TWI675915B - Cell exfoliation method, cell support substrate, and cell culture method - Google Patents

Abstract

An object of the present invention is to provide a technique capable of detaching an adherent cell from a support under mild conditions.

When peeling off the cells 2 attached to the surface of the substrate 1 as a support, the cells 2 are detached from the substrate 1, the substrate 1 contains a light-responsive gas generating agent that generates a gas by irradiation of light, The gas bubbles 7 generated by irradiating the substrate 1 with light are brought into contact with the cells 2 to peel the cells 2 from the substrate 1. According to the method of the present invention, the burden on the surface of the cell 2 is small, and the cell 2 can be peeled and recovered in a nearly non-damaged state. The cells 2 may be detached in a colony state. The present invention also provides a substrate for supporting a cell containing a photo-responsive gas generating agent.

Description

Cell detachment method, cell support substrate, and cell culture method

The present invention relates to a method for detaching cells, a substrate for supporting cells, and a method for culturing cells. The present invention is useful for the cultivation, recovery, screening, and analysis of iPS cells (induced pluripotent stem cells).

Since previously, when the adherent cells that have proliferated on the support are detached, trypsin treatment has generally been performed. That is, the cells adhered to the support are exposed to a trypsin solution, and the cell surface is treated to detach the cells. By trypsin treatment, the cells are detached from the support, and the attachment of the cells to each other is also destroyed, and basically each cell is suspended independently (single cell suspension).

On the other hand, damage to cells due to trypsin treatment is considered a problem. For example, there is a case where a receptor on the cell surface is damaged by exposing the cell to trypsin. As a result, the survival rate of the cells is reduced. Therefore, in the case of recovering the cells attached to the substrate, it is desirable to recover cells that have not been damaged on the cell surface as much as possible. When substituting iPS cells or ES cells (embryonic stem cells), etc., it is preferable to perform them in the state of colonies rather than single cells. Therefore, there is a desire to perform cell detachment under conditions where the intercellular binding can be maintained to a certain degree of mildness.

As a technique for removing cells from a support without using trypsin, there is a technique described in Patent Document 1, for example. Patent Document 1 discloses irradiation of cells attached to an optical fiber A technology that removes cells from optical fibers, such as ultraviolet light. However, this technique is used to remove unwanted cells and does not consider the damage of the detached cells.

In addition, there is a desire to efficiently screen specific cells from a cell population. For example, there is a case where a cell community is divided, and an interval containing a desired cell is specified, and cells are to be recovered from the interval. In this case, it is preferable that only a specific cell (group) is detached from the support instead of the entire population of cells. In the technology described in Patent Document 1, a configuration is possible in which cells can be detached for each optical fiber.

[Prior technical literature] [Patent Literature]

[Patent Document 1] International Publication No. 2008/114828

However, the technique described in Patent Document 1 requires a plurality of types of optical fibers, and the configuration is complicated. Therefore, an object of the present invention is to provide a technique for removing cells from a support with a simpler structure and under mild conditions.

One aspect of the present invention is a method for removing cells, which is characterized in that it is a method of removing cells attached to the surface of a substrate as a support from the substrate, and the substrate contains a material irradiated with light. Those who generate a gas-responsive photo-responsive gas generator contact the cells with the bubbles of the gas generated by irradiating the substrate with light, thereby peeling the cells from the substrate.

In the method for exfoliating cells in this aspect, the substrate as a support contains a photo-responsive gas generating agent. Therefore, by irradiating the substrate with light, gas can be generated from the substrate. In this aspect, the cells are detached from the substrate by contacting the bubbles of gas generated by irradiating the substrate with light. In this aspect, since the cells are detached by the gas bubbles, the detachment is performed gently, and the burden on the cell surface is small. because Therefore, it will not cause an excessive burden on the cell surface like trypsin treatment, for example, it will not damage the receptors on the cell surface. Furthermore, because the conditions for detachment are gentle, the cell-to-cell binding can be maintained to a certain degree, and thus the cells can be easily detached and recovered in the state of colonies. On the other hand, even when single cells are recovered, cells without a large burden on the cell surface can be recovered.

Preferably, light is selectively irradiated to a part of the substrate, that is, a part to which cells to be peeled are attached.

According to this preferred aspect, only required cells can be efficiently detached. For example, in a plurality of cells uniformly attached to the same plane, only a specific cell (group) can be targeted and peeled off.

