JP5765763B2 - Cell automatic sorting apparatus and cell automatic sorting method - Google Patents

Description

  The present invention relates to an automatic cell sorting device and an automatic cell sorting method, and more particularly to an automatic cell sorting device and an automatic cell sorting method for sorting and sorting desired living cells using a cell array sorter.

  In recent years, regenerative medicine that regenerates damaged or lost tissues or organs by cell culture or promotes self-renewal by supplying cells from the outside due to advances in medicine has attracted much attention. In addition, gene therapy is also attracting attention after performing gene repair on cells taken out of the body and then returning them to the body again.

  In order to realize such regenerative medicine and gene therapy, it is essential to select a specific target cell and collect it alive. For example, in the process of collecting and culturing embryonic stem cells (ES cells), universal cells (iPS cells), undifferentiated cells, etc., cell differentiation proceeds, or cells are damaged or damaged. It is necessary in order to advance regenerative medicine and the like to remove cells that have undergone differentiation, cells that have been damaged or damaged, and sort out safe universal cells.

  As a method for selecting and collecting cells, a flow cell sorter using flow cytometry or the like is used. The flow cell sorter performs analysis by passing cells dispersed in a fluid one by one in front of a sensor using flow cytometry, and then applying an electric field or a magnetic field to a droplet containing target cells. Only target cells are collected (see, for example, Patent Document 1).

JP 2010-216992 A

  However, the flow cell sorter has a problem in cell sorting accuracy. If one's own normal hematopoietic stem cells can be isolated and transplanted, it is expected that leukemia and myeloma can be treated without causing rejection or the like. On the other hand, in a cell sorting apparatus using flow cytometry or the like, it is difficult to isolate only hematopoietic stem cells, and contamination of tumor cells is unavoidable. For this reason, there are only a limited number of cases where hematopoietic stem cell autotransplantation can be applied.

  In addition, devices using flow cytometry are generally large and expensive, require a large amount of sample, have a high risk of damaging cells at the stage of creating droplets, There are also problems such as inability to observe.

  An object of the present invention is to solve the above-described problems and to realize an automatic cell sorting apparatus that automatically and accurately sorts cells even with a small amount of sample.

  In order to achieve the above object, the present invention automatically determines whether a cell is an unnecessary cell based on the optical data of the cell adhered to the adhesion spot of the cell array sorter, and kills the unnecessary cell. The configuration is to be

  Specifically, the automatic cell sorting apparatus according to the present invention includes a culture unit that houses a cell array sorter having a plurality of adhesion spots to which cells are adhered, and a sorting unit that sorts and removes cells adhered to the adhesion spots. And a control unit that controls the culture unit and the selection unit, and the adhesion spot has a stimulus-responsive polymer that is changed between a cell adhesive state and a cell non-adhesive state by stimulation, fixed on the surface. Drives the sorting unit to obtain optical data of the adhered cells for each of the plurality of adhesion spots, and determines whether the cells are target cells or unnecessary cells based on the optical data. The cells determined to be target cells are selectively collected.

  In the automatic cell sorting apparatus of the present invention, the control unit drives the sorting unit to obtain optical data of cells adhered to each of the plurality of adhesion spots, and the cells are selected as target cells based on the optical data. It is determined whether it is a non-required cell or not, and the cell determined to be a target cell is selectively recovered. For this reason, it is possible to automatically select cells with high accuracy and high speed. Moreover, since the stimulus-responsive polymer is fixed to the adhesion spot, it is possible to control cell adhesion and release. Therefore, the cells can be collected without damaging the target cells after sorting.

  In the automatic cell sorting apparatus of the present invention, the stimulus-responsive polymer is a temperature-responsive polymer that becomes cell-adhesive at the first temperature and non-cell-adhesive at the second temperature. A selection unit that selects one adhesion spot from the spot; an optical system that obtains an optical image of a cell adhered to the selected adhesion spot; and an optical system that focuses light from the light source onto the selected adhesion spot; A data conversion unit that converts the optical image into optical data, and the control unit uses the temperature of the culture unit as a first temperature to adhere cells to the adhesion spot, drives the sorting unit, For each, obtain optical data, make a determination, and if it is determined that it is a waste cell, irradiate light from the light source to selectively remove the waste cell from the adhesion spot and remove the waste cell Shi After the temperature of the culture part as the second temperature, it was adhered to the adhesive spots cell may be configured to perform an operation to recover by free.

  In this case, by irradiating light to raise the temperature of the unnecessary cells and killing them, the temperature of the adhesion spot where the unnecessary cells are adhered by irradiating light is set to the second temperature even if the unnecessary cells are removed. By doing so, unnecessary cells may be removed.

