CN107164325A - MSCs来源的少突胶质细胞的制备方法及试剂盒 - Google Patents
MSCs来源的少突胶质细胞的制备方法及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种MSCs来源的少突胶质细胞制备试剂盒,包括OPCs无血清基础培养基、OPCs培养添加剂1、OPCs培养添加剂2及OPCs培养表面包被液,其中,所述OPCs无血清基础培养基为含有B27,N2,L‑谷氨酰胺,1,25‑二羟维生素D3,三碘甲状腺氨酸,人褪黑素的DMEM/F12培养基;所述OPCs培养添加剂1为含有重组人碱性成纤维细胞生长因子、重组人表皮生长因子的DMEM/F12培养基;所述OPCs培养添加剂2为含有重组人血小板来源生长因子AA、重组人神经营养因子3的DMEM/F12培养基;所述OPCs培养表面包被液为含有重组人层黏连蛋白、重组人玻连蛋白的DMEM/F12培养基。
Description
技术领域
本发明涉及细胞制备技术领域,具体来说,涉及一种MSCs来源的少突胶质细胞的制备方法及试剂盒。
背景技术
多发性硬化症、白血病、营养不良等多种神经退行性疾病,以及中枢神经***损伤等都会导致髓鞘损失,目前缺乏有效的治疗手段。少突胶质前体细胞(oligodendrocyteprecursor cells,OPCs)在中枢神经***中分化产生少突胶质细胞形成髓鞘结构,其异常会导致中枢神经***脱髓鞘病变,继而导致神经元损伤和精神类疾病,是重要的治疗靶点。少突胶质前体细胞替代治疗或再生修复治疗是维持和修复损伤的轴突、重建轴突电传导功能的最新进展和最有前景的治疗手段。
现有技术中的少突胶质细胞的制备采用胚胎干细胞(embryonic stem cells,ESCs)、诱导多能干细胞(induced pluripotent stem cells,iPSCs)体外分化获得。ESCs来源的少突胶质细胞细胞制剂关系到严重的伦理问题和体内移植致瘤的严重临床风险,iPSCs来源的少突胶质细胞制剂关系到外源基因转染和更严重的体内移植致瘤性的严重临床风险。而且ESCs或iPSCs体外诱导分化制备OPCs工艺复杂,使用血清等动物源成分,对OPCs制剂质量和临床安全性产生不可预估的影响,同时限制了OPCs制剂产率,影响了临床研究和产业化转化潜力。
针对相关技术中的问题,目前尚未提出有效的解决方案。
发明内容
针对相关技术中的上述技术问题,本发明提出一种MSCs来源的少突胶质细胞的制备方法及试剂盒,既避免了采用ESCs来源的少突胶质细胞细胞制剂而产生的的严重的伦理问题和体内移植致瘤的严重临床风险,也避免了采用iPSCs来源的少突胶质细胞制剂产生的外源基因转染和更严重的体内移植致瘤性的严重临床风险,操作简单,OPCs收率高、无动物源成分。
为实现上述技术目的,本发明的技术方案是这样实现的:
一种MSCs来源的少突胶质细胞制备试剂盒,包括OPCs无血清基础培养基、OPCs培养添加剂1、OPCs培养添加剂2及OPCs培养表面包被液,其中,
所述OPCs无血清基础培养基为含有 B27,N2,L-谷氨酰胺,1,25-二羟维生素D3,三碘甲状腺氨酸,人褪黑素的DMEM/F12培养基;
所述OPCs培养添加剂1为含有重组人碱性成纤维细胞生长因子、重组人表皮生长因子的DMEM/F12培养基;
所述OPCs培养添加剂2为含有重组人血小板来源生长因子AA、重组人神经营养因子3的DMEM/F12培养基;
所述OPCs培养表面包被液为含有重组人层黏连蛋白、重组人玻连蛋白的DMEM/F12培养基。
进一步地,
所述OPCs无血清基础培养基含有1×B27,1×N2,2mM L-谷氨酰胺,1uM 1,25-二羟维生素D3,40ng/mL三碘甲状腺氨酸,0.5ug/mL人褪黑素;
