Cell culture apparatus, cell culture method, cell culture program and cell culture system

The invention intends to provide a cell culture apparatus which is able to realize an adequate culture according to the culture state of cells while alleviating the labor of an operator. The cell culture apparatus includes a culture bag for causing the cells to proliferate, a cell inoculation cassette (or culture bag as an antibody stimulating and proliferation culture vessel) for stimulating the cells by an inducer for the proliferation, a culture medium cassette for storing a culture medium supplied to the culture bag and the cell inoculation cassette, a CCD camera 88 for acquiring images of the cells in the cell inoculation cassette, and an image processing computer and an operation control computer for determining the culture state (proliferation capability and proliferation ability of the cells) of the cells from the images of the cells acquired by the CCD camera, and causing a culture operation to be carried out on the basis of the determination.

This application is a U.S. National Phase Application under 35 USC 371 of International Application PCT/JP2006/321901 filed Nov. 1, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a cell culture apparatus, a cell culture method, a cell culture program and a cell culture system for evaluating the culture state of a cell and culturing the cell.

2. Description of the Related Art

In the related art, cell culture, in particular, suspension cell culture used for an immune cell therapy is executed by human operation in most cases. For example, cells sampled from a patient are inoculated to a flask having an antibody (inducer) attached thereto together with a culture medium, and stored in an incubator. Then, the flask is taken out from the incubator every day to observe the culture state (for example, the state of proliferation) using a microscope or the like. When the proliferation is found, or when a predetermined time period has elapsed from the cell inoculation, the culture medium is added to the flask to culture the cell (for example, proliferation).

In the manual cell culture as described above, the daily operation is executed on the basis of a culture evaluation depending on the experience of a culture engineer, or the culture operation is executed homogenously for all the cells on the basis of a predetermined manual. Therefore, the culture (for example, proliferation) might not be sufficient depending on the sampled cells. In order to induce the proliferation ability of the cell which is different from patient to patient, to a maximum extent, an objective culture evaluation and the culture operation by the hour on the basis of this evaluation are necessary.

Part of such problem is solved in the culture of adherent cells as described in JP-A-2002-218995. In other words, in JP-A-2002-218995, the proliferation ability of the entire cell population is understood by observing the form of the individual adherent cells in a non-invasive and non-destructive manner through the images.

As an example which is used for the manual cell culture as described above, JP-A-2005-73566 discloses a cell culture system in which a plurality of canisters (storing units) are arranged in a single incubator (culture chamber) and culture vessels are stored in the canisters one by one for culturing cells. In this culture system, the culture environment is adapted to be the same for all the canisters in the single incubator.

However, in the case of the culture of suspension cells used for the immune cell therapy, devices for evaluating the culture state of the cells objectively or for culturing the cells on the basis of the evaluation do not exist.

In the manual cell culture including the cell culture system disclosed in JP-2005-73566, an operator who is engaged in the culture, is required to collect culture environment data in the incubator or the canisters and record the log of the culture operation such as change of the culture medium, which is a very complicated work. There is a probability that trouble in the cell culture cannot be known adequately.

SUMMARY OF THE INVENTION

In view of such circumstances as described above, it is an object of the invention to provide a cell culture apparatus, a cell culture method and a cell culture program which realize an adequate culture according to the state of the cell culture with an alleviated labor for an operator.

It is another object of the invention to provide a cell culture system, a cell culture method and a cell culture program which are able to collect and accumulate cell culture-related data which relates to the cell culture automatically, and are able to monitor and control the cell culture on the basis of the cell culture-related data.

A cell culture apparatus according to a first aspect of the invention includes a culture vessel for culturing cells; a culture medium storage unit for storing a culture medium supplied to the culture vessel; an image acquiring unit for acquiring images of the cells in the culture vessel; and a control unit for determining the culture state of the cells from the images of the cells acquired by the image acquiring unit and causing a culture operation to be carried out on the basis of the determination.

Preferably, the culture vessel includes a proliferation culture vessel for causing the cells to proliferate and a function expressing culture vessel for causing the cells to express the functions, and the image acquiring unit acquires the images of the cells in the function expressing culture vessel.

Preferably, the function expressing culture vessel is an inducer stimulating culture vessel for stimulating cells by an inducer for the proliferation, and the control unit determines the proliferation capability of the cells and the proliferation ability of the cells on the basis of the images of the cells in the inducer stimulating culture vessel and controls timing of transferring the cells from the inducer stimulating culture vessel to the proliferation culture vessel and the culture operations such as supply of the culture medium from the culture medium storage unit to the proliferation culture vessel.

Preferably, the function expressing culture vessel is a differentiation-inducing culture vessel for causing the cells to differentiate, and the control unit controls a differentiation-inducing operation on the basis of the images of the cells in the differentiation-inducing culture vessel.

Preferably, the culture medium storage unit has a cassette structure and is connected to the culture vessel.

Preferably, the function expressing culture vessel has a cassette structure, and is connected to the proliferation culture vessel and the culture medium storage unit.

Preferably, the culture medium storage unit, the function expressing culture vessel and the proliferation culture vessel make up a closed system.

Preferably, a liquid reservoir is selectively formed in the proliferation culture vessel, and the cell and the culture medium are stored in the liquid reservoir in an initial stage of culture in the proliferation culture vessel.

Preferably, the cells proliferated in the proliferation culture vessel are introduced into the function expressing culture vessel and the image acquiring unit acquires images of the introduced cells.

Preferably, the culture medium storage unit includes a used culture medium storage vessel which is able to store a used culture medium installed therein together with a culture medium storage vessel which is able to store a culture medium to be supplied to the culture vessel, and the used culture medium in the proliferation culture vessel is discharged to the used culture medium storage vessel and is stored therein.

Preferably, the culture medium storage unit is able to mount a cell collecting vessel for collecting the cells mounted thereto, and the cells condensed in the proliferation culture vessel are collected in the cell collecting vessel.

Preferably, the cells are suspension cells.

Preferably, the cells are used in an immune cell therapy.

A cell culture method according to a second aspect of the invention includes: acquiring images of cells in a culture vessel for culturing cells by an image acquiring unit; determining the culture state of the cells from the images of the cells acquired by the image acquiring unit; and carrying out a culture operation in the culture vessel on the basis of the determination.

Preferably, the culture vessel includes a proliferation culture vessel for causing the cells to proliferate and a function expressing culture vessel for causing the cells to express the functions, and the image acquiring unit acquires the images of the cells in the function expressing culture vessel.

Preferably, the function expressing culture vessel is an inducer stimulating culture vessel for stimulating cells by an inducer for the proliferation, and the proliferation capability of the cells and the proliferation ability of the cells are determined on the basis of the images of the cells in the inducer stimulating culture vessel, the timing of transferring the cells from the inducer stimulating culture vessel to the proliferation culture vessel is controlled and culture operations such as supply of the culture medium from the culture medium storage unit to the proliferation culture vessel is carried out.

Preferably, the function expressing culture vessel is a differentiation-inducing culture vessel for causing the cells to differentiate, and the differentiation-inducing operation is controlled on the basis of the images of the cells in the differentiation-inducing culture vessel.

Preferably, the cells and the culture medium are stored in a liquid reservoir in the proliferation culture vessel in an initial stage of culture in the proliferation culture vessel.

Preferably, the cells proliferated in the proliferation culture vessel is introduced into the function expressing culture vessel, and the image acquiring unit acquires images of the introduced cells.

Preferably, a used culture medium in the proliferation culture vessel is discharged to a used culture medium storage vessel in the culture medium storage unit.

Preferably, the cells condensed in the proliferation culture vessel are collected in the cell collecting vessel in the culture medium storage unit.

Preferably, the cells are suspension cells.

Preferably, the cells are used in an immune cell therapy.

A cell culture program stored in a computer for carrying out the cell culture according to a third aspect of the invention includes: a step of acquiring images of cells in a culture vessel for culturing cells by an image acquiring unit; a step of determining the culture state of the cells from the images of the cells acquired by the image acquiring unit; and a step of carrying out a culture operation in the culture vessel on the basis of the determination.

A cell culture program stored in a computer for culturing cells according to a fourth aspect of the invention includes; a step of acquiring images of cells in a function expressing culture vessel for causing the cells to express the functions thereof by an image acquiring unit; a step of determining the culture state of the cells from the images of the cells acquired by the image acquiring unit; and a step of carrying out a culture operation in at least one of the function expressing culture vessel and the proliferation culture vessel for causing the cells to proliferate on the basis of the determination.

According to the first aspect of the invention, preferably, the culture vessel is placed on a platform and a culture area is changed by moving part of the platform upward and downward.

Preferably, one of a simple feeding culture and a perfusion culture is selectable.

Preferably, one of an intermittent perfusion culture and a consecutive perfusion culture is selectable.

According to the second aspect of the invention, preferably, the culture vessel is placed on the platform and the culture area is changed by moving part of the platform upward and downward.

Preferably, one of the simple feeding culture and the perfusion culture is selectable.

Preferably, one of the intermittent perfusion culture and the consecutive perfusion culture is selectable.

A cell culture system for culturing cells and monitoring and controlling the culture according to a fifth aspect of the invention includes: an incubator having a plurality of culture units in which culture vessels for culturing cells are disposed respectively so as to be apart from each other for culturing the cells under culture environments independent for the individual culture unit; and a control unit for collecting and accumulating cell culture-related data relating to the cell culture for the individual culture unit in the incubator, monitoring the culture state of the cells for the individual culture unit on the basis of the cell culture-related data and controlling a culture operation of the cells.

