Establishment and characterization of cell clones from the Papilio cell line RIRI-PaDe-3 by a high-efficiency clonal method
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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.
KeywordsCell cloning Semisolid culture Micromanipulation Inter-simple sequence repeat
This work was supported by “the Fundamental Research Funds for the Central Non-profit Research Institution of CAF (No. CAFYBB2016ZD005)” and “the Fundamental Research Funds for the Central Non-profit Research Institution of Research Institute of Resource Insects, CAF (No. riricaf2012003M)”.
Compliance with ethical standards
Conflict of interest
The authors report no conflicts of interest.
- Allieri MA, Douay L, Deloux J, Smadja N, Najman A, Gorin NC (1990) The role of methylcellulose on colony growth of human myeloid leukemic progenitors (AML-CFU). Exp Hematol 18:911–915Google Scholar
- Dharshanan S, Chong H, Hung CS, Zamrod Z, Kamal N (2011) Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity. Electron J Biotechnol 14:1–10. https://doi.org/10.2225/vol14-issue2-fulltext-7 CrossRefGoogle Scholar
- Freshney RI (2004) Cloning and selection culture of animal cells: a manual of basic technique. Wiley-Liss, Hoboken, pp 217–222Google Scholar
- Grasela JJ, McIntosh AH (2003) Application of inter-simple sequence repeats to insect cell lines: Identification at the clonal and tissue-specific level. In Vitro Cell Dev Biol Anim 39:353–363. https://doi.org/10.1290/1543-706X(2003)039%3C0353:AOISRT%3E2.0.CO;2 CrossRefGoogle Scholar
- Kasai F, Hirayama N, Ozawa M, Iemura M, Kohara A (2016) Changes of heterogeneous cell populations in the ishikawa cell line during long-term culture: proposal for an in vitro clonal evolution model of tumor cells. Genomics 107:259–266. https://doi.org/10.1016/j.ygeno.2016.04.003 CrossRefGoogle Scholar
- Nguyen Q, Ying-Mei Q, Yang W, Chan LCL, Nielsen LK, Reid S (2011) In vitro production of Helicoverpa baculovirus biopesticides—automated selection of insect cell clones for manufacturing and systems biology studies. J Virol Methods 175:197–205. https://doi.org/10.1016/j.jviromet.2011.05.011 CrossRefGoogle Scholar
- Wen FY, Zhang YA, Qu LJ, Zhang H, Yang ZQ, Qin QL, Wang YZ, Wang WH (2009) Two new cell lines originated from the embryos of Clostera anachoreta (Lepidoptera: Notodontidae): characterization and susceptibility to baculoviruses. In Vitro Cell Dev Biol Anim 45:409–413. https://doi.org/10.1007/s11626-009-9196-3 CrossRefGoogle Scholar