所述OPCs培养添加剂1为含有1 ug /mL重组人碱性成纤维细胞生长因子、1 ug /mL重组人表皮生长因子的DMEM/F12;
所述OPCs培养添加剂2为含有1 ug 重组人血小板来源生长因子AA、100ng/mL重组人神经营养因子3的DMEM/F12;
所述OPCs培养表面包被液为含有100ng/mL重组人层黏连蛋白、100ng/mL重组人玻连蛋白的DMEM/F12。
根据本发明的另一方面,提供了一种MSCs来源的少突胶质细胞制备方法,包括如下步骤:
S1.用OPCs培养表面包被液室温包被培养表面1小时,吸弃包被液,经PBS漂洗后置于4℃保存不超过1周;
S2.P2代的MSCs按6000个/cm2接种,以含10%无动物源成分血清替代品的MSCs培养基培养至60-70%汇合;
S3.换含OPCs培养添加剂1的OPCs无血清基础培养基,培养至80-90%汇合,用胰蛋白酶溶液消化收获细胞,记为神经上皮预诱导细胞;
S4.以含OPCs培养添加剂1和OPCs培养添加剂2的OPCs无血清基础培养基重悬神经上皮预诱导细胞,调整细胞密度至1.5×104/cm2,接种至被OPCs培养表面包被液包被的培养表面,培养至60-80%汇合时传代,记为P0代OPCs。
进一步地,
所述OPCs培养表面包被液为经DMEM/F12稀释10倍的OPCs培养表面包被液;
所述含OPCs培养添加剂1的OPCs无血清基础培养基中OPCs培养添加剂1含量为2%;
所述含OPCs培养添加剂1和OPCs培养添加剂2的OPCs无血清基础培养基中OPCs培养添加剂1的含量为2%,OPCs培养添加剂2的含量为2%。
进一步地,所述步骤S4后还包括如下步骤:将P0代OPCs按常规贴壁细胞传代方法传代,收获P2代细胞。
进一步地,所述常规贴壁细胞传代方法传代过程中的接种密度为1.5×104/cm2,培养体系为含1%OPCs培养添加剂1和1% OPCs培养添加剂2的OPCs无血清基础培养基;培养表面为经OPCs培养表面包被液包被的培养表面。
进一步地,所述MSCs来源于人脐带、羊膜、羊水、胎盘、脐带血、宫膜、牙髓、骨髓、皮肤、肌腱、骨骼肌。
本发明的有益效果:本发明提供了以MSCs为来源制备少突胶质细胞的方法,并提供了一种以MSCs为来源制备少突胶质细胞的试剂盒。MSCs是组织来源最丰富的成体干细胞之一,使用MSCs制备少突胶质细胞既避免了采用ESCs来源的少突胶质细胞细胞制剂而产生的的严重的伦理问题和体内移植致瘤的严重临床风险,也避免了采用iPSCs来源的少突胶质细胞制剂产生的外源基因转染和更严重的体内移植致瘤性的严重临床风险,并且操作简单,OPCs收率高、无动物源成分。
附图说明
图1是P2代MSCs的细胞形态图;
图2是P2代MSCs细胞表达nestin形态图;
图3是P2代MSCs细胞表达A2B5形态图;
图4是神经上皮预诱导细胞形态图;
图5是神经上皮预诱导细胞表达nestin形态图;
图6是神经上皮预诱导细胞表达A2B5形态图;
图7是P0代OPCs的细胞形态图;
图8是P0代OPCs细胞表达nestin形态图;
图9是P0代OPCs细胞表达A2B5形态图;
图10是P2代OPCs的细胞形态图;
图11是P2代OPCs细胞表达nestin形态图;
图12是P2代OPCs细胞表达A2B5形态图;
图13是不同培养阶段细胞标志蛋白的表达的统计图;
图14是不同培养阶段细胞得率统计图;
图15是少突胶质细胞形态图;
图16是少突胶质细胞表达nestin形态图;
图17是少突胶质细胞表达A2B5形态图;
图18是少突胶质细胞表达MBP形态图。
具体实施方式
