A new preclinical 3-dimensional agarose colony formation assay
A new preclinical 3-dimensional agarose colony formation assay
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DOI：
10.1177/153303460800700407
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发表时间：
2008-08-01
影响因子：
2.8
通讯作者：
Levin, Victor A.
中科院分区：
文献类型：
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作者：
Kajiwara, Yoshinori;Panchabhai, Sonali;Levin, Victor A.
神经胶质瘤和其他癌症的新药治疗和联合治疗的评价需要一种稳健的方法来询问宽剂量范围和不同的药物暴露时间，而不会使细胞（集落）染色失活。为此，我们开发了一种三维（3D）集落形成测定法，该测定法利用GelCount（TM）技术，一种用于凝胶和软琼脂的新细胞集落计数器。使菌落在双层琼脂糖中生长，所述双层琼脂糖在底部具有0.7%琼脂糖并且在顶部具有0.3%琼脂糖。然后，我们研究了DFMO、卡铂和SAHA在3-log剂量范围内和多天药物暴露的影响。使用GelCount，我们将集落体积的曲线下面积（AUC）近似为每个平板中集落体积（μ m（2）× OD）的总和，以计算IC 50值。MiaPaCa和SW 480细胞生长速度快，5-6天计数到100个集落;胶质瘤细胞生长较慢，9-10天计数到100个集落。可靠的对数剂量与AUC曲线观察所有药物studied.In结论，GelCount（TM）的方法，我们描述的是更定量比传统的集落测定，并允许精确的研究药物的剂量和暴露时间使用更少的培养板。
The evaluation of new drug treatments and combination treatments for gliomas and other cancers requires a robust means to interrogate wide dose ranges and varying times of drug exposure without stain-inactivation of the cells (colonies). To this end, we developed a 3-dimensional (3D) colony formation assay that makes use of GelCount (TM) technology, a new cell colony counter for gels and soft agars.We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas adenocarcinoma and SW480 colon adenocarcinoma cell lines. Colonies were grown in a two-tiered agarose that had 0.7% agarose on the bottom and 0.3% agarose on top. We then studied the effects of DFMO, carboplatin, and SAHA over a 3-log dose range and over multiple days of drug exposure. Using GelCount we approximated the area under the curve (AUC) of colony volumes as the sum of colony volumes (mu m(2) x OD) in each plate to calculate IC50 values.Adenocarcinoma colonies were recognized by GelCount (TM) scanning at 3-4 days, while it took 6-7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid, with 100 colonies counted in 5-6 days; glioma cells grew more slowly, with 100 colonies counted in 9-10 days. Reliable log dose versus AUC curves were observed for all drugs studied.In conclusion, the GelCount (TM) method that we describe is more quantitative than traditional colony assays and allows precise study of drug effects with respect to both dose and time of exposure using fewer culture plates.