CN114058573B - 一种含有生物素的培养基 - Google Patents
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
本发明公开了一种含有生物素的培养基。所述的培养基还包含氰钴胺和/或硫辛酸;所述的培养基用于培养间充质干细胞;所述氰钴胺在所述的培养基中的浓度为0.8μmol/L~1.2μmol/L,所述硫辛酸在所述培养基中的浓度为0.1μmol/L~0.4μmol/L,所述生物素在所述培养基中的浓度为0.41μmol/L~0.82μmol/L。使用本发明的培养基独对间充质干细胞进行培养时,培养后所得细胞在保证表面特异性蛋白稳定、分化能力与现有技术相当的前提下,细胞培养时间段、细胞得率以及扩增倍数高。
Description
技术领域
本发明属于生物技术领域,具体涉及一种含有生物素培养基,特别是含有生物素,以及氰钴胺和/或硫辛酸的培养基。
背景技术
干细胞是一种能够长期自我更新并维持自身特性的多潜能细胞,在一定的生理或实验条件下可分化为多种类型的细胞。根据发育阶段,干细胞可分为胚胎干细胞和成体干细胞。胚胎干细胞(ES)可以分化成几乎所有细胞系,但由于伦理和畸胎瘤等问题,ES在临床研究上的使用受到限制。
与ES不同,间充质干细胞(MSCs)属于成体干细胞,不存在伦理和畸胎瘤等问题,临床研究使用广泛。据不完全统计,临床试验使用MSCs的疾病种类多达493种,其中移植物抗宿主病(GVHD)、脊髓损伤、心肌梗死、糖尿病和慢性间质性肝炎占60%。
MSCs几乎存在于所有组织中,它们很容易从骨髓、脐带、胎盘、脂肪、牙髓中分离,并在体外扩增。目前体外培养的方式按培养液类型大致分为两种:胎牛血清(FBS)培养和人血小板裂解液(hPL)培养(即第一代无血清培养液)。
FBS在四十年前就被证明能够培养骨髓中的成纤维样细胞。迄今为止,FBS仍是使用最广泛的MSC培养补充物。但作为培养液补充物,FBS对于临床研究仍是一种潜在威胁。首先,FBS成分不明确,异源蛋白可能干预细胞功能、细胞生长、细胞表型和基因型的稳定性。其次,FBS的来源(未出生胎牛)及取样过程(活体取牛血清)决定FBS可能存在污染风险,内毒素、支原体、朊病毒等一旦存在,便难以去除。此外,FBS制作过程复杂、批间差异大,成分不能保持一致,使得实验和生产标准化困难。最后,FBS价格昂贵,保质期短。尽管它作为化学培养液的添加剂被广泛而持久地使用,但它有其经济、伦理和科学上的缺陷。这就产生了开发比FBS更好的替代品的必要性,从而克服这些缺点。
hPL作为FBS培养MSC的替代物,它取样和制作过程简单(单采人血清,离心反复冻融再次离心即可),不存在伦理问题。此外hPL含有丰富的内皮细胞和成纤维细胞生长因子,适宜MSC的扩增,且扩增数量和倍增时间都优于FBS。最重要的是hPL没有异源蛋白,临床副作用小。但hPL并不是完美无缺的,它来源同样稀缺,制作过程难以标准化,存在异体使用。最重要的是因为直接取材于人,hPL也不能避免被已知或未知病毒和支原体等感染的威胁。
因此,研发一款成分明确、来源稳定且体外扩增能力媲美FBS和HPL无血清体外扩增培养液对于临床研究和医学转化而言尤为重要。
发明内容
本发明为克服现有技术的不足,提供一种含有生物素培养基,特别是含有生物素,以及氰钴胺和/或硫辛酸的培养基。
本发明通过以下技术方案解决上述技术问题。
本发明的技术方案之一为:一种含有生物素的培养基,所述的培养基还包含氰钴胺和/或硫辛酸;所述的培养基用于培养间充质干细胞;所述氰钴胺在所述的培养基中的浓度为0.8μmol/L~1.2μmol/L,所述硫辛酸在所述培养基中的浓度为0.1μmol/L~0.4μmol/L,所述生物素在所述培养基中的浓度为0.41μmol/L~0.82μmol/L。
其中,所述氰钴胺在所述的培养基中的浓度较佳地为1μmol/L。
所述硫辛酸在所述的培养基中的浓度较佳地为0.1μmol/L。
所述的生物素在所述培养基中的浓度较佳地为0.41μmol/L。
本发明中所述的间充质干细胞可为本领域中常规,较佳地选自脐带干细胞、胎盘干细胞、脂肪干细胞以及羊膜干细胞。
