All six cell lines (numbered 1 to 6 in Figure 4C) retained their phenotype after at least 30 passages. Thus, having started with the transfection of 30,000 cells, we obtained six stable cell lines after two rounds of selection in about 2 weeks. The first and most efficient round comprised the isolation of microcolonies that grew up, fluoresced, and were homogenous. This eliminated any cells that could not proliferate, or did not produce GFP, or produced mosaic colonies after a number of generations. In the second round, we verified the viability and homogeneity of the selected colonies. As the only purpose of this experiment was the demonstration of cell cloning, the clones were propagated and passaged in a conventional 2-D culture. Of course, if there was also a need to maintain the cell physiology, the merged gels described here could be used in the second round, too.
Here, colonies were selected by visual assessment of their homogeneity and fluorescence intensity, and manually picked. In a special experiment, the ability of this simple approach to yield high-quality cell preparations was confirmed by a flow cytometry analysis of the selected colonies (Supplementary Figure S6). Yet it is conceivable that the throughput and reliability of the procedure could be further improved by using automated devices for colony imaging and collection (36).
Cloning of eukaryotic cells is required in many basic and applied research areas (37, 38), in particular for isolating cell lines stably producing the proteins of interest (39), but it still remains a challenging task. As pointed out by Clarke et al. (38) in regard to the existing cloning methods, “there is one problem common to them all: there is no way to be certain that the derived population originated from a single cell.” The procedure reported here is essentially “cloning by micromanipulation,” but it differs from the earlier published protocol (38) in that the cells are presented as a monolayer and immobilized in a gel, rather than provided as a suspension in which cells can freely migrate at different depths. Hence, the potential problems of cloning by micromanipulation (limited depth of microscopy field and eventual adherence of cells to the outside of the micropipet; see Reference 38) can be overcome. Moreover, by enabling monitoring the entire process of the formation of individual colonies, the present method allows the colonies that have originated from single cells to be precisely identified. This feature is illustrated in Figure 3A; it can be seen that colonies numbered 2 to 8 have each originated from a single cell, while colonies 1 and 9 have originated from two and four cells, respectively. Therefore, we believe that the approach described here enables, at least potentially, the true cloning of eukaryotic cells.
In conclusion, it should be noted that the method reported here enables cloning from rather dense cell populations. In the experiment described in Figure 4, the cells were immobilized at a surface density of ╛100 cells/mm2, which, assuming that the layer thickness was 10 m (compare Figure 1B), corresponded to the volume concentration of 107 cells/mL. Although still lower than the cell concentration in solid tissues of up to 109 cells/mL (38), it equals the concentration of leukocytes in the peripheral blood, with the same mean cell-to-cell distances. This largely overcomes the negative effects of the very low population density at which cells occur during the conventional cloning procedures (38).
The authors thank Drs. F.K. Gioeva and T.R. Samatov for their help and advice, I.A. Eliseeva for assistance in the acquisition of confocal images, and N.A. Nikitenko for carrying out flow cytometer analyses. This work was supported in part by program “Molecular and Cell Biology” of the Russian Academy of Sciences and by the Russian Foundation for Basic Research.
A.B.C. and H.V.C. are co-inventors and co-owners of a patent (no. RU2394915) and patent applications (nos. WO2007111639, US2009105082, and EP1999268) disclosing the method of merged gels. A.A.G. declares no competing interests.
Address correspondence to Alexander B. Chetverin, Institute of Protein Research of the Russian Academy of Sciences, 4 Institutskaya Street, Pushchino, Moscow Region, Russia. Email: [email protected]
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