JP2003304867A - Method for rapidly aliquoting aimed cell by flow cytometry without fluorescent labeling - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly aliquoting live aimed cells from among a plurality kind of cell mixtures without carrying out any modification treatment at all, particularly to provide such a method for rapidly aliquoting a high-purity nerve stem cell system usable in the fundamental research of the reproduction medical treatment hopeful for encephalopathy and in therapies including transplantation as an application thereof. <P>SOLUTION: The method for isolating or aliquoting the aimed cells comprises the steps of irradiating cells with laser beams by flow cytometry, measuring the forward scattering light and the sideward scattering light of the irradiation beams without using any fluorescent labeling, and discriminatorily displaying the measurements as position information on a two-dimensional screen. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for rapidly collecting living cells of interest from a mixture of plural kinds of cells without any modification treatment. In particular, it relates to basic research on regenerative medicine expected for brain diseases and, as an application thereof, a method for rapidly separating high-purity neural stem cells and neural progenitor cells that can be used for treatment such as transplantation.

[0002]

2. Description of the Related Art In recent years, regenerative medicine has attracted attention as a new method for treating brain diseases. Regenerative medicine uses the regenerative capacity of neural stem cells in the living body for treatment to generate new nerve cells that have been said to be unable to regenerate. Neural stem cells and neural progenitor cells (hereinafter referred to as neural stem cell line) have been proved to differentiate into cells such as neurons and glia under optimal conditions, and from the viewpoint of regenerative medicine as described above, Alzheimer's disease, It is expected to be useful in the treatment of brain diseases such as Parkinson's disease.

Under such circumstances, attempts have been made to purify and separate neural stem cell lines, and in particular, it has been expected to rapidly isolate highly pure neural stem cell lines. The following method can be mentioned as an example of such an attempt.

First, a method using cell culture (Isolation an
d characterization of mouse neural precurser cells
in primary culture, in vitro cell. Dev. Bio1. 27
A, 615-624 (1991), HIROSHI KITANI et al.). According to this method, it is difficult to obtain a high-purity neural stem cell line, and further it is necessary to culture the target cells in 10% fetal bovine serum (FBS). However, there is a drawback that the property changes due to. In addition, the method using specific gravity centrifugation (Fibroblast growth factor-2 activates a latent ne
urogenic program in neural stem ce11s from diverse
regions of theadult CNS. J Neurosci 19 (19): 8487-
97 (1999), Palmer TD, et a1.) Had the drawback that chemical substances could affect the separation operation, and it was difficult to secure a large amount of cells by centrifugation.

By the way, there is a report on the fractionation of blood cells using flow cytometry. Blood is composed of multiple types of cells, but since each blood cell can be clearly distinguished from its structure and properties, isolation of each blood cell can be achieved relatively easily. Recently, we have tried to purify and separate neural stem cell line from a brain tissue extraction mixture containing a lot of unknown structurally and qualitatively by using a cell sorter (cell sorter) utilizing the cell analysis ability of flow cytometry. Attempts have been made to do so. As an example, a method using flow cytometry for fluorescently labeled neurons (Direct iso
relation of human central nervous system stem cell
s., Proc Natl AcadSci USA 19; 97 (26): 14720-5 (2000).
Uchida N, et a1.) Has been reported. In the neural stem cell system, it is essential to maintain and obtain the properties of the cells themselves. However, the above method using a combination of fluorescence intensity and forward scattered light requires pre-labeling with an antibody, which adversely affects the properties of the neural stem cell line. That is, prior to the cell sorting operation, it is necessary to bind a fluorescent dye to the cell surface molecule of the target cell and label an antibody specific to the molecule, and maintain the properties of the neural stem cell system. Difficult to obtain.

[0006] Further, a method for separating a neural stem cell line from a transgenic animal by flow cytometry (Direct iso
lation of committed neuronal progenitor cells from
transgenic mice coexpressing spectrally distinct
fluorescent proteins regulated by stage-specific n
eural promoters., J Neurosci Res, 65, 220-227 (200
1) Sawamoto K, et al.) Has been reported. But,
This method is a technique applied to an animal in which a fertilized egg is labeled in advance, that is, a recombinant animal, and it is not possible to fractionate a live neural stem cell line that has not undergone recombination and is intact.

[0007]

Therefore, an object of the present invention is to provide a method for rapidly collecting live cells of interest from a mixture of a plurality of types of cells without any modification treatment. It is an object of the present invention to provide a method for rapidly collecting a high-purity neural stem cell line that can be used for basic research of regenerative medicine expected for brain diseases and its application as a treatment such as transplantation.

[0008]

Means for Solving the Problems As a result of intensive studies on the above-mentioned objects, the inventors of the present invention have used a cell sorter for flow cytometry, without using fluorescence, from a neural stem cell line itself when irradiating with laser. By setting the two scattered light of the above in appropriate conditions, differentiating it from other cells, and visualizing it as two-dimensional positional information, the neural stem cell system in early development that survived without any modification treatment can be rapidly performed. Further, they have found that they can be collected with high purity and have completed the present invention.

That is, according to the present invention, (1) a cell is irradiated with laser light by flow cytometry, forward scattered light and side scattered light of the irradiated light are measured without using a fluorescent label, and the measured value is measured. Is displayed as position information on the secondary screen, and thereby the target cells are separated and sorted, and (2) a laser is applied to the cells by flow cytometry. Irradiate light, measure forward scattered light and side scattered light of the irradiated light, identify and display the measured value as position information on the secondary screen, and thereby separate / separate neural stem cell system The present invention provides a method for sorting a neural stem cell line characterized by the above.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below.

In flow cytometry, only scattered light emitted from the cells themselves when a laser is irradiated to individual cells can be used as a detection parameter for cell sorting without using fluorescence.

FIG. 10 shows an outline of a preferred example of the flow cytometer system used in the present invention. The procedure of flow cytometry will be described with reference to FIG.
First, the sheath liquid and the sample liquid are introduced into the flow cell 7 from the sheath liquid container 2 and the sample liquid container 14, respectively, while air pressure is applied to adjust the sheath pressure adjuster 1 and the sample pressure adjuster 4. Here, the sheath liquid and the sample liquid do not mix in the flow cell 7. The sample core is surrounded by the sheath liquid flow as it flows through the flow cell 7. A laser beam 8 is applied to the sample core flow surrounded by the introduced sheath liquid flow, and the generated scattered light 9 is detected. The scattered light 9 observes the cells in the sample core.

Next, the scattered light 9 is emitted by the irradiation of the laser light 8.
Sort the cells observed in. Therefore, a positive or negative electric charge is applied to the entire water stream just before the water stream is divided into water droplets, and the water droplets containing the target cells in the set area are positively or negatively charged. The charged sample cores are sorted by the positive voltage plate 10 and the negative voltage plate 11 and divided into a right sort 12 and a left sort 13, respectively. Those that are not charged fall vertically and become waste liquid.

The detection parameters used in the present invention are forward scattered light (FLS; 1.5 ° to 19 °) and side scattered light (P
LS; 90 °). It is known that the forward scattering has a correlation with the size of the cell (cell surface area), and the side scattering has a correlation with the intracellular fine structure, which represents the characteristics of the cell. One dot on the two-dimensional coordinate represented by these two parameters represents an individual cell, and the shading represents the cell density. The distribution also reflects differences in cell size and intracellular structure.

FIG. 1 shows the results of sorting by flow cytometry at standard sensitivity, and FIG. 2 shows the results of further highly sensitive sorting of the nestin-positive cell population of Gate III obtained thereby. FIG. 1a is a two-dimensional screen in which cell clusters are differentiated, and FIG. 1b is a two-dimensional screen in which a sorting gate is created for the cell clusters differentiated in a. 2a is a two-dimensional screen with standard sensitivity for Gate III, FIG. 2b is a two-dimensional screen with high sensitivity for Gate III, and FIG. 2c is Gate III, IIIA and IIIB.
Is a two-dimensional screen in which is displayed on one screen.

As shown in FIGS. 1 and 2, I ': cell debris, II': dead cells, IIIA: neural stem cell line, II
IB: Nestin weakly positive cells, IV ': Supporting cells containing a mixture of nestin weakly positive cells and fibroblast-like cells, V': Fibroblast-like cells, VI ': Doublet (two cells are connected) clusters Existing 2
A dimension screen is obtained, and individual cell populations can be sorted by setting gates (I to VI, IIIA, and IIIB) on the cell clusters displayed by these two parameters.

The cells to be sorted by the method of the present invention are not particularly limited, such as neural stem cell lines and ES cells. The source of cells to be sorted by the method of the present invention may be a cell mixture of early embryo, inner cell mass of early embryo, fetal tissue, adult tissue and the like. When the neural stem cell line is sorted by the method of the present invention, the source of the neural stem cell line is fetal brain,
Any cell mixture may be used as long as it includes a neural stem cell line such as adult brain tissue. The mixed cell suspension may be prepared by any known method such as an enzymatic method using trypsin and a mechanical pulverization method using a pipette. In the enzymatic method using trypsin described above, hyaluronic acid and kynurenic acid may be simultaneously contained in the solution. Further, in the above-mentioned enzyme method using trypsin, type 3 collagenase may be contained in the solution.

In the case of the enzyme method using only trypsin, PBS (phosphate buffered saline), Hanks balanced salt (HBSS), or HEPES buffer solution may be used as the solvent.
The solvents may be used in combination. At this time, the concentration of trypsin is preferably 0.025 to 0.1 (v / v)%. In the case of the method by mechanical crushing with a pipette, the solvent is PB
S, HBSS, HEPES buffer solution may be used.
The solvents may be used in combination.

The above cell suspension is preferably prepared as a single (non-assembled) cell suspension, and the cells are passed through the laser one by one. For that purpose, a sample liquid consisting of a sample liquid flow in which a single cell flows continuously and a sheath liquid enclosing it
The core is formed, the flow is adjusted so that one cell in the sample core passes through the center of the laser beam, and the laser beam is focused so that multiple cells are not irradiated at one time to increase the intensity. It is preferable to increase. The size of cells treated in the present invention is 7 to 18 μm. Since the intensity of the laser light is lower at the edges than at the beam center, it is preferred that all cells pass through the laser beam center in the same path to enhance sensitivity and resolution. Therefore, the smaller the diameter of the sample core is, the more preferable.
The diameter of the sample core is preferably 25 to 50 μm,
28-38 μm is particularly preferable.

For the reasons mentioned above, the slower the flow of the sample core, the better. Specifically, the sample flow is preferably 25 to 40, and particularly preferably 28 to 38. EPICS as a flow cytometer
(Registered trademark, manufactured by COULTER), the value of the sample flow is as follows: sheath pressure (pressure against the container containing the sheath liquid) 7.5 psi = 100 (sample pressure (containing the cell suspension) Pressure on container): 7.5 ps
As i), 7.5 psi-1 psi = 0 (sample pressure: 6.5 psi) is correlated to a value obtained by dividing 1 psi of the difference into 100 equal parts. Flow rate during measurement is 100-90
0 cells / second is preferable, and 400 to 700 cells / second is particularly preferable.

The flow of the sample core is controlled by the sheath pressure, and the diameter of the sample core is controlled by the difference between the sheath pressure and the sample pressure. For example, while keeping the sample pressure constant, increasing the sheath pressure accelerates the flow of the sample core, and the diameter of the sample core becomes smaller.

Specifically, the difference between the sheath pressure and the sample pressure is 0.5 to 0.75 and the sample pressure is 6.75 psi.
It is preferably 7.0 psi. The difference between the sheath pressure and the sample pressure is 0.62 to 0.72 and the sample pressure is 6.7.
More preferably, it is between 8 psi and 6.88 psi.

The laser used as the light source of the present invention may be any known laser such as an argon laser, as long as it does not deteriorate the neural stem cell system and generates scattered light from the cells, but the argon laser is preferable. Wavelength 48
Irradiation light 10 to 2 when using an 8 nm argon laser
5 mW is preferable, and irradiation light of 15 mW is more preferable.
The argon laser may be focused using a beam shaping lens. The cross section of the argon laser with which the sample core is irradiated is preferably close to the size of the cross section of cells to be treated, and particularly preferably has an elliptical shape with a major axis of 57 μm and a minor axis of 15 μm.

When an argon laser having a wavelength of 488 nm is used, the wavelength of the forward scattered light to be detected is preferably 488 nm. The forward scattered light is so bright that it may be detected through a filter that reduces the intensity of the light. The scattered light detector may be a photomultiplier or a silicon photodiode. Each of the photomultiplier tube and the silicon photodiode tube may vary in sensitivity depending on the voltage applied to itself. The detector for the forward scattered light is preferably a silicon photodiode. The detector for side scattered light is preferably a photomultiplier tube.

When the detector of the forward scattered light is a silicon photodiode, the voltage of the forward scattered light applied to the detector through the filter is 130 V to 170 V when sorting by the standard sensitivity. It is preferable that it is detected at, and it is particularly preferable that it is detected at 145 to 155 volts. When the detector for the forward scattered light is a silicon photodiode, the forward scattered light is detected at a voltage applied to the detector through the filter at 220 V to 330 V during high-sensitivity sorting. It is preferable that the detection is performed at 245 to 255 volts.

When an argon laser having a wavelength of 488 nm is used, the side scattering wavelength to be detected is preferably 488 nm. When the side scattered light detector is a photomultiplier,
When sorting with standard sensitivity, the side scattered light is preferably detected at a voltage applied to the detector of 350 V to 410 V, and particularly preferably at 395 V to 405 V. preferable. When the detector of the side scattered light is a photomultiplier tube, the voltage applied to the detector is 400 times when the side scattered light is sorted by high sensitivity.
It is preferable to detect the voltage from volt to 520 volt, and it is particularly preferable to detect the voltage from 430 to 450 volt. The current generated by the detector may be converted into voltage pulses as a signal. The voltage pulse as a signal may be subjected to processing such as amplification (amplification ratio: gain) and analog-digital conversion.

When individual cell populations are sorted by setting gates on the cell clusters on the two-dimensional screen displayed for the above two parameters (forward scattered light and side scattered light), the gate setting is performed. Alternatively, the droplets derived from the sample core corresponding to the region may be charged to be collected.

In the method using flow cytometry of forward scattering and side scattering according to the present invention, the optimum conditions of sensitivity and resolution are set by microspheres which are beads labeled with a fluorescent substance (Flowcheck, trade name, manufactured by COULTER). Alternatively, single (non-aggregated) cells derived from E12 forebrain which have been previously fixed with 4% (w / v) paraformaldehyde and prepared by the trypsin method can be used.

As the antibody used for detecting the neural stem cell line in the present invention, as the primary antibody, any known antibody specific to the neural stem cell line such as an anti-nestin antibody or an anti-musashi 1 antibody can be used. Often, it is preferable to use an anti-nestin antibody.

The anti-musashi 1 antibody is an RNA-binding protein and is an antibody to the neural stem cell line ((1) Mo
use-Musashi-1, a neural RNA-binding protein highly
enriched in the mammalian CNS stem cell., Develop
mental Biology., 176 (2), 230-242 (1996) Sakakibara
S, et al .; (2) Musashi1: an evolutionally conserve
d marker for CNS progenitor cells including neural
stem cells., Dev. Neuroscience, 22, 138-152 (200
0) Kaneko Y, et al.). The secondary antibody may be any known antibody as long as it has an affinity for the primary antibody, and FITC (fluorescein isothiocyanat) may be used.
e) Labeled goat anti-rabbit IgG (Fab ′) 2 antibody is preferred.

[0031]

EXAMPLES The present invention will be described in detail below with reference to Examples.
The present invention is not limited to this.

Reference Example 1 1) Preparation of cell suspension: Rat fetus day 12 (E12)
Fetal brains (including forebrain and midbrain) were separated from gauze by a 10 cm Petri dish (SH90-15, product number, Corning) and a 6 cm dish (15020,
It was performed under a stereoscopic microscope using a product number, manufactured by Corning Inc.). Then, use a 15 ml conical tube (209
5, product number, manufactured by Falcon) and incubated with 0.1% trypsin / PBS for 30 minutes, and then 0.0
4% DNase (DN-25, product number, manufactured by Sigma) and 10% fetal bovine serum were added and the mixture was allowed to stand for 5 minutes. Then D
F (DMEM / F-12 = 1: 1) medium (GibcoB
After washing with RL) and pipetting with a 1 ml pipette (7503, product number, Falcon) equipped with a tip for 200 μl in DF medium containing 10% fetal bovine serum, a cell suspension was prepared.

2) Aseptic sorting by flow cytometry: The flow cytometry uses an argon laser as a light source (488 nm, 15 mW output) and a diameter of 100 μm.
The chip is attached to the flow cell. Microspheres, which are fluorescent-labeled beads, were used to set the optimum conditions for sensitivity and resolution of flow cytometry. The liquid feeding line of the flow cytometer was previously sterilized with 70% (v / v) ethanol. The sheath liquid is sterilized by an autoclave,
Filtered PBS was used.

The analysis of the cells in the cell suspension was carried out by the scattered light analysis using the intensity of scattered light (488 nm) generated when the individual cells were irradiated with a laser as a parameter. This analysis method uses forward scatter and 90 ° side scatter of laser-irradiated cells, and the measurement results consist of the distribution and density of cells as two parameters of FLS and PLS on the workstation monitor, 64 channels. It is displayed as a dotgram on a two-dimensional screen divided into two parts.

Since the target cells can be determined from the cytogram from the examination in the preliminary experiment conducted by the above-mentioned method of cell culture by HIROSHI KITANI et al., An area on the screen was designated and fractionated. As shown in FIG. 3, the designated sorting areas were E and F, E was sorted into the right tube, and F was sorted into the left tube. FIG. 3a shows the results of applying a cell suspension prepared by enzymatic treatment of fetal brain to a flow cytometer. FIG. 3b shows the cytogram after sorting the sorting range E.

The conditions were set as follows in order to obtain data reproducibility. FLS: 150 V (gain 5), PLS: 405 V (gain 10), difference between sheath pressure and sample pressure: 0.60 to 0.64 (sample pressure /
Sheath pressure = 6.88 / 7.5), sample flow: 34
~ 37, flow rate: 800-1000 cells / sec.

At the time of sorting, the cell suspension was preliminarily filtered through a nylon mesh of 50 μm (Koushinko, 50N) to remove aggregated cells. Filtered single (unassembled)
The cell suspension was divided into sample tubes (2063, product number, manufactured by Falcon), stored at 4 ° C., and sequentially subjected to sorting. Sorted cells are 10% fetal bovine serum / DF
Aseptically collect in a 4.5 ml tube containing
After centrifugation, the cells were collected in a 6 cm dish.

3) Cell culture: Aseptically collected cells were centrifuged, washed and cultured in a serum-containing medium and a serum-free medium. Culture 24-well microplate (25820MP, product number, manufactured by Corning) and chamber slide (Lab)
-The surface of Tek, trade name, manufactured by Nunc) is polyethyleneimine (PEI, P-3143, product number, manufactured by Sigma) in advance.
Two types were used: one coated with (PEI-treated dish) or untreated (untreated dish).
As the culture medium, DF (DMEM / F-12 = 1: 1) medium was used, and in the examination of serum-free medium, sodium selenite (manufactured by Wako Pure Chemical Industries, Ltd.) was added and apo-transferrin (T-114) was used.
7, product number, manufactured by Sigma), insulin (manufactured by CBP), and heparin (H-3149, product number, manufactured by Sigma) are added in combination, and a 24-well plate or a chamber slide is used at 37 ° C., 5 The cells were cultured in% CO ₂ , and survival and proliferation differentiation were examined by detection of specific antigen by immunofluorescence method. The medium was replaced every 3 days.

As a known nutritional factor, aFGF (F
ibroblast growfactor) (G
F-01-010, trade name, manufactured by Biosource), bFGF (233-FB, trade name, R & B Sy
Stems), TGF-α (3247SA, product number, GibcoBRL), EGF (3247SA,
Product number, GibcoBRL, IGF-I (13
245-014, product number, Life technology
OGIES), IGF-II (3255SA, product number, GibcoBRL), NGF (NC011, product name, Chemicon) and LIF (250-
L, trade name, manufactured by R & B Systems), and culture supernatants of neuronal tumor cells, gliomas and neuroblastomas were added to the culture system of the neural stem cell system to examine the proliferation and differentiation ability (Bottenstein). , JE, and Sato, GH P
roc. Natl. Acad. Sci. USA 76, 514-517 (1979)).
The five cell lines were donated by the Cancer Research Foundation Cell Bank (JCRB).

Analysis of Cell Population in Cell Suspension A cell suspension was prepared by an enzymatic method to obtain a neural stem cell line from a neuroepithelial cell layer. When this cell suspension was observed under a microscope, a cell population containing a relatively large number of neural stem cell lines and having multiple properties with different morphology was observed, and mainly fibroblast,
These were matrix cells, blood cells, dead cells and cell debris. Next, in order to identify which position each of these different cell populations occupies in the display on the monitor of the workstation, in particular, where in this, the neural stem cell line is distributed was investigated. Monitoring of the distribution of cell suspensions revealed almost 5 cell populations.

First, a preliminary experiment was carried out using a blood cell line. Blood cells were collected from the abdominal aorta with a syringe containing heparin when the fetus was removed, diluted with an equal volume of PBS, and then separated with a lymphocyte specific gravity separation liquid (manufactured by Japan Antibody Research Institute). Flow cytometric analysis of the separated hematopoietic cells revealed that lymphocytes were distributed almost at the lower left of the neural stem cell population, and granulocytes and macrophage-like cells were scattered at the upper left and right sides, and were separated from the neural stem cell population. It was found that

Further, the neural stem cell lines, fibroblasts, and matrix cells in which the cell suspensions were individually separated by the culture method depending on the difference in cell adhesion were analyzed by flow cytometry to identify the position of each cell. (Mari Yamanobata, Yuzo Tsukada, Neurochemistry 446-447
(1994)).

From these results, the fibroblast-like cells are located on the upper right side of the neural stem cell line population in the E region of FIG. 3, that is, in the F region, and the matrix cells are located slightly below the right region. Was away from. The dead cells were scattered on the band from the lower left to the upper right and could be completely separated from the neural stem cell line population. Cell debris was found in the lower left.

Results of Sorting The cell suspension prepared by enzymatic treatment of fetal brain (1.7 × 10 ⁷ cells / ml) was immediately applied to a flow cytometer, and the results are shown in FIG. 3a. The position of the neural stem cell system was judged from the result of the preliminary experiment by the culture method, and the sorting range was set to E shown in FIG. In addition, F was also tried to sort. The cells set in E and F were sorted into the right and left tubes, respectively. In the analysis of the cell population on the display, the occupancy of E to the whole cells (range A in FIG. 3) was 37%, and F
Was 23%. In order to examine the recovery rate of the sorted cells, they were analyzed again by cytometry. The result is shown in Figure 3.
b and Table 1. FIG. 3b shows the cytogram after sorting the sorting range E, which was separable from the sorting range F. The recovery rate of cells after fractionation of E is 77.5.
%Met.

[0045]

[Table 1]

Results of culturing the sorted cells: The results of culturing the sorted cell populations in FIG. 3 show that in E, many blue circular cells are present, in F, yellow and deformed cells and in E, the blue round cells are observed. Many large cells were found among the cells.
This cell population was cultured in a PEI-treated dish and an untreated dish, respectively. In the untreated dish, the E cells proliferate while converging, but in the PEI-treated dish, the E cells remarkably spread, and when the culture is continued, the colonies form and grow while concentrating between the expanded cells. I found out that The proliferating cells maintained their proliferative ability even in the secondary culture. In F, cells proliferating while adhering like fibroblasts were observed. The fibroblast-adherent cells can be separated by weak pipetting, but when they are transferred to a new untreated dish and subcultured, they grow while converging.

Therefore, the E cell population was used as a cell population containing a neural stem cell line in the following examination of culture / proliferation ability.

Examination of Cell Culture Table 2 shows the results of examination of serum-free culture of a cell population containing the above neural stem cell line. Among them, survival of the neural stem cell line was observed in DF medium containing transferrin, insulin and heparin.
By adding bFGF to this, survival was changed to proliferation. It was also found that the addition of heparin has a synergistic effect and plays an important role in survival of the neural stem cell line.

[0049]

[Table 2]

Cell growth ability of neurotrophic factor FIG. 4 shows aFGF (20 ng / ml) and bFGF (1) in a DF medium having a culture density of 24-well plate 1 × 10 ⁴ cells / cm ^2.
0 ng / ml), TGF-α (50 ng / ml), EG
F (50 ng / ml), IGF-I (100 ng / m
1) and the results of examining addition of IGF-II (100 ng / ml) are shown. aFGF and bFGF showed strong proliferative capacity of neural stem cell lines. In particular, bFGF showed a strong proliferation ability. In the evaluation, absorption at wavelengths of 570 and 600 nm after 6 days of culture was monitored by Alamar Blue Assay (manufactured by Alamar Biosciences).

Effect of culture supernatant of glioma and neuroblastoma As shown in Table 3, the culture supernatant of glioma had almost no effect, but the degree of difference was recognized in the three types of neuroblastoma. Both promoted proliferation. These were cultured in serum-free medium.

[0052]

[Table 3]

Example 1 Based on the findings such as the sorting result and the result of examination of culture conditions obtained in Reference Example 1, first, fractionation with standard sensitivity was tried. 1-1) Preparation of cell suspension Preparation with 0.1% (v / v) trypsin (manufactured by GibcoBRL): As a fetal brain, an SD rat brain containing a forebrain at embryonic day 12 (E12) was used. The extracted fetal brain is 0.1% trypsin / PBS at 37 ° C.
And incubated for 30 minutes. After that, 0.04% (v
/ v) DNase (manufactured by Sigma) and 0.07% (v / v) ovomucoid (manufactured by Worthington) were added, and the mixture was allowed to stand for 5 minutes and washed with DF medium.

The supernatant was removed by centrifugation, and the precipitated brain tissue was 100 μg / ml transferrin (manufactured by Sigma).
5 μg / ml insulin (Collaborative Biochemical Products), 25 μg / ml heparin (Sigma), 30 nM sodium selenate (Wako Pure Chemical Industries), 50 units / ml penicillin and 50
DF medium containing μg / ml streptomycin (TIH
-DF), pipetting was repeated 20 times with a 1 ml pipette equipped with 200 μl of a yellow tip to obtain a cell suspension.

Preparation by mechanical crushing: Part of the excised fetal brain was trypsin-free but 1 ml with 200 μl yellow tip in 1 ml PBS containing 0.04% (v / v) DNase / DF. The pipette was repeatedly pipetted up and down 20 times and allowed to stand, and only a few minutes later, only the submerged tissue fragment was pipetted again. This operation was repeated three times in total, and each cell suspension was collected, washed with DF medium and stored in TIH-DF. Each cell suspension prepared by these two methods was sequentially applied to a flow cytometer. Moreover, all the operations were performed in a serum-free medium.

1-2) Cell culture The culture was 4 × by adding 5 ng / ml bFGF (manufactured by R & D) to TIH-DF medium.
The cell density was 10 ³ / cm ² and a 3.5 cm dish (Falcon) was used at 37 ° C. under 5% CO ₂ . Half of the cultivated medium was replaced with fresh medium or separately prepared medium at intervals of 3 to 4 days. Nerve cell clusters (sp
The quantity of "heres" was 3.5 cm dish under an inverted microscope.

For culturing the secondary nerve cell mass, the floating nerve cell mass formed in the primary culture was transferred to a tube with a transfer pipette, and the suspension of single (non-aggregated) cells was carried out by the mechanical crushing described above. The cells were recultured as a suspension at a culture density of 4 × 10 ³ cells / cm ² . The degree of differentiation of cultured cells is
200μ of the nerve cell mass formed by the determined culture day
The sample was picked up with a yellow chip, transferred to an 8-well chamber slide (manufactured by Nunc), and cultured for 1 week in a differentiation medium, and then judged from the degree of expression of the differentiation antigen.
As the differentiation medium, TIH-DF medium containing 1 ng / ml bFGF, 1% fetal bovine serum and 1 μM retinoic acid was used.

1-3) Preparation of nestin peptide antibody In order to select a region suitable as an antigen from the primary structure of nestin molecule, Hyphil value, SurfPr value and Al-Ind value were used.
Regions that meet these setting criteria were selected (using the computer of Genome Analysis Center, Institute of Medical Science, University of Tokyo). A homology search was performed on this selected region using BLAST. Finally, 515-533 (ESGLDTE on the N-terminal side of the amino acid sequence of the nestin molecule)
ETQDSQGPLQKE (SEQ ID NO: 1)) and C-terminal side 1723-1737 (DIGGQSPNLDSEQ).
Two sites of VN (SEQ ID NO: 2)) were selected. In synthesizing the two types of peptides, one cysteine residue was added to the C-terminal side of the former (N20) and one cysteine residue was added to the N-terminal side of the latter (C16). Hemocyanin (KLH) was used as a carrier protein, and each synthetic peptide was combined with it to obtain an antigen. Rabbits were immunized subcutaneously at multiple sites.

The initial immunization was performed with Freund's complete adjuvant (FCA), and after 3 weeks, booster immunization with incomplete adjuvant (FIA) was performed every other week, and blood was appropriately collected to check the antibody titer. Antiserum uses two peptides as carriers (FMP activated cellulofine, manufactured by Seikagaku Corporation)
And purified by affinity chromatography. Two purified antibodies (Ig
G) was positive for the cells in the primary culture, but C16
I decided to use the antibody. -30 until antibody is used
Stored at ° C. The specificity of C16 as an anti-nestin antibody is the anti-nestin antibody donated by Dr. Masato Nakafuku, University of Tokyo (Cattaneo, E. and McKay, R., Nature 347, 76).
2-765 (1990)) and the cell specificity of the primary culture system.

1-4) Flow Cytometry with Standard Sensitivity As in Reference Example 1, analysis was performed from the cytogram of a two-dimensional screen divided into 64 channels by FLS and PLS. 15mW at 488nm for flow cytometer
An EPICS (registered trademark, COUL) equipped with a flow cell equipped with an output argon laser and a chip with a diameter of 100 μm.
TER) was used. The liquid supply line was sterilized with 70% ethanol in advance, and the sheath liquid was sterilized and filtered PBS. The conditions were set as follows in order to obtain the reproducibility of the data. FLS: 150 V (gain 5), PL
S: 405 volts (gain 10), difference between sheath pressure and sample pressure: 0.60 to 0.64 (sample pressure / sheath pressure =
6.88 / 7.5), sample flow: 34-37, flow rate: 800-1000 cells / sec.

1-5) Identification of Nestin-Positive Cells in Cell Suspension by Flow Cytometry The cell suspension prepared by trypsin had a nylon filter (pore size:
After filtering with 50 μm), a part of the suspension was applied to a flow cytometer and analyzed by FLS and PLS. As shown in FIG. 1a, cells could be differentiated into 6 cell clusters (I ′ to VI ′) based on the nature of scattered light. In order to identify the cells contained in each of these cell clusters, a sorting gate for analysis was created so as to surround the clusters as shown in (I to VI) of FIG. 1b.

The cells sorted for each sorting gate were collected in a tube (2063, product number, manufactured by Falcon). Pre-fill the tube with 0.5 ml of TIH-
DF medium was added, and the sorted cells were replaced with a new tube when the volume became 4 ml. The tube was centrifuged to collect cells, and the cells were fixed and then stained with an anti-nestin antibody. The respective results are shown in FIG. Figure 5
Is a histogram in which each gate is stained with an anti-nestin antibody and the fluorescence intensity thereof is used as a parameter. As shown in FIG. 5, nestin-positive cells were again analyzed by flow cytometry, and this time, analyzed and displayed by a histogram using the fluorescence intensity as a parameter. This result shows that nestin-positive cells are gates III and IV, mainly gate I.
It converged to II. Gate III corresponds to a cell population containing the neural stem cell line E of Reference Example 1 as can be seen from the cytogram.

1-6) Separation of Nestin-Positive Cells from Other Cells The cells sorted from each gate were collected by centrifugation and immediately cultured in DF-TIH medium containing bFGF. The results of observing the cultured cells with a phase contrast microscope are shown in FIG. 6 (scale bar: 25 μm). 6a is for cells from gate III, FIG. 6b is for cells from gate VI, FIG. 6c is for cells from gate V, and FIG. 6d is for cells from gate IV. The observation results are shown.

As shown in FIG. 6a, the nestin-positive cells obtained from gate III form a nerve cell mass, whereas in gate IV shown in FIG. 6d, they adhere to the culture dish surface together with some nerve cell mass. The supporting cells, and the gate V shown in FIG. 6c, were found to have fibroblast-like cells having a different morphology from IV and widely adhering to the surface of the culture dish. In addition, cell debris is on Gate I, dead cells are on Gate II,
And feeder cells such as doublets and histiocytes in Fig. 6b were found in gate VI.

1-7) 2 of cells sorted from Gate III
The morphology of the cells collected from the subculture gate III is shown in FIG. As described above, clusters were recognized, and the collected gate III was collected in serum-free medium containing bFGF (T
After culturing in IH-DF) for 6 days, formation of neuronal cell mass shown in FIG. 7a was observed. In addition, a single (non-aggregated) cell suspension was prepared from this nerve cell mass by an enzymatic method to expect the formation of a secondary nerve cell mass. However, as shown in FIG. Many flat type cells adhering to the surface were observed. The scale bar in FIG. 7 indicates 25 μm. Flat type cells that adhere to the dish surface are anti-nestin antibody / FITC
It was weakly positive when stained with.

1-8) Immunostaining Cells were fixed with 4% (w / v) paraformaldehyde-0.1M PBS (pH 7.3) at room temperature for 20 minutes, and then immunostaining was carried out using each antibody described below. Single or double staining was performed, positive cells were identified by a fluorescence microscope, and the positive rate was calculated.

The antibodies used were anti-nestin antibody (secondary antibody: FITC-labeled goat anti-rabbit IgG (Fab ') 2 antibody (manufactured by Cappel)), anti-β-tubulin isotype III antibody (manufactured by Sigma-Aldrich), Anti-MA
P-2 antibody (Chemicon) and anti-GFAP antibody (Chemicon) (each secondary antibody: TRITC-labeled goat anti-mouse IgG antibody), anti-O4 antibody (Chemicon) (secondary antibody: FITC-labeled goat) Anti-rabbit IgM antibody).
As controls for these, the primary antibodies were an anti-rabbit IgG antibody (manufactured by Sigma), an anti-mouse IgG antibody (manufactured by Sigma), and an anti-mouse IgM antibody (manufactured by Sigma), respectively.
Was used. The anti-O4 antibody is an antibody against oligodendrocytes.

2-1) Sorting with high sensitivity 1-
From the results of 7), FLS and P used in flow cytometry with standard sensitivity in order to separate the neural stem cell line from the flat type cells adhering to the dish surface.
The sensitivity of LS was changed, and as shown in Table 4, it was set to be stronger by 45% (FLS) and 10% (PLS) on average, respectively. The conditions were set as follows in order to obtain the reproducibility of the data. FLS: 250 volts (gain 5), PL
S: 440 volts (gain 10), difference between sheath pressure and sample pressure: 0.62 to 0.72 (sample pressure / sheath pressure =
6.78 / 6.88), sample flow: 28-38,
Flow rate: 400-700 cells / sec.

[0069]

[Table 4] 2-2) Results of sorting with high sensitivity When a cell suspension prepared in the same manner was analyzed using this method with high sensitivity, two cluster III cell populations were obtained as shown in the cytogram of FIG. 2b. separated. That is, nestin-positive cells were fractionated into cells of two different morphologies. These clusters are newly referred to as clusters IIIA and IIIB.

Next, the cells of clusters IIIA and IIIB were sorted, and bFGF-containing serum-free medium (TIH-
The results of culturing in DF) for 6 days are shown in FIG. 8 (scale bar:
25 μm). Cluster II as shown in Figure 8
Regarding the cell morphology in the regions of IA and IIIB, the former formed a nerve cell mass, and the latter showed flat type cells adhering to the dish surface. The region of fibroblast-like cells has shifted further to the right of IIIB, II
It was different from the IB area.

Gates III, IIIA and III were used as position markers with 30 channels for FLS and PLS, respectively.
As you can see from Figure 2c, which shows B on one screen, this I
It is considered that the cell group observed in the IIA region is a higher-shifted neural stem cell line in the III region. Then, the cells mixed in cluster III are newly added to cluster III.
Since it was recognized as B, it was possible to fractionate a highly pure living neural stem cell line from cluster IIIA. Therefore, it was possible to fractionate a highly pure living neural stem cell line by the method of the present invention.

Separately, a cell suspension prepared by treating the forebrain with mechanical crushing was analyzed by flow cytometry in the same manner. As with the enzymatic method, cluster III showed two regions, cluster IIIA and cluster IIIB. Was recognized by.

2-3) Cell recovery rate and purity by sorting The sorting was tried 5 times independently from the gate IIIA. As shown in Table 5, when the cells of the enzyme-treated cell suspension were directly stained with an anti-nestin antibody, the number of positive cells was 52.5% with respect to the whole cells. When the cell suspension was applied to flow cytometry and the cells of Gate IIIA were sorted and stained with an anti-nestin antibody, it was 38.6% based on the total cells. The number of anti-nestin antibody-positive cells obtained by this method is 3 to 5 per fetus.
There were 10 ⁴ cells. Further, as shown in Table 6, the cell suspension of fetal brain treated by mechanical pulverization was calculated in the same manner.

[0074]

[Table 5]

[0075]

[Table 6]

2-4) Examination of Viability of Highly Purified Neural Stem Cell Line The neural stem cell line sorted from Gate IIIA was 5 × 10 ³ cells / cm ² with DF-TIH containing 5 ng / ml of bFGF. It was cultured in. The cultivated cells proliferated up to the 8th day of culturing, and an increase in their size was observed while forming a nerve cell mass, but they all died on the 9th day of culturing. Therefore, the conditions for survival after the 10th day of culture were examined as shown in FIG.

In FIG. 9, A + / B-indicates that the cells of gate IIIA are contained but the cells of gate IIIB are not contained.
A + / B + is shown to include cells of gate IIIA and gate IIIB. The number of cell clusters was calculated with an inverted microscope. To investigate the effect of concentration of addition of bFGF, 20
Although ng / ml bFGF was used, no survival was observed after the 10th day. Next, when cells of the flat type having dish surface adhesiveness were sorted from the gate IIIB and the sorted neural stem cell lines were added thereto from the gate IIIA and cultured, survival on the 10th day or later was confirmed. Survival was observed even when the neural stem cell line was added on each culture day from the 1st to 8th day of culture. However, as shown in FIG.
No survival of the neural stem cell line was observed when the medium was DF-TIH containing no FGF.

2-5) Long-Term Proliferation of Neural Stem Cell Line From the above-mentioned examination of viability, the highly purified neural stem cell line obtained by the method of the present invention was killed by bFGF alone on the 10th day of culture, and long-term culture was performed. It was found necessary to add IIIB adherent cells or culture supernatant of adherent cells as the condition.

The nerve cell mass that proliferated after 10 days of co-culture was pipetted with 5 ng / ml of bFG.
When transferred to TIH-DF medium containing F, survival and growth were observed. Nerve cell masses on the 20th day of culture were made into single cells by an enzyme treatment method, and cultured in TIH-DF medium containing bFGF to form only secondary nerve cell masses.

2-6) Analysis of degree of differentiation of neural stem cell system From the expression of the differentiation antigen, the degree of differentiation of the neural stem cell system obtained by the present invention was analyzed. After culturing in DF-TIH containing bFGF for 6 days according to the culture conditions of 1-2), 4
When the cells were fixed with% (w / v) paraformaldehyde and stained, the constituent cells were nestin-positive, but the obtained neuronal cell mass also showed anti-β tubulin III antibody specific for neural progenitor cells. Positive cells were found. After switching to a differentiation medium on day 6 of culture for these nerve cell masses and culturing for 7 days, they were also positive for MAP-2, which is a nerve cell marker. At that time, neither GFAP-positive cells of astrocytes nor O4-positive cells of oligodendrocytes were observed, but GFAP-positive cells and further O4-positive cells were found in the neuronal cell mass cultured after switching for 15 days. Admitted.

From the analysis of the degree of differentiation of the neural stem cell line,
It was found that the neural stem cell line obtained by the method of the present invention is a living neural stem cell line in early development.

Therefore, according to the method using flow cytometry of the present invention, by displaying the position information on the secondary screen as a positional information, a living neural stem cell line in early development can be obtained without adding any foreign matter to the cells. It has become possible to collect with high purity.

[0083]

INDUSTRIAL APPLICABILITY As described above, the present invention makes it possible to rapidly fractionate a living target cell from a mixture of a plurality of types of cells without any modification treatment. The present invention enables rapid fractionation of a living neural stem cell line in early development, which can be used for research such as transplantation and therapy.

[0084]

[Sequence list]                                SEQUENCE LISTING <110> Banri, YAMANOHA <120> A method of rapidly sorting nerve stem cell and neuronal precursor  cell using flow cytometry <130> PT2002013 <140> JP <141> 2002-4-17 <160> 2 <170> <210> 1 <211> 19 <212> PRT <213> Unknown <400> 1 Glu Ser Gly Leu Asp Thr Glu Glu Thr Gln Asp Ser Gln Gly Pro Leu   1 5 10 15 Gln Lys Glu       <210> 2 <211> 15 <212> PRT <213> Unknown <400> 2 Asp Ile Gly Gly Gln Ser Pro Asn Leu Asp Ser Glu Gln Val Asn   1 5 10 15

[Brief description of drawings]

FIG. 1 is a diagram showing a cytogram at standard sensitivity. a: Two-dimensional screen that differentiates cell clusters. b: A two-dimensional screen in which a sorting gate is created for the cell clusters differentiated by a.

FIG. 2 is a diagram showing cytograms at standard sensitivity and high sensitivity for gate III in FIG. 1.
a: Two-dimensional screen with standard sensitivity for Gate III.
b: Two-dimensional screen with high sensitivity for Gate III. c:
A two-dimensional screen that displays Gates III, IIIA, and IIIB on one screen.

FIG. 3a is a diagram showing the results of applying a cell suspension prepared by enzymatic treatment of fetal brain to a flow cytometer. FIG. 3b is a diagram showing a cytogram after sorting the sorting range E.

FIG. 4 is a graph showing the effects of neurotrophic factors.

FIG. 5 is a diagram showing a histogram in which each gate is stained with an anti-nestin antibody and the fluorescence intensity thereof is used as a parameter.

FIG. 6 is a diagram showing the culture of cells sorted from Gates III to VI and the difference in their cell morphology according to standard sensitivity.

FIG. 7 is a diagram showing the morphology of cells sorted from Gate III. a: Shown after culturing for 6 days in a serum-free medium (TIH-DF) containing bFGF after fractionation. b: Secondary culture after making a single cell suspension from a.

FIG. 8 is a diagram showing a difference in morphology of cells sorted from Gates IIIA and IIIB.

FIG. 9 is a graph showing the effect on the survival of neural stem cell lines in adherent cells sorted from Gate IIIB.

FIG. 10 is a schematic diagram of a system of a flow cytometer that can be used in the present invention.

[Explanation of symbols]

1 Sheath pressure regulator 2 Sheath liquid container 3 sheath line 4 Sample pressure controller 5 pinch valve 6 transducer 7 flow cell 8 laser light 9 scattered light 10 Positive voltage plate 11 Negative voltage plate 12 right sort 13 Left sort 14 Sample solution container

Claims (2)

[Claims] 1. A cell is irradiated with laser light by flow cytometry, and forward scattered light and side scattered light of the irradiated light are measured without using a fluorescent label, and the measured value is 2
Identification information is displayed on the next screen as position information,
A method for sorting cells, which comprises separating and sorting target cells. 2. The cells are irradiated with laser light by flow cytometry, the forward scattered light and the side scattered light of the irradiated light are measured without using a fluorescent label, and the measured value is 2
Identification information is displayed on the next screen as position information,
A method for sorting neural stem cells, which comprises separating and sorting neural stem cells and neural progenitor cells.