JPWO2011102385A1 - Image recognition cell recovery device - Google Patents

Abstract

The present invention is a method in which a bottom surface of a culture dish is coated with a polymer gel that undergoes polymerization / depolymerization change depending on the concentration of calcium ions in a solution in a thin layer, and a chamber for culturing cells in units of one cell is provided on the top surface thereof. The cell culture can be performed on a cell basis, and the chelating agent of calcium ions can be applied to the chamber position of the cell to be collected to dispose and collect the cultured cells locally on a cell unit basis. Provide a method.

Description

  The present invention relates to an apparatus and method for observing cultured cells, non-invasively detaching and re-culturing target cells from the observation evaluation results.

  Various types of cells are mixed in cells that are collected from tissues of multicellular organisms and are primarily cultured. In this way, identification of specific cells in tissues and culture media is an important technique in biological and medical analysis, and various methods have been developed. When there is almost no difference, it was necessary to identify the cells one by one based on information stained with fluorescent antibodies or visual information. For example, in recent years, research and development of this technique has progressed as a technique called Quantitative Imaging Cytometry (Non-patent Document 1: Harnett, M., “Laser scanning cytometry: understanding the immune system in situ”. Nature Review Immunology, Vol. 7, pp. 897-904 (2007)). This imaging cytometry is a fusion of the traditional optical microscope imaging method and the concept of cytometry. From imaging, a reference value is set for a specific index such as cell shape and fluorescent labeling, and all cells in the tissue are measured and statistically processed. Even if a target cell is found by an observation technique, it is difficult to detach and re-culture only a specific cell.

  Currently, established cells such as cancer cells and fibroblasts are detached from the culture dish, subcultured, and maintained because of their own growth potential. For this peeling, an enzyme such as trypsin, which is a proteolytic enzyme, or collagenase, which is an extracellular matrix, is used. However, such cell detachment agents are highly toxic to cells, and it is generally impossible to detach primary cultured cells collected from tissues and cultured. For this reason, once the cells are cultured and the target cells are found, it is difficult to recover and reuse them.

  In recent years, attempts have been made to regenerate organs by isolating stem cells from tissues or culturing and differentiating ES cells in the field of regenerative medicine. However, in order to use the differentiated cells on the culture dish like transplantation, it is essential to exfoliate non-invasively. The development of technology to non-invasively dissociate cells once differentiated on the culture dish will lead to the physiological nature of the cells. It becomes possible to collect and reuse the cells with different cycle periods once confirmed. Isolating and recovering specific cells in the culture medium is an important technique in biological and medical analysis.

  One example of a technique for non-invasively peeling and collecting cells in culture is poly N-isopropylacrylamide (PIPAAm) (Patent Document 1: JP 2006-238707, Patent Document 2: JP 2000-219708, Patent) Reference 3: JP 2000-219709, Non-Patent Document 2: JR Soc Interface. 6 Suppl 3: S293-309 (2009)). This substance has a hydrophobic property near 37 ° C., which is a temperature for culturing, and a hydrophilic property near 20 ° C. By coating PIPAAm on a culture dish and changing the temperature of the cultured cell to about 20 ° C. for about 30 minutes, the cells have been successfully detached non-invasively. In a petri dish coated with this substance, cells can be detached without using an enzyme, and therefore, it can be recovered from the petri dish without removing the bonds between the cells. Therefore, it becomes possible to produce and collect a cell sheet in which cells are bound to each other. This means that it becomes possible to collect various cells differentiated from primary cultured cells and stem cells. However, when cells are detached by this means, the entire culture dish is cooled. Therefore, all the cells in the culture dish are peeled off, and only the target cells have not been recovered. Furthermore, even cells cultured at 20 ° C., such as cells collected from nematodes, cannot be cultured. In the future, with the development of regenerative medicine, the demand for technology for non-invasively collecting cells with the same properties from a single cell unit is increasing, so new technology development is required.

JP 2006-238707 JP2000-219708 JP2000-219709

Harnett, M., “Laser scanning cytometry: understanding the immune system in situ. Nature Review Immunology, Vol. 7, pp. 897-904 (2007) Nagase K, Kobayashi J, Okano T. J R Soc Interface. 6 Suppl 3: S293-309 (2009)

  The present inventors have provided a noninvasive culture cell exfoliation apparatus and a noninvasive method for recovering specific cells in culture from a single cell unit in a noninvasive manner and without changing the temperature while maintaining an arbitrary culture temperature. In order to provide a cultured cell detachment method, the bottom of the culture dish is coated with a polymer gel that changes polymerization and depolymerization depending on the concentration of calcium ions in the solution in a thin layer, and cells are cultured in units of 1 cell. Place the chamber on the top surface, and after culturing the cells in 1-cell units in the chamber, let the chelating agent of calcium ion act on the chamber position of the cells you want to collect, so that the cultured cells are locally located in 1-cell units. An apparatus and method for peeling and collecting have been developed. In order to effectively utilize cell collection technology that can selectively collect specific cells at the single cell level, a threshold value is set quantitatively by analyzing the cell state and observing the cell quantitatively. Depending on the situation, a technique for recovering cells is required.

  Primary cells separated from living tissue are cultured in a culture dish, and cells differentiated to a state where phenotype can be observed are very difficult to remove non-invasively by a normal culture method. For this reason, even though physiological analysis of cells can be performed, research to peel them off for reuse in another application has not yet been made. This problem is greatly related to regenerative medicine research.

  Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are usually adhered to a culture dish in order to induce them into differentiated cells such as nerve cells and cardiomyocytes. Therefore, it is necessary to exfoliate noninvasively in order to transplant differentiated cells on a culture dish. In addition, it is known that differentiated cells do not differentiate into only one type of cell, but differentiate into various types of cells. Therefore, it is necessary to collect noninvasively from one cell unit while confirming necessary cells. In addition, the temperature conditions for culturing cells vary depending on the type of cell, and depending on the cell, changing the temperature may change the state of the cell, so it can be cultured in any temperature environment. It is desirable that the cells can be recovered while maintaining this temperature even when the cells are recovered.

  Thus, it is required to collect specific cells in culture from one cell unit non-invasively and without changing temperature while maintaining an arbitrary culture temperature.

