JP3494535B2 - Cell detaching device and detaching method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus and method for adhering or adhering and proliferating microorganisms or animal / plant cells, and then exfoliating the cells, and more particularly to damaging the cells. Can be peeled off without a cell wall,
The present invention relates to a cell peeling device and a peeling method capable of reliably collecting useful substances in cell membranes and cells.

[0002]

2. Description of the Related Art It is also known that there are cells that grow by adhering to a certain kind of material, and among these adherent cells, those of animal origin do not grow until after adhering. When a useful substance is produced on the wall, membrane or inside of such a cell and is to be collected, after adhering or adhering / proliferating the cell to a predetermined material, induction is applied or the cell is peeled off as it is. In some cases, it has been necessary to physically peel it off or to peel it off with a chemical such as enzyme or EDTA.

[0003]

However, in such a conventional method, a physical peeling method is inefficient, and a drug-based method may damage cells, so that a peeling device or method that does not damage cells can be used. Development is desired. Moreover, since cells are negatively charged, a method of promoting cell adhesion or controlling production of intracellular useful substances by positively charging a material for adhering cells has also been proposed. However, there is no technology that positively attaches cells to the conductive plate and that can be easily and gently peeled off. The present invention has been made by paying attention to such conventional problems, and the purpose is to:
It is an object of the present invention to provide a cell peeling device and a peeling method that can easily peel cells without damaging the adhered cells.

[0004]

As a result of intensive studies to achieve the above object, the present inventors noticed that the cells themselves were negatively charged, and the cells adhered or adhered and proliferated. Later, I thought that the cells might be easily peeled off by negatively charging the adhesion part, and when I carried out this, I found that the above-mentioned object was achieved,
The present invention has been completed. That is, the cell detachment device of the present invention is characterized by including a conductive material for adhering cells and a charging means for negatively charging the conductive material.
In addition, the cell exfoliation method of the present invention is characterized by immersing a conductive material to which cells are adhered in a cell immersion liquid and negatively charging the conductive material.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The cell detachment apparatus and cell detachment method of the present invention will be described in detail below. As described above, this cell detachment device has a configuration in which the conductive material for adhering cells and the charging means are provided, and the conductive material is negatively charged by the charging means. Here, as the conductive material, it is sufficient if it is a material capable of adhering cells and having conductivity, and specifically, a conductor such as carbon, metal and metal oxide, and a semiconductor or these Any combination can be mentioned.

Examples of the carbon include glassy carbon, activated carbon and graphite. Further, the metal is preferably one which has low toxicity to cells and may be selected in consideration of compatibility with cells, but in general, stainless steel, titanium, gold, platinum and the like are preferable because they have no toxicity. Further, titanium oxide or indium oxide can be used as the metal oxide.

The conductive material may be formed of only the above-mentioned conductor or semiconductor, but is not limited to this, and it is sufficient if at least the surface to which cells adhere has conductivity. For example, the surfaces of non-conductors such as glass, ceramics and plastics may be coated with the above-mentioned conductors or semiconductors.

Further, the conductive material preferably has transparency in order to facilitate microscopic observation of adhered cells, and from such a viewpoint, conductive transparent plastic, transparent glass or transparent plastic, etc. A transparent base material coated with an optically transparent electrode can be preferably used.
As such an optically transparent electrode, a metal such as platinum or gold or a metal oxide such as titanium oxide or indium oxide deposited on a thin layer of 500 nm or less can be used, but tin or nickel is doped. Since the indium oxide described above has excellent transparency and low electric resistance, it is particularly preferable to use such an indium oxide thin film coated with a transparent substrate. In addition, cell adhesion can be easily performed.

The shape of the conductive material is not particularly limited and various shapes can be adopted, for example, plate shape, rod shape, powder shape and particle shape. In the case where the conductive material is in the form of powder or particles, for example, a plate-shaped or rod-shaped current collector may be added. The material may be brought into contact with the material and the current collector may be negatively charged.
Further, it is possible to use such a current collector as a container filled with the cell immersion liquid.

