JP2012080869A - Culture reactor and tool for culture reactor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture reactor usable for culturing and observing cells or the like, and to provide a tool for the same.SOLUTION: The culture reactor includes: a storage portion for culturing cells inside; a transparent plate 5 constituting at least part of a cell contact surface inside the storage portion; a first transparent conductive film 10d constituting a sensor portion and an electric stimulation portion or heater portion continuously disposed from the inside to the outside of the storage portion on the cell contact surface side of the transparent plate 5; and at least one layer of a second transparent conductive film and a third transparent conductive film constituting a heater portion or a sensor portion, respectively, on the opposite side of the first transparent conductive film setting surface. The tool for the culture reactor for fixing the culture reactor is also provided.

Description

  The present invention relates to a cell culture reactor that can be used for culturing and observing cells and the like, and a jig therefor.

  In recent years, for example, in the field of regenerative medicine, basic research, clinical trials, drug development, disease treatment methods, and the like regarding universal cells and proteins have been promoted. In general, in research using cultured cells, cell culture tests, cell observation, or analysis of the obtained cells are performed using a culture vessel for culturing cells. Such evaluation is performed directly on a microscopic basis. It may be desirable to do so by observing. In order to enable such observation, a culture container having a slide glass as a cell culture surface is known.

  In addition, warming or electrical stimulation may be applied to observe cell changes or to differentiate cells. In order to perform such a test, a heater for adjusting the temperature, an electrode for applying electrical stimulation to the cell, or an electrode for detecting an electrical signal from the cell or the cell temperature is arranged in the cell culture container. There is a case.

  In order to simultaneously achieve direct observation of cells with a microscope and control of culture temperature, the present inventor has reported a culture vessel in which a transparent conductive film functioning as a heater is formed on a slide glass substrate or petri dish (Patent Document) 1). In the main culture container, by providing the transparent conductive film on the transparent substrate, the cells in culture can be directly observed on the culture container with a microscope.

  Furthermore, in a cell evaluation test for nerve cells or the like, the reaction of cells when an electrical stimulus is applied may be observed. The inventors of the present invention have proposed a cell culture container capable of applying electrical stimulation to cells by forming a transparent conductive film on the cell contact surface (see Patent Document 2). Since the container is provided with a transparent conductive film on the cell culture surface, in addition to applying electrical stimulation to the cells, the transparent conductive film is used as a heater to directly heat the cells, An electrochemical culture experiment is possible through a transparent conductive film. In another transparent conductive film, the temperature of the cell culture surface in the culture vessel can be measured from the detected current.

JP 2009-093126 A (Claims etc.) JP 2009-201509 A (claim 6 and the like)

  However, since the culture containers described in Patent Document 1 and Patent Document 2 use a petri dish usually used for cell culture, in order to install an electrode for a transparent conductive film placed on a cell contact surface, the lid of the culture container It is necessary to provide a probe electrode in contact with the transparent electrode film layer disposed on the cell contact surface from the through hole. Therefore, there exists a problem that workability | operativity, such as opening and closing of the lid | cover during culture | cultivation, is bad. Furthermore, since it is necessary to install an electrode in the through hole, there is a problem that contamination is likely to occur. Furthermore, there is a problem that impurities may be mixed inside the culture vessel due to elution of impurities from the probe electrode.

  The present invention has been made to respond to the above-described problems, and an object thereof is to provide a culture reactor that has good workability and reduces the possibility of contamination.

  In order to achieve the above object, an embodiment of a culture reactor according to an embodiment of the present invention includes a container for culturing cells therein, and a transparent constituting at least a part of a cell contact surface in the container. It has a plate and the 1st transparent conductive film provided in the cell contact surface side of a transparent plate continuously from the inner side of an accommodating part to the outer side.

  Furthermore, the portion of the first transparent conductive film described above that extends outside the accommodating portion can constitute an electrode extraction terminal portion.

  Further, the accommodating portion is formed by joining the cylindrical portion and the flange portion, and the first transparent conductive film can be formed on the same plane that continues to the inside and the outside of the cylindrical portion.

  The first transparent conductive film may constitute a sensor unit, an electrical stimulation unit, or a heater unit. Further, the second transparent conductive film and the third transparent conductive film can also constitute a sensor unit, an electrical stimulation unit, or a heater unit.

  Furthermore, the outer periphery of the cylindrical portion has a corner portion that protrudes outward when viewed in the axial direction of the cylindrical portion, and the portion of the first transparent conductive film that extends outside the accommodating portion is from the corner portion. Can protrude.

  In the culture reactor of the present invention, the transparent plate may have at least one transparent conductive film on the side opposite to the first transparent conductive film installation surface. In the culture reactor of the present invention, when two or more transparent conductive films are provided on the opposite side of the first transparent conductive film installation surface of the transparent plate, an insulating layer is provided between the transparent conductive films. Moreover, in the culture reactor of the present invention, when a transparent conductive film is installed on the opposite side of the first transparent conductive film installation surface of the transparent plate, the transparent conductive film is second transparently in order from the transparent plate. The conductive film is referred to as a third transparent conductive film.

  In the culture reactor according to the embodiment of the present invention, the film thickness of at least one of the first transparent conductive film, the second transparent conductive film, and the third transparent conductive film is 80 nm or less. Is preferred.

  In the culture reactor of the present invention, at least one of the second transparent conductive film and the third transparent conductive film may have a film thickness of 500 nm or less.

  In one embodiment, the first transparent conductive film constitutes an electrical stimulation part or a sensor part, and its film thickness is 1000 nm or less, and the second transparent conductive film constitutes a heater part, and its film The thickness is preferably 500 nm or less, and the third transparent conductive film preferably constitutes a sensor unit for measuring temperature and has a film thickness of 80 nm or less. Alternatively, the first transparent conductive film constitutes an electrical stimulation part or a sensor part, and the film thickness thereof is 1000 nm or less, and the second transparent conductive film constitutes a sensor part for measuring temperature. In addition, it is preferable that the film thickness is 80 nm or less, and the third transparent conductive film constitutes a heater part and the film thickness is 500 nm or less.

  In addition, the culture reactor jig of the present invention includes an accommodating portion for culturing cells therein, a transparent plate constituting at least a part of the cell contact surface in the accommodating portion, and a cell contacting surface side of the transparent plate. A culture reactor having a transparent conductive film continuously provided from the inside to the outside of the housing portion is fixed. The culture reactor jig is for fixing the culture reactor, for supplying power to the transparent conductive film, or for irradiating light to the area where the transparent plate is overlapped with the electrode for detecting current. It has a translucent part.

  ADVANTAGE OF THE INVENTION According to this invention, workability | operativity is good, the possibility of contamination is reduced, and the culture reactor which an impurity is hard to mix can be provided.

It is a perspective view of the culture reactor which concerns on embodiment of this invention. It is a perspective view at the time of removing the cover part of the culture reactor shown in FIG. 1 from a dish part. It is a disassembled perspective view of the culture reactor shown in FIG. It is a top view at the time of seeing the culture reactor shown in FIG. 1 from the upper surface. It is the sectional view on the AA line of the culture reactor shown in FIG. FIG. 5 is a cross-sectional view of the culture reactor shown in FIG. 4 taken along the line BB. It is sectional drawing at the time of cut | disconnecting the top transparent plate which a culture reactor has with the AA of FIG. It is sectional drawing at the time of cut | disconnecting the bottom transparent plate which a culture reactor has with the AA of FIG. It is a top view at the time of seeing the transparent conductive film as a heater part of the lower surface side of a bottom transparent plate from the lower surface side. It is a top view at the time of permeate | transmitting in the thickness direction the heater part of a bottom transparent plate, and the transparent conductive film as a sensor part provided in the lower surface side of the heater part. It is a perspective view of the jig for culture reactors concerning an embodiment of the invention. It is a perspective view which shows the state which mounted the culture reactor in the jig | tool for culture reactors of FIG. It is a perspective view which shows the state which mounted the culture reactor on the jig | tool for culture reactors of FIG. 11, and attached the electrode etc. It is the schematic which shows the state in the case of mounting a culture reactor on the jig | tool for culture reactors of FIG. 11, and observing with a microscope. It is a block diagram at the time of culturing a cell using the culture reactor concerning an embodiment of the invention. In the modification of this invention, it is a top view at the time of seeing from the upper side of the bottom transparent plate which has a transparent conductive film as a sensor part on the upper side. It is sectional drawing at the time of cut | disconnecting the bottom transparent plate of FIG. 16 with the same line as the AA of FIG. It is a graph which shows the temperature rise at the time of using the transparent conductive film produced in a present Example as a heater part compared with a simulation result.

  Next, an embodiment of the culture reactor of the present invention will be described with reference to the drawings.

(Composition of culture reactor)
FIG. 1 is a perspective view of a culture reactor 1 according to the present embodiment. FIG. 2 is a perspective view when the lid 2 of the culture reactor 1 shown in FIG. FIG. 3 is an exploded perspective view of the culture reactor 1 shown in FIG. Hereinafter, when the lid 2 is removed from the culture reactor 1, the direction in which the lid 2 is detached from the dish 3 is referred to as “up”, and the opposite direction is referred to as “down”. The distance between the vertical directions is called the thickness.

  The culture reactor 1 is mainly used for culturing adherent cells. The culture reactor 1 is preferably made of a transparent material so that the state of the contained cells and the like can be seen. For example, the culture reactor 1 is preferably composed of polystyrene having high transparency and a low shrinkage rate upon curing. The culture reactor 1 has, for example, a substantially square shape with a piece of 40 mm when viewed from the top, a height of 15 mm in the thickness direction, and a so-called substantially rectangular parallelepiped shape.

  As shown in FIGS. 1 and 2, the culture reactor 1 mainly has a lid portion 2 and a dish portion 3. The dish part 3 has an accommodating part (which will be described in detail later) that can accommodate cells for culturing in an inner package, and the accommodated cells can be isolated from the outside by closing the opening part of the accommodating part with the lid part 2. .

  FIG. 4 is a plan view when the culture reactor 1 shown in FIG. 1 is viewed from above. FIG. 5 is a cross-sectional view taken along line AA of the culture reactor 1 shown in FIG. FIG. 6 is a cross-sectional view of the culture reactor 1 shown in FIG. Hereinafter, in each cross-sectional view, the scales in the thickness direction and the width direction are appropriately changed for easy viewing.

  The lid part 2 mainly has a frame part 2a, a top transparent plate 4 and a cylinder part 2b from the upper surface side. By joining, fitting, or insert molding the frame portion 2a, the cylindrical portion 2b, and the top transparent plate 4, the lid portion 2 is integrally configured.

  The frame part 2a is a frame-like member having a substantially square outer periphery when viewed from above, and having a substantially square opening inside thereof. The cylindrical part 2b is a substantially square cylindrical member whose outer periphery is smaller than the outer periphery of the frame part 2a when viewed from above. The cylinder part 2b is fixed to the frame part 2a so that the cylinder inner side overlaps with the opening part of the frame part 2a when viewed from above. The window part constituted by the frame part 2 a and the cylinder part 2 b includes a top transparent plate 4.

  The top transparent plate 4 is a substantially square transparent plate-like member having an outer periphery smaller than the outer periphery of the frame portion 2a and larger than the outer periphery of the cylindrical portion 2b. The top transparent plate 4 is exposed to the outer surface of the lid 2 from the opening of the frame 2a and the inside of the cylinder 2b. Therefore, when the user views from the upper side of the lid portion 2, an object to be observed (for example, cells in culture) existing below the top transparent plate 4 can be visually recognized through the top transparent plate 4. The top transparent plate 4 preferably has optical characteristics equivalent to those of the cover glass so that microscopic observation can be easily performed through the top transparent plate 4. Further, the top transparent plate 4 is required to have a high total light transmittance, a low refractive index, and uniform optical characteristics. For example, optical characteristics equivalent to borosilicate glass and borosilicate glass are required. It is mainly comprised from the resin etc. which have this. For example, the top transparent plate 4 is a substantially square member having a thickness of 150 μm and a length of one piece of 35 mm.

  Furthermore, the lid 2 may have a carbon dioxide injection hole 7, a perfusion injection hole 8, and a perfusion discharge hole 9. The carbon dioxide injection hole 7, the perfusion injection hole 8 and the perfusion discharge hole 9 are through-holes penetrating the upper side surface and the lower side surface of the lid part 2, and are located on the inner side of the inner periphery of the cylindrical part 2b when viewed from above. Provided. The carbon dioxide injection hole 7, the perfusion injection hole 8, and the perfusion discharge hole 9 are used for injecting or discharging carbon dioxide gas, oxygen, ozone, perfusion, or the like into the accommodating portion. For example, carbon dioxide, oxygen, ozone, or the like can be supplied from the carbon dioxide injection hole 7 to the cells being cultured. Alternatively, a medium or the like may be supplied from the perfusion injection hole 8 and an old medium or the like may be discharged from the perfusion discharge hole 9. The diameters of the carbon dioxide injection hole 7, the perfusion injection hole 8, and the perfusion discharge hole 9 are, for example, around 4 mm, and each supply substance may be supplied using a tube having a diameter that can be fitted to these hole diameters. Moreover, as a tube fitted to the carbon dioxide injection hole 7, the perfusion injection hole 8, and the perfusion discharge hole 9, a tube made of silicone resin, polyamide resin, or fluorine resin can be used.

  The dish part 3 mainly has a cylinder part 3a, a bottom transparent plate 5 and a flange part 3b from above. The lid portion 2 is integrally formed by joining, fitting, or insert molding the cylindrical portion 3a, the bottom transparent plate 5, and the flange portion 3b. The inner surface of the tube portion 3a, the upper surface of the flange portion 3b, the upper surface of the bottom transparent plate 5, and the lower surface of the lid portion 2 are spaces for storing cells to be cultured, that is, a storage portion. Constitute.

  The cylindrical portion 3a is a cylindrical member having a substantially square opening when viewed from above. Moreover, the cylinder part 3a has the flange part 6 extended in an outward direction so that the outer periphery may be surrounded in the lower surface side edge part. The flange part 6 has the notch part 7 in the corner | angular part of the cylinder part 3a. When the dish part 3 and the lid part 2 are fitted, the outer periphery of the cylinder part 3a is fitted in a form surrounded by the inner periphery of the cylinder part 2b. In that case, it is preferable that the outer periphery of the cylinder part 3a is slightly smaller than the inner periphery of the cylinder part 2b so that the inner periphery of the cylinder part 2b and the outer periphery of the cylinder part 3a are in close contact with each other.

  The flange 3b is a member having a substantially square outer periphery and a substantially square opening as viewed from above. The collar part 3b has an opening part inward rather than the cylinder inner side of the cylinder part 3a. The bottom transparent plate 5 is composed of the same members as the top transparent plate 4. Further, since the bottom transparent plate 5 has an outer periphery that is larger than the outer periphery of the cylindrical portion 3 a when viewed from above, the end portion of the bottom transparent plate 5 is exposed from the notch portion 7 of the flange portion 6. Therefore, the bottom transparent plate 5 is exposed to the outside of the dish portion 3 from the inside of the tube portion 3 a, the opening of the flange portion 3 b, and the inside of the notch portion 7. In the dish part 3 having such a structure, the user can illuminate the object to be observed on the bottom transparent plate 5 by irradiating light from the lower side of the dish part 3.

  FIG. 7 is a cross-sectional view of the top transparent plate 4 taken along the line AA. FIG. 8 is a cross-sectional view of the bottom transparent plate 5 taken along the line BB.

  The top transparent plate 4 and the bottom transparent plate 5 are referred to as transparent conductive films 10a, 10b, 10c, and 10d (hereinafter referred to as “transparent conductive film 10” when referring to all of the transparent conductive films 10a, 10b, 10c, and 10d). ) Is formed. The transparent conductive film 10 includes tin-doped indium oxide (ITO), tin oxide (TO), fluorine-doped tin oxide (FTO), and antimony-doped tin oxide (ATO). It can be composed of a semiconductor ceramic layer mainly composed of a conductive metal oxide such as Antimony-doped Tin Oxide (Zinc Oxide). The semiconductor ceramic layer mainly composed of these conductive metal oxides has both excellent transparency to visible light and high electrical conductivity. Among these transparent conductive layers, the transparent conductive film 10 mainly composed of ITO is particularly preferable since it has high transparency and heat resistance and has an antibacterial action.

  The transparent conductive film 10 functions as a heater part, a sensor part, or an electrical stimulation part. For example, when the transparent conductive film 10 has a predetermined electric resistance value, when the transparent conductive film 10 is energized, part of the electrical energy is released as thermal energy, and the transparent conductive film 10 generates heat. Due to the heat generation, the transparent conductive film 10 can function as a heater unit for heating the object to be observed.

  The transparent conductive film 10 can function as a sensor for measuring temperature. Generally, with respect to the temperature change ΔT, the electric resistance changes in a relationship of ΔR [Ω] = kΔT (k: coefficient). ΔR can be measured by detecting a current change ΔI [A] when a voltage V [V] is applied. Therefore, the temperature change can be measured by detecting the current value I flowing through the transparent conductive film 10. As described above, the transparent conductive film 10 functions as a sensor unit that detects the amount of current, whereby the temperature can be measured based on the obtained amount of current.