Preferably, the control unit includes an operation control panel being installed in a culture room together with the incubator and having a function to control the culture in the respective culture units in the incubator, and a monitoring computer installed out of the culture room for receiving and displaying data in the operation control panel.

Preferably, the cell culture-related data is at least one of identification signs of the cells, the culture medium, the culture vessel, the incubator, the culture unit and an operator, culture environment data in the incubator and the culture unit, and image data of the cells in the culture vessel.

Preferably, a plurality of the incubators are installed, and the operation control panels are connected to the respective incubators and a single monitoring computer is connected to the plurality of operation control panels.

Preferably, a remote monitoring computer is connected to the monitoring computer via a public network.

Preferably, canisters in the incubators, which constitute the culture units, are isolated from each other so as to avoid incoming and outgoing of bacteria or the like.

Preferably, the canisters in the incubator, which constitute the culture units, each include a fan installed therein for introducing air in the incubator toward the canister, and the fans of all the canisters in the single incubator are adapted to stop when a door of the incubator is opened.

Preferably, the canisters in the incubator, which constitute the culture units, are adapted in such a manner that only one of doors of the canisters in the single incubator is opened at a time.

Preferably, the cells are suspension cells.

Preferably, the cells are used in an immune cell therapy.

A cell culture method according to a sixth aspect of the invention for culturing cells and monitoring and controlling the culture including: disposing a incubator having a plurality of culture units in which culture vessels for culturing cells respectively are disposed so as to be apart from each other for culturing the cells under culture environments independent for the individual culture units; collecting and accumulating cell culture-related data relating to the cell culture for the individual culture unit in the incubator, monitoring the culture state of the cells for the individual culture unit on the basis of the cell culture-related data; and controlling a culture operation of the cells.

Preferably, the cell culture-related data is at least one of identification signs of the cells, the culture medium, the culture vessel, the incubator, the culture unit and an operator, culture environment data in the incubator and the culture unit, and image data of the cells in the culture vessel.

Preferably, the cells are suspension cells.

Preferably, the cells are used in an immune cell therapy.

A cell culture program stored in a computer for culturing cells and monitoring and controlling the culture according to a seventh aspect of the invention including: a step of collecting and accumulating cell culture-related data relating to the cell culture individually for a plurality of culture units in a incubator, the culture unit each having a culture vessel for culturing cells disposed therein, the incubator having the plurality of culture units arranged therein so as to be apart from each other for culturing the cells under culture environments independent for the individual culture units; a step of monitoring the culture state of the cells for the individual culture unit on the basis of the cell culture-related data; and a step of controlling a culture operation of the cells for the individual culture unit on the basis of the cell culture-related data.

A cell culture apparatus according to an eighth aspect of the invention is characterized in that a plurality of culture vessels for culturing cells are connected in series, and the respective culture vessels are adapted to culture the cells in different culture environments, transfer the cultured cells to the culture vessel on the downstream side to culture the same.

Preferably, the two culture vessels are installed, and one of the culture vessels is a function expressing culture vessel having a culture environment for causing the cells to express the functions and the other culture vessel is a proliferation culture vessel having a culture environment for causing the cells to proliferate.

Preferably, the function expressing culture vessel is an inducer stimulating culture vessel having a culture environment for stimulating the cells by an inducer for the proliferation.

Preferably, the function expressing culture vessel is a differentiation-inducing culture vessel having a culture environment for causing the proliferated cells to differentiate.

Preferably, the cells are suspension cells.

Preferably, the cells are used in an immune cell therapy.

A cell culture method according to a ninth aspect of the invention includes: culturing cells in different culture environments in a plurality of culture vessels and transferring the cells cultured in one of the culture vessels to different culture vessels on the downstream side in one-to-one correspondence in sequence to carry out the culture.

Preferably, the plurality of culture vessels are two culture vessels and the cells are stimulated by the inducer for the proliferation in one of the two vessels and then the cells are proliferated in the other culture vessel.

Preferably, the plurality of culture vessels are two culture vessels and the cells are proliferated in one of the two culture vessels and then the cells are differentiated in the other culture vessel.

Preferably, the cells are suspension cells.

Preferably, the cells are used in an immune cell therapy.

A cell culture apparatus according to a tenth aspect of the invention includes: a culture vessel for culturing cells; and a platform for placing the culture vessel, and is characterized in that the platform includes a portion which is able to move upward and downward, and the upward and downward movement of the portion which is able to move upward and downward changes the area of the culture vessel placed thereon which is able to be used for the culture.

Preferably, the cells are suspension cells.

Preferably, the cells are used in an immune cell therapy.

Preferably, a culture medium storage unit for storing a culture medium to be supplied to the culture vessel is connected to the culture vessel.

Preferably, the culture medium storage unit and the culture vessel make up a closed system.

Preferably, the culture medium storage unit includes a used culture medium storage vessel which is able to store the used culture medium installed therein together with a culture medium storage vessel which is able to store culture medium to be supplied to the culture vessel, and the used culture medium in a proliferation culture vessel is discharged to the used culture medium storage vessel and is stored therein.

Preferably, an image acquiring unit for acquiring images of cells in the culture vessel and a control unit for determining the culture state of the cells from the images of the cells acquired by the image acquiring unit and causes a culture operation to be carried out on the basis of the determination are provided.

Preferably, the culture vessel includes the proliferation culture vessel for causing the cells to proliferate and a function expressing culture vessel for causing the cells to express the functions, and the image acquiring unit acquires images of the cells in the function expressing culture vessel.

Preferably, the function expressing culture vessel is an inducer stimulating culture vessel for stimulating the cells by an inducer for the proliferation, and the control unit determines the proliferation capability of the cells and the proliferation ability of the cells on the basis of the images of the cells in the inducer stimulating culture vessel and controls timing of changing the area which is available for the culture and the culture operations such as supply of the culture medium from the culture medium storage unit to the proliferation culture vessel.

According to the invention, the culture state of cells are determined from the images of the cells in the culture vessel, and the culture operation according to the culture state is carried out. Therefore, the culture state of the cells is determined in a non-contact state, and hence the cells are prevented from getting damaged, and the operator does not need to carry out the culture operation one by one, whereby the labor of the operator is alleviated. Furthermore, since the adequate culture operation according to the culture state of the cell is realized, an adequate culture operation is achieved. Since the adequate culture operation according to the culture state of the cells is achieved, the culture operation by the hour is enabled, and hence the culture term is shortened.

According to the invention, the culture medium storage unit has a cassette structure and is connected to the culture vessels (the function expressing culture vessel and the proliferation culture vessel). Therefore, the culture vessels are always maintained in an environment optimal for the culture, so that damage to the cells in the culture vessels in association with the change in environment is alleviated, and the aseptic operation for supplying the culture medium to the culture vessel in the clean bench or the like is omitted.

According to the invention, the culture storage unit, the function expressing culture vessel and the proliferation culture vessel make up a closed system. Therefore, the culture storage unit, the function expressing culture vessel and the proliferation culture vessel are maintained under the aseptic conditions.

According to the invention, the cells and the culture medium are stored in the liquid reservoir in the proliferation culture vessel in the initial stage of the culture in the proliferation culture vessel. Therefore, the efficient proliferation of the cells is achieved by maintaining the cell density per area to a density preferable to the proliferation.

According to the invention, the cells proliferated in the proliferation culture vessel are introduced into the function expressing culture vessel, and the images of the cells are acquired by the image acquiring unit. Therefore, the number of cells and the form of the cells are observed by acquiring the cells proliferated in the proliferation culture vessel as the images.

According to the invention, the used culture medium in the proliferation culture vessel is discharged and accumulated in the used culture medium storage vessel in the culture medium storage unit, and hence the cells in the proliferation culture vessel is condensed to increase the cell density. Therefore, the number of times of the operation of centrifugation for collecting the cells is reduced. Consequently, the labor for collecting the cells is saved, and the damage of the cells in association with the centrifugation is reduced.

According to the invention, all the cells condensed in the proliferation culture vessel are collected into the cell collecting vessel in the culture medium storage unit, and hence the cells are collected by mounting the cell collecting vessel directly to the centrifuge. Therefore, the labor for collecting the cells is saved.

According to the invention, the area of the liquid reservoir for storing the cells and the culture medium in the culture vessel is able to be changed by the predetermined area, and hence the cell density per area during the culture is maintained at a density preferable for the proliferation. Therefore, efficient proliferation of the cells is achieved.

According to the invention, the operation control panel and the monitoring computer collect and accumulate the cell culture-related data relating to the cell culture individually for the each culture unit in the incubator. Therefore, the culture log relating to the cells cultured in the arbitrary culture unit is adequately known. The operation control panel and the monitoring computer monitor the culture state of the cells on the basis of the cell culture-related data for the individual culture unit in the incubator. Therefore, trouble in the culture state may be monitored individually for the each culture unit. Furthermore, the operation control panel and the monitoring computer control the operation of cell culture such as the change of the culture medium (replacement of the culture medium cassette) and collection of the cells after having completed the culture (collection of the cells by the culture bag or the cell collecting bag) individually for the each culture unit (the canister and the culture cassette) in the incubator. Therefore, the operation schedule for the operation (work) carried out by the operator individually for the each culture unit in the incubator is easily prepared. The control unit includes the operation control panel being installed in the culture room together with the incubator and having a function to control the culture in the each culture unit (the canister and the culture cassette) in the incubator and the monitoring computer being installed in the monitor room other than the culture room and receiving and saving the data that the operation control panel has and making the same available for display (inspection). Therefore, the culture state of the cells in the culture unit in the incubator in the culture room may be observed, monitored and controlled in the monitor room other than the culture room by the monitoring computer. With this control, the log or the like is automatically recorded and stored, and occurrence of intentional alteration or erroneous recording is prevented.