As described above, the surface of the conductive material needs to be conductive, but it is also possible to coat the surface with another material as long as it does not impair the proper conductivity. When it is difficult for the cells to adhere to each other, a cell adhesion treatment for covering the surface with collagen or protein can be performed to improve the adhesion of the cells.

Next, the charging means will be described. As a typical example of the charging means, there is a device that includes an anode for the conductive material (cathode) and can apply a voltage between the both because of the necessity of negatively charging the conductive material. Examples of such an anode include conductors or semiconductors such as platinum, gold, and other metals that are not susceptible to oxidation, metal oxides, and carbon. Further, the container itself filled with the cell immersion liquid may be formed of the above conductor or semiconductor and used as the anode.

Next, the cell exfoliation method of the present invention will be described in detail. This cell exfoliation method is, as described above, immersing the conductive material to which the cells are adhered in a cell immersion liquid to negatively charge the conductive material, which is also effective for cells derived from microorganisms or plants, Particularly, it is effective for cells that have no cell wall and are physically and chemically weak, such as animal cells. Here, as the cell immersion liquid, various media, physiological saline, P
It is possible to use a solution in which EDTA and / or a surfactant is added to BS and PBS, but among these, the use of PBS and a solution in which EDTA and a surfactant are added to PBS improves cell detachability. Therefore, it is preferable to use these.

The negative charge on the conductive material is
As described above, an anode may be provided for the conductive material (cathode) and a voltage may be applied between both electrodes. It should be noted that reproducibility can be easily obtained by providing a reference electrode such as a silver / silver chloride electrode at the time of this application, measuring the potential of the conductive material to which cells are adhered, and adjusting so that the potential becomes constant. It is not always necessary to measure the potential of the conductive material, and an appropriate voltage obtained from preliminary experiments or the like may be applied between the conductive material and the anode.

The voltage applied to the cell detachment, that is, the potential difference between the conductive material and the anode varies depending on the material of the conductive material, the degree of adhesion of cells, the type of cell immersion liquid, etc., and can be appropriately changed. There is a case where the silver / silver chloride electrode peels off even at around 0.0V, but if it exceeds 1.2V, an electrochemical reaction other than the purpose such as gas generation or electrodeposition of a cationic substance occurs, and the It is not preferable because it may cause damage.

[0015]

EXAMPLES The present invention will now be described in more detail with reference to examples. (Embodiment 1) FIG. 1 is a side view showing an embodiment of the cell detachment apparatus of the present invention. In the figure, this cell dissociation device comprises a conductive plate 1 which is an example of a conductive material, an anode plate 2, and a silver / silver chloride electrode 3 which is an example of a comparison electrode, and these members are filled in a container 5. It is configured by immersing in the cell immersion liquid 4.

The following cell exfoliation test was carried out using the cell exfoliation device shown in FIG. That is, a transparent plate is prepared by depositing indium oxide doped with tin on one surface of a glass plate,
This transparent plate is placed on a Petri dish with the vapor deposition surface facing up, and CH
The O-Kl strain was cultured and adhered to the vapor deposition surface. After culturing, the transparent plate was used as a conductive plate 1 and was separated from the carbon plate 2 which was an anode plate, and both were immersed in PBS4 which was a cell immersion liquid, and a voltage was applied between both plates with the carbon plate 2 being positive. . At this time, the potential of the transparent plate 1 to which the cells adhered was measured by the silver / silver chloride electrode 3.

As a result, when the potential of the transparent plate was -50 mV with respect to the silver / silver chloride electrode, the cells did not detach even after 10 minutes, but at -200 mV, it was observed that the cells detached visually. did it. When the voltage was further increased, the peeling became fast and reliable, but it was found that when the voltage exceeds -1.2 V, the transparent plate surface becomes dirty due to the electrodeposition reaction. Also, P
When EDTA and a surfactant (produced by Asahi Kasei Co., Ltd .: trade name Prolonic F68) were added to BS, peeling was accelerated.
In this example, although charged in PBS, it was -20 in the medium.
The cells were not detached even when charged to 0 mV. Further, the potential of the transparent plate was measured with a silver / silver chloride electrode, but this is not always necessary, and similar results were obtained even when a voltage of -300 mV was applied between both electrodes in PBS.