  In the automatic cell sorting apparatus of the present invention, the stimulus-responsive polymer is a temperature-responsive polymer that becomes cell-adhesive at the first temperature and non-cell-adhesive at the second temperature. A selection unit that selects one adhesion spot from the spot; an optical system that obtains an optical image of a cell adhered to the selected adhesion spot; and an optical system that focuses light from the light source onto the selected adhesion spot; A data conversion unit that converts the optical image into optical data, and the control unit uses the temperature of the culture unit as a first temperature to adhere cells to the adhesion spot, drives the sorting unit, For each, optical data is acquired and determined. If it is determined to be a target cell, light from the light source is irradiated and the temperature of the adhesion spot is set to the second temperature, and the target cell is determined to be the adhesion spot. Et selectively liberated may be configured to perform an operation to recover the liberated cells.

  In the automatic cell sorting apparatus of the present invention, the light source may be a laser light source.

  In the automatic cell sorting apparatus of the present invention, the culture unit has a supply unit that supplies a labeling reagent that specifically labels the cell according to the state of the cell to the cell adhered to the adhesion spot, and the control unit includes: The determination may be made based on the presence or absence of a sign. In this case, the labeling reagent may be configured to include a primary antibody that specifically recognizes unnecessary cells and a secondary antibody that is labeled with a dye that absorbs light and specifically recognizes the primary antibody.

  The cell automatic sorting method according to the present invention uses a cell array sorter having a plurality of adhesion spots to which a stimulus-responsive polymer that changes between a cell adhesion state and a cell non-adhesion state in response to a stimulus is fixed. A method of attaching cells to an adhesion spot with a stimulus-responsive polymer in a cell adhesive state, and a step of selecting and removing unnecessary cells from cells adhered to the adhesion spot. Recovering cells adhered to the adhesion spot by removing the unwanted cells and then bringing the stimuli-responsive polymer into a non-cell-adhesive state, and the step of selecting the unwanted cells comprises a plurality of adhesion spots. For each of the above, the process of obtaining optical data of the adhered cells and automatically determining whether the adhered cells are target cells or unnecessary cells based on the optical data It includes a determining step, and a step of selectively removing the unnecessary cells from the adhesive spot.

  In the automatic cell sorting method of the present invention, the step of selecting unnecessary cells includes a step of labeling unnecessary cells with a labeling reagent that specifically labels unnecessary cells, and confirms the presence or absence of labeling based on optical data. What is necessary is just to determine whether it is a useless cell.

  According to the automatic cell sorting apparatus according to the present invention, it is possible to sort cells automatically with high accuracy even with a small amount of sample.

It is a block diagram which shows the cell automatic sorting apparatus which concerns on one Embodiment. It is a schematic diagram which shows the structure of the cell automatic sorting apparatus which concerns on one Embodiment. It is sectional drawing which shows the cell array sorter used for the automatic cell sorter concerning one embodiment. It is a block diagram which shows the structure of the control part of the cell automatic sorting apparatus which concerns on one Embodiment. It is sectional drawing which shows the modification of the cell array sorter used for the automatic cell sorter concerning one embodiment. It is a top view which shows the modification of the cell array sorter used for the cell automatic sorting apparatus which concerns on one Embodiment.

  As shown in FIG. 1, the automatic cell sorting apparatus of the present embodiment includes a culture unit 101 for culturing cells adhered to the cell array sorter 105, a sorting unit 102 for sorting cells adhered to the cell array sorter 105, It has the control part 103 which drives the culture | cultivation part 101 and the selection part 102. FIG.

As shown in FIG. 2, the culture unit 101 contains a cell array sorter 105 and holds it at a predetermined temperature, and a supply unit that supplies a cell suspension, a labeling reagent, and the like to the cell array sorter 105 housed in the chamber 111. 112, a recovery unit 113 that recovers target cells, and a discard unit 114 that discards unnecessary cells and reagents after use. The upper surface of the chamber 111 is transparent, and the cell array sorter 105 accommodated in the chamber 111 can be observed from above. The chamber 111 has a carbon dioxide (CO ₂ ) introduction function and a CO ₂ concentration adjustment function for keeping the pH of the medium constant. The supply unit 112 may be divided into a block that supplies target cells and the like, a block that supplies labeling reagents, and the like. The unnecessary cells may not be discarded, but may be collected by a collecting unit different from the target cells. Moreover, you may collect | recover the surplus cells which were not adhere | attached on the cell array sorter.

  As shown in FIG. 3, the cell array sorter 105 has a plurality of adhesion spots 502 made of gold or the like formed on a substrate 501 made of glass. A temperature-responsive polymer 503 that is cell-adhesive at the first temperature and non-cell-adhesive at the second temperature is fixed to the surface of the adhesion spot 502. The characteristics of the temperature-responsive polymer 503 may be appropriately selected according to the type of cells to be sorted, but in the case of normal cells, for example, the first temperature is about 37 ° C., and the second temperature is 25. What is necessary is just to set it as about ℃. A cell non-adhesive polymer 504 having a small interaction with cells is fixed in a region excluding the adhesion spot 502 on the substrate 501.