According to the invention, the canisters in the incubator which constitutes the culture units are each isolated to avoid entry of the cells and/or bacteria by the air-intake filter and the exhaust filter. Therefore, the cells in the culture bag and the cell inoculation cassette stored in the each canister are prevented from being contaminated by bacteria. The blast fans of all the canisters in the single incubator are configured to stop when the main body door of the incubator is opened. Therefore, the sealed state of the each canister in the incubator is maintained even when the main body door of the incubator is opened, so that the independent culture environment for the each canister is preferably secured, so that the fluctuation of the culture environments in the canisters are restrained. Only one of the canister doors of the canisters in the single incubator is allowed to be opened. Therefore, there is no probability that the two or more canister doors of the canisters are opened at a time in the single incubator, so that the mix-up of the culture bags and the cell inoculation cassettes among the canisters is prevented, and the cross-contamination of the cells is avoided.

According to the invention, the antibody stimulation and the cell proliferation in the initial stage of culture in the cell culture bag are carried out in the same culture bag, and the area of the liquid reservoir for storing the cells and the culture medium in the culture bag is changed by a predetermined area. Therefore, efficient proliferation of the cells is achieved by maintaining the cell density per area during the proliferation to a density preferable for the proliferation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to the drawings, best modes for carrying out the invention will be described below.

[A] First Embodiment

FIG. 1Ais a configuration drawing showing a cell culture apparatus according to a first embodiment of the invention andFIG. 1Bis a configuration drawing showing a cell culture system according to the first embodiment of the invention.FIG. 2is a perspective view illuartaring an incubator inFIGS. 1A and 1B.FIG. 5is a layout drawing showing a configuration of a culture unit including one canister in the incubator and a culture cassette stored in the canister.

A cell culture apparatus10shown inFIG. 1Ais specifically adapted to culture suspension cells used for an immune cell therapy, and includes an incubator11provided with a plurality of (eight, for example) culture units12, an operation control panel13for controlling an operation of the incubator11and the culture units12, an image processing computer14for processing images of cells, and a monitoring computer15being connected to the operation control panel13and the image processing computer14for monitoring the incubator11and the culture units12. The operation control panel13and the image processing computer14function as a control unit.

The cell culture apparatus10is also implemented as a cell culture system. The cell culture system10is specifically adapted to culture the suspension cells used for the immune cell therapy, and includes the incubator11, the operation control panel13, the image processing computer14and the monitoring computer15like the cell culture apparatus10shown above. As shown inFIG. 1B, the incubator11, the operation control panel13and the image processing computer14are installed in a culture room (clean room)94suitable for the cell culture, and the monitoring computer15is installed in a monitor room95other than the culture room94. The incubator11includes a plurality of (eight, for example) the culture units12in which a culture vessel for culturing cells (a proliferation culture vessel, an inducer stimulating culture vessel described later) arranged so as to apart from each other. In this incubator11, cells in the culture vessels are cultured under culture environments independent for the individual culture units12.

The operation control panel13controls the operation of the incubator11and the culture units12, and monitor and control the cell culture for the individual culture units12in the incubator11. The image processing computer14processes images of the cells under culture for the individual culture units12in the incubator11, and functions as a control unit for controlling the culture operation. The monitoring computer15is connected to the operation control panel13and the image processing computer14, receives and stores data from the operation control panel13and the image processing computer14, makes the data available for display (inspection), and serves as a control unit for monitoring and controlling the cell culture.

The known suspension cells include peripheral blood mononuclear cells, LAK cells (Lymphokine Activated killer cells), neural stem cells, and ES cells. These types of suspension cells are referred simply to as “cells”, hereinafter. These cells are stimulated and cultured with inducers which are suitable for the individual cells. The inducers differ from cell to cell. For example, the suitable inducer for LAK cells is anti-human CD3 antibody, the suitable inducer for the neural stem cells is epidermal growth factor such as EGF, and the suitable inducer for ES cells is fibroblast growth factor such as FGF-8b. The cell culture apparatus10is also applicable to a case of culturing adherent cells other than the suspension cells.

The culture unit12includes a plurality of (eight, for example) canisters16(FIG. 2andFIG. 5) separated so as to be apart from each other in the incubator11, and culture cassettes17(FIG. 2andFIG. 3) to be stored in the canisters16individually. The culture cassette17includes, a culture bag18as a proliferation culture vessel, a cell inoculation cassette19as an inducer stimulating culture vessel, and a culture medium cassette20as a culture medium storage unit. Cells in the culture vessels (culture bag18and cell inoculation cassette19) are cultured under the independent culture environments for the individual canisters16.

As shown inFIG. 2, the incubator11includes a plurality of tiers of shelves23in an incubator body22having an openable-closable main body door21installed therein, and the canisters16are arranged on the shelves23one by one. The incubator11maintains the environment (temperature, humidity, and CO2concentration) in the incubator body22in an environment required for the cell culture in as state in which the main body door21is closed.

Therefore, as shown inFIG. 5andFIG. 6, a temperature sensor24, a CO2sensor25, a door sensor26and a heater27are arranged in the incubator body22, and a gas cylinder28installed outside is coupled to the incubator body22. Signals from the temperature sensor24, the CO2sensor25and the door sensor26are transmitted to the operation control panel13. The operation control panel13controls the heater27on the basis of the temperature signal from the temperature sensor24, and controls the amount of the CO2gas supplied from the gas cylinder28to the incubator body22on the basis of the CO2concentration signal from the CO2sensor25.

As shown inFIG. 5andFIG. 6, the canister16has an openable-closable canister door31mounted to a canister body30, and an air-intake filter32and an exhaust filter33are installed to the canister body30and a blast fan34is installed on the air-intake filter32side of the canister body30.

The air-intake filter32and the exhaust filter33are filters for removing bacteria, and prevent entry of bacteria from the incubator body22into the canister16when air and CO2gas in the incubator body22is taken into the canister16by the operation of the blast fan34. The operation of the blast fan34is controlled by the operation control panel13and, when a signal indicating that the main body door21of the incubator11is opened is transmitted from the door sensor26to the operation control panel13, the operation of the blast fan34is stopped and the sealed state of the canister16is ensured.

The function of the air-intake filter32and the exhaust filter33to prevent entry of bacteria from the incubator body22into the canister16, and the securement of the sealing property of the canister16due to the operation stop of the blast fan34bring the interiors of the individual canisters16to independent culture environment. Accordingly, cells in the culture cassette17stored in one single canister16in the incubator11are isolated from cells in other canisters16, and cells in the culture cassette17stored in the canister16is prevented from being contaminated by bacteria. So-called cross-contamination, which is the contamination by cells of other patients, is also prevented.

The canister body30is further provided with a door sensor35, a door lock sensor36, a temperature sensor37, a humidity sensor38, a door lock mechanism39, a heater40and a circulation fan41. The operation control panel13controls the heater40on the basis of the temperature signal from the temperature sensor37. The operation control panel13controls the operation of the circulation fan41, and circulates air and CO2gas in the canister16. The humidity sensor38detects the humidity in the canister16, transmits the same to the operation control panel13, and detects the trouble therein. In this manner, the interior of the canister16is maintained in an environment optimal for the cell culture.

The operation control panel13controls the operation of the door lock mechanism39so as to prevent two or more canister doors31from opening at the same time in the single incubator11. Accordingly, erroneous transportation of cells or the culture medium between the different canisters16is prevented. The locking operation of the door lock mechanism39is detected by the door lock sensor36, and is transmitted to the operation control panel13. The opened or closed state of the canister door31is detected by the door sensor35, and is transmitted to the operation control panel13.

A stage42for supporting the culture cassette17to be stored in the canister16is provided at the lower portion in the canister body30shown inFIG. 5, a weight meter43is installed on the stage42. The weight meter43is adapted to weigh the culture bag18of the culture cassette17stored in the canister16and, actually, measures the amount of culture medium to be supplied from the culture medium cassette20to the culture bag18. The measured value of the weight meter43is also transmitted to the operation control panel13. A indication lamp44is provided in the canister body30. When culture is carried out automatically in some canisters16, the indication lamps44of the corresponding canisters16are illuminated, for example, in red, and the indication lamps44of the canisters16to which the operation instruction is issued and the canister16in which the incubation cassette17is not mounted are illuminated in green. Accordingly, the culture environment independent from other canisters16is secured for the canisters16in which an automatic culture is carried out, so that the cross contamination or mix-up with specimen (cells) stored in other canisters16may be prevented.

The culture cassette17will now be described.