Preferably, the lens is used to selectively irradiate the above-mentioned portion with a lens that focuses light.

Preferably, the substrate has a plurality of sections, and light is irradiated only to sections including cells to be detached.

According to this preferable aspect, only cells existing in a specific interval can be efficiently detached.

The gas generating agent is preferably contained in an adhesive resin having adhesiveness.

Another aspect of the present invention is a substrate for cell support, which is used to support cells by attaching cells to the surface, and contains a light-responsive gas generating agent that generates gas by irradiating light, which can be obtained from the above The bubbles of the gas generated by the photo-responsive gas generating agent contact the cells.

The substrate for supporting a cell in this aspect contains a photo-responsive gas generating agent that generates a gas by irradiating light. Therefore, the substrate can be irradiated with light to generate a gas. Furthermore, in this aspect, the cells of the gas bubbles generated from the photo-responsive gas generating agent can be brought into contact with the cells. Therefore, the cells adhered to the surface of the substrate can be gently peeled off.

Preferably, the substrate for supporting cells has a plurality of sections, and can support cells in each section.

The gas generating agent is preferably contained in an adhesive resin having adhesiveness.

Preferably, the gas generating agent contains an azo compound or an azide compound.

Another aspect of the present invention is a method for culturing cells, which is characterized in that the cells are adhered to the above-mentioned substrate for supporting cells, and the cells are cultured.

According to this aspect, the cultured cells can be detached and recovered under gentle conditions.

According to the present invention, cells can be detached and recovered without placing a large burden on the cell surface. Furthermore, it is easy to recover cells in a colony state, and it is useful for recovery of iPS cells or ES cells.

1‧‧‧ Substrate (Substrate for Cell Support)

2‧‧‧ cells

3‧‧‧Gas generating film

5‧‧‧Gas barrier

6‧‧‧Mask

7‧‧‧ Bubble

8‧‧‧ liquid

10‧‧‧ lens

FIG. 1 is an explanatory view schematically showing a method for exfoliating a cell according to an embodiment of the present invention. (A) shows a state before light irradiation, and (b) shows a state after light irradiation.

FIG. 2 is an explanatory view schematically showing a method for exfoliating a cell according to another embodiment of the present invention.

FIG. 3 is a microscope photograph showing the results of experiments performed in the examples, (a) shows the state of cells before light irradiation, and (b) shows the state of cells after light irradiation.

Hereinafter, embodiments of the present invention will be described.

The cell peeling method of the present invention is one in which cells adhered to the surface of a substrate containing a photoresponsive gas generating agent are peeled by using bubbles of a gas generated from the photoresponsive gas generating agent. First, the basic concept of the present invention will be described while referring to the embodiment of the present invention shown in FIG. 1. FIG. 1 schematically shows an embodiment of the present invention.

FIG. 1 (a) shows a state of the substrate (cell supporting substrate) 1 and the cells 2 before the cells are detached (before irradiation with light). Before peeling, a plurality of cells 2 are uniformly adhered to the surface of the substrate 1. The cell 2 line is immersed in a liquid 8 such as a culture medium.

The substrate 1 contains a photo-responsive gas generator. In this embodiment, the substrate 1 It has a structure in which a gas generating film 3 containing a photo-responsive gas generating agent and a light-permeable gas barrier layer 5 are laminated. In addition, the gas generating film 3 is used as an upper side and the gas barrier layer 5 is used as a lower side, and the cells 2 are attached to the surface of the gas generating film 3. The details of the photo-responsive gas generating agent will be described later.

A light-impermeable photomask 6 is provided on the back side of the gas barrier layer 5. A part of the gas barrier layer 5 is covered by the photomask 6. Conversely, only the portion not covered by the photomask 6 is exposed on the back side of the gas barrier layer 5.

When the cells 2 are detached from the state shown in FIG. 1 (a), light is irradiated from the back surface side of the substrate 1, that is, the gas barrier layer 5 side. At this time, light is selectively irradiated to only the portion of the base material 1 that is not covered by the photomask 6 (see arrow). The irradiated light passes through the gas barrier layer 5 and reaches the gas generating film 3.

