JP2016021969A - Cell culture apparatus and cell culture test observation system with the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus and system which makes it possible to perform microscopic observation of a cell culture experiment in real time.SOLUTION: A cell culture apparatus 20 includes: a cell culture device 30 in which 100 wells arranged in 10 rows and 10 columns in 1 square centimeter are formed by piling up a chamber communication passage member, a chamber member, a thin film, a culture solution flow passage member, and a heating sheet member, which are made of transparent material, on a glass substrate and joining them together; and a case box 23 and a case lid which are made of a transparent material and hermetically store the cell culture device together with a cotton material 28 containing sterilized water and a carbon dioxide concentration adjustment agent 29. By applying a voltage to the heating sheet member to maintain a temperature suitable for cell culture, how cells and cell masses of embryoid bodies and the like are cultured in the wells arranged in 10 rows and 10 columns can be observed through the case in real time for a long time.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture device and a cell culture test observation system including the same, and more specifically, a well is formed in each of a plurality of chambers having an effective diameter of 1 mm or less arranged in M rows and N columns, and cells are placed in each well. A cell culture device for culturing or aggregating cells in each well to form an embryoid body and performing various operations on the embryoid body, and cells and embryoid bodies using this cell culture device The present invention relates to a cell culture test observation system for microscopically observing the culture state.

  Conventionally, as this type of cell culture apparatus, an apparatus in which a heater transparent plate is attached to the upper surface of a petri dish lid and the bottom surface of the petri dish has been proposed (for example, see Patent Document 1). In this device, a transparent conductive film made of indium tin oxide (ITO) is used as the transparent plate, and a heater and a temperature sensor are attached to the transparent plate attached to the bottom of the petri dish. A circuit for configuring is configured. Such a transparent plate enables microscopic observation of cell culture while controlling the temperature in the petri dish.

JP 2009-201509 A

  However, although the above-described apparatus is suitable for microscopic observation for a relatively short time, it is not suitable for microscopic observation over a long period of time because humidity management and carbon dioxide concentration management cannot be sufficiently performed. In petri dishes, the production of embryoid bodies, which are three-dimensional stem cell aggregates of stem cells, is not suitable for operations such as mass production of uniform embryoid bodies, differentiation induction, and recovery.

  The inventors of the present application have developed a pressure-driven multiwell plate (Pressure Actuated Shapable Microwell) that enables simultaneous production of 100 uniform embryoid bodies, introduction experiment of up to 10 reagents, and selective recovery of embryoid bodies. Array, PASMA) was developed and used to conduct embryoid body production and differentiation induction experiments using human stem cells, to produce uniform embryoid bodies using human adipose-derived stem cells, and to differentiate into multiple cell types It has succeeded in producing embryoid bodies using induced, human induced pluripotent stem cells (iPS cells). For such embryoid body experiments, real-time observation over a long period of time is desired.

  The cell culture device of the present invention and the cell culture test observation system equipped with the same are mainly intended to provide an apparatus and a system capable of microscopic observation of cell culture experiments in real time.

  The cell culture device of the present invention and the cell culture test observation system equipped with the same employ the following means in order to achieve the above-mentioned main object.

The cell culture device of the present invention comprises:
A chamber portion having a plurality of chambers having an effective diameter of 1 mm or less arranged in M rows and N columns and a stretchable and non-cell-adhering treatment on the upper surface, and joining the upper surface of the chamber portion so as to cover the plurality of chambers A chamber layer having a thin film portion formed therein, and a chamber communication path layer formed so as to be in close contact with the lower surface of the chamber layer, and having N communication paths that communicate with the plurality of chambers for each column, A culture fluid channel layer, which is arranged so as to be in close contact with the upper surface of the chamber layer, and in which M culture fluid channels for supplying culture fluid to the thin film portions on the upper surface of the plurality of chambers are provided for each row; A thin film portion of a plurality of chambers arranged in the M rows and N columns by discharging the fluid of the N communication passages to discharge the fluid, and extending the thin film portions to the chamber side. A more M rows and N to form a plurality of wells arranged in rows, the cell culture apparatus for culturing cells supplied by the plurality of wells from the M broth flow path from the culture liquid flow path layer,
The cell culture device is formed of a material that is transparent and heated when a voltage is applied, and has a heating layer disposed so as to cover the upper surface of the culture fluid channel layer,
Formed from a transparent material, the cell culture device is provided with a case that can be hermetically stored together with auxiliary agents necessary for cell culture such as water and carbon dioxide concentration adjusting agent,
This is the gist.