  The selection unit 102 includes an optical system 121 that forms an optical image of a cell adhered to the adhesion spot 502, a camera 122 that is a data conversion unit that converts the optical image into optical data such as image data, and the optical system 121. The laser light source 123 for generating a laser beam focused on the adhesive spot 502 by the above and the position selection unit 124 for moving the arbitrary adhesive spot 502 within the field of view of the optical system 121. In the present embodiment, the optical system 121 is a fluorescence microscope, and includes an objective lens 211, a dichroic mirror 212, and a fluorescence filter 213. The laser light source 123 serves as both the excitation light source of the fluorescence microscope and the light source for killing unnecessary cells. The output of the laser light source 123 may be guided to the optical system 121 by an optical fiber or the like. The position selection unit 124 is an XY stage on which the culture unit 101 is mounted. The resolution of the XY stage may be about several tens nm to several hundreds nm although it depends on the size and pitch of the adhesion spot. The moving speed of the XY stage may be several ms to several s / pitch, and may be faster than this.

  The control unit 103 drives the culture unit 101 and the selection unit 102, controls the temperature of the chamber 111, controls the supply of the cell suspension and the labeling reagent to the chamber 111, and determines whether the cell adhered to the adhesion spot 502 is a target cell. It is judged whether it is a waste cell, control of laser light irradiation to the waste cell, control of recovery of the target cell, and the like.

  Below, operation | movement of the cell automatic sorting apparatus of this embodiment is demonstrated. First, the cell suspension is supplied from the supply unit 112 to the cell array sorter 105 accommodated in the chamber 111. At this time, the temperature of the chamber 111 is set to the first temperature so that the adhesion spot 502 becomes cell adhesive. As a result, the cells in the cell suspension are adhered to the adhesion spot 502 having cell adhesion.

Cells adhered to the adhesion spot 502 are cultured as necessary. The culture is usually performed in a carbon dioxide (CO ₂ ) atmosphere. When culturing, a chemical solution necessary for culturing may be supplied from the supply unit 112 or a reagent for promoting differentiation may be supplied as necessary. A plurality of supply units 112 may be provided according to the type of cell suspension and reagent to be supplied. After culturing the cells, a labeling reagent that specifically recognizes the unnecessary cells is supplied from the supply unit 112 to label the unnecessary cells. Any labeling reagent may be used as long as it can specifically label unnecessary cells. For example, a primary antibody that specifically recognizes unnecessary cells and a secondary antibody that specifically recognizes the primary antibody and has a dye bound thereto may be used.

  Next, an optical image of a cell adhered to the adhesion spot 502 is taken into the control unit 103 as data, and it is determined whether or not it is a useless cell. In the present embodiment, when the dye is labeled, it is determined as an unnecessary cell, and when the dye is not labeled, it is determined as a target cell. Specifically, the cells adhered to the adhesion spot 502 are observed by the optical system 121 that is a fluorescence microscope, and it is determined whether the cells are unnecessary cells based on the presence or absence of fluorescence by the dye. The data conversion unit that converts an optical image into image data may be an image sensor such as a cooled CCD (Charge Coupled Device) camera. The data converter may be a photomultiplier tube or the like, and the presence or absence of fluorescence may be determined by counting photons. When observing the presence or absence of the label, the output of the laser light source 123 may be kept low to excite the fluorescent dye so as not to damage the cells.

  When it is determined that the cells adhered to the adhesion spots are unnecessary cells, the laser light source 123 is driven to irradiate the cells adhered to the adhesion spots 502 with laser light. Absorption of the laser light by the labeled dye raises the temperature of the unwanted cells and kills the unwanted cells. When killing the unwanted cells, the output of the laser light source 123 is increased to greatly increase the temperature of the unwanted cells. Dead dead cells are detached from the adhesion spot 502.

  In some cases, a plurality of cells are adhered to one adhesion spot. Also in this case, it is possible to make a determination by acquiring optical data for each of a plurality of cells that are adhered. Although it depends on the size of the adhesion spot, it is possible to kill only unnecessary cells among a plurality of cells adhered to one adhesion spot by adjusting the spot diameter of the laser beam and the like. It is also possible to kill a plurality of cells adhered to one adhesion spot at a time.

  The position selection unit 124 is driven to acquire optical data, determine whether or not the cell is a waste cell, and irradiate the unnecessary cell with a laser beam on all the adhesion spots 502, so that the cell array sorter 105 has a waste cell. It can be set as the state which was not adhere | attached but only the target cell was adhere | attached. In addition, it is not necessary to continuously acquire optical data, determine whether or not the cell is a waste cell, and irradiate the waste cell with a laser beam for each adhesion spot. After taking in optical data and determining whether or not the cell is a waste cell, the adhesion spot 502 determined to be a waste cell may be irradiated with a laser beam.

  Next, after dead dead cells are washed away, the temperature of the chamber 111 is set to a second temperature at which the adhesion spot 502 becomes non-cell-adhesive, and target cells adhered to the adhesion spot 502 are released. The released target cells are collected in the collection unit 113.