A gas is generated in the light-irradiated region in the gas generating film 3. The generated gas becomes a bubble 7 and is mainly supplied to the adhesion surface of the cell 2 to the substrate 1 and touches (contacts) the cell 2. Thereby, as shown in FIG.1 (b), the cell 2 floats from the base material 1, and peels from the base material 1.

Here, in this embodiment, since the generation of gas is limited to the area irradiated with light, only the cells 2 located in the area irradiated with light are detached. On the other hand, no gas is generated in the area covered by the photomask 6, and the cells 2 located in the area are not peeled off.

According to this embodiment, since the cells are detached by bringing the gas bubbles into contact with the cells, the burden on the cell surface is small. Therefore, there is no risk of damaging receptors on the cell surface, and cells that are closer to no damage can be recovered. In addition, since the detachment conditions are gentler than trypsin treatment, the cell-to-cell binding can be maintained to a certain degree, and the cells can be easily recovered in a state of colonies.

In addition, in this embodiment, by limiting the irradiation area of light, gas is generated only in a specific area. Therefore, only the required cells (groups) can be efficiently detached from the plurality of cells (groups) attached to the same plane.

In the above embodiment, the photomask 6 is used to selectively irradiate light to a desired portion. but However, the method of selectively irradiating light to a desired portion is not limited to this. For example, in the embodiment shown in FIG. 2, the lens 10 is used to focus light on a desired portion and selectively irradiate the light. The lens 10 is a convex lens and collects incident light. According to this embodiment, since the photomask 6 is not required, the cell can be detached with a simpler structure. In this embodiment, only one lens 10 may be used, or a plurality of lenses 10 may be used.

Examples of the material constituting the gas barrier layer 5 include polyethylene terephthalate, polyethylene naphthalate, polybutylene terephthalate, glass, quartz glass, and Pyrex glass. )Wait.

The light source used when the substrate is irradiated with light is not particularly limited as long as it does not adversely affect the cells. For example, a light emitting diode (LED) can be used. Examples of other light sources include lasers, electroluminescence elements (EL elements), plasma light-emitting elements, external electrode-type fluorescent lamps (EEFL), miniature halogen lamps, optical fibers, and combinations with optical selectors. Emitted light source, etc.

In the embodiment described above, the “base material containing a photoresponsive gas generating agent” is configured using a gas generating film, but the aspect containing the photoresponsive gas generating agent is not limited to this. For example, a resin molded article containing a light-responsive gas generator may constitute part or all of the substrate.

In the case where it is desired to peel off only a part of the cells by dividing the surface of the substrate to which the cells are attached, it is possible to easily specify an area to be irradiated with light. As a state of division, for example, when a plurality of cells are attached to the same plane, a visible grid-like mark is given to the plane. In this case, a square or rectangular region that is a repeating unit of the lattice becomes an interval.

The above-mentioned interval may be constituted by a plurality of recessed portions (pits, recessed holes) provided on the flat plate. In this case, since each section (recess) is independent, it is particularly easy to irradiate light in a specific section and recover cells that have been detached. For example, a plate body having a plurality of cylindrical recesses, such as a known microtiter plate for cell culture, can be used as the substrate. In this case, the bottom surface of the recessed hole may be formed of a material containing a photo-responsive gas generator. The size of the recessed hole can be, for example, about 2 to 35 mm in diameter, about 10 to 12 mm in depth, and about 0.1 to 5 cm ^{2 in} bottom area.

Furthermore, the above substrate may be constituted by a microarray wafer. For example, the above-mentioned substrate having a plurality of sections can be constituted by fabricating a microarray wafer having a plurality of microcavities that can accommodate one or a plurality of cells from a material containing a light-responsive gas generator. According to this aspect, a desired cell can be easily specified on a microarray wafer by fluorescence analysis or the like. In addition, after that, only the microcavity containing the required cells can be irradiated with light, and the required cells or the cell population containing the required cells can be separated and recovered. By applying the present invention to a microarray wafer, cell analysis such as CTC (Circulating Tumor Cell) inspection is facilitated.

The size or shape of the microcavity may be, for example, a cylindrical shape having a diameter of about 20 to 1000 μm and a depth of about 7 to 2000 μm.

In the case where the substrate for cell support of the present invention contains a photo-responsive gas generating agent, bubbles of a gas generated from the photo-responsive gas generating agent can be brought into contact with the supported cells.