下面将结合具体实施例对本发明实施例中的技术方案进行清楚、完整地描述。实施例中所使用的各种试剂、机械未经特殊说明均可从商业途径获得。
首先,对本申请中出现的英文名称及实施例中所具体使用的各种产品的生产厂家、货号进行简要说明。
OPCs:少突胶质前体细胞,oligodendrocyte precursor cells。
ESCs:胚胎干细胞,embryonic stem cells。
MSCs:间充质基质细胞,mesenchymal stroma cells。
DMEM/F12:是一种细胞培养基,实施例中所使用的DMEM/F12货号是12400-024,生产厂家是GIBCO。
B27:人体白细胞抗原。
N2: 是一种细胞培养添加剂,实施例中所使用的N2货号是17502-048,生产厂家是GIBCO。
PBS:磷酸盐缓冲液。
无动物源成分血清替代品:实施例中所使用的无动物源成分血清替代品货号是C08003C,生产厂家是Stemboscience。
0.25%胰蛋白酶溶液:实施例中所使用的0.25%胰蛋白酶溶液货号是T6424-1VL,成产厂家是Biological industries。
抑肽酶溶液:实施例中所使用的抑肽酶溶液货号是A1250000,成产厂家是Sigma。
FBS:胎牛血清,货号04-400-1A,生产厂家BI。
T3:三碘甲状腺氨酸,货号709611,生产厂家Sigma-Aldrich。
sonic hedgehog:音猬因子,实施例中所使用的sonic hedgehog货号是SRP3156,生产厂家是Sigma。
Noggin:头蛋白,实施例中所使用的Noggin货号是H6416,生产厂家是Sigma。
双丁酰c-AMP:实施例中所使用的双丁酰 c-AMP货号是D0627,生产厂家是Sigma。
IGF-1:类胰岛素生长因子1,货号cyt-216,生产厂家Prospec-Tany。
NT3:神经营养因子3,货号cyt-257,生产厂家Prospec-Tany。
实施例一:试剂盒的构成
MSCs来源的少突胶质细胞制备试剂盒包括OPCs无血清基础培养基(OBM)、OPCs培养添加剂1(OS1)、OPCs培养添加剂2(OS2)及OPCs培养表面包被液(OCB)。
其中,OPCs无血清基础培养基(OBM)为包含以下物质的DMEM/F12培养基:
1×B27,
1×N2,
2mM L-谷氨酰胺,
1uM 1,25-二羟维生素D3(1,25(OH)2D3),
40ng/mL三碘甲状腺氨酸(3,3',5-tri-iodo-thyronine,T3),
0.5ug/mL人褪黑素(Melatonin)。
OPCs培养添加剂1(OS1)为包含以下物质的DMEM/F12培养基:
1 ug /mL重组人碱性成纤维细胞生长因子(rh b-FGF),
1 ug /mL重组人表皮生长因子(rh EGF)。
OPCs培养添加剂2(OS2)为包含以下物质的DMEM/F12培养基:
1 ug 重组人血小板来源生长因子AA(rh PDGF-AA),
100ng/mL重组人神经营养因子3(rh NT-3)。
OPCs培养表面包被液(OCB)为包含以下物质的DMEM/F12培养基:
100ng/mL重组人层黏连蛋白(liminin,LN),
100ng/mL重组人玻连蛋白(vitronectin (VN))。
实施例二:OPCs的制备
OPCs的制备使用的试剂,除PBS、消化液、冻存保护液外,均为实施例一中所述的MSCs来源的少突胶质细胞制备试剂盒提供。
S1. 4℃条件下将-20℃以下冻存的OS1、OS2、OCB过夜解冻备用;