本发明中,所述培养基较佳地还包含烟酰胺二核苷酸激活剂,所述烟酰胺二核苷酸激活剂的浓度为0.2μmol/L~0.5μmol/L;较佳地为0.3μmol/L。
在本发明一较佳实施例中,所述的培养基包含如下组分:
0.15~0.25mmol/L甘氨酸、0.15~0.25mmol/L丙氨酸、0.15~0.25mmol/L天冬酰胺酸、0.15~0.25mmol/L天冬氨酸、0.15~0.25mmol/L谷氨酸、0.15~0.25mmol/L脯氨酸、0.15~0.25mmol/L丝氨酸、0.25~0.35mmol/L抗坏血酸、0.41~0.82μmol/L生物素、0.8~1.2μmol/L氰钴胺、0.08~0.12%脂质浓缩液、55~56.5μmol/L丁二胺、0.015~0.021μmol/L黄体酮、80~120nmol/L氢化可的松、0.08~0.12%胰岛素-转铁蛋白-硒、0.8~1.2μg/cm2人源纤连蛋白、3.5~4.5g/L重组人血白蛋白、18~22μg/L重组人碱性成纤维生长因子以及8~12μg/L重组人表皮生长因子;较佳地,所述的培养基还包含0.1~0.4μmol/L硫辛酸;更佳地,所述的培养基还包含0.2~0.5μmol/L烟酰胺二核苷酸激活剂。
在本发明一具体实施例中,所述的培养基包含如下组分:0.2mmol/L甘氨酸、0.2mmol/L丙氨酸、0.2mmol/L天冬酰胺酸、0.2mmol/L天冬氨酸、0.2mmol/L谷氨酸、0.2mmol/L脯氨酸、0.2mmol/L丝氨酸、0.3mmol/L抗坏血酸、0.41μmol/L生物素、1μmol/L氰钴胺、0.10%脂质浓缩液、55.9μmol/L丁二胺、0.018μmol/L黄体酮、100nmol/L氢化可的松、1%胰岛素-转铁蛋白-硒、1μg/cm2人源纤连蛋白、4g/L重组人血白蛋白、20μg/L重组人碱性成纤维生长因子以及10μg/L重组人表皮生长因子;较佳地,所述的培养基还包含0.1μmol/L硫辛酸;更佳地,所述的培养基还包含0.3μmol/L烟酰胺二核苷酸激活剂。
在本发明另一较佳实施例中,所述的培养基包含如下组分:0.15~0.25mmol/L甘氨酸、0.15~0.25mmol/L丙氨酸、0.15~0.25mmol/L天冬酰胺酸、0.15~0.25mmol/L天冬氨酸、0.15~0.25mmol/L谷氨酸、0.15~0.25mmol/L脯氨酸、0.15~0.25mmol/L丝氨酸、0.25~0.35mmol/L抗坏血酸、0.41~0.82μmol/L生物素、0.08~0.12%脂质浓缩液、55~56.5μmol/L丁二胺、0.015~0.021μmol/Lμmol/L黄体酮、80~120nmol/L氢化可的松、0.1~0.4μmol/L硫辛酸、0.08~0.12%胰岛素-转铁蛋白-硒、0.8~1.2μg/cm2人源纤连蛋白、3.5~4.5g/L人血白蛋白、18~22μg/L重组人碱性成纤维生长因子以及8~12μg/L重组人表皮生长因子。
在本发明一更佳实施例中,所述的培养基包含如下组分作为营养添加物:0.2mmol/L甘氨酸、0.2mmol/L丙氨酸、0.2mmol/L天冬酰胺酸、0.2mmol/L天冬氨酸、0.2mmol/L谷氨酸、0.2mmol/L脯氨酸、0.2mmol/L丝氨酸、0.3mmol/L抗坏血酸、0.41μmol/L生物素、0.10%脂质浓缩液、55.9μmol/L丁二胺、0.018μmol/L黄体酮、100nmol/L氢化可的松、0.1μmol/L硫辛酸、1%胰岛素-转铁蛋白-硒、1μg/cm2人源纤连蛋白、4g/L重组人血白蛋白、20μg/L重组人碱性成纤维生长因子以及10μg/L重组人表皮生长因子。
本发明的技术方案之二为:一种人源间充质干细胞无血清培养基的添加物,所述添加物包含生物素,以及氰钴胺和/或硫辛酸;
其中:所述生物素在所述人源间充质干细胞无血清培养基中的浓度为0.41μmol/L~0.82μmol/L,优选0.41μmol/L;所述氰钴胺在所述人源间充质干细胞无血清培养基中的浓度为0.8μmol/L~1.2μmol/L,优选1μmol/L;所述硫辛酸在所述人源间充质干细胞无血清培养基中的浓度为0.1μmol/L~0.4μmol/L,优选0.1μmol/L。
