JP6795765B2 - Cell membrane perforation container and cell membrane perforation method using this - Google Patents

Description

The present invention relates to, for example, a cell membrane perforation container provided with a perforator for perforating a cell membrane and a cell membrane perforation method using the same.

In recent years, with the development of the field of regenerative medicine, the importance of technology for introducing a target substance into cells has increased. Conventionally, as such a substance introduction technique, a virus vector method, an electroporation method, a microinjection method and the like are known. In the viral vector method, the target substance can be introduced into an unspecified cell group, and the electroporation method is widely used as a relatively easy method. The microinjection method forms perforations only in specific cells.

Japanese Unexamined Patent Publication No. 2001-523725

However, it has been pointed out that the viral vector method may cause canceration of cells, and the electroporation method has a problem of low cell survival rate after perforation. Further, the microinjection method has a drawback that the work efficiency is poor.

The present invention has been made in view of the above-mentioned problems, and an object thereof is a cell membrane perforation container having a high cell survival rate and a high work efficiency in introducing a target substance into the cell. It is to provide the cell membrane perforation method using this.

The present invention has a first invention and a second invention.
The first invention comprises a first bag body formed of a flexible material and a second bag body formed of a flexible material and laminated on the first bag body. The bag body is provided on the first communication port capable of communicating the inside and the outside, the first inner surface facing the second bag body, the first outer surface located on the opposite side, and the first inner surface. The second bag body has a first opening, and the second bag body is located on a side opposite to a second communication port capable of communicating the inside and the outside, a second inner surface facing the first bag body, and the opposite side. It has a second outer surface and a second opening provided on the second inner surface, and the first bag body and the second bag body are provided in the first opening and the second opening. At the same time, the cells communicate with each other through a cell-supporting filter having a mesh size in which the liquid can permeate and the cells cannot permeate, and the cells are perforated inside the second outer surface at a position facing the cell-supporting filter. The perforated body comprises a possible perforated body, the perforated body includes a base portion joined to the second outer surface, and a plurality of protrusions protruding from the base portion toward the cell-supporting filter, and the protrusions include a plurality of protrusions. It is characterized by comprising an oxidation treatment means for a cell membrane .

In the first invention, the oxidation treatment means is a photosensitizer, and the first bag may be light transmissive.

In the first invention, the perforated body is pressed from the second outer surface toward the cell-supporting filter, and pressure is dispersed to disperse the pressing force on the outer surface side of the cell-supporting filter. Means may be provided.

The second invention comprises a first bag body formed of a flexible material and a second bag body formed of a flexible material and laminated on the first bag body. The bag body is provided on the first communication port capable of communicating the inside and the outside, the first inner surface facing the second bag body, the first outer surface located on the opposite side, and the first inner surface. The second bag body has a first opening, and is located on a side opposite to a second communication port capable of communicating the inside and the outside thereof, a second inner surface facing the first bag body, and the opposite side thereof. It has a second outer surface to be formed and a second opening provided on the second inner surface, and the first bag body and the second bag body are provided in the first opening and the second opening. At the same time, the cells communicate with each other through a cell-supporting filter having a mesh size in which the liquid can permeate and the cells cannot permeate, and the cells are perforated inside the second outer surface at a position facing the cell-supporting filter. The perforated body comprises a possible perforated body, the perforated body includes a base portion joined to the second outer surface, and a plurality of protrusions protruding from the base portion toward the cell-supporting filter, and the protrusions include a plurality of protrusions. A cell membrane perforation method using a cell membrane perforation container provided with an oxidation treatment means for the cell membrane, wherein a cell culture solution containing a plurality of cells is injected into the second bag through the second communication port, and the cell support. It is characterized by including a step of pressing the perforated body against a filter to form a perforated cell .

The second invention is characterized in that the oxidation treatment means is a photosensitizer and includes a step of supplying a light stimulus to the perforated body in a state where the perforated body is pressed against the cell-supporting filter. ..

In the second invention, the perforated body is light transmissive, and the light stimulus may be supplied through the perforated body.

In the present invention, it is possible to form perforations in a plurality of cells simply by holding cells with a cell-supporting filter and pressing a perforator against the cells, and it is possible to improve work efficiency. In addition, since the perforation formed by the perforator is closed by the repairing force of the cell itself, it is possible to maintain a high survival rate of the cell.

