CAJ | 학술논문

背景：大多哺乳类细胞在正常生理状态下多以三维结构存在，为还原细胞本身在体内的自然状态，很多研究者开始尝试在体外对细胞进行三维培养，球形培养是一种常见的三维培养模式。 　　目的：尝试用3种不同的方法在体外对人脂肪干细胞进行球形培养，并观察其生物学特征。 　　方法：对提取的脂肪来源细胞进行表面 Marker 流式检测以及成脂成骨诱导鉴定后，证实为脂肪干细胞。先后用低黏附培养法、hanging-drop培养法和Eppendorf管培养法3种方法在体外对脂肪干细胞进行球形培养，对3种方法形成球形的特点进行比较分析，并通过 Imagej 软件计算出3组多细胞球体的平均面积，利用Viability/Cytotoxicity Assay Kit for Animal Live&Dead Cel s试剂盒对3组多细胞球体进行活力检测。 　　结果与结论：①通过流式细胞术鉴定细胞表型标志物，其中CD29，CD44，CD59完全阳性，同时成脂成骨诱导也为阳性，证实提取的细胞为脂肪干细胞。3代以内脂肪干细胞多呈长梭形，并克隆状生长。②低黏附培养法、hanging-drop 培养法、Eppendorf 管培养法均可使脂肪干细胞聚集成球形生长，但所成多细胞球形有所差异。低黏附培养法所形成的细胞球形大小不一，不易控制；而hanging-drop培养法和Eppendorf管培养法均可使脂肪干细胞形成大小均一的球形，但在大小和形成时间上有所不同。③3种不同方法所形成的球形细胞均保持较好的细胞活力。
배경：대다포유류세포재정상생리상태하다이삼유결구존재，위환원세포본신재체내적자연상태，흔다연구자개시상시재체외대세포진행삼유배양，구형배양시일충상견적삼유배양모식。 　　목적：상시용3충불동적방법재체외대인지방간세포진행구형배양，병관찰기생물학특정。 　　방법：대제취적지방래원세포진행표면 Marker 류식검측이급성지성골유도감정후，증실위지방간세포。선후용저점부배양법、hanging-drop배양법화Eppendorf관배양법3충방법재체외대지방간세포진행구형배양，대3충방법형성구형적특점진행비교분석，병통과 Imagej 연건계산출3조다세포구체적평균면적，이용Viability/Cytotoxicity Assay Kit for Animal Live&Dead Cel s시제합대3조다세포구체진행활력검측。 　　결과여결론：①통과류식세포술감정세포표형표지물，기중CD29，CD44，CD59완전양성，동시성지성골유도야위양성，증실제취적세포위지방간세포。3대이내지방간세포다정장사형，병극륭상생장。②저점부배양법、hanging-drop 배양법、Eppendorf 관배양법균가사지방간세포취집성구형생장，단소성다세포구형유소차이。저점부배양법소형성적세포구형대소불일，불역공제；이hanging-drop배양법화Eppendorf관배양법균가사지방간세포형성대소균일적구형，단재대소화형성시간상유소불동。③3충불동방법소형성적구형세포균보지교호적세포활력。
BACKGROUND:Many types of mammalian cells aggregate and display three-dimensional multicellular spheroids when they are in normal physiological conditions. In order to observe and explore cellular natural states, many researchers try to use spherical cellculture in vitro, a common three-dimensional culture pattern. OBJECTIVE:To use three different methods for spherical culture in vitro of adipose-derived stem cells and to observe their biological features. METHODS:Adipose-derived stem cells were confirmed by the analysis of the markers for cellphenotypes as wel as adipogenic and osteogenic differentiation potential assays. Three different methods of sphere cultures were used as fol ows:(1) ultra low attachment culture;(2) hanging-drop culture and (3) Eppendorf tube culture. The sphere formation was compared among above three methods. We used Imagej to calculate mean areas of these spheres. And we used Viability/Cytotoxicity Assay Kit for Animal Live&Dead cells to detect their vitality. RESULTS AND CONCLUSION:(1) Adipose-derived stem cells were confirmed by the analysis of the markers for cellphenotypes, CD29, CD44, CD59 were positive, as wel as adipogenic and osteogenic differentiation potential assays were positive. The conventional monolayer cultures of adipose-derived stem cells showed spindle and cloning growth within three passages. (2) Ultra low attachment culture, hanging-drop culture, Eppendorf tube culture al could elicit adipose-derived stem cells spherical growth. However, spherical size, shape and uniformity differed depending on cellnumbers, culture time and spherical culture methods. The ultra low attachment culture was comparatively difficult to control spherical shape and uniformity of adipose-derived stem cells. But hanging-drop culture and Eppendorf tube culture were able to form even cellspheres. (3) Spherical formation of adipose-derived stem cells using our three methods displayed good cellvitality.