S2.用10×OCB(即将试剂盒中的OCB经DMEM/F12稀释至终浓度为10%)按每毫升包被35cm2培养表面室温包被培养表面1小时,吸弃包被液,以2-8℃PBS漂洗3次,置于4℃保存不超过1周;
S3.人脐带、羊膜、羊水、胎盘、脐带血、宫膜、牙髓、骨髓、皮肤、肌腱、骨骼肌等组织来源的P2代MSCs,按6000个/cm2接种,以含10%无动物源成分血清替代品的MSCs培养基培养至60-70%汇合;
S4.换含2%OS1的OBM培养基,培养至80-90%汇合,用0.25%胰蛋白酶溶液消化收获细胞,记为神经上皮预诱导细胞;
S5.以含2%OS1和2% OS2的OBM培养基重悬神经上皮预诱导细胞,调整细胞密度至1.5×104/cm2,接种至OCB包被的培养表面培养4-6天60-80%汇合时传代,记为P0代OPCs;
S6.P0代OPCs按常规贴壁细胞传代方法传代,消化液为AccutaseTM消化液(货号40506ES60,生产厂家Innovative Cell Technologies, Inc.),接种密度为1.5×104/cm2,培养体系为含1%OS1和1% OS2的OBM培养基;培养表面为OCB包被的培养表面;
S7.收获P2代细胞,可以直接制备制剂,也可以低温冻存,冻存保护液使用货号为CELLBANKER-II,生产厂家为ZENOAQ 的冻存保护液,冻存细胞密度为2×106/mL。
实施例三:脐带MSCs来源的OPCs体外制备
3.1脐带MSCs分离培养
以组织块法原代培养脐带MSCs,培养体系为MSCs培养基,即含10%无动物源成分血清替代品的DMEM/F12培养基,培养至12天时,轻轻震荡培养瓶,悬浮残余组织块,小心吸弃培养上清,PBS洗涤培养表面1次,加入0.25%胰蛋白酶溶液,室温消化5分钟,加入1mL抑肽酶溶液终止消化;400g离心5min,弃上清,收获沉淀物;以PBS洗涤2次,以100um尼龙细胞筛过滤,收集滤液;400g离心5min,弃上清,收获沉淀细胞,记为P0代MSCs;P0代MSCs以MSCs培养基重悬,调整细胞密度为6000/cm2,接种至175cm2组织培养瓶(EasyFlask,NUNC),培养至70-80%汇合时收获P1代MSCs;按细胞密度6000个/cm2继续传代,收获P2代MSCs。
3.2MSCs源OPCs制备
以MSCs培养基重悬P2代脐带来源的MSCs,按6000个/cm2接种至175cm2组织培养瓶,培养至60-70%汇合,换含2%OS1的OBM培养基,培养至80-90%汇合,吸弃培养上清,PBS洗涤培养表面1次,加入0.25%胰蛋白酶溶液,室温消化5分钟,加入1mL抑肽酶溶液终止消化;400g离心,5min,弃上清,收获沉淀细胞,记为神经上皮预诱导细胞;
以含2%OS1和2% OS2的OBM培养基重悬神经上皮预诱导细胞,按1.5×104/cm2接种至OCB包被的175cm2组织培养瓶,隔天换液,60-80%汇合时,吸弃培养上清,PBS洗涤培养表面1次,加入AccutaseTM消化液(货号40506ES60,生产厂家Innovative Cell Technologies,Inc.)5mL,室温消化5分钟,加入1mL抑肽酶溶液终止消化;400g离心5min,弃上清,收获沉淀细胞,记为P0代OPCs;
以含2%OS1和2% OS2的OBM培养基重悬P0代OPCs,按1.5×104/cm2接种至OCB包被的175cm2组织培养瓶,培养至60-80%汇合时收获,记为P1代OPCs;P1代OPCs继续传代培养至P2代,收获。
实施例四:P2代MSCs来源的OPCs分化潜能检测
4.1少突胶质细胞分化培养
按5×104/mL接种P2代OPCs于OCB包被的24孔组织培养板培养,每两天半量换液,第8天时行组织学检测。少突胶质细胞分化培养基为含有如下成分的DMEM/F12培养基:
