JPH048291A - Cell fusion using light trapping by laser - Google Patents

Abstract

PURPOSE:To obtain syncytia in a sterile state in high probability, by maintaining different cells with plural laser beams, respectively, transferring the laser beams and contacting the cells mutually. CONSTITUTION:Cells a1 and cells b2 are optically trapped by two laser beams from light sources 3 of each laser beams, position relationship between the light sources 3 is maintained constant. Then the light sources are transferred in parallel by a micromanipulator, etc., and the cells a1 are brought into contact with the cells b2. Then 400-700V high-voltage pulses are applied from a high- voltage pulse generator 4 to electrodes 5 in the vicinity of the cells a1 and b2 and the cells 1 and 2 are fused in the order of (a) (b) (c).

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for fusion of two specific cells without contact.

[Conventional technology]

Conventional cell fusion methods include the PEG method and the electrofusion method. In the PEG method, polyethylene glycol (PEG)
In this method, a solution containing a high concentration (40 to 50%) of CaCl is mixed with a solution containing cells, and then PEG is removed with a solution of high pH and high concentration of CaC1□ to obtain fused cells. According to cell fusion (Cell Engineering Vol. I No. 31982),
The PE method does not require a large-scale fusion device and is easy to operate.However, it is not possible to fuse specific cells with each other, and since a high concentration of PEG is used, caution should be taken in terms of residual drug efficacy. There's something I have to do.

Next, the electric fusion method will be explained. According to the literature; Electrofusion and Selection (Cell Engineering Vol. 1.3 No. 61984), (1) Microelectrode method (2) Parallel electrode method (3) D
There are currently methods such as electrophoresis.

The microelectrode method is a method of obtaining fused cells by manipulating a pair of microelectrodes with a micromanipulator, lightly touching the electrodes to the surface of each cell to be fused, and applying an electric pulse. This method has the advantage of being able to fuse the specific cells that you want to fuse by stimulating them with electrical pulses, but because the electrodes touch the cells, they cannot fuse under completely sterile conditions. In addition, in the case of fusion using the same method, the fused cells often adhere to the tip of the microelectrode.In the parallel electrode method, the cells to be fused are placed between a pair of parallel metal plate electrodes and a high voltage pulse is applied. There is a parallel electrode method that fuses the two. In this electrofusion method, the fusion operation is simple, but the microelectrode method, Di
The fusion efficiency is lower than the electrophoresis method. In the dielectrophoresis method, by applying an alternating current voltage between parallel electrodes, cells are lined up in a string from an electrode plate in a direction perpendicular to the direction of the electric field, and then a high voltage pulse is applied to fuse them. This method has higher fusion efficiency than the parallel electrode method because the cells are aligned in contact with each other compared to the parallel electrode method. However, the fused cells tend to adhere to the electrodes, and if they are forcibly removed, the survival rate of the fused cells is poor. It has the disadvantage of becoming.

In this way, in the case of cell fusion, ■The fused cells are non-toxic; ■The ability to perform fusion reactions under sterile conditions; ■ High probability of fusion, ■The ability to fuse specific cells together, A fusion method that satisfies the following is needed.

[Problem to be solved by the invention]

From the above-mentioned conventional techniques, it can be said that the electrofusion method is better than the chemical fusion method (PEG method) when considering the influence of the medicinal efficacy of the fused cells. In addition, in the electric fusion method, as in the microelectrode method and the dielectrophoresis method, the fusion probability is higher if the cells to be fused are in contact with each other and an electric pulse is applied, so it is necessary to bring the cells to be fused into contact with each other. be.

When bringing cells into close contact with each other, it is possible to use microelectrode methods or direct contact with cells using microtubules, but this does not result in a completely sterile state, and depending on the degree of manipulation, the cells themselves may be exposed. There is a possibility of injury. Therefore, a non-contact cell handling method has been desired.

[Means to solve the problem]

In order to solve the above problems, in cell fusion, which involves bringing different cells into contact with each other and applying high voltage pulses to fuse them, one cell is irradiated with multiple laser beams from multiple directions. The cell is held at a specific position within a laser beam, and other different cells are similarly held at specific positions within multiple other laser beams. This holding property is called optical trapping by a laser). A cell fusion method using optical trapping using a laser is performed, which is characterized by bringing both cells into contact with each other by moving a laser beam at the same time.