As shown inFIG. 3andFIG. 5, the culture cassette17is composed of the culture bag18, the cell inoculation cassette19, and the culture medium cassette20mounted to a large tray45, and is a culture vessel in which the culture bag18and the cell inoculation cassette19culture cells. The cell inoculation cassette19is a function expressing culture vessel for causing the cells to express the function (for example, to cause the cells to proliferate, to cause the cells to differentiate (described later)), and is an inducer stimulating culture vessel for stimulating the cells by the inducer for proliferation in this embodiment. The culture bag18is a proliferation culture vessel for causing the cells to proliferate stimulated by the inducer in the cell inoculation cassette19.

The culture bag18is a flexible disposable vessel for storing culture suspension in which cells are inoculated, is placed on a culture bag tray47shown inFIG. 4A, interposing a platform46(FIG. 5) between the culture bag18and the culture bag tray47, and the culture bag tray47is detachably mounted to the large tray45shown inFIG. 3andFIG. 5. The culture bag18is a bag formed of, for example, an oxygen permeable material. As shown inFIG. 5, a first pump48, a second pump49and a third pump50are arranged on the large tray45. One end of the culture bag18is connected to a seventh connector57via the second pump49using a tube63. The other end of the culture bag18is connected to a first connector51using a tube60. The first pump48, the second pump49and the third pump50are preferably of a peristaltic-type pump from the convenience of replacement of a sterile tube.

In the large tray45, one end of the tube61is connected to the third connector53and the other end thereof is connected to a fifth connecter55. The tube61is disposed on a rotating portion, not shown, of the first pump48. The tube63is connected to the seventh connector57at one end as described above and to the culture bag18at the other end. The tube63is disposed on a rotating portion, not shown, of the second pump49. One end of the tube62is connected to the tube61via the connecter X and the other end thereof is connected to the tube63via connecter Y. The tube62is disposed on a rotating portion, not shown, of the third pump50.

As shown inFIG. 4C, the cell inoculation cassette19is formed into a cassette structure by immobilizing the inducer on the inner side of the bottom surface of an inducer stimulation vessel65, adding the culture medium into the inducer stimulation vessel65, inoculating cells on the culture medium, and installing the inducer stimulation vessel65in a set frame66. The operation to add the inducer, the culture medium and the cells into the inducer stimulation vessel65is carried out under aseptic conditions in a clean bench or a safety cabinet (hereinafter, referred to as clean bench etc. in this embodiment). The mixed liquid of the culture medium and cells is referred to as culture suspension.

As shown inFIG. 3, the cell inoculation cassette19is detachably mounted to the large tray45. At this time, as shown inFIG. 5, the second connector52and the fourth connector54of the cell inoculation cassette19are coupled to the first connector51and the third connector53of the large tray45under the aseptic conditions, respectively. In other words, the first connector51and the second connector52are coupled, for example, by inserting a rubbery joint on one of those to a needle joint on the other one of those under the aseptic conditions. The coupling of the third connector53and the fourth connector54is the same.

By the coupling between the first connector51and the second connector52, the cell inoculation cassette19and the culture bag18in the different culture environment, that is, the cell inoculation cassette19having a culture environment for stimulating cells by the inducer to cause the cells to express the proliferating function and the culture bag18having a culture environment for causing the cells to proliferate are connected using the tube60. Therefore, the cells which are stimulated by the inducer in the cell inoculation cassette19and has started the proliferation may be transferred to culture bag18to cause the cells only to proliferate in the culture bag18.

As shown inFIG. 4B, the culture medium cassette20is formed into a cassette structure with mounting a culture medium bag67as a culture medium storage vessel and a used culture medium bag68as a used culture medium storage vessel on a culture medium bag tray69. The culture medium bag67is adapted to store the culture medium to be supplied to the culture bag18and the cell inoculation cassette19. The used culture medium bag68is adapted to store the used culture medium (supernatant) discharged from the culture bag18. With the culture medium cassette20having the cassette structure, change and supply of the culture medium are enabled only by mounting the culture medium cassette20to the culture cassette17in the canister16in a state of maintaining the culture bag18in the canister16.

The culture medium cassette20is detachably mounted to the large tray45(FIG. 3). At this time, as shown inFIG. 5, a sixth connector56and an eighth connector58of the culture medium cassette20are coupled to the fifth connecter55and the seventh connector57of the large tray45under the aseptic conditions respectively in the same manner as the case of the first connector51and the second connector52. By the coupling between the sixth connector56and the fifth connecter55, the culture medium bag67and the cell inoculation cassette19are connected. By the coupling between the seventh connector57and the eighth connector58, the used culture medium bag68and the culture bag18are connected.

With the connection among the culture bag18, the cell inoculation cassette19and the culture medium cassette20as described above, a closed system in which the culture medium in the culture medium bag67in the culture medium cassette20is supplied to the culture bag18via the cell inoculation cassette19when the first pump48is activated, and the used culture medium in the culture bag18is discharged to the used culture medium bag68in the culture medium cassette20when the second pump49is activated is established. With the establishment of the closed system, the system (the culture bag18, the cell inoculation cassette19and the culture medium cassette20) is maintained under the aseptic conditions.

The cell inoculation cassette19described above is replaced by a dummy cassette70in a stage where there is no more cell therein. Accordingly, the inducer in the cell inoculation cassette19is prevented from being transferred to the interior of the culture bag18. The culture medium cassette20is replaced by a new culture medium cassette20having the culture medium bag67filled with the culture medium and an empty used culture medium bag68in a stage where the culture medium bag67becomes empty. This replacement is carried out by the operator. The dummy cassette70simply has a function as a flow channel for allowing the culture medium to flow.

The culture (cell proliferation) in the culture bag18includes a feeding culture in which the first pump48is activated to supply (feed) the culture medium in the culture medium bag67in the culture medium cassette20to the culture bag18so that the proliferation of cells is achieved, a perfusion culture in which the first pump48and the second pump49are activated to discharge the used culture medium in the culture bag18to the used culture medium bag68in the culture medium cassette20and to supply the culture medium in the culture medium bag67to the culture bag18so that the proliferation of cells is achieved, a shaking culture using a shaking device80described later and a static culture in which the shaking operation is not carried out. The perfusion culture includes an intermittent perfusion culture in which discharge of the used culture medium and supply of the culture medium are carried out alternately, and a consecutive perfusion culture in which the discharge of the used culture medium and the supply of the culture medium are carried out simultaneously. In the consecutive perfusion culture, a filter71for preventing transfer of cells is normally disposed between the culture bag18and the second pump49in the tube63to prevent the cells in the culture bag18from being discharged to the used culture medium bag68.

The third pump50is activated, for example, in the case of observing the cells proliferating in the culture bag18using the images. In other words, the cells in the culture bag18is introduced from the culture bag18to the cell inoculation cassette19or the dummy cassette70via the tube63, the tube62and the tube61when the filter71is not disposed in the tube63, and is shot by a CCD camera88, described later, so that the number of proliferated cells or the like is observed. When the filter71is disposed in the tube63, the upstream side of the third pump50is connected to the culture bag18via a bypass tube73, and the cells in the culture bag18are introduced into the cell inoculation cassette19or the dummy cassette70via the bypass tube73and the tube62, and are observed using the CCD camera88.

The used culture medium in the culture bag18is discharged to the used culture medium bag68in the culture medium cassette20after having completed the proliferation of cells in the culture bag18, and the cells in the culture bag18is condensed. The discharge of the used culture medium is carried out by activating the second pump49until the amount of the culture suspension in the culture bag18is reduced to about ½ to ⅓ in quantity by the control of the operation control panel13on the basis of the value measured by the weight meter43. By the condensation of the cells in the culture bag18, the number of times of centrifugation carried out by a centrifuge for the purpose of wash and condensation of cells, carried out later, is reduced.

In the culture medium cassette20, it is also possible to replace the used culture medium bag68by a cell collecting bag72as a cell collecting vessel which is attachable to the centrifuge after having condensed the cells in the culture bag18as described above, and then supply the culture suspension in which the cells are condensed in the culture bag18(the culture medium and the cells) into the cell collecting bag72by activating the second pump49provided that the filter71is not disposed in the tube63. Accordingly, collection of the cells in the bag which is attachable to the centrifuge may be carried out within the canister16, which is a space in the closed system, so that the labor for collecting the cells is saved.

As shown inFIG. 5andFIG. 6, the large tray45of the culture cassette17stored in the canister16is provided with a cell inoculation cassette sensor74which detects the fact that the cell inoculation cassette19or the dummy cassette70is mounted to the large tray45and a culture medium cassette sensor75for detecting the fact that the culture medium cassette20is mounted on the large tray45. Signals from the sensors74and75are transmitted to the operation control panel13. The operation control panel13confirms the fact that the cell inoculation cassette19or the dummy cassette70is mounted to the large tray45of the culture cassette17and the fact that the culture medium cassette20is mounted, and then activates the first pump48, the second pump49and the third pump50.

As shown inFIG. 5, the culture cassette17is stored within the canister16, and the culture cassette17is supported on the stage42of the canister16, and the stage42is provided with an inclined motor76, a cam mechanism79and a positioning sensor77below a position where the culture bag tray47of the culture cassette17is installed. A part (elevating unit, not shown), of the platform46for placing the culture bag18on the culture bag tray47directly is adapted to be able to be moved upward and downward. The inclined motor76rotates the cam mechanism79to move the elevating unit of the platform46upward and downward. The position of the elevating unit is detected by the positioning sensor77, and is transmitted to the operation control panel13. The inclined motor76is controlled by the operation control panel13so as to move the elevating unit of the platform46downward in an initial stage of culture in the culture bag18. Accordingly, a liquid reservoir78(FIGS. 8A and 8B,FIGS. 9A and 9B) is formed in the culture bag18.