较佳地,所述添加物还包含:0.2μmol/L~0.5μmol/L烟酰胺二核苷酸激活剂,优选0.3μmol/L烟酰胺二核苷酸激活剂;
更佳地,还包含0.15~0.25mmol/L甘氨酸、0.15~0.25mmol/L丙氨酸、0.15~0.25mmol/L天冬酰胺酸、0.15~0.25mmol/L天冬氨酸、0.15~0.25mmol/L谷氨酸、0.15~0.25mmol/L脯氨酸、0.15~0.25mmol/L丝氨酸、0.25~0.35mmol/L抗坏血酸、0.08~0.12%脂质浓缩液、55~56.5μmol/L丁二胺、0.015~0.021μmol/L黄体酮、80~120nmol/L氢化可的松、0.08~0.12%胰岛素-转铁蛋白-硒、0.8~1.2μg/cm2人源纤连蛋白、3.5~4.5g/L重组人血白蛋白、18~22μg/L重组人碱性成纤维生长因子以及8~12μg/L重组人表皮生长因子;
在本发明一具体实施例中,所述的添加物还包含0.2mmol/L甘氨酸、0.2mmol/L丙氨酸、0.2mmol/L天冬酰胺酸、0.2mmol/L天冬氨酸、0.2mmol/L谷氨酸、0.2mmol/L脯氨酸、0.2mmol/L丝氨酸、0.3mmol/L抗坏血酸、0.10%脂质浓缩液、55.9μmol/L丁二胺、0.018μmol/L黄体酮、100nmol/L氢化可的松、1%胰岛素-转铁蛋白-硒、1μg/cm2人源纤连蛋白、4g/L重组人血白蛋白、20μg/L重组人碱性成纤维生长因子以及10μg/L重组人表皮生长因子。
对本方案中的所述的人源间充质干细胞的进一步限定同技术方案之一,即:较佳地选自脐带干细胞、胎盘干细胞、脂肪干细胞以及羊膜干细胞。
本发明的技术方案之三为:一种包含如技术方案之二所述的人源间充质干细胞无血清培养基的添加物的培养基。
本发明的技术方案之四为:硫辛酸在制备含有生物素的培养基中的应用。
本发明的技术方案之五为:氰钴胺在制备含有生物素的培养基中的应用。
本发明的技术方案之六为:烟酰胺单核苷酸在制备含有生物素的培养基中的应用。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
使用本发明的培养基独对间充质干细胞进行培养时,培养后所得细胞在保证表面特异性蛋白稳定、分化能力与现有技术相当的前提下,细胞培养时间段、细胞得率以及扩增倍数高。
附图说明
图1为生物素、氰钴胺和硫辛酸对脐带间充质干细胞增殖的影响。
图2为NMN对脐带间充质干细胞传代增殖的影响。
图3为不同培养基与hMSC supplement组合下脐带间充质干细胞原代细胞形态。
图4为不同培养基与hMSC supplement组合下脐带间充质干细胞传代培养。
图5A和图5B为不同培养基与hMSC supplement组合下脐带间充质干细胞P2代流式表型;阴性表型CD34、CD11b、CD19、CD45、HLA-DR总和<0.1%,阳性表型CD73、CD90、CD105、CD44均>95%。
图6为不同培养基与hMSC supplement组合下脐带间充质干细胞P1和P10代细胞形态。
图7为不同培养基与hMSC supplement组合下脐间充质干细胞P5成脂成骨染色图;绿色荧光为成脂诱导后的脂滴,红色为成骨诱导后形成的钙结节。
图8为不同培养基与hMSC supplement组合下胎盘间充质干细胞原代细胞形态。
图9为不同培养基与hMSC supplement组合下胎盘间充质干细胞传代培养。
图10为不同培养基与hMSC supplement组合下胎盘间充质干细胞P2代流式表型;其中A为:10%FBS-MEM-a,B为:hMSC supplement-MEM-a;C为:hMSC supplement-DMEM;D为:hMSC supplement-MSCBM。
图11为不同培养基与hMSC supplement组合下胎盘间充质干细胞P5成脂成骨染色图;绿色荧光为成脂诱导后的脂滴,红色为成骨诱导后形成的钙结节。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
本发明实施例所用营养添加物中各组分的购买来源以及工作浓度。
表1
实施例1 生物素,以及硫辛酸、氰钴胺对MSC传代增殖的影响