  FIG. 4 shows the configuration of the control unit 103. FIG. 4 shows a case where the presence or absence of fluorescence is determined by counting photons using a photomultiplier tube 127. The control unit 103 includes a plurality of interfaces 133, a counting unit 134, and a calculation unit 131 connected to the bus line 132. The culture unit 101 and the selection unit 102 are controlled via the interface 133. In addition, information is output to the display unit 135 and an operation is input from the input unit 136. For the selection unit 102, driving of the position selection unit 124 that is an XY stage, magnification switching and focus adjustment of the optical system 121 that is a fluorescence microscope, driving of the laser light source 123, driving of the camera 122, capturing of image data, and photoelectron The output of the multiplier tube 127 is taken in. The output of the photomultiplier tube 127 is input to the counting unit 134 via the interface 133, and photons are counted. Based on the image data from the camera 122 and the photon count result, the calculation unit 131 determines whether the cell is a waste cell. When it is determined that the cells are unnecessary, the laser light source 123 is driven to irradiate the unnecessary cells with high-power laser light, thereby killing the unnecessary cells. When the process is completed for one adhesive spot, the position selection unit 124 is driven so that the next adhesive spot is within the field of view of the optical system 121. This is repeated, and all the adhesion spots are selected.

  The automatic cell sorting apparatus according to the present embodiment can automatically determine whether a cell adhered to each of the adhesion spots is a target cell or an unnecessary cell. In addition, it is possible to determine whether or not the cell is a target cell by optical observation. Therefore, the determination accuracy is high, and the possibility that unnecessary cells other than the target cells are mixed can be extremely reduced. Further, since laser light can be irradiated at each adhesion spot at a pinpoint, there is almost no risk of damaging the target cells adhered to the surrounding adhesion spots when killing unnecessary cells. Since the target cells can be collected only by changing the temperature, there is almost no risk of damaging the target cells during the collection. For this reason, it is possible to improve the recovery efficiency by a factor of three or more compared to the conventional flow cell sorter. Further, it is not necessary to form a large flow path, the apparatus can be miniaturized, and the manufacturing cost can be greatly reduced.

  Since the cell array sorter can be made to have a size of several mm square to several cm square, it can be processed even with a small amount of sample of several hundred μl to several ml. On the other hand, selection of adhesion spots to be determined, determination of whether or not the cells are unnecessary, and destruction of unnecessary cells are automatically performed by the control unit. For this reason, even when thousands to tens of thousands of adhesion spots are provided on the substrate of the cell array sorter, the time required for the selection and recovery of the target cells can be reduced to about one hour.

  In this embodiment, an example of specifically labeling unnecessary cells has been shown, but target cells may be specifically labeled. In this case, the unlabeled cells may be killed by irradiating them with laser light. Different labeling may be performed on the target cells and the unnecessary cells. Further, labeling may be performed by cell staining or the like instead of labeling using an antibody. Furthermore, labeling is not always necessary, and it is possible to determine whether a target cell or an unnecessary cell is based on a difference in cell shape. The target cell depends on differences in infrared or near-infrared absorption, Raman scattering, chemiluminescence, refractive index, plasmon absorption on the cell adhesion surface, photothermal conversion efficiency on the cell adhesion surface, redox current, etc. You may determine whether it is a useless cell. It is also possible to combine a plurality of determination methods.

  In this embodiment, the position selection unit 124 for selecting the adhesion spot is an XY stage on which the culture unit 101 is mounted, and the position of the optical system 121 is fixed and the position of the cell array sorter 105 is moved. However, the position of the optical system 121 may be moved while the position of the cell array sorter 105 is fixed.

  The optical system 121 may have a magnification that allows one adhesive spot 502 to be within the field of view. However, even if the magnification is such that a plurality of adhesive spots 502 exist in the field of view, the determination can be made for each adhesive spot 502 by performing image processing in the control unit 103. Moreover, it is good also as a structure which can switch a low magnification and a high magnification. For example, it is possible to switch between a 10 × objective lens and a 50 × objective lens, move the stage in a low magnification state, and perform detailed observation in a high magnification state.

The wavelength, output, and the like of the laser light source 123 may be appropriately determined according to the purpose. The spot size of the laser beam may be about several tens of μm to several hundreds of μm that matches the size of the adhesion spot. Further, a pulse laser may be used. For example, unnecessary cells could be killed by irradiating laser light having a wavelength of 532 nm and a spot diameter of 50 μm for 1 second at an intensity of 5.82 mJ / pulse / cm ² . At this time, the death of the target cells adhered to the surrounding adhesion spots was not observed. The laser light source 123 serves as both a laser light source for killing cells and a light source for exciting fluorescent dyes. However, the laser light source for killing cells and fluorescent dyes are used. A light source for exciting the light source may be provided separately. Further, when there is no need to observe fluorescence, a light source for exciting the fluorescent dye is unnecessary. The optical system 121 may be a phase contrast microscope or a laser scanning microscope.

  The light source does not need to be a laser light source. If a wavelength and intensity necessary for cell diagnosis and cell death such as fluorescence observation can be obtained, a halogen lamp, a xenon lamp, a mercury lamp, or a light emitting diode (LED) A lamp light source or the like may be used.

