JP7443762B2 - How to retain cells contained in the sample - Google Patents

Description

The present invention relates to a method for retaining cells contained in a sample.

In the field of life science research, it is required to detect and recover target cells in a single living state from samples containing multiple cell types. An example of the method is a method using a substrate provided with a holding portion capable of holding microparticles (for example, Patent Document 1).

JP2019-100940A

In the substrate described in Patent Document 1, electrodes are provided on the top plate surface arranged in parallel with the holding part for the purpose of generating dielectrophoretic force in the cell holding space. Therefore, when attempting to collect target cells held in the holding unit by suction using a capillary, etc., a step is required to remove the electrode (top plate), and the cells held in the holding unit are There is a problem in that the holder moves outside the holding section or to another holding section.
Furthermore, in the above-described method, since cells are retained in the holding portion regardless of whether they are alive or dead, there is also the problem that dead cells may be mixed in when trying to detect and collect living cells.
An object of the present invention is to suppress the contamination of dead cells and to stably maintain the state in which the cells are held in the holding portion.

In order to solve the above problems, the present inventors have made extensive studies and have arrived at the present invention.

That is, one aspect of the present invention is
A method for holding cells using a cell holding device equipped with a substrate having a holding part capable of holding cells, an inlet, and an outlet, the sample containing the cells being passed through the inlet. The method includes the steps of introducing the sample into the holding part, holding the cells in the holding part, and introducing a replacement solvent from the inlet and discharging the sample solvent from the outlet. do.

The present invention will be explained in detail below.

Examples of cells retained in the holding unit include human-derived induced pluripotent stem cells (human iPS cells) or embryonic stem cells (human ES cells), differentiated cells derived from these cells, and human-derived mesenchymal stem cells (human MSCs). CAR-T cells) and chimeric antigen receptor T cells (CAR-T cells).

The cell holding device includes a substrate provided with a holding part capable of holding cells, an inlet, and an outlet. The holding part has a shape with a recess or a through hole, and when the recess is used as the holding part, one end has an opening large enough to hold all or a part of the cells to be held, and the other end has an opening large enough to hold all or a part of the cells to be held. When the end forms the bottom and the through hole is used as the holding part, one end has an opening large enough to hold all or a part of the cells to be held, and the other end has the opening. The opening may be narrower than the opening, or the opening may be large enough to hold a portion of the cells to be held at both ends. If the size (diameter) of the holding part is set to a size that can hold only one cell, the detected cells can be collected and analyzed in single cell units (morphological analysis, tissue type analysis, genetic analysis, etc.) This is preferable because it can be easily performed.
Although there is no particular restriction on the shape or size of the inlet and the outlet, it is preferable that the inlet and the outlet are arranged so as to be opposite to each other on the substrate.

The solvent used for samples containing cells is not particularly limited as long as it is isotonic to the cells and can maintain the viability of the cells; examples include physiological saline, mannitol, glucose, and sucrose. an aqueous solution containing sugars such as, an aqueous solution further containing an electrolyte such as calcium chloride or magnesium chloride, and/or a protein such as BSA (bovine serum albumin), or a culture medium used for culturing the cells. Can be mentioned. In particular, it is preferable to use a liquid in which a sample containing cells is suspended in an aqueous solution containing sucrose as the liquid containing cells, since damage to the cells is reduced. The concentration of sucrose to be added may be a concentration that provides an isotonic solution, and specifically, it is preferably 200 mM or more and 350 mM or less. Furthermore, if the cells are cells that can be contained in blood, such as CTCs (circulating tumor cells), blood samples such as blood (whole blood), diluted blood, serum, plasma, cerebrospinal fluid, umbilical cord blood, blood component collection, etc. In addition, samples that may contain blood components such as urine, saliva, semen, feces, sputum, amniotic fluid, and ascites are also included in the sample solvent in the present invention.