实验材料:UCMSC-P4(脐带间充质干细胞)。
实验试剂耗材:12孔板(Thermo,150628)、DMEM细胞培养液(Gibco,10567-014)、PBS(Hyclone,SH30256.01)、tryple细胞消化酶(Gibco,12605-028)、0.4%台盼蓝(Gibco,15250-061)、细胞计数板(Nexcelom,SD100)。
实验设备:生物安全柜(Thermo,1300series A2)、二氧化碳培养箱(Thermo,steri-cycle i160)、细胞计数仪(Nexcelom,cellometer mini)、低温冷冻离心机(Thermo,ST16R)。
取12孔板两块,设立空白对照组(不含硫辛酸、生物素、氰钴胺的营养添加物加入DMEM培养基中,混匀),实验组依次为硫辛酸、生物素、氰钴胺、硫辛酸+生物素、硫辛酸+氰钴胺、生物素+氰钴胺、硫辛酸+生物素+氰钴胺,每组设立平行孔,按孔板培养底面积1μg/cm2的量依次添加fibronectin,然后加入最小体积的PBS缓冲液与之充分混匀并润湿孔板底部,37℃孵育2h,轻轻吸去上清,PBS轻轻润洗一遍,吸去上清,按组分别加入DMEM细胞培养液和相应营养添加物(含表1中除NMN的所有组分,浓度亦同上表)。
取生长融合度达90%的UCMSC-P4,弃培养上清,PBS洗涤一遍,加适量tryple后,消化5-10min。加等体积DMEM细胞培养液混匀终止消化,400×g,4℃离心5min,弃上清,DMEM细胞培养液重复洗涤两次,台盼蓝计数。每组按10000cells/cm2接种,细胞融合度达90%以上消化收集细胞,台盼蓝染色计数,计算每组细胞增殖倍数。细胞连续传代5次。
根据图1可知:硫辛酸和氰钴胺为长期维持细胞活性所必需,生物素不仅能够维持细胞活性而且有利于细胞增殖。
实施例2 NAD+boosters(烟酰胺二核苷酸激活剂)——NMN(Nicotinamidemononucleotide)对MSC增殖和传代影响
实验材料:UCMSC-P4(脐带间充质干细胞)。
实验试剂耗材:12孔板(Thermo,150628)、DMEM细胞培养液(Gibco,10567-014)、PBS(Hyclone,SH30256.01)、tryple细胞消化酶(Gibco,12605-028)、0.4%台盼蓝(Gibco,15250-061)、细胞计数板(Nexcelom,SD100)。
实验设备:生物安全柜(Thermo,1300series A2)、二氧化碳培养箱(Thermo,steri-cycle i160)、细胞计数仪(Nexcelom,cellometer mini)、低温冷冻离心机(Thermo,ST16R)
实验方法:取12孔板,按NMN浓度设立实验组(0ng/ml、50ng/ml、100ng/ml、1000ng/ml),按孔板培养底面积1μg/cm2的量依次添加fibronectin,然后加入最小体积的PBS缓冲液与之充分混匀并润湿孔板底部,37℃孵育2h,轻轻吸去上清,PBS轻轻润洗一遍,吸去上清,按组加DMEM细胞培养液、营养添加物(含表1所列的所有组分,浓度亦同)。
取生长融合度达90%的UCMSC-P4,弃培养上清,PBS洗涤一遍,加适量tryple后,消化5-10min。加等体积DMEM完全培养液混匀终止消化,400×g,4℃离心5min,弃上清,DMEM完全培养液重复洗涤两次,台盼蓝计数。每组按12000cells/cm2接种,细胞融合度达90%以上消化收集细胞,台盼蓝染色计数,计算每组细胞增殖倍数。细胞连续传代4次。
根据图2可知:100ng/ml NMN添加组细胞增殖明显。
实施例3 脐带间充质干细胞(umbilical cord derived mesenchymal stemcell)原代和传代培养
实验材料:脐带组织
