于培养容器中于37℃、饱和湿度、CO2体积分数为5%的培养箱内培养2~6h,然后采用缓冲液进行洗涤,洗去未贴壁的细胞,再向培养容器中加入权利要求1~6任一所述的间充质干细胞培养基进行培养。
10.根据权利要求9所述的干细胞分离培养方法,其特征在于,步骤(1)中所述消化酶为Liberase酶,基础培养基中消化酶的浓度为1mg/ml;步骤(3)中所述缓冲液为HBSS溶液。
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