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. 2018 Aug;70(4):1235-1245.
doi: 10.1007/s10616-018-0216-6. Epub 2018 Apr 9.

Establishment and characterization of cell clones from the Papilio cell line RIRI-PaDe-3 by a high-efficiency clonal method

Zhi-Gang Liu  1 , Wei-Feng Ding  1 , Shi-Cong Xie  1 , Na Sun  1 , Xin Zhang  1 , Xian Li  1 , Ying Feng  2
Affiliations

Establishment and characterization of cell clones from the Papilio cell line RIRI-PaDe-3 by a high-efficiency clonal method

Zhi-Gang Liu et al. Cytotechnology. 2018 Aug.

Abstract

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 × 105-6.10 × 105 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.

Keywords: Cell cloning; Inter-simple sequence repeat; Micromanipulation; Semisolid culture.

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Conflict of interest statement

The authors report no conflicts of interest.

Figures

Fig. 1
Fig. 1
Schematic presenting the configuration of feeder cell layers supporting culture of single cells
Fig. 2
Fig. 2
Schematic presenting the semisolid cloning protocol. RIRI-PaDe-3 cells were cultured in semisolid medium, and single cells proliferated in clonal plaques that were then transferred into a 96-well plate using a micromanipulator system
Fig. 3
Fig. 3
Photomicrographs of RIRI-PaDe-3 and cell clones (Scale bar: 50 μm). a RIRI-PaDe-3; b RIRI-PaDe-3-C4; c RIRI-PaDe-3-C8; d RIRI-PaDe-3-C15; e RIRI-PaDe-3-C26; f RIRI-PaDe-3-C27; g RIRI-PaDe-3-C28; h RIRI-PaDe-3-C30; i RIRI-PaDe-3-C31; j RIRI-PaDe-3-C41
Fig. 4
Fig. 4
Number of chromosomes in cells clonally derived from the RIRI-PaDe-3 cell line
See this image and copyright information in PMC

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