实验试剂耗材:T25 FLASK(Thermo)PBS(Hyclone,SH30256.01)、tryple细胞消化酶(Gibco,12605-028)、MEM-a(gibco,32561037)、DMEM(gibco,10567022)、1640(gibco,72400120)、MSCBM(DAKEWE)、FBS(gibco,1099141)、Collagenase Type I(gibco,17100017)、StemProadipogenesis Differentiation Kits(gibco,A1007001)、StemProOsteogenesisDifferentiation Kits(Gibco,A1007201)、4%多聚甲醛(Solarbio,P1112)、LipidTOXTM Green neutral lipids stain(Life Technologis,H34475)、茜素红(Sigma,A5533)、0.4%台盼蓝(Gibco,15250-061)、细胞计数板(Nexcelom,SD100)
实验设备:生物安全柜(Thermo,1300series A2)、二氧化碳培养箱(Thermo,steri-cycle i160)、细胞计数仪(Nexcelom,cellometer mini)、低温冷冻离心机(Thermo,ST16R)、荧光倒置显微镜(Olympus,IX73-DP80)
原代、传代方法:取20cm人脐带组织(donor 1),挤血,PBS或生理盐水洗涤4次,剔除组织中的动脉和静脉,剪碎至1cm2大小,按体积加一定浓度的I型胶原酶消化2小时。8倍生理盐水终止消化,600×g离心10分钟,弃上清,加PBS或生理盐水悬浮沉淀,400×g离心10min,弃上清,加入一定体积PBS定容,AOPI染色计数,按20000cells/cm2分别接种到含有hMSC无血清营养添加物(表1中所有组分,浓度亦同)的不同培养基中(MEM-a、DMEM、1640和MSCBM)和10%FBS-MEM-a中,37℃,5%CO2进行原代培养。传代培养按10000cells/cm2进行。
流式检测方法:取1×106P1代细胞悬浮至700μlPBS中待用。取流式细胞管分别设立阴性对照管、阴性管(CD34、CD11b、CD19、CD45、HLA-DR)、阳性对照管、阳性(CD105、CD90、CD73)管、CD44阳性对照管、CD44阳性管并按操作说明书加入相应的抗体,及100μl待检细胞悬液,混匀,避光孵育30min。加入2ml PBS终止反应,400×g离心5min。弃上清,再次洗涤离心2次,弃上清,加入200μl PBS悬浮细胞,上机检测。
成脂成骨诱导染色方法:取P5代细胞,成脂按10000cells/cm2和成骨按6000cells/cm2接种以上各组细胞至24孔板,10%FBS-MEM-a孵育过夜,PBS洗涤一遍,分别加入成脂成骨诱导培养基,成脂诱导8天后,吸取上清,PBS洗涤一遍,加入4%多聚甲醛固定30min,在洗涤一遍,加入绿色中性脂肪染色液孵育20min,去染色液并洗涤一次,加入PBS至荧光显微镜下观察。成骨诱导21天后,吸取上清,PBS洗涤一遍,加入4%多聚甲醛固定30min,在洗涤一遍,加入茜素红染色液孵育3min,光学显微镜下观察。图3显示:与FBS相比,hMSC supplement培养的细胞更小。图4显示:与FBS相比,hMSC supplement扩增倍数较高。图5A和图5B显示:与FBS相比,hMSCsupplement培养的细胞表面特异性蛋白同样稳定(阳性蛋白CD105、CD90、CD73>95%,阴性蛋白CD34、CD11b、CD19、CD45、HLA-DR总和<0.1%)。图6显示:与FBS相比,hMSC supplement培养的细胞P10代细胞仍呈涡旋生长细胞短小长梭型。图7显示:与FBS相比,hMSC supplement培养的细胞具有同样的成脂成骨分化能力。
表2.不同培养基与hMSC supplement组合下脐带间充质干细胞原代细胞得率与FBS相比,hMSC supplement培养时间更短,细胞得率较高
实施例4胎盘间充质干细胞(placenta tissue derived mesenchymal stemcell)原代和传代培养
实验材料:胎盘组织(placental tissue)
实验试剂耗材:MEM-a(gibco,32561037)DMEM(gibco,10567022)1640(gibco,72400120)MSCBM(DAKEWE)FBS(gibco,1099141)CollagenaseType I(gibco,17100017)Dispase(gibco,17105041)Tryple Express(gibco,12605028)。