2%无动物源成分血清替代品,
2%FBS,
1×N2,
40ng/mL T3,
100ng/mL sonic hedgehog,
100 ng /ml Noggin,
10μM 双丁酰,
100 ng /ml IGF-1,
10ng/NT3。
4.2MSCs、神经上皮预诱导细胞、OPCs、少突胶质细胞的一般生物学检测
4.2.1免疫组织学检测
贴壁细胞免疫组织学检测时,吸弃培养基,以PBS漂洗2次,以4%多聚甲醛(PFA)固定细胞10分钟,以PBS漂洗2次,滴加一抗,4℃过夜;PBS洗涤2次,滴加FITC标记二抗,室温孵育1小时;PBS漂洗2次,滴加1μg/ ml 4',6-二脒基-2-苯基吲哚(DAPI),孵育5分钟,PBS漂洗2次,镜检。
免疫组织化学检测使用抗体包括:
mouse anti-nestin【clone 10C2 (1:400),生产厂家Millipore】;
mouse anti-A2B5【clone 105 (1:500) ,生产厂家R&D Systems】;
rabbit anti-MBP 【(1:200),生产厂家Sigma-Aldrich】;
FITC标记二抗:猴抗鼠IgG (1:200)和羊抗兔IgG (1:200)(生产厂家LifeTechnologies)。
4.2.2细胞得率统计
以细胞成像***(Cytell Cell Imaging System,GE Healthcare)行活细胞计数和免疫组织学染色计数,包括:P2代MSCs、nestin+DAPI+神经上皮预诱导细胞数量,nestin+DAPI+、A2B5+DAPI+的P0代OPCs和P2代OPCs数量,MBP+、DAPI+的P2代OPCs和少突胶质细胞数量。
统计学分析采用SPSS17.0进行统计学处理。数据以±S表示,均值比较采用独立样本t-test,P<0.05有统计学意义。
实施例五:统计结果
5.1神经上皮预诱导细胞培养
不同培养阶段细胞标志蛋白的表达统计结果如图13所示。
如图1~3所示,
P2代MSCs随培养时间延长呈典型的漩涡状生长,多数细胞为长梭形,少数为三角形、多角形(图1);
11.87±1.15%的细胞表达nestin(图2);
7.84±0.56%的细胞表达A2B5(图3);
如图4~6所示,
经含2%OS1的OBM培养基培养后,细胞逐渐相互平行, 缩回细胞质, 形成明显拉长的细胞体(图4);
95.84±8.61%的细胞表达nestin(图5);
21.84±5.56%的细胞表达A2B5(图6)。
5.2P0代OPCs培养
如图7~9所示
以含2%OS1和2% OS2的OBM培养基培养神经上皮预诱导细胞4-6天,部分贴壁细胞获得双极性的OPCs形态(图7);
45.21±4.66%的细胞表达nestin(图8);
46.22±7.39%的细胞表达A2B5(图9)。
5.3P2代OPCs培养
以含1%OS1和1% OS2的OBM培养基继代培养P0代OPCs至P2代,多数细胞表现出双极性或三极性,少数细胞表现出多极性形态(图10);
41.83±3.91%的细胞表达nestin(图11);
92.09±11.43%的细胞表达A2B5(图12)。
5.4OPCs细胞得率
不同培养阶段细胞得率统计结果如图14所示。
1.05×106P2代MSCs按6000个/cm2接种到175cm2组织培养瓶(Easyflask,NUNC)中,获得5.74±1.48×106神经上皮预诱导细胞;
神经上皮预诱导细胞约按1.5×104/cm2接种至OCB包被的2个175cm2组织培养瓶(Easyflask,NUNC)中,获得15.66±3.39 ×106 P0代OPCs;
