在线微载体肝细胞荧光示踪研究

目的拟探讨SYTO/EB在微载体细胞培养中活体细胞原位示踪的应用价值。方法采用活细胞核酸染色剂(SYTO13)和溴化乙啶(EB)2种核酸染色剂对微载体生长的细胞进行双重染色,荧光显微镜下通过颜色不同判断细胞的活力状态并计算出活力百分比。以间接活力测定的四氮唑盐法同时对肝细胞进行活力的检测,并将结果进行比较。结果 2种方法测定的结果均可显示微载体生长的肝细胞。但SYTO/EB法更灵敏,可精确计数。结论 SYTO/EB测定微载体肝细胞的活力快速、灵敏且简便,并可原位观察细胞的活力,不失为一种可取的微载体活力检测方法。

Improved microscopic observation of mammalian cells on microcarriers by fluorescent staining

Objective To develop an assay for viability detection of liver cell cultured on the Cytodex 3 in situ.Methods Two nucleic acid dyes,SYTO 13 and ethidium bromide(EB) were used to assess the viability of liver cells.Cells were stained with SYTO 13/EB.The viability of cells was determined by fluorescence microscope,and compared with MTT assay.Results Although the cell number and morphology could be clearly seen with the Cytodex 3,but cells viability could not be discerned.No cell was obviously visible inside the gelatin beads,although some cells were seen on the external surface in MTT assay.When cells were cultivated as aggregates,this FDA/EB staining technique could also be applied to such a system.High viability could be seen in large clumps of cells.Conclusion This staining technique is to be simple,quick and reliable when used in microcarrier liver cell culture.This technique has been applied to a number of microcarrier.The use of this technique with macroporous microcarriers makes it a potential visualization technique in other immobilized mammalian cell systems.