实验设备:生物安全柜(Thermo,1300series A2)、二氧化碳培养箱(Thermo,steri-cycle i160)、细胞计数仪(Nexcelom,cellometer mini)、低温冷冻离心机(Thermo,ST16R)、荧光倒置显微镜(Olympus,IX73-DP80)
原代、传代方法:洗净胎盘血污,脐带连接面朝上,去除表面羊膜,避开血管,剪取近脐带周边的组织3-4块至50ml离心管,PBS洗涤4遍,剪刀剪碎组织至1cm2左右,定容,加入一定浓度的I型胶原酶和分离酶消化1小时。8倍生理盐水终止消化,400×g瞬时离心收集上清,弃沉淀,加PBS或生理盐水悬浮沉淀,400×g离心5min,弃上清,加入一定体积PBS在洗涤一次,弃上清,定容,AOPI染色计数,按20000cells/cm2分别接种到含有hMSC无血清营养添加物(含表1中所有组分,浓度亦同)的不同培养基中(MEM-a、DMEM、1640、MSCBM)和10%FBS-MEM-a中,37℃,5%CO2进行原代培养。传代培养按10000cells/cm2进行。
流式检测方法:取1×106P2代细胞悬浮至700μl PBS中待用。取流式细胞管分别设立阴性对照管、阴性管(CD34、CD11b、CD19、CD45、HLA-DR)、阳性对照管、阳性(CD105、CD90、CD73)管、CD44阳性对照管、CD44阳性管并按操作说明书加入相应的抗体,及100μl待检细胞悬液,混匀,避光孵育30min。加入2ml PBS终止反应,400×g离心5min。弃上清,再次洗涤离心2次,弃上清,加入200μl PBS悬浮细胞,上机检测。
成脂成骨诱导染色方法:取P5代细胞,成脂按10000cells/cm2和成骨按6000cells/cm2接种以上各组细胞至24孔板,10%FBS-MEM-a孵育过夜,PBS洗涤一遍,分别加入成脂成骨诱导培养基,成脂诱导8天后,吸取上清,PBS洗涤一遍,加入4%多聚甲醛固定30min,在洗涤一遍,加入绿色中性脂肪染色液孵育20min,去染色液并洗涤一次,加入PBS至荧光显微镜下观察。成骨诱导21天后,吸取上清,PBS洗涤一遍,加入4%多聚甲醛固定30min,在洗涤一遍,加入茜素红染色液孵育3min,光学显微镜下观察。图8显示:与FBS相比,hMSCsupplement培养的细胞更小。图9显示:与FBS相比,hMSC supplement扩增倍数较高。图10显示:与FBS相比,hMSC supplement培养的细胞表面特异性蛋白同样稳定(阳性蛋白CD105、CD90、CD73>95%,阴性蛋白CD34、CD11b、CD19、CD45、HLA-DR总和<2%)。图11显示:与FBS相比,hMSC supplement培养的细胞具有同样的成脂成骨分化能力。
表3
表3显示:不同培养基与hMSC supplement组合下胎盘间充质干细胞原代细胞得率与FBS相比,hMSC supplement培养时间更短,细胞得率较高。
Claims (1)
1.一种含有生物素的脐带间充质干细胞的无血清培养基,其特征在于,所述的培养基用于培养间充质干细胞,所述的培养基包含如下组分:0.2 mmol/L甘氨酸、0.2 mmol/L丙氨酸、0.2 mmol/L天冬酰胺酸、0.2 mmol/L天冬氨酸、0.2 mmol/L谷氨酸、0.2 mmol/L脯氨酸、0.2 mmol/L丝氨酸、0.3 mmol/L抗坏血酸、0.41 μmol/L生物素、1 μmol/L氰钴胺、0.10%脂质浓缩液、55.9 μmol/L丁二胺、0.018 μmol/L黄体酮、100 nmol/L氢化可的松、1%胰岛素-转铁蛋白-硒、1 μg/cm2人源纤连蛋白、4 g/L重组人血白蛋白、20 μg/L重组人碱性成纤维生长因子、10 μg/L重组人表皮生长因子、0.1μmol/L硫辛酸以及0.3μmol/L烟酰胺二核苷酸激活剂,所述烟酰胺二核苷酸激活剂为NMN。
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