中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
10期
1539-1546
,共8页
干细胞%脐带脐血干细胞%培养%间充质干细胞%血小板裂解液%细胞培养%成骨分化%成脂分化
榦細胞%臍帶臍血榦細胞%培養%間充質榦細胞%血小闆裂解液%細胞培養%成骨分化%成脂分化
간세포%제대제혈간세포%배양%간충질간세포%혈소판렬해액%세포배양%성골분화%성지분화
stem cells%mesenchymal stem cells%culture media%adipogenesis%actihaemyl
背景：目前国内外大多数实验仍采用经典的培养基和胎牛血清进行脐带间充质干细胞培养，血清培养潜在的不安全因素限制了未来临床应用的可行性。<br> 目的：以人血小板裂解液替代胎牛血清培养、鉴定人间充质干细胞，以期进一步应用于临床低密度扩增人间充质干细胞。<br> 方法：人血小板裂解液通过反复冻融、离心、过滤、浓缩等方法制备。以 IMDM 为基础培养基，添加5%浓缩血小板裂解液作为实验组培养基；以添加体积分数10%胎牛血清为对照组培养基。采用酶消化法分离培养人脐带间充质干细胞，以3000/cm2的浓度进行传代培养，待细胞扩增至P5代后，进行细胞形态与直径、免疫表型、成骨成脂分化、克隆形成率等细胞生物学特性检测，并比较两者之间的差别。<br> 结果与结论：人血小板裂解液扩增的脐带间充质干细胞形态细长，有更高的细胞累积群倍数；克隆形成检测结果显示两者形成的克隆效率差异无显著性意义，流式细胞术检测结果显示两者有相似的细胞表型，体外诱导分化显示两者都具有成骨、成脂分化能力，但人血小板裂解液扩增的间充质干细胞成骨分化能力更强。提示人血小板裂解液可以取代胎牛血清用于临床低密度扩增人间充质干细胞。
揹景：目前國內外大多數實驗仍採用經典的培養基和胎牛血清進行臍帶間充質榦細胞培養，血清培養潛在的不安全因素限製瞭未來臨床應用的可行性。<br> 目的：以人血小闆裂解液替代胎牛血清培養、鑒定人間充質榦細胞，以期進一步應用于臨床低密度擴增人間充質榦細胞。<br> 方法：人血小闆裂解液通過反複凍融、離心、過濾、濃縮等方法製備。以 IMDM 為基礎培養基，添加5%濃縮血小闆裂解液作為實驗組培養基；以添加體積分數10%胎牛血清為對照組培養基。採用酶消化法分離培養人臍帶間充質榦細胞，以3000/cm2的濃度進行傳代培養，待細胞擴增至P5代後，進行細胞形態與直徑、免疫錶型、成骨成脂分化、剋隆形成率等細胞生物學特性檢測，併比較兩者之間的差彆。<br> 結果與結論：人血小闆裂解液擴增的臍帶間充質榦細胞形態細長，有更高的細胞纍積群倍數；剋隆形成檢測結果顯示兩者形成的剋隆效率差異無顯著性意義，流式細胞術檢測結果顯示兩者有相似的細胞錶型，體外誘導分化顯示兩者都具有成骨、成脂分化能力，但人血小闆裂解液擴增的間充質榦細胞成骨分化能力更彊。提示人血小闆裂解液可以取代胎牛血清用于臨床低密度擴增人間充質榦細胞。
배경：목전국내외대다수실험잉채용경전적배양기화태우혈청진행제대간충질간세포배양，혈청배양잠재적불안전인소한제료미래림상응용적가행성。<br> 목적：이인혈소판렬해액체대태우혈청배양、감정인간충질간세포，이기진일보응용우림상저밀도확증인간충질간세포。<br> 방법：인혈소판렬해액통과반복동융、리심、과려、농축등방법제비。이 IMDM 위기출배양기，첨가5%농축혈소판렬해액작위실험조배양기；이첨가체적분수10%태우혈청위대조조배양기。채용매소화법분리배양인제대간충질간세포，이3000/cm2적농도진행전대배양，대세포확증지P5대후，진행세포형태여직경、면역표형、성골성지분화、극륭형성솔등세포생물학특성검측，병비교량자지간적차별。<br> 결과여결론：인혈소판렬해액확증적제대간충질간세포형태세장，유경고적세포루적군배수；극륭형성검측결과현시량자형성적극륭효솔차이무현저성의의，류식세포술검측결과현시량자유상사적세포표형，체외유도분화현시량자도구유성골、성지분화능력，단인혈소판렬해액확증적간충질간세포성골분화능력경강。제시인혈소판렬해액가이취대태우혈청용우림상저밀도확증인간충질간세포。
BACKGROUND:Classic media and fetal bovine serum are commonly used in the culture of umbilical cord mesenchymal stem cells, but the potential risk of serum culture limits its clinical application. OBJECTIVE:To use human platelet lysate alternative to fetal bovine serum for large-scale production of mesenchymal stem cells for therapeutic applications. METHODS:Human platelet lysate was prepared by repeated freezing and thawing, centrifugation, filtration, and concentration. Human umbilical cord mesenchymal stem cells were cultured in Iscove’s Modified Dulbecco’s Medium containing 5%concentrated platelet lysate (experimental group) or 10%fetal bovine serum (control group). After separation and enzymatic digestion, human umbilical cord mesenchymal stem cells were subcultured at a concentration of 3 000/cm2 up to the fifth generation. Then, cellmorphology and diameter, immune phenotype, osteogenic and adipogenic differentiation, and cloning efficiency were detected and compared between two groups. RESULTS AND CONCLUSION:Human umbilical cord mesenchymal stem cells cultivated in human platelet lysate-supplemented media showed a smal er size and more elongated morphology than those in fetal bovine serum-supplemented media. Colony forming unit-fibroblast analyses further showed no significant differences in colony efficiency. Human umbilical cord mesenchymal stem cells cultivated in human platelet lysate-supplemented media showed an increase of proliferation capacity;whereas, similar immunophenotypes remained in the two groups. In vitro assays revealed intact differentiation potential. Moreover, human umbilical cord mesenchymal stem cells cultivated in human platelet lysate-supplemented media showed a significantly higher capacity to differentiate towards osteocytes, indicating human platelet lysate is an alternative to fetal bovine serum for low-density production of mesenchymal stem cells for therapeutic applications.