Perspective view of cell membrane perforation container Exploded-view of the cell membrane perforation container of FIG. FIG. 1 is a cross-sectional view taken along the line III-III of the cell membrane perforation container of FIG. 1 and shows an outline thereof. Partial enlarged view of the perforated body An image of an embodiment according to the present invention and an image processed image taken in an experimental example. Image of control experiment taken in Experimental Example and image processed image

1 to 3 show an embodiment of the cell membrane perforation container of the present invention. Although FIG. 3 is a cross-sectional view taken along the line III-III of FIG. 1, the thickness dimension is shown large for easy understanding, which is different from the dimensional relationship of FIGS. 1 and 2.
The cell membrane perforation container 1 includes a first bag body 10 and a second bag body 20 that are arranged in an overlapping manner. The first bag body 10 is formed of a light-transmitting flexible material, and has a first communication port 11 capable of communicating the inside and the outside thereof, a first inner surface 12 facing the second bag body, and vice versa. It has a first outer surface 13 located on the side and a first opening 14 provided near the center of the first inner surface 12. The first inner surface 12 and the first outer surface 13 are watertightly fixed around them.

Similarly, the second bag body 20 is formed of a light-transmitting flexible material, and has a second communication port 21 capable of communicating the inside and the outside thereof, and a second inner surface 22 facing the first bag body 10. It has a second outer surface 23 located on the opposite side thereof, and a second opening 24 provided near the center of the second inner surface 22. The second inner surface 22 and the second outer surface 23 are watertightly fixed around them. These fixations can be realized by, for example, heat welding or adhesion using an adhesive.

As the first inner surface 12, the first outer surface 13, the second inner surface 22, and the second outer surface 23, a resin such as polyethylene (PE) or ethylene vinyl acetate copolymer (EVA) or a combination material thereof can be used.

The first inner surface 12 and the first outer surface 13 and the second inner surface 22 and the second outer surface 23 have substantially the same shape and size as a rectangle, and the first communication port 11 and the second communication port 21 are provided on one side of the rectangle. Each is provided. The connectors 15 and 16 are watertightly connected to the first communication port 11 and the second communication port 21, respectively, and the caps 17 and 18 are attached to the connectors 15 and 16. The inside and the outside of the first bag body 10 and the second bag body 20 can communicate with each other via the connectors 15 and 16. Specifically, the tip of the syringe can be attached to the connectors 15 and 16 from which the caps 17 and 18 have been removed, and the liquid or gas can be injected into the first bag body 10 or the second bag body 20 by the syringe. Further, the liquid or gas of the first bag body 10 or the second bag body 20 can be sucked by reducing the pressure using a syringe. In this embodiment, a syringe is used for pressurizing and depressurizing the first bag body 10 and the second bag body 20, but the present invention is not limited to this, and any device capable of pressurizing and depressurizing is used. Just do it.

The connectors 15 and 16 are provided at positions where the first bag body 10 and the second bag body 20 do not overlap in the stacking direction. By not overlapping in this way, for example, when a syringe is connected to one connector for injection / suction, it is possible to prevent the other connector from interfering with the work.

The first bag body 10 and the second bag body 20 are laminated with the first opening 14 and the second opening 24 facing each other. A cell-supporting filter 3 is provided between the first opening 14 and the second opening 24, and the first bag body 10 and the second bag body 20 communicate with each other through the cell-supporting filter 3. The cell-supporting filter 3 is larger than the diameter of the first opening 14 and the second opening 24. The end of the cell-supporting filter 3 is watertightly fixed to the peripheral edge of the first opening 12 of the first inner surface 12 on the surface facing the first bag body 10. Similarly, on the surface facing the second bag body 20, the end portion thereof is watertightly fixed to the peripheral edge of the second opening 22 of the second inner surface 22. Further, the first inner surface 12 and the second inner surface 22 are watertightly fixed around the cell-supporting filter 3.

The cell-supporting filter 3 has a mesh size that allows liquid to permeate and does not allow cells to permeate. Inside the second outer surface 23 of the second bag body 20, the cells are placed at positions facing the cell-supporting filter 3. A perforating body 4 capable of perforating is provided. The perforated body 4 is light-transmitting, and more specifically, it is desirable that the perforated body 4 is made of a transparent material. With reference to FIG. 4, the perforated body 4 includes a base portion 41 joined to the second outer surface 23 and a plurality of protrusions 42 protruding from the base portion 41 toward the cell-supporting filter 3, and the protrusions. Part 42 is provided with means for oxidizing the cell membrane. The protrusion 42 can be set to have a diameter of about 0.3 to 1 μm, a height of about 0.3 to 2 μm, and a pitch of about 0.3 to 3 μm.

