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1、人脐带华通氏胶间充质干细胞的分离、培养、鉴定及冻存、复苏南通大学(医学版)?413?JournalofNantongUniversity(MedicalScien!)Q!(鱼人脐带华通氏胶间充质干细胞的分离,培养,鉴定及冻存,复苏杨晓清,张沐,杨兵,张华,张玉泉f?南通大学附属医院妇产科,z南通大学医学院I临床071班,南通226001,江苏省干细胞库)【摘要】目的:探讨从人脐带华通氏胶(Whaaon'Sjelly,WJ)中分离,培养,鉴定间充质干细胞(mesenchymalstemce11s,
2、MSCs)及其冻存,复苏的方法.方法:采用植块法分离,培养间充质干细胞,流式细胞仪检测P3代细胞免疫表型,鉴定其向成骨,成脂方向诱导分化的能力;将Pl细胞冻存6个月后复苏,鉴定复苏后细胞的特性.结果:植块法容易从人脐带华通氏胶中获得间充质干细胞;组织块贴壁后6d可见组织块周围细胞爬出,原代培养14-18d细胞融合70%-80%:P3代细胞强烈表达CD73,CD90,CD105,不表达CD14,CD34,CD45,CD79a和HLA—DR;成骨诱导分化后10d,可见明显钙结节;成脂诱导14d,有明
3、显的脂滴出现,油红O染色阳性.冻存再复苏细胞活力达80%,细胞免疫表型及成骨,成脂诱导显示与冻存前细胞呈相同的特性.结论:组织块培养法可从人脐带华通氏胶中分离,培养出纯度较高间充质干细胞,冻存,复苏不改变其特性.『关键词]人脐带间充质干细胞;华通氏胶;细胞培养;诱导分化;冻存[中图分类号】R394.2【文献标志码]A[文章编号]1674-7887(2010)06—0413—04Isolation,culture,identification,frozennessandthawofmesenchy
4、malstemcellsfromWharton'sjellyofhumanumbilicalcordYANGXiaoqing,ZHANGMu,YANGBin,ZHANGHua3,ZHANGYuquan(DepartmentofGynaecologyandObstetrics,AffiliatedHospitalofNantongUniversity;DepartmentofClinicalMedical,Class071,MedicalCollegeofNantongUniversity,Nan
5、tong226001;DepartmentofStemCellBank,JiangsuProvince)[Abstract】Objective:Toexploretheapproachofisolating,culturing,identifing,frozeningandthawingMSCsfromWhartonSjellyofhumanumbilicalcord.Methods:Humanumbilicalcordmesenchymalstemcells(hUCMSCs)weresepar
6、atedbycultureoftissueadherence.Thesurfaceofpassage3cellsurfaceantigenwasmeasuredbyflowcytometry.Theosteogenicandadipogenicdifferentiationwastestedandevaluatedbyspecificstainingmethods.Passage1cells,frozen6months,werethawed,thentheirbiologicalcharacte
7、ristiswereidentified.Results:MSCswereeasilyobtainedfromWharton'Sjetlyofhumanumbilicalcordviatheproposedapproachoftissueadherence.After6daysofincubation,cellswerepresented.Theprima~cellsgrewupto70%-80%confluenceafter14-18daysofculture.Flowcytometryana
8、lysisrevealedthatCD73,CD90andCD105werehighlyexpressedonthesurfaceofpassages3cells,buttheexpressionwasnegativeforCD14,CD34,CD45,CD79aandHLA—DR.Thesecellsshowedcalciumnodeaftercultureinductionofosteogenicdifferentiation10dayslater.Furthermore,liquidvac