  In the cell culturing apparatus of the present invention, the cell device includes a chamber portion having a plurality of chambers having an effective diameter of 1 mm or less arranged in M rows and N columns and a stretchable and non-cell-adhering treatment on the upper surface. N communication paths that are arranged in close contact with the lower surface of the chamber layer and communicate with each of the plurality of chambers for each column, the chamber layer having a thin film portion joined so as to cover the plurality of chambers on the upper surface of the portion Are formed so as to be in close contact with the upper surface of the chamber layer, and M culture fluid channels for supplying the culture fluid to the thin film portions on the upper surface of the plurality of chambers are formed. The thin film portions of a plurality of chambers arranged in M rows and N columns are extended to the chamber side by discharging fluid of N communication channels and arranged in M rows and N columns. Multiple to Forming a E le, it can be cultured cells supplied from the M broth flow path from the culture liquid flow path layer (including a cell mass such as embryoid bodies) in a plurality of wells. Then, a cell culture device that is transparent and is formed of a material that is heated when a voltage is applied and has a heating layer disposed so as to cover the upper surface of the culture fluid flow path layer, and this cell culture device is a cell such as a water or carbon dioxide concentration adjusting agent. It is hermetically stored in a case formed of a transparent material together with auxiliary agents necessary for culture. Therefore, by applying an appropriate voltage to the heating layer of the cell culture device, the temperature can be adjusted to a temperature suitable for cell culture in a plurality of wells arranged in M rows and N columns, and sufficient humidification in the case can be achieved. An appropriate carbon dioxide concentration can be adjusted. In addition, since the heating layer and the case are made of a transparent material, the state of cell culture in a plurality of wells arranged in M rows and N columns through the case, the state of creation of cell masses such as embryoid bodies, It is possible to observe the state of changes to the cell mass and the reagent in real time over a long period of time. Here, air, oil, or the like can be used as the fluid supplied to and discharged from the N communication paths of the chamber communication path layer. When air is used as the fluid, fluid discharge means a negative pressure. When oil is used as the fluid, it is necessary to fill each chamber and the communication path with oil in advance. The chamber communication channel layer, chamber layer, culture fluid channel layer, and heating layer may be formed as a single member, or a part or all of them may be formed as different members and stacked. It doesn't matter. The auxiliary agent is not limited to the above-mentioned water or carbon dioxide concentration adjusting agent, and any auxiliary agent may be used as long as it is necessary for cell culture.

  In such a cell culture apparatus of the present invention, the case is provided with N fluid supply / discharge pipes for supplying and discharging fluid to and from each of the N communication paths of the chamber communication path layer, and the heating. A power line may be attached that applies a voltage to the layer. In addition, the case may be configured such that a culture solution supply / discharge tube for supplying and discharging the culture solution is attached to each of the M culture solution channels of the culture solution channel layer. In this state, the formation of the well in column j is canceled by supplying fluid to the communication channel corresponding to column j in row M and column N of the N communication channels of the chamber communication channel layer. By flowing the culture fluid through the culture fluid channel corresponding to i row in M rows and N columns among the M culture fluid channels of the culture fluid channel layer, it is located in i row and j column in M rows and N columns. A cell mass such as a cell or embryoid body cultured in a well (hereinafter referred to as “cell or the like”) can be taken out. Then, after the cells are taken out, the flow of the culture solution to the culture solution channel corresponding to i row of the culture solution channel layer is stopped, and the communication channel corresponding to the j row of the chamber communication channel layer is passed through. By discharging the fluid to form the well in the j-th row, it is possible to continue the culture of the cells in the well except for the i-th row among the cells cultured in the j-th row well. That is, it is possible to take out a desired cell in the i-th row and j-th column from the cells cultured in the M-row and N-column wells, and then continue to culture the cells in other wells. Here, “culture of cells and the like” includes administration of a reagent to cells and the like, and so on.

  Further, in the cell culture device of the present invention, N cultures arranged at upper portions of both ends of the M culture fluid channels in the culture fluid channel layer and respectively communicating with both ends of the M culture fluid channels. A pair of culture solution supply / discharge members having a solution supply / discharge unit may be provided, and the case may house the pair of culture solution supply / discharge members.

The cell culture test observation system of the present invention comprises:
A cell culture test observation system for observing a cell culture test,
A cell culture device of the present invention according to any one of the above-described aspects;
A microscopic observation apparatus for microscopically observing wells formed in the plurality of chambers by placing the cell culture apparatus;
It is a summary to provide.

  Since the cell culture test observation system of the present invention includes the cell culture device of the present invention and the microscopic observation device according to any one of the above-described aspects, the cell culture test in a plurality of wells arranged in M rows and N columns through the case is performed. It is possible to microscopically observe in real time over a long period of time, how the cell mass such as the embryoid body is created, and how the cell or cell mass is changed with respect to the reagent.

  In such a cell culture test observation system of the present invention, fluid supply / discharge means for individually supplying and discharging fluid to and from the N communication paths of the chamber communication path layer, and the M cultures of the culture fluid flow path layer When a culture solution supply / discharge unit for supplying / discharging the same or different culture solution to / from the solution flow path and a well located in the i row and j column among the plurality of wells arranged in the M row and N column are designated , Supply fluid to j rows of the N communication passages to release formation of the j rows of wells, and supply i rows of culture fluid channels to the M culture fluid channels. Cell discharge control for controlling the fluid supply / discharge unit and the culture solution supply / discharge unit to supply the culture solution, and after the cell discharge control, supply of the culture solution to the i-th culture solution channel is stopped. Before forming a j-row well by draining the fluid in the j-row communication passage May be intended to comprise a control means for executing the well re-formation control for controlling said culture solution supply and discharge means and the fluid supply and discharge means, and the. In this way, by simply designating the well located in the i row and j column among the plurality of wells arranged in the M row and the N column, the cells cultured in the i row and j column wells are taken out, and then the other The cells can be continuously cultured in the wells. In the cell culture test observation system of the present invention of this aspect, the designation buttons for designating the plurality of wells arranged in the M rows and N columns are arranged in the M rows and N columns using a display screen displaying in a matrix. Designating means capable of designating one or a plurality of wells among a plurality of wells, and the control means executes the cell discharge control and the well re-formation control for the designated well. It can also be a means. In this way, it is possible to easily designate and take out one or a plurality of wells in which cells or the like to be taken out are cultured. In this case, the display screen may display images of a plurality of wells arranged in the M rows and N columns observed by the microscope observation device on the designation button. By doing this, it is possible to specify cells to be taken out while viewing images of cells or the like cultured in a plurality of wells arranged in M rows and N columns that are observed in real time.

