Liu Zhi-Gang, Ding Wei-Feng, Xie Shi-Cong, Sun Na, Zhang Xin, Li Xian, Feng Ying
Key Laboratory of Cultivating and Utilization of Resource Insects of State Forestry Administration, Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming, 650233, Yunnan, China.
Cytotechnology. 2018 Aug;70(4):1235-1245. doi: 10.1007/s10616-018-0216-6. Epub 2018 Apr 9.
Cell cloning is of great importance in keeping particular properties of cultured cells, and interesting cells can be selected by cloning from heterogeneous cell populations. In addition, continuous cell lines usually from primary culture are prone to heterologous constitution and genetic instability, so that supplementary cloning steps are necessary for achieving a homogenous cell population. In this study, limiting dilution culture and feeder layer culture were originally used for cloning RIRI-PaDe-3 cell line, but both failed. Afterward, we designed a cloning protocol which was composed of two steps: cells in semisolid medium with seeding density in the range of 3.05 × 10-6.10 × 10 cells/mL formed colonies from monodispersed cell suspensions; 40 well-dispersed colonies were removed from the suspended state by using micromanipulator system and finally scaled up. To determine whether this method can isolate cell lines possessing characteristics different from the parent population, we made an evaluation of cells monoclonal in biological characteristics. Significant differences have been found among clones isolated from the RIRI-PaDe-3 insect cell line in cell morphology, chromosome numbers, and genetic background. Thus the indicated modified semisolid medium cloning protocol was advantageous to the convenient and genuine cloning from the previously heterogeneous population.
细胞克隆对于保持培养细胞的特定特性非常重要，并且可以通过从异质细胞群体中克隆来选择感兴趣的细胞。此外，通常源自原代培养的连续细胞系易于出现异源组成和遗传不稳定性，因此需要补充克隆步骤以获得同质细胞群体。在本研究中，最初使用有限稀释培养和饲养层培养来克隆RIRI-PaDe-3细胞系，但均失败了。随后，我们设计了一种由两个步骤组成的克隆方案：细胞在半固体培养基中，接种密度在3.05×10 - 6.10×10个细胞/毫升范围内，从单分散细胞悬液形成菌落；使用显微操作仪系统从悬浮状态中取出40个分散良好的菌落，最后进行扩大培养。为了确定该方法是否能够分离出具有与亲本群体不同特征的细胞系，我们对细胞的生物学特性进行了单克隆评估。从RIRI-PaDe-3昆虫细胞系分离出的克隆在细胞形态、染色体数目和遗传背景方面存在显著差异。因此，所示的改良半固体培养基克隆方案有利于从先前的异质群体中方便且真实地克隆。