There are no particular limitations on the method for holding cells contained in the introduced sample in the holding unit; it is possible to simply introduce the sample containing cells through the introduction port, or by using centrifugal force after introducing the sample containing cells. The cells may be forcefully held in the holding section. Among these, it is preferable to introduce a sample containing cells and then use dielectrophoretic force to hold the cells in the holding part, since the cells can be efficiently held in the holding part. In either method, the cells are not fixed to the holding part, but are in a state of sedimentation within the holding part. When using dielectrophoretic force, specifically, dielectrophoretic force may be generated by applying an alternating current voltage, and cells may be held in the holding portion. The applied AC voltage is preferably an AC voltage having a waveform in which charging and discharging of the cells in the holding part are periodically repeated, the frequency is between 100 kHz and 3 MHz, and the electric field strength is between 1 x 10 ^{and 5} x. It is particularly preferable to set it between 10 ⁵ V/m (see WO2011/149032 and JP2012-013549). Examples of preferable substrates include the substrate 100 shown in FIGS. 1 and 2, which will be described later, and the substrate disclosed in Japanese Patent Application Publication No. 2019-100940.

The replacement solvent, unlike the sample solvent, is a solvent that has a lower density than the cells in the sample and a higher kinematic viscosity, specifically, 3 mm ² /s (3 cSt) or more. Preferred examples include mineral oil, liquid paraffin, and isopropyl palmitate.

When the replacement solvent is introduced from the inlet of the substrate, the sample solvent in the holding part is pushed out and discharged from the outlet, so that the sample solvent in the holding part is replaced by the replacement solvent. It is preferable to introduce a sufficient amount of replacement solvent in consideration of the volume of the holding section.

The cells held in the holding part can be detected based on the characteristics of the cells. For example, detection may be based on the size of the cell or the presence or absence of granules or organelles present within the cell. It may be detected based on. A preferred example of the cell nucleus staining reagent is Hoechst 33342 (trade name), which can permeate cell membranes. The index of whether or not a cell is a detection target does not need to be determined by a clear numerical value. ) may be used.

After detecting the cells contained in the sample using the method described above, the detected cells are collected by suction and discharge using a capillary, thereby making it possible to collect specific cells contained in the sample in units of cells. The capillary is not particularly limited as long as it is capable of collecting specific cells in single cell units, and examples thereof include a nanopipette, a picopipette, a glass capillary, and a microparticle collection device disclosed in JP-A No. 2016-142616. Can be mentioned.

According to the present invention, dead cells contained in a sample can be selectively removed and the state of cell retention in the holding part can be maintained, so that detection and collection of cells held in the holding part can be facilitated.

FIG. 1 is a diagram (exploded view) showing an example of a substrate constituting an apparatus capable of implementing the method of the present invention. 2 is a front view of the substrate shown in FIG. 1. FIG. FIG. 3 is a diagram showing an example of the cell detection and recovery method of the present invention using a device equipped with the substrate shown in FIGS. 1 and 2. FIG. 3 is a diagram showing the results of Example 1. FIG. FIG. 3 is a diagram showing the results of Example 2. FIG. 7 is a diagram showing the results of Example 3.

Hereinafter, the present invention will be explained 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 a front view thereof is shown in FIG.

The substrate 100 shown in FIG.
Consists of 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 part 13c. Cell introduction and holding means 10;
electrodes 21 and 22 provided to closely sandwich the cell introduction and holding means 10 in the vertical direction;
A conductive wire 30 connecting the electrodes 21 and 22,
a signal generator 40 that applies signals to the electrodes 21 and 22;
It is equipped with 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 so that the positions of the respective through-holes match. . A holding part 50 is constituted by the through hole 11a, the through hole 12a, and the electrode substrate 21 provided in close contact with the lower part of the light shielding member 11. When a liquid containing cells is introduced from the introduction port 13a, it enters the holding part 50 through the through hole 13c. Cells 200 are introduced. The electrode 22 is provided in close contact with the upper part of the spacer 13 to prevent the liquid containing the cells 200 introduced from the introduction port 13a from scattering or evaporating. Note that, in order to facilitate recovery of the cells 200 held in the holding section 50, the electrode 22 has a structure that is removable from the spacer 13.