As the cell supporting substrate of the present invention, for example, the substrate of the above embodiment can be directly used. That is, as shown in FIG. 1, the cell-supporting substrate can be formed using the gas generating membrane 3. Furthermore, a plurality of sections may be provided, and only a specific cell (group) can be easily separated. A plurality of recesses (pits, recesses) may be provided on the flat plate. The above-mentioned microarray wafer can also be used as a substrate for cell support of the present invention.

The substrate for supporting a cell of the present invention can be used as a substrate for cell culture. For example, the above-mentioned cell culture substrate can be prepared by constituting a cell attachment surface of a culture plate, a petri dish, a flask, or the like generally used in the culture of adherent cells with a material containing a photoresponsive gas generating agent. Specifically, the gas-generating membrane having the above-mentioned configuration can be provided on the bottom surface of a petri dish or flask to prepare the above-mentioned cell culture substrate.

The invention includes a method for culturing cells, which is to attach cells to cells containing light. The cells are cultured in response to the substrate of the gas generating agent. The present invention also includes a method for supporting a cell by attaching the cell to a substrate containing a photoresponsive gas generating agent to support the cell.

The present invention includes the use of a substrate for using a substrate containing a photo-responsive gas generating agent as a substrate for cell support. In addition, the present invention includes the use of a substrate for using a substrate containing a photoresponsive gas generating agent as a substrate for cell culture.

The cells to be the subject of the present invention are not particularly limited, and any cells can be used as long as they are adherent cells. Examples include the iPS cells and ES cells. These cells are preferably subcultured in the state of colonies, so the method for exfoliating the cells of the present invention can be particularly preferably used. Examples of other cells include: Hela derived from human cervical cancer; MCF-7 derived from human breast cancer; nucleated red blood cells or fetal white blood cells derived from fetal cells; Jurkat, HD-Mar2 derived from T-cell leukemia, SKW-3; NALM-6 from B-cell leukemia; HL-60 from leukemia; PC12 from rat adrenal medulla; COS-1 or COS-7 from African green monkey kidney; from China CHO / HGPRT and other cells of hamster ovary.

Next, a photo-responsive gas generating agent used in the present invention will be described. Examples of the compound constituting the photoresponsive gas generator include an azo compound, an azide compound, and a polyoxyalkylene compound. Among them, an azo compound or an azide compound can be preferably used because the gas generation efficiency is high.

Examples of the azo compound include 2,2'-azobis- (N-butyl-2-methylpropylamidamine) and 2,2'-azobis (N-butyl-2-methyl) Propylpropylamine), 2,2'-azobis (N-cyclohexyl-2-methylpropylamine) and the like. Examples of the azide compound include 3-azidomethyl-3-methyloxetane, and a glycidyl azide polymer. These azo compounds or azide compounds may be used alone or in combination of two or more.

The photo-responsive gas generator may be contained in a binder resin. The adhesive resin exhibits a function of fixing a light-responsive gas generating agent or a material containing a light-responsive gas generating agent. The material has adhesiveness, or adds various functions. For example, the photo-responsive gas generating agent may be dispersed in the binder resin, or may be compatible with the binder resin. By using a binder resin, it is easy to process a material containing a gas generating agent into a desired shape. For example, it can be easily formed into a shape such as a film.

Preferred examples of the binder resin include, but are not limited to, acrylic resins and epoxy resins. In addition, the adhesive resin itself may have a property of generating gas by irradiation of light. This adhesive resin may be used alone or in combination of two or more.

Further, the adhesive resin may be one having adhesiveness, and may include, for example, an adhesive resin. With this configuration, for example, when used in a film-like shape (gas-generating film, etc.), handling becomes easy. Moreover, it is preferable that the adhesive resin has a property that the adhesiveness does not decrease by irradiation of light. In this case, even after light irradiation is started on the gas generating agent, high adhesiveness can be maintained. Moreover, it is preferable that an adhesive resin has the property which does not crosslink by light irradiation, for example.

Examples of the adhesive resin include a rubber-based adhesive resin, a (meth) acrylic-based adhesive resin, a polysiloxane adhesive resin, a urethane-based adhesive resin, and benzene. Ethylene-isoprene-styrene copolymer-based adhesive resins, styrene-butadiene-styrene copolymer-based adhesive resins, epoxy-based adhesive resins, and isocyanate-based adhesive resins.

