JP2019100940A - Method of detecting and recovering undifferentiated cells with high sensitivity - Google Patents

Abstract

To provide a method of highly sensitively detecting and recovering single-state undifferentiated cells contained in a sample together with shape information.SOLUTION: A detection method comprises introducing a sample containing undifferentiated cells and a label protein that recognizes protein of the undifferentiated cells into 10or more retainers capable of retaining the cells to form a complex of the label protein and the undifferentiated cells contained in the sample, and then detecting the undifferentiated cells on the basis the labels on the complex.SELECTED DRAWING: Figure 5

Description

  The present invention relates to a method for sensitively detecting and recovering undifferentiated cells contained in a sample.

  In the regenerative medicine field, when trying to obtain tissue by differentiating from stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), undifferentiated stem cells may be cancerous. It is necessary to obtain differentiated cells which underlie the tissue without including the undifferentiated stem cells as much as possible.

As a method of distinguishing undifferentiated cells from differentiated cells, a method using a fluorescence microscope (see, for example, Patent Document 1) and a method using a flow cytometer (see, for example, Patent Document 2) are conventionally known. . However, the method using a fluorescence microscope has poor detection sensitivity to aggregated cells, and the method using a flow cytometer can detect single cells, but the detection sensitivity is 1/10 ³ and the sensitivity is low. (See, for example, Non-Patent Document 1).

  In addition, when cells are dropped into the fine pores of the substrate and aligned by using dielectrophoresis, they can be detected as single cells (see, for example, Non-Patent Document 2). However, the number of micropores in the substrate is about 300,000, the number of detected cells is limited, and it is difficult to detect with high sensitivity.

International Publication 2010/101225 International Publication No. 2006/126574

Kuroda et al. , PLoSONE. 2012 Morimoto et al. , PLoSONE. 2015

  An object of the present invention is to provide a method for sensitively detecting and recovering undifferentiated cells contained in a sample in a single state and in a form including shape information.

  MEANS TO SOLVE THE PROBLEM In order to solve the said subject, the present inventors arrived at this invention, as a result of repeating earnest examination.

Specifically, a first aspect of the present invention introduces recognizing labeled protein for a protein having the sample and the undifferentiated cells including undifferentiated cells to a substrate having a ¹⁰⁶ or more holding portion capable of holding cells, the A complex is formed between a labeled protein and undifferentiated cells contained in a sample, and then the undifferentiated cells are detected based on the label in the complex.
Next, a second aspect of the present invention is characterized in that the fine holes in the substrate are arranged in a triangular lattice.
Furthermore, in the third aspect of the present invention, a sample containing undifferentiated cells and a labeled protein that recognizes a protein possessed by the undifferentiated cells are mixed to form the complex and then introduced to the substrate. It features.
Furthermore, the fourth aspect of the present invention is characterized in that dielectrophoretic force is used in holding on the substrate.
Furthermore, the fifth aspect of the present invention is characterized in that the labeled protein is a labeled antibody or a labeled lectin against a protein possessed by the undifferentiated cells.
Furthermore, the sixth aspect of the present invention is characterized in that undifferentiated cells detected by any of the above-mentioned methods are recovered by aspiration and discharge with a nozzle.

  Hereinafter, the present invention will be described in detail.

  In the present invention, undifferentiated cells refer to cells one stage before differentiated cells, and examples thereof include stem cells such as ES cells, iPS cells, and mesenchymal stem cells. The size of stem cells such as iPS cells is about 10 μm to 20 μm.

  In the present invention, a protein possessed by an undifferentiated cell is a protein or glycoprotein which is expressed on the surface or in the cell of an undifferentiated cell and not expressed in a differentiated cell, and is appropriately selected depending on the target undifferentiated cell / differentiated cell. It should be selected. As an example, in the case where the undifferentiated cells are stem cells such as human ES cells and human iPS cells, a preferred embodiment of the protein possessed by the undifferentiated cells includes a glycoprotein recognized by the anti-SSEA4 antibody.