  Further, when there are cells in contact with the transparent conductive film 10, the transparent conductive film 10 can function as a sensor for detecting an electrical signal generated by the cells. For example, the electrical signal may be detected in order to measure the activity of a nerve cell or the like that generates the electrical signal.

  Further, when cells in contact with the transparent conductive film 10 are present, the cells can be electrically stimulated by energizing the transparent conductive film 10. In some cases, it is possible to control cell differentiation induction and the like by applying electrical stimulation to the cells. In addition, a direct current electric pulse signal may be used to perforate a partition wall such as a cell membrane to introduce a physiologically active substance such as a gene or a protein. Thus, the transparent electrode film 10 provided on the cell contact surface can function as an electrical stimulation unit.

  In the present embodiment, a transparent conductive film 10a serving as a heater portion is formed on the upper surface of the top transparent plate 4 (that is, the cell non-contact surface). The transparent conductive film 10a has a film thickness of 1000 nm or less, preferably 500 nm or less. The transparent conductive film 10a is formed so that the electric resistance value per layer of the transparent conductive film 10a is 150Ω or less. By providing the transparent conductive film 10a as a heater part on the upper surface of the top transparent plate 4, it can prevent that the cover part 2 becomes cloudy by dew condensation.

  Also, as shown in FIG. 8, a transparent conductive film 10d as a first transparent conductive film is formed on the upper surface of the bottom transparent plate 5. The transparent conductive film 10d is continuous from the inner side (that is, the cell contact surface) of the cylindrical part 3a and is formed on the outer side of the cylindrical part 3a and exposed from the notch 7 of the flange part 6 (that is, outside the reactor). Is formed. The portion of the transparent conductive film 10d that is exposed from the cutout portion 7 is hereinafter referred to as “electrode extraction portion 8”. Since the upper surface of the bottom transparent plate 5 is a cell contact surface that comes into contact with the cells to be cultured, by applying a voltage to the transparent conductive film 10d on the upper surface of the bottom transparent plate 5, it is possible to apply electrical stimulation to the cells. The transparent conductive film 10d may be a smooth film or a film having an uneven shape. When the transparent conductive film 10d has a concavo-convex shape, it becomes a cell scaffold, and thus the cell may be easily attached.

  FIG. 9 is a plan view showing the transparent conductive film 10b when the bottom transparent plate 5 is viewed from below.

  As shown in FIGS. 8 and 9, the bottom transparent plate 5 has a lower surface (that is, a cell non-contact surface), from the bottom transparent plate 5 side, a second transparent conductive film and a transparent conductive as a heater part. A film 10b, an insulating layer 11, and a transparent conductive film 10c as a third transparent conductive film as a sensor portion are formed.

  The transparent conductive film 10b functioning as a heater part has a film thickness of 1000 nm or less, preferably 500 nm or less. The transparent conductive film 10b is formed so that the electric resistance value per layer of the transparent conductive film 10b is 150Ω or less. The transparent conductive film 10b preferably has a groove 12 in a part thereof. When the transparent conductive film 10b has the groove 12 at an appropriate position, the transparent conductive film 10b has a uniform temperature distribution regardless of the distance from the electrode. The groove 12 has, for example, a width of 1 mm and extends from the ends of the two sides facing each other toward the other side. The groove 12 has a length in the longitudinal direction that is about half of the length in the same direction of the transparent conductive film 10b, and is formed so as not to intersect with the other grooves 12. The groove 12 is formed, for example, by removing the transparent conductive film 10b where the groove 12 is provided by laser processing or the like. The transparent electrode film 10 b is provided with an anode terminal 13 and a cathode terminal 14 at positions that do not overlap with the grooves 12.

The insulating layer 11 is a layer for insulation provided so that the transparent conductive film 10b and the transparent conductive film 10c are not electrically connected. The insulating layer 11 is an insulating material such as silicon dioxide (SiO ₂ ) or aluminum oxide (Al ₂ O ₃ ), and is mainly composed of a material having high heat resistance. The insulating layer 11 is preferably formed with a thickness that can be sufficiently insulated. For example, the insulating layer 11 is formed with a thickness of 2000 nm. In the present specification, “insulating” means substantially not conducting electricity, for example, having an electric conductivity of 1.0 × 10 ⁻⁶ S / m or less.

  FIG. 10 is a plan view showing the transparent conductive film 10b and the transparent conductive film 10c in an overlapping manner when the bottom transparent plate 5 is viewed from the lower side. In addition, illustration of the insulating layer 11 is abbreviate | omitted in FIG.

  The transparent conductive film 10b includes a transparent conductive film 10c as a sensor part on the lower surface side through an insulating layer 11. The transparent conductive film 10c has a film thickness of 18 to 80 nm, and particularly preferably a film thickness of 18 to 50 nm or less. When the film thickness of the transparent conductive film 10c is 18 nm or more, it is easy to form a uniform film. When the film thickness of the transparent conductive film 10c is 80 nm or less, the electrical resistance value per sheet of the transparent conductive film 10c is 10 kΩ or more. When the electrical resistance value of the transparent conductive film 10c is 10 kΩ or more, the magnitude of the current flowing through the transparent conductive film 10c can be changed to a level that can be measured by a temperature change. Therefore, the transparent conductive film 10c can function as a sensor. Moreover, when the film thickness of the transparent conductive film 10c is 80 nm or less, since the rate of change of the electrical resistance value according to the environmental temperature is sufficiently large, the transparent conductive film 10c becomes a sensor unit with excellent detection sensitivity. In particular, a sensor part having a film thickness of 50 nm or less of the transparent conductive film 10c is more excellent in detection sensitivity because the change rate of the electric resistance value according to the environmental temperature is 10% or more.

  The transparent conductive film 10c functioning as the sensor unit is formed to have a width of 1 mm and a total length of 10.8 mm, for example. Specifically, the transparent conductive film 10c is configured by providing six transparent conductive films having a width of 1 mm and a length (SL2 in FIG. 10) of 20 mm in parallel and connecting them in a bellows shape. Can do. By increasing the overall circuit length of the transparent conductive film 10c and arranging it in a bellows shape, the area of the detectable region can be increased. Moreover, the edge part of the transparent conductive film 10c is connected to the terminal for anodes and the terminal for cathodes whose length (SL3, SL4 of FIG. 10) is 9 mm. The bellows-shaped part of the transparent conductive film 10c is arranged in the heat generating part of the transparent conductive film 10b as a heater part. Therefore, since the resistance value of the transparent conductive film 10c changes according to the temperature of the heater part, by detecting the amount of current that flows when a certain voltage is applied to the transparent conductive film 10c, A temperature change can be detected. That is, by using the transparent conductive film 10c having the above-described film thickness and shape, the sensor function of the transparent conductive film 10c can be optimized.

  Moreover, in the culture reactor 1 as described above, it is easy to apply a voltage to the transparent conductive film 10c. This is because a voltage can be applied from the electrode extraction portion 8 exposed from the cutout portion 7 in the transparent conductive film 10c. Therefore, it is not necessary to provide a through hole in the lid portion 2 or provide a through hole between the cylindrical portion 3a and the flange portion 3b to insert the electrode into the storage portion. Thus, since there is no need to provide a through-hole in the lid of the culture vessel, workability is good and contamination is unlikely to occur. In addition, by extending the transparent conductive film 10c from the inside to the outside of the storage unit, the electrode does not contact the culture, and the substance constituting the electrode is compared with the case where the probe electrode is inserted into the storage unit. Since there is no eluate from the impurities, contamination of impurities can be reduced.

  In the culture reactor 1 as described above, the temperature can be detected with higher detection sensitivity using the transparent conductive film 10c. This is because, in order to improve the resistance value of the transparent conductive film 10c as the sensor unit, the planar shape of the transparent conductive film 10c as viewed from above is not only bellows but also the film thickness is 80 nm or less. The transparent conductive film 10c having such a structure has a change rate of the electric resistance value corresponding to the temperature of 10% or more, and becomes a sensor unit having excellent detection sensitivity. Moreover, you may use the control part which adjusts the emitted-heat amount of a heater part according to the temperature detected with the sensor part. Moreover, since the transparent conductive film 10c as a sensor part and the transparent conductive film 10b as a heater part adjoin only through an insulating layer, temperature detection is easy. Furthermore, since the transparent conductive film 10b is adjacent to the cells in culture only through the bottom transparent plate 5, the cells can be efficiently heated.

  Moreover, in the culture reactor 1 as described above, the groove 12 is provided in the transparent conductive film 10b as the heater portion, so that it can be heated uniformly regardless of the place. In particular, since the transparent conductive film 10b is adjacent to the cell contact surface via the bottom transparent plate 5, it is preferable that the heater portion has a more uniform temperature distribution.

(Culture reactor jig)
Next, the culture reactor jig 20 according to the present embodiment will be described. FIG. 11 is a cross-sectional view when the culture reactor 1 according to the present embodiment is fixed to the culture reactor jig 20 and observed with an inverted microscope.

  FIG. 12 is a perspective view showing a culture reactor jig 20 according to the present embodiment. FIG. 13 is a perspective view showing a state in which the culture reactor 1 is placed on the culture reactor jig 20 of FIG. FIG. 14 is a perspective view showing a state in which the culture reactor 1 is fixed to the culture reactor jig 20 of FIG. In FIG. 12, illustration of a probe electrode 25 (to be described later) and a member for holding the probe electrode 25 are omitted for ease of viewing.

  The culture reactor jig 20 has a base 21 on which the culture reactor 1 is placed. The pedestal 21 is a surface that comes into contact with the lower surface of the dish portion 3. The pedestal 21 has a fixing holder 22 that functions as a fixing means for fixing the culture reactor 1 from the side surface side. The fixed holder 22 is preferably movable in a direction in which the culture reactor 1 is sandwiched.

  The culture reactor jig 20 includes a sensor electrode 24, probe electrodes 25 and 25, electrodes 26 and 26, heater electrodes 27 and 27, and the like as electrodes for supplying power to the transparent conductive film 10 or detecting current. Have. The sensor part electrode 24 and the heater part electrodes 27, 27 are provided on the upper surface of the base 21. Therefore, when the culture reactor 1 is placed on the pedestal 21 in a predetermined direction, the sensor electrode 24 is electrically connected to the anode electrode and the cathode electrode of the transparent conductive film 10c. Similarly, when the culture reactor 1 is placed on the pedestal 21 in a predetermined direction, the heater electrodes 27 and 27 are electrically connected to the anode terminal 13 and the cathode terminal 14. Therefore, by fixing the culture reactor 1 at a predetermined location on the pedestal 21, it is possible to detect the value of the current flowing through the transparent conductive film 10 c via the sensor electrode 24, and via the heater electrodes 27 and 27. A voltage can be applied to the transparent conductive film 10b. The pedestal 21 or the fixed holder 22 is preferably provided with a guide groove or the like so that the culture reactor 1 can be placed only in a predetermined direction.

  The probe electrodes 25, 25 are provided to electrically connect to the electrode extraction portion 8 and apply a voltage to the transparent conductive film 10d. Note that the probe electrodes 25 and 25 may be electrodes for flowing a current or detecting a current, not for applying a voltage. In addition, the electrodes 26 and 26 are provided to supply power to the transparent conductive film 10 a provided on the lid 2. Therefore, after placing the culture reactor 1 on the pedestal 21 with the fixed holder 22 or the like, the probe electrodes 25 and 25 are connected to the electrode take-out portion, and the electrodes 26 and 26 are respectively attached to the transparent conductive film 10a provided on the lid portion 2. Can connect.

  A through hole 28 serving as a translucent portion is provided in a region of the pedestal portion 21 that overlaps the opening. Therefore, the light from the illumination member provided inside the culture reactor jig 20 can illuminate the cells being cultured inside the culture reactor 1 through the through hole 28 and the opening.

  FIG. 15 is a block diagram showing an outline of a system for culturing cells and the like in the culture reactor 1.

  The culture reactor jig 20 is used as follows, for example. First, the culture reactor 1 is fixed by the fixed holder 22. It is placed on a frame directly above the objective lens of the inverted microscope 30. Next, the electrode is connected to a power source (illustrated), and carbon dioxide is supplied from the carbon dioxide supply unit 31 to the controller 33 through the tube 32. The controller 33 senses a concentration suitable for culture, finely adjusts the sensing value to a concentration suitable for culture (for example, about 5%) by the carbon dioxide supply unit 32, and the culture reactor 1 through the tube 32. Carbon dioxide gas is supplied into the storage section. The method used for perfusion is the same, and is performed using the perfusion unit device 34 to be circulated.

  By using the culture reactor jig 20 as described above, each electrode for connection to the culture reactor 1 can be easily connected. Moreover, since the culture reactor 1 can be fixed, it is easy to observe the cultured cells. Further, by using the culture reactor 1 as described above, the supply from each gas cylinder can be appropriately supplied by the controller 33. Therefore, without preparing gas cylinders for each culture condition, the culture conditions can be changed by supplying the supply to the controller via a tube or the like. Therefore, it is possible to reduce the size of the culture apparatus and to effectively use the space on the desk for microscopic observation.

(Other embodiments)
While the embodiments of the culture reactor 1 and the apparatus for culture reactor of the present invention have been described above, the present invention is not limited to the above-described embodiments, and can be implemented with various modifications.

  For example, in the present embodiment, the transparent conductive film 10c as a sensor unit functions as a sensor for detecting temperature, but the sensor unit may be used for detecting other than temperature.

  In the present embodiment, the dimensions of each member have been illustrated, but the present invention is not limited to the illustrated dimensions, and may include errors. For example, the culture reactor 1 is a square having a side of 40 mm when viewed from the top and has a thickness of 15 mm, but is not limited to this value. Further, the culture reactor 1 does not have to be substantially square when viewed from above. These values can be appropriately changed depending on the cell to be measured. However, the culture reactor 1 having a size capable of efficiently observing using the inverted microscope 30 and capable of culturing cells has an area equivalent to a square having a side of 20 to 50 mm in a plan view seen from above. It is preferable to have a shape.

  Moreover, in this Embodiment, although the culture reactor 1 shall be substantially square by the planar view seen from the top, it is not restricted to such a form. For example, the shape when viewed from above may be any other shape such as a circle, a polygon other than a square, an ellipse, or the like. When the outer periphery of the cylindrical portion 3b has a corner portion when viewed from above, the electrode extraction portion 8 can be easily formed by providing the notch portion 7 at the corner portion. In the present specification, the “corner portion” may be chamfered with a flat surface or a curved surface regardless of whether it is an acute angle or an obtuse angle. In addition, when the shape when viewed from above the culture reactor 1 is a rectangle, observation is easy when the culture reactors 1 are observed side by side. Further, when the shape of the culture reactor 1 when viewed from the upper side is a regular polygon, it has an advantage that it can be easily stacked when it is stacked and stored.

  Moreover, in this Embodiment, although the electrode extraction part 8 shall be provided in a corner | angular part, it is not restricted to such a form. For example, by providing the notch portion 7 other than the corner portion in the flange portion 6, the electrode extraction portion 8 can be provided at a portion other than the corner portion. However, by providing the electrode take-out portion 8 at the corner, the electrode can be easily brought into contact.

  Moreover, in this Embodiment, although the cover part 2 shall sandwich the top transparent plate 4 between the frame part 2a and the cylinder part 2b, it is not restricted to such a form. Any form may be used as long as the lid 2 can support the top transparent plate 4. However, since the top transparent plate 4 is thin glass and is brittle, it can be suitably fixed by fixing it so as to be sandwiched between the frame portion 2a and the cylindrical portion 2b. Similarly, although the plate part 3 is integrally molded so that the bottom transparent plate 5 may be pinched | interposed between the cylinder part 3a and the collar part 3b, it is not restricted to such a form.

  Moreover, in this Embodiment, although the cover part 2 has the carbon dioxide injection hole 7, the perfusion injection hole 8, and the perfusion discharge hole 9, these are not essential. The lid 2 may have only the carbon dioxide injection hole 7 or only the perfusion injection hole 8 and the perfusion discharge hole 9, or all of the carbon dioxide injection hole 7, the perfusion injection hole 8, and the perfusion discharge hole 9. It may not have. Moreover, the cover part 2 may have a hole for inserting a substance other than carbon dioxide and perfusion. Further, the substance may be injected from the carbon dioxide injection hole 7, the perfusion injection hole 8, and the perfusion discharge hole 9 by something other than a tube. For example, you may inject | pour with a syringe etc.

  In the above-described embodiment, the frame portion 2a and the flange portion 3b have substantially square openings. However, the present invention is not limited to such a configuration. For example, the openings provided in the frame portion 2a and the flange portion 3b are not square but may be various shapes such as a circle, a polygon, and an ellipse. However, by making the openings of the frame portion 2a and the flange portion 3b the same shape as the inner periphery of the cylindrical portion 2b and the cylindrical portion 3a, a wider area that can be observed with a microscope can be secured.

  Moreover, although the above-mentioned embodiment has all the transparent conductive films 10a, 10b, 10c, and 10d, some of them may not be present. For example, when the culture reactor 1 is used in a place where condensation does not occur, the transparent conductive film 10a is not essential. If the cells do not need to be heated, the transparent conductive films 10b and 10c need not be provided. Further, when it is not necessary to detect the temperature when heated by the transparent conductive film 10b, the transparent conductive film 10c may not be provided. Moreover, you may provide another transparent conductive film 10 as a sensor part on the upper side of the transparent conductive film 10a through an insulating layer. Or the transparent conductive film 10 as a sensor part may be provided in a cell contact surface.

  FIG. 16 is a plan view of the transparent conductive film 10e as a sensor unit provided on the cell contact surface when viewed from the upper side of the bottom transparent plate 5. FIG. FIG. 17 is a cross-sectional view of the transparent conductive film 10e taken along the line AA in FIG.

  As shown in FIG. 16, when the transparent conductive film 10e as a sensor part is provided on the cell contact surface, the transparent conductive film 10e has a width of 1 mm and a total length of 10. It is formed to be 8 mm or more. Specifically, it can be configured by providing six transparent conductive films having a width of 1 mm and a length (SL2 in FIG. 16) of 20 mm in parallel and connecting them in a bellows shape. Further, the two ends of the transparent conductive film 10 e extend to different electrode extraction portions 8 along the sides of the bottom transparent plate 5. In the transparent conductive film 10 e shown in FIG. 16, the current can be measured by the probe electrode 25. By having the structure shown in FIG. 16, since the transparent conductive film 10e is provided on the cell contact surface, the cell temperature can be directly detected. Therefore, when it is required to measure the cell temperature more accurately, it is preferable to provide the transparent conductive film 10e on the upper side of the bottom transparent plate 5 as shown in FIG. In addition, when providing the transparent conductive film 10e as a sensor part in a cell contact surface, it is preferable to provide an insulating layer above the transparent conductive film 10e so that an electric current may not flow into a cell. However, if the cells to be cultured can withstand electrical stimulation of the current level for functioning as a sensor unit, or if there is no particular change in response to electrical stimulation, an insulating layer is not provided on the transparent conductive film 10e. May be.

  Moreover, the transparent conductive film 10 may be provided in the cylinder part 3b etc. However, since the adherent cells generally adhere to the upper surface of the bottom transparent plate 5, it is preferable to provide the bottom transparent plate 5 with the transparent conductive film 10 as a heater portion so that the cells can be directly heated. Moreover, since it is necessary to provide the transparent conductive film 10 as an electric stimulation part in the surface to which a cell adheres, it is preferable to provide in the upper surface of the bottom transparent plate 5 normally. Moreover, you may provide the transparent conductive film which functions also as a heater part or a sensor part in a cell contact surface.

  Moreover, in this Embodiment, although the transparent conductive film 10a provided in a cell contact surface shall function as an electrical stimulation part, it is not restricted to such a form. For example, the transparent conductive film 10a may function as a sensor unit for receiving an electrical signal generated by a cell. When the transparent conductive film 10a functions as a sensor unit for receiving an electric signal from a cell, the transparent conductive film 10a may be divided so that the electric signal can be observed for each region.

  In the above-described embodiment, the culture reactor 1 is made of a transparent member. Here, transparent refers to a material that substantially transmits visible light, for example, total light transmittance. Of 60% or more, preferably 80% or more. Further, in the present specification, “transparent” is not only colorless and transparent, but may be colored and transparent in the case of the above-described total light transmittance. Moreover, as the top transparent plate 4 and the bottom transparent plate 5, it is more preferable to use a plate having a higher transmittance than other members.

  Next, examples of the present invention will be described.

(Heater design)
A substantially square culture reactor having a piece of 40 mm as viewed from above was prepared. The heater used for culturing cells is usually required to have the ability to raise the temperature in the culture reactor by about 20 ° C. Therefore, the specification of the transparent conductive film as a heater part that can be heated from room temperature to 20 ° C. by a transparent conductive film having a length of 2.0 cm and a width of 2.3 cm was designed by calculation. The calculation results are shown in Table 1.

  Next, Table 2 shows the result of calculating the inter-terminal resistance value when the groove is provided in the transparent conductive film and calculating the power consumed thereby.

  Further, Table 3 shows the result of comparison between the resistance value voltage and the actual measurement value in consideration of the rising temperature. Since the thickness of the transparent conductive film differs depending on the processing lot, taking into account the error of the electrical resistance value of the transparent conductive film, the value of 80% of the electrical resistance value in Table 2 is the actual electrical resistance value. Assuming that

  Based on the above study, the design specifications of the heater were determined. Table 4 shows the specifications. Further, FIG. 18 shows a graph showing the temperature characteristics with respect to the current of the heater manufactured according to the specifications shown in Table 4. In addition, the transparent conductive film was formed into a film with the vacuum evaporation apparatus (BMC-1400, the product made by Shincron Co., Ltd.). Moreover, in order to provide a groove | channel in a transparent conductive film, the transparent conductive film of the groove part was removed with the YAG laser using the laser micro cutter (LR-3100FSUV, the product made by HOYA Corporation).

(Sensor design)
Next, sensor design will be described. First, the insulating layer was provided in the transparent conductive film as a heater part produced as mentioned above. As the insulating layer, a SiO ₂ film was formed using a vacuum deposition apparatus (BMC-1400, manufactured by Shincron Co., Ltd.). Next, a transparent conductive film made of ITO was formed as a sensor part on the formed insulating layer.

  Here, when the resistance value per transparent conductive film (hereinafter referred to as sheet resistance value) was 5 to 150 kΩ / sheet, the transparent conductive film could function as a sensor. Therefore, in order for the sensor unit to function as a temperature sensor, it is preferable that there is a sufficient change in resistance value between terminals with respect to the measured temperature. In this example, in order to set the resistance value between terminals to 10 kΩ or more, six layers each having a width of 1 mm and a length of 20 mm are provided in parallel, connected in a bellows shape, and the total circuit length is 10.8 mm. A transparent conductive film was formed. In FIG. 10, a transparent conductive film as a sensor portion was formed so that SL1 was 20 mm, SL2 was 18 mm, SL3 was 9 mm, and SL4 was 9 mm.