Next, an example of the cell detection and recovery method of the present invention using an apparatus equipped with the substrate shown in FIGS. 1 and 2 will be described using FIG. 3.

(1-1) Step of labeling cells A solution containing a labeled protein that recognizes a protein possessed by a cell to be detected is added to a sample to form a complex between the cell and the labeled protein.

(1-2) Step of introducing cells into the holding part A sample containing detection target cells 210 bound to labeled protein 300 and contaminant cells 220 is introduced from the introduction port 13a provided in the substrate 100 shown in FIG. 1, and subjected to dielectrophoresis. These cells are introduced into the holding part 50 using the 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, and the detection target cells 210 and the contaminant cells 220 are introduced into the holding part 50. The AC voltage applied from the signal generator 40 to the electrodes 21 and 22 is preferably an AC voltage having a waveform in which charging and discharging of the cells held in the holding unit 50 are periodically repeated, and the frequency is set to be 100 kHz or more and 3 MHz or less. It is particularly preferable that the electric field strength is 1×10 ⁵ or more and 5×10 ⁵ V/m or less.

(1-3) Solvent Replacement Step By introducing the replacement solvent from the inlet 13a, the remaining sample is discharged from the outlet 13b, and the space inside the holding part 50 is replaced with the replacement solvent. Through this step, dead cells among the detection target cells 210 can be selectively removed.

(1-4) Step of detecting detection target cells 210 While moving the substrate in the XY axis directions using means for moving the substrate (not shown), the label bound to the detection target cells 210 is detected by the detection unit 400 and the measurement unit. Protein-derived fluorescence or luminescence and position information are acquired, and the position of the detection target cell 210 is detected.

(1-5) Step of collecting the detection target cells 210 In order to collect the detection target cells 210 detected by the detection unit 400 and the measurement unit, after removing the electrode 22 from the spacer 13, by suctioning with the capillary 500, Detection target cells 210 are collected from the substrate 100. When removing the electrode 22, it is necessary to remove the spacer 13 without peeling it off. This is because if the spacer 13 were to peel off from the insulator 12, the solution held within the device would flow out of the system and the detection target cells 210 would be destroyed.
The detection target cells 210 are sucked into the holding part in which the detection target cells 210 detected in the step (1-3) are held using a means for moving the substrate and a means for moving the nozzle (both not shown). The detection target cells 210 are collected by moving the center of the capillary 500 and sucking the liquid through the capillary 500. Note that if the position at which the capillary 500 aspirates the detection target cells 210 is shifted by a certain distance in the horizontal direction from the center of the holding part 50 holding the detection target cells 210, the detection target cells 210 can be easily aspirated. preferable. Specifically, the suction position of the detection target cell 210 is set horizontally from the center of the holding part 50 by a distance of 0.1 to twice the diameter of the holding part 50 (however, the distance between adjacent holding parts 50 is It is preferable that the height of the holding part 50 is shifted by 0.01 times to twice the height of the holding part 50 in the vertical direction from the height of the holding part 50.

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

Example 1 Effect of dead cell removal by solvent replacement (1) Preparation of iPS cells (1-1) Human iPS cell line 201B7 strain (manufactured by iPS Academia Japan) on a polystyrene plate coated with iMatrix (manufactured by Nippi) (culture substrate) using StemFit AK02N medium (manufactured by Takara Bio Inc.).
(1-2) After washing with PBS (phosphate buffered saline), a mixed solution of TrypLE select (manufactured by Thermo Fisher) and Versene Solution (manufactured by Thermo Fisher) was added and left at 37°C for 10 minutes. By doing so, the iPS cells were detached from the culture substrate.
(1-3) The detached iPS cells were collected in a StemFit AK02N medium to which Y-27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: concentration 10 μM) was added (hereinafter referred to as StemF+Y medium) (iPS cell sample).

(2) Staining with fluorescent labeling (2-1) The “iPS cell sample” prepared in (1) was treated with cell nucleus staining reagent Hoechst 33342 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 1:500) and dead cell staining reagent. The cells were stained by soaking in StemF+Y medium containing SYTOX Green (Thermo Fisher, 1:2000) for 30 minutes at room temperature.
(2-2) After staining, 250mM Sucrose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 10mM HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid, manufactured by Dojindo Laboratories) and 1% (w/v ) An isotonic solution with low electrical conductivity (hereinafter referred to as DEPB1) containing BSA (manufactured by Merck) was added and centrifuged (160×g, 5 minutes).
(2-3) Washing was performed by removing the supernatant, adding DEPB1 again, and centrifuging twice, and the resulting pellet was suspended in DEPB1.

(3) Implementation of dielectrophoresis and acquisition of cell images before solvent replacement (3-1) The suspension obtained in (2-3) was introduced into the substrate 100 having the configuration shown in FIGS. 1 and 2. In the cell holding means 10 of the substrate 100 used in this example, holding portions 50 each having a diameter of 30 μm and a depth of 40 μm are provided in a lattice shape with a center-to-center distance of 50 μm.
(3-2) Cells were retained in the holding part 50 using dielectrophoretic force by applying an AC voltage with a frequency of 1 KHz from the signal generator 40 to the electrodes 21 and 22 for 10 minutes.
(3-3) Fluorescence (Hoechst 33342 and SYTOX Green) possessed by the cells held in the holding part 50 was photographed with a CMOS camera using a fluorescence microscope (IX83, manufactured by Olympus).

(4) Solvent Replacement By introducing DEPB1 or mineral oil (manufactured by Merck) from the inlet 13a of the substrate 100 and discharging the surplus solution inside the substrate from the outlet 13b, the solution in the space inside the substrate including the holding part 50 is removed. Replaced everything.

(5) Cell image acquisition after solvent replacement Fluorescence (Hoechst 33342 and SYTOX Green) possessed by cells held in the holding unit 50 was photographed with a CMOS camera using a fluorescence microscope (IX83, manufactured by Olympus).

(6) Calculation of cell migration rate before and after solvent replacement The cell numbers obtained in (3) and (5) were compared, and the removal rate shown below was calculated. Furthermore, Hoechst 33342-positive cells indicate live cells, and SYTOX Green-positive cells indicate dead cells.
Removal rate [%] = 100 × [1-{(5) Number of cells after solvent replacement/(3) Number of cells before solvent replacement}]
The results are shown in Figure 5. By replacing the solvent in (4) with mineral oil, dead cells could be selectively removed (dead cell removal rate: 27.61%, live cell removal rate: 3.13%). On the other hand, when the solvent was replaced with DEPB1, almost no dead cells or live cells were removed (dead cell removal rate: 0.59%, live cell removal rate: 0.24%).

Example 2 Cell retention effect in the holding part by solvent replacement (1) Preparation of MCR-5 cells MRC-5 cells (human fetal lung-derived fibroblasts: provided by JCRB cell bank) as cells larger in size than iPS cells ( 30 μm in diameter) was selected. After culturing MRC-5 cells in DMEM (Dulbecco's Modified Eagle Medium) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% (w/v) FBS (Fetal Bovine Serum), they were cultured in Example 1 (1). It was detached in the same manner as in Example 1 (1-3) and collected in the same manner as in Example 1 (1-3) (MRC-5 cell sample).

(2) Staining with a cell nucleus staining reagent (2-1) The “iPS cell sample” prepared in Example 1 (1) or the “MRC-5 cell sample” prepared in this Example (1) was stained with a cell nucleus staining reagent Hoechst. The cells were stained by immersion in StemF+Y medium containing 33342 (trade name) for 30 minutes at room temperature.
(2-2) After the staining operation, DEPB1 was added and centrifuged (160×g, 5 minutes).
(2-3) Washing was performed by removing the supernatant, adding DEPB1 again, and centrifuging twice, and the resulting pellet was suspended in DEPB1.

(3) Cell retention in the holding part (3-1) The suspension obtained in (3) was introduced into the substrate 100 shown in FIGS. 1 and 2. The substrate 100 has holding portions 50 each having a diameter of 30 μm and a depth of 40 μm arranged in a lattice shape with a center-to-center distance of 50 μm.
(3-2) Cells were retained in the holding part 50 using dielectrophoretic force by applying an AC voltage with a frequency of 1 KHz from the signal generator 40 to the electrodes 21 and 22 for 10 minutes.

(4) Solvent Replacement DEPB1 or mineral oil (manufactured by Merck) was introduced from the inlet 13a provided on the substrate 100, and the excess solution was discharged from the outlet 13b to replace the solvent in the space inside the holding part 50.

(5) Cell image acquisition The fluorescence derived from the cell nucleus staining reagent (Hoechst 33342) possessed by the cells held in the holding unit 50 was photographed with a CMOS camera using a fluorescence microscope. After the photographing, the electrode 22 was removed, and a fluorescence-stained image was photographed again using a fluorescence microscope. The positions of the cells before and after the electrode 22 was removed were compared, and the migration rate was calculated using the formula shown below.
Migration rate [%] = 100 × [1 - {number of cells whose position did not move before and after electrode removal/number of cells detected before electrode removal}]
The results are shown in FIG. When the solvent replacement in (4) was performed with DEPB1, 3.79% of iPS cells and 23.65% of MRC-5 cells migrated due to the electrode 22 removal operation. On the other hand, when the solvent replacement in (4) was performed with mineral oil, there was almost no migration, 0.1% for iPS cells and 1.67% for MRC-5 cells, even after the electrode 22 removal operation. .

Example 3 Effect of liquid paraffin replacement on cell migration rate due to top plate removal (1) Cell nucleus staining of the “iPS cell sample” prepared in Example 1 (1) by the method described in Example 2 (2) The cells were stained with a reagent and held in the holding part by the method described in Example 2 (3).
(2) The substitution solvent was an electrical conductivity solution containing 250mM Sucrose, 2.5mM HEPES, 0.5mM MgCl ₂ (manufactured by Nacalai Tesque) and 1% (v/v) Pluronic F-68 (manufactured by Thermo Fisher). The solvent was replaced in the same manner as in Example 2 (4), except that a low isotonic solution (hereinafter referred to as DEPB2) or liquid paraffin (low viscosity type: manufactured by Nacalai Tesque) was used.
(3) In the same manner as in Example 2 (5), fluorescence images before and after removal of the electrode 22 were obtained, and the migration rate was calculated.
The results are shown in FIG. When the solvent replacement in (2) was performed with DEPB2, 6.65% was transferred due to the electrode 22 removal operation, but when the solvent replacement in (2) was performed with liquid paraffin, the electrode 22 removal operation was performed. However, the migration rate was 0.15%.

100: Substrate 10: Cell introduction and holding means 11: Light shielding member 12: Insulator 11a, 12a: Through hole 13: Spacer 13a: Inlet port 13b: Outlet port 13c: Penetration portion 21 and 22: Electrode substrate 30: Conductive wire 40: Signal Generator (AC voltage)
50: Holding part 60: Dielectrophoretic force 200: Cell 210: Detection target cell 220: Contaminant cell 300: Labeled protein 400: Detection part (fluorescence microscope)
500: Capillary

Claims (3)

A method for holding cells using a cell holding device equipped with a substrate having a holding part capable of holding cells, an inlet, and an outlet, the method comprising:
introducing the sample containing the cells into the holding part from the introduction port;
holding the cells in the holding section using dielectrophoretic force ;
Introducing a displacement solvent such as mineral oil or liquid paraffin from the inlet and discharging the sample solvent from the outlet;
The method comprising: A method for analyzing cells, comprising analyzing cells held in the holding section by the method according to claim 1 . A method for collecting cells, comprising collecting the cells analyzed by the method according to claim 2 by suction and ejection using a capillary.