[Function and Examples]

In this cell fusion method, cells to be fused are irradiated with multiple laser beams from multiple directions, and the cells are held at specific positions within the multiple laser beams. The cell is held in a specific position within the laser beam by pressure caused by the momentum of the cell and changes in momentum caused by refraction, reflection, and scattering of light as it passes through the cell. When performing optical trapping with multiple laser beams, the number of laser beams is larger than when performing optical trapping with a single laser beam (although there is a disadvantage that one laser beam must be focused at a steep angle). This is because when light trapping is performed with a single laser beam, the light is focused at a steep angle, so it may be difficult to bring cells into contact with each other because the cells are located near the laser light source. Since a laser beam is used, it has the advantage of stably optically trapping cells.The optical output of the laser beam needs to be within a range that will not kill the cells, due to the absorption characteristics of the wavelength of light unique to the cells to be fused. The optical output is not so large, so the laser light source may be a semiconductor laser or the like, and the light spectrum can be adjusted within a range that takes into account the absorption characteristics of cells for the wavelength of light. Optical trapping of cells is possible even if the cell is not monochromatic.It is possible to irradiate a single cell with multiple laser beams that meet these conditions and hold the cell at a specific position within the multiple laser beams. Figure 1),
Furthermore, by performing the same method on different cells and moving each laser light source at the same time, it is possible to reliably bring specific cells into contact without contact (Figure 2).

In this cell fusion method, first, each cell to be fused is optically troughed using multiple laser beams. FIG. 1 shows a state in which cells A and C are optically traversed with two laser beams. Next, while keeping the cell in the optically tra-noved state, that is, keeping the positional relationship between the plurality of laser light sources constant, the plurality of laser beams 5 are moved in parallel using a micromanipulator etc. contact. This state is shown in FIG. 2. At this time, the two pairs of laser light sources may be moved together, or the cell a
The cells may be brought into contact with each other. Furthermore, as shown in Figure 2, a high voltage pulse (
400 to 700 V), and cells A and C are fused in the order of FIGS. 5(a)-(b)-(C).

Above, we have explained this fusion method using an example in which two laser beams face each other and optically perform optical tracking on one cell as shown in Figure 1, but the number of laser beams and the angle between the laser beams are It is sufficient that cells can be optically trapped, and it is possible to optically trap cells in addition to the example shown in FIG. In Figure 3, three laser beams are used and the angle between the laser beams is 120 degrees each.
Figure 2 shows separate light trapping at degrees. To summarize this method, as shown in Fig. 4, a plurality of laser beams 7I to 7 (m is an integer such as m>1) are used to optically trough cells A and C to bring them into contact with each other, and from nearby electrodes. Cell fusion is performed by applying high voltage pulses.

〔Effect of the invention〕

Compared to conventional cell fusion methods that simply place cells to be fused in the same container and apply high voltage pulses, this method allows cells to be treated individually, making it possible to select specific cells and fuse them. In addition, compared to fusion methods that use microelectrodes or microtubules to handle cells, this method
Since a laser beam is used, it is possible to handle cells without contact, that is, in a sterile state. therefore,
Compared to conventional fusion methods, cells can be brought into contact with each other more reliably under sterile conditions, resulting in a higher probability of fusion.

[Brief explanation of drawings]

Fig. 1 is a schematic diagram showing that cell a and cell s are optically trapped by a laser for cell fusion, and Fig. 2 is a schematic diagram of a cell a and cell s that have been optically trapped by a laser brought into contact for cell fusion. A schematic diagram showing a state in which a high voltage pulse is applied to an electrode near the cell. Figure 3 is a schematic diagram showing that three laser beams are used to optically trap cells 1 and 2. Figure 4 is a diagram showing m cells. (m is an integer where m>1) is a schematic diagram showing a state in which a cell a and a cell are optically trapped and in contact with each other by a plurality of laser beams, and FIG. 5 is a schematic diagram showing changes in cells in this method. , the same figure (
Figure (a) shows the state in which the cells are in contact with each other, and the figure (b) shows the state in which the cells are in contact with each other.
), high voltage pulses are applied, and cell fusion has begun. Figure (c) shows a state in which the state shown in figure (b) has progressed and cell fusion has been completed. DESCRIPTION OF SYMBOLS 1... Cell a, 2... Cell b, 3... Laser light source, 4... High voltage pulse generator, 5... Electrode, 6... Moving direction, 7... Laser Beam (1〈05m m integer). n is

Claims (1)

[Claims] In cell fusion, which involves bringing different cells into contact with each other and applying high voltage pulses to fuse them, a single cell is irradiated with multiple laser beams from multiple directions to hold the cell in a specific position within the laser beam. Laser-based light is characterized in that other different cells are similarly held at specific positions within a plurality of other laser beams, and both cells are brought into contact with each other by simultaneously moving the plurality of laser beams. Cell fusion method using trapping.