The cell membrane perforation method using the cell membrane perforation container 1 as described above will be described. In this embodiment, a photosensitizer is used as the oxidation treatment means. Specifically, a protrusion 42 of the perforated body containing a photosensitizer is used. A sterilized container 1 for perforating the cell membrane is used, and at least the work of removing the caps 17 and 18 is performed in a sterilized state in, for example, a clean room. Whether or not the work is performed in a sterilized state can be appropriately selected according to the purpose.

The cell dispersion containing the cells and the target substance to be introduced is injected into the second bag body 20 in the cell culture medium. Specifically, the cell dispersion is filled in a syringe, the tip of the syringe is connected to the connector 16 from which the cap 18 is removed, and the cell dispersion is injected into the second bag body 20. After injection, the cap 18 is fitted to the connector 16. In this state, the cells can be cultured in the second bag body 20.

With the cell dispersion liquid injected into the second bag body 20, an empty syringe is connected to the connector 15 of the first bag body 10 to suck the inside of the first bag body 10. By reducing the pressure inside the first bag body 10, the cell dispersion liquid of the second bag body 20 moves toward the cell-supporting filter 3. Since the cell-supporting filter 3 permeates the liquid but does not permeate the cells, the cells are retained in the cell-supporting filter 3. By holding the cells on the cell-supporting filter 3 in this way, it is possible to ensure the contact between the protrusions 42 of the perforator 4 and the cells in a later step. That is, in the state where the cells are suspended in the medium, when the perforator 4 is pressurized, the cells are dispersed so as to escape from the protrusion 42, and it is difficult to bring the protrusion 42 into contact with the cells. This can be made possible by holding the cell-supported filter 3 in place. The suction in the first bag body 10 is not an essential step. This step can be omitted if the cells can be retained by the cell-supporting filter 3 without aspiration.

In the first bag body 10, a spacer may be provided so that the cell-supporting filter 3 and the first outer surface 13 do not come into close contact with each other and block the first communication port 11 when the inside of the first bag body 10 is depressurized. Spacers may be separately provided inside and outside the first bag body 10, or the first bag body 10 and the first communication port 11 and the like may be reinforced so that at least a part of them does not come into close contact with each other.

After holding the cells with the cell-supporting filter 3, the syringe is disconnected and the cap 17 is fitted to the connector 15. At this time, a liquid such as a culture solution may be injected into the first bag body 10 and the liquid may be filled between the first outer surface 13 and the cell-supporting filter 3. In this embodiment, the first bag body 10 filled with the liquid functions as a pressure distribution means. By filling the first bag body 10 with a liquid, when the perforated body 4 described later is pressed against the cell-supporting filter 3, the pressure is dispersed and the contact pressure between the perforated body 4 and the cell-supported filter 3 is equalized. be able to. When the pressing force from the perforator 4 is non-uniform, there arises a problem that the cells are crushed in a certain part and no perforation is formed in another part. However, by equalizing the pressure as described above, the cells Perforations can be formed uniformly in the group. Further, by separately arranging a liquid pack or the like as a pressure dispersion means below the drawing of the cell membrane perforation container 1, it is possible to obtain the same pressure dispersion effect as when the first bag body 10 is filled with the liquid.

Next, the perforator 4 is pressed against the cells held by the cell-supporting filter 3, and the protrusion 42 of the perforator 4 is brought into contact with the cells. In a state where the protrusion 42 is in contact with the cell, light irradiation is performed from the base 41 side of the perforator 4 toward the contact portion between the cell and the protrusion 42. Since the perforated body 4 is light-transmitting, it is possible to irradiate cells through it. Since the protrusion 42 contains a photosensitizer, perforation is formed in the membrane of the cell in contact with the protrusion 42 by light irradiation. The target substance can flow in from the perforation formed in this way and be taken up into the cell. The perforation is closed by the repairing power of the cell itself in a few seconds to a few minutes.

As described above, the target substance can be introduced into cells by using the cell membrane perforation container 1. According to this embodiment, since the cells can be perforated by using the plurality of protrusions 42, the cell group can be perforated. It is possible to form perforations in a plurality of cells at the same time, and it is possible to improve work efficiency. Further, by putting the cells and the target substance to be introduced together in the cell dispersion, perforation and introduction of the target substance can be performed at the same time, and the work efficiency is further improved.

By using this cell membrane perforation container 1, the perforated cells are closed by the repairing force of the cells themselves, so that a high cell viability can be maintained. Moreover, since perforation is formed only by mechanically pressurizing the cell membrane perforation container with a set pressure, it can be easily and easily realized.