  In addition, in order to selectively recover specific target cells, a technique that combines cytometry technology, which is a cell observation element that identifies target cells, and noninvasive cell recovery elements for cells is required. Yes.

  In view of such circumstances, the present invention provides the following cell culture device, method for producing the cell culture device, noninvasive culture cell detachment method, selective culture cell recovery / reculture method, and culture cell.

[1] a culture dish;
A cell culture carrier layer containing a polymer gel formed on the surface of the culture dish and capable of being solated with a solubilizing agent;
A cell culture apparatus comprising:
[2] The cell culture device according to [1], further comprising an electrode or an array of a plurality of electrodes capable of measuring a cell potential between the surface of the culture dish and the cell carrier layer.
[3] A cell potential measuring unit connected to the electrode and capable of measuring the cell potential;
The cell culture device according to [2], further comprising a cell discrimination unit that discriminates cells to be collected by judging a change in cell type and cell state based on the cell potential measured by the cell potential measurement unit.
[4] The cell culture carrier layer according to any one of [1] to [3], wherein the cell culture carrier layer is formed of a thin film-like alginate gel layer containing calcium alginate or magnesium alginate mixed with a cell adhesion substrate. Cell culture equipment.
[5] The solubilizing agent is a chelating agent of calcium ions or magnesium ions,
By applying the chelating agent in the vicinity of the cells to be collected, the portion of the cell culture carrier layer to which the chelating agent is applied is made into a sol, thereby peeling and collecting the target cultured cells locally in units of one cell. The cell culture device according to any one of [1] to [4], wherein
[6] The cell culture device according to any one of [1] to [5] above,
It is an agarose layer formed on the upper surface of the carrier layer, and a part of the agarose layer can be solated with the above-mentioned solubilizer and removed to enable cell culture in a single cell unit. An agarose layer comprising a microchamber having a volume to be extended and a microchannel extending from the microchamber,
A cell culture apparatus comprising:
[7] The cell culture device according to [6], wherein the part to which the solubilizing agent is applied is a microchamber containing the cells to be collected.
[8] The cell culture device according to [6] or [7], wherein the microchamber is formed corresponding to a position where the electrode is disposed.
[9] The cell culture device according to [6] or [7], wherein the microchannel does not allow cell migration but can pass cell protrusions extended from the cell.
[10] Furthermore,
Means capable of spraying the sol-agent under the cells cultured in the microchamber and macrochannel in units of one microchamber and one microchannel;
A means of measuring the state of the cells;
A cell culture device according to any one of the above [6] to [9], comprising: means capable of recovering detached cells.

[11] A step of coating the surface of the culture dish with a polymer gel that can be solated with a solubilizing agent in a thin layer to form a cell culture carrier layer;
A step of forming an agarose layer on the cell culture carrier layer, and removing a part of the agarose layer by laser irradiation to form a microchamber having a volume capable of cell culture in units of one cell and a microchannel extending from the microchamber. The process of
A method for producing a cell culture device, comprising:
[12] before the step of forming the cell culture carrier layer, further comprising a step of arranging an electrode or an array of a plurality of electrodes capable of measuring a cell potential on the surface of the culture dish;
The method according to [11] above, wherein in the step of forming the microchamber and the microchannel, the microchamber is formed corresponding to the position of the electrode.
[13] The production method according to [11], wherein the polymer gel is an alginate gel containing calcium alginate or magnesium alginate mixed with a cell adhesion substrate.
[14] In the step of forming the microchamber and the microchannel,
By irradiating the agarose layer with focused infrared light having a wavelength of water absorption and changing the temperature to agarose in a gel state,
The production method according to any one of [11] to [13], which comprises constructing a chamber by optically specifying the agarose region to be in a sol state while confirming its position.
[15] Using neurons as the cultured cells,
The microchamber has a diameter of about 50 μm so that a single cell can just adhere, and the microchannel has a width of about 10 μm and a length of about 100 μm such that a nerve process can extend straight from the nerve cell. The manufacturing method according to any one of [11] to [14], comprising forming the microchannel and the microchamber so as to have a thickness.

[16] A step of preparing the cell culture device according to any one of [1] to [10] above,
Culturing cells in the cell culture apparatus,
A step of allowing the solubilizing agent to act on a portion of the cell culture carrier layer in the vicinity of the cells to be collected; and
A step of peeling and collecting the cultured cells locally in units of one cell;
A non-invasive method for detaching cultured cells.
[17] In the cell culturing step,
The state of the cells is optically or electrically observed, and the culture carrier layer is solated by adding a solubilizer in one chamber unit based on the observation result,
In the step of peeling and collecting the cultured cells,
The method according to [16] above, which comprises collecting the target cells that have floated as a result of the lower culture carrier layer being made into a sol.
[18] As the cell culture carrier layer of the cell culture device, an alginate gel layer containing calcium alginate or magnesium alginate gel formed in a thin film and mixed with a cell adhesion substrate is used.
In the step of peeling and collecting the cultured cells,
Prepare a hollow microneedle with an inner diameter that allows several cells to be recovered simultaneously, filled with a culture solution containing ethylenediaminetetraacetic acid sodium salt (EDTA · 2Na) as a solubilizing agent inside the needle,
After a few days of culture, the above-mentioned culture solution containing EDTA · 2Na is locally sprayed on the cells that adhere and have phenotypes revealed by optical or electrical measurement techniques. Solubilize calcium alginate or magnesium alginate gel that is a scaffold for cultured cells,
The method according to [16] or [17] above, comprising non-invasively exfoliating a target cell.
[19] The method according to [18] above, comprising collecting, with a microneedle, cells suspended by the alginate gel layer serving as a scaffold for the cultured cells formed into a sol.

[20] A cultured cell isolated noninvasively by the method according to any one of [16] to [19].
[21] Cultured nerve cells that are non-invasively detached and collected from the cell culture medium while maintaining the shape of the artificially created nerve network by the method according to any one of [16] to [19].
[22] Cultured nerve cells exfoliated and collected non-invasively from the cell culture medium while maintaining the shape of the artificially created nerve network.

