WO2011102385A1 - Collecteur de cellules par reconnaissance d'image - Google Patents

Collecteur de cellules par reconnaissance d'image Download PDF

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Publication number
WO2011102385A1
WO2011102385A1 PCT/JP2011/053281 JP2011053281W WO2011102385A1 WO 2011102385 A1 WO2011102385 A1 WO 2011102385A1 JP 2011053281 W JP2011053281 W JP 2011053281W WO 2011102385 A1 WO2011102385 A1 WO 2011102385A1
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Prior art keywords
cell
cells
culture
cultured
cell culture
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English (en)
Japanese (ja)
Inventor
英之 寺薗
賢二 安田
明弘 服部
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ON-CHIP CELLOMICS CONSORTIUM Co Ltd
Kanagawa Academy of Science and Technology
Tokyo Medical and Dental University NUC
ON CHIP CELLOMICS CONSORTIUM CO Ltd
Original Assignee
ON-CHIP CELLOMICS CONSORTIUM Co Ltd
Kanagawa Academy of Science and Technology
Tokyo Medical and Dental University NUC
ON CHIP CELLOMICS CONSORTIUM CO Ltd
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Priority to JP2012500625A priority Critical patent/JPWO2011102385A1/ja
Publication of WO2011102385A1 publication Critical patent/WO2011102385A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

Definitions

  • the present invention relates to an apparatus and method for observing cultured cells and non-invasively detaching and re-culturing the target cells from the observation evaluation results.
  • the cells collected from the tissue of multicellular organisms and cultured primarily contain various types of cells. 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. With regard to this technique, for example, research and development has recently been progressing 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.97897-904 (2007)) This imaging cytometry is a fusion of conventional optical microscope imaging methods and cytometry concepts.
  • 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.
  • Patent Document 1 JP 2006-238707
  • Patent Document 2 JP 2000-219708
  • Patent Document 3 Japanese Patent Laid-Open No. 2000-219709
  • Non-Patent Document 2 J R 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.
  • 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.
  • 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.
  • 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.
  • Embryonic stem cells and induced pluripotent stem cells (iPS cells) are usually adhered to a culture dish in order to induce 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.
  • 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.
  • 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.
  • the present invention provides the following cell culture device, method for manufacturing the cell culture device, noninvasive culture cell detachment method, selective culture cell recovery / reculture method, and culture cells.
  • [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.
  • 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.
  • 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 polymer gel is an alginate gel containing calcium alginate or magnesium alginate mixed with a cell adhesion substrate.
  • 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 microchamber has a diameter of about 50 ⁇ m so that a single cell can just adhere, and the microchannel has a width of about 10 ⁇ m and a length of about 100 ⁇ m such that a nerve process can extend straight from the nerve cell.
  • 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 a solubilizer to act on a portion of a cell culture carrier layer in the vicinity of a cell to be collected, and a step of locally peeling and collecting the cultured cells in units of one cell A non-invasive method for detaching cultured cells.
  • 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,
  • 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.
  • 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 as the cell culture carrier layer of the cell culture device.
  • 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.
  • 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 is formed on the surface of the culture dish and can be solated with a solubilizing agent;
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a step of preparing a culture dish on which a cell culture carrier layer containing a polymer gel that can be solated by a solating agent 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.
  • 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.
  • the production method according to [32] wherein nerve cells are used as the cultured cells.
  • the manufacturing method according to [32], wherein the pattern of the mold is a tile pattern.
  • a non-invasive exfoliation culture dish produced by the production method according to any one of [32] to [35].
  • 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.
  • cultured cells to be reused can be efficiently selected and collected.
  • the present invention provides a cell culture device.
  • the cell culture apparatus includes a culture dish (culture plate) and a cell culture carrier layer containing a polymer gel that is formed on the surface of the culture dish and can be solated by a solubilizing agent. .
  • an agarose layer formed on the upper surface of the carrier layer is further provided.
  • 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.
  • a metal ion such as EDTA or EGTA
  • 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.
  • the “solating agent” of alginic acid gel includes, 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.
  • calcium ion chelating agents eg, EDTA ⁇ 2Na, EGTA, NTA (Nitrilo Triacetic Acid), citric acid, phytic acid, etc.
  • “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 causing a calcium chloride solution to act on sodium alginate mixed with a cell-adhesive substrate. it can.
  • 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.
  • a chamber for arranging cells is constructed by locally dissolving the agarose layer.
  • 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.
  • 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.
  • an alginate layer no agarose gel layer
  • 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.
  • 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.
  • 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
  • a non-invasive exfoliation method after clarifying the degree of differentiation of nerve cells by culturing the nerve cells.
  • a small-scale chamber is created by dissolving agarose (hereinafter referred to as a microchamber) with a diameter of about 50 ⁇ m to which one cell can just adhere, and as a modification, the nerve process can be extended in a straight line.
  • a microchannel Create a groove (hereinafter referred to as a microchannel) with a width of approximately 10 ⁇ m and a length of approximately 100 ⁇ m 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.
  • 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.
  • 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 Cells can be cultured in cell units, and calcium ion chelating agents (eg, EDTA-2Na, EGTA, NTA® (Nitrilo® Triacetic® Acid), citric acid, phytic acid, etc.) are to be recovered.
  • calcium ion chelating agents eg, EDTA-2Na, EGTA, NTA® (Nitrilo® Triacetic® Acid), citric acid, phytic acid, etc.
  • 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).
  • EDTA ⁇ 2Na sodium ethylenediaminetetraacetate
  • cells suspended by 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.
  • FIG. 1 is a diagram conceptually showing an example of the configuration of the cell culture device of the present invention used for non-invasive 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.
  • cell adhesion substrates examples include cationic reagents such as polylysine and polyethyleneimine, collagen, vitronectin, laminin, fibronectin, and lectin.
  • cationic reagents such as polylysine and polyethyleneimine, collagen, vitronectin, laminin, fibronectin, and lectin.
  • 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.
  • cells can be cultured on an alginate sheet for each cell. Take measures to peel off with a chelating agent (see FIGS. 7 to 9). Further, as other adhesion substrate, it is preferable to mix a reagent that hardly denatures alginate gel (eg, neutralized collagen type IV, vitronectin, laminin, fibronectin, lectin, etc.).
  • a reagent that hardly denatures alginate gel eg, neutralized collagen type IV, vitronectin, laminin, fibronectin, lectin, etc.
  • 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.
  • 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.
  • step 2 the alginate sheet 201 of the cell adhesion substrate under the cells 801 is locally applied by spraying the EDTA-containing medium 1001 near the target cells with the microneedles 901. To sol. Thereby, the cells that have adhered and differentiated are non-invasively detached.
  • 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.
  • 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 exfoliation and re-cultivation after dispersion culture in a non-invasive culture dish without an agarose gel layer in the upper stage (A1, A2), and culture with an agarose layer in the lower stage (B1, B2).
  • A1, A2 the state of exfoliation and re-cultivation after dispersion culture in a non-invasive culture dish without an agarose gel layer in the upper stage
  • 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 that automates the process of separating and collecting only characteristic cells after analyzing the physiological properties of cultured cells.
  • 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.
  • 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.
  • 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.
  • the cultured cells on the non-invasive peeling culture dish are analyzed by the CCD camera 704 and the control personal computer 2001.
  • parameters for confirming cell characteristics eg, cell shape, cell cycle, cell colony shape and size analysis over time, degree of cell differentiation
  • the region (or threshold) having the characteristics to be collected is controlled.
  • 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 exfoliation culture dish for exfoliating nerve cells.
  • a template for stamping the PLL on an alginate gel eg, calcium alginate gel, magnesium alginate gel
  • PDMA polydimethylsiloxane
  • FIG. 8A shows a part of the shape of PLL micro-printed 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.
  • 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.
  • the joint portion of the tile pattern is the alginate gel 4002 that is not insolubilized.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 by 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.
  • 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 selectively and non-invasively detaching and reusing cultured cells on a cell-by-cell basis.
  • 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 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.