  In the present embodiment, an example has been shown in which the temperature of unnecessary cells is increased by light irradiation, thereby killing the unnecessary cells and removing the unnecessary cells from the adhesion spots. However, instead of killing the unwanted cells by heat, a configuration may be adopted in which the unwanted cells are killed by disassembling the scaffold to which the unwanted cells are adhered by irradiation of light or the accompanying heat. Moreover, if unnecessary cells can be removed, it is not necessary to kill them. For example, the temperature of the adhesion spot may be set to a second temperature at which the temperature-responsive polymer is brought into a cell non-adhered state by light irradiation, and unnecessary cells may be released from the adhesion spot. In this case, a temperature-responsive polymer that switches from cell adhesion to cell non-adhesion by raising the temperature may be used. Moreover, you may fix the photoresponsive polymer which switches cell adhesiveness and cell non-adhesiveness by irradiating the light of a specific wavelength instead of a temperature-responsive polymer to an adhesion spot. In the case of using a photoresponsive polymer, it is not necessary to change the temperature, and therefore a low output light source can be used.

  In this embodiment, the example which collect | recovers a target cell after removing an unnecessary cell previously was shown. However, it is also possible to leave the target cells fixed and release the target cells for recovery. In this case, a cell array sorter in which a temperature-responsive polymer or a photo-responsive polymer that can be switched from the cell adhesion state to the cell non-adhesion state under a condition that does not damage the target cells is fixed to the adhesion spot may be used. By irradiating the target cells with light, the target cells can be collected without damaging the target cells.

  As an example of the cell array sorter 105, the adhesion spot 502 which is a convex gold film formed on the substrate 501 is shown. However, as shown in FIG. 5, the first layer 505 made of gold or the like is formed on the first layer 505. The formed opening may be a bonding spot 502a. As shown in FIG. 5, when the opening is the adhesion spot 502a, the cells can be held in the adhesion spot, so that it is difficult to cause the cells once adhered to fall off. In addition, it is possible to make it difficult for cells that protrude from the adhesion spot to be deformed due to the influence of the area other than the adhesion spot. In addition, by using a transparent material as the substrate 501, cells can be observed with a phase contrast microscope or the like. For this reason, it is possible to observe a more detailed form of the cells adhered to the adhesion spot 502a, and it is possible to further improve the determination accuracy. By making the adhesion spot a convex portion or a concave portion formed on the substrate 501, the position of the adhesion spot can be easily determined. However, if the position of the adhesion spot can be clarified, the adhesion spot to which the temperature-responsive polymer 503 is fixed and the region to which the cell non-adhesive polymer 504 is fixed may be formed flat.

  The substrate 501 of the cell array sorter 105 may be a transparent material such as a glass substrate or an opaque material such as a silicon substrate. Moreover, it is good also as organic materials, such as a plastics. As a material for forming the convex adhesive spot 502 or the concave adhesive spot 502a, a gold film or the like can be used. Also, other metal materials, inorganic materials such as diamond-like films, and organic materials such as plastics can be used. The diameter of the adhesion spot may be about 20 μm to 500 μm, and the adhesion spot may be formed in a matrix shape with a pitch of about 30 μm to 500 μm. The thickness of the convex adhesive spot 502 may be about 10 nm to 100 μm, the depth of the concave adhesive spot 502 a may be about 10 nm to 100 μm, and if it can be formed, it can be made thicker or deeper. Good.

  The temperature responsive polymer 503 may be hydrophobic at high temperature and phase separated and dissolved at low temperature, or may be hydrophobic at low temperature and phase separated and dissolved at high temperature. In the case of phase separation at high temperatures, it is generally a reversible phase that becomes cloudy when heated to a temperature above the lower critical solution temperature (LCST), and dissolves again when it cools to a temperature lower than that. The separation behavior is shown. Conversely, in the case of phase separation at a low temperature, the phase separation occurs when the temperature falls below the upper critical solution temperature (UCST), and re-dissolves when heated to a temperature above the upper critical temperature. Shows behavior. Examples of the temperature-responsive polymer showing the lower critical solution temperature include poly (N-substituted acrylamide), poly (N-substituted methacrylamide), polyethers and methylcellulose. Examples of the temperature-responsive polymer showing the upper critical solution temperature include a sulfobetaine polymer that is a bipolar polymer. Either type of temperature-responsive polymer may be used depending on the purpose.

  More specifically, poly (N-alkyl (meth) acrylamide) represented by poly-N-isopropylacrylamide (PIPAAm) (wherein the alkyl group is ethyl, n-propyl, isopropyl, 3-ethoxypropyl, isobutyl) Cyclohexylacrylamide, hydroxymethyl, hydroxyethyl, 2-hydroxypropyl, acetyl, trans- (2-ethoxy-1,3-dioxane-5-yl, pyrenyl, etc.).