P0代OPCs约按1.5×104/cm2接种至OCB包被的6个175cm2组织培养瓶(Easyflask,NUNC)中,获得46.51±8.82 ×106 P1代OPCs,继而约按1.5×104/cm2接种至OCB包被的18个175cm2组织培养瓶(Easyflask,NUNC)中,获得139.89±36.22 ×106 P2代OPCs。
P2代OPCs按5×104/mL接种于OCB包被的24孔组织培养板培养8天,诱导少突胶质细胞分化。结果表明,培养第4天开始,细胞停止增殖,大量细胞死亡,至第8天时,平均存活细胞数为2.24±0.88×104/孔,所有细胞分化成典型少突胶质细胞样形态(图15)。
免疫组织化学检测结果表明,2.83±0.52%存活细胞表达nestin(图16),9.95±2.22%存活细胞表达A2B5(图17),而98.31±11.42%存活细胞表达MBP(图18)。
实施例六:结果分析
哺乳动物中枢神经***损伤,轴突脱髓鞘和少突胶质细胞细胞死亡引起髓鞘损伤、缺失后引起的动作电位传播中断是造成神经传导功能丧失的重要原因,损伤发生后细胞难以再生阻碍了神经功能恢复。既往的研究表明,细胞移植,特别是神经前体细胞移植诱导产生髓磷脂能促进轴突髓鞘再生,恢复完整动作电位传导和神经功能。胚胎干细胞、神经干细胞等多种胚胎、成体干细胞能分化为OPCs,为髓鞘损失的细胞治疗提供了潜在的细胞来源。但是,胚胎干细胞临床应用的伦理障碍和体内移植致瘤性成为胚胎干细胞来源的OPCs治疗脱髓鞘疾病的最大障碍之一。出生后的成体获取神经干细胞极其困难,细胞量难以保证,因此成体神经干细胞多来源于流产胎儿,伦理障碍更大。所以更多的研究关注于成体多能干细胞体外诱导、扩增产生的OPCs。
MSCs是存在于多种组织的多能干细胞,具有三胚层分化潜能。除成体骨髓、脂肪、皮肤等组织外,围产期胎儿附属物,包括脐带、羊膜、羊水、胎盘、脐带血等组织中均能分离出大量的MSCs。其中脐带来源的MSCs最易于规模化培养和扩增,细胞均一性好,且具有成骨、成脂肪、成软骨、成内皮祖细胞、成神经干细胞等分化潜能,免疫原性低,移植后没有明显的不良反应和致瘤性。目前,国内已经建立了多个MSCs库,从而MSCs成为除造血干细胞之外,最适于用于细胞治疗的种子细胞,开发MSCs来源的OPCs制备体系是OPCs移植治疗脱髓鞘疾病产业化途径中最重要的工艺问题。既往的研究多集中与胚胎干细胞、诱导多能干细胞的OPCs诱导体系,即采用“拟胚体培养—神经球—OPCs诱导分化”三步制备体系,近几年来,有团队采用这个体系制备MSCs来源的OPCs,但因工艺复杂,很难满足MSCs来源的OPCs标准化、规模化制备。
基于上述情况,申请人开发了一种试剂盒,用于MSCs来源的OPCs产业化制备,无需经过神经球培养阶段,按常规传代即可,工艺简单,而且本试剂盒不含任何血清等动物源成分,满足临床研究对培养基的要求。细胞制剂用于临床研究和临床应用,其细胞数量不仅需要满足临床剂量的要求,还需要满足质量控制的要求。既往的研究表明,除了胚胎干细胞能大量诱导分化产生神经干细胞,再诱导分化产生OPCs外,很难通过神经球培养和诱导获得数量足够的、A2B5阳性率高的OPCs。使用本试剂盒制备MSCs来源的P2代OPCs,除了A2B5表达率高达92.09±11.43%(附图13),体外少突胶质细胞诱导得率高达44.04%(附图14)外,相比于P2代MSCs,扩增倍率高达133.23倍(附图14)。以本实验室制备P2代脐带来源的MSCs为例,一根10cm的脐带, P2代MSCs得率平均为1.34±1.10 ×109,体外制备应可收获1.79×1011P2代OPCs,完全满足建立OPCs库的产业化制备需求。因此,本试剂盒是一款使用方便、无血清等动物源成分的、高效制备A2B5+OPCs的试剂盒。
