JPH02242664A - Chamber for carrying out electric treatment of cell - Google Patents

Abstract

PURPOSE:To produce a chamber having simple structure and capable of readily operating at a low cost by opening upper face and lower face of spaces of a pair of facing electrodes. CONSTITUTION:A pair of electrodes 4 and 6 is attached to a body 2. The facing faces of the electrodes 4 and 6 are formed to parallel planes and the interval is kept to proper value in the range of e.g. 0.5-2mm capable of being retained by surface tension when a cell suspension is charged. The edge part in the cross section shape of facing faces of the electrodes 4 and 6 is preferably made acute angel so that the cell suspension is stably retained by surface tension. Spaces 10 and 12 are provided in both ends of spaces opposite to the electrodes 4 and 6 not so as to bring into contact with the body 2. Electricity is applied to the electrodes 4 and 6 by terminals 14 and 16 connected to a lead of electric source device.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a cell electroprocessing chamber used to fuse cells or introduce genes into cells using electrical stimulation. It is.

Cell fusion and gene transfer technologies are used for propagation and research in the fields of agriculture and fisheries, and for the development of new species in the fields of medicine and food.

(Prior Art) Most cell electroprocessing chambers used for cell fusion and gene transfer have counter electrodes arranged on a flat bottom surface.

To cause cell fusion, put the cell suspension in the chamber, wait for the cells to settle on the bottom, apply an AC voltage to cause electrophoresis, bring the cells into contact with the chamber, and then Apply voltage pulses to cause fusion.

After applying the pulse, it is left standing for about 15 minutes, for example, to promote fusion.

(Problem M to be solved by the invention) Some cells have the property of adhering to the vessel wall. In particular, many eggs and animal cells are attached to the organ wall. In the case of cell fusion, fusion is caused after precipitation, so in the case of cells that adhere to the organ wall, electrophoresis occurs and fusion is difficult to occur.

In addition, after fusion or gene transfer treatment, cells adherent to the vessel wall adhere to the bottom surface while the vessel is left standing, making it difficult to collect them.

An object of the present invention is to provide a cell electroprocessing chamber in which even cells that adhere to the vessel wall do not adhere to the vessel wall and become difficult to fuse or collect.

(Means for Solving the Problems) In order to prevent cells from adhering to the bottom surface, the bottom surface of the chamber is eliminated in the present invention.

Therefore, in the cell electroprocessing chamber of the present invention, the pair of electrodes are held by an insulating member so that the opposing surfaces are parallel planes, and the upper and lower surfaces of the space where the pair of electrodes are facing are open. There is.

(Operation) When a cell suspension is placed in a space where a pair of electrodes face each other, the cell suspension is held within this chamber by surface tension. Cells settle on the lower interface of the cell suspension and are not caught, allowing sufficient electrophoresis to occur and forming crowbar chains or arrays.

(Example) Figure 1 is a plan view showing one example, and Figure 2 is an A-A thereof.
It is a sectional view at a line position. FIG. 2 further shows the objective lens of the microscope along with the petri dish and lid.

Reference numeral 2 denotes a body made of an insulating material, and a pair of electrodes 4 and 6 are attached to the body 2 with an adhesive and held therein.

Opposing surfaces 4a and 6a of electrodes 4 and 6 are parallel planes. The distance between the opposing surfaces 4a and 6a is such that when the cell suspension 8 is placed between the opposing surfaces 4a and 6a, the cell suspension 8 can be held by surface tension. It is set to an appropriate value.

The edges of the cross-sectional shapes of the opposing surfaces 4a and 6a of the electrodes 4 and 6 must be 90 degrees or less so that the cell suspension 8 is stably held by surface tension, as shown in Figure 1. It is desirable that the angle be acute.

A space 10° 12 is provided in the body 2 so that both ends of the space where the electrodes 4 and 6 face do not come into contact with the body 2.

14 is a terminal connected to the electrode 4, and 16 is a terminal connected to the electrode 6.

