Cells Fusion

 

There are several known methods how to fuse living cells. The first one uses immersion of cells into chemical solution (e.g. 50% PEG 1500)¹, the second method employs external electric field for the cell perforation². Unfortunately both these methods cannot be easily used to fuse ndividually selected cells. The third method of cell fusion uses focused laser beams that evaporate tiny volume of the cell membrane^{3, 4}. Laser induced cells fusion takes place under an objective of a microscope and easily enables the study of the fusion dynamics.

 

The principle of the laser-induced cell fusion:

 

 

 

Two selected cells (No. 1 and 2) are brought into contact by optical tweezers (see subfigure a). Bigger cells, which cannot be dragged by optical tweezers, are transported by mechanical micromanipulator (Eppendorf TransferManâ NK) and micropipette (Eppendorf CellTram Air). A sequence of pulses is applied at the point of cells contact (subfigure b denoted by arrow), the membranes in contact are perforated and the content of both cells is mixed (subfigure c). A few minutes later the fusion product takes round shape again and cell nuclei start to mix.

 

Experiment:

The experiments were performed in cooparation with the group of Prof. S. Kozubek from Institute of Biophyscis ASCR, Prof. M. Kozubek from Faculty of Informatics Masaryk University in Brno. The first experiment with cells fusion we performed with human lymphocyte cells HL60. Later on we worked with adherent MCF 7 cells that fused easier. They were placed on the micro-grid cover slip (CELLocate, square size 55 mm) for easier localization under the microscope.

 

 

Figure shows the employment of the trapping and cutting beam for the cell fusion HL60. Two human lymphocyte cells (No. 1 and 2) were brought into touch by optical tweezers (see subfigure a). Laser pulses of the cutting beam (dark spot in subfigure b denoted by the arrow) perforated the outer cell membrane and both cells fused together (c, d). Subfigure c is taken 40s and subfigure d  160s  after the wall perforation.

 

 

Two MCF 7 cells (denoted as 1, 2) form a cluster (see subfigure a). Laser pulses are applied to perforate the cell membrane at the point of contact (light spot in subfigure b denoted by circle). The content of both cells is mixed (see subfigure c).

 

 

Further studies with fused cells

The cell nuclei were dyed by low flourochrome concentration for their easier identification in the fused cell. One of MCF7 cell was colored by Hoechst 33342 which provides blue fluorescence and the other by Propidium Iodide which provides red fluorescence. Optical tweezers or micropipette was used to bring both cells to contact and series of 4-5 pulses from UV laser (an average energy per pulse was equal to 8 mJ) perforated the membrane. To achieve this, it was necessary to move the sample vertically so that the point of contact coincided with focal plane of the UV beam.   The plasmatic membranes disrupted by thermal ablation and their ends immediately joined to form a single hybrid cell. After the fusion, the cells were cultured in a fresh medium. In the intervals of 4, 8, 12, and 24 hours fused cells were fixed by paraformaldehyde and afterwards they were studied by fluorescence in situ hybridization using the specific DNA probe for chromosomes 12 and 7 centromere. We found out that non-fused single MCF7 cells had three signals corresponding to the chromosomes 12 and 7 (trisomia of chromosomes 12 and 7), meanwhile six signals were found in fused cells (see the figures below). Using high-resolution cytomery⁵, the dynamics of the chromosome arrangement in the progress of time after the cell fusion was studied. We observed that the homologous chromosomes in the fused cells do not merge together but occupy their separate positions in the fused nucleus.

               

Unfortunately we have not observed division of fused cell even if we observed it for several days. Moreover they usually died within this period. 

Further reading:

J. Jezek, S. Palsa, E. Lukasova, S. Kozubek, P. Jakl, M. Sery, A. Jonas, M. Liska, P. Zemanek: "Employment of laser induced fusion of living cells for the study of spatial structure of chromatin",
       Proceedings of  SPIE 5259, 336-340, 2003.

J. Jezek, S. Palsa, E. Lukasova, S. Kozubek, P. Jakl, M. Sery, A. Jonas, M. Liska, P. Zemanek: "Spatial structure of chromatin in hybrid cells produced by laser induced fusion studied by optical microscopy",
       Proceedings of SPIE 5036, 630-634, 2002.

References:

 

1.       G. A. Neil and U. Zimmermann, "Electrofusion", Methods Enzymol. 220, pp. 174-196, 1993.

2.       A. Strömberg et al, " Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion", PNAS 97 (1), pp. 7-11, 2000.

3.       S. Sato et al,"Achievement of laser fusion of biological cells using UV pulsed dye laser beams", Appl. Phys. B 54, pp. 531-533, 1992.

4.       R. Wiegand et al, "Laser-induced fusion of cells and plant protoplasts", Journal of Cell Science 88, pp. 145-149, 1987.

5.       M. Kozubek et al, "Combined confocal and wide-field high-resolution cytometry of FISH-stained cells" Cytometry 45, pp.1-12, 2001.