[23] a stage unit including an XY stage on which a cell culture dish provided with a cell culture carrier layer including a polymer gel that can be solated by a solating agent, formed on the surface of the culture dish;
An optical microscope system for microscopic observation of the cultured cells in the cell culture dish,
An imaging unit optically connected to the optical microscope system and including an optical camera that captures an optical image from the optical microscope;
A display unit including a display for displaying an image captured by the imaging unit;
A first micropipette part including a micropipette for discharging a solubilizing agent that discharges a solubilizing agent capable of solating the polymer gel;
A second micropipette part including a micropipette for cell recovery for recovering cultured cells that have become detachable due to the polymer gel being sol;
A recording unit for recording the captured image;
A control / analysis unit that controls the operation of each unit and analyzes the captured image;
A culture cell separation and recovery system comprising:
[24] Furthermore,
Measure the cell potential of the cultured cells in the cell culture dish, including an electrode or an array of a plurality of electrodes that can measure the cell potential disposed between the surface of the cell culture dish and the cell culture carrier layer Equipped with a cell potential measurement system
The display unit further includes a second display unit that can display the cell potential measured by the cell potential measurement system or display the cell potential measured by the cell potential measurement system,
The cell potential can be recorded by the recording unit, or a second recording unit for recording the cell potential is provided,
The cell potential data analysis unit for analyzing the cell potential of the cell measured by the cell potential measurement system, or the cell potential of the cell measured by the cell potential measurement system can be analyzed by the control / analysis unit The system for separating and collecting cultured cells according to [23] above.
[25] Target cells are selectively collected based on the optical image of the cells cultured on the surface of the culture dish imaged by the imaging unit and the cell potential of the cells measured by the cell potential measurement system. The cultured cell separation and recovery system according to [23] or [24], which can be performed.
[26] The system according to [23], wherein the polymer gel is an alginate gel containing calcium alginate or magnesium alginate gel, and the solubilizing agent is a chelating agent of calcium ions or magnesium ions.
[27] The system according to [19], further comprising a culture dish for re-culture for re-culturing the cells collected with the micropipette for cell collection.

[28] A method for separating cells using the cultured cell separation and recovery system according to any one of [23] to [27],
Quantifying the cultured cell image data acquired by the imaging unit according to desired parameters, and selecting the cultured cells based on a predetermined threshold;
Dissolve the cell culture carrier layer by discharging a solubilizer using a first micropipette part in the vicinity of the sorted cell or cell group, and the sorted cell or cell group that has become detachable is the second micropipette. Recovering using a pipette part,
A cell separation method comprising:
[29] Presence / absence and / or expression of each ion channel based on the release time after depolarization of each ion channel expressed on the cell surface based on cell potential data of the cultured cells acquired by the measuring unit that measures the cell potential Quantifying as a parameter the approximate amount and / or the presence and / or degree of transmission of depolarization stimuli between cells to be joined, and sorting the cultured cells based on a predetermined threshold,
The cell separation method according to [28], further comprising:
[30] The method according to [28] or [29] above, further comprising the step of re-culturing the collected cells.
[31] The method described in [28] or [30] above, wherein the parameter is cell shape, cell cycle, stem cell colony analysis, or cell differentiation degree.

[32] A step of preparing a culture dish on which a cell culture carrier layer containing a polymer gel that can be solated by a solubilizer is formed,
Applying the polymer gel denaturing agent to a template having a desired pattern, stamping the cell culture carrier layer surface with the template, and placing the denaturant on the cell culture carrier layer surface;
The manufacturing method of the culture dish for noninvasive peeling including this.
[33] The production method according to [32], wherein the polymer gel is an alginate gel containing calcium alginate or magnesium alginate gel, and the modifier is polylysine.
[34] The production method according to [32], wherein nerve cells are used as the cultured cells.
[35] The manufacturing method according to [32], wherein the pattern of the mold is a tile pattern.
[36] A non-invasive exfoliation culture dish produced by the production method according to any one of [32] to [35].

[37] A method for noninvasively collecting cells in culture,
A step of culturing cells using the noninvasive exfoliation culture dish described in [36] above,
A step of adding a solubilizing agent to the culture dish, and a step of detaching cultured cells from the cell culture carrier layer solated by the solubilizing agent,
Including a method.

  According to the present invention, it is possible to selectively detach a cultured cell from a cell dish in a non-invasive manner and reuse it.

  Further, according to the present invention, cultured cells to be reused can be efficiently selected and recovered.

It is a schematic diagram which shows notionally an example of a structure of the cell culture apparatus of this invention. It is an apparatus block diagram which shows typically an example of the apparatus which processes the agarose layer in the cell culture apparatus of FIG. It is a figure which shows typically the procedure of an example which culture | cultivates, peels, collect | recovers, and re-cultures a cell using the culture apparatus in FIG. It is a figure which shows the result of having actually performed cell culture, peeling, collection | recovery, and re-culture according to the procedure in FIG. 3, and having image | photographed. It is a schematic diagram which shows notionally about an example of the apparatus which optically observes a cell, collect | recovers a cell based on the result, and re-cultures to another culture dish. It is a schematic diagram which shows notionally about an example of the technique and procedure which observes a cell optically, collect | recovers a cell based on the result, and re-cultures to another culture dish. It is a schematic diagram which shows notionally about an example of the processing method of a substrate surface. It is a microscope picture which shows an example of the result of a process of a board | substrate surface. After culturing neurons using a non-invasive exfoliation culture dish as shown in FIG. 8, the state (B) in which the cultured neurons are exfoliated non-invasively is compared with the conventional method (A). It is the figure typically shown by contrast. It is a figure which shows typically the procedure of an example which measures the electric potential of a cultured cell and culture | cultivates, peels, collect | recovers, and re-cultures a cell. A micrograph (A) showing an example of culturing cultured cells using a cell culture apparatus in which the electrode array 5001 of the present invention is placed on a culture dish, a diagram (B) schematically showing a configuration in a cross section thereof, and It is a graph (C) which shows an example of the time change of the cell potential actually acquired.