It is a block diagram which shows the outline of a structure of the cell culture apparatus 20 as one Example of this invention. 3 is a configuration diagram showing an outline of the configuration of a case lid 21. FIG. It is a fragmentary sectional view which expands and shows a part of AA cross section in FIG. It is explanatory drawing which shows an example of a structure of the communicating path member 40 for chambers. 4 is an explanatory diagram showing an example of a configuration of a chamber member 50. FIG. 4 is an explanatory diagram showing an example of a configuration of a culture fluid flow path member 60. FIG. It is explanatory drawing which shows an example of a structure of the culture solution supply / discharge member 65,66. FIG. 5 is an explanatory diagram showing an outline of a configuration of a heating sheet member 70. It is explanatory drawing which shows an example of the relationship between the voltage (V) applied in the heating sheet member 70, and temperature. 4 is an explanatory diagram schematically showing each state of an embryoid body (cell mass) creation process in the cell culture device 20. FIG. It is explanatory drawing which shows typically each state of the collection | recovery process of the embryoid body cultivated in the cell culture apparatus 20. FIG. It is a block diagram which shows the outline of a structure of the cell culture test observation system 120 of an Example. It is a block diagram which shows the outline of a structure of the fluid supply / discharge device 140. FIG. It is a block diagram which shows the outline of a structure of the culture solution supply / discharge apparatus 150 with the supply / discharge holes 65a-65j and 66a-66j of the culture solution supply / discharge member 65,66. 4 is a flowchart illustrating an example of a collection control routine executed by a computer 160. It is explanatory drawing which shows an example of the well designation | designated screen displayed on the display. It is explanatory drawing which shows a part of well designation | designated screen of a modification.

  Next, the form for implementing this invention is demonstrated using an Example.

  FIG. 1 is a configuration diagram showing an outline of the configuration of a cell culture apparatus 20 as an embodiment of the present invention, and FIG. 2 is a configuration diagram showing an outline of the configuration of a case lid 21 of the cell culture apparatus 20 of the embodiment. FIG. 3 is a partial cross-sectional view showing a part of the AA cross section in FIG. 1 in an enlarged manner. The cell culture apparatus 20 of the embodiment includes a cell culture device 30 for culturing a cell mass such as cells and embryoid bodies (hereinafter referred to as “cells”), a material 28 containing sterilized water, and carbon dioxide gas. The density adjusting agent 29 is hermetically stored by the case lid 21 and the case box 23. The outer dimensions of the case are about 7 cm wide, 4 cm deep, and 1.5 cm high.

  The case box 23 is formed of a transparent resin. As shown in FIG. 1, the device storage unit 25a, the sterilized water storage unit 25b, and the carbon dioxide concentration adjusting agent storage unit 25c are separated by partition walls 24a and 24b. Is defined. Through holes 26a and 26b for taking out the power line to the outside are formed on the left side surface of the case box 23 in the drawing. Further, ten supply / discharge tubes 46 a to 46 j for the well formation release fluid are attached to the upper side surface of the case box 23 in the drawing. Similarly to the case box 23, the case lid 21 is formed of a transparent resin, and as shown in FIG. 2, substantially rectangular through holes 22a and 22b for supplying and discharging the culture solution are formed.

  As shown in FIG. 3, the cell culture device 30 includes a glass substrate 32, a chamber communication channel member 40, a chamber member 50, a thin film 58, a culture fluid channel member 60, and an insulating sheet 68 in order from the bottom. And the heating sheet member 70 are laminated using an adhesive, and culture solution supply / discharge members 65 and 66 are disposed on the upper surfaces of both ends thereof. As the adhesive, polydimethylsiloxane (PDMS) used for forming the chamber communication path member 40, the chamber member 50, the thin film 58, and the culture fluid flow path member 60 can be used.

  FIG. 4 is an explanatory diagram showing an example of the configuration of the chamber communication path member 40. The chamber communication passage member 40 is formed in a plate shape from a transparent material, for example, silicon rubber such as polydimethylsiloxane (PDMS), and has ten groove-like communication passages 42a each having a width of 600 μm on the upper surface thereof. To 42j, and tube attachment portions 44a to 44j for supplying and discharging fluid to and from the communication passages 42a to 42j are formed on the upper side in the drawing. The tube attachment portions 44 a to 44 j are connected to supply / discharge tubes 46 a to 46 j attached to the case box 23.