In the initial stage of culture in the culture bag18, the culture suspension from the cell inoculation cassette19(the culture medium and the cells) is retained in the liquid reservoir78, so that the cell density per area in the culture bag18is maintained at a density suitable for the proliferation and hence the cells efficiently proliferate in the initial stage of the culture. In the middle stage and the later stage of the culture in which the amount of the culture suspension in the culture bag18is increased to a level at least equal to a predetermined amount a (described later), the inclined motor76moves the elevating unit of the platform46upward via the cam mechanism79to bring the culture bag18into a horizontal state to eliminate the liquid reservoir78.

As shown inFIG. 5, a shaking mechanism91of the shaking device80is provided in the canister16above the position where the culture bag tray47of the culture cassette17is installed as a pressing unit. The shaking device80includes the shaking mechanism91, an operating motor81, a cam mechanism90and a positioning sensor82. As shown inFIG. 7, the shaking mechanism91includes an operating plate85disposed in an apparatus frame83so as to be capable of moving upward and downward via a guide rod84, and a plurality of projections86provided on the bottom surface of the operating plate85. When the operating plate85is moved upward and downward alternately on the basis of the action of the cam mechanism90(FIG. 5) by the operating motor81, the projections86of the operating plate85presses the culture bag18positioned below the shaking mechanism91repeatedly, that is, repeats pressing and releasing with respect to the culture bag18. Accordingly, the culture suspension in the culture bag18is stirred, and the cells in the culture bag18float and move in the culture suspension, so that the distribution of the cells and the concentration of the components of the culture medium in the culture bag18are homogenized, whereby the oxygen supply capacity is improved and hence the proliferation of the cells is promoted.

As shown inFIG. 6, the position of the operating plate85is detected by the positioning sensor82, and is transmitted to the operation control panel13, whereby the operating motor81is controlled by the operation control panel13. The cell culture (shaking culture) in the culture bag18using the shaking device80described above may be carried out before the culture suspension is filled in the culture bag18to approximately a maximum level, or may be carried out after having filled therein to approximately the maximum level.

As shown inFIG. 5, an lighting LED87and the CCD camera88as an image acquiring unit are installed in the canister16above and below the position where the cell inoculation cassette19or the dummy cassette70of the culture cassette17is arranged, respectively. The lighting LED87illuminates the cell inoculation cassette19or the dummy cassette70from above. The CCD camera88shoots the cells in the cell inoculation cassette19or the dummy cassette70from below to acquire the image thereof. The illuminating operation of the lighting LED87and the shooting operation of the CCD camera88are controlled by the operation control panel13(FIG. 6), and images of the cells in the cell inoculation cassette19or the dummy cassette70are acquired at predetermined time intervals (every 6 hours, for example). The cell images shot at the predetermined time intervals are stored in an image memory circuit89of the image processing computer14.

The image processing computer14carries out image processing, for example, binarization or multithresholding, for the cell images shot at the predetermined time intervals stored in the image memory circuit89, so that an average value of the projected areas of the single cells and the increasing rate of the non-single cell, which is a cell aggregate formed of a plurality of single cells, are calculated as evaluation parameters of the cell culture. The average value of the projected areas of the single cells (the average projected area of the single cell) is calculated from the cell image taken when twenty-four hours has elapsed from the moment when the culture is started after having mounted the cell inoculation cassette19to the culture cassette17and stored the culture cassette17in the canister16.

Determination of whether the cell is the non-single cell described above or not is achieved in such a manner that the cell having a projected area at least equal to 100 μm2is determined as the non-single cells since the projected area of the single cell in the initial stage of culture is smaller than 100 μm2. The change of the ratio of the non-single cells with respect to all the cells is computed from follow-up images of the cells (for example, images of the cells after having elapsed 24 hours, 48 hours and 72 hours from the start of culture) to calculate the increasing rate of the non-single cell.

The image processing computer14calculates a lag time from the average projected area of the single cell and estimates the start time of the proliferation of the cells. The lag time means a time length of an induction phase required from inoculation of cells in the inducer stimulation vessel65in the cell inoculation cassette19until the proliferation is started. The image processing computer14determines whether the culture state of the cells, that is, whether the cell has a capability to proliferate by the stimulation from the inducer or not, from the timing when the cells has started to proliferate. Then, the image processing computer14transmits the result of determination of the cells (for example, “YES” when the cells have a capability to proliferate and “NO” when the cells have no capability to proliferate) to the operation control panel13. When the operation control panel13receives a signal indicating that the capability of proliferation of the cells in the inducer stimulation vessel65in the cell inoculation cassette19is determined to be remarkably low, the operation control panel13displays the state of the corresponding cells. The cells demonstrating too long lag time are cells which are remarkably hard to get stimulated by the inducer, and hence are determined that the capability of proliferation is low.

The image processing computer14calculates a minimum doubling time of the cells from the increasing rate of the non-single cell. The doubling time in this specification means a time period required for the number of cells at a certain time instant to be increased to two times the number of cells. The image processing computer14determines the culture state of the cells, that is, the proliferation ability of the cells from the minimum doubling time and transmits the same to the operation control panel13. Then, the operation control panel13that has received the signal from the image processing computer14decides the timing for moving the cells from the cell inoculation cassette19to the culture bag18or the feeding velocity for feeding the culture medium to the culture bag18on the basis of the proliferation ability of the cells. The cells demonstrating a too long minimum doubling time is determined to be cells having a remarkably low proliferation ability.

The operation control panel13and the image processing computer14functioning as control units include a CPU for executing computation or control, a storage device (memory) for storing a processing program or data, and a input/output circuit for the connection with the input devices such as a keyboard, a mouse or a touch panel for supplying data or commands and the output devices such as a monitor. The image processing computer14also includes the image memory circuit89for storing the image data from the CCD camera88.

The storage device of the image processing computer14, stores a program for processing (for example, binarization or multithresholding) the images of the cells in the cell inoculation cassette19shot at the predetermined time intervals by the CCD camera88, calculating evaluation parameters for the cell culture (the average projected area of the single cell, the increasing rate of the non-singe cell), and determining the culture state of the cells (the proliferation capability of the cells, and the proliferation ability of the cells) from the evaluation parameters of the cell culture.

The storage device of the operation control panel13stores a equipment control program for controlling equipment (for example, the first pump48and the second pump49) relating to the incubator11, the canister16and the culture cassette17according to the culture state of the cells and carrying out the culture operation. The storage device of the operation control panel13also stores a program for controlling the equipment relating to the incubator11, the canister16and the culture cassette17on the basis of the signals from respective sensors in the incubator11, the canister16and the culture cassette17, such as controlling the CCD camera88at predetermined time intervals and acquiring the images of the cells.

In the cell culture system10, the storage device of the operation control panel13, which is one of the operation control panel13and the monitoring computer15as the control units, stores a program for collecting and accumulating cell culture related-data relating to the cell culture for the each canister16in the incubator11and for the each culture cassette17stored in the canister16, a program for monitoring the culture state of the cells for the each canister16and the each culture cassette17on the basis of the cell culture-related data, and a program for controlling the culture operation of the cells for the each canister16and for the each culture cassette17on the basis of the cell culture-related data.

The cell culture-related data in this specification is at least one of the identification codes which are readable by, for example, a barcode reader such as individual IDs of the cells, the culture medium, the inducer, the culture bag18, the large tray45, the culture medium bag67, the culture medium cassette20, the inducer stimulation vessel65, the cell inoculation cassette19, the cell collecting bag72and the operator, or individual addresses of the incubator11and the canister16, culture environment data detected by the respective sensors in the incubator11, the canister16and the culture cassette17(the temperature sensor24, the door sensor35, the culture medium cassette sensor75the weight meter43, etc.) and indicating the operating state of the respective pieces of equipment (the first pump48, the second pump49, the blast fan34, the operating motor81, etc.), and the image data of the cells acquired by the image processing computer14(the image data from the CCD camera88, the image data acquired after having applied with processing such as binarization to the images, the evaluation parameter calculated from the image data, etc.).

Therefore, the operation control panel13accumulates and collects the cell culture-related data at predetermined time intervals (every minute, for example) for the each canister16or the each culture cassette17in the incubator11on the basis of the above-described program. Accordingly, the culture log relating to the cells cultured by the arbitrary canister16and the culture cassette17in the incubator11is acquired automatically, not by the operator. Likewise, the operation control panel13observes the culture state of the cells on the basis of the culture-related data for the each canister16and the each culture cassette17in the incubator11to monitor whether there is a trouble or not. Likewise, the operation control panel13also controls the various culture operations such as replacement of the culture medium cassette20(change of the culture medium), replacement by the dummy cassette70, and replacement by the cell collecting bag72for the each canister16and the each culture cassette17in the incubator11, and prompts the operator to carry out these operations. With this control, the log is automatically recorded and stored, and occurrence of intentional alteration or erroneous recording is prevented.

The monitoring computer15receives the cell culture-related data, the culture log data, data relating to whether there is a trouble or not, and data relating to the culture operation of the each canister16and the each culture cassette17in the incubator11stored in the operation control panel13from the operation control panel13and store the same, and make the data available for display (inspection) on the monitor. Accordingly, the operator is able to observe, monitor and control the cell culture carried out in the incubator11in a monitor room95other than the culture room94in which the incubator11is installed. With this control, the log is automatically recorded and maintained, and occurrence of intentional alteration or erroneous recording is prevented.