(Experimental example)
By the following experiment, perforation was formed in the cell group using the cell membrane perforation container 1. As a cell group, neural lineage cell PC12 was used. The culture of this cell is TK Saito, M. Takahashi, H. Muguruma, E. Niki and K. Mabuchi, “Phototoxic process after rapid photosensitive membrane damage of 5,5 ″ -bis (aminomethyl) -2,2 ′: 5 ′, 2 ″ -terthiophene dihydrochloride ”, J. Photochem. Photobiol., Vol.61, Issue 3, pp.114-121 (2001) regarding cell culture.

The perforated body 4 had a diameter of 85 mm and a thickness of 1.5 mm, and polydimethylsiloxane (PDMS, a type of silicone rubber) was used as a material. The protrusion 42 has a diameter of about 1 μm, a height of about 2 μm, and a pitch of about 3 μm. In the first bag body 10 and the second bag body 20, the material is polyethylene (thickness 140 μm corresponding to γ-ray sterilization), and the dimensions are about 170 mm in length and about 150 mm in width. As the cell-supporting filter 3, an omnipore (Merck Millipore, pore diameter 0.2 μm, film thickness 65 μm, diameter 90 mm) was used. The filter is welded between the bags using a self-made circular heating sealing device (Marudai Kiko).

5 ml of phosphate buffered saline (PBS) solution was added to the first bag body 10 with a 10 ml syringe, air was evacuated, and the cap was closed. Subsequently, 40 ml of PBS solution was added to the second bag body 20 with a 10 ml syringe, air was evacuated, and the cap was closed. PC12 cells were exfoliated and dispersed from the cell culture vessel and placed in a 15 ml centrifuge tube. A mixture of 3000 μl of cell dispersion + 30 μl of 10-fold diluted solution of fluorescent dye Alexa Fluor 594 for Microinjection (Life Technologies) was put into the upper layer with a 5 ml pipette. It was visually confirmed that the dye was spread over the first bag body 10.

The cell membrane perforation container 1 was set in a pressurizing / light irradiation device (CP-02, Marudai Kiko) to perform perforation treatment. The pressurization was 98.9 N and the processing time was 60 seconds. After perforation, the upper layer cell dispersion was collected with a 10 ml syringe and placed in a 15 ml centrifuge tube. When sufficient recovery was not possible, 5 ml of PBS was added to the second bag body 20 and shaken lightly to recover. Centrifuge to remove all supernatant. Then, 1 ml of imaging medium (Neuro Basal without Phenol Red) was added to redisperse the cells. 30 μl of the cell dispersion was dropped on a slide glass, covered with a cover glass, and observed and photographed with a fluorescence microscope (IX71, Olympus). The result is shown in FIG. FIG. 6 shows the results of a control experiment, which was not irradiated with light in the same experiment as above. In this experimental example, since the cells could be retained by the cell-supporting filter 3 without sucking the culture solution from the first bag body 10, this step was omitted.

The transmitted images and fluorescent images in FIGS. 5 and 6 were image-processed by an image processing program, and the cell viability was calculated. The basic part of the image processing program was created by NI Vision Assistant 2015 SP1 (National Instruments), and finally completed by LabVIEW 2015 SP1 (National Instruments).

The image processing program operates as follows.
1. 1. Read the transmission image and fluorescence image files and specify the output image file.
2. 2. The region where cells exist (determined to exclude foreign substances smaller than cells) is automatically calculated from the transmission image, only the brightness information of the same region in the fluorescence image is valid, and the region where cells are determined not to exist is set to 0 brightness. ..
3. 3. In this way, the image in which only the fluorescence derived from the cell region is automatically recognized is saved as an output image file.
4. Then, the average brightness in the area where the cells are present is calculated and displayed.

Comparing the values obtained in this way, the results of 22.8, 29.7, 21.6 and the average value of 24.7 were obtained in the regions 1-3 of the perforable conditions with light irradiation and pressurization shown in FIG. 5, respectively. Was done. Results of 15.7, 16.5, 10.3 and mean value 14.2 were obtained in the control areas 1-3, which were shown in Fig. 6 and could not be perforated with no light irradiation and with pressure, respectively.

When the ratio of atrophic dead cells detected by the cell counter before the experiment was 40% and the tendency of these cells to be fluorescently stained regardless of the conditions was taken into consideration, the brightness was significantly increased under the perforable condition, and the cells were raw. It was judged that intracellular introduction of fluorescent dye by perforation of cell membrane and restoration of membrane was observed in cells.

