JP2009201509A - Culture vessel formed by transparent electroconductive film processing and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture vessel for more precise culture environment by solving the problems that the cell culture environment using a conventional culture vessel has an apparatus including microscope observation system and functions including temperature control or the like of the culture chamber on an observation stand thereof, and the temperature control is carried out with a measure to control the atmospheric temperature in the culture chamber without controlling temperature in the vicinity of a cell sample, therefore, explication concerning the basis associated with proliferation, life or death of cells has been recently expected. <P>SOLUTION: The method includes forming a resistive element and an electroconductive film by making use of each characteristic of materials for the transparent electroconductive film, making use of transparency, forming the film to have a scaffold structure and making use of antimicrobial actions as physical properties. In addition, the temperature control, one of factors in culture, is carried out by processingly installing a heater made of a transparent electroconductive film allowed to control temperature by bringing the heater to directly heat a sample, and a sensor by using the film to cell culture vessels such as petri dishes. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell culture vessel having functionality for improving the accuracy of a culture environment. A heater part made of a transparent conductive film is attached to a culture vessel, and the local part of the vessel is directly conducted at an arbitrary temperature to control the temperature, and is provided in a simple and inexpensive form. is there. One of the purposes is to accurately control the temperature of the culture sample part by providing a temperature sensor for temperature measurement and monitoring in the vicinity of the heater heating part or on the same pattern. The second is to provide a top transparent plate with an anti-fogging function on the lid of the culture vessel so as not to cause any trouble during microscopic observation. The third is to form the heater part and the sensor part described above on the surface of the petri dish, well plate, test tube or flask, or on the outer peripheral surface or bottom surface on the body side. As a method, a transparent conductive film is formed on the culture vessel main body, and then pattern processing is performed in the heater unit or the sensor unit in order to appropriately function.

  In the field of regenerative medicine, basic research related to universal cells and proteins, clinical development, drug discovery, and disease treatment are being developed, and cell culture tests, observation evaluations, and analytical methods have evolved. In addition, the culture vessel is also required to have high accuracy and high functionality.

Among the culture environments, the temperature environment is important. What responds to the temperature environment which can control temperature correctly also about a culture container is desired. There is a proposal using a conductive polymer as a heating means in the container.
Special table 2003-512809 However, this proposal does not overcome the problem of non-uniform heat distribution on the heat generation surface, which is a conventional problem, and this method cannot cope with high-magnification observation with a microscope.

There is also a proposal for temperature control using a heating resistor attached to the bottom surface of the culture vessel and a temperature-responsive polymer attached to the bottom surface.
Published 2007-174982 In this proposal as well, the bottom surface is not formed so that high magnification observation of 40 to 100 times is possible from the limit of the wall thickness due to molding problems due to being a polymer material as in the case of (0003). In addition, since the heating means is in the vicinity of the cell container and the sensor for monitoring the temperature has not been proposed, there is a problem as a method for accurately controlling the cell sample surface. For this purpose, cell culture cells are detached and collected, and more precise control and high magnification observation are not required. Further, the heating resistor used for the heating means is also proposed to use a large manufacturing means using an exposure apparatus or the like which is a semiconductor manufacturing method. This is to heat the polymer material substrate using an external heating means, which is different from the proposal of simultaneously forming a sensor on a heat generating substrate which is a transparent plate as in the present invention.

As another aspect, temperature control is important when observing cultured cells under a microscope, and a transparent heater is used so as not to obstruct the microscope observation.
Published 2004-206069 Published 2007-212633 However, in these methods, since heating is performed through a heating plate installed on the microscope stage, the culture vessel such as a petri dish becomes indirect heating in which heating is performed through a chamber constituting the heating plate that houses the vessel. There was a limit to heating the cultured sample at a truly predetermined temperature. That is, in order to achieve a predetermined temperature directly under the culture sample, a culture vessel having a method for reducing heat conduction loss is required.

On the other hand, a sensor is indispensable for temperature control, and a transparent thin film sensor is expected for observation with an optical microscope. In the next report, ITO sensors have been proposed. Since these sensors are also manufactured using an expensive apparatus such as a photolithographic method, they have been awaited to be manufactured by an even cheaper and simpler method. The present inventors use a laser processing method, which is one of laser processing methods, to use a heavy equipment and a complicated manufacturing method by converting the same ITO thin film forming the heater heating surface into a temperature sensor. We have proposed a manufacturing method that can be used at low cost. .
Development of DNA purification device for deep sea using microfabrication technology (Production Research 2004, Vol. 50, No. 6, pp. 29-32)

Another example of a transparent thin film temperature sensor is the use of a temperature sensor on the opposite side of the heater rather than on the same substrate. For the same reason as the above item (0006), it is a proposal to form a sensor independently, in addition to cost such as forming the heater part and the sensor part on separate surfaces. This is different from the structure of the present invention in which a heater mainly for heating is formed using a transparent conductive film and a sensor is provided on the same substrate to function as a cell culture vessel.
Open hei 10-340802

  In addition, during cell culture, the cell container is always heated, so the inside of the petri dish becomes clouded, and the object lens is used on the bottom of the dish on the observation side in the inverted microscope observation. From the lid surface of the vessel, it was necessary to irradiate transmitted light, which sometimes hindered microscopic observation.

  In the actual registration No. 3142507, the present inventors proposed a resin or glass slide glass substrate and a bio substrate in which a heater having a transparent conductive film formed thereon is integrated, and the heater part can uniformly generate heat. Thus, it is disclosed that a resistor circuit provided with a slit shape processing can be provided and functioned, and the heater pattern circuit can be easily processed by a laser device.

  Further, in Japanese Patent Application No. 2007-285543, a processing proposal is made for a heater pattern in which a heat generation area is further fractionated and different heat generation is performed for each fractionation area. A plurality of different temperatures are generated simultaneously on one substrate.

  However, these devices and inventions depend on the conventional sensing method for temperature control as a method for more accurately controlling the direct heating method, which is the original feature, and there remains a problem in dealing with it.

  The objective of this invention is invention of the cell culture container which has the function which enables it to exhibit the electrical and optical and physical property which are each characteristic of a transparent conductive film material. For electrical properties, use resistors and conductive films; for optical properties, make use of their transmittance; for shapes, use scaffold structures for extracellular matrix; and for physical properties, use antibacterial action. It is.

  In order to make the resistor act as a heat generating heater, it is the same as forming a pattern part to be a heater using ITO, which is a mixture of indium oxide and tin oxide, which is one of transparent conductive film materials. A transparent temperature sensor portion is formed on a transparent plate using the same apparatus used for forming the pattern portion. By attaching this to the culture vessel itself, the temperature environment of the cell sample surface can be accurately constructed. Furthermore, it is possible to eliminate the need for a heating chamber for accommodating the culture vessel. The transparent conductive film material may be a transparent oxide material such as ZnO or TiO 2.

  Using this temperature sensor, the temperature control is easily performed by detecting and feeding back the temperature of the heat generating part on the heater surface.

  Further, as a processing method for manufacturing the sensor at a low cost, it can be dealt with by adopting a laser processing method used for forming a slit processing circuit used for the purpose of uniformizing the heat generating portion.

  In order to control the temperature of a culture sample directly and accurately, there is a problem of measuring the temperature of the heater surface. A bottom transparent plate with the heater and sensor formed on the same surface is attached to a petri dish or well plate as a culture vessel. Appropriately attaching to the pan.

  Furthermore, a transparent conductive film pattern serving as a heater and a sensor is appropriately attached to the surface of the culture vessel, the outer peripheral surface of the trunk, or the bottom.

  The second object of the present invention is to use the top transparent plate on which the transparent conductive film pattern to be the heater is formed and mount it on the portion that becomes the lid of the petri dish or well plate, so that it can also be used for anti-fogging applications. It is to provide what was made. In this case, the transparent conductive film pattern can be directly formed on each lid of the petri dish or well plate.

  Since the transparent plate according to claim 1 also serves as a cover glass for microscopic observation, it provides quartz or borosilicate glass that satisfies the performance of low refractive index and high transmittance, or a resin that has optical performance. Is to do.

  Furthermore, another object is to provide a culture vessel corresponding to an electrochemical method by paying attention to the electrical conductivity of a transparent conductive film formed on the cell adhesion surface side of the culture vessel. At the same time, it is to provide the culture vessel having a scaffold structure which is one of functions as an adhesion factor serving as an extracellular matrix on the cell attachment surface side.

  On the other hand, the cell container of the present invention has a feature that it becomes a small culture container that does not require a culture chamber for storing the cell container. There was a demand for miniaturization that requires gas furan vessels and uses them as built-in culture devices.

  According to claim 1 of the present invention, there is provided a cell culture vessel comprising a transparent conductive film formed on a cell culture vessel main body for culturing adherent cells, wherein the transparent conductive film comprises a heater part and a current detection sensor. The cell culture vessel is characterized by having a portion. The cell culture vessel main body has a shape in which a bottom transparent plate is attached to a hole-performed one and a shape that is not perforated. .

According to the second aspect of the present invention, the bottom plate of the dish is formed by punching a dish portion of a petri dish, which is a cell culture container, so as to close the hole. The bottom transparent plate has a transparent conductive film formed on the back surface, further includes a resistance circuit formation using the transparent conductive film as a heater, and a sensor for detecting the current to obtain a desired resistance value. is there. By providing the heater pattern circuit and the sensor circuit on the same substrate, more accurate temperature information of the heater area can be obtained. A characteristic of the circuit pattern of the bottom transparent plate is a cell culture vessel characterized in that a slit on each of an anode side and a cathode side made of a pair of opposing longitudinal grooves is provided. The cell petri dish used here is made of resin such as polystyrene.