Referring now toFIG. 18toFIG. 20, acquisition of the cell culture-related data before the cell culture, during the cell culture and after the cell culture will be described.

As shown inFIG. 18, when adding the inducer and the culture medium to the inducer stimulation vessel65to inoculate cells in a clean bench or the like before the cell culture, the operation control panel13acquires the ID of the inducer stimulation vessel65, the IDs allocated to the storage vessels for the inducer and the culture medium, the ID of sample cells and the operator's ID together with the date and time of operation using the barcode reader and transmits the same to the monitoring computer15. When mounting the inducer stimulation vessel65to the cell inoculation cassette19, the operation control panel13acquires the ID of the inducer stimulation vessel65, the ID of the cell inoculation cassette19and the operator's ID together with the date and time of operation using the barcode reader, and transmits the same to the monitoring computer15. When mounting the culture bag18to the large tray45, the operation control panel13acquires the ID of the culture bag18, the ID of the large tray45and the operator's ID together with the date and time of operation using the barcode reader, and transmits to the monitoring computer15. When mounting the culture medium bag67and the used culture medium bag68to the culture medium cassette20, the operation control panel13acquires the ID of the culture medium bag67, the ID of the culture medium cassette20and the operator's ID together with the date and time of operation, and transmits the same to the monitoring computer15using the barcode reader.

Likewise, when carrying the large tray45into the canister16in the incubator11before the cell culture, the operation control panel13acquires the addresses of the incubator11and the canister16, the ID of the large tray45and the operator's ID together with the date and time of operation using the barcode reader, and transmits the same to the monitoring computer15. When mounting the culture medium cassette20to the large tray45stored in the canister16in the incubator11before the cell culture, the operation control panel13acquires the addresses of the incubator11and the canister16, the ID of the culture medium cassette20and the operator's ID together with the date and time of operation using the barcode reader, and transmits the same to the monitoring computer15. When mounting the cell inoculation cassette19to the large tray45stored in the canister16in the incubator11before the cell culture, the operation control panel13acquires the addresses of the incubator11and the canister16, the ID of the cell inoculation cassette19and the operator's ID together with the date and time of operation using the barcode reader, and transmits the same to the monitoring computer15.

As shown inFIG. 19, during the cell culture, the operation control panel13acquires the date and time as well as the address of the incubator11together with the operating/stopping state of the incubator11and measured data from the various sensors (temperature sensor24, etc.) in the incubator11, and transmits the same to the monitoring computer15. During the cell culture, the operation control panel13acquires the date and time and the address of the canister16together with the operating/stopping state of the incubator11, the canister16and the culture cassette17in the canister16, the operating/stopping states of various equipment (the blast fan34, the first pump48, etc.) in the canister16and the culture cassette17, and measured data of the various sensors (the temperature sensor37, the weight meter43, etc.) of the canister16and the culture cassette17, and transmits the same to the monitoring computer15. Also, during the cell culture, the operation control panel13acquires the date and time and the address of the canister16, the image data of the cells shot by the CCD camera88, the processed data acquired by processing such as binarization the image data and the evaluation parameter from the image processing computer14, and transmits the same to the monitoring computer15. At this time, the operation control panel13outputs the control signal for controlling the various equipment to the incubator11, the canister16and the culture cassette17as described above.

When the cell inoculation cassette19has no more cell left therein during the cell culture and hence is replaced by the dummy cassette70, the operation control panel13acquires the addresses of the corresponding incubator11and the canister16, the ID of the large tray45, the ID of the cell inoculation cassette19and the operator's ID together with the date and time of operation, and is transmitted to the monitoring computer15. When the culture medium bag67in the culture medium cassette20is emptied during the cell culture and hence is replaced by a new culture medium cassette20having the culture medium bag67filled with the culture medium (when replacing the culture medium), the operation control panel13acquires the addresses of the corresponding incubator11and the canister16, the ID of the culture medium cassette20, the ID of the culture medium bag67and the operator's ID together with the date and time of operation, and transmits the same to the monitoring computer15.

Furthermore, when the cells are collected not in the culture bag18, but in the cell collecting bag72, the cells are condensed in the culture bag18during the cell culture, and the operation control panel13acquires the addresses of the corresponding incubator11and the canister16, the ID of the large tray45, the ID of the culture medium cassette20, the ID of the cell collecting bag72and the operator's ID together with the date and time of operation and transmits the same to the monitoring computer15when the used culture medium bag68in the culture medium cassette20is replaced by the cell collecting bag72. At the time of the replacement by the dummy cassette70, the replacement of the culture medium cassette20(change of the culture medium), and replacement by the cell collecting bag72, the operation control panel13activates the door lock mechanism39in the canister16of the corresponding incubator11to release the door lock, and confirms the replacement by the dummy cassette70, the replacement of the culture medium cassette20(displacement of the culture medium), and the replacement by the cell collecting bag72by the signals from the cell inoculation cassette sensor74or the culture medium cassette sensor75.

As shown inFIG. 20A, when collecting the cells in the culture bag18having completed the cell culture, the operation control panel13acquires the addresses of the corresponding incubator11and the canister16, the ID of the large tray45and the operator's ID together with the date and time of operation when the large tray45is removed, and transmits the same to the monitoring computer15. Subsequently, when taking the culture bag18out from the large tray45in the clean bench or the like, the operation control panel13acquires the ID of the large tray45, the ID of the culture bag18and the operator's ID together with the date and time of operation, and transmits the same to the monitoring computer15. When removing the large tray45and the culture bag18, the operation control panel13activates the door lock mechanism39in the canister16of the corresponding incubator11to release the door lock.

As shown inFIG. 20B, when the cells are collected in the cell collecting bag72after having completed the cell culture, the operation control panel13acquires the addresses of the corresponding incubator11and the canister16, the ID of the culture medium cassette20, the ID of the cell collecting bag72and the operator's ID together with the date and time of operation when removing the culture medium cassette20provided with the cell collecting bag72, and transmits the same to the monitoring computer15. Subsequently, when removing the cell collecting bag72from the culture medium cassette20in the clean bench or the like, the operation control panel13acquires the ID of the culture medium cassette20, the ID of the cell collecting bag72and the operator's ID together with the date and time of operation, and transmits the same to the monitoring computer15.

Referring now to process drawings fromFIG. 8toFIG. 11and flowcharts shown inFIG. 12toFIG. 17, a process followed by the operation control panel13and the image processing computer14to carry out the above-described program to culture the cells will be described. In this cell culture process, the operation control panel13and the image processing computer14set and control the culture environment independently for the each canister16in the incubator11, and culture cells in the culture cassette17stored in the each canister16.

FIG. 8toFIG. 13show an inducer stimulating intermittent perfusion culture process carried out in the case of collecting the cells in the culture bag18. As shown inFIG. 8AandFIG. 12, the operator places the empty culture bag18on the culture bag tray47interposing the platform46between the culture bag18and the culture bag tray47, mounts the culture bag tray47to the large tray45, and connects the culture bag18to the first pump48, the second pump49and the third pump50.

Subsequently, the operator puts the large tray45into the single canister16whose indication lamp44is illuminated, for example, in green, in the incubator11, and makes the stage42to support the same. Then, the operator immobilizes the inducer in the inducer stimulation vessel65in the clean bench or the like, puts the culture medium therein, and mounts the cell inoculation cassette19to which cells are inoculated to the large tray45in the canister16, as shown inFIG. 8B. Subsequently, the operator mounts the culture medium cassette20having the culture medium bag67to which the culture medium is put in the clean bench or the like to the large tray45in the canister16(S1inFIG. 12).

Subsequently, the operator confirms the output of the image from the CCD camera88in the canister16in which the culture cassette17having the culture bag18, the cell inoculation cassette19and the culture medium cassette20mounted to the large tray45is stored (S2inFIG. 12). Before or after the confirmation of the image output, the operator activates the inclined motor76of the canister16to move the elevating unit of the platform46downward and form the liquid reservoir78in the culture bag18. The operator then measures the weight of the empty culture bag18by the weight meter43of the canister16.

Then, as shown inFIG. 9A, the operator closes the canister door31and starts the cell culture in the corresponding canister16(S3inFIG. 12). Accordingly, in the inducer stimulation vessel65in the cell inoculation cassette19, the cells are stimulated by the inducer for proliferation (S4inFIG. 12). The CCD camera88of the corresponding canister16takes images of the cells in the inducer stimulation vessel65in the cell inoculation cassette19at predetermined time intervals (every six hours, for example), and the image processing computer14calculates the evaluation parameter of the cell culture from the picked-up images. In addition, the image processing computer14calculates a lag time from the evaluation parameter to determine whether there is a capability that the cells proliferate by the stimulation from the inducer or not, and further calculates the minimum doubling time to determine the proliferation ability of the cells (S5inFIG. 12).

