JP2009122016A - Determination system, selection system, determination method, cell production method, program, and recording medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a determination system, etc. suited to determine a polarized state of measured objects in contact with or adjacent to substrates, etc. by a surface plasmon resonance system. <P>SOLUTION: The determination system 17 determines a polarized state of mitochondria 9, 11 in living cells 5, 7 on a substrate 3 by using a surface plasmon resonance phenomenon and includes a polarized state determination section 21 to determine three-dimensional distribution/intensity/morphology of the polarized state of mitochondria 9, 11 based on at least two kinds of incident light 13 to the substrate 3. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a determination device, a selection device, a determination method, a cell production method, a program, and a recording medium, and more particularly to a determination device that determines the polarization state of mitochondria in a living cell using a surface plasmon resonance phenomenon.

  The inventors have studied monitoring the polarization state of intracellular mitochondria using a surface plasmon resonance (SPR) device (see Patent Document 1).

  In addition, with the aim of regenerating human biological tissue from embryonic stem cells (ES cells), we are conducting research on marker molecules for identifying factors that induce differentiation into specific biological tissues and monitoring the differentiation state.

  Cell biology and molecules for early human development by creating totipotent stem cells that can be maintained in differentiated or undifferentiated states or that can be induced to differentiate into extraembryonic or somatic cell lines in vitro It enables the generation of differentiated cells from stem cells for use in biological research, functional genomic engineering, transplantation or drug screening and drug discovery in vitro.

  In general, stem cells are undifferentiated cells that can give rise to a series of mature functional cells. For example, hematopoietic stem cells can give rise to any of the different types of terminally differentiated blood cells. Embryonic stem cells (ES cells) are embryonic and pluripotent, so have the ability to develop and form any organ, cell type or tissue type, or perhaps a complete embryo.

  In order to obtain specific cell lines differentiated from totipotent stem cells, an in vivo mechanism was used that led differentiation to specific cell lines. For example, neural lineage stem cells were isolated after modifying totipotent stem cells with a reporter construct and then reintroducing them into early embryos (Non-Patent Document 1). The reporter construct is expressed during neurogenesis and cells expressing the reporter gene are isolated and placed in culture. This method allows the isolation of cells involved in the nervous system through in vivo mechanisms, but cells that have been separated and placed once in culture proceed to terminal differentiation.

Patent Document 2 teaches a method for isolating lineage-specific stem cells in vitro as follows: Lineage-specific binding to totipotent embryonic stem cells operably to DNA encoding a reporter protein Transfecting constructs containing regulatory regions of genetic genes; culturing totipotent embryonic stem cells under conditions such that the totipotent embryonic stem cells differentiate into lineage-specific stem cells; and a reporter protein in the culture Is isolated from other cells; wherein the cell expressing the reporter protein is an isolated, lineage-specific stem cell.

  To date, induction of differentiation from totipotent embryonic stem cells to specific tissues has been reported.

World Publication No. 2007/069692 Pamphlet US Pat. No. 5,639,618 Ott, etal. (1994) J. Cell. Biochem. Supplement 18A: 187

  The surface plasmon resonance apparatus uses a surface plasmon resonance phenomenon to measure a change in resonance angle. This change in resonance angle depends on the change in dielectric constant in the vicinity of the gold film surface of the sensor unit. Each biomolecule has a specific dielectric constant, but when binding to a target ligand immobilized on a gold film occurs, a complex is formed and the dielectric constant changes.

  However, the surface plasmon resonance apparatus normally uses light of a specific wavelength. Therefore, various information was mixed in the information obtained about the mitochondria to be measured. This applies not only to intracellular mitochondria but also to the measurement of the polarization state of an object that is in contact with or close to the substrate.

  The methods described in Patent Document 2 and Non-Patent Document 1 introduce a non-biological substance into stem cells and monitor the state of the stem cells based on the response of the substance, and are intended for regeneration of living tissue and regenerative medicine. There is a problem that it cannot be used for induction of differentiation of stem cells.

  Induction of differentiation from totipotent embryonic stem cells to specific tissues reported so far, the differentiation state is characterized based on in vitro analysis of intracellular molecule expression profiles, cell surface antigen expression profiles, etc. Yes. For example, the differentiation state can be determined by analyzing the expression of Oct-3 / 4, which is a transcription factor. In order to perform these in vitro analyses, it is necessary to collect the differentiating cells and adjust the extract, and it was impossible to continuously monitor the differentiation state.

  Accordingly, an object of the present invention is to provide a determination device and the like suitable for determining the polarization state of a measurement object using a surface plasmon resonance device.

  The invention according to claim 1 is a determination device that determines a polarization state of a mitochondrion in a living cell by using a surface plasmon resonance phenomenon, and determines the polarization state of the mitochondrion based on at least two types of incident light State determination means.

  The invention according to claim 2 is the determination apparatus according to claim 1, wherein the determination by the polarization state determination means is at least one of distribution, intensity and form of the polarization state of the mitochondria.

  The invention according to claim 3 is the determination apparatus according to claim 1 or 2, wherein the at least two types of incident light are different from each other in at least one of a wavelength and an evanescent wave to be generated. The polarization state determination means determines the polarization state of the mitochondria by spectral analysis.

  The invention according to claim 4 is the determination apparatus according to any one of claims 1 to 3, comprising a property determination unit that determines the property of the living cell based on a determination result by the polarization state determination unit. It is.

  The invention according to claim 5 is a selection device including a selection unit that selects a living cell to be cultured based on the property of the living cell determined by the property determination unit according to claim 4.