  FIG. 5 is an explanatory diagram showing an example of the configuration of the chamber member 50. The chamber member 50 is also formed in a plate shape from a transparent material, for example, silicon rubber such as polydimethylsiloxane (PDMS), and has a square side with a side of 1 cm in the center and a square with a side of 600 μm in cross section. 100 chambers 52aa to 52jj configured as holes are formed. The 100 chambers 52aa to 52jj are regularly arranged in 10 rows and 10 columns so as to be aligned with the 10 communication passages 42a to 42j when they are stacked on the chamber communication passage member 40. A thin film 58 formed of the same material is bonded to the upper surface of the chamber member 50 so as to cover the chambers 52aa to 52jj. As will be described later, the thin film 58 extends toward the chambers 52aa to 52jj. Thus, wells 59aa to 59jj for culturing cells are formed. The upper surface of the thin film 58 is subjected to cell non-adhesion treatment to prevent cell adhesion.

  FIG. 6 is an explanatory diagram showing an example of the configuration of the culture fluid channel member 60. The culture fluid channel member 60 is also formed in a plate shape by a transparent material, for example, silicon rubber such as polydimethylsiloxane (PDMS), and a groove-like width of 600 μm for flowing the culture fluid is formed on the lower surface thereof. Ten culture fluid channels 62a to 62j are formed, and communication holes 63a to 63j and 64a to 64j communicating with the culture fluid channels 62a to 62j are formed on the left and right sides in the drawing. The culture fluid flow paths 62a to 62j are adjusted so that the central portions thereof are aligned with the chambers 52aa to 52jj formed in 10 rows and 10 columns when overlapped with the chamber member 50, and both ends thereof are wide. It is formed to spread.

  FIG. 7 is an explanatory diagram showing an example of the configuration of the culture solution supply / discharge members 65 and 66. The culture fluid supply / discharge members 65 and 66 are also formed in a substantially rectangular parallelepiped by a transparent material, for example, silicon rubber such as polydimethylsiloxane (PDMS), and have ten supply / discharge holes 65a to 65a penetrating in the vertical direction. 65j, 66a to 66j are formed. The supply / discharge holes 65a to 65j of the culture solution supply / discharge member 65 and the supply / discharge holes 66a to 66j of the culture solution supply / discharge member 66 are arranged as shown in FIG. It is adjusted so as to be aligned with the communication holes 63a to 63j and the communication holes 64a to 64j of the member 60. The culture fluid supply / discharge members 65, 66 are adhered to the culture fluid flow path member 60 by using polydimethylsiloxane (PDMS) as an adhesive, and the communication holes 63a-63j, 64a-64j are connected to the supply / discharge holes 65a-65j, 66a to 66j are performed so as to match.

  The insulating sheet 68 is formed of a transparent insulating material, for example, a PET film (polymer film formed of polyethylene terephthalate resin).

  FIG. 8 is an explanatory diagram showing an outline of the configuration of the heating sheet member 70. The heating sheet member 70 is transparent and is formed of a material that heats when a voltage is applied (indium tin oxide (ITO) in the embodiment), and is formed on the transparent conductive film 72 and the upper and lower portions of the transparent conductive film 72 in the figure. It comprises electrodes 74a and 74b formed by gold vapor deposition and power lines 76a and 76b attached to the electrodes 74a and 74b. FIG. 9 shows an example of the relationship between the voltage (V) applied to the electrodes 74 a and 74 b through the power lines 76 a and 76 b and the temperature of the transparent conductive film 72. As illustrated, the temperature of the transparent conductive film 72 increases as the applied voltage increases. Therefore, in the heating sheet member 70 of the embodiment, in order to set the temperature of the transparent conductive film 72 to a temperature suitable for cell culture (for example, about 38 ° C.), a voltage of less than 5 V may be applied to the electrodes 74a and 74b. I understand that.

  Next, a process for creating a cell mass such as an embryoid body and a process for recovering a cell mass such as the created embryoid body in the cell culture device 20 of the above-described embodiment will be described. FIG. 10 is an explanatory diagram schematically showing each state of the process of creating an embryoid body in the cell culture device 20 of the example. FIG. 11 is an embryoid body cultured in the cell culture device 20 of the example. It is explanatory drawing which shows typically each state of this collection process. In the process of creating an embryoid body, first, cells are seeded by flowing a cell suspension obtained by suspending cells in the culture solution into the culture solution channels 62a to 62j of the culture solution channel member 60 (FIG. 10). (A)). Next, the fluid in the communication passages 42a to 42j of the chamber communication passage member 40 is discharged, and the thin film 58 of the chambers 52aa to 52jj of the chamber member 50 is expanded (inflated) to the chambers 52aa to 52jj. 59aa to 59jj are formed (FIG. 10B). As the fluid in the communication passages 42a to 42j, for example, air or oil can be used. In the case of air, the discharge of air means a negative pressure. If left in this state for a while, the cells settle by gravity, and the cells aggregate in each well to form an embryoid body (cell mass) (FIG. 10 (c)). Since the upper surface of the thin film 58 is subjected to cell non-adhesion treatment, the created embryoid body does not adhere to the thin film 58. For the embryoid body (cell mass) thus prepared, the same reagent or different reagents are supplied to the culture fluid flow paths 62a to 62j of the culture fluid flow path member 60 for each flow path, Differentiated cells can be cultured.

  In the process of recovering embryoid bodies, the case of recovering embryoid bodies cultured in well 59ed out of embryoid bodies cultured in wells 59aa to 59jj arranged in 10 rows and 10 columns will be described as an example. . First, a fluid (for example, air) is supplied to the communication passage 42d of the chamber communication passage member 40 to release the formation of the wells 59ad to 59jd in the row d, and the embryoid body cultured in the wells 59ad to 59jd is removed from the culture solution. It floats in the culture fluid flow paths 62a to 62j of the flow path member 60 (FIG. 11 (b)). Next, by supplying the culture solution to the culture solution channel 62e, the embryoid body cultured in the well 59ed floating in the culture solution channel 62e is caused to flow, and the supply / discharge hole 66e of the culture solution supply / discharge member 66 is flown. (Fig. 11 (c)). At this time, embryoid bodies are cultured in the wells 59ea to 59ec and 59ee to 59ej to which the culture solution is supplied by the culture solution flow path 62e, but these embryoid bodies float on the culture solution flow path 62e. Therefore, by paying attention to the flow rate (feed rate) of the culture solution in the culture solution channel 62e, only the embryoid bodies cultured in the well 59ed are collected while remaining in the wells 59ea to 59ec and 59ee to 59ej. be able to. When the embryoid body is recovered in this way, the supply of the culture solution in the culture solution channel 62e of the culture solution channel member 60 is stopped (FIG. 11 (d)), and the fluid is discharged to the communication channel 42d of the chamber communication channel member 40. Then, wells 59ad to 59jd are formed again in the ten chambers 52ad to 52jd in the d row (FIG. 11E). Of the wells 59ad to 59jd, the embryoid bodies floating in the culture fluid flow paths 62a to 62d and 62f to 62j are settled in each well except the well 59ed, and the culture of the embryoid bodies is continued.

  According to the cell culture device 20 of the embodiment described above, since the case lid 21, the case box 23, and the heating sheet member 70 are formed of a transparent material, the heating sheet member 70 and the culture solution supply / discharge member are added to the cell culture device 30. 65, 66 are placed and hermetically sealed with a case 28 and a case lid 21 together with a material 28 containing sterilized water and a carbon dioxide concentration regulator 29, and a voltage is applied to the heating sheet member 70 to be suitable for cell culture. By adjusting the temperature, it is possible to observe the state of culturing cells and the like in the wells 59aa to 59jj arranged in 10 rows and 10 columns over the case in real time over a long time. Naturally, cells can be cultured in wells 59aa to 59jj arranged in 10 rows and 10 columns within 1 cm square, embryoid bodies can be prepared, and reagents can be supplied to the prepared embryoid bodies.

  In the cell culture device 20 of the embodiment, the chambers 52aa to 52jj having 10 rows and 10 columns are formed as the cell culture device 30. Any number of columns may be used.

  In the cell culture device 20 of the example, the chambers 52aa to 52jj are formed as square through holes having a side of a cross section of 600 μm as the shape of the chambers 52aa to 52jj, but may be formed in any shape as long as the effective diameter is 1 mm or less.

  In the cell culture device 20 of the embodiment, the thin film 58 is bonded to the chamber member 50. However, the thin film 58 may be formed as a single member in a state where the thin film 58 is bonded to the chamber member 50.

  In the cell culture device 20 of the example, the chamber communication channel member 40, the chamber member 50, the thin film 58, and the culture fluid channel member 60 are all formed of silicon rubber such as polydimethylsiloxane (PDMS). As long as it is a transparent material, these may be formed of other materials. In this case, the thin film 58 is required to be a stretchable material. Further, when the culture fluid channel member 60 is formed of an insulating material, the insulating sheet 68 is not necessary.

  In the cell culture device 20 of the embodiment, the chamber communication passage member 40, the chamber member 50, the thin film 58, the culture fluid flow path member 60, the insulating sheet 68, and the heating sheet member 70 are formed as different members. Since a passage layer, a chamber layer, a culture fluid channel layer, and a heating sheet layer may be formed, all or a part of them may be formed as a single member.

  In the cell culture device 20 of the embodiment, the case box 23 is provided with a device storage portion 25a, a sterilized water storage portion 25b, and a carbon dioxide concentration adjusting agent storage portion 25c by partition walls 24a and 24b, and includes the cell culture device 30 and sterilized water. The wadding material 28 and the carbon dioxide concentration adjusting agent 29 are accommodated. However, in addition to the water and the carbon dioxide concentration adjusting agent, other auxiliary agents necessary for cell culture are accommodated together with the cell culture device 30. , Or one of the water and carbon dioxide concentration regulators and other supplements necessary for cell culture, or it is necessary for cell culture instead of water and carbon dioxide concentration regulators. Other adjuvants may be stored.

  In addition, in the cell culture apparatus 20 of an Example, not only can it observe in real time over a long time the state of culture | cultivation of the cell etc. in well 59aa-59jj arranged in 10 rows and 10 columns over a case, but also cell culture. It is also possible to use the device 20 for conveying cells. In this case, the cells can be transported in a cultured state.

  Next, a cell culture test observation system 120 including the cell culture device 20 of the example will be described. FIG. 12 is a configuration diagram illustrating an outline of a configuration of the cell culture test observation system 120 according to the embodiment. The cell culture test observation system 120 according to the example is attached to a cell culture device 20, a microscopic observation device 130 on which the cell culture device 20 is mounted, and a case box 23 of the cell culture device 20, as illustrated. Connected to the supply and discharge tubes 46a to 46j and connected to the supply / discharge holes 140a and 65j of the culture solution supply / discharge member 65 and the fluid supply / discharge device 140 for supplying and discharging fluid to and from the chamber communication passage member 40 communication passages 42a to 42j The culture medium supply / discharge device 150, a computer 160 for controlling the whole, and a display 170 connected to the computer 160 are provided.

  The microscope observation device 130 is configured as a device using a well-known microscope, and scans the wells 59aa to 59jj of the cell culture device 30 arranged in 10 rows and 10 columns in a 1 cm square by a computer 160 for microscopic observation. So that it is controlled.

  FIG. 13 is a configuration diagram showing an outline of the configuration of the fluid supply / discharge device 140. The fluid supply / discharge device 140 includes a vacuum pump 141, a branch pipe 142 connected to the vacuum pump 141 and branched into 11 branches, 10 of 11 branches of the branch pipe 142, and a supply / discharge tube attached to the case box 23. Connection tubes 146a to 146j connected to 46a to 46j, an electromagnetic valve 143 attached to the branch pipe 142 on the vacuum pump 141 side, and one of the 11 branches of the branch pipe 142 not connected to the supply / discharge tubes 46a to 46j A solenoid valve 144 for opening the branch pipe 142 to the atmosphere by being attached to the individual, and solenoid valves 145a to 145j attached to ten of the 11 branches of the branch pipe 142 connected to the supply / discharge tubes 46a to 46j; . The fluid supply / drainage device 140 opens the solenoid valve 143 and the solenoid valves 145a to 145j with the solenoid valve 144 closed, and operates the vacuum pump 142, whereby the communication passages 42a to 42 of the chamber passage member 40 are opened. The air of 42j is discharged, and wells 59aa to 59jj are formed in the respective chambers 52aa to 52jj. The solenoid valve 144 is opened while the solenoid valve 143 is closed, and any one or more of the solenoid valves 145a to 145j are opened, so that the communication path connected to the opened solenoid valve is opened. Air is supplied to release the well formation. These solenoid valves 143, 144, 145 a to 145 j and the vacuum pump 142 are driven and controlled by a computer 160.

  FIG. 14 is a configuration diagram showing a schematic configuration of the culture solution supply / discharge device 150 together with supply / discharge holes 65a to 65j and 66a to 66j of the culture solution supply / discharge members 65 and 66. The culture fluid supply / discharge device 150 includes ten syringes 153a to 153j filled with culture fluid and reagents, actuators 152a to 152j that drive the plungers of the syringes 153a to 153j, syringes 153a to 153j, and culture fluid supply / discharge Connection tubes 154a to 154j connecting the supply and discharge holes 65a to 65j of the member 65 are provided. The supply / discharge holes 66a to 66j of the culture solution supply / discharge member 66 are connected to the trays 156a to 156j by connection tubes 155a to 155j. The culture fluid supply / discharge device 150 drives the plungers of the ten syringes 153a to 153j filled with the cell suspension, thereby causing the cell suspension to flow into the culture fluid channels 62a to 62j of the culture fluid channel member 60. To form embryoid bodies in the wells 59aa to 59jj. Thereafter, by driving the plungers of the ten syringes 153a to 153j filled with the same or different reagents, the desired reagent can be supplied to the desired culture fluid channel among the culture fluid channels 62a to 62j. it can. As the actuators 152a to 152j, for example, hydraulic drive can be used. The actuators 152a to 152j are driven and controlled by the computer 160.

  Since the cell culture test observation system 120 of the embodiment configured in this way uses the cell culture device 20 of the embodiment, the state of culturing cells and the like in the wells 59aa to 59jj arranged in 10 rows and 10 columns over the case is shown for a long time. It is possible to perform microscopic observation in real time. Although not described in detail, the state of culture of the cells of the wells 59aa to 59jj can be sequentially displayed on the display 170 by scanning with the microscopic observation device 130, or can be stored as an image in a storage device (not shown). .

  In the cell culture test observation system 120 of the embodiment, a collection control routine illustrated in FIG. 15 is executed by the computer 160 to collect cells or the like cultured in the designated well. When the collection control routine is executed, the computer 160 first displays a well designation screen on the display 170 (step S100), and executes a process of waiting until the “execute” button is operated on the well designation screen (step S110). To do. FIG. 16 shows an example of the well designation screen. In this well designation screen, designation buttons are arranged in a matrix so as to correspond to wells 59aa to 59jj of 10 rows and 10 columns a to j, and one or a plurality of desired wells are selected using an instruction device such as a mouse. It can be operated over a range of hours. In addition to the “execute” button, there is also provided an “ALL cancel” button that cancels the designated operation of the well that has been designated and returns to the initial state in which no well is designated.

  When the “execute” button is operated in a state where one or a plurality of wells have been designated on the well designation screen, a well from which cells or the like are to be collected is determined (step S120). The determination of the well is performed, for example, by determining in the order in which the designation button is operated or by checking whether the designation button is operated in order from the a column. Now, as a method for determining wells, a method of sequentially checking from column a will be described, and as shown in FIG. 16, a case where a well 59cb and a well 59ed are designated and the “execute” button is operated will be described as an example. In this case, first, the well 59cb is determined as a well from which cells and the like are to be collected. When the wells are determined, air is supplied to the communication passage 42b of the chamber communication passage member 40 corresponding to the row b which is the well row to release the wells of the wells 59ab to 59jb in the row b (step S130). . Specifically, the operation is performed by opening the electromagnetic valve 144 of the fluid supply / discharge device 140 and opening the electromagnetic valve 145b. Subsequently, the culture solution is supplied to the culture solution channel 62c of the culture solution channel member 60 corresponding to the determined row c and the cells and embryoid bodies floating in the culture solution channel 62c are obtained. The liquid is poured into the tray 156c and collected (step S140). Specifically, this is performed by driving the actuator 152c of the culture solution supply / discharge device 150 to supply the culture solution filled in the syringe 153c to the supply / discharge hole 65c of the culture solution supply / discharge member 65.

  When the cells in the wells thus determined are collected, the supply of the culture solution to the culture solution channel 62c is stopped by stopping the driving of the actuator 152c (step S150), and the solenoid valve 145b is opened and the solenoid valve is opened. 144 is closed, the electromagnetic valve 143 is opened, and suction is performed by the vacuum pump 141, so that the wells 59 ab to 59 jb in the row b are formed again (step S 160), and there is still a well in which the designated button is operated. It is determined whether or not there is a well in which the designation button is operated (step S170), and the process returns to the well determination in step S120. In the example now considered, since the well 59ed remains, an affirmative determination is made in step S170, the process returns to step S120, and the well 59ed is determined as a well from which cells and the like are to be collected. Also for the well 59ed, when cells and the like are collected by the same process as the well 59cb (steps S130 to S160) and the well is re-formed and the process reaches step S170, it is determined that there is no well in which the designated button is operated. This routine is terminated.

  In the cell culture test observation system 120 of the embodiment described above, one desired well is added to the wells 59aa to 59jj arranged in 10 rows and 10 columns displayed on the well designation screen by executing the collection control routine. Alternatively, cells or the like cultured in a desired well can be collected simply by operating the “execute” button by operating the designation buttons of a plurality of wells.

  In the cell culture test observation system 120 of the example, a plurality of designation buttons arranged in a matrix so as to correspond to the wells 59aa to 59jj of 10 rows and 10 columns a to j are displayed on the well designation screen. As shown in a part of the well designation screen of the modified example of FIG. 17, images of the cells of the wells 59aa to 59jj observed by the microscopic observation device 130 are displayed on a plurality of designation buttons arranged in a matrix. It is good also as what to do. FIG. 17 shows a state where the well 59cb in the c row and the b column is designated. In this way, it is possible to designate and take out cells and the like to be taken out while viewing images of cells and the like cultured in the respective wells 59aa to 59jj arranged in 10 rows and 10 columns that are observed in real time.

  In the cell culture test observation system 120 of the embodiment, the trays 156a to 156j are used for the collection of cells and the like through the connection tubes 155a to 155j, but a petri dish or a test tube may be used instead of the tray. .

  In the cell culture test observation system 120 of the embodiment, air is used as a fluid to be supplied to and discharged from the communication passages 42 a to 42 j of the chamber communication passage member 40, and a vacuum pump 141 or the like is provided as the fluid supply / discharge device 140 for that purpose. However, liquid such as oil may be used as the fluid to be supplied to and discharged from the communication passages 42a to 42j of the chamber communication passage member 40, and a hydraulic circuit may be provided as the fluid supply / discharge device.

  In the cell culture device 20 of the embodiment, the connection tubes 154a to 154j and the connection tubes 155a to 155j are connected to the supply / discharge holes 65a to 65j and 66a to 66j of the culture solution supply / discharge members 65 and 66, respectively. The through holes 22a and 22b are formed in the case lid 21 in order to attach the trays 156a to 156j, but the through holes 22a and 22b are not formed in the case lid 21, and the culture solution supply / discharge members 65 and 66 are formed. May be stored in a case.

  As mentioned above, although the form for implementing this invention was demonstrated using the Example, this invention is not limited at all to such an Example, In the range which does not deviate from the summary of this invention, it is with various forms. Of course, it can be implemented.

  The present invention can be used in the manufacturing industry of cell culture devices and cell culture test observation systems.

  20 cell culture device, 21 case lid, 22a, 22b through hole, 23 case box, 24a, 24b partition wall, 25a device storage unit, 25b sterilized water storage unit, 25c carbon dioxide concentration adjusting agent storage unit, 26a, 26b through hole , 28 Cotton material, 29 Carbon dioxide concentration adjusting agent, 30 Cell culture device, 32 Glass substrate, 40 Chamber communication channel member, 42a-42j Communication channel, 44a-44j Tube mounting part, 46a-46j Supply / exhaust tube, 50 Chamber layer, 52aa-52jj chamber, 58 thin film, 59aa-59jj well, 60 culture fluid flow path member, 62a-62j culture fluid flow path, 63a-63j, 64a-64j communication hole, 65, 66 culture fluid supply / discharge member, 65a to 65j, 66a to 66j Supply / exhaust hole, 68 Insulating sheet, 70 Heating Member, 72 transparent conductive film, 74a, 74b electrode, 76a, 76b power line, 120 cell culture test observation system, 130 microscope observation device, 140 fluid supply / discharge device, 141 vacuum pump, 142 branch pipe, 143, 144 145a to 145j Solenoid valve, 146a to 146j connection tube, 150 culture fluid supply / discharge device, 152a to 152j actuator, 153a to 153j syringe, 154a to 154j, 155a to 155j connection tube, 156a to 156j saucer, 160 computer, 170 display.

Claims (8)

A chamber portion having a plurality of chambers having an effective diameter of 1 mm or less arranged in M rows and N columns and a stretchable and non-cell-adhering treatment on the upper surface, and joining the upper surface of the chamber portion so as to cover the plurality of chambers A chamber layer having a thin film portion formed therein, and a chamber communication path layer formed so as to be in close contact with the lower surface of the chamber layer, and having N communication paths that communicate with the plurality of chambers for each column, A culture fluid channel layer, which is arranged so as to be in close contact with the upper surface of the chamber layer, and in which M culture fluid channels for supplying culture fluid to the thin film portions on the upper surface of the plurality of chambers are provided for each row; A thin film portion of a plurality of chambers arranged in the M rows and N columns by discharging the fluid of the N communication passages to discharge the fluid, and extending the thin film portions to the chamber side. A more M rows and N to form a plurality of wells arranged in rows, the cell culture apparatus for culturing cells supplied by the plurality of wells from the M broth flow path from the culture liquid flow path layer,
The cell culture device is formed of a material that is transparent and heated when a voltage is applied, and has a heating layer disposed so as to cover the upper surface of the culture fluid channel layer,
Formed from a transparent material, the cell culture device is provided with a case that can be hermetically stored together with auxiliary agents necessary for cell culture such as water and carbon dioxide concentration adjusting agent,
Cell culture device. The cell culture device according to claim 1,
The case has N fluid supply / discharge pipes for supplying and discharging fluid to and from each of the N communication paths of the chamber communication path layer, and a power line for applying a voltage to the heating layer. Attached,
Cell culture device. The cell culture device according to claim 1 or 2,
The case is provided with a culture solution supply / discharge tube for supplying and discharging the culture solution to each of the M culture solution channels of the culture solution channel layer.
Cell culture device. The cell culture device according to claim 1 or 2,
A pair of cultures having N culture fluid supply / discharge portions that are arranged on both ends of the M culture fluid channels in the culture fluid channel layer and communicate with both ends of the M culture fluid channels, respectively. A liquid supply / discharge member;
The case houses the pair of culture solution supply / discharge members,
Cell culture device. A cell culture test observation system for observing a cell culture test,
A cell culture device according to any one of claims 1 to 4;
A microscopic observation apparatus for microscopically observing wells formed in the plurality of chambers by placing the cell culture apparatus;
A cell culture test observation system. The cell culture test observation system according to claim 5,
Fluid supply / discharge means for individually supplying and discharging fluid to and from the N communication paths of the chamber communication path layer;
A culture fluid supply / discharge means for supplying / discharging the same or different culture fluid individually to the M culture fluid channels of the culture fluid flow path layer;
When a well located in i row and j column among a plurality of wells arranged in M rows and N columns is designated, fluid is supplied to j communication channels of the N communication channels to provide j columns For controlling the fluid supply / discharge means and the culture liquid supply / discharge means to release the wells and supply the culture liquid to the i-th culture liquid flow path among the M culture liquid flow paths. After the discharge control and the cell discharge control, the fluid is stopped so that the supply of the culture medium to the i-th culture medium flow path is stopped and the fluid in the j-column communication path is discharged to form the well in the j-row. Control means for performing well re-formation control for controlling the supply / discharge means and the culture solution supply / discharge means;
A cell culture test observation system. The cell culture test observation system according to claim 6,
One or a plurality of wells of the plurality of wells arranged in the M rows and N columns using a display screen in which designation buttons for designating the plurality of wells arranged in the M rows and N columns are displayed in a matrix. There is a specification means that can specify
The control means is means for executing the cell discharge control and the well re-formation control for the designated well.
Cell culture test observation system. The cell culture test observation system according to claim 7,
The display screen displays images of a plurality of wells arranged in the M rows and N columns observed by the microscope observation device on the designation button.
Cell culture test observation system.

Images