  Further, poly (methacryloyl D, L-alanine alkyl ester (wherein the alkyl group is methyl, ethyl, propyl, etc.), (Nα-acetyl-Nε-methacrylamide-D, L-lysine methyl ester), poly ( Nε-acetyl-Nα-methacrylamide-D, L-lysine methyl ester, and poly (N- (meth) acryloyl-D, L-proline) (provided that the ester alkyl group is ethyl, methyl, propyl, etc.) In this case, one of the L-form and D-form of optical isomers or a mixture of D-form and L-form may be used. A repeating unit with the L-form may be formed.

  Poly (vinyl caprolactam), polyvinyl ethers, alkyl celluloses (alkyl groups are ethyl, methyl, propyl, etc.), poly (N-alkyl fumarate), poly (ethyl ethylene phosphate), poly (N, N-dimethylacrylamide) , Random copolymer having a repeating unit of a polymer comprising poly (γ-glutamate), poly (styrenesulfonate), poly (vinylisobutyramide), poly (vinylcaprolactam), and poly (sulfobetaine), block It may be a copolymer or a graft copolymer.

  Further, the various polymers described above may be gelled by a crosslinking agent such as N, N-methylenebisacrylamide, or may be an interpenetrating polymer network. Any of these polymers may be a structure contained in at least one of the core-shell type polymer structures. A structure in which these polymers are graft-polymerized on the surface of an inorganic carrier such as silica or mica or the surface of polyphosphazene may be used. A mixture of these polymers and a polymer carrier such as hydroxyethyl cellulose or hydroxypropyl cellulose or an inorganic carrier may be used.

  Furthermore, the following components may be included as a copolymer component. Acrylic acid, (meth) acrylic 3- (trialkoxysilyl) propyl, N-vinylpyrrolidone, (3- (meth) acrylamidopropyl) trimethylammonium chloride, (meth) acrylic acid methoxytriethylene glycol, (meth) acrylic acid alkyl (Methyl, ethyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, 2-hexylhexyl, etc.), 2-vinylpyridine, 4-vinylpyridine styrene, fumaric acid alkyl ester (methyl, ethyl, propyl, butyl, Isobutyl, sec-butyl, tert-butyl, etc.), N- (meth) acryloylsuccinimide, vinylformamide, vinyl acetate, 2-methoxyethyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, (meth) acrylic Acid 2-phenoxyethyl , (Meth) acrylate, 3-sulfopropyl methacrylate, 5-methacryloyloxyundecanoate, 11-methacryloyloxyundecanoate, undecenoic acid, metalloporphyrin complex, O- (meth) acryloyl-D, L-serine, O- (meth) acryloyl-D, L-threonine, ND, L-amino acid (meth) acrylamide, D, L-amino acid (meth) acrylic acid ester (however, the amino acid is glycine, alanine, etc.) 2-methacryloyloxyethyl phosphorylcholine, polyferrocene-block-polysiloxane, polyphenylene vinylene-block-polystyrene, polybutadiene-block-polyethylene oxide, polystyrene-block-polyacrylic acid, polyethylene oxide-block-poly ((me T) 2-tetrahydropyranyl acrylate), polyethylene oxide-block-polypropylene oxide-block-polyethylene oxide, poly (L-lactide-star block-poly (N-isopropylacrylamide), dextran-graft-poly (N-isopropyl) Acrylamide-co-N, N-dimethylacrylamide), polyethylglycidyl ether-block-polyethylene glycol, and the like.

  In order to facilitate fixing of the temperature-responsive polymer, polymerization is performed using an initiator in which an amino group or a carboxyl group is introduced at the terminal, or polymerization is performed by adding a copolymer component. May be.

  A temperature-responsive polymer can form a scaffold that allows cells to adhere using cell adhesion proteins such as collagen and peptides as auxiliary substances during cell culture, and the adhered cells are released from the surface due to temperature changes. It only has to be. Release of cells from temperature-responsive polymers is caused by changes in the solubility of polymer chains in the medium due to temperature changes, changes between the hydrophilicity and hydrophobicity of the polymer surface, or changes in the ionic state of the polymer surface, etc. Can do. Therefore, a polymer in which at least one of these changes occurs can be used as a temperature-responsive polymer. When cells are released from the temperature-responsive polymer, cell adhesion auxiliary substances such as proteins and peptides may be released together, and no auxiliary substances are released or auxiliary substances are released at different times. Also good. Further, the temperature-responsive polymer may be free from the adhesion spot.

  The temperature-responsive polymer can sharply change at the phase transition temperature between the cell-adhesive state that serves as a scaffold to which cells can adhere when cultured cells adhere or proliferate, and the non-cell-adherent state that the cells release. preferable. The phase transition temperature is preferably in a temperature range in which cell adhesion and proliferation can be effectively performed according to the target cultured cells. When the target cell is a general cell, it is preferable that the phase transition temperature is 20 ° C. to 40 ° C., and the cell adhesion state is obtained at 37 ° C. suitable for culture. In addition, when the adhesiveness of a cell is changed by a change in hydrophobicity, the contact angle is about 50 ° to 80 ° when the cell is in an adhesive state, and the contact angle is less than that when the cell is in a free state. What should be done.