Reference numeral 18 indicates a petri dish or slide glass placed on the lower side of the chamber 1, and 20 is a lid placed on the upper side of the chamber 1. By sandwiching the chamber 1 between the petri dish 18 and the lid 20, the cell suspension is kept in the cell suspension during electrical processing. 8 can be protected from germs. 22 is an eyepiece of an inverted microscope.

Next, operations for performing cell fusion using this example will be explained.

Chamber 1 is held in the air in a clean bench, and electrode 4 is
, 6, the cell suspension 8 is gently injected using a fine-tipped pipette or the like. The cell suspension 8 is collected on the parallel plane portions of the opposing electrodes due to surface tension. As long as you don't apply a strong shock, you can turn chamber 1 in any direction! ! i Do not drip the suspension 8.

Confirm that the cell suspension 8 is sufficiently contained on the bottom surface between the electrodes 4 and 6, place the chamber 1 on the Petri dish 18, and connect the terminal 1.
4. Connect 16 to the power supply lead. A lid 20 is placed on the lid 20, and a voltage is applied between the electrodes 4 and 6 while observing with a microscope 22. In the case of fusion, cells are first brought into contact with each other by electrophoresis using alternating current voltage, and then a direct current high voltage pulse is applied to fuse them.

FIG. 3 shows a state in which the chamber 1 of this embodiment is attached to a stand 24.

The stand 24 has a pair of leads 26 (only one lead is shown, and the other lead has the same shape) that sandwich the terminals 14, 16 of the chamber 1.

terminals 14, 16 of chamber 1 can each be moved vertically along a board 26. 28 is lead 2
6, and the other lead is also provided with a terminal of the same shape.

In the illustrated state, the chamber 1 is placed horizontally. The chamber 1 can be raised upward along the lead 26 and held at the position of the widened portion 26a of the lead 26. In that position the chamber 1 can be oriented in any direction, for example vertically as indicated by the dashed line 1'. In the vertical chamber 1' condition,
The part containing the cell suspension 8 between the electrodes 4.6 is arranged vertically.

When the chamber 1 is arranged horizontally, the lower surface of the cell suspension 8 may curve between the electrodes 4 and 6, as shown in FIG. If the eggs or cells 8a settle on the curved interface, the electric lines of force may not act sufficiently on the eggs or cells 8a when a voltage is applied between the electrodes 4 and 6. In such a case, hold the chamber 1 vertically as shown by the dashed line 1' in Figure 3, allow a large amount of time for the cells to settle along the vertical space, and arrange the cells during that time. When an electric pulse is applied, electric lines of force can be effectively activated.

Further, the chamber 1 can be held not only vertically but also at any angle, and the cell arrangement can be adjusted by controlling the sedimentation rate of the cells.

(Effects of the Invention) In the chamber of the present invention, there is no bottom surface of the container, so even if the cells are adherent to the container wall.

There is no problem of cells adhering to the bottom surface as in the past.

Since the upper and lower surfaces between the electrodes are open, cleaning is extremely easy and contamination due to poor cleaning is reduced.

Since the structure is simple, it can be manufactured at low cost.

In addition, the cell suspension can be injected and collected from both the top and bottom of the chamber, making it easy to operate.

[Brief explanation of drawings]

Fig. 1 is a plan view showing one embodiment, Fig. 2 is its A-A
A cross-sectional view at a line position is shown together with a petri dish, a lid, and an objective lens of a microscope. FIG. 3 is a sectional view showing the chamber of one embodiment mounted on a stand, and FIG. 4 is a sectional view showing the case where the interface of the cell suspension between the electrodes is curved. 1...Chamber, 2...Body, 4,6
... Electrode, 4a, 6a ... Electrode facing surface, 8 ... Cell suspension. Patent applicant: Shimadzu Corporation

Claims (1)

[Claims] (1) A cell electroprocessing chamber in which a pair of electrodes are held by an insulating member so that opposing surfaces are parallel planes, and the upper and lower surfaces of the space where the pair of electrodes are facing are open.