HepG2细胞在不同琼脂糖浓度下克隆形成能力的比较

Comparison of colony-forming capacity of HepG2 cells in different soft agar systems

目的:探讨分离细胞克隆的最佳细胞密度和最适琼脂浓度,为肿瘤干细胞的筛选提供更广阔的思路.方法:将HepG2单细胞悬液与10g/L的琼脂糖相混合,在2g/L(A组)和3g/L(B组)琼脂中接种不同密度的HepG2细胞,14d后观察克隆形成.结果:100-1000个/孔的各个实验组中,A组细胞的克隆数高于B组,差异有统计学意义(t=4.36,P<0.05);在细胞密度为100-500个/孔时,A、B组细胞克隆形成率随着细胞密度的增加逐渐增加;600-1000个/孔,A、B组均随着细胞密度的逐渐加大,克隆形成率呈现下降趋势.结论:在进行软琼脂克隆形成实验时,可以采用2g/L的上层琼脂浓度,并采用10g/L的琼脂糖凝胶作为储备胶,HepG2细胞在500个/孔时的克隆形成率最高.

AIM： To determine the optimal cell density and agar concentration for separation of cell clones and thereby provide a reference for screening of tumor stem cells. METHODS： HepG2 cells were mixed with 10 g/L agarose and inoculated in 2 g/L （group A） and 3 g/L （group B） soft agar at different densities. After 14 days of culture, the number of cell clones was counted and the rate of colony formation was calculated. RESULTS： The number of clones formed in group A was significantly higher than that in group B （t = 4.36, P 〈 0.05）. The rates of colony formation observed in both group A and group B increased with the increase in cell density that varied between 100 and 500 cells per well. In contrast, the rate of colony formation decreased with the increase in cell density that varied between 600 and 1000 cells per well. CONCLUSION： The optimal concentration of agar in the upper layer for colony formation is 3 g/L, with 10% agarose as a stock agar. The maximum rate of colony formation is achieved when ceils are inoculated at a density of 500 cells per well.