  In one embodiment, the present invention provides a cell culture device. The cell culture apparatus includes a culture plate and a cell culture carrier layer that is formed on the surface of the culture plate and includes a polymer gel that can be solated by a solubilizing agent. .

  Further embodiments of the present invention further comprise an agarose layer formed on the upper surface of the carrier layer. In this embodiment, a part of the agarose layer is removed to form a microchamber having a volume that enables cell culture in a single cell unit and a microchannel extending from the microchamber.

  The “polymer gel” used in the present invention is typically a polymer gel that forms a sol by chelating a metal ion with a chelating agent of a metal ion such as EDTA or EGTA, that is, for example, calcium alginate. Examples include, but are not limited to, gels, alginic acid gels such as magnesium alginate gel. The insolubilized (or modified) alginate gel includes, for example, an alginate polylysine gel.

  In the “solubilizing agent” of alginic acid gel, as a chelating agent for metal ions, for example, calcium ion chelating agents (eg EDTA · 2Na, EGTA, NTA (Nitrilo Triacetic Acid), citric acid, phytic acid, etc.) (particularly , Calcium alginate gel), magnesium ion chelating agents (example: EDTA · 2Na) (particularly in the case of magnesium alginate gel), but not limited thereto.

  “Insolubilizers” or “denaturing agents” of polymer gels or alginic acid gels include, for example, cationic reagents such as polylysine (poly-L-lysine: PLL), and acidic reagents (reagents that are stronger than alginic acid). However, it is not limited to these.

  The cell culture device of the present invention can be prepared, for example, by preparing a thin layer of calcium alginate gel on a culture dish by allowing a calcium chloride solution to act on sodium alginate mixed with a cell adhesive substrate. it can. In the cell culture device of a further embodiment of the present invention, a thin film of agarose gel is further laminated and adhered on the calcium alginate gel layer, and a part of the agarose layer is locally formed into a shape suitable for cell culture. The calcium alginate mixed with the cell adhesion substrate is exposed to construct a microchamber and a microchannel extending therefrom. Therefore, this invention also provides the manufacturing method of a cell culture apparatus in one Embodiment.

  In the present invention, a chamber for placing cells is constructed by locally dissolving the agarose layer. According to the present invention, the agarose layer is irradiated with focused infrared light having a certain wavelength of water absorption to change the gel state of agarose into a sol state, and optically confirms the position of the agarose layer. A chamber can be constructed by specifying the agarose region to be used.

  The present invention also provides a non-invasive method for detaching cultured cells.

  In one embodiment, the present invention creates a population cell group in which cells are dispersed and cultured using only an alginate layer (no agarose gel layer) coated on a culture dish, and the cells are detached. Thus, a method for easily and non-invasively collecting cells on a sheet is provided.

  In a further embodiment, the present invention provides an agarose gel layer on an alginate layer, and a part of the agarose gel layer is removed to form a microchamber and a microchannel, and a nerve is formed in the microchamber and the microchannel. Provided is a method for non-invasively detaching and reusing cultured nerve cells in which a neural network is artificially created by culturing cells while maintaining the shape of the network.

  More specifically, in the present invention, a part of an agarose layer on an alginate sheet formed on the surface of a culture dish is dissolved with infrared focused light to form a certain shaped chamber and channel, Provided is a non-invasive exfoliation method after clarifying the degree of differentiation of nerve cells by culturing the nerve cells. Specifically, a small-scale chamber is created by dissolving agarose (hereinafter referred to as a microchamber) with a diameter of about 50 μm to which one cell can just adhere, and as a modification, the nerve process can be extended in a straight line. Create a groove (hereinafter referred to as a microchannel) with a width of approximately 10 μm and a length of approximately 100 μm so as to be connected to the microchamber (hereinafter referred to as patterning), place cells on the patterned area, and continue to culture for a while. Since the neurites extend along the axis, a selective peeling method is provided by confirming and peeling the neurite.

  In the microchamber of the cell culture device according to one embodiment of the present invention, the cells are cultured in units of one cell and observed by means of optical or electrical observation of the state of the cells. The alginate gel (eg, calcium alginate gel) layer was solated by adding a solution containing a solubilizer (eg, a drug capable of chelating calcium ions) in chamber units, and the lower layer was solated The cultured cells can be isolated non-invasively by collecting the target cells suspended by the above.

  That is, for example, the bottom surface of a culture dish is coated with a polymer gel that changes in polymerization / depolymerization depending on the concentration of calcium ions in the solution in a thin layer, and a chamber for culturing cells in units of one cell is formed on the top surface of the chamber. Cells can be cultured in 1-cell units and calcium ion chelating agents (eg, EDTA-2Na, EGTA, NTA (Nitrilo Triacetic Acid), citric acid, phytic acid, etc.) are to be recovered. By acting on the chamber position, the cultured cells can be peeled and collected locally in a single cell unit.

  Furthermore, according to the present invention, a hollow microneedle having an inner diameter sufficient to collect several cells from one cell at a time is prepared, and the inside of the needle is, for example, a culture solution containing sodium ethylenediaminetetraacetate (EDTA · 2Na). It can be solated by chelating calcium in the calcium alginate gel with EDTA · 2Na in this culture solution, so that it adheres after several days of culture and the phenotype is revealed by optical or electrical measurement techniques By subjecting the above-mentioned cells to the above-mentioned EDTA · 2Na-containing medium locally by microneedles, the calcium alginate that is locally used as a scaffold for cultured cells is solated in units of chambers, Only target cells can be exfoliated invasively.

  Furthermore, in the present invention, cells suspended due to the formation of calcium alginate, which is a scaffold for cultured cells, can be collected with a microneedle and transferred to another cell culture dish.

  Hereinafter, although embodiments of the present invention will be described in more detail with reference to the drawings, these are merely examples, and the scope of the present invention is not limited to these embodiments.