  When the sheet resistance value was 5 to 150Ω / sheet, the film thickness of the transparent conductive film and the inter-terminal resistance value were calculated by simulation according to the sheet resistance value. The results are shown in Table 5.

  Table 6 shows the results of calculating the inter-terminal resistance values of the transparent conductive films having different film thicknesses by the same calculation method as the specification calculation of the transparent conductive film as the heater section.

  From the results of Table 6, when the film thickness was 80 nm or less, the resistance value between terminals was 10 kΩ or more. Therefore, it can be said that a transparent conductive film having a thickness of 80 nm or less is suitable for use as a sensor portion.

  Here, a transparent conductive film having a sheet resistance value of 120Ω / sheet and a film thickness of 18 nm was actually created, and the resistance value between terminals with respect to temperature was measured by a resistance meter (Digital Multitester R6552, manufactured by Advantest Corporation). It measured using. The temperature was measured using a thermistor thermometer (RF-100, manufactured by Electronic Temperature Instruments Ltd.). Table 7 shows the measurement results.

  As shown in the data in Table 7, in a transparent conductive film having a sheet resistance value of 120Ω / sheet and a film thickness of 18 nm, there is a correlation between an increase in temperature and an increase in resistance value between terminals. In addition, a sensor having a sheet resistance value of 120Ω / sheet and a film thickness of 18 nm had sufficient detection ability as a sensor because the inter-terminal resistance value per 1 ° C. changed by 10% or more.

(Comparative example)
Here, the resistance value between terminals when the temperature was changed was measured using a transparent conductive film having a sheet resistance value at 23 ° C. of 5Ω or less and a film thickness of 400 nm. Table 8 shows the measurement results.

  From the results in Table 8, in the comparative example in which the film thickness was 400 nm, the correlation function between the temperature increasing tendency and the inter-terminal resistance value was lower than in the case where the film thickness was 18 nm. Moreover, since the change rate of the resistance value between terminals per 1 degreeC was 6%, it was difficult to use as a sensor part.

  The present invention can be used, for example, for culturing and observing cells and the like.

1 Culture Reactor 2, 2a, 2b Lid 3, 3b Dish 3a Tube (Part of Dish)
4 Top transparent plate (transparent plate, part of lid)
5 Bottom transparent plate (transparent plate, part of the pan)
6 Buttocks (part of the plate)
7 Notch (part of plate)
8 Electrode extraction part (transparent conductive film, sensor part or part of electrical stimulation part)
11 Insulating layers 10a, 10 Transparent conductive film (heater part)
10b, 10 transparent conductive film (heater part, second transparent conductive film)
10c, 10 transparent conductive film (sensor part, third transparent conductive film)
10d, 10 transparent conductive film (electric stimulation part, first transparent conductive film)

Claims (11)

A container for culturing cells inside,
A transparent plate constituting at least a part of the cell contact surface in the accommodating portion;
A culture reactor comprising a first transparent conductive film provided continuously from the inside to the outside of the housing portion on the cell contact surface side of the transparent plate. The culture reactor according to claim 1,
A portion of the first transparent conductive film that extends outside the accommodating portion constitutes an electrode lead-out terminal portion. In the culture reactor according to claim 1 or 2,
The culture reactor according to claim 1, wherein the first transparent conductive film constitutes a sensor unit, an electrical stimulation unit, or a heater unit. In the culture reactor according to any one of claims 1 to 3,
The accommodating portion is formed by joining a cylindrical portion and a collar portion,
The culture reactor according to claim 1, wherein the first transparent conductive film is formed on the same plane continuous to the inside and the outside of the cylindrical portion. The culture reactor according to claim 4,
The outer periphery of the cylindrical portion has a corner portion that protrudes outward when viewed in the axial direction of the cylindrical portion,
A portion of the first transparent conductive film that extends outside the accommodating portion protrudes from the corner portion. The culture reactor according to any one of claims 1 to 5,
On the opposite side of the first transparent conductive film installation surface of the transparent plate, it has at least one layer of the second transparent conductive film and the third transparent conductive film,
In the case where both the second transparent conductive film and the third transparent conductive film are provided, the second transparent conductive film and the third transparent conductive film are laminated via an insulating layer. Feature culture reactor. The culture reactor according to claim 6,
A culture reactor, wherein the thickness of at least one of the first transparent conductive film, the second transparent conductive film, and the third transparent conductive film is 80 nm or less. In the culture reactor according to claim 6 or 7,
The first transparent conductive film constitutes a sensor part or an electrical stimulation part,
The culture reactor according to claim 2, wherein the second transparent conductive film and the third transparent conductive film constitute a heater part or a sensor part, respectively. In the culture reactor according to any one of claims 6 to 8,
At least one of the second transparent conductive film and the third transparent conductive film has a film thickness of 500 nm or less. A culture reactor according to any one of claims 6 to 9,
The first transparent conductive film constitutes the electrical stimulation part or the sensor part, and has a film thickness of 1000 nm or less,
The second transparent conductive film constitutes the heater part and has a film thickness of 500 nm or less,
The third transparent conductive film constitutes the sensor unit for measuring temperature and has a film thickness of 80 nm or less. A container for culturing cells inside,
A transparent plate constituting at least a part of the cell contact surface in the accommodating portion;
On the cell contact surface side of the transparent plate is a culture reactor jig for fixing a culture reactor having a transparent conductive film continuously provided from the inside to the outside of the housing part,
Fixing means for fixing the culture reactor;
An electrode for supplying power to the transparent conductive film or for detecting a current;
A culture reactor jig having a light transmitting portion for irradiating light to a region overlapping with the transparent plate.

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