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Abstract

L'invention concerne un dispositif de séparation et de recueil de cellules cultivées localement dans des unités de cellule unique et son procédé, comprenant : le revêtement de la partie inférieure d'une boîte de culture avec un gel polymère subissant la polymérisation et la dépolymérisation en fonction de la concentration en ions calcium d'une solution de celui-ci pour former une couche fine; et la disposition sur sa partie supérieure, d'un compartiment de culture de cellules en unités de cellule unique afin que les cellules puissent être cultivées en unités de cellule unique et un traitement avec un agent de chélation des ions calcium peut être réalisé à l'emplacement d'une cellule, ladite cellule devant être recueillie dans le compartiment.
PCT/JP2011/053281 2010-02-16 2011-02-16 Collecteur de cellules par reconnaissance d'image Ceased WO2011102385A1 (fr)

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JP2013055911A (ja) * 2011-09-08 2013-03-28 Dainippon Printing Co Ltd 細胞培養容器とその製造方法
WO2013146971A1 (fr) * 2012-03-29 2013-10-03 公益財団法人神奈川科学技術アカデミー Dispositif et procédé de placement cellulaire
JP2014079171A (ja) * 2012-10-12 2014-05-08 Yamaguchi Univ 足場依存性細胞の培養方法
JP2014103857A (ja) * 2012-11-23 2014-06-09 Kansai Univ 生体材料パターニング用樹脂基材およびその製造方法ならびに生体材料パターニング材およびその製造方法
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JP2013055911A (ja) * 2011-09-08 2013-03-28 Dainippon Printing Co Ltd 細胞培養容器とその製造方法
WO2013146971A1 (fr) * 2012-03-29 2013-10-03 公益財団法人神奈川科学技術アカデミー Dispositif et procédé de placement cellulaire
JP2014079171A (ja) * 2012-10-12 2014-05-08 Yamaguchi Univ 足場依存性細胞の培養方法
JP2014103857A (ja) * 2012-11-23 2014-06-09 Kansai Univ 生体材料パターニング用樹脂基材およびその製造方法ならびに生体材料パターニング材およびその製造方法
WO2024251858A1 (fr) * 2023-06-07 2024-12-12 The Cultivated B. Gmbh Surfaces antimicrobiennes et leurs utilisations dans des cultures cellulaires

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