When the operation control panel13receives a signal determining that the proliferation capability cannot be confirmed even a predetermined time length (72 hours, for example) has elapsed since the cells are stimulated by the inducer in the inducer stimulation vessel65in the cell inoculation cassette19from the image processing computer14, the operation control panel13displays so. When the operation control panel13receives a signal determining that the cells in the inducer stimulation vessel65in the cell inoculation cassette19has a probability to proliferate from the image processing computer14, the operation control panel13decides the timing to transfer the cells to the culture bag18or the feeding velocity of the culture medium into the culture bag18on the basis of the proliferation ability of the cells. The operation control panel13activates the first pump48on the basis of these decision and, as shown inFIG. 9B, to feed the culture medium in the culture medium bag67of the culture medium cassette20to the cell inoculation cassette19, transfers the cells in the inducer stimulation vessel65in the cell inoculation cassette19to the culture bag18, and feeds the culture medium in the culture medium bag67to the culture bag18(S6inFIG. 12).

By the operation of the first pump48, the static culture of the cells in the liquid reservoir78of the culture bag18is started (S7inFIG. 12). The operation control panel13follows the program to carry out the feeding culture while feeding the culture medium, and to determine whether the weight of the culture suspension in the culture bag18measured by the weight meter43is increased to a level at least equal to a predetermined value α or not (S8inFIG. 12). The operation control panel13activates the inclined motor76at a timing when the predetermined value α is reached, moves the elevating portion of the platform46upward via the cam mechanism79and, as shown inFIG. 10A, brings the culture bag18into a horizontal state to eliminate the liquid reservoir78(S9inFIG. 12).

Then, the operation control panel13determines whether the weight of the culture suspension in the culture bag18measured by the weight meter43is increased to a level at least equal to a predetermined value β (S10inFIG. 12) or not, and activates the operating motor81at a timing when the predetermined value β is reached. Accordingly, as shown inFIG. 10B, the shaking device80is activated, and the shaking culture in which the operating plate85in the shaking mechanism91of the shaking device80presses the culture bag18repeatedly is started (S11inFIG. 12). The operation control panel13continues to determine whether the weight of the culture suspension in the culture bag18measured by the weight meter43is increased to a level at least equal to a predetermined value χ (S12inFIG. 12) or not. When the predetermined value χ is reached, the first pump48is stopped, feeding of the culture medium from the culture medium bag67in the culture medium cassette20to the culture bag18is stopped, and the operating motor81is stopped to stop the shaking culture in the culture bag18(S13inFIG. 13).

The operation control panel13activates the second pump49after the cells are settled in the culture bag18to discharge the used culture medium in the culture bag18(supernatant in the culture bag18) to the used culture medium bag68in the culture medium cassette20as shown inFIG. 11A(S14inFIG. 13). Then, the operation control panel13activates the first pump48and feeds the culture medium from the culture medium bag67in the culture medium cassette20to the culture bag18, and activates the operating motor81to carry out the shaking culture in the culture bag18by the shaking device80(S15inFIG. 13). After having elapsed a predetermined time length, the operation control panel13stops the first pump48, stops the feeding of the culture medium from the culture medium bag67in the culture medium cassette20to the culture bag18, and stops the operating motor81to stop the shaking culture in the culture bag18(S16inFIG. 13).

The operation control panel13determines whether the desired culture term depending on the date and time of usage of the cells to proliferate is reached or not, or the image processing computer14determines whether the cells in the culture bag18reaches the desired number of cells or not (S17inFIG. 13). When determining whether the cells in the culture bag18reaches the desired number of cells or not, the operation control panel13activates the third pump50(FIG. 11A), transfers a part of the cells in the culture bag18to the cell inoculation cassette19or the dummy cassette70(the dummy cassette70in most cases), and makes the CCD camera88to take an image of the cells. The image processing computer14processes the image of the cells, determines whether the number of cells is at least equal to a specified value or not, and transmits the same to the operation control panel13. When the culture term or the number of cells is not reached, the operation control panel13repeats the processing operation from Step S14to Step S17.

The Steps S13to S17are steps for the intermittent perfusion culture in which discharge of the used culture medium in the culture bag18and supply (feed) of new culture medium to the culture bag18are carried out alternately.

In Steps S9to S17, the presence or absence of the cells in the inducer stimulation vessel65in the cell inoculation cassette19is confirmed by the CCD camera88and, when there is no cell in the inducer stimulation vessel65, the operation control panel13prompts the operator to replace the cell inoculation cassette19by the dummy cassette70so as to prevent the inducer from flowing from the inducer stimulation vessel65into the culture bag18. When replacing the cell inoculation cassette19by the dummy cassette70, the operation control panel13stops feeding of the culture medium from the culture medium cassette20to the culture bag18and the shaking culture by the shaking device80temporarily until the replacement of the cell inoculation cassette19by the dummy cassette70is completed.

Then, in Steps S9to S17, when the operation control panel13determines that there is no more culture medium in the culture medium bag67in the culture medium cassette20from the measured value of the weight meter43, the operation control panel13prompts the operator to replace the culture medium cassette20by a new culture medium cassette20. When replacing the culture medium cassette20by the new culture medium cassette20as well, the operation control panel13stops feeding of the culture medium from the culture medium cassette20to the culture bag18and the shaking culture by the shaking device80temporarily until the replacement of the culture medium cassette20is completed.

At the time point when the desired culture term is reached or when the desired number of cells is reached in Step S17, the operation control panel13stops shaking. When the cells are settled, the operation control panel13activates the second pump49as shown inFIG. 11B, discharges the used culture medium in the culture bag18to the used culture medium bag68of the culture medium cassette20and condenses the cells until the culture suspension in the culture bag18is reduced to about ½ to ⅓ on the basis of the measured value by the weight meter43(S18inFIG. 12).

The operation control panel13then stops the second pump49to complete the cell culture (S19inFIG. 12). After having completed the culture, the cells in the culture bag18are transferred to a vessel for the centrifuge in the clean bench or the like by the operator, and then the cells are collected by the centrifugation (S20inFIG. 12).

Subsequently, a case in which the process of collecting the cells by the cell collecting bag72(FIG. 5) in the inducer stimulating intermittent perfusion culture process is include will be shown inFIG. 14andFIG. 15. Therefore, Steps S21to S38in the process shown inFIG. 14andFIG. 15are the same as Steps S1to S18inFIG. 12andFIG. 13, and hence description is omitted.

In Step S38shown inFIG. 15, after having condensed the cells in the culture bag18by the activation of the second pump49, the operation control panel13stops the second pump49, and prompts the operator to replace the used culture medium bag68in the culture medium cassette20by the cell collecting bag72(FIG. 5) (S39inFIG. 13). The cell collecting bag72is a bag which may be used for the centrifugation by being mounted to the centrifuge.

After having replaced the used culture medium bag68in the culture medium cassette20by the cell collecting bag72, the operation control panel13activates the second pump49and the operating motor81, and transfers the cells in the culture bag18to the cell collecting bag72mounted to the culture medium cassette20together with the culture medium while shaking the interior of the culture bag18by the shaking device80(S40inFIG. 15). The operation control panel13then stops the second pump49and the operating motor81and stops the cell culture (S41inFIG. 15). After having completed the culture, the operator mounts the cell collecting bag72to the centrifuge and collects the cells by the centrifugation (S42inFIG. 15).

Referring now toFIG. 16andFIG. 17, the inducer stimulating consecutive perfusion culture process will be described. Steps S51to S62in the inducer stimulating consecutive perfusion culture process shown inFIG. 16andFIG. 17are the same as Steps S1to S12in the inducer stimulating intermittent perfusion culture process inFIG. 12andFIG. 13, and hence description is omitted. In the inducer stimulating consecutive perfusion culture process, the filter71is disposed between the culture bag18and the second pump49.

The operation control panel13activates the second pump49and discharges the used culture medium in the culture bag18to the used culture medium bag68in the culture medium cassette20at a moment when the weight of the culture suspension in the culture bag18is increased to a level at least equal to the predetermined value X (S62inFIG. 16) during the shaking culture by the shaking device80in the culture bag18(S61inFIG. 16) while feeding the culture medium from the culture medium bag67in the culture medium cassette20into the culture bag18(S56inFIG. 16). Accordingly, the consecutive perfusion culture which carries out feeding of the culture medium to the culture bag18and discharge of the used culture medium from the culture bag18simultaneously is started in the culture bag18(S63inFIG. 17). At this time, the cells in the culture bag18are prevented from flowing by the filter71and hence do not flow into the used culture medium bag68. The shaking culture by the shaking device80is carried out simultaneously during the consecutive perfusion culture.

The operation control panel13determines whether the desired culture term depending on the date and time of usage of the cells to proliferate is reached or not, or the image processing computer14determines whether the cells in the culture bag18reaches the desired number of cells or not (S64inFIG. 17). When determining whether the cells in the culture bag18reaches the desired number of cells or not, the operation control panel13activates the third pump50, transfers a part of the cells in the culture bag18to the cell inoculation cassette19or the dummy cassette70(the dummy cassette70in most cases), and makes the CCD camera88to take an image of the cells. The image processing computer14processes the image of the cells, determines whether the number of cells is at least equal to a specified value or not, and transmits the same to the operation control panel13. When the culture term or the number of cells as described above is not reached, the operation control panel13repeats the consecutive perfusion culture in Step S63.

In Steps S59to S64, the presence or absence of the cells in the inducer stimulation vessel65in the cell inoculation cassette19is confirmed by the CCD camera88and, when there is no cell in the inducer stimulation vessel65, the operation control panel13prompts the operator to replace the cell inoculation cassette19by the dummy cassette70so as to prevent the inducer from flowing from the inducer stimulation vessel65into the culture bag18. When replacing the cell inoculation cassette19by the dummy cassette70, the operation control panel13stops feeding of the culture medium from the culture medium cassette20to the culture bag18and the shaking culture by the shaking device80temporarily until the replacement of the cell inoculation cassette19by the dummy cassette70is completed.