The cell membrane perforation container 1 as described above can form perforations while maintaining the sterilized state inside the first bag body 10 and the second bag body 20. Therefore, it is also possible to directly return the cells into which the target substance has been introduced from the cell membrane perforation container 1 into the living body, for example. Although the cells are held in the cell-supporting filter 3 in the cell membrane perforation container 1, the cells can be suspended in the culture medium simply by stirring the cell membrane perforation container 1 in the presence of the culture medium. Recovery is also easy.

In this embodiment, a transparent material is used as the first bag body 10 and the second bag body 20, so that the inside of the cell membrane perforation container 1 can be easily visually recognized. Therefore, the work can be performed while visually recognizing the state of the cell dispersion liquid, the state of contact between the perforated body 4 and the cell-supporting filter 3, and the like.

In the cell-supporting filter 3, the size, the size of the pores, and the material thereof may be those exemplified in this example, as well as various ones used in the field of the present technology depending on the purpose. Similarly, the material of the perforated body 4, the size of the protrusion 42, the pitch, and the like can be arbitrarily changed.

In this embodiment, a photosensitizer is used as an oxidation treatment means to form perforations in the cell membrane by light irradiation, but in addition to this, for example, an acoustic sensitizer is used as an oxidation treatment agent and ultrasonic irradiation is performed. It is also possible to form a perforation.

In this embodiment and the invention, the terms "first" and "second" are used merely to distinguish similar components and do not indicate permutations.

1 Cell membrane perforation container 3 Cell-supporting filter 4 Perforation body 10 1st bag body 11 1st communication port 12 1st inner surface 13 1st outer surface 14 1st opening 20 2nd bag body 21 2nd communication port 22 2nd inner surface 23 2nd outer surface 24 2nd opening 41 Base part 42 Projection part

Claims (6)

The first bag made of flexible material and
A second bag body formed of a flexible material and laminated on the first bag body is provided.
The first bag body includes a first communication port capable of communicating the inside and the outside, a first inner surface facing the second bag body, a first outer surface located on the opposite side thereof, and the first inner surface. Has a first opening provided in the
The second bag body includes a second communication port capable of communicating the inside and the outside, a second inner surface facing the first bag body, a second outer surface located on the opposite side thereof, and the second inner surface. Has a second opening provided in
The first bag body and the second bag body are provided in the first opening and the second opening, and have a mesh size that allows liquid to permeate and cells cannot permeate. Communicate through
Inside the second outer surface, a perforator capable of perforating cells is provided at a position facing the cell-supporting filter .
The perforated body includes a base portion bonded to the second outer surface and a plurality of protrusions protruding from the base portion toward the cell-supporting filter, and the protrusions are provided with means for oxidizing a cell membrane. A container for perforating cell membranes. The oxidation treatment means is a photosensitizer and
The cell membrane perforation container according to claim 1 , wherein the first bag is light-transmitting. The perforated body is pressed from the second outer surface toward the cell-supporting filter, and is provided with a pressure distribution means for dispersing the pressing force on the outer surface side of the cell-supporting filter. The cell membrane perforation container according to claim 1 or 2 . The first bag made of flexible material and
A second bag body formed of a flexible material and laminated on the first bag body is provided.
The first bag body includes a first communication port capable of communicating the inside and the outside, a first inner surface facing the second bag body, a first outer surface located on the opposite side thereof, and the first inner surface. Has a first opening provided in the
The second bag body includes a second communication port capable of communicating the inside and the outside, a second inner surface facing the first bag body, a second outer surface located on the opposite side thereof, and the second inner surface. Has a second opening provided in
The first bag body and the second bag body are provided in the first opening and the second opening, and have a mesh size that allows liquid to permeate and cells cannot permeate. Communicate through
Inside the second outer surface, a perforator capable of perforating cells is provided at a position facing the cell-supporting filter, and the perforator has a base portion joined to the second outer surface and the base portion to the above. A cell membrane perforation method using a cell membrane perforation container provided with a plurality of protrusions protruding toward a cell-supporting filter, and the protrusions are provided with means for oxidizing a cell membrane.
A step of injecting a cell culture solution containing a plurality of cells into the second bag through the second communication port, and
A method for perforating a cell membrane, which comprises a step of pressing the perforated body against the cell-supporting filter to form perforations in cells. The oxidation treatment means is a photosensitizer and
The cell membrane perforation method according to claim 4 , further comprising a step of supplying a light stimulus to the perforated body in a state where the perforated body is pressed against the cell-supporting filter. The perforated body is light transmissive and
The cell membrane perforation method according to claim 5 , wherein the photostimulation is supplied through the perforator.