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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
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 18:911–915
Barnes LM, Bentley CM, Dickson AJ (2001) Characterization of the stability of recombinant protein production in the GS-NS0 expression system. Biotechnol Bioeng 73:261–270. https://doi.org/10.1002/bit.1059
Barnes LM, Moy N, Dickson AJ (2006) Phenotypic variation during cloning procedures: analysis of the growth behavior of clonal cell lines. Biotechnol Bioeng 94:530–537. https://doi.org/10.1002/bit.20856
Browne SM, Al-Rubeai M (2007) Selection methods for high-producing mammalian cell lines. Trends Biotechnol 25:425–432. https://doi.org/10.1016/j.tibtech.2007.07.002
Calles K, Eriksson U, Haggstrom L (2006a) Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures. Biotechnol Prog 22:653–659. https://doi.org/10.1021/bp050252+
Calles K, Svensson I, Lindskog E, Haggstrom L (2006b) Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity. Biotechnol Prog 22:394–400. https://doi.org/10.1021/bp050297a
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
Ding WF, Feng Y, Zhang X, Li X, Wang CY, Xie SC (2015) Establishment and characterization of four cell lines developed from Papilio demoleus (Lepidoptera: Papilionidae). Acta Entomol Sin 58:826–835. https://doi.org/10.16380/j.kcxb.2015.08.002
Drugmand J, Schneider Y, Agathos SN (2012) Insect cells as factories for biomanufacturing. Biotechnol Adv 30:1140–1157. https://doi.org/10.1016/j.biotechAdv.2011.09.014
Freshney RI (2004) Cloning and selection culture of animal cells: a manual of basic technique. Wiley-Liss, Hoboken, pp 217–222
Grace TDC (1968) The development of clones from lines of Antheraea eucalypti cells grown in vitro. Exp Cell Res 52:451–458. https://doi.org/10.1016/0014-4827(68)90487-4
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
Gross A, Schoendube J, Zimmermann S, Steeb M, Zengerle R, Koltay P (2015) Technologies for single-cell isolation. Int J Mol Sci 16:16897–16919. https://doi.org/10.3390/ijms160816897
Hou JJ, Hughes BS, Smede M, Leung KM, Levine K, Rigby S, Gray PP, Munro TP (2014) High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system. N Biotechnol 31:214–220. https://doi.org/10.1016/j.nbt.2014.02.002
Hu YC (2005) Baculovirus as a highly efficient expression vector in insect and mammalian cells. Acta Pharmacol Sin 26:405–416. https://doi.org/10.1111/j.1745-7254.2005.00078.x
Ikonomou L, Schneider YJ, Agathos SN (2003) Insect cell culture for industrial production of recombinant proteins. Appl Microbiol Biotechnol 62:1–20. https://doi.org/10.1007/s00253-003-1223-9
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
Kioukia N, Nienow AW, Emery AN, Al-Rubeai M (1995) Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus. J Biotechnol 38:243–251. https://doi.org/10.1016/0168-1656(94)00128-Y
Lindgren K, Salmén A, Lundgren M, Bylund L, Ebler Å, Fäldt E, Sörvik L, Fenge C, Skoging-Nyberg U (2009) Automation of cell line development. Cytotechnology 59:1–10. https://doi.org/10.1007/s10616-009-9187-y
Llames S, Garcia-Perez E, Meana A, Larcher F, Del RM (2015) Feeder layer cell actions and applications. Tissue engineering. Tissue Eng Part B Rev 21:345–353. https://doi.org/10.1089/ten.TEB.2014.0547
Lynn DE (2001) Novel techniques to establish new insect cell lines. In Vitro Cell Dev Biol Anim 37:319–321. https://doi.org/10.1007/BF02577564
Mcintosh AH, Rechtoris C (1974) Insect cells: colony formation and cloning in agar medium. In Vitro 10:1–5
Mitsuhashi J (2002) Karyotype analysis invertebrate tissue culture methods. Springer, Tokyo, pp 321–323
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
Pan MH, Cai XJ, Liu M, Lv J, Tang H, Tan J, Lu C (2010) Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori. Tissue Cell 42:42–46. https://doi.org/10.1016/j.tice.2009.07.002
Sokolenko S, George S, Wagner A, Tuladhar A, Andrich JM, Aucoin MG (2012) Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: benefits and drawbacks. Biotechnol Adv 30:766–781. https://doi.org/10.1016/j.biotechadv.2012.01.009
Swiech K, Picanço-Castro V, Covas DT (2012) Human cells: new platform for recombinant the rapeutic protein production. Protein Expr Purif 84:147–153. https://doi.org/10.1016/j.pep.2012.04.023
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
Yamaji H, Manabe T, Watakabe K, Muraoka M, Fujii I, Fukuda H (2008) Production of functional antibody Fab fragment by recombinant insect cells. Biochem Eng 41:203–209. https://doi.org/10.1016/j.bej.2008.04.017
Zhang X, Feng Y, Ding WF, Chen XM, Wang CY, Ma T (2011) Establishment and characterization of an embryonic cell line from Gampsocleis gratiosa (Orthoptera: Tettigoniidae). In Vitro Cell Dev Biol Anim 47:327–332. https://doi.org/10.1007/s11626-011-9397-4
Acknowledgements
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)”.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors report no conflicts of interest.
Rights and permissions
About this article
Cite this article
Liu, ZG., Ding, WF., Xie, SC. et al. Establishment and characterization of cell clones from the Papilio cell line RIRI-PaDe-3 by a high-efficiency clonal method. Cytotechnology 70, 1235–1245 (2018). https://doi.org/10.1007/s10616-018-0216-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-018-0216-6