Claims (7)
1.一种MSCs来源的少突胶质细胞制备试剂盒,其特征在于,包括OPCs无血清基础培养基、OPCs培养添加剂1、OPCs培养添加剂2及OPCs培养表面包被液,其中,
所述OPCs无血清基础培养基为含有 B27,N2,L-谷氨酰胺,1,25-二羟维生素D3,三碘甲状腺氨酸,人褪黑素的DMEM/F12培养基;
所述OPCs培养添加剂1为含有重组人碱性成纤维细胞生长因子、重组人表皮生长因子的DMEM/F12培养基;
所述OPCs培养添加剂2为含有重组人血小板来源生长因子AA、重组人神经营养因子3的DMEM/F12培养基;
所述OPCs培养表面包被液为含有重组人层黏连蛋白、重组人玻连蛋白的DMEM/F12培养基。
2.根据权利要求1所述的一种MSCs来源的少突胶质细胞制备试剂盒,其特征在于,
所述OPCs无血清基础培养基含有1×B27,1×N2,2mM L-谷氨酰胺,1uM 1,25-二羟维生素D3,40ng/mL三碘甲状腺氨酸,0.5ug/mL人褪黑素;
所述OPCs培养添加剂1为含有1 ug /mL重组人碱性成纤维细胞生长因子、1 ug /mL重组人表皮生长因子的DMEM/F12;
所述OPCs培养添加剂2为含有1 ug 重组人血小板来源生长因子AA、100ng/mL重组人神经营养因子3的DMEM/F12;
所述OPCs培养表面包被液为含有100ng/mL重组人层黏连蛋白、100ng/mL重组人玻连蛋白的DMEM/F12。
3.一种MSCs来源的少突胶质细胞制备方法,其特征在于,包括如下步骤:
S1.用OPCs培养表面包被液室温包被培养表面1小时,吸弃包被液,经PBS漂洗后置于4℃保存不超过1周;
S2.P2代的MSCs按6000个/cm2接种,以含10%无动物源成分血清替代品的MSCs培养基培养至60-70%汇合;
S3.换含OPCs培养添加剂1的OPCs无血清基础培养基,培养至80-90%汇合,用胰蛋白酶溶液消化收获细胞,记为神经上皮预诱导细胞;
S4.以含OPCs培养添加剂1和OPCs培养添加剂2的OPCs无血清基础培养基重悬神经上皮预诱导细胞,调整细胞密度至1.5×104/cm2,接种至被OPCs培养表面包被液包被的培养表面,培养至60-80%汇合时传代,记为P0代OPCs。
4.根据权利要求3所述的一种MSCs来源的少突胶质细胞制备方法,其特征在于,
所述OPCs培养表面包被液为经DMEM/F12稀释10倍的OPCs培养表面包被液;
所述含OPCs培养添加剂1的OPCs无血清基础培养基中OPCs培养添加剂1含量为2%;
所述含OPCs培养添加剂1和OPCs培养添加剂2的OPCs无血清基础培养基中OPCs培养添加剂1的含量为2%,OPCs培养添加剂2的含量为2%。
5.根据权利要求3所述的一种MSCs来源的少突胶质细胞制备方法,其特征在于,所述步骤S4后还包括如下步骤:将P0代OPCs按常规贴壁细胞传代方法传代,收获P2代细胞。
6.根据权利要求5所述的一种MSCs来源的少突胶质细胞制备方法,其特征在于,所述常规贴壁细胞传代方法传代过程中的接种密度为1.5×104/cm2,培养体系为含1%OPCs培养添加剂1和1% OPCs培养添加剂2的OPCs无血清基础培养基;培养表面为经OPCs培养表面包被液包被的培养表面。
7.根据权利要求3所述的一种MSCs来源的少突胶质细胞制备方法,其特征在于,所述MSCs来源于人脐带、羊膜、羊水、胎盘、脐带血、宫膜、牙髓、骨髓、皮肤、肌腱、骨骼肌。
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