  The cell non-adhesive polymer 504 may be any material as long as it has a small interaction with cells and less non-specific cell adhesion. In general, since cells are difficult to adhere to a hydrophilic surface, the cell non-adhesive polymer 504 is preferably a polymer having high hydrophilicity. For example, a synthetic phospholipid polymer such as poly-2-methacryloyloxyethyl phosphorylcholine, poly-2-ethoxyacrylate, polyethylene glycol, or the like may be used. The synthetic phospholipid-based polymer may be a copolymer of methacryloyloxyethyl phosphorylcholine and 2- (meth) acryloyloxyethyl isocyanate. Polyethylene glycol may be poly (meth) acrylic acid polyethylene glycol or poly (meth) acrylic acid polyethylene glycol alkyl ester or the like.

  Also, a mixed polymer comprising polyvinyl alcohol and at least one of polyethylene glycol, polyacrylamide, water soluble salt of polystyrene sulfonic acid, water soluble salt of polyacrylic acid, water soluble salt of polyglutamic acid, polyvinyl pyrrolidone and the like. A hydrogel molded product or the like may be used. Polymers of zwitter structure ions such as poly (MPC), polymers of zwitterion structures such as poly (O-methacryloyl-L-serine), and polyion complexes such as polyacrylic acid and polyethyleneimine may be used. Furthermore, it is good also as a structure by which the other copolymer segment is contained in these polymer segments at random, a block, or the graft form. Not only synthetic polymers but also natural polymers such as non-cell-adhesive proteins may be used. Furthermore, the cell non-adhesiveness may be realized by making the surface highly hydrophobic using a fluororesin or the like instead of making it hydrophilic. Further, when the substrate 501 or the first layer 505 is made of a cell non-adhesive material such as a titanium alloy or titanium oxide, the cell non-adhesive polymer 504 may not be used.

  The temperature-responsive polymer 503 and the cell non-adhesive polymer 504 may be fixed by a known method. For example, it may be fixed using a bifunctional or trifunctional or higher linker. When the temperature-responsive polymer 503 or the cell non-adhesive polymer 504 is fixed to a surface such as glass or silicon, a silane coupling agent such as ω-aminopropyltetraalkoxysilane may be used as a linker. What is necessary is just to select the organic functional group of a silane coupling agent suitably according to the kind of polymer to fix. The silane coupling agent may be fixed to the surface of glass or the like in advance and then coupled to the polymer, or the silane coupling agent may be previously coupled to the polymer and then fixed to the surface of glass or the like.

  When a polymer is fixed on the surface of a gold film or the like, dithiobissuccinimidyl undecanoate (DSU) having a thiol group may be used as a linker. In this case, the linker may be fixed on the surface of a gold film or the like before coupling the polymer, or the polymer and the linker may be coupled in advance and then fixed on the surface of the gold film or the like. Alternatively, a thiol group may be introduced at the end of the polymer, and the introduced thiol group may react with a gold film or the like.

  Instead of fixing a preformed polymer, a polymerization initiator is fixed to the surface of a glass or gold film using a linker or the like, and a monomer is polymerized using the fixed polymerization initiator to temperature-responsive polymer or cell A non-adhesive polymer may be formed.

  Furthermore, the polymer does not need to be fixed to the surface of the substrate or the gold film by a covalent bond, and may be fixed by an ionic bond or a hydrophobic bond generated between an amino group and a carboxyl group.

  Although the cell array sorter in which the temperature-responsive polymer is fixed to the adhesion spot has been described, it is sufficient that the target cell can be released from the adhesion spot without damaging the target cell. You may fix to an adhesion spot. For example, it is also possible to use a photoresponsive polymer that switches between cell adhesion and cell non-adhesion by irradiating with light of a specific wavelength. Examples of the photoresponsive polymer include a polymer obtained by terpolymerizing a copper chlorophyllin complex with a poly (2-N-hydroxypropylmethacrylamide-co-methyl methacrylate) chain. The photoresponsive polymer can be fixed to the adhesion spot in the same manner as the temperature responsive polymer. Further, it may be a polymer that responds to pH or ionic strength. Rather than fixing a polymer that switches between cell adhesion and cell non-adhesiveness by stimulation such as heat or light to the adhesion spot, cell adhesion polymer is attached via a polymer that peels off from the surface of the adhesion spot by stimulation. It may be fixed. Moreover, the site | part cut | disconnected by irritation | stimulation is introduce | transduced, You may fix the cell adhesive polymer which peels from an adhesion spot by irritation | stimulation. Even with such a configuration, the target cells can be recovered from the adhesion spot without damaging the target cells.

  The automatic cell sorting apparatus of this embodiment automatically performs all operations of cell seeding on a cell array sorter, cell culture, labeling of unnecessary cells, cell sorting and cell recovery. However, operations other than the cell sorting operation may be performed manually or by another apparatus. When only the cell sorting operation is performed, cell culturing is not necessary. Therefore, the chamber 111 only needs to be able to maintain the temperature of the cell array sorter 105 at the first temperature, and may be a heater or the like disposed below the cell array sorter 105. Can do. Further, as shown in FIG. 6, the cell array sorter 105 may have a configuration in which a heater 506 is embedded below each adhesion spot 502. In this case, the unwanted cells may be killed by heating the unwanted cells with a heater 506 embedded under the adhesive spot 502 instead of the laser beam. Further, the unnecessary cells may be removed by releasing them from the adhesion spot instead of killing them, or the target cells may be selectively released and recovered.

  The automatic cell sorting apparatus according to the present invention can sort cells automatically with high accuracy even with a small amount of sample, and in particular, automatic cell sorting for sorting and automatically sorting stem cells, universal cells, etc. Useful as a sorting device.

DESCRIPTION OF SYMBOLS 101 Culture | cultivation part 102 Selection part 103 Control part 105 Cell array sorter 111 Chamber 112 Supply part 113 Collection | recovery part 114 Discard part 121 Optical system 122 Camera 123 Laser light source 124 Position selection part 127 Photomultiplier tube 131 Calculation part 132 Bus line 133 Interface 134 Counting Part 135 display part 136 input part 211 objective lens 212 dichroic mirror 213 fluorescent filter 501 substrate 502 adhesion spot 502a adhesion spot 503 temperature-responsive polymer 504 cell non-adhesive polymer 505 first layer 506 heater

Claims (8)

A culture unit containing a cell array sorter having a plurality of adhesion spots to which cells are adhered;
A sorting section for sorting and removing cells adhered to the adhesion spot;
A control unit for controlling the culture unit and the selection unit,
In the adhesion spot, a stimulus-responsive polymer in which a cell adhesive state and a cell non-adhesive state are changed by stimulation is fixed to the surface,
The stimulus-responsive polymer is a temperature-responsive polymer that becomes cell-adhesive at a first temperature and non-cell-adhesive at a second temperature;
The sorting unit
A selection unit for selecting one adhesive spot from the plurality of adhesive spots;
An optical system for obtaining an optical image of the cells adhered to the selected adhesion spot, and condensing light from a light source on the selected adhesion spot;
A data converter that converts the optical image of the cell into the optical data,
The controller is
With the temperature of the culture part as the first temperature, cells are adhered to the adhesion spot,
Drive the sorting unit to obtain optical data of cells adhered for each of the plurality of adhesion spots,
Based on the optical data, it is determined whether the cell is a target cell or a waste cell. When it is determined that the cell is a waste cell, the waste cell is irradiated with light from the light source. And killing the unwanted cells selectively from the adherent spots,
An automatic cell sorting apparatus characterized in that after removing the unnecessary cells, the temperature of the culture section is set to the second temperature, and the cells adhered to the adhesion spots are released and collected . The automatic cell sorting apparatus according to claim 1 , wherein the light source is a laser light source. The culture unit has a supply unit that supplies a labeling reagent that specifically labels the cell according to the state of the cell to the cell adhered to the adhesion spot,
Wherein the control unit, automated cell sorting apparatus according to claim 1 or 2, characterized in that the determination by the presence or absence of said label. The labeling reagent includes a primary antibody that specifically recognizes the unnecessary cells, and a secondary antibody that is labeled with a dye that absorbs light from the light source and specifically recognizes the primary antibody. The automatic cell sorting device according to claim 3 . 5. The automatic cell sorting apparatus according to claim 4, wherein, when the unnecessary cells are killed, the output of the light source is made higher than when the determination is made based on the presence or absence of the label. A cell automatic separation method using a cell array sorter having a plurality of adhesion spots to which a stimulus-responsive polymer in which a cell adhesive state and a cell non-adhesive state change in response to a stimulus is fixed,
Adhering cells to the adhesion spot with the stimulus-responsive polymer in a cell-adhesive state;
Selecting and removing unwanted cells from the cells adhered to the adhesion spots;
Recovering the cells adhered to the adhesion spots by removing the unwanted cells and then bringing the stimulus-responsive polymer into a non-cell-adhesive state,
The step of selecting and removing the unwanted cells includes:
Obtaining optical data of adhered cells for each of the plurality of adhesion spots;
Automatically determining whether the adhered cells are target cells or the unwanted cells based on the optical data;
Irradiating the unnecessary cells with light to raise the temperature of the unnecessary cells, thereby killing the unnecessary cells and selectively removing the unnecessary cells from the adhesion spot. Cell sorting method. The step of selecting and removing the unwanted cells includes:
Labeling the waste cells with a labeling reagent that specifically labels the waste cells,
The automatic cell sorting method according to claim 6 , wherein it is determined whether or not the cell is a waste cell by confirming the presence or absence of a label based on the optical data. 8. The cell according to claim 7, wherein when the unnecessary cell is killed, the light output to the cell is made higher than when the presence or absence of a label is confirmed based on the optical data. Automatic sorting method.

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