  The invention according to claim 6 is a determination method for determining a mitochondrial polarization state in a living cell using a surface plasmon resonance phenomenon, wherein the mitochondrial polarization state is determined based on at least two types of incident light. , Including.

  The invention according to claim 7 is a cell production method for producing cells by culturing, and the determination result of the polarization state of mitochondria in living cells using surface plasmon resonance phenomenon and incident light of at least two kinds of light And a production step of culturing a living cell to be cultured and producing the cell.

  The invention according to claim 8 is a determination device for determining a mitochondrial polarization state in a living cell using a surface plasmon resonance phenomenon, wherein the mitochondrial polarization state is determined based on at least two types of incident light. It is a program for functioning as a determination device including a determination unit that determines at least one of distribution, intensity, and form.

  The invention according to claim 9 is a recording medium on which the program according to claim 8 is recorded.

  The invention according to claim 10 is a determination device that determines a polarization state of a determination object that is in contact with or close to the surface using a surface plasmon resonance phenomenon, and the polarization is based on at least two types of incident light. And determining means for determining at least one of state distribution, intensity and form.

  In addition, the living cell used as determination object may be on a board | substrate. The incident light may be on the substrate. The determination target may be in contact with or close to the substrate. In addition, the living cell to be determined may be one that determines the polarization state while maintaining a differentiable state.

  In addition, living cells to be determined include, for example, stem cells, embryonic stem cells (ES cells), differentiated cells derived from ES cells, mesenchymal stem cells (eg, adipose stem cells, blood stem cells), and undifferentiated tissues. It may be a cell, a cell that holds a multi-differentiation function, or the like. Moreover, normal cells, cancer cells, fertilized eggs, animal and plant clonal cells, ES cells, cancer stem cells, and the like, or cultured cells thereof may be used.

  Furthermore, the distribution, intensity, morphology, etc. of the mitochondrial polarization state may be measured by imaging mapping.

  Further, at least two types of light are different in at least one of wavelength and generated evanescent wave, for example, as described in claim 3. For example, white light (wavelength has a constant width). Some), or light having different wavelengths may be used as incident light continuously.

  According to the invention according to each claim of the present application, by making the incident light into a plurality of types of light, the distribution (three-dimensional distribution), intensity, and form of the mitochondria can be unlabeled and nondestructive. Can be monitored.

  Further, for example, as in the inventions according to claim 4 and claim 5, even when there are a plurality of living cells, the distribution, strength, and form of the polarization state of each mitochondrion are determined, and the properties of each living cell ( For example, mitotic activity, aging status, malignancy (whether or not it is a cancer cell), etc. can be specified.

  That is, for example, the following is known about the polarization state of mitochondria. In aging cells, the degree of mitochondrial polarization decreases. Cells with high cell division activity have a high degree of polarization. The mitochondrial polarity changes greatly during cell growth and differentiation. Regarding mitochondria and differentiation, it has been reported that when the activity of the inner mitochondrial membrane component (cytochrome bc1 (complex III)) is suppressed and the membrane potential is changed, the initial differentiation from ES cells to myocardium is inhibited. Also, at the early stage of ontogeny, endogenous membrane potential changes occur and normal embryonic development is maintained. The role of mitochondria in central nervous cells changes from energy metabolism to synaptic extension and maintenance as nerves mature. Furthermore, changes in membrane polarization are also used to evaluate the functionality of differentiated cells, such as depolarization by KCl and changes in membrane polarization for cardiomyocyte potential conduction in neurons differentiated from human and mouse ES cells. It has been.

  The present invention makes it possible to specify the properties of a plurality of living cells, for example, early selection of cells in the differentiation stage from ES cells to mature functional cells at the single cell level, to a specific cell lineage It becomes possible to select a cell population whose differentiation is destined. It can be used to establish an efficient differentiation-inducing method for a target functional cell population or tissue. That is, identification and purity of tissue stem cells can be increased, and three-dimensional (three-dimensional) things such as organs can be produced. The differentiation induction method using this surface plasmon resonance is useful, for example, in regenerative medicine and also in regenerative medicine as practice.

  Embodiments of the present invention will be described below with reference to the drawings. However, the following is an example, and the present invention is not limited to this.

  First, an outline of a surface plasmon resonance measurement method that is an embodiment of the present invention will be described.

  The preprocessing will be described with reference to FIG. FIG. 1 is a diagram showing an outline of pretreatment, where (a) shows a 12-well cell culture plate (Falcon # 353225), and (b) shows a thermonox cover slip (Nunc # 174950) set therein. Show. Each well of the 12-well cell culture plate is circular with a diameter of 22 mm. The Thermonox coverslip is circular with a diameter of 13 mm, and cells such as ES cells are cultured on it. For example, the cells may be cultured after the coverslip is coated with the coating agent, and the ES cells may be cultured on the feeder cells. The Thermonox coverslip is coated on the 12-well cell culture plate and coated with a coating agent in the same manner as cell culture.

Next, except for the coating agent on the plate, the cells detached from the petri dish are seeded on the well one by one. Then, the lid of the plate is closed, and the cells are cultured at 37 ° C. and a CO ₂ concentration of 5%.

  Subsequently, the measurement will be described. FIG. 2 is a schematic block diagram of the determination / culture system 1 according to the embodiment of the present invention. The determination / culture system 1 includes a surface plasmon resonance device 2, a determination device 17, a selection device 23, and a culture device 26.

  Surface plasmon resonance is a phenomenon that occurs when, for example, a prism is used, a metal surface plasmon is resonantly excited using an evanescent wave obtained by light such as laser light (see Patent Document 1).