Then, in Steps S59to S64, when the operation control panel13determines that there is no more culture medium in the culture medium bag67in the culture medium cassette20from the measured value of the weight meter43, the operation control panel13prompts the operator to replace the culture medium cassette20by a new culture medium cassette20. When replacing the culture medium cassette20by the new culture medium cassette20as well, the operation control panel13stops feeding of the culture medium from the culture medium cassette20to the culture bag18and the shaking culture by the shaking device80temporarily until the replacement of the culture medium cassette20is completed.

The operation control panel13stops the first pump48, the second pump49and the operating motor81and stops the perfusion culture and the shaking culture at a moment when the desired culture term in Step S64is reached, or when the desired number of cells is reached (S65inFIG. 17).

The operation control panel13then activates the second pump49to discharge the used culture medium in the culture bag18to the used culture medium bag68in the culture medium cassette20and condense the cells until the amount of the culture suspension in the culture bag18is reduced to about ½ to ⅓ on the basis of the measured value by the weight meter43(S66inFIG. 17). To stop the shaking device in the condensing process is to prevent the large number of cells from flowing into the tube, and from clogging the filter.

The operation control panel13then stops the second pump49to complete the cell culture (S67inFIG. 17). After having completed the culture, the cells in the culture bag18are transferred into the vessel for the centrifuge in the clean bench or the like by the operator and then the cells are collected by the centrifugation (Step S68inFIG. 17).

In this configuration, according to the embodiment shown above, the following effects (1) to (8) are achieved.

(1) The image processing computer14processes the image of the cells in the cell inoculation cassette19picked up by the CCD camera88to acquire the evaluation parameters (the average projected area of the single cell, the increasing rate of the non-single cell) of the cell culture, and to determine and evaluate the culture state of the corresponding cells (the proliferation capability of the cells and the proliferation ability of the cells). The operation control panel13carries out the culture operation according to the culture state (the timing of transfer of the cells from the cell inoculation cassette19to the culture bag18, the feeding of the culture medium at the predetermined feeding velocity from the culture medium cassette20to the culture bag18, etc.). Consequently, since the culture state of the cells is determined in a non-contact, non-invasive state, the cells are prevented from getting damaged, and the risk of contamination and the loss of the cells due to the sampling are avoided.

The labor of the operator is alleviated as the operator needs not to carry out the culture operation one by one. And it is not necessary to open and close the main body door21of the incubator11and the canister door31of the canister16for observing the culture state. Therefore, the culture environment in the incubator11and the canister16is maintained in a desirable state.

In addition, the cells of one patient may be inoculated to the cell inoculation cassette19in the culture cassette17which is stored in the single canister16, and the culture operation according to the culture state of the cells may be carried out on the individual bases. Therefore, the culture operation suitable for each patient is achieved, and the cross-contamination is avoided. Since the culture operation suitable for the culture state of the cells is achieved, the culture operation by the hour is enabled, and the culture is accelerated to shorten the culture term.

(2) The culture medium cassette20has a cassette structure, and is connected to the culture bag18and the cell inoculation cassette19, which is also has a cassette structure. Therefore, the culture bag18, in particular, is constantly retained in the canister16which provides an environment suitable for the culture. The change of the culture medium is enabled only by opening and closing the main body door21of the incubator11and the canister door31of the canister16, and the number of times of opening and closing operation of the both doors21and31may be reduced. Therefore, fluctuation of the culture environment in the incubator11and the canister16may be restrained. Consequently, damages to the cells in the culture bag18in association with the environment change is reduced, and aseptic manipulation for supplying the culture medium to the culture bag18in the clean bench or the like may be omitted.

(3) The culture medium cassette20, the cell inoculation cassette19and the culture bag18are connected to form a closed system. Therefore, the culture medium cassette20, the cell inoculation cassette19and the culture bag18are maintained under the aseptic conditions.

(4) The culture suspension is stored in the liquid reservoir78of the culture bag18in the initial stage of the culture in the culture bag18, and hence the cell density per area may be maintained at a suitable density. Therefore, the cells may proliferate efficiently in the initial stage of culture.

(5) When the cells proliferated in the culture bag18is introduced into the cell inoculation cassette19or the dummy cassette70and the images of the cells are acquired by the CCD camera88, the number of cells and the form of the cells are observed by acquiring the cells proliferated in the culture bag18as the images without sampling.

(6) The used culture medium in the culture bag18is discharged and stored in the used culture medium bag68in the culture medium cassette20, and hence the cells in the culture bag18may be condensed to increase the cell density. Therefore, the number of times of the operation of centrifugation for collecting the cells may be reduced. Consequently, the labor for collecting the cells is saved, and the damage of the cells in association with the centrifugation is reduced.

(7) When all the cells condensed in the culture bag18are collected to the cell collecting bag72mounted to the culture medium cassette20, the cells may be collected by mounting the cell collecting bag72directly to the centrifuge. Therefore, the labor for collecting the cells may be saved.

(8) The culture suspension in the culture bag18is stirred by repeatedly pressing the flexible culture bag18having the culture medium to which the cells are inoculated stored therein by the projections86of the operating plate85in the shaking mechanism91of the shaking device80. Therefore, the distribution of the cells and the concentration of the components of the culture medium in the culture bag18are homogenized, and the oxygen supply capacity is improved, so that the proliferation of the cells is accelerated and the efficiency of the cell culture is improved.

Since the cells are simply suspended in the culture suspension stirred by being pressed repeatedly by the operating plate85of the shaking device80, the cells are prevented from getting damaged.

(9) The operation control panel13and the monitoring computer15collect and accumulate the cell culture-related data relating to the cell culture individually for the each culture unit12in the incubator11(the canister16and the culture cassette17). Therefore, the culture log relating to the cells cultured in the arbitrary culture unit12is adequately known.

The operation control panel13and the monitoring computer15monitor the culture state of the cells on the basis of the cell culture-related data for the individual culture unit12in the incubator11(the canister16and the culture cassette17). Therefore, trouble in the culture state may be monitored individually for the each culture unit12.

Furthermore, the operation control panel13and the monitoring computer15control the operation of cell culture such as the change of the culture medium (replacement of the culture medium cassette20) and collection of the cells after having completed the culture (collection of the cells by the culture bag18or the cell collecting bag72). Therefore, the operation schedule for the operation (work) carried out by the operator individually for the each culture unit12in the incubator11is easily prepared.

(10) The control unit includes the operation control panel13being installed in a culture room94together with the incubator11and having a function to control the culture in the each culture unit12(the canister16and the culture cassette17) in the incubator11and the monitoring computer15being installed in a monitor room95other than the culture room94and receiving and saving the data that the operation control panel13has and making the same available for display (inspection). Therefore, the culture state of the cells in the culture unit12in the incubator11in the culture room94may be observed, monitored and controlled in the monitor room95other than the culture room94using the monitoring computer15. With this control, the log or the like is automatically recorded and stored, and occurrence of intentional alteration or erroneous recording is prevented.

(11) The canisters16in the incubator11which constitutes the culture units12are each isolated to avoid entry of the cells and/or bacteria by the air-intake filter32and the exhaust filter33. Therefore, the cells in the culture bag18and the cell inoculation cassette19stored in the each canister16are prevented from being contaminated by bacteria.

(12) The blast fans34of all the canisters16in the single incubator11are configured to stop when the main body door21of the incubator11is opened. Therefore, the sealed state of the each canister16in the incubator11is maintained even when the main body door21of the incubator11is opened, so that the independent culture environment for the each canister16is preferably secured, and the fluctuation of the culture environments in the canisters16are restrained.

(13) Only one of the canister doors31of the canisters16in the single incubator11is allowed to be opened. Therefore, there is no probability that the two or more canister doors31of the canisters16are opened in the single incubator11, so that the mix-up of the culture bags18and the cell inoculation cassettes19among the canisters16is prevented, and the cross-contamination of the cells is avoided.

[B] Second Embodiment

FIG. 21is a layout drawing showing a configuration (differentiation-inducing state) of the culture unit of the cell culture apparatus according to a second embodiment of the invention.FIG. 22is a layout drawing showing the configuration of the culture unit inFIG. 21in a state before the differential inducement). In the second embodiment, the same parts as in the first embodiment are designated by the same reference numerals and description is omitted.

A cell culture apparatus100according to the second embodiment is an apparatus in which the cell inoculation cassette19(FIG. 5) in the cell culture apparatus10in the first embodiment is replaced by a differentiation-inducing cassette101as a differentiation-inducing culture vessel in which differentiation-inducing factor is added in advance. Then, the cell culture apparatus100activates the second pump49to introduce the culture medium from the culture medium bag67in the culture medium cassette20to the culture bag18to cause the cells to proliferate, and then activates the first pump48to introduce the proliferated cells to the differentiation-inducing cassette101to give a role to the corresponding cells, that is, differentiates the corresponding cells. The term “differentiation” in this specification means to give a role to the cells as cells of a heart, or to give a role to the same as cells of liver. As shown inFIG. 22, a dummy cassette102is arranged instead of the differentiation-inducing cassette101before the cells proliferated in the culture bag18are differentiated in the differentiