Patent Application
20140186941
The present invention relates to a cell culture vessel and a culture device. In particular, the present invention relates to a method for improving incubation efficiency.
Regenerative medicine has gotten a lot of attention as an innovative medical treatment, which enables basic remedy for damaged and/or defective cells, tissues, and organs. The regenerating tissue used for regenerative medicine, which is produced through the steps of collecting cells from the body of a patient or the other person; separating and purifying the collected cells in vitro, and growing and organizing the cells into tissue, is transplanted into the body of the patient. Tissue engineering, making advances yearly, has enabled the methods for forming one kind of cells into a sheet and for arranging several kinds of cells sterically to assemble an organ by artificial means to be developed.
To amplify therapeutic cells, in particular adherent cells in large quantities, an incubator large in area is useful. It is because adherent cells are amplified while expanding in the planar direction. On the other hand, it has a problem that as the area of an incubator becomes larger, its culture surface increasingly deforms; thereby, cells aggregate in a lower area, leading to deteriorated usage efficiency. As an effective technique for manipulating cells, electrophoresis has gotten attention. The systematic study and theoretical analysis of electrophoresis were set out by Pohl in 1970s (see Nonpatent Literature 1). Micro biological substances, such as bacteria and cells, have been already employed as a principal target to be manipulated since the initial study; accordingly, biotechnology is one of key applications of electrophoresis.
A dielectrophoretic force FDEP exerted on dielectric particles is given by the following equation 1 (see Nonpatent Literature 1). In the following paragraph, how to calculate is explained taking an example of dielectric particles being cells. [Mathematical formula 1]
F
DEP=2πα3∈0∈mRe[K]∇E2 (Formula 1)
Where a is the radius of a cell approximated to a spherical shape, ∈0: electric permittivity in vacuum, ∈m: specific electric permittivity in medium, E: electric field intensity, and ∇ is an operator representing a gradient. In this case, ∇E2, which is the gradient for the square of an electric field intensity (E2), indicates how degree E2 inclines at that point, namely, how suddenly the electric field spatially changes. K is called a Claudius-Mossotti number and is represented by an equation 2. Herein, assuming that ∈b* and ∈m* be complex dielectric constants for cells and a medium, respectively, and Re [K] be the real part of the Claudius-Mossotti number, Re [K]>0 represents positive electrophoresis and the cells migrate in the same direction as that of the electric field gradient, namely toward an electric field concentration part. Re [K]<0 represents negative electrophoresis and cells migrate in the direction apart from the electric field concentration part, namely toward a weak electric field part.
Formula 3 generally represents complex dielectric constant ∈r
Where, ∈r is the specific electric permittivity for a cell or medium, σ is the electric conductivity of a cell or medium, and ω is the angular frequency of an applied electric field. As known from Formulae 1, 2, and 3, a dielectrophoretic force depends on the radius of a cell, the real part of a Claudius-Mossotti number, and an electric field intensity. Moreover, it is known that the real part of the Claudius-Mossotti number varies depending on the complex electric permittivity and electric field frequency of a cell and medium.
The DEPIM method, combining dielectrophoresis and impedance measurement, has been proposed as a method for measuring microbial counts using dielectrophoresis. The DEPIM method is characterized in that these parameters are appropriately selected and a positive dielectrophoretic force exerted on microorganisms is sufficiently increased to collect the microorganisms into an electrode gap, making electrical measurement to determine a microbial count in the sample solution (see Nonpatent Literature 2).
In addition, a culture device, which eliminates unnecessary cells from a cell suspension using negative dielectrophoresis to culture necessary cells at high concentrations, is disclosed (see Patent Literature 1 and Patent Literature 3).
Moreover, a method and apparatus, for collecting cells efficiently in a target area without losing the activity of functional cells using positive dielectrophoresis, is disclosed (see Patent Literature 2).
However, it is difficult to manipulate cells and microorganisms directly in an ion-rich culture solution (namely, high electric conductivity) using dielectrophoresis described in BACKGROUND. For this reason, generally, the target cells are moved in an ion-poor buffer solution, manipulated, and then returned back in the original culture solution. As a result, a cell manipulation process is complicated, causing a problem of increased stress on the cells due to a change in culture environment. In addition, this method has another problem that generally, an enzyme is used to detach the cells grown during surface culture from the surface of culture medium, increasing load on the cells.
An object of the present invention is to simplify the cell manipulation process to reduce the stress on the cells, as well as the load on the grown cells exerted when detached from the surface of the culture medium in order to solve these problems. This makes possible to improve culture efficiency of a cell culture vessel and determine cell distribution and growth via electric signals.
To address the aforementioned problems, the key characteristics of the cell culture vessel of the present invention are as described below.
A cell culture vessel for supporting and culturing cells is composed of a space enclosed by a housing for supporting a medium and a cell attachment part disposed on the bottom surface of the space for attaching and supporting the cells therein. The cell attachment part has a cell immobilizing mechanism for guiding the cells to the cell attachment part from the cell space and immobilizing them therein, and a cell detachment mechanism for detaching the cells attached in the cell attachment part. The cell immobilizing mechanism includes a step of applying voltage in an electrode to generate an inhomogeneous electric field in the space, and the cell detachment mechanism includes a step of applying voltage in an electrode disposed in the cell attachment part to induce electrolysis in the space.
A cell culture device of the present invention is mainly characterized as described below. The cell culture device equipped with the cell culture vessel for supporting and culturing the cells therein is composed of a feeding/discharging part for feeding/discharging the medium into/from the cell culture vessel, and a power source for applying voltage to an electrode disposed in the cell culture vessel. A cell culture vessel is composed of a space enclosed by a housing for supporting a medium and a cell attachment part disposed on the bottom surface of the space for attaching and supporting the cells therein. The cell attachment part has a cell immobilizing mechanism for guiding the cells into the cell attachment part from the cell space and immobilizing them therein, and a cell detachment mechanism for detaching the cells attached in the cell attachment part. The cell immobilizing mechanism includes a step of applying voltage in an electrode to generate an inhomogeneous electric field in the space, and the cell detachment mechanism includes a step of applying voltage in an electrode disposed in the cell attachment part to induce electrolysis in the space.
It has been known that in the ion-rich environment with the electric conductivity of the medium equal to or less than 1000 mS/m, dielectrophoresis becomes negative always at the frequency equal to or less than 109 Hz. Taking advantage of cell migration in the direction apart from the electric field concentration, namely toward the weak electric field by negative electrophoresis, the present invention enables the cells to be immobilized in a desired location.
The cells may be detached from the surface of the culture medium by applying a direct current (DC) field; thereby, the need for the use of any enzyme (e.g., trypsin) in cell detachment, as with traditional apparatuses, is eliminated.
The present invention enables the culture efficiency of the cell culture vessel to be improved and the cell distribution and growth to be determined via electric signals.
Hereinafter, by reference to the accompanying drawings, the embodiments of the present invention will be explained. It should be noted that the same signs are assigned to the same components in the drawings and the explanation of these components are omitted.
Hereinafter, by reference to the accompanying drawings, the embodiments will be explained.
One example of the cell culture vessel of the present invention will be explained by reference to
In
The aforementioned the ceiling substrate 1 and the bottom substrate 2 may be formed using any of insulating materials, as their base material, such as glass, silicone, quartz plastics, polymers. Preferably, the ceiling substrate 1 and the bottom substrate 2 are formed using, as their material, any of materials with light transmittance to the degree that the cells are enabled to be observed under an optical microscope, and more preferably, for the surface of the bottom substrate 2, a material is used, which may be modified through cleaning and preprocessing processes before the cells are attached thereon.
Generally, an ion-rich, highly-conducting medium (1000 mS/m) is used for cell culture, in particular for animal cell culture.
As shown in
The present invention is not limited to the electrodes according to the aforementioned first embodiment but may be the electrodes formed into the shape capable of generating the weal electric field shown in
In the case of the cells culture on the surface of the medium, it is desired that to grow the cells, a layer for facilitating cell attachment capacity, for example a polymeric membrane, is coated between the bottom surface of the incubator between the electrodes, as well as the surfaces of the electrodes.
Hereinafter, by reference to
As shown in
During the step of culturing the cells (for example, animal cells) for growth, the cells are attached to the culture surface at 37° C. for growth. During the step of exchanging a mixed gas for culture (composed of air, 5% Co2, and 100% water), as shown in
To detach the grown cells from the culture surface, as shown in
The cells contained in the medium precipitate, when left as it is, spontaneously down toward the bottom of the cell culture vessel under its own weight. However, it tales long time, about several hours, for the cells to reach the bottom of the incubator and initiate their growth, especially for light cells; thereby they are likely to die before they initiate their growth. To solve this problem, it is required that an appropriate voltage is applied to facilitate cell. However, even though the voltage is applied, the precipitated cells are eccentrically deposited; thereby it is not expected that the cells grow homogeneously over a wide range.
According to the first embodiment of the present invention, it is expected that the death of the cells may be avoided.
Moreover, according to the first embodiment of the present invention, the cell culture vessel of the present invention enables the cells to be incubated more efficiently, the distribution and growth progress of the cells to be estimated, and the cells to be detached from the culture surface through electrophoresis. In other words, the cell culture vessel according to the first embodiment of the present invention has advantages of improving cell culture efficiency and reducing the running cost of the apparatus using the cell culture vessel.
With regard to the second embodiment of the present invention, explained is a method for estimating the distribution and growth progress of the cells by measuring the impedance between the lower electrodes of the present invention.
Hereinafter, assuming that the impedance between the lower electrodes be Z, capacitance be C, reactance be x, resistance be r, and resistor be R, the aforementioned method is explained using formulas 4 to 8 by reference to
The formula 4 represents a synthetic impedance Z in a CR parallel equivalent circuit, the formula 5 represents a resistance r in the CR parallel equivalent circuit, the formula 6 represents a reactance x in the CR parallel equivalent circuit, the formula 7 represents a resistor R in the CR parallel equivalent circuit, and the formula 8 represents capacitance C in the parallel equivalent circuit.
With respect to the third embodiment of the present invention, the gap distance between the electrodes of the cell immobilizing mechanism, applied voltage, and applied frequency are explained.
An electric field intensity E between the electrodes of the cell immobilizing mechanism may be represented by the formula 9.
Where, E is the electric field intensity, V is the applied voltage, and d is the gap distance. Water, which is a principal component of the medium for the cell culture, undergoes electrolysis theoretically at 1.23 V; thereby, the applied voltage V need to be set to 1.23 V and preferably, it is equal to and higher than 1 V. However, a lower applied voltage has a disadvantage that it induces only a weak dielectrophoretic force, taking long time for cell growth; accordingly, the lower limit of the applied voltage is preferably approx. 20 mV from the practical view. Moreover, when the cells are manipulated using the thin film electrodes, the electric field intensity E need to be equal to or higher than 1×104 V/m; thereby gap distance d between electrodes becomes equal to or lower than 123 μm. Furthermore, in the case of the cells, the average diameter of them is 10 μm; accordingly, the gap distance between the electrodes is preferably 20 to 30 μm. The formula 10 represents the amplitude of the impedance between the aforementioned electrodes.
Where S is the facing surface areas of the electrodes. As known from the formula 10, with d between the electrode gaps being constant, the larger the applied frequency f, the smaller the impedance. Specifically, applying high frequency decreases the resistance between the electrodes, causing a larger current to flow. This elevates the medium temperature, causing the environment appropriate for cell culture to be deteriorated or a current control system to be complicated. In addition, considering the technique for achieving a high frequency apparatus, to gain a practical dielectrophoretic force, the applied frequency is preferably equal to or lower than 10 MHz. However, with a lower applied frequency, electrolysis of water occurs readily; accordingly, the lower limit is preferably approx. 100 Hz.
With respect to the fourth embodiment, another cell culture vessel of the present invention is explained by reference to
In the fourth embodiment, an expansion mechanism 3A shown in
In the cell culture vessel configured as shown in
In the cell culture vessel shown in
In the example 1, a castle-wall electrodes are used for cell immobilization, cell count measurement, and cell growth progress measurement.
With respect to the example 1, the result of the use of the 3T3 cells and the DMEM medium have been explained; however, the use of the cells derived from any other animal of comparable size and another medium with electric conductivity equivalent to that of the DMEM medium may give the same result.
As known from
With respect to the example 2, the result of an experiment, in which the cultured cells are detached from the medium surface using the detachment mechanism of the present invention. Since the experimental conditions are the same as those of the example 1, the explanation of them is omitted. After 24-hour culture, 0.5 V of voltage was applied between the upper electrode 3 and lower electrode 4 of the cell culture vessel from the DC power source 12. Two hours after applying the voltage, it was observed that the cells were gradually detached away from the medium surface. To facilitate this detachment step, the applied voltage may be increased; however, it is concerned about the possibility of damage to the cells due to strong electrolysis. Taking advantage of this phenomenon, the cells may be detached with no need for an enzyme for detachment as with conventional techniques, for example trypsin, reducing the running cost.
On the other hand, no technique method for concentrating the cells using dielectrophoresis has been reported.
With respect to the example 3 and its succeeding examples, an apparatus for concentrating the cells in the medium efficiently with less load on the cells using negative dielectrophoresis is explained.
One configuration of the cell concentrating apparatus according to the example 3 of the present invention is explained by reference to
In
The concentrating electrode 102 disposed on the bottom surface of the piston-type incubator 101 may be formed directly of, for example metal wire, or may be formed by evaporating or fixing a metal material on a solid insulating substrate made of any of materials such as glass, silicone, quartz, plastics, and polymers and then forming a through hole between the electrodes. Moreover, it is desired that any of materials capable of suppressing the chemical reaction with the medium and the influence on the cells is used for the aforementioned electrodes and the support member. The allowable materials for the electrodes include platinum, gold, chromium, palladium, silver, aluminum, tungsten, and ITO, or any combination of them. It goes without saying that the cross-sectional shape of the concentering electrode is preferably circular but may be other shapes such as rectangle and polygon.
Generally, ion-rich media with high conductivity (1000 mS/m or higher) is used for culturing the cells, especially animal cells.
By reference to
With respect to the example 3, the technique for inserting the concentrating electrode 102 down into the cell suspension vessel 105 vertically from the top side has been described; however, it goes without saying that the concentrating electrode 102 may be inserted from the bottom side or the lateral side. Insertion of the concentrating electrode 102 into the cell suspension vessel 105 from the top side is preferable because it improves cell concentration efficiency by exerting the gravity force G and the dielectrophoretic force from their individual directions.
By reference to
Herein, the gap distance between the concentrating electrodes 102, and the applied voltage and applied frequency are explained.
The electric field intensity E between cell concentrating electrodes is represented by the above formula 9.
Water, which is a principal component of the medium for cell culture, undergoes electrolysis theoretically at 1.23 V; thereby, the applied voltage need be set to 1.23 V or lower. Moreover, as shown in the formula 1, since the dielectrophoretic force is proportional to the applied voltage. At the applied voltage lower than 20 mV, the dielectrophoretic force becomes smaller, namely the force for driving the cells is decreased; accordingly, the applied voltage is preferably equal to or higher than 20 mV.
To manipulate the cells, the electric field intensity E equal to or higher than 1×104 V/m is required, resulting in the gap distance d between the electrodes being equal to or lower than 123 μm. Furthermore, for the animal cells, of which average diameter is 10 μm, the gap distance between the electrodes is preferably within the range from 20 to 30 μm.
The above formula 10 represents the amplitude of the impedance between the aforementioned electrodes.
In the formula, S is the area between the opposing electrodes. As known from the formula 10, assuming that the gap distance d between the electrodes is constant, the larger the applied frequency f, the smaller the impedance. Specifically, when high frequency is applied, the resistance between the electrodes, increasing flowing current. This elevates the medium temperature, causing the environment appropriate for cell culture to be deteriorated or a current control system to be complicated. In addition, considering the technique for achieving a high frequency apparatus, the applied frequency is preferably equal to or lower than 10 MHz. However, with higher electric conductivity of the medium, electrolysis of water occurs even when low-frequency AC voltage is applied; accordingly, the applied voltage is preferably equal to or higher than 100 MHz.
According to the example 3 of the present invention, the cell concentrating apparatus of the present invention is capable of concentrating the cells easily and efficiently by means of dielectrophoretic force, rather than the conventional membrane separation process, centrifuge separation method, and precipitation separation method.
The example 4 of the present invention measures the impedance between the concentrating electrode and the bottom surface electrode of the present invention to determine the cell concentration. The example 4 is explained by reference to
113 is a bottom surface electrode disposed on the bottom surface of the cell suspension vessel, 111 is an impedance measuring apparatus electrically connected to the bottom surface electrode 113, and 112 is a switch connecting the concentrating electrode 102 and the impedance measuring apparatus. 114 is a position sensor of the concentrating electrode, and 115 is magnetic sheet for the position sensor.
The flow of a process of measuring the cell concentration according to the example 4 of the present invention is explained by reference to
Herein, the method for determining the cell concentration by measuring the impedance between the concentrating electrode 102 and the bottom surface electrode 113 is explained.
Hereinafter, the impedance Z between the concentrating electrode and the bottom surface electrode is explained by reference to
In the formulas, the capacitance is represented by C, the reactance is represented by x, the resistance is represented by r, and the resistor is R.
The formula 4 represents the synthetic impedance Z in the CR parallel equivalent circuit, the formula 5 represents the resistance r in the CR parallel equivalent circuit, the formula 6 represents the reactance x in the CR parallel equivalent circuit, the formula 7 represents the resistor R in the CR parallel equivalent circuit, and the formula 8 represents the capacitance C in the CR parallel equivalent circuit.
The medium is homogenous liquid. In contrast, the cell is enclosed with an almost insulating cell membrane and therefore, large differences in capacitance and resistance are observed between the cell and the medium. Specifically, the capacitance and resistance of the medium have been measured in advance and when the cells are seeded in the medium, the cell count is determined based on the changes in capacitance and resistance. Since the impedance may be assessed based on the capacitance and resistance, the cell count may be estimated based on the impedance by assessing, in advance, the relationship between the cell count and the impedance. In other words, the cell count may be estimated based on the impedance between the concentrating electrode and the bottom surface electrode.
By reference to
In
The cell concentration system shown in
Taking advantage of the above functions, the cells may be concentrated to the target level. The flow sheet of controlling the process is shown in
A cell concentration apparatus according to the example 6 of the present invention is explained by reference to
According to the example 6, the concentrating electrode 102 is disposed on the wall of the cell suspension vessel 105 in the form of multi-layer electrodes 102A to 102C. The corresponding one of switches 104A to 104C is disposed between each of the concentrating electrode 102A to 102C and the AC power source 103.
The flow of the process for concentrating the cells according to the example 6 is explained by reference to
Focusing on cell concentration, the present invention has been explained. The principle of the apparatus and the system of the present invention may be inversely used to dilute the cell suspension to the target concentration.
As long as the features of the present invention are not lost, the present invention is not limited to the aforementioned embodiments and examples and includes other embodiments, which may be considered within the scope of the technical idea of the present invention.
Hereinafter, the embodiments of the present invention are summarized and recited.
(1) A cell concentration apparatus for concentrating cells contained in a medium, which includes a cell suspension vessel that supports the media containing the cells, a piston-type incubator moving toward the cell suspension vessel, an electrode disposed on the bottom surface of the piston-type vessel, a through-hole, which pierces through the bottom surface of the piston-type incubator disposed between the electrodes, a power source that applies an AC voltage to the electrode, a driving mechanism that moves the piston-type incubator up from and down to the cell suspension vessel, and a discharge mechanism that discharges the medium, which enters the piston-type vessel through the through-hole.
(2) The cell concentrating apparatus described in (1) which is characterized in that the electrode disposed on the bottom surface of the piston-type incubator presses the cells in a cell suspension against the bottom of the cell suspension vessel by means of a diectrophoretic force.
(3) The cell concentrating apparatus described in (1) which further includes an impedance measuring apparatus that measures the impedance between the electrode disposed on the bottom surface of the piston-type incubator and the electrode disposed on the bottom surface of the cell suspension vessel to estimate the count of the cells in the medium based on the measured impedance.
(4) The cell concentrating apparatus described in (3), which further includes a position sensor that measures the position of the electrode, in which it finds the volume of the cell suspension based on the measured electrode position to determine the cell concentration.
(5) The cell concentration apparatus for concentrating the cells contained in the medium, characterized by including a cell suspension vessel that supports the medium contained the cells, a plurality of electrodes disposed on the inner wall of the cell suspension vessel in the multi-layer form, a through-holes formed between the electrodes, a power source that applies an AC voltage to the electrode, a switch that switches among the plurality of electrodes to which the AC voltage is applied, and a discharge mechanism that discharges the medium which enters the cell suspension vessel.
(6) The cell concentrating apparatus described in (5) which is characterized in that the plurality of electrodes disposed on the inner wall of the cell suspension vessel in the multi-layer form presses the cells in the cell suspension against the bottom of the cell suspension vessel by means of a negative dielectrophoretic force.
(7) The cell concentrating apparatus described in any one of (1) to (6) which is characterized in that the voltage applied for generating an electric field between the electrodes is within the range from 20 mV to 1.23 V.
(8) The cell concentrating apparatus described in any one of (1) to (6) which is characterized in that the frequency applied for generating an electric field between the electrodes is within the range from 100 Hz to 10 MHz.
(9) The cell concentrating apparatus described in any one of (1) to (6) which is characterized in that the gap distance between the electrodes is equal to or less than 123 μm.
(10) The cell concentrating apparatus described in any one of (1) to (6) which is characterized in that the electrodes are made of any one of platinum, gold, chrome, palladium, rhodium, solver, aluminum, tungsten, and ITO, or any combination of them.
(11) A cell concentrating system composed of a cell concentrating apparatus described in any one of (1) to (6); and a control processor that controls the individual parts of the cell concentrating apparatus.
(12) A method for concentrating cells using a cell concentrating apparatus described in one of (1) and (2), which includes: supplying a medium containing cells in the cell suspension vessel; moving the piston-type incubator downward while applying an AC voltage to the electrode; and discharging the medium, which enters the piston-type incubator through the through-holes.
(13) A method for concentrating the cells using the cell concentrating apparatus described in (4), which includes: supplying the medium containing the cells in the cell suspension vessel; moving the piston-type incubator downward while applying an AC voltage to the electrode; and discharging the medium, which enters the piston-type incubator through the through-holes; measuring the impedance between the electrode disposed on the bottom surface of the piston-type incubator and the electrode disposed on the bottom surface of the cell suspension vessel using the impedance gauge and finding the volume of the cell suspension using the position sensor; finding the cell concentration based on the measured impedance and volume of the cell suspension; and ending the cell concentration process if the found cell concentration has reached the target level, and going to the step of moving the piston-type incubator downward if it is lower than the target level.
(14) A method for concentrating the cells using the cell concentrating apparatus described in one of (5) and (6), which includes: supplying the medium containing the cells in the cell suspension vessel; switching among the electrodes arranged in the multi-layer form to apply the AC voltage to them sequentially from one end; and discharging the medium, which enters the cell suspension vessel through the through-holes.
Note that according to the above-recited embodiments of the present invention, the cells contained in the medium may be concentrated efficiently with less load on them. The cell concentration may be measured by means of electric signals.
1 . . . Incubator ceiling substrate, 2 . . . Incubator bottom substrate, 3 . . . Upper electrode, 3A . . . Expansion mechanism, 3B . . . Side electrode, 4 . . . Lower electrode, 5 . . . Inside of the incubator, 5A . . . Culture medium, 5B . . . Cell, 6 . . . Medium inlet, 6A . . . Medium inlet valve, 7 . . . Medium outlet, 7A . . . Medium outlet valve, 8 . . . Mixed-gas inlet, 8A . . . Mixed-gas inlet valve, 9 . . . Mixed-gas outlet, 9A . . . Mixed-gas outlet valve, 10 . . . AC power source, 11 . . . Impedance measuring apparatus, 12 . . . DC power source, 13A . . . Switch, 13B . . . Switch, 14 . . . Switching element, 15A . . . Driving circuit, 15B . . . Driving circuit, 15C . . . Driving circuit, 16. Electrode, 17 . . . Capacitance C, 18 . . . Resistor R, 101 . . . Piston-type incubator, 102 . . . Concentrating electrode, 103 . . . AC power source, 104 . . . Switch, 105 . . . Cell suspension vessel, 106. Medium, 107 . . . Cell, 108 . . . Driving mechanism, 108A . . . Support mechanism, 109 . . . Discharge mechanism, 110 . . . Discharge tube, 111 . . . Impedance measuring apparatus, 112 . . . Switch, 113 . . . Bottom surface electrode, 114 . . . Position sensor, 115 . . . Magnetic sheet, 116 . . . Electrode, C17 . . . Capacitance C, R18. Resistor R, 119 . . . Control processor, 120 . . . Monitor.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-130197 | Jun 2011 | JP | national |
| 2011-181369 | Aug 2011 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2012/064466 | 6/5/2012 | WO | 00 | 1/16/2014 |