  The surface plasmon resonance device 2 has a substrate 3. The substrate 3 is, for example, a light transmission type such as a glass waveguide. On the substrate 3 is a metal layer 4. There are live cells 5 and 7 on the substrate 3. There are mitochondria 9 and 11 in living cells 5 and 7.

  By making the incident light 13 enter the substrate 3 and causing total reflection in the substrate 3, an evanescent wave can be generated on the opposite side of the reflecting surface. When the thin metal film 4 is present on the substrate 3, the evanescent wave passes through the metal layer 4 and resonantly excites surface plasmons on the opposite surface of the metal. Factors that determine the conditions that cause resonance include the dielectric constant of a substance that forms an interface with the metal layer 4. The polarization state of mitochondria 9 and 11 in the living cells 5 and 7 can be a factor that determines the conditions that cause resonance as the dielectric constant of the measurement object. By analyzing the light 15 based on the incident light 13, It can be detected.

  In this example, the measurement was specifically performed as follows.

  First, set up the machine. An existing surface plasmon resonance device can be used to detect using an area detector (see Patent Document 1). The setting software is SPR spectra, and the analysis software is BW Spec. First, cover the detection side with a rubber cap and aluminum, and take dark data. Next, reference data is taken using each culture medium of the cells to be measured. Remove the medium from 4 ° C and leave it for at least 15 minutes. The light source is measured for 15 minutes after starting.

  Next, the cover slip with the cells attached to the top is turned over on the gold substrate and set in the machine with the cells adhered to the substrate. FIG. 3A shows a glass waveguide which is an example of the substrate 3 of FIG. 2 used in this embodiment, and FIG. 3B shows the use of the thermonox cover slip of FIG. An example in which cultured cells are adhered to a substrate is shown. As shown in FIG. 3A, the substrate used in this example is a BK7 glass waveguide, which is 65 mm wide and 20 mm long, and has a square metal film with a side of 3 mm at the center. And as shown in FIG.3 (b), an example which makes the state which adhere | attached the cell cultured using the thermonox cover slip of FIG.1 (b) on a board | substrate is shown. As described above, the measurement can be performed even if the cultured cells are directly adhered to the substrate without fixing the cells. In addition, about a metal plate, as shown in FIG. 2, you may make it all the living cell groups exist on the metal plate 4, and you may make it be a part as shown in FIG. .

  Then, spectrum data of each cell is taken from the light 15. Hereinafter, in the drawings, Spectrum indicates raw waveform data, and t / r% is obtained by calculating (spectrum-dark) / (reference-dark) * 100.

  In this way, for example, spectral data as shown in FIGS. 4 and 5A can be obtained. 5-9, (a) is a spectrum waveform of a cell and a culture medium. Cells are indicated by solid lines, and culture medium is indicated by dotted lines. (B) is obtained by further differentiating t / r% obtained by standardizing cell resonance at each wavelength with a reference. The positive peak indicates the reference, and the negative peak indicates the maximum resonance angle of the cell.

  Analysis parameters include, for example, the shift of the maximum resonance angle between the medium and the cell, the resonance intensity at the maximum resonance angle of the cell, the width of the resonance curve, and the intensity difference from the reference at the wavelength with the least resonance. .

  2 is based on the determination result of the polarization state determination unit 19 and the polarization state determination unit 19 that determine at least one of the distribution, intensity, and form of the mitochondrial polarization state based on the light 15. A property determining unit 21 that determines the property of each living cell 5 and 7 is provided. For example, as shown in FIG. 2, the light 15 is light that can be observed from the substrate 3 based on the incident light 13.

  For example, Japanese Patent Application Laid-Open No. 2004-271337 discloses simultaneous analysis of cells using the surface plasmon resonance phenomenon. However, the number of cells, adhesion area, size, etc. The present invention is also different from the present invention which is also intended for those that do not adhere to the substrate such as the polarization state of mitochondria inside the cell. Japanese Patent Application Laid-Open No. 2004-279041 describes that spectrum analysis is performed in a surface plasmon resonance apparatus. This is fluorescence observation, and detection is performed by illuminating an evanescent wave that is fluorescently labeled as illumination light. This method is different from the present invention.

  2 includes a selection unit 25 that selects live cells to be cultured from the live cells 5 and 7 based on the properties of the live cells 5 and 7 determined by the property determination unit 21. The culture apparatus 26 includes a culture unit 27 that cultures the living cells selected by the selection unit 25. Cells are produced by the culture in the culture unit 27.

  Next, an evaluation method for mitochondrial staining will be described.

As pretreatment, each well of a 4-well culture slide (Falcon # 354114) was coated with a coating agent in the same manner as in cell culture. Next, the cells detached from the petri dish were seeded 1 well at a time except for the coating agent. Then, the lid of the culture slide was closed, and the cells were cultured at 37 ° C. and CO ₂ concentration of 5%.

As a method for staining mitochondria, the culture medium was removed, replaced with 500 μl of a new culture medium containing 2 μM JC-1 iodide, and placed in a CO ₂ incubator (37 ° C., CO ₂ concentration 5%) for 30 minutes.

  Then, after 30 minutes of incubation, the fluorescence intensity was observed. The microscope used is a Nikon ECLIPSE 80i. The objective lens is Nikon Japan L Plan SLWD 20 × 0.35 WD24. The fluorescent filters are B-2A (excitation filter 450-490, dynamic mirror 505, absorption filter 520) and G-2A (excitation filter 510-560, dynamic mirror 575, absorption filter 590). The analysis software is NIS-Elements BR2.30. The exposure is 400-600ms.

  Accumulation of the cationic dye JC-1 inside the mitochondria depends on the membrane potential. Little pigment accumulates when the mitochondrial membrane potential is low. At low concentrations, the dye is present as a monomer and emits green fluorescence. When the membrane potential of mitochondrion is high, a large amount of dye accumulates. At such a high concentration, the dye forms a J-aggregate, and the fluorescence shifts from green to red. Various ratiometric analyzes are possible by combining the signal of the green fluorescent JC-1 monomer and the J-aggregate. The ratio of red / green JC-1 fluorescence depends on the membrane potential and is independent of the size, shape and density of mitochondria.

  5-9, (c) is a diagram showing an image (merge) in which green and red colors are combined, (d) is a diagram showing an image of J-aggregate alone, (e) is a diagram It is a figure which shows the enlarged view (x22.5) of a J-aggregate.

  Analytical parameters include mitochondrial action potential, distribution, density, shape (spherical, fibrous, etc.), cell-specific morphological changes, and the like.

  Next, the operation of the determination device 17 in FIG. 2 will be specifically described with reference to FIGS.

  FIG. 4 is a diagram showing a surface plasmon resonance spectrum by a culture medium. The test medium is an ES medium, a neural stem cell medium, an insulin-producing cell medium, a nerve cell medium, and a skin cell medium, and five types of complete medium are set with ultrapure water as a reference. The effect of the growth promoter and serum contained in each culture medium on the surface plasmon resonance signal is evaluated. In this measurement, there are two types of medium containing serum: ES medium (10% v / v) and nervous system cell medium (5% v / v).

  The five types of culture media used for the measurement are as follows. ES medium is a liquid medium containing MEM non-essential amino acids 100 μM, penicillin 40 units / ml, streptomycin 40 μg / ml, 2-mercaptoethanol 0.1 mM, FBS 14% (v / v), mLIF (mouse leukemia inhibitory factor) 500 units / ml DMEM. The neural stem cell medium is DMEM / F-12 (1: 1) containing insulin 5 μg / ml, transferrin 50 μg / ml, sodium selenite 30 nM, fibronectin 5 μg / ml. Insulin-producing cell medium is DMEM / F-12 containing 5 μg / ml insulin, transferrin 100 μg / ml, progesterone 6.3 ng / ml, putrescine 16.11 μg / ml, selenite 5.2 ng / ml, nicotinamide 10 mM, LY294002 10 μM. 1: 1) (see PNAS 2002; 99: 16105-16110). The nerve cell medium is a medium for nerve cell culture containing B27 supplement 1 ×, FBS 14% (v / v) (see Journal of Neurochemistry 2005; 92: 1265-1276). The skin cell medium is a serum-free basal medium for hematopoietic stem cell research, 2-mercaptoethanol 0.1 mM (see Nature Biotechnology 2005; 23: 1542-1550).

  First, evaluation of the culture medium by surface plasmon resonance will be described. As shown in FIG. 4, measurement was performed using five types of complete culture media as test media: ES medium, neural stem cell culture medium, insulin-producing cell culture medium, nerve cell culture medium, and skin cell culture medium, with ultrapure water set as a reference. Place the substrate on the prism on the surface plasmon resonance sensor, fill the substrate with 1 ml of each medium, and measure. As a result, as shown in FIG. 4, no characteristic difference was observed in the surface plasmon resonance signal due to the medium.

  Subsequently, evaluation of the mitochondrial polarization state of undifferentiated ES cells will be described.

Mouse ES cells were used as test cells. MEM non-essential amino acid 100 μM, penicillin 40 units / ml, streptomycin 40 μg / ml, 2-mercaptoethanol 0.1 mM, FBS 14% (v / v), mLIF (mouse leukemia inhibitory factor) at 37 ° C. and CO ₂ concentration 5% A liquid medium DMEM containing 500 units / ml was used as a culture medium, and co-cultured on feeder cells (mouse embryonic skin cells) in a T25 flask coated with 0.2% gelatin.

  FIG. 5 is a diagram showing a surface plasmon resonance spectrum and a mitochondrial image of ES cells. With reference to FIG. 5 (a), (b), evaluation of the mitochondrial polarization state of the undifferentiated ES cell by surface plasmon resonance can be performed by the following procedures, for example.

As test cells, ES cells grown to 90% confluence were added with trypsin 0.25% / EDTA 1 mM and incubated at 37 ° C. for 10 minutes to detach the cells. The detached cells were collected in a medium containing FBS, centrifuged at 1000 rpm for 5 minutes, and the obtained cell suspension was seeded again in a petri dish. ES cells were incubated on a gelatin-coated petri dish at 37 ° C. for 5 hours at a CO ₂ concentration of 5% to separate them from feeder cells (co-cultured cells), and the supernatant was collected and coated with feeder cells first. The seeds were seeded on a cover slip (Nunc Thermonox cover slip). In this state, the cells were cultured at 37 ° C. under a concentration of 5% CO ₂ until they grew to 90% confluence. After culturing for about 48 hours, the cover slip with the cells adhered to the substrate was placed so as to face each other, and placed directly on the prism on the surface plasmon resonance sensor to start measurement.

  FIG. 5A is a diagram showing a spectrum when ES cells are placed (solid line) using only ES medium as a reference (dotted line). The horizontal axis is wavelength, and the vertical axis is rerlative intensity. As shown in FIG. 5 (a), the maximum resonance angle of the resonance curve when the ES cells were loaded was shifted toward the upper right direction as compared with the resonance curve using only the ES medium as a reference. FIG. 5B is obtained by further differentiating the t / r% obtained by standardizing the cell resonance at each wavelength with the reference.

  As analysis parameters, in this example, as indicated by symbols A to D in FIG. 5A, the shift of the maximum resonance angle between the medium and the cell (A), the resonance at the wavelength at which the cell exhibits the maximum resonance. The intensity (B), the width of the resonance curve (C), and the intensity difference (D) from the medium at the minimum resonance wavelength are used.

  Subsequently, the monitoring of the mitochondrial polarization state by the fluorescent reagent JC-1 will be described with reference to FIGS.

As test cells, ES cells grown to 90% confluence were added with trypsin 0.25% / EDTA 1 mM and incubated at 37 ° C. for 10 minutes to detach the cells. The detached cells were collected with a medium containing FBS, centrifuged at 1000 rpm for 5 minutes, and the obtained cell suspension was seeded on a 4-well chamber previously coated with supporting cells. In this state, the cells were cultured at 37 ° C. under a concentration of 5% CO ₂ until they grew to 90% confluence. After culturing for about 48 hours, 500 μl of ES medium containing 2 μM JC-1 iodide was exchanged and placed in a CO ₂ incubator (37 ° C., CO ₂ concentration 5%) for 30 minutes. After 30 minutes of incubation, the medium was replaced with an ES medium containing no reagent, and the fluorescence intensity was observed.

  FIG.5 (c) is what merged the J-aggregate (red) with the monomer pigment | dye (green) as mentioned above. The monomer dye exhibits a green fluorescence intensity representing a single portion. The J-aggregate exhibits a red fluorescence intensity representing a solidified part. FIG. 5 (d) is a diagram showing red fluorescence intensity representing J-aggregates. FIG. 5 (e) is an enlarged view (× 22.5) of the J-aggregate. As shown in FIGS. 5C to 5E, mitochondria with very high membrane potential are located so as to surround the nucleus and aggregate around the nucleus. The mitochondrial morphology is small and globular and numerous. In addition, each cell is in close proximity, and the mitochondrial distribution is very close.

  Next, evaluation of cells that have been induced to differentiate stepwise from ES cells into neural stem cells and nervous system cells will be described.

Test cells include mouse ES cells and Ronald DG. Cells that had been induced to differentiate into nervous system cells using the method of Mckay et al. Were used (see Journal of Neurochemistry 2005; 92: 1265-1276). The procedure for inducing differentiation from ES cells to nervous system cells at 37 ° C. and 5% CO ₂ is briefly described below.

For the test cells, ES cells grown to 90% confluence were detached, and the cell suspension was seeded on one gelatin-coated 10 cm petri dish. After incubation for 4 hours at 37 ° C. under a CO ₂ concentration of 5%, supernatant ES cells were collected and cultured in ES medium excluding mLIF for 2 days. Meanwhile, Petri formed embryoid bodies using an ultra-low adhesion surface dish (Corning), and then switched to neural stem cell medium for selection of neural stem cells. Cells differentiated to this stage were used as neural stem cells and used for measurement of mitochondrial polarization by surface plasmon resonance and JC-1. In addition, these cells were then grown on mN3FL medium (insulin 25 μg / ml, transferrin 50 μg / ml, sodium selenite 30 nM, progesterone 20 μM, putrescine 100 μM, basic fibroblast growth on a petri dish pre-coated with laminin and ornithine. After growth using DMEM / F-12 (1: 1) containing 5ng / ml of factor and 1μg / ml of laminin, the medium is changed to neuronal medium for 12 days (1/4 volume of medium once every 5 days) Was exchanged) and cultured for use.

  FIG. 6 is a diagram showing a surface plasmon resonance spectrum and a mitochondrial image of a neural stem cell. With reference to FIG. 6 (a), (b), evaluation of the polarization state of the mitochondria of the neural stem cell by surface plasmon resonance can be performed by the following procedures, for example.

  Cells differentiated to neural stem cells as described above were seeded on coverslips. After adhering to the coverslip with DMEM containing FBS, the medium was replaced with neural stem cell medium and cultured for 2 days. Then, the measurement was started by placing the cover slip with the cells adhered to the substrate so as to face each other and placing it directly on the prism on the surface plasmon resonance sensor.

  FIG. 6A is a diagram showing a spectrum in a case where neural stem cells are placed (solid line) using only neural stem cell culture medium as a reference (dotted line). The horizontal axis is wavelength, and the vertical axis is rerlative intensity. As shown in FIG. 6 (a), the resonance curve when the neural stem cell was placed was wider than the resonance curve using only the neural stem cell medium as a reference, and the maximum resonance angle was shifted to the right. . FIG. 6B is obtained by further differentiating t / r% obtained by standardizing cell resonance at each wavelength with a reference.

  Subsequently, monitoring of the mitochondrial polarization state using the fluorescent reagent JC-1 will be described with reference to FIGS.

As test cells, cells differentiated to neural stem cells were adhered to a 4-well chamber with DMEM containing FBS, and then replaced with neural stem cell culture medium. In this state, the cells were cultured at 37 ° C. under a CO ₂ concentration of 5%. Cultured for days. The neural stem cell medium containing 2 μM of JC-1 iodide was replaced with 500 μl and placed in a CO ₂ incubator (37 ° C., CO ₂ concentration 5%) for 30 minutes. After 30 minutes of incubation, the medium was changed to a neural stem cell medium containing no reagent, and the fluorescence intensity was observed.

  The relationship between FIGS. 6C to 6E is the same as the relationship with FIGS. 5C to 5E. 6C to 6E show that mitochondria of neural stem cells are localized around the nucleus, but, unlike ES cells, mitochondria with high membrane potential and low mitochondria are scattered. Further, the form includes spherical and fibrous forms. As the cell shape changes, the distribution of mitochondria is not uniform.

  Subsequently, evaluation of the mitochondrial polarization state of mature nervous system cells by surface plasmon resonance will be described.

  Cells differentiated to mature nervous system cells by the above method were seeded on ornithine / laminin-coated coverslips (Nunc Thermonox coverslips). After culturing for one week, the measurement was started by placing the cover slip with the cells adhered to the substrate facing each other and placing it directly on the prism on the surface plasmon resonance sensor.

  FIG. 7 is a diagram showing a surface plasmon resonance spectrum and a mitochondrial image of a nervous system cell. As shown in FIG. 7 (a), the resonance curve when the mature nervous system cell is placed is wider than the resonance curve using only the nerve cell medium as a reference, and the maximum resonance angle shifts to the upper right part. Was. FIG. 7B is obtained by further differentiating t / r% obtained by standardizing cell resonance at each wavelength with a reference.

  Subsequently, monitoring of the mitochondrial polarization state using the fluorescent reagent JC-1 will be described with reference to FIGS.

The test cells were cultured for 12 days after changing the medium to a nerve cell medium and used for measurement. Cells differentiated to mature nervous system cells were seeded onto a 4-well chamber coated with ornithine / laminin. In this state, the cells were cultured for 7 days at 37 ° C. and 5% CO ₂ , and then replaced with 500 μl of neuronal cell culture medium containing 2 μM JC-1 iodide in a CO ₂ incubator (37 ° C., 5% CO ₂ concentration). For 30 minutes. After 30 minutes of incubation, the medium was replaced with a neuronal medium containing no reagent, and the fluorescence intensity was observed.

  The relationship between FIGS. 7C to 7E is the same as the relationship between FIGS. As shown in FIGS. 7 (c) to (e), the nervous system cells were dotted with red indicating hyperpolarization on the synapse. In nervous system cells, mitochondria are scattered on the transcellular mass and axons. In addition, the morphology is often fibrous and is dotted with heterogeneous mitochondria of different sizes. Mitochondria with high membrane potential are localized in the cell mass.

  Next, evaluation of cells induced to differentiate from ES cells into insulin producing cells will be described.

Test cells include mouse ES cells from Seung K. et al. Insulin-producing cells differentiated using the method of Kim et al. (See PNAS 2002; 99: 16105-16110). A procedure for inducing differentiation from ES cells to insulin-producing cells at 37 ° C. and 5% CO ₂ is briefly described below.

For the test cells, ES cells grown to 90% confluence were detached, and the cell suspension was seeded on one gelatin-coated 10 cm petri dish. After incubation for 4 hours at 37 ° C. and 5% CO ₂ , supernatant ES cells were collected and cultured for 2 days in ES medium excluding mLIF. Meanwhile, Petri formed embryoid bodies using an ultra-low adhesion surface dish (Corning), and then switched to neural stem cell medium for selection of neural stem cells. Cells differentiated to this stage were used as neural stem cells. The cells were then placed on a petri dish pre-coated with fibronectin and ornithine on stage 4 medium (insulin 5 μg / ml, transferrin 100 μg / ml, progesterone 6.3 ng / ml, putrescine 16.11 μg / ml, selenite 5.2 ng / ml, B27 supplement 1 ×, DMEM / F-12 (1: 1) with basic fibroblast growth factor 10ng / ml), stage5NB (insulin 5μg / ml, transferrin 100μg / ml, progesterone 6.3ng / ml, putrescine 16.11μg / ml, Selenite 5.2 ng / ml, B27 supplement 1 ×, DMEM / F-12 (1: 1) containing nicotinamide 10 mM), stage5NL medium (above) and cells prepared by exchanging the medium every 6 days Was used for the measurement.

  FIG. 8 is a diagram showing a surface plasmon resonance spectrum and a mitochondrial image of insulin-producing cells. Cells that had been differentiated into insulin-producing cells and clamped according to the above method were seeded on fibronectin-coated coverslips. After adhering to the coverslip with DMEM containing FBS, the medium was replaced with an insulin-producing cell medium and cultured for 2 days. Then, the measurement was started by placing the cover slip with the cells adhered to the substrate so as to face each other and placing it directly on the prism on the surface plasmon resonance sensor. As a result, as shown in FIG. 8 (a), the resonance curve (solid line) when the insulin-producing cells are placed is wider than the resonance curve (dotted line) using only the insulin-producing cell medium as a reference. The resonance angle was shifted to the lower right. FIG. 8B is obtained by further differentiating t / r% obtained by standardizing cell resonance at each wavelength with a reference.

  Subsequently, monitoring of the mitochondrial polarization state using a fluorescent reagent will be described with reference to FIGS. The relations of FIGS. 8C to 8E are the same as the relations of FIGS. 5C to 5E. As shown in FIGS. 8C to 8E, the mitochondria of the insulin-producing cell spread throughout the cell, the form and size are not constant, and there are many mitochondria with a low membrane potential in the cell mass.

  Subsequently, evaluation of cells induced to differentiate from ES cells to skin-like cells will be described.

As test cells, skin-like cells differentiated from mouse ES cells using the method of Shin-Ichi Nishikawa et al. Were used (see Nature Biotechnology 2005; 23: 1542-1550). A procedure for inducing differentiation from ES cells to skin-like cells at 37 ° C. and 5% CO ₂ is briefly described below.

For the test cells, ES cells grown to 90% confluence were detached, and the cell suspension was seeded on one gelatin-coated 10 cm petri dish. After incubation for 4 hours at 37 ° C. and 5% CO ₂ , supernatant ES cells were collected, and cells were seeded at a concentration of 10 ⁵ / ml in a petri dish coated with fibronectin. It was cultured for 10 days until skin-like cells were formed using skin cell medium, and then used for measurement.

  FIG. 9 is a diagram showing a surface plasmon resonance spectrum and a mitochondrial image of skin-like cells. With reference to FIGS. 9A and 9B, evaluation of the mitochondrial polarization state of skin-like cells by surface plasmon resonance can be performed as follows.

  Cells differentiated to skin-like cells according to the above method were seeded on a coverslip coated with fibronectin. After adhering to the coverslip with DMEM containing FBS, it was replaced with skin cell medium and cultured for 2 days. Then, the measurement was started by placing the cover slip with the cells adhered to the substrate so as to face each other and placing it directly on the prism on the surface plasmon resonance sensor. As a result, as shown in FIG. 9A, the resonance curve when the skin-like cells are placed is wider than the resonance curve using only the skin cell culture medium as a reference, and the maximum resonance angle is in the upper right direction. It was moving towards. FIG. 9B is obtained by further differentiating the t / r% obtained by standardizing the cell resonance at each wavelength with the reference.

  Subsequently, monitoring of the mitochondrial polarization state using a fluorescent reagent will be described with reference to FIGS. The relations of FIGS. 9C to 9E are the same as the relations of FIGS. 5C to 5E. As shown in FIGS. 9C to 9E, mitochondria aggregate and cells with a very high membrane potential exist. In addition, these mitochondria are elongated and dense around the nucleus. Mitochondria are interspersed with the size of each cell.

  Subsequently, the consistency between the surface plasmon resonance spectrum and the mitochondrial image will be described with reference to FIGS.

  In stem cells, mitochondria incline the energy of cell division and proliferation, while in differentiated cells, mitochondria maintain their role as mature cells, such as maintaining cell morphology and, for example, axon extension in the nervous system. It is known that the function of will change. From that perspective, stem cell mitochondria have very active energy as a whole cell, while differentiated cells are expected to emit locally active energy, and the overall energy level is higher than that of stem cells. Inferred to be low.

  Therefore, with respect to the spectrum waveform for each cell, the shift of the maximum resonance angle between the culture medium and the cell (see A in FIGS. 5A and 5B), the strength of resonance at the maximum resonance angle of the cell (FIG. 5 (a), (b) B), resonance curve width (see C in FIG. 5 (a)), intensity difference from the reference at the wavelength with the least resonance (see D in FIG. 5 (a)) To consider.

  First, the shift of the maximum resonance angle between the medium and the cells is examined. The maximum resonance angle is most shifted in ES cells and skin-like cells, and the difference is smallest in neural cells. In ES cells and skin-like cells, mitochondria with high action potentials are aggregated. On the other hand, neural cells are localized in a part of the cell mass. Therefore, for example, “the density and distribution of mitochondria in a polarized state” can be known from the shift of the maximum resonance angle between the medium and the cells.

  Next, the resonance intensity at the wavelength at which the cell exhibits the maximum resonance is examined. The resonance intensity is equally large in ES cells, neural stem cells, and skin-like cells, and the smallest in insulin-producing cells. In ES cells, neural stem cells, and skin-like cells, the distribution and shape of mitochondria are different, but the number of polarized mitochondria is very large. Therefore, for example, “total amount of mitochondrial polarization = energy level” can be known from the resonance intensity at the wavelength at which the cell exhibits the maximum resonance.

  Next, the width of the resonance curve will be examined. The width of the resonance curve is equally wide in neural stem cells and skin-like cells, and the narrowest in nervous system cells. The mitochondrial morphology of neural stem cells and skin-like cells is scattered in many spherical and fibrous forms, but many of the nervous system cells are fibrous. Therefore, for example, “mitochondrial morphology uniformity” can be known from the width of the resonance curve.

  Next, the difference in intensity from the reference at the minimum resonance wavelength will be examined. Skin-like cells have the widest difference, and ES cells and nervous system cells have the smallest difference. Since ES cells are not in contact with each other, and axons develop even in nervous system cells, both cells have areas where the cells are not in contact on the substrate. On the other hand, there are few gaps in cells that spread all over like skin-like cells. Therefore, for example, the “cell adhesion area” can be known from the difference in strength from the medium at the minimum resonance wavelength.

  In order to quantify the above four points, for example, mitochondrial morphology such as the number of mitochondria in one cell and the degree and size of aggregation, measurement of cell number and adhesion area on the substrate and observation of morphological changes, gene transfer It is considered effective to utilize consistency with real-time monitoring by cells and construction of differentiation system models from somatic stem cells.

  FIG. 10 is a diagram showing a comparison of spectra in the case of various cells, taking into account the effects on the reference, (a) showing a comparison between ES cells and neural stem cells, and (b) showing nervous system cells and insulin production. (C) shows a comparison of neural stem cells, neural cells, and insulin-producing cells, and (d) shows ES cells, neural stem cells, neural cells, insulin-producing cells, and skin-like cells. A comparison of is shown.

  Referring to FIG. 10A, the maximum resonance angle is such that ES cells are located on the upper right and neural stem cells are located on the lower left. Further, although the minimum resonance angle is the same for ES cells and neural stem cells, the value of t / r% is different. As shown in FIGS. 5 (e) and 6 (e), ES cells and neural stem cells differ in the three-dimensional distribution, intensity, morphology, etc. of the mitochondrial polarization state. Therefore, as shown in FIG. 7A, it can be seen that the results of spectrum analysis differ depending on the three-dimensional distribution, intensity, morphology, etc. of the mitochondrial polarization state. Therefore, it can be seen that the three-dimensional distribution, intensity, morphology, etc. of the mitochondrial polarization state can be detected by performing spectral analysis on surface plasmon resonance.

  In addition, referring to FIG. 10 (b), the mitochondrial polarization state as shown in FIG. 7 (e), FIG. 8 (e), and FIG. 9 (e) also in the nervous system cells, insulin producing cells, and skin-like cells. It can be seen that the results of the spectrum analysis are different due to differences in the three-dimensional distribution, intensity, form, and the like. Therefore, it can be seen that the three-dimensional distribution, intensity, morphology, etc. of the mitochondrial polarization state can be detected by performing spectral analysis on surface plasmon resonance.

  Furthermore, as shown in FIGS. 10C and 10D, even when ES cells, neural stem cells, nervous system cells, insulin producing cells, and skin-like cells are compared, the three-dimensional distribution of the mitochondrial polarization state・ It can be seen that the spectrum analysis results for the surface plasmon resonance are different when the intensity and the form are different. Therefore, it can be seen that the three-dimensional distribution, intensity, and morphology of the mitochondrial polarization state can be detected.

  FIG. 11 is a diagram showing (a) La Jade Island-like structure (insulin-producing cells, glucagon-producing cells), (b) nerve-like cells, and (c) skin-like cells.

  FIG. 12 and FIG. 13 are diagrams showing differentiation induction from ES cells into nerve cells, pancreatic β cells, cardiomyocytes, and mesendoderm cells.

It is a figure which shows the outline | summary of a pre-processing, (a) shows a 12 well cell culture plate, (b) shows the thermonox cover slip set in it. 1 is a schematic block diagram of a determination / culture system 1 according to an embodiment of the present invention. (A) shows the glass waveguide which is an example of the board | substrate 3 of FIG. 2 used in a present Example, (b) is cultured using the thermonox cover slip of FIG.1 (b). An example in which cells are adhered to a substrate is shown. It is a figure which shows the surface plasmon resonance spectrum by a culture medium. It is a figure which shows the surface plasmon resonance spectrum and mitochondrial image of ES cell. It is a figure which shows the surface plasmon resonance spectrum and mitochondrial image of a neural stem cell. It is a figure which shows the surface plasmon resonance spectrum and mitochondria image of a nervous system cell. It is a figure which shows the surface plasmon resonance spectrum and mitochondrial image of an insulin producing cell. It is a figure which shows the surface plasmon resonance spectrum and mitochondrial image of a skin-like cell. It is a figure which shows the comparison which considered the influence with respect to the reference of the spectrum of the light in the case of various cells based on FIGS. It is a figure which shows (a) insulin producing cells, (b) nerve-like cells, and (c) skin-like cells. It is FIG. 1 which shows the differentiation induction from an ES cell to a neuron-like cell, a pancreatic beta cell, and a cardiac muscle cell. FIG. 2 is a second diagram showing induction of differentiation from ES cells to mesendoderm cells.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 Determination and culture system, 2 Surface plasmon resonance apparatus, 3 Substrate, 4 Metal layer, 5, 7 Living cell, 9,11 Mitochondria, 13 Incident light, 15 Light, 17 Determination apparatus, 19 Polarization state determination part, 21 Property determination Part, 23 selection device, 25 selection part, 26 culture device, 27 culture part

Claims (10)

A determination device for determining a mitochondrial polarization state in a living cell using a surface plasmon resonance phenomenon,
Polarization state determining means for determining the polarization state of the mitochondria based on at least two types of incident light;
A determination apparatus comprising:   The determination apparatus according to claim 1, wherein the determination by the polarization state determination means is at least one of distribution, intensity, and form of the polarization state of the mitochondria. The at least two types of incident light have different wavelengths and at least one of evanescent waves to be generated,
The polarization state determination means determines the polarization state of the mitochondria by spectral analysis;
The determination apparatus according to claim 1 or 2.   The determination apparatus according to claim 1, further comprising a property determination unit that determines a property of the living cell based on a determination result by the polarization state determination unit.   5. A selection apparatus comprising selection means for selecting live cells to be cultured based on the properties of live cells determined by the property determination means according to claim 4. A determination method for determining a mitochondrial polarization state in a living cell by using a surface plasmon resonance phenomenon,
Determining the polarization state of the mitochondria based on at least two types of incident light;
A determination method including A cell production method for producing cells by culture,
Production step of producing cells by culturing living cells to be cultured based on the determination result of the polarization state of mitochondria in living cells using surface plasmon resonance phenomenon as incident light with at least two kinds of light ,
A cell production method comprising: Computer
A determination device for determining a mitochondrial polarization state in a living cell using a surface plasmon resonance phenomenon,
Determining means for determining at least one of distribution, intensity and morphology of the mitochondria polarization state based on at least two types of incident light;
A program for functioning as a determination device.   A recording medium on which the program according to claim 8 is recorded. A determination device that determines a polarization state of a determination object that is in contact with or at least close by using a surface plasmon resonance phenomenon,
Determination means for determining at least one of the distribution, intensity and form of the polarization state based on at least two types of incident light;
A determination apparatus comprising: