JP4638255B2 - Cell culture device, instrument and system - Google Patents

Description

  The present invention relates to a cell culture apparatus, an instrument, and a system for culturing living cells while maintaining an external environment.

  In the conventional cell culture technique, as a cell culture device, for example, a culture solution is generally stored in each petri dish, and is maintained at 37 ° C. for a certain period of time in an incubator for cell proliferation. When the grown cells are peeled off in the form of a sheet, the cells are peeled off and collected from the bottom of the petri dish using an enzyme when the grown cells are uniformly attached to the bottom of the petri dish.

In addition, as a means for separating and collecting cells uniformly adhered to the petri dish bottom surface in a sheet form, a method of forming and using a polymer that reversibly switches hydrophilicity and hydrophobicity depending on temperature on the petri dish bottom surface is known. . According to this method, cells on the polymer surface exhibiting hydrophobicity grow and adhere at a culture temperature of 37 ° C., and then the polymer surface changes to hydrophilic when the temperature is lowered to 30 ° C. or lower. Can be recovered.
JP2004-236547

  However, in the case where a plurality of cell types are cultured and grown in the same plane on the petri dish bottom surface, it is necessary to provide a region where cells adhere and a region where the cells cannot adhere with a polymer whose hydrophilicity and hydrophobicity are switched by the above temperature. . In that case, a method was adopted in which materials having different temperatures at which the hydrophilicity and hydrophobicity of the polymer were switched were set so that both hydrophilic and hydrophobic regions were created on the petri dish bottom surface at a certain temperature. . For this reason, it becomes complicated to set an arbitrary area, and it is difficult to set an area during cell culture or after cell growth.

  In view of the above-described conventional problems, the present invention provides a cell culture device, an instrument, and a system thereof that can grow different types of cells in an arbitrary pattern on the same sheet. Also provided are a cell culture apparatus, instrument and system capable of peeling and collecting a cell sheet at an arbitrary location.

  In order to solve this problem, the cell culture instrument of the present invention is a cell culture instrument for culturing biological cells, and is arranged on at least one flat plate to control the surface temperature of the flat plate. Two heaters, temperature measuring means paired with the heater, a connection terminal of a control circuit that autonomously controls the temperature of the heater based on the temperature detected by the temperature measuring means, and a back surface of the flat plate are cooled The cell culture medium is applied to the cooling means, a thin film polymer layer formed on the upper surface of the heater and the temperature measurement means, which switches between hydrophilicity and hydrophobicity according to temperature, and a region containing the surface temperature control means and the thin film polymer. And storing means for storing.

  Here, the area | region of the said storage means contains the area | region where the said heater does not exist. Further, the heater is arranged in a spiral shape in the area of the storage means. The heaters are arranged in a grid pattern in the area of the storage means. The heater is patterned using semiconductor process technology. The thin film polymer layer whose hydrophilicity and hydrophobicity are switched depending on the temperature contains isopropyl acrylamide. Further, a second polymer layer for promoting the formation of the thin film polymer layer is further provided between the surface temperature control means and the thin film polymer layer. The second polymer layer includes polystyrene formed by melt coating.

  The cell culturing apparatus of the present invention is a cell culturing apparatus for culturing biological cells, and is at least one surface temperature control means arranged on at least one flat plate to control the surface temperature of the flat plate. Surface temperature control means comprising at least one heater, temperature measurement means paired with the heater, and a control circuit for autonomously controlling the temperature of the heater based on the temperature detected by the temperature measurement means, A culture vessel comprising a cooling means for cooling the back surface of the flat plate, a chamber capable of gas exchange, covering the surface temperature control means, and having a thin film polymer layer on the bottom surface that switches between hydrophilicity and hydrophobicity depending on temperature. It is characterized in that cell culture is carried out.

  Here, the bottom region of the culture vessel includes a region where the heater does not exist. Further, the heater is arranged in a spiral shape in the bottom area of the culture vessel. The heaters are arranged in a grid pattern in the bottom area of the culture vessel. The heater is patterned using semiconductor process technology. The thin film polymer layer whose hydrophilicity and hydrophobicity are switched depending on the temperature contains isopropyl acrylamide. The culture vessel further includes a second polymer layer for promoting the formation of the thin film polymer layer between the surface temperature control means and the thin film polymer layer. The second polymer layer includes polystyrene formed by melt coating.

  The cell culture system of the present invention is a cell culture system for managing cell culture of organisms, comprising a cell culture device for culturing cells of organisms and a computer for monitoring and managing the cell culture device, The cell culture device is at least one surface temperature control means arranged on at least one flat plate to control the surface temperature of the flat plate, and includes at least one heater and a temperature measuring means paired with the heater, Covering the surface temperature control means having a control circuit for autonomously controlling the temperature of the heater by the temperature detected by the temperature measurement means, cooling means for cooling the back surface of the flat plate, and the surface temperature control means, A gas exchange chamber, and a culture vessel having a thin film polymer layer on its bottom surface, which switches between hydrophilicity and hydrophobicity depending on the temperature. And performing cell culture according to monitoring and management by chromatography data.

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a cell culture device, an instrument, and a system thereof that can grow different types of cells in an arbitrary pattern on the same sheet. In addition, it is possible to provide a cell culture apparatus, instrument, and system thereof that can peel and collect a cell sheet at an arbitrary location.

  Hereinafter, a cell culture device according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings. This embodiment is an example of the present invention and is not limited to this. Further, the dimensions of the respective parts in the drawings do not limit the present invention.

<Configuration Example of Cell Culture System Including Cell Culture Device of Present Embodiment>
FIG. 1 is an external perspective view showing a configuration example of the cell culture system of the present embodiment.

  In FIG. 1, reference numeral 100 denotes a culture chamber in which the cell culture container of this embodiment is installed. In addition, in order to sterilize the culture chamber 100, the sterilization lid | cover 200 shut off from the outside is provided. During cell culture, the culture chamber 100 is filled with the culture gas 300.

  Reference numeral 400 denotes a heating means control device (also referred to as a temperature controller) disposed in a housing that supports the culture chamber 100. The heater at the lower part of the cell culture container in the culture chamber 100 is supplied from the temperature measurement means (including the thermistor). Based on the measured temperature, the heater is controlled to a target temperature. Reference numeral 500 denotes a cooling means controller for controlling a cooling means for cooling the cell culture container in the culture chamber 100. The cooling means is related to the heater even if it is fixed in hardware including a fan and a heat sink. It may be configured to be controlled as described above. The cooling means is disposed further below the heater below the cell culture container.

  Reference numeral 600 denotes a temperature control / monitoring personal computer that manages and monitors the cell culture apparatus. The personal computer 600 may be a general-purpose computer, and functions to input and output temperature control parameters to the heating means control device 400, monitor the cell culture environment and history, and notify the operator.

<Configuration example of culture chamber and cooling means>
FIG. 2 is an external perspective view from two directions showing a configuration example of the culture chamber and the cooling means. FIG. 2A is an external perspective view seen from an angle at which the exhaust heat fan 130 can be seen, and FIG. 2B shows the exhaust heat fin 150 on the opposite side of the exhaust heat fan 130. It is the external appearance perspective view seen from the angle.

  In the culture chamber 100, there are petri dish type cell culture containers 120a, 120b,. The bottom surface of the cell culture vessel 120 is in contact with the common cooling means 110. A Peltier element and a heat sink 140 are used at the contact portion between the cell culture container 120 and the cooling means 110. The cooling means 110 includes a heat exhaust fan 130 and a heat exhaust fin 150.

  FIG. 2 shows four examples of the cell culture container 120, but the number is not limited.

<Schematic configuration example of cell culture container and production example thereof>
FIG. 3 is a diagram schematically showing a cut surface of the cell culture container 120. FIG. 3 illustrates one cell culture vessel illustrated in FIG.

  In FIG. 3, reference numeral 160 denotes a substrate made of silicon or glass. The entire lower surface of the substrate 160 is in contact with the cooling means 110. A heater 121 is patterned on the surface of the substrate 130 by a semiconductor process technique (examples of heater patterns will be described below). Note that chromium (Cr) is suitable as the heater material. Reference numeral 121a denotes a wiring pattern and input / output terminals for connecting the heater 121 to the external heating means control device 400.

Reference numeral 122 denotes an insulating layer that covers the heater 121 and is insulated from the thermistor 123, and is a layer of, for example, silicon nitride (Si ₃ N ₄ ). Reference numeral 123 denotes a thermistor formed on the heater 121 with the insulating layer 122 interposed therebetween. As the thermistor material, germanium (Ge) is suitable. Reference numeral 123 a denotes a wiring pattern and input / output terminals for connecting the thermistor 123 to the external heating means control device 400.

  A polymer layer 124 is applied in order to suitably form (polymerize) the polymer layer 125 whose hydrophilicity / hydrophobicity is switched depending on the temperature, but is not an essential layer in the present embodiment. For example, polystyrene is preferable. Such polystyrene is melted and applied, and also functions as a water-resistant protective film for protecting the heater pattern / thermistor pattern. Reference numeral 125 denotes a polymer layer whose hydrophilicity / hydrophobicity is switched depending on temperature, and is, for example, isopropyl acrylamide. In this embodiment, since the temperature can be controlled locally, it is easy to use other temperature-responsive polymers that switch at different temperatures as long as the polymer switches between hydrophilicity and hydrophobicity. It is.

  126 is a culture solution used for cell culture, and the culture solution 126 is stored in an enclosure 127 that fractionates the cell culture vessel 120. 128 is a silicone resin for fixing the enclosure 127 and may be any resin that does not have biotoxicity. For the enclosure 127, for example, polystyrene is preferably used. Here, the enclosure 127 is formed after the wiring patterns of the heater 121 and the thermistor 123 are created, for example, and the polymer layers 124 and / or 125 are formed after the enclosure 127 is created.

  In the above, a schematic configuration example of a cell culture container and a manufacturing example thereof have been shown. However, such an example is an example, and it is important to form a pattern by a semiconductor process technology, and a material and a manufacturing method thereof are described in this example. It is not limited.

<Pattern example of heater / thermistor of this embodiment>
Examples of the heater / thermistor pattern of the present embodiment are shown below. The pattern examples are not limited to the following examples, and any pattern can be used as long as the culture medium temperature of the present invention can be controlled. include.

(Example 1 of heater / thermistor pattern)
FIG. 4 shows an example of the heater / thermistor pattern in the present embodiment. Note that reference numerals in the following drawings and description correspond to heaters (thermistors), and each element is represented by heater 121 and thermistor 123. .

  FIG. 4 is a diagram showing an example of a spiral / heater thermistor that warms the entire cell culture container (petri dish) 120. For example, FIG. 4 shows a spiral pattern 121-1 and thermistor 123-1 of a heater having a diameter of 35 mm, and has a lead-out wiring pattern 121a-1 (123a-1). In the upper part of FIG. 4, an enlarged view of the thermistor 123-1 is shown. FIG. 4 shows an example in which the thermistor 123-1 is arranged at the center of the spiral pattern 121-1 of the heater. However, the present invention is not limited to this example, and the thermistors 123-1 are arranged in a distributed manner. Alternatively, it may be a multiple arrangement overlapping each heater.

  In this spiral pattern, the temperature of each cell culture container (petri dish) 120 is controlled as a whole. Therefore, in such a cell culture device, the same type of cells are cultured in each cell culture container (petri dish) 120 unit, and detachment / recovery is performed. Is one. In addition, if the spiral pattern 121-1 is formed only on a part (for example, the center) of each cell culture container (petri dish) 120, only the part can be cultured.

(Example of heater / thermistor pattern 2)
FIG. 5 shows another example of the heater / thermistor pattern in this embodiment.

  FIG. 5 shows that each cell culture container (petri dish) 120 is formed as a region by forming a heater / thermistor in a dot pattern inside each cell culture container (petri dish) 120 and making each heater / thermistor controllable independently. It is a figure which shows the heater / thermistor example which can be temperature-controlled separately. For example, FIG. 5 shows a 5 × 5 dot 0.5 mm square heater / thermistor pattern.

  In the enlarged view, 121-2 (123-2) is a 5 × 5 dot heater / thermistor, 121a-2 is a lead-out wiring and input / output terminal of the heater 121-2, and 123a-2 is the thermistor 123-2. Lead wiring and input / output terminals.

  As is apparent from the enlarged view of the heater / thermistor section, each heater / thermistor is controlled independently, and culture, detachment, and recovery can be controlled for each heater / thermistor region. That is, cells can be cultured only in the area where the heater is turned on, and only cells in the area where the heater is turned off can be detached.

<Specific configuration example of cell culture vessel>
FIG. 6 is an external perspective view of the cell culture container (petri dish) of this embodiment, which is an example of the cell culture container (petri dish) using the heater / thermistor of FIG. 5 described above.

  The reference numbers in FIG. 6 are the same as the same numbers in FIGS. 3 and 5. That is, 121-2 (123-2) is a 5 × 5 dot heater / thermistor, 121a-2 (123a-2) is a wiring and input / output terminal, 127 is enclosed, and 128 is a silicone resin. In FIG. 6, the polymer layer and the cell culture solution are not shown.

<Example of control block of cell culture system of this embodiment>
FIG. 7 is a diagram illustrating an example of a control block of the cell culture system of the present embodiment. FIG. 7 shows only parts related to the present embodiment, and does not show known and general-purpose elements such as the OS of the personal computer 600 and the temperature controller of the temperature controller 400.

(Temperature control monitor PC)
The temperature control / monitor personal computer 600 includes a CPU 610 for operation control, a ROM 620 for storing fixed programs and parameters, a RAM 630 used as a temporary storage when the CPU 610 executes a program, and an image storage unit 640 such as a disk or CD.

  In the present embodiment, the RAM 630 stores a control parameter 631 that is set in the temperature controller 400. Control parameters 631 include cultured cell type 631a, cell culture container position and / or heater number 631b, heater on / off flag 631c, heater area set temperature 631d, thermistor measured temperature 631e, heater input set time 631f, the current elapsed time 631g of the heater input is included. A control parameter 631 is set for each cell culture container or each heater. The control parameter 631 is set when the control information input / output module 642b is executed, an input screen is displayed on the display unit 653, and an operator inputs the input screen. Note that the measurement temperature 631e is necessary for maintaining the environmental history during cell culture, and it is not necessary to read it if the environmental history is not required. Further, it is preferable that the operator inputs / selects the setting of the control parameter 631 in accordance with the contents stored or learned in advance in the control parameter table 641.

  The program load area 632 of the RAM 630 is an area where the CPU 610 loads an application program related to the present embodiment stored in the external storage unit 640.

  The external storage unit 640 stores a control parameter table 641 that stores standard control parameters, a set history, and the like. The program storage area 642 includes a temperature control control module 642a for controlling the temperature control 400, a control information input / output module 642b for controlling the input of control parameters or the output of the current status and history, and an optional cooling means in this example. And a culture history storage module 642d for storing a history of cell culture.

  The temperature control / monitor personal computer 600 further includes an input / output interface 650 for interfacing with a keyboard 651 and a mouse 652 as input devices and a display unit 653 as an output device, and with a temperature controller 400 and optionally controlling cooling means. The apparatus 500 and other peripheral devices, for example, various sensors for controlling the room temperature, various controls such as controlling the culture gas, and a camera for photographing the progress of cell culture are interfaced.

(Temperature controller)
The temperature controller 400 is an existing temperature control, that is, a temperature control that controls a heater based on a measured temperature from a thermistor to a set target temperature (the temperature control is hardware or software). In other words, the control parameter storage unit 410 has a control parameter storage unit 410 configured to store the measured temperature set by the temperature control / monitor personal computer 600 or from the thermistor. The control parameter 410 is stored in a register if hardware control is performed, and is stored in a memory if software control is performed.

  The control parameter 410 is stored corresponding to each heater / thermistor. For example, assume that the cell culture container 120a in FIG. 2 has the spiral pattern in FIG. 4 and the cell culture container 120b has a dot pattern.

  Corresponding to the cell culture vessel 120a, the parameters of the culture vessel (in this case, the same as the heater) a411 include the heater on / off flag 411a for the heater 121a of the culture vessel a, the set temperature 411b, and the thermistor 123a of the culture vessel a. Measurement temperature 411c.

  Corresponding to one 5 × 5 heater b1 (413) of the cell culture vessel 120b, parameters include a heater on / off flag 413a for the heater b1 (121b1) of the culture vessel b, a set temperature 413b, and a culture vessel b It has a measurement temperature 413c from the thermistor b1 (123b1). Corresponding to one 5 × 5 heater b2 (414) of the cell culture vessel 120b, parameters include a heater on / off flag 414a for the heater b2 (121b2) of the culture vessel b, a set temperature 414b, and a culture vessel b It has a measurement temperature 414c from the thermistor b2 (123b2).

(Heater / Thermistor)
Within the culture chamber 100, the cell culture container a has a spiral heater 121a and the thermistor 123a, while the cell culture container b has a 5 × 5 dot pattern heater b1, heater b2,. There are thermistors b2,..., The measured temperature of the thermistor is sent to the temperature controller 400, and the heater is controlled by the temperature controller 400.

<Control procedure example of temperature control / monitor personal computer of this embodiment>
FIG. 8 is a flowchart showing a control procedure example of the temperature control / monitoring personal computer 600 in the present embodiment. The operation of each step in the control procedure can be realized by a general-purpose computer.

  First, in step S81, an input screen for inputting a control parameter is displayed on the display unit 653, and an input from the operator is awaited in step S82. If necessary input is made and the operator inputs OK, the process proceeds to step S83, and the necessary control parameters are transmitted to the temperature controller 400 from the control parameters input in step S81 and set.

  In step S84, the status of cell culture start from the temperature controller 400 is confirmed, and if it is OK (for example, the cell culture device is ready: power supply, circuit operation, culture chamber, sterilization lid set, culture gas status, etc. Etc.), start monitoring cell culture.

  In step S85, culture history information (temperature history, growth state if a camera is installed, etc.) is acquired from the temperature controller 400, and the culture state is accumulated and displayed in step S86. In addition to the culture history information, the occurrence of an event (device failure, abnormal growth state, etc.) is monitored in step S85, and an alarm or warning message is displayed in step S86. In step S87, the end of the cell culture is determined. If not, the process returns to step S85 and steps S85 to S87 are repeated.

  In this embodiment, when detaching and collecting cells in units of cell culture containers or in partial areas in the containers, the culture gas is stopped and the culture chamber is opened. At that time, turn off the cell culture container to be peeled and collected from the temperature control / monitor personal computer 600 or the heater in the partial area in the container, and confirm that the temperature has become 30 ° C. or less. In 400, temperature control of another cell culture container or a partial region in the container is continued. Such an operation is also included in the event, and after the operation is completed, the ready of the cell culture apparatus is reconfirmed.

It is an external appearance perspective view which shows the structural example of the cell culture system of this embodiment. It is an external appearance perspective view from 2 directions which shows the structural example of a culture chamber and a cooling means. It is a figure which shows the cut surface of the cell culture container 120 typically. It is a figure which shows an example of the pattern of the heater / thermistor in this embodiment. It is a figure which shows the other example of the pattern of the heater / thermistor in this embodiment. It is an external appearance perspective view of the cell culture container (petri dish) of this embodiment. It is a figure which shows the example of the control block of the cell culture system of this embodiment. It is a flowchart which shows the example of a control procedure of the personal computer for temperature control and monitoring in this embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Culture chamber 110 Cooling means 120 Cell culture container 121 Heater pattern 121a Heater wiring and input / output terminal / pattern 122 Insulating layer 123 Thermistor pattern 123a Thermistor wiring and input / output terminal / pattern 124 Polystyrene layer 125 Isopropyl acrylamide layer 126 Cell culture fluid 127 Enclosure 128 Silicone resin 130 Heat exhaust fan 140 Peltier element and heat sink 150 Heat exhaust fin 160 Substrate (silicon or glass)
200 Sterilization lid 300 Cultivation gas 400 Heating means control device 500 Cooling means control device 600 PC for temperature controller / minita

Claims (17)

A cell culture device for culturing cells of a living organism,
At least one heater disposed on at least one flat plate to control the surface temperature of the flat plate;
Temperature measuring means paired with the heater;
A connection terminal with a control circuit that autonomously controls the temperature of the heater based on the temperature detected by the temperature measuring means;
Cooling means for cooling the back surface of the flat plate;
A thin film polymer layer formed on the upper surface of the heater and the temperature measuring means, wherein hydrophilicity and hydrophobicity are switched depending on temperature,
A cell culture device comprising: a storage means for storing a cell culture solution in a region containing the surface temperature control means and the thin film polymer.   2. The cell culture instrument according to claim 1, wherein the region of the storage means includes a region where the heater is not present.   The cell culture instrument according to claim 1 or 2, wherein the heater is arranged in a spiral shape in the region of the storage means.   The cell culture instrument according to claim 1 or 2, wherein the heater is arranged in a grid pattern in the area of the storage means.   The cell heater according to claim 3 or 4, wherein the heater is patterned using a semiconductor process technology.   The cell culture device according to claim 1, wherein the thin film polymer layer whose hydrophilicity and hydrophobicity are switched depending on the temperature contains isopropyl acrylamide.   The cell culture instrument according to claim 1, further comprising a second polymer layer for promoting the formation of the thin film polymer layer between the surface temperature control means and the thin film polymer layer.   The cell culture device according to claim 7, wherein the second polymer layer includes polystyrene formed by melt coating. A cell culture device for culturing cells of a living organism,
At least one surface temperature control means arranged on at least one flat plate to control the surface temperature of the flat plate, comprising at least one heater, a temperature measuring means paired with the heater, and the temperature measuring means. A surface temperature control means having a control circuit for autonomously controlling the temperature of the heater according to the detected temperature;
Cooling means for cooling the back surface of the flat plate;
A chamber covering the surface temperature control means and capable of gas exchange;
A cell culture apparatus for carrying out cell culture by placing a culture vessel having a thin film polymer layer on its bottom surface, which switches between hydrophilicity and hydrophobicity depending on temperature.   The cell culture device according to claim 9, wherein a bottom region of the culture vessel includes a region where the heater is not present.   The cell culture device according to claim 9 or 10, wherein the heater is spirally arranged in a bottom area of the culture vessel.   The cell culture device according to claim 9 or 10, wherein the heaters are arranged in a grid pattern in a bottom region of the culture vessel.   The cell culture device according to claim 11 or 12, wherein the heater is patterned using a semiconductor process technology.   The cell culture device according to claim 9, wherein the thin film polymer layer whose hydrophilicity and hydrophobicity are switched depending on the temperature contains isopropyl acrylamide.   The said culture container is further provided with the 2nd polymer layer for accelerating formation of the said thin film polymer layer between the said surface temperature control means and a thin film polymer layer. Cell culture device.   The cell culture device according to claim 15, wherein the second polymer layer includes polystyrene formed by melt coating. A cell culture system for managing cell culture of organisms,
A cell culture device for culturing living cells and a computer for monitoring and managing the cell culture device;
The cell culture device comprises:
At least one surface temperature control means arranged on at least one flat plate to control the surface temperature of the flat plate, comprising at least one heater, a temperature measuring means paired with the heater, and the temperature measuring means. A surface temperature control means having a control circuit for autonomously controlling the temperature of the heater according to the detected temperature;
Cooling means for cooling the back surface of the flat plate;
A chamber covering the surface temperature control means and capable of gas exchange;
A cell culture system characterized in that a cell culture is carried out in accordance with monitoring and management by the computer, with a culture vessel on which a thin film polymer layer whose hydrophilicity and hydrophobicity are switched depending on temperature is formed.

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