  FIG. 1 is a diagram conceptually showing an example of the configuration of a cell culture device of the present invention used for noninvasive detachment of cultured cells. FIG. 1A shows an example of a cell culture device in which an alginate gel (eg, calcium alginate gel or magnesium alginate gel) in which a thin cell adhesion substrate 201 is mixed in a culture dish is prepared on the culture dish 101. FIG. 1B shows an example of a cell culture apparatus comprising a micropatterned culture dish in which an agarose sheet 301 is coated on a cell adhesion substrate 201 laid on a culture dish 101, and agarose is partially dissolved by local heating. Show. The cell adhesion substrate 201 can be changed depending on the cells to be cultured. Examples of cell adhesion substrates that can be used in the present invention include cationic reagents such as polylysine and polyethyleneimine, collagen, vitronectin, laminin, fibronectin, and lectin. However, when a cationic reagent such as polylysine (poly-L-lysine: PLL) or an acidic reagent (a reagent with a stronger acid than alginic acid) is mixed in the alginic acid gel, it will not be solated by a chelating agent such as EDTA · 2Na. Therefore, by making micro-areas on the tiles that do not make the insolubilized region into a wide sheet, such as by microcontact printing, on the tiles, cells can be cultured on an alginate sheet for each cell. A device that can be peeled off by a chelating agent is applied (see FIGS. 7 to 9). In addition, as another adhesion substrate, it is preferable to mix a reagent (eg, neutralized collagen type IV, vitronectin, laminin, fibronectin, lectin, etc.) that is difficult to denature alginate gel. Alternatively, 201 may be alginic acid whose adhesion ability is increased by cross-linking a protein composed of arginine-glycine-aspartic acid, which is a cell binding domain, with alginic acid. The microchamber 302 and the microchannel 303 are manufactured by an apparatus for performing agarose processing shown in FIG.

  FIG. 2 is a schematic diagram of the overall configuration of the agarose processing apparatus when processing the agarose layer of the cell culture apparatus of the present invention. The agarose processing apparatus includes a 1480 nm Raman fiber laser generator 501, a laser shutter 702, a dichroic mirror 703, an objective lens 701, a mirror 706, a CCD camera 704, and an xy stage 601. The Raman fiber laser 502 generated from the Raman fiber laser generator 501 is reflected by the dichroic mirror 703 and focused by the objective lens 701. The agarose on the noninvasive peeling dish 401 is locally heated by the focused 1480 nm laser focused light 503 to create the microchamber 302 and the microchannel 303. The micro chamber 302 and the micro channel 303 are created by changing the laser output of the Raman fiber laser generator 501 and the magnification of the objective lens 701 and the shutter speed of the shutter 702, and the output of the laser focused light 503, the focal radius, and the irradiation time. This can be done by changing the shape and can be processed into various shapes. The microchannel 303 can be created by moving the xy stage 601 while continuing to irradiate the irradiation light 503. Such processing is performed while observing the photographed image 705 using the objective lens 701, the mirror 706, and the CCD camera 704. Processing can be automatically performed by automatically controlling the shutter 702 and the xy stage 601 with a computer.

  FIG. 3 is a schematic diagram showing a series of steps from cell culture on a non-invasive exfoliation culture dish to cell exfoliation, cell recovery, and re-culture over time. First, as step 1, cell culture is started on the noninvasive peeling culture dish 401. The cells do not adhere on the agarose sheet 301 but adhere on the alginate sheet 201 of the cell adhesion substrate. After culturing for several days, when the cells differentiated and expressed the phenotype, as step 2, the EDTA-containing medium 1001 is sprayed to the vicinity of the target cells by the microneedles 901 to locally dispose the alginate sheet 201 of the cell adhesion substrate under the cells 801. To sol. Thereby, the cells that have adhered and differentiated are non-invasively detached. As step 3, the microneedle 901 used in step 2 is collected. The cells collected in step 3 are re-cultured by transferring them to another culture dish in step 4. As the re-culture in step 4, other than the culture dish, it may be transplanted as it is to a living tissue.

  FIG. 4 is a diagram showing a result obtained by actually performing the process in FIG. 3 and observing the process. Fig. 4 shows the state of detachment and re-culture after dispersive culture in a non-invasive culture dish without an agarose gel layer in the upper (A1, A2), and culture with an agarose layer in the lower (B1, B2). This is a time series photograph of the state of non-invasive detachment, recovery and re-culture of cells cultured on a microchamber / microchannel after agarose processing in a dish. In the re-culture, a state of adhesion after 30 minutes from peeling is observed.

  FIG. 5 shows a schematic diagram of an apparatus system in which the process of separating and collecting only characteristic cells after analyzing the physiological properties of cultured cells is automated. A non-invasive cell detachment culture dish 401 and a cell recovery culture dish 402 are prepared on the XY stage 601, and a physiological characteristic extraction of a cell population is performed using a CCD camera 704 and a control personal computer 2001, and then an alginate solution. Discharge the lysate in the vicinity of the target cells with the discharge micropipette 902 to locally peel off the target cells, and collect the target cells peeled off with the cell recovery micropipette 903. Although not shown, the apparatus system of the present invention includes an optical microscope system for observing cells in the culture dish on the XY stage 601, and the CCD camera 704 is optically connected to the optical microscope system. It is connected. Examples of the optical microscope that can be used in the present invention include, but are not limited to, a phase contrast microscope, a differential interference microscope, a bright field microscope, a dark field microscope, and a scanning optical microscope.

  The collected cells are re-cultured in the cell collection culture dish 402. The control computer 2001 automatically performs cell image analysis by CCD, XY stage control, alginate solution discharge control, and cell recovery necessary for this operation. Cell shape, cell cycle, cell colony analysis (cell color reagent such as Hoechst reagent, characteristics of spatial distribution of intracellular molecules by scanning confocal Raman spectroscopy, cell colony size, or cell colony size Etc.), or the ratio of cell nucleus to cell size, the degree of cell differentiation by analyzing the amount and distribution of cytoplasmic organelles, etc., can be used for the separation and collection of cells.

  FIG. 6 shows the procedure of cell sorting by image analysis of cultured cells cultured on a culture dish. First, the cultured cells on the non-invasive peeling culture dish are analyzed by the CCD camera 704 and the control personal computer 2001. Next, parameters for confirming cell characteristics (eg, cell shape, cell cycle, cell colony shape and size analysis over time, cell differentiation degree) are measured, and the region (or threshold) having the characteristics to be collected is controlled. Set on a PC. Next, the cells having the set characteristics are peeled off by the micropipette 902 for discharging the alginic acid solution, recovered by the micropipette 903 for cell recovery, and transferred to the culture dish 402 for cell recovery. The micropipette part including the micropipette 902 for discharging the solution may further include a solution tank for holding the solution, means for feeding the solution (eg, pump, syringe, motor, etc.) and the like. . The micropipette part including the cell recovery micropipette 903 may further include means for aspirating cells (eg, pump, syringe, motor, etc.).

  FIG. 7 shows the process of producing a non-invasive exfoliating culture dish for exfoliating nerve cells. A template for stamping the PLL on an alginate gel (eg, calcium alginate gel, magnesium alginate gel) is made of polydimethylsiloxane (PDMA), and the PLL is dried at the place to stamp. Next, it is stamped on the alginic acid by lightly pressing it on the alginic acid, and an insolubilized micropattern is formed only at the point of contact by the reaction of alginic acid and PLL.

  FIG. 8A shows a part of the shape of PLL microprinted on an alginate sheet. FIG. 8B is a diagram when primary hippocampal neurons are cultured on an alginate sheet on which PLL is microcontact-printed. In this way, by using a template of a desired design, a minute area where about one cell can adhere can be appropriately produced (for example, in a tile shape) as an insolubilized region, and as a result, While cells are cultured on an alginate sheet, the cells can be detached with a chelating agent.

  FIG. 9 shows a state in which nerve cells are cultured using a non-invasive peeling culture dish as shown in FIGS. 7 and 8 and then the cultured nerve cells are peeled non-invasively for each cell. It is the figure typically shown in contrast with. 9A shows a case where PLL is applied to the entire surface of the alginate gel layer on the culture dish, and FIG. 9B shows a case where PLL is applied in a tile shape. As shown in FIG. 9A, when the entire culture carrier is an insolubilized alginate gel 4001, even if the solubilizing agent 1002 is acted using the micropipette 904, it is non-invasively removed from the culture dish for each cell. It is difficult to peel off. On the other hand, as shown in FIG. 9B, when the PLL is applied in a tile shape as shown in FIGS. 7 and 8, the joint portion of the tile pattern is the alginate gel 4002 that is not insolubilized. When 1002 is allowed to act, it can be made into a sol, whereby the cultured cells can be detached from the culture dish non-invasively cell by cell.

  FIG. 10 is a schematic diagram showing, over time, a series of steps from cell culture on a non-invasive exfoliation culture dish in which multiple electrodes for measuring the cell potential of cultured cells are arranged on the bottom surface, cell exfoliation, cell recovery, and reculture. FIG. First, as schematically shown in FIGS. 10A and 10B, as step 1, cell culture is started on a non-invasive peeling culture dish 401. An electrode array 5001 capable of measuring a cell potential is disposed on the culture dish 101, and an alginate layer 201 is disposed thereon. Further, the cells do not adhere on the agarose sheet 301 arranged in a specific shape on the alginic acid layer 201, and the cells adhere on the alginic acid sheet 201 of the cell adhesion substrate. After culturing for several days, the cells differentiated, and from the measured cell potential pattern, the expression of the protein such as membrane ion channel protein expressed in the cells represents the phenotype of the cell type to be recovered, as shown in FIG. 10C. In step 2, the EDTA-containing medium 1001 is sprayed to the vicinity of the target cell by the microneedle 901, so that the alginate sheet 201 of the cell adhesion substrate under the cell 801 is locally solated. Thereby, the cells that have adhered and differentiated are non-invasively detached. As shown in FIG. 10D and FIG. 10E, the microneedle 901 used in step 2 as step 3 is collected. Furthermore, as shown in FIG. 10F or FIG. 10G, the cells collected in step 3 are re-cultured by transferring them to another culture dish or cell culture dish with electrodes in step 4. As the re-culture in step 4, other than the culture dish, it may be transplanted as it is to a living tissue. In this apparatus, data acquired for cell identification by measuring cell potential is, for example, the presence / absence of each ion channel based on the release time after depolarization of each ion channel expressed on the cell surface and / or Alternatively, the approximate amount of expression and / or the presence / absence and / or degree of transmission of depolarization stimulation between cells to be joined and the transmission speed, etc. shall be obtained as parameters, and threshold values are determined for each data. The cell differentiation state or phenotypic change is continuously measured from the combination of cells to confirm whether the cells are to be recovered, or after the cell culture starts, the cells adhere to the electrode and the cell potential is measured. When it becomes possible, it is possible to simply identify cell types and phenotypes based on the acquired data. Determining the cell to yield.

  FIG. 11 specifically shows an example of how to use the present technology taking human ES cell-derived cardiomyocytes as an example. FIG. 11A is a photomicrograph of culturing cardiomyocyte clusters on an example of a non-invasive exfoliated culture dish in which the electrode array 5001 for measuring the cell potential shown in FIG. 10A is arranged. FIG. 11B is a schematic diagram specifically showing the configuration of FIG. 11A. Each electrode 5002 for measuring a cell potential is connected to a cell potential measuring unit 5004 by an electric wire 5003. The upper cell potential can be continuously measured in real time. Cell potential measuring unit 5004 is, for example, an analog amplifier array unit capable of parallel processing as many as the number of electrodes for amplifying analog signals of electrode potentials, and an AD conversion array unit capable of simultaneously processing amplified analog signals in parallel Digital arithmetic circuit unit that processes the obtained digital signal, DA conversion type stimulation current application unit that can give stimulation to each electrode in units of one electrode in order to give forced stimulation to the cell, the stimulation signal from the measurement signal Feedback control unit that automatically instructs the application timing under preset conditions, switching circuit array unit that switches the circuit between applying a stimulus to a cell and receiving a cell signal, and if necessary, analog at the first stage It can be composed of a noise filter array or the like. The cell potential measuring unit is connected to a cell potential data analyzing unit (or cell discriminating unit or control / analyzing unit) 5005 composed of a personal computer or the like, for example, and analyzes the cell potential data on each electrode. Analyze cell types and conditions, and display the results on a monitor. FIG. 11C is a graph of an example of the obtained cell potential data. As shown in this graph, by analyzing cell potential data, the expression state of various ion channels expressed on the cell surface can be analyzed, and cells having specific desired cell potential data can be selected. . In particular, long-term continuous measurement using this technology not only analyzes cell characteristics, but also changes in cell potential data characteristics that gradually change due to changes in proteins expressed in cells during cell culture. It is also possible to collect the cells after confirming that the desired state has been achieved. Also. In this example, cardiomyocytes differentiated from human stem cells were used, but nerve cells can be similarly measured to confirm their characteristics. Furthermore, for cells that do not have spontaneous firing characteristics, a circuit that applies an electric field to each electrode 5002 can be added to stimulate the cells on each electrode, and the response can be measured by each electrode 5002.

  The present invention is useful for realizing detachment and reuse of cultured cells selectively and non-invasively on a cell-by-cell basis. In particular, the cultured cells obtained by the noninvasive cultured cell exfoliation method of the present invention are used when cells (eg, stem cells, ES cells) separated from tissues are cultured and differentiated to regenerate organs in the field of regenerative medicine. Useful as cultured cells. In addition, in order to enable sorting of cells using parameters, cell shapes, etc., which are different from FACS and flow cyto sorter that float cells and flow into the flow path to sort cells, more detailed information is available. The cells can be used in industries such as regenerative medicine.

DESCRIPTION OF SYMBOLS 101,102 ... Culture dish 201 ... Alginic acid sheet 301 with adhesion substrate ... Agarose sheet 302 ... Micro chamber 303 ... Micro channel 401 ... Non-invasive peeling culture dish 402 ... Cell recovery culture dish 501 ... 1480nm Raman fiber laser generator 502 ... 1480 nm Raman fiber laser 503 ... 1480 nm laser focused light 601 ... xy stage 701 ... objective lens 702 ... shutter 703 ... dichroic mirror 704 ... CCD camera 705 ... photographed image 706 ... mirror 801 ... cell 802 ... nerve cell 901 ... microneedle 902 ... alginic acid Micropipette 903 for discharging lysate ... Micropipette 904 for recovering cells ... Micropipette 1001 for discharging solubilizing agent ... Medium 1002 with EDTA ... Sollating agent 2001 ... PC for control 3001 ... PMDS stamp 002 ... PLL
3003 ... Alginic acid gel 4001 ... Insolubilized alginate gel 4002 ... Non-insolubilized alginate gel 5001 ... Electrode array 5002 ... Electrode 5003 ... Electric wire 5004 ... Cell potential measurement unit 5005 ... Cell potential data analysis unit

Claims (37)

A culture dish,
A cell culture carrier layer comprising a polymer gel formed on the surface of the culture dish and capable of being solated with a solubilizing agent;
A cell culture apparatus comprising:   The cell culture device according to claim 1, further comprising an electrode or an array of a plurality of electrodes capable of measuring a cell potential between the surface of the culture dish and the cell carrier layer. A cell potential measuring unit connected to the electrode and capable of measuring the cell potential;
The cell culture device according to claim 2, further comprising: a cell discriminating unit that discriminates cells to be collected by judging a change in cell type and cell state based on the cell potential measured by the cell potential measuring unit.   The cell culture device according to any one of claims 1 to 3, wherein the cell culture carrier layer is formed of a thin film-like alginate gel layer containing calcium alginate or magnesium alginate mixed with a cell adhesion substrate. The solubilizing agent is a chelating agent of calcium ion or magnesium ion,
By applying the chelating agent in the vicinity of the cells to be recovered, the portion of the cell culture carrier layer to which the chelating agent is applied is made into a sol, thereby peeling and recovering the target cultured cells locally in units of one cell. The cell culture device according to any one of claims 1 to 4, wherein The cell culture device according to any one of claims 1 to 5, further comprising:
An agarose layer formed on the upper surface of the carrier layer, wherein a part of the agarose layer is sol-solubilized and removed by the solubilizing agent, enabling cell culture in a single cell unit An agarose layer comprising a microchamber having a volume to be extended and a microchannel extending from the microchamber,
A cell culture apparatus comprising:   The cell culture device according to claim 6, wherein the part to which the solubilizing agent is applied is a microchamber containing the cells to be collected.   The cell culture device according to claim 6 or 7, wherein the microchamber is formed corresponding to a position where the electrode is disposed.   The cell culture device according to claim 6 or 7, wherein the microchannel does not allow movement of a cell, but can pass a cell process extended from the cell. further,
Means for spraying the solubilizer under the cells cultured in the microchamber and macrochannel in units of 1 microchamber and 1 microchannel;
A means of measuring the state of the cells;
The cell culture device according to any one of claims 6 to 9, comprising means capable of recovering the detached cells. Coating the surface of the culture dish with a polymer gel that can be solated with a solubilizing agent in a thin layer to form a cell culture carrier layer;
A step of forming an agarose layer on the cell culture carrier layer, and removing a part of the agarose layer by laser irradiation to form a microchamber having a volume capable of cell culture in units of one cell and a microchannel extending from the microchamber. The process of
A method for producing a cell culture device, comprising: Before the step of forming the cell culture carrier layer, further comprising the step of arranging an electrode or an array of a plurality of electrodes capable of measuring a cell potential on the surface of the culture dish,
The manufacturing method according to claim 11, wherein in the step of forming the microchamber and the microchannel, the microchamber is formed corresponding to the position of the electrode.   The manufacturing method according to claim 11, wherein the polymer gel is an alginate gel containing calcium alginate or magnesium alginate mixed with a cell adhesion substrate. In the step of forming the microchamber and the microchannel,
By irradiating the agarose layer with focused infrared light having a wavelength of water absorption and changing the temperature to agarose in a gel state,
The manufacturing method according to any one of claims 11 to 13, comprising constructing a chamber by optically specifying an agarose region to be in a sol state while confirming its position. Using nerve cells as the cultured cells,
The microchamber has a diameter of about 50 μm that allows just one cell to adhere, and the microchannel has a width of about 10 μm and a length of about 100 μm such that a nerve protrusion can extend straight from the nerve cell. The manufacturing method according to claim 11, comprising forming the microchannel and the microchamber so as to have a thickness. Preparing the cell culture device according to any one of claims 1 to 10,
Culturing cells in the cell culture device,
A step of allowing the solubilizing agent to act on a portion of the cell culture carrier layer in the vicinity of the cells to be collected; and
A step of peeling and collecting the cultured cells locally in units of one cell;
A non-invasive method for detaching cultured cells. In the cell culturing step,
The state of the cells is optically or electrically observed, and the culture carrier layer is solated by adding a solubilizer in one chamber unit based on the observation result,
In the step of peeling and collecting the cultured cells,
The method according to claim 16, comprising collecting the target cells that have floated due to the lower culture carrier layer being made into a sol. As a cell culture carrier layer of the cell culture device, an alginate gel layer containing calcium alginate or magnesium alginate gel mixed with a cell adhesion substrate formed in a thin film shape is used.
In the step of peeling and collecting the cultured cells,
Prepare a hollow microneedle with an inner diameter that allows several cells to be recovered simultaneously, filled with a culture solution containing ethylenediaminetetraacetic acid sodium salt (EDTA · 2Na) as a solubilizing agent inside the needle,
After a few days of culture, the cells that have adhered and whose phenotype has been clarified by optical or electrical measurement techniques are locally sprayed by the microneedle with the culture solution containing EDTA · 2Na, thereby locally Solubilize calcium alginate or magnesium alginate gel that is a scaffold for cultured cells,
18. A method according to claim 16 or 17, comprising non-invasively exfoliating cells of interest.   The method of Claim 18 including collect | recovering the cell which floated when the said alginic acid gel layer used as the scaffold of the said cultured cell was sol-ized with a microneedle.   A cultured cell noninvasively isolated by the method according to claim 16.   20. Cultured nerve cells peeled and collected non-invasively from a cell culture medium while maintaining the shape of an artificially created nerve network by the method according to any one of claims 16 to 19.   Cultured neurons that have been exfoliated and collected non-invasively from cell culture medium while maintaining the shape of the artificially created neural network. A stage unit including an XY stage on which a cell culture dish having a cell culture carrier layer including a polymer gel containing a polymer gel that can be solated by a solubilizing agent is formed on the surface of the culture dish;
An optical microscope system for microscopic observation of cultured cells in the cell culture dish;
An imaging unit including an optical camera optically connected to the optical microscope system and capturing an optical image from the optical microscope;
A display unit including a display for displaying an image captured by the imaging unit;
A first micropipette part including a micropipette for discharging a solubilizing agent that discharges a solubilizing agent capable of solating the polymer gel;
A second micropipette part including a micropipette for cell collection that collects cultured cells that have become detachable due to the polymer gel being sol;
A recording unit for recording the captured image;
A control / analysis unit that controls the operation of each unit and analyzes the captured image;
A culture cell separation and recovery system comprising: further,
Measure the cell potential of the cultured cells in the cell culture dish, including an electrode or an array of a plurality of electrodes that can measure the cell potential disposed between the surface of the cell culture dish and the cell culture carrier layer Equipped with a cell potential measurement system
The display unit can further display a cell potential measured by the cell potential measurement system, or a second display unit that displays a cell potential measured by the cell potential measurement system,
The cell potential can be recorded by the recording unit, or comprises a second recording unit for recording the cell potential,
A cell potential data analysis unit for analyzing the cell potential of the cell measured by the cell potential measurement system, wherein the cell potential of the cell measured by the cell potential measurement system can be analyzed by the control / analysis unit The cultured cell separation and recovery system according to claim 23, comprising:   Based on the optical image of the cells cultured on the surface of the culture dish imaged by the imaging unit, and the cell potential of the cells measured by the cell potential measurement system, target cells can be selectively recovered, The cultured cell separation and recovery system according to claim 23 or 24.   24. The system of claim 23, wherein the polymer gel is an alginate gel comprising calcium alginate or magnesium alginate gel, and the solubilizer is a chelating agent of calcium ions or magnesium ions.   The system of Claim 19 provided with the culture dish for reculture for recultivating the cell collect | recovered with the said micropipette for cell collection. A method for separating cells using the cultured cell separation and recovery system according to any one of claims 23 to 27, comprising:
Quantifying the cultured cell image data acquired by the imaging unit according to desired parameters, and selecting the cultured cells based on a predetermined threshold;
In the vicinity of the sorted cells or cell groups, the solubilizing agent is discharged by using a first micropipette unit to dissolve the cell culture carrier layer, and the sorted cells or cell groups that have become detachable are taken as the second micro Recovering using a pipette part,
A cell separation method comprising: The cell potential data of the cultured cells acquired by the measurement unit that measures the cell potential is based on the release time after depolarization of each ion channel expressed on the cell surface. Quantifying the amount and / or presence and / or extent of depolarization stimulation between cells to be joined as parameters, and sorting the cultured cells based on a predetermined threshold;
The cell separation method according to claim 28, further comprising:   30. The method of claim 28 or 29, further comprising the step of re-culturing the collected cells.   The method according to claim 28 or 30, wherein the parameter is a cell shape, a cell cycle, a stem cell colony analysis, or a cell differentiation degree. Preparing a culture dish in which a cell culture carrier layer containing a polymer gel that can be solated by a solubilizer is formed on the surface thereof;
Applying the denaturing agent of the polymer gel to a template having a desired pattern, stamping the cell culture carrier layer surface with the template, and disposing the denaturant on the cell culture carrier layer surface;
The manufacturing method of the culture dish for noninvasive peeling including this.   The production method according to claim 32, wherein the polymer gel is an alginate gel containing calcium alginate or magnesium alginate gel, and the modifying agent is polylysine.   The production method according to claim 32, wherein nerve cells are used as the cultured cells.   The manufacturing method according to claim 32, wherein the pattern of the mold is a tile pattern.   A culture dish for noninvasive exfoliation produced by the production method according to any one of claims 32 to 35. A non-invasive method for recovering cells in culture,
A step of culturing cells using the non-invasive exfoliation culture dish according to claim 36;
A step of adding a solubilizing agent to the culture dish, and a step of detaching cultured cells from the cell culture carrier layer solated by the solubilizing agent,
Including a method.