The substrate containing the photoresponsive gas generating agent may further contain an amine compound. With this configuration, the positive charge on the surface of the substrate can be enhanced, and cell attachment can be made more reliable. The amine compound is not particularly limited, and various primary, secondary, or tertiary amine compounds can be used.

、二苯甲酮、苯乙酮類及卟啉等。 The substrate containing the photo-responsive gas generator may further contain a photosensitizer. Examples of the photosensitizer include 9-oxysulfur

, Benzophenone, acetophenone and porphyrin.

The substrate containing the photo-responsive gas generator can also contain various additives as required Agent. Examples of the additive include a coupling agent, a plasticizer, a surfactant, a tackifier, a crosslinking agent, and a stabilizer. Examples of other additives include particles such as porous bodies, fillers, metal foils, and microcapsules. If these particles are dispersed in a substrate containing a gas generating agent, the diffusion of the gas will become faster.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the examples.

[Example]

The cell peeling method schematically shown in FIG. 1 was performed using cells and a light-responsive gas generating film.

者澆鑄於PET(polyethylene terephthalate，聚對苯二甲酸乙二酯)膜(厚度50μm)上，以乾燥後之厚度成為50μm之方式製作。 36 recessed holes (through-holes) with a diameter of 7 mm were made on an acrylic substrate (50 mm × 100 mm × 2 mm), and a gas generating film was attached to the bottom surface to prepare a substrate for cell support. The gas-generating membrane system will include a glycidyl azide polymer, an acrylic polymer, and 9-oxysulfur

It was cast on a PET (polyethylene terephthalate) film (thickness: 50 μm), and was prepared so that the thickness after drying became 50 μm.

40 μL of a medium containing breast cancer cells (1 × 10 ⁶ cells / mL) was added to each well, and the cells were allowed to stand for 20 minutes in a CO ₂ incubator (37 ° C., 5% CO ₂ ) to allow the cells to adhere to the membrane surface. Fig. 3 (a) is a micrograph showing the cells on the membrane at this time point.

Then, a part of the back side of the gas generating film was covered with a photomask, and then the LED was irradiated with light for 10 seconds to generate gas. The amount of gas generated was about 2 μL. The state of the cells after gas generation is shown in Fig. 3 (b). That is, cells uniformly attached to the gas generating film before light irradiation are peeled from the gas generating film by light irradiation. In addition, the cells covered with the photomask remain attached to the gas generating membrane, and only the cells exposed to the light are peeled off.

According to the above, by irradiating light to a substrate containing a photogas-responsive gas generating agent to generate gas, cells on the surface of the substrate can be peeled off. In addition, by limiting the irradiation range of light, only cells located in a desired area can be detached.

Claims (11)

A method for exfoliating a cell, which is characterized in that the cell is detached from the substrate by attaching cells attached to the surface of the substrate as a support, and the substrate includes a light-responsive gas that generates a gas by irradiating light. In the generator, the cells are detached from the substrate by contacting bubbles of a gas generated by irradiating the substrate with light. For example, the method for removing cells from claim 1, wherein a part of the substrate, that is, a part to which cells to be separated are attached is selectively irradiated with light. The method of exfoliating cells according to claim 2, wherein the above-mentioned portion is selectively irradiated with light using a lens for focusing light. In the method for removing cells according to any one of claims 1 to 3, wherein the substrate has a plurality of sections, and only the sections containing cells to be detached are irradiated with light. The cell exfoliation method according to any one of claims 1 to 3, wherein the gas generating agent is contained in an adhesive resin having adhesiveness. The method for removing cells according to claim 4, wherein the gas generating agent is contained in an adhesive resin having adhesiveness. A substrate for cell support, characterized in that it is used to support cells by attaching cells to the surface, and contains a light-responsive gas generating agent that generates gas by irradiating light, and can make the light-responsive gas The bubbles of the gas generated by the generating agent contact the above-mentioned cells. For example, the substrate for supporting cells according to claim 7, which has a plurality of sections, can support cells in each section. The cell supporting substrate according to claim 7 or 8, wherein the gas generating agent is contained in an adhesive resin having adhesiveness. A method for culturing cells, characterized in that the cells are cultured by attaching the cells to a substrate for supporting cells as claimed in claim 7 or 8. A method for culturing a cell, characterized in that the cell is cultured by attaching the cell to a cell supporting substrate as claimed in claim 9.