  According to the third aspect of the present invention, a lid is formed on a petri dish serving as a cell culture container, and is manufactured from a top transparent plate that forms an upper lid so as to close the hole. The top transparent plate is a cell culture vessel characterized in that a transparent conductive film is formed on the surface and a resistance circuit is formed using the transparent conductive film as a heater. The purpose of this section is that if the lid condenses, it will interfere with the observation of an upright microscope, and in the inverted microscope observation method, the illumination light from the upper side of the microscope will be obstructed and the observation will be hindered. However, the provision of a heater circuit in the lid part is to prevent fogging. Unlike the above-mentioned item 0023, since only the anti-fogging function is provided, the sensor circuit and the heater circuit for uniformly generating heat are not necessary, and the anti-fogging function is higher than the heater temperature by the bottom transparent plate that forms the bottom of the dish. It only has to generate heat.

  In the cell culture container according to claim 4 of the present invention, when it is necessary to observe the cells cultured in the incubator with time under a microscope, the culture environment to survive is pH other than temperature. Carbon dioxide supply and perfusion may be required as a means of managing (hydrogen concentration ion index). In order to cope with these, a carbon dioxide injection cylinder, a discharge cylinder, a perfusion injection cylinder, and a discharge cylinder can be easily attached to the lid portion of the petri dish which is the cell culture container. The injection and discharge cylinders are made of a corrosion-resistant material having an outer diameter of around 4 mm. It is used by connecting it to a silicon or nylon tube (not shown).

  According to a fifth aspect of the present invention, compared to the structure in which a transparent conductive film is formed on the lower side of the bottom transparent plate according to the second aspect, heat conduction and heating to the local area of the cell In this cell culture container, a transparent conductive film is formed on the upper side of the surface of the bottom transparent plate to which the cells adhere so as to make it possible to make the accuracy more accurate.

According to a sixth aspect of the present invention, in the cell culture container according to the second aspect, a pair of electrode needles of an anode and a cathode is provided to allow a current to flow, and a transparent conductive film surface is formed on the upper side of the bottom transparent plate The cell culture vessel is characterized by being formed so as to allow a weak current to flow using a weak current generator (not shown). As the electrode needle, a contact probe having a small diameter with a contact terminal diameter of 1 mm is used. The purpose of this section is to pioneer a new electrochemical culture experiment by applying a weak current to the transparent conductive film on the upper side of the bottom transparent plate, which is a surface for cell culture.

  According to the seventh aspect of the present invention, a transparent conductive film is formed and patterned directly on the culture vessel without using the top transparent plate or the bottom transparent plate. Although it is not suitable for high-magnification observation of 60 to 100 times, each transparent plate processing becomes unnecessary, so that it can be supplied at low cost.

  According to the eighth aspect of the present invention, the same processing is performed on a microwell plate, which is another cell culture container of a petri dish, or a culture container of a test tube or a flask.

  According to the above-mentioned respective claims of the present invention, a transparent conductive film is formed on a cell culture vessel, and a heater pattern and a temperature sensor pattern serving as a heat generation area are formed by a laser processing apparatus. By paying attention to the conductivity, the heater pattern circuit and the sensor circuit are provided on the same substrate, whereby more accurate temperature information of the heat generation area can be obtained. The shape of the transparent plate corresponds to a rectangle or a circle.

  The function of the heater is that the electric load is applied to the resistance value between the heater terminals due to the applied voltage control, and the current value fluctuates.To set the heater temperature, the proportional relationship between the applied current and voltage is used. Is set. The correlation between the current value at that time and the heat generation temperature that is proportional to the current value is used. A voltage necessary to obtain the predetermined temperature is applied in advance, and at this time, the current is measured and detected by a sensor, converted into temperature, and the voltage is adjusted to perform temperature control. Since the resistance value between the terminals is measured, this relationship is used as a temperature sensor.

  Although the present invention is a proposal of a transparent temperature sensor, the conventional manufacturing method has been a proposal to manufacture using a heavy equipment photolithographic equipment accompanied by mask manufacturing as described in the paragraph (0006). By using the laser processing apparatus to be described, there is an advantage that it can be manufactured at low cost. Below, we propose the formation of a transparent sensor part using a laser method for forming a sensor circuit at low cost. The formation includes (1) a method of providing the sensor unit alone (FIG. 10), (2) a method of providing the sensor unit inside the heater pattern (FIG. 11), and (3) the heater heating unit and the heating unit. A method of using the terminal part as a sensor control part (FIG. 12) is conceivable, but it may be selected according to the purpose of use or the purpose of manufacture. In this proposal, the laser processing pattern is a proposal for forming slits on the anode side and the cathode side each consisting of a pair of opposing vertical grooves.

  Since the transmittance of ITO, which is a transparent conductive film to be formed, is about 80%, in order to improve this, an antireflection film can be laminated on the transparent conductive film to improve the transmittance.

  The present invention is not an indirect heating method using an external heating device, but can directly heat an observation cultured cell, and for a local direct heating method that efficiently conveys the heat conduction, a predetermined culture temperature is set directly below the cultured cell. Can be made.

  In the present invention, although the lid portion is condensed due to heating during cell culture, a transparent heater is formed for the purpose of anti-fogging, so observation without obstructing upright microscope observation or inverted microscope observation method Can do.

  The present invention facilitates temperature control by attaching a heater to a petri dish or the like that is a culture vessel and integrating it with the culture vessel, and further providing a transparent temperature sensor for temperature feedback to improve its function. Therefore, there is no need to open the petri dish lid to measure the temperature, there is no need to add an external temperature sensor, and the configuration is simple. Further, since the sensor pattern is made of the same material as the heater formed, transparency can be obtained, so that there is no trouble with observation with a microscope.

  The heater patterning of the heater part by the slit processing for the purpose of uniform heat generation and the sensor pattern processing use the same laser processing apparatus, and further, the manufacturing cost is low because it is manufactured in the same process.

  The present invention allows new electrochemical culture experiments such as cell control by electrical stimulation or electrophysiological experiments such as nerve cells by passing a weak current through the transparent conductive film surface formed on the surface where cell culture is performed. It can be done under an optical microscope. Furthermore, a scaffold structure which is one of the functions as an adhesion factor serving as an extracellular extracellular matrix is formed on the conductive film surface formed on the cell culture surface.

  The following description is based on non-limiting examples of the present invention with reference to the drawings.

  FIG. 1A is a perspective view of the above-described top transparent plate 1 attached to a petri dish lid part and the bottom transparent plate 35 attached to a dish part. (B) is a sectional view thereof. The petri dish container 3 which is a culture container has a structure in which the lid and the dish are previously perforated and have the top transparent plate 1 and the bottom transparent plate 35 so as to close it.

  The petri dish 3 has a cylindrical shape having an outer diameter of 35 mm and a depth of 10 mm. The container is made of a resin such as polystyrene. The outer diameter of the container is increased so that as many cells to be cultured (not shown) can be attached to the central part of the lid and the dish as much as possible. Is provided.

  Further, the top transparent plate 1 and the bottom transparent plate 35 are connected to the lid portion 12 and the dish portion 13 via a silicon-based curable adhesive in order to close each opening 31 on the outside. . The shapes of the top transparent plate 1 and the bottom transparent plate 35 are 170 μm in thickness and inside and outside the outer diameter of 30 mm, which are the same as a cover glass for microscopic observation, and are made of borosilicate glass or resin with high optical quality. As the adhesive material, a silicone-based photocurable or thermosetting resin having a refractive index (1.6) comparable to that of glass can be used. For example, a nano hybrid silicone material manufactured by ADEKA Corporation is an example.

  In addition, a slit pattern is provided on the lower surface 35b of the bottom transparent plate so as to perform a heater action and a sensor action by a laser processing method so that local heating to the cell can be performed accurately.

  In order to prevent the condensation of the lid 12, the top transparent plate 1 is placed on the upper surface 12 of the lid in the same manner as the above-described items 0038 and 0039 in order to close the opening 31 a. Connect through. A slit pattern is provided on the upper surface 1a of the top transparent plate to form a heater by a laser processing method.

  FIG. 2 (A) is a perspective view of a petri dish having the above-mentioned petri dish provided with a carbon dioxide gas injection cylinder and its discharge cylinder, and a culture medium perfusion cylinder and its discharge cylinder. (B) is a sectional view thereof. The perfusion culture system for circulating carbon dioxide and culture medium using the main culture vessel 3 is shown in FIG. 15, but the carbon dioxide injection cylinder 37 and the perfusion injection cylinder 39 and its carbon dioxide discharge cylinder 38 and perfusion discharge cylinder 40 are provided. It is a culture vessel provided. A small-diameter tube (not shown) is connected and used for inflow into these. The shape of the injection cylinder and its discharge cylinder is made of a corrosion-resistant material having an outer diameter of around 4 mm. It is used by being connected to silicon, nylon, or a silicone tube (not shown). Gases such as oxygen and ozone that have physiological activity can be supplied through this injection cylinder.

  FIG. 3 shows a transparent conductive film formed on the upper surface 35a of the bottom transparent plate and the inner surface 13a, the lower surface 13b, and the outer peripheral portion 13c of the container, which are attached to the opening 31b of the dish portion 13 of the container 3. FIG. 3 is a cross-sectional view of a film formed first, then formed by laser processing to form a heater pattern and a sensor pattern, and the lid portion of the container 3 is formed in the same manner as in the item 0042. On the petri dishes 13a, 13b and 13c, conductive electrode terminals (not shown) are formed by laser processing. This is to provide a heat generation area on the cell attachment surface which is the upper surface 35a of the bottom transparent plate of the dish portion 13.

  FIG. 4A shows the inner surface 12b of the lid portion of the container 3, the inner surface 13a and the lower surface 13b of the plate portion to which the bottom transparent plate 35 is attached, and the outer peripheral side surface portion 13c. FIG. 3 is a perspective view of a transparent conductive film first formed on the upper surface 35a and the lower surface 35b of the bottom transparent plate and then formed by laser processing and heater pattern and sensor pattern processing. (B) is a sectional view thereof.

  In FIG. 4A, the inner surface 12b of the lid portion of the container 3 is processed with a transparent conductive film. The outer surface 12a of the lid portion is coated with a transparent antibacterial agent 60. It is a perspective view. (B) is a sectional view thereof. The polystyrene resin used in the container is easy to attach cells, and conversely, titanium oxide TiO2 is applied to a part of the container main body, in this example, the outer surface 12a of the lid, so as to deal with invasion of other cells. Etc. are used for coating.