  The protein that recognizes the protein possessed by the undifferentiated cell used in the method of the present invention is not limited in its aspect as long as it has the function of recognizing the protein possessed by the undifferentiated cell. And lectins that recognize proteins possessed by undifferentiated cells. Specifically, an anti-Nanog antibody, an anti-Oct4 antibody, an anti-TRA-160 antibody, an anti-TRA-180 antibody, an anti-SSEA3 antibody, an anti-SSEA4 antibody, which is an antibody against a protein possessed by undifferentiated cells, or an undifferentiated cell And BC2 LCN, which are lectins that recognize proteins possessed by

One aspect of the present invention, means for detecting the cells of interest from the cells micropores is holding unit capable of holding is held on the substrate and the holding portion arranged 10 ⁶ or more triangular lattice cells And a device provided with

  As a detection method of the present invention, a method using a labeled protein that recognizes a protein (for example, a labeled antibody against a protein possessed by undifferentiated cells or a labeled lectin) is generally used from the viewpoint of versatility. The labeling substance in the labeling protein may be appropriately selected from substances usually used in the field of measurement using an antigen-antibody reaction, and for example, a fluorescent substance such as fluorescein, an enzyme such as alkaline phosphatase, a radioactive substance Can be mentioned. There is no limitation on the binding mode of the protein and the labeling substance in the labeled protein, and the protein may be directly bound to the labeling substance by a chemical bond or the like, and the protein may be the protein to which the labeling substance is bound. In one embodiment, the antibody is bound indirectly by an antibody against the antibody (when the protein is an antibody, this corresponds to a labeled secondary antibody). The timing at which the labeled protein is added is also not particularly limited, and may be added before introducing a sample containing undifferentiated cells into a substrate, or after holding undifferentiated cells in a holding unit provided on a substrate You may add.

  The shape of the holding portion provided on the substrate used in the present invention is not particularly limited, and it may be a flat holding portion as long as it can maintain a liquid containing cells by interfacial tension or the like. Is preferable in that it can stably hold a liquid containing the

The arrangement of the micropores, which are holding portions capable of holding cells, used in the present invention is preferably in the form of a triangular lattice. By changing in a triangular lattice shape, increasing the number of micropores per unit area, it is possible to analyze the more cells, it is possible to increase the detection sensitivity to 1/10 ⁶ order.

  In the present invention, the size of the opening of the holding portion is preferably 10 μm to 30 μm, and the depth of the holding portion is preferably 10 μm to 40 μm.

  Also, there is no particular limitation on the method of introducing the liquid containing the undifferentiated cells into the substrate and holding the cells in the holding part, and it is sufficient to simply introduce the liquid containing the cells into the holding part. After introducing the liquid containing the cells, centrifugal force may be used to forcibly introduce the cells into the holder. Among them, it is preferable to introduce a liquid containing undifferentiated cells and then introduce the cells into the holding part using dielectrophoretic force, in that the undifferentiated cells can be efficiently held in the holding part.

  The device for detecting undifferentiated cells described above is further provided with a recovery means for recovering the undifferentiated cells by aspiration and discharge with a nozzle, whereby the undifferentiated cells contained in the sample can be recovered.

  The present invention can detect undifferentiated cells contained in a sample in a single state and in a form including shape information. In addition, undifferentiated cells held in the holding unit can be recovered by suction and discharge using a nozzle.

It is the figure (exploded view) which comprised the apparatus which can implement the method of this invention, and showed an example of the board | substrate used conventionally. It is a front view of the board | substrate shown in FIG. It is the figure which showed an example of the detection and collection | recovery method of an undifferentiated cell using the apparatus provided with the board | substrate shown in FIG. It is the figure which showed another example of the detection and collection | recovery method of an undifferentiated cell using the apparatus provided with the board | substrate shown in FIG. It is a figure of the board | substrate with which the fine hole used conventionally was arrange | positioned in the grid | lattice form, and the board | substrate with which the fine hole used by this invention was arrange | positioned in triangular lattice shape.

  Hereinafter, the present invention will be described in more detail using the drawings.

An example of a substrate constituting an apparatus capable of carrying out the method of the present invention is shown in FIG. 1, and its front view is shown in FIG.
The substrate 100 shown in FIG.
It comprises a flat light shielding member 11 having a through hole 11a, a flat insulator 12 having a through hole 12a, and a flat spacer 13 having an inlet 13a, an outlet 13b and a through portion 13c. Cell introduction and holding means 10,
Electrodes 21 and 22 provided in close contact with the cell introduction and holding means 10 in the vertical direction;
Conducting wire 30 connecting the electrodes 21 and 22 together;
A signal generator 40 for applying a signal to the electrodes 21 and 22;
Is equipped. The through hole 11a of the light shielding member 11 and the through hole 12a of the insulator 12 have the same size and shape, and the light shielding member 11 and the insulator 12 are provided such that the positions of the through holes coincide with each other. . The holding portion 50 is formed of the through hole 11a, the through hole 12a, and the electrode substrate 21 provided in close contact with the lower portion of the light shielding member 11, and when the liquid containing cells is introduced from the inlet 13a, Cells 200 are introduced. The electrode 22 is provided in close contact with the upper portion of the spacer 13 to prevent scattering and evaporation of the liquid containing the cells 200 introduced from the inlet 13a. In addition, in order to facilitate recovery of the cells 200 held by the holding unit 50, the electrode 22 is configured to be removable from the spacer 13.

  Next, an example of a method for detecting and recovering undifferentiated cells using an apparatus provided with the substrate shown in FIG. 1 will be described with reference to FIG. In the present invention, as shown in FIG. 5, a substrate in which fine holes are arranged in a triangular lattice is used.

(1-1) Step of labeling undifferentiated cells (not shown)
By adding a solution containing a labeled protein that recognizes a protein possessed by undifferentiated cells to a sample, a complex of the undifferentiated cells contained in the sample and the labeled protein is formed.

(1-2) Step of Introducing Cells into Holding Portion Undifferentiated cells bound to differentiated cells 220 (when contained in the sample) and labeled protein 300 from the inlet 13a provided on the substrate 100 shown in FIG. A sample containing 210 is introduced, and these cells are introduced into the holder 50 using the dielectrophoretic force 60. Specifically, the dielectrophoretic force 60 is generated by applying an alternating voltage from the signal generator 40 to the electrodes 21 and 22 to introduce the undifferentiated cells 210 and the differentiated cells 220 into the holding unit 50. The liquid containing cells to be introduced into the substrate 100 shown in FIG. 1 may be any liquid suspended so that the cells can be moved by dielectrophoretic force, for example, an aqueous solution containing saccharides such as mannitol, glucose and sucrose, A liquid in which a sample containing cells is suspended in an aqueous solution further containing an electrolyte such as calcium chloride or magnesium chloride and / or a protein such as BSA (bovine serum albumin) in the aqueous solution may be mentioned. In particular, it is preferable to use a liquid obtained by suspending a sample containing cells in an aqueous solution containing mannitol as a liquid containing cells, since damage to the cells is reduced. The concentration of mannitol to be added may be an isotonic solution concentration, specifically, 250 mM to 350 mM.

The alternating voltage applied from the signal generator 40 to the electrodes 21 and 22 is preferably an alternating voltage having a waveform in which charge and discharge of cells held in the holding unit 50 are periodically repeated, and the frequency is from 100 kHz to 3 MHz. It is particularly preferable that the electric field strength be in the range of 1 × 10 ⁵ to 5 × 10 ⁵ V / m.

(1-3) Step of Detecting Undifferentiated Cells 210 After introducing the washing solution from the inlet 13a and discharging the remaining sample, the substrate is moved in the XY axis direction by means (not shown) for moving the substrate. The detection unit 400 and the measurement unit acquire fluorescence or luminescence and positional information derived from the labeled protein bound to the undifferentiated cell 210, and detect the position of the undifferentiated cell 210.

(1-4) Step of Collecting Undifferentiated Cells 210 In order to collect the undifferentiated cells 210 detected by the detection unit 400 and the measurement unit, the electrode 22 is removed from the spacer 13 and then aspirated by the nozzle 500, Undifferentiated cells 210 are recovered from the substrate 100. When removing the electrode 22, it is necessary to remove the spacer 13 so as not to remove it. If the spacer 13 is detached from the insulator 12, the solution held in the device will flow out of the system and the undifferentiated cells 210 will be destroyed.

  The aspirating of undifferentiated cells 210 is performed by means for moving the substrate and means for moving the nozzle (both not shown). The center of the nozzle 500 is moved and the liquid is aspirated by the nozzle 500 to recover the undifferentiated cells 210. In addition, when the suction position of the undifferentiated cells 210 by the nozzle 500 is shifted from the center of the holding unit 50 holding the undifferentiated cells 210 by a predetermined distance in the horizontal direction, suction of the undifferentiated cells 210 can be easily performed. preferable. Specifically, the suction position of the undifferentiated cells 210 is 0.1 to 2 times the diameter of the holding portion 50 in the horizontal direction from the center of the holding portion 50 (however, the distance between adjacent holding portions 50 is It is preferable to shift the position by a factor of 2 or less and to increase the height of the holding portion 50 by 0.01 to 2 times the height of the holding portion 50 from the height of the holding portion 50 in the vertical direction.

  Another example of the method for detecting and recovering undifferentiated cells using the apparatus provided with the substrate shown in FIG. 1 will be described with reference to FIG.

(2-1) Step of Introducing Cells into Holding Portion A sample including undifferentiated cells 210 and differentiated cells 220 (when contained in the sample) is introduced from the inlet 13a provided on the substrate 100 shown in FIG. These cells are introduced into the holding unit 50 using the dielectrophoretic force 60. The introduction method and conditions may be performed by the same method and conditions as the above (1-2).

(2-2) Step of Introducing Labeled Protein 300 After introducing the washing solution from the inlet 13a and discharging the remaining sample, a reagent containing the labeled protein 300 is introduced from the inlet 13a to introduce the undifferentiated cells 210 and the labeled protein. Form a complex with 300. The labeled protein may be the same as the above (1-1).

(2-3) Step of Detecting Undifferentiated Cell 210 Performed by the same method as (1-3) above.

(2-4) Step of Collecting Undifferentiated Cells 210 Performed by the same method as (1-4) above.

  Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to the examples.

Example 1
The detection sensitivity was examined using two types of fluorescent microparticles as model cells by the method described below.
(1) 170 million green fluorescent particles and 1,005 red fluorescent particles were suspended and mixed in ultrapure water.
(2) A suspension containing 17 million equivalent of the fine particles was introduced to the substrate 100 having a cell holding portion. The substrate is a substrate in which the fine holes have a diameter of 15 μm, the distance between the fine hole centers is 25 μm, and are arranged in a triangular lattice.
(3) By applying an alternating voltage with a frequency of 1 KHz to the electrodes 21 and 22 for 10 minutes, the microparticles are held in the holding portion 50 of the substrate 100 using the dielectrophoretic force.
(4) The fluorescence of the fluorescent fine particles held by the holding unit 50 was observed by photographing with a CMOS camera using a fluorescence microscope (IX 83, manufactured by Olympus Corporation), and the detection sensitivity of the red fluorescent particles was calculated.
(5) The number of detected green fluorescent particles and red fluorescent particles and the detection rate [× 10 ⁻⁶ ] calculated from the number of detected particles by the following equation are shown in Table 1.

Detection rate [× 10 ^-6 ] = number of red fluorescent particles / (number of red fluorescent particles + number of green fluorescent particles)
The theoretical detection rate calculated from the number of introduced particles is 5.91 [× 10 ^-6 ], and the average actual detection rate is comparable to 6.98 [× 10 ^-6 ]. Using this device, it was possible to detect with 1/10 ⁶ order sensitivity.

(Example 2)
Since detection sensitivity of 1/10 ⁶ order was confirmed using fluorescent particles, detection sensitivity of 1/10 ⁵ and 1/10 ⁶ order was examined using human iPS cells using the following method.

(1) Human iPS cells were prepared by the following method.
(1-1) Seed a human iPS cell 201B7 strain (purchased from iPS Academia Japan) on a petri dish coated with iMatrix (manufactured by Takara Bio Inc.), and use a StemFitAK02N medium (manufactured by Takara Bio Inc.) under a 5% CO ₂ atmosphere And cultured at 37 ° C.
(1-2) The iPS cells cultured for 7 days are washed with PBS (phosphate buffered saline) and then in RPMI medium containing 10 μM CellTracker Orange (manufactured by Thermo Fisher) at a final concentration of 37 ° C. in a 5% CO ₂ atmosphere. Culture for 30 minutes.
(1-3) After 30 minutes, the medium was changed to StemFitAK02N medium and then washed with PBS. Next, the cells were detached with a mixture of TrypLE select (manufactured by Thermo Fisher) and Versene Solution (manufactured by Thermo Fisher).
(1-4) The detached cells were collected, washed with PBS, and then immersed in a 4% (w / v) paraformaldehyde solution at room temperature for 40 minutes to fix the cells. After fixation, it was washed with PBS and stored.

(2) MRC-5 cells were prepared by the following method.
(2-1) Human fetal lung-derived fibroblasts MRC-5 strain (purchased from JCRB Cell Bank, Institute of Health and Nutrition Research and Nutrition) 10% DMEM containing fetal bovine serum (FBS) (manufactured by Thermo Fisher) The cells were cultured at 37 ° C. in a 5% CO ₂ atmosphere.
(2-2) The MRC-5 cells cultured for 7 days were washed with PBS, and then the cells were detached using 0.05% Trypsin EDTA (manufactured by Thermo Fisher).
(2-3) The detached cells were collected, washed with PBS, and then immersed in a 4% (w / v) paraformaldehyde solution at room temperature for 40 minutes to fix the cells. After fixation, it was washed with PBS and stored.

(3) The solution for dielectrophoresis was produced using the method shown below.
(3-1) Polyethylene glycol (mPEG-NHS) having a molecular weight of 5,000, one end of which is a methoxy group and the other end of which is an N-hydroxysuccinimide ester group, and bovine serum albumin (BSA) (300 mg, 0 After dissolving .3 mmol) in sodium hydrogencarbonate buffer (0.1 M, 15 mL), the solution was stirred at room temperature for 3 hours to prepare polyethylene glycol conjugated BSA (PEG-BSA). In addition, when preparing, it was made for the molar ratio (mPEG-NHS / BSA) of mPEG-NHS and BSA to be 2. After preparation, using a dialysis membrane with a molecular weight cut off of 10000, the solution was replaced with pure water for 3 days. A solution containing PEG-BSA (1% (w / v) as BSA) prepared in (3-1) and 300 mM mannitol was prepared and used as a solution for dielectrophoresis.

(4) The cell sample was prepared using the method shown below.
(4-1) The stored iPS cells and MRC-5 cells were suspended in a solution for dielectrophoresis containing 2 μg / mL DAPI (made by Dojindo Laboratories) at a final concentration, reacted at room temperature for 30 minutes, and the cell nuclei were stained.
(4-2) After 30 minutes, it was washed twice with a solution for dielectrophoresis.
(4-3) Count the number of iPS cells stained with DAPI and the number of MRC-5 cells, and spike 1 or 10 iPS cells against 1 million MRC-5 cells to prepare a total volume of 850 μL. , This was a sample.

  (5) The sample is introduced into the substrate used in Example 1, and an alternating voltage of 1 MHz frequency is applied to the electrodes 21 and 22 for 10 minutes to use the dielectrophoretic force to hold the cells in the holding portion 50 of the substrate 100. I kept it.

  (6) The fluorescence of the cells held in the holder 50 is observed by photographing with a CMOS camera using a fluorescence microscope (IX 83, manufactured by Olympus), and the number of cells recognized as DAPI positive is the total number of cells. The number of DAPI-positive and CellTracker Orange-positive cells was determined as the number of iPS cells, and the detection sensitivity was calculated.

(7) Tables 2 and 3 show the total cell number and iPS cell number, and the detection rate [× 10 ⁻⁶ ] calculated by the following equation from the number of detections.

Detection rate [× 10 ⁻⁶ ] = the number of iPS cells / the total number of cells introduced The theoretical value of detection rate calculated from the number of cells [1 × 10 ⁻⁶ ], 10 [× 10 ⁻⁶ ], actual detection rate The results were comparable with the average of 1.58 [× 10 ^-6 ] and 11.1 [× 10 ^-6 ], so it was included in the sample with 1/10 ⁶ order sensitivity using this device. Were able to detect iPS cells.

100: substrate 10: cell introduction and holding means 11: light shielding member 12: insulator 11a, 12a: through hole 13: spacer 13a: introduction port 13b: discharge port 13c: penetration part 21 · 22: electrode substrate 30: conducting wire 40: signal Generator 50: Holding part 60: Dielectrophoretic force 200: Cell 210: Undifferentiated cell 220: Differentiated cell 300: Labeled protein 400: Detection part 500: Nozzle

Claims (6)

Undifferentiated cell cells by introducing recognizing labeled protein for a protein having the sample and the undifferentiated cells including undifferentiated cells to a substrate having a ¹⁰⁶ or more holding portion capable of holding, contained in the labeled protein and the sample After forming a complex with, and detecting undifferentiated cells based on the label in the complex.   The detection method according to claim 1, wherein the holding portion of the substrate is arranged in a triangular lattice shape.   The sample according to claim 1 or 2, wherein a sample containing undifferentiated cells and a labeled protein that recognizes a protein possessed by the undifferentiated cells are mixed to form the complex and then introduced into the substrate. Detection method.   The detection method according to any one of claims 1 to 3, wherein a dielectrophoretic force is used at the time of holding on the substrate.   The detection method according to any one of claims 1 to 4, wherein the labeled protein is a labeled antibody or a labeled lectin against a protein possessed by the undifferentiated cell.   A collection method characterized in that undifferentiated cells detected by the method according to any one of claims 1 to 5 are collected by suction and discharge using a nozzle.

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