Example 2 The same operation as in Example 1 was repeated except that glassy carbon was used as the cell contact plate instead of the glass plate. In the case of this example, peeling was performed at a lower voltage of −30 mV with an electrode against silver / silver chloride. In Examples 1 and 2, a part of the exfoliated cells was collected and re-cultivated to proliferate, so that it could be demonstrated that the exfoliated cells were not dead.

(Example 3) Glacier Carbon particles having a diameter of 0.5 mm were placed in a petri dish and cultured to obtain a glass.
The cells were allowed to adhere on the carbon particles. The obtained particles are P
It is transferred into a stainless petri dish in which BS is injected, and while gently stirring, the stainless petri dish is used as a cathode,
When a carbon plate arranged so as not to contact the above particles was used as an anode and a voltage was applied between both electrodes, it was peeled in the same manner as above.
At this time, when the voltage between both electrodes was set to −100 mV or more, peeling could be performed in a short time.

Although the present invention has been described in detail with reference to some embodiments, the present invention is not limited to these embodiments and various modifications can be made within the scope of the gist of the present invention. For example, although animal cells were used in the above examples, plant cells typified by microorganisms and moss can also be exfoliated in the same manner. However, in general, it is necessary to make the applied voltage higher than that of animal cells or to lengthen the time of applying the voltage.

The conductive material and the anode may be of any shape as long as they can apply a voltage. Further, the conductive material may be one that can adhere to cells and has conductivity. When the cells do not directly adhere to the conductive material, the same effect can be obtained by coating the surface with a material such as collagen, cellulose, gelatin or the like that easily adheres to the cells so that the cells adhere to the surface.

[0022]

As described above, according to the present invention, after the cells adhere or adhere and proliferate, the adhesion part is negatively charged. Therefore, the adhered cells can be easily charged without damage. A cell peeling device and a peeling method capable of peeling can be provided. The present invention is also effective for collecting physiologically active substances in the cell wall or inside the cells, since the cells can be separated from the adhesive surface without damaging the cells.

[Brief description of drawings]

FIG. 1 is a side view showing an embodiment of a cell detachment device of the present invention.

[Explanation of symbols]

1 conductive plate 2 Anode plate 3 Silver / silver chloride electrode 4 PBS 5 containers

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Claims (11)

(57) [Claims] 1. A cell exfoliating device comprising: a conductive material for adhering cells, and a charging means for negatively charging the conductive material. 2. A comparison electrode comprising a silver / silver chloride electrode is added, and the potential difference between the comparison electrode and the conductive material is 1.
The cell detachment device according to claim 1, which has a voltage of 2 V or less. 3. The cell detachment device according to claim 1, wherein the conductive material is subjected to a cell adhesion treatment. 4. The conductive material comprises one or two or more kinds selected from the group consisting of carbon, metal and metal oxide, according to any one of claims 1 to 3. The described cell detachment apparatus. 5. The cell detachment device according to claim 1, wherein the conductive material is formed by coating an optically transparent electrode on a transparent base material. 6. The cell detachment device according to claim 5, wherein the optically transparent electrode is indium oxide doped with tin or nickel. 7. The cell detachment device according to any one of claims 1 to 6, wherein the cells are animal cells. 8. A method for cell detachment, which comprises immersing a conductive material to which cells are adhered in a cell immersing liquid to negatively charge the conductive material. 9. The cell detachment method according to claim 8, wherein the conductive material is charged to −1.2 V or less with respect to a reference electrode composed of a silver / silver chloride electrode. 10. The method for detaching cells according to claim 8, wherein the cell immersion liquid is a medium, physiological saline, PBS, or PBS to which EDTA and / or a surfactant has been added. 11. The cell detachment method according to any one of claims 8 to 10, wherein the cells are animal cells.