  In addition, in FIG. 3 or FIG. 4, the transparent conductive film 15 used for the upper surface 35a of the bottom transparent plate can use ITO ink (fine particles) by a coating method. The transparent conductive film is characterized by a particle size distribution state of a nano-unit particle diameter, and in addition to the property of conductivity, its adhesion to become an extracellular extracellular matrix by utilizing the irregularity of fine particles in the surface state. The scaffold structure is a factor.

  Similarly, in FIG. 3 or FIG. 4, a DLC film that is a diamond-like carbon film can be used as the conductive film used for the upper surface 35a of the bottom transparent plate. As in the above item 0049, this DLC membrane is also used as a scaffold structure for cell culture.

  5 includes a pair of electrode needles of an anode 52 and a cathode 53 in order to pass an electric current, and forms a transparent conductive film surface on the side 35a of the bottom transparent plate attached to the dish portion to form a weak current generator ( It is sectional drawing of what enables a weak electric current to flow using (not shown). As the electrode needle, a contact probe having a small diameter with a contact terminal diameter of 1 mm is used. In addition, a slit pattern for forming a heater and a sensor by a laser processing method is provided on the lower surface 35b of the bottom transparent plate so as to accurately perform local heating on the cell.

  FIG. 6 shows an antireflection film formed on the surface on which the slit pattern is provided in order to serve as a heater and a sensor on the lower surface 35b side of the bottom transparent plate attached to the dish portion 13 of the container 3 shown in FIG. Is.

  FIG. 7A is a perspective view of what is formed by first forming a transparent conductive film on the upper surface 12a of the lid portion of the container 3 and the lower surface 13b of the dish portion, and then processing the heater pattern and sensor pattern by laser processing. is there. In this method, a heat generating area and a sensor are provided on the petri dish itself without using a transparent plate. (B) is a sectional view thereof.

  FIG. 8A is a plan view of a well plate 41 for 6 holes in which the bottom transparent plate 35 is attached to the bottom surface of the dish portion and the top transparent plate 1 is attached to the top surface of the lid portion. . (B) is a sectional view thereof. The shape of a general well plate 41 has an outer diameter of 85 mm × 127 mm, and the well plate is an example in which six wells having a diameter of 35 mm are arranged.

  FIG. 9A is a top view of a film formed by first forming a transparent conductive film on the surface and peripheral side surface of a test tube, and then forming a heater pattern and a sensor pattern by laser processing. (B) is the perspective view.

  FIG. 10 is an example of a method of providing the temperature sensor unit 5 alone, and is a plan view of a pattern in which the heat generating unit 4 and the sensor unit 5 including the anode side terminal 6 and the cathode side terminal 7 to be applied are mounted. When the slit pattern portion 30 is formed by a laser processing method, heat generation can occur at a desired place. Heat generation at the center of the heater 4 can be increased.

  The method of forming the transparent heater part 4 is performed by the present inventors by the method described in the actual application 2007-3637 and Japanese Patent Application No. 2007-285543, and a laser is used to make the temperature of the heat generating part to be the heater uniform. Patterning is performed by processing the anode side slit 30a and the cathode side slit 30b using a processing apparatus.

  The sensor unit 5 is processed and arranged in the vicinity of a part that is most often used as a heater, and a detection terminal A (8) and a detection terminal B (9) are provided at both ends of the sensor. The manufacturing method which becomes a transparent temperature sensor using ITO which is a transparent conductive film material is shown below.

  The bottom transparent plate 35 or the container main body tray portion 13 that forms the pattern is designed so as to be the heat generating portion 4 and the sensor portion 5, and a circuit pattern in which the range shown in FIG.

  For example, in order to form a heating resistor circuit that aims to keep the living cells at about 40 ° C., ITO, which is a transparent conductive film, is first formed with a sheet resistance value of 10Ω / □ or less, and the film formation surface. A processing circuit to be the slit pattern 30 is made by a laser processing apparatus so as to obtain a resistance value between terminals required for the above.

  Table 1 shows the ability of a 35 mm diameter petri dish in which about 80% of the aqueous solution is sealed to function as a heating container. As can be seen, the heater W required for the heat generating part may be 10 W or less. At this time, the amount of the culture solution in the 35 mm diameter petri dish is 7065 mm 3, and the amount of heat (Wx seconds sec) required at 10 W is 578 J (joules). On the other hand, it is 150J at 2.2W. Table 1 shows the simulation for determining the heater capacity required for the heat generating part. This determines the resistance value between terminals to be set, and is a correlation between the number of W, the voltage, and the current value required at that time. Further, the temperature raising performance of the heater is determined by a factor of 1 minute rising temperature.

  In the case of a heater capacity of 2.2 W and an inter-terminal resistance value of 10Ω, the rising temperature for 1 minute is obtained from J / water (specific heat x volume) and is 5.08 ° C. The measured values at this time are shown in Table 2. The measured temperature positions are 32 (1), 33 (2), and 34 (3) in FIG.

  Table 3 data shows the relationship between the measured value and the set value of the heater capacity of 2.2 W at this time.

  From this data, it can be seen that if the heater capacity is low, the actual temperature reaches the saturation temperature after about 7 minutes, and the comparison with the actual temperature is about 5 ° C. lower than the set value. This is expected to cause heat dissipation loss as another factor, and when the heater performance is important, the following items are recommended.

  Next, the laser processing apparatus is used to form slits 6a, 6b, 7a, and 7b and slit portions 30a and 30b of the heat generating portion that divide the anode side terminal portion 6 and the cathode side terminal portion 7 that are both electrodes to be applied into heat generating portions. Use to remove. The gray part in the figure is a heat generating part made of a transparent conductive film that is not removed, and the removed part is a slit part indicated by a blank.

  Furthermore, when the size of the bottom transparent plate 35 is assumed to be 20 × 20 mm and the applied power source is assumed to be 3V, the pattern that the temperature sensor unit 5 acts is as shown in FIG. When a circuit composed of the horizontal circuit 10 and the waveform circuit 11 is formed and based on the application of 3 V and 150 mA, the resistance value between terminals is 20Ω at an exothermic temperature of 20 ° C., 30Ω at 30 °, and 50Ω at 40 °. A parameter is obtained. To achieve this, as shown in FIG. 10, the detection terminal length (L1 + L2 + L3) is 20 mm, the terminal width (W1) is 3 mm, the horizontal terminal portion (W2) is 1.5 mm, and the length (L1 or L2). It is preferable to form a circuit having a terminal width (W3) of 0.5 m, with 5 waveforms having 5 mm and peaks and valleys. A detection terminal A (8) and a detection terminal B (9) are provided at both ends.

  FIG. 11 is a plan view of a method of providing the temperature sensor unit 5 inside the heat generating unit 4. This arranges and forms the temperature sensor part closer to the heater heat generating part 4.

In this example, this can be dealt with by designing in the same manner as the above-mentioned items 0059 to 0069.

  FIG. 12 is a plan view of a method of using the heat generating portion 4 and the terminal portion to which the heat generating portion is applied as a sensor control portion in this example. Two application methods for heat generation and sensor control are adopted. At this time, the anode side terminal 6 and the cathode side terminal 7 which are heat generating terminals are also used as the detection terminal A (8) and the detection terminal B (9) which are sensor control terminal parts. That is, the resistance of the terminal / detection unit A (56) and the terminal / detection unit B (57) is a high resistance (for example, 50Ω), and the resistance of the heat generating unit 4 is a lower resistance (for example, 30Ω). I do. In the drawing, the slit 30a on the anode side, the slit 30b on the cathode side, the slits 30c1 and 30c2 on the anode side, and the slits 30d1 and 30d2 on the cathode side are shown.

  FIG. 13 is a circuit diagram of the two systems described in FIG. The opposing power supply circuit is used by alternately switching between the heat generation circuit 58 and the control circuit 59. Application is performed using a heater power source (not shown), and the voltage is controlled in order to obtain a desired temperature of the heat generating portion 4. A voltage is applied to the opposing anode-side terminal 6 and cathode-side terminal 7 to generate heat, and the current of the terminal / detection unit A (56) and terminal / detection unit B (57) is monitored and measured to generate the heat generation unit. The voltage of 4 is fed back.

  Based on the above design, laser cutting (also referred to as trimming) of the heater pattern or sensor pattern is performed using a laser device.

  FIG. 14 is a cross-sectional view showing a method of using the petri dish container 3 for heating. However, the power supply unit to which the voltage is applied is not shown. The petri dish 3 shown in FIG. 1 in which the heater and the sensor are integrated is put into the dish fixing holder 17 integrated with the sample mounting holder ring portion 23 for the inverted microscope through the electrode path 22 connected to the power source. Insertion is performed, and the electrode is used by being pressed and fixed at a predetermined position corresponding to the electrode. In this example, an inverted microscope is assumed, and an objective lens 24 is disposed in the lower part.

  To the electrode part composed of the holder side electrode A (18) and the holder side electrode B (19) in the dish fixing holder 17, the anode side terminal (6) and the cathode side terminal (7) of the heat generating part on the petri dish 3 Is applied by pressing and fixing.

  Next, the lid fixing holder 25 is put over and the petri dish lid 12 is pressed and fixed. At this time, the upper / lid portion fixing holder electrode A (26) and the upper / lid portion fixing holder electrode B (27) and the electrode pad A (20) and the electrode pad B (21) are pressure-contacted to each other. A fixed application is performed.

  Detection is performed using the holder-side detection terminal A (28) and the holder-side detection terminal B (29) 0 in the dish-fixing holder 17.

  When the setting is completed, application is performed using a power supply unit (not shown).

  FIG. 15 is a temperature characteristic diagram of a heater capacity of 2.2 W and a resistance value between terminals of 10Ω.

  FIG. 16 is a schematic perspective view when the culture vessel in which the heater and sensor of the present invention are incorporated is used under an inverted microscope. In a conventional apparatus using a large gas cylinder, it is complicated to cope with each experiment in order to change the culture conditions, that is, the apparatus is naturally enlarged because it is derived from handling of the high-pressure gas container. By using the present invention, it is possible to reduce the size of the culture test environment device, and the space on the desk for microscopic observation can be used effectively.

  The petri dish 3 is housed in the holder 17 for fixing the petri dish 3 and placed on a stand just above the objective lens of the inverted microscope, connected to a power source (not shown), and carbon dioxide gas is supplied from the carbon dioxide supply unit 46 through the tube 47 to the controller. 50, the concentration suitable for the culture is sensed, and the carbon dioxide gas supply unit 46 performs fine adjustment so that the sensed value is suitable for the culture (about 5%). It supplies to the petri dish 3. The tube 49 is for discharge. A circulator used for perfusion is not shown.

  The gas source used for the carbon dioxide supply unit 46 is a small mini gas cylinder 61. As an example, a mini gas cartridge No. 6 (74 g) is used. This dimension is as small as 134 mm long x 40 mm barrel diameter, and its capacity is about 37 L when 100% concentrated compressed gas is released. In cell culture, in order to obtain a carbon dioxide gas having a concentration of about 5%, it is mixed with air and a purge meter type flow meter 62 is used, and control is performed by a regulator or a flow controller not shown. The tube to be introduced is about 4 mm in diameter, and the material is nylon or flexible tube.

  When the carbon dioxide gas pressure is around 0.1 MPa and the mixed gas flow rate around 5% is 0.2 L / min, the air gas capacity (190 mL / min) and carbon dioxide gas (10 mL / min) are mixed. Inflow. Under this condition, the small mini gas cylinder can be used for several days, and the gas source can be used for a long time by installing a plurality of gas sources because of its small size.

  For long-term cell culture, it is important to keep the pH of the culture medium constant. For example, in order to maintain the pH at 7.4, the carbon dioxide concentration needs to be 5%. In the controller 50, temperature control is performed by applying as described in the paragraph 0072, but the concentration of carbon dioxide can be monitored using a sensor. As an example, the concentration sensor 63 can use model GMM111 manufactured by VAISARA.

(A) is a perspective view of the petri dish container with the transparent plate attached to the petri dish lid and dish. (B) is a sectional view thereof. (A) is a perspective view of a container provided with a carbon dioxide injection cylinder and its discharge cylinder, and a culture medium perfusion cylinder and its discharge cylinder on the lid of the petri dish of the above-mentioned petri dish. (B) is a sectional view thereof. It is sectional drawing of the cell culture container in which the transparent conductive film was formed in the surface upper surface to which the cell of the bottom transparent plate of the dish part of this petri dish container adheres. FIG. 4A shows a transparent conductive film, a heater, and a sensor processed on both the inner surface of the lid of the petri dish, the inner and lower surfaces of the dish, the outer peripheral side, and the bottom transparent plate attached to the dish. FIG. 2 is a perspective view of the outer surface of the lid portion coated with an antibacterial agent. (B) is a sectional view thereof. FIG. 5 is a cross-sectional view of the petri dish container provided with electrode needles and allowing a weak current to flow through the upper surface of the bottom transparent plate attached to the dish portion. FIG. 6 is a cross-sectional view of the petri dish container in which an antireflection film is formed on the bottom transparent plate lower surface side. FIG. 7A is a perspective view of the petri dish container provided with a heat generating part and a sensor on the petri dish itself without using a transparent plate. (B) is a sectional view thereof. FIG. 8 is a plan view of the transparent plates attached to the bottom surface of the plate portion and the top surface of the lid portion of the well plate for 6 holes, respectively. (B) is a sectional view thereof. FIG. 9 (A) is a top view of what is formed by processing the heat generating part and the sensor pattern on the surface and the peripheral side surface of the test tube. (B) is the perspective view. FIG. 10 is a method of providing a sensor unit alone, and is a plan view of a pattern including a heat generation unit and a sensor. FIG. 11 is a plan view of a method for providing the sensor portion inside the heat generating portion. FIG. 12 is a plan view of a method of using a heat generating portion and a terminal portion to which the heat generating portion is applied as a sensor control portion. FIG. 13 is a circuit diagram of the two systems described in FIG. FIG. 14 is a schematic cross-sectional view of a holder for fixing and applying and detecting the petri dish shown in FIG. FIG. 15 is a temperature characteristic diagram of the petri dish having the performance of the heater capacity of 2.2 W and the resistance value between terminals of 10Ω shown in FIG. FIG. 16 is a reference perspective view when the culture vessel shown in FIG. 1 in which the heater and sensor of the present invention are incorporated is used under an inverted microscope. A circulating device such as a pump is not shown.

1. Top transparent plate 1a. Top transparent plate upper surface 1b. 1. Top transparent plate bottom surface Rectangular slide glass substrate 3 Petri dish 4 Heat generation part (transparent heater part)
5. 5. Temperature sensor unit 6. Anode side terminal Cathode side terminal 8. Detection terminal A
9. Detection terminal B
10. Horizontal circuit 11. Waveform circuit 12. Petri dish lid 12a. Petri dish top surface 12b. Petri dish lower surface 13. Petri dish 13a. Petri dish inner surface 13b. Petri dish lower surface 13c. Petri dish outer peripheral surface 14. Culture medium15. Transparent conductive film 16. Antireflection film 17. Dish unit fixing holder 18. Holder side electrode A
19. Holder side electrode B
20. Electrode pad A
21. Electrode pad B
22. Power supply circuit 23. Microscope sample mounting holder ring 24. Objective lens 25. Upper / lid fixing holder 26. Upper and lid fixing holder electrode A
27. Upper and lid fixing holder electrode B
28. Holder side detection terminal A
29. Holder side detection terminal B
30. Slit pattern portion 30a. Slit 30b on the anode side. Cathode side slits 30c1, 30c2. Slits 30d1, 30d2. Slit on the cathode side 31. Petri dish opening 31a. On the petri dish opening 31b. Below petri dish opening part 32. Temperature measurement point (bottom edge)
33. Temperature measurement point (bottom center)
34. Temperature measurement point (aqueous solution center)
35. Bottom transparent plate 35a. Bottom transparent plate upper surface 35b. Bottom transparent plate lower surface 36. Transparent conductive film 37. Carbon dioxide injection cylinder 38. Carbon dioxide discharge tube 39. Perfusion tube 40. Perfusion discharge tube 41. Well plate 42. Well 43. Test tube 44. Test tube barrel 45. Inverted microscope 46. Carbon dioxide supply tube 47. Carbon dioxide supply tube 1
43. Test tube 44. Test tube barrel 45. Inverted microscope 46. Carbon dioxide supply tube 47. Carbon dioxide supply tube 1
48. Carbon dioxide supply tube 2
49. Carbon dioxide discharge tube 50. Controller 51. Transparent conductive surface 52. Microelectrode needle (+)
53. Microelectrode needle (-)
54. Detection terminal C
55. Detection terminal D
56. Terminal / Detector A
57. Terminal / detector B
58. Heat generation circuit 59. Control circuit 60. Antibacterial coat 61. Small mini gas cylinder 62. Flow meter 63. Carbon dioxide concentration sensor

Claims (8)

  A cell culture container comprising a transparent conductive film formed on a cell culture container body for culturing adherent cells, wherein the transparent conductive film comprises a heater part and a current detection sensor part. container.   The cell culture container according to claim 1, wherein the dish part of the container body includes a bottom transparent plate part for attaching the cells, and a transparent conductive film is formed below the bottom transparent plate, It is possible to provide a resistance circuit formation using a conductive film as a heater and a sensor for detecting the current to obtain a desired resistance value for the resistance, thereby enabling accurate local heating of the cells. A cell culture vessel characterized by comprising a slit on each of an anode side and a cathode side formed of a pair of formed longitudinal grooves. The cell culture container according to claim 1, wherein the lid of the container body includes a top transparent plate portion, a transparent conductive film is formed on the top transparent plate, and the transparent conductive film serves as a heater. A cell culture container characterized in that it is formed so that the lid portion of the cell container can be fogged.   The cell culture container according to claim 1, wherein the lid of the container is provided with a carbon dioxide gas injection cylinder and its discharge cylinder, and a culture medium perfusion injection cylinder and its discharge cylinder. container.   The cell culture container according to claim 2, wherein the dish portion of the main body includes a bottom transparent plate portion for attaching the cells, and a transparent conductive film is formed on an upper surface of the bottom transparent plate to which the cells adhere, A resistance circuit formation using the transparent conductive film as a heater and a sensor for detecting a current for obtaining a desired resistance value of the resistance are further provided, and the transparent conductive film is provided below the transparent plate according to claim 2. Each of the anode side and the cathode side formed of a pair of opposed longitudinal grooves formed so as to enable accurate heat conduction and heating to the local area of the cell as compared with the structure in which the cell is formed A cell culture vessel characterized by providing a slit.   6. The cell culture vessel according to claim 1, 2 or 5, comprising a pair of anode and cathode electrode needles for passing a current, and forming a transparent conductive film surface on the upper side of the bottom transparent plate to allow a weak current to flow. A cell culture vessel characterized by being formed as described above.   The cell culture container main body according to claim 1, wherein a transparent conductive film is directly formed on the bottom of the outer surface of the dish part of the container, and a resistance circuit is formed using the transparent conductive film as a heater, and a resistance for which the resistance is desired. An anode side and a cathode side consisting of a pair of opposed vertical grooves formed so as to enable accurate heating of the cells to the local area by providing a sensor unit for current detection to obtain a value In addition, the lid portion of the main body is formed with a transparent conductive film directly on the outer surface, and a resistance circuit is formed using the transparent conductive film as a heater. A cell culture container characterized by being formed so that the lid of the cell container can be anti-fogged.   A transparent conductive film is formed around the cell culture vessel main body according to claim 1, a resistor circuit is formed using the transparent conductive film as a heater, and a sensor for detecting current that obtains a desired resistance value is provided. And provided with slits on the anode side and the cathode side, each of which is formed of a pair of opposed longitudinal grooves formed so as to enable accurate heating of the sample in the culture vessel body. A culture vessel which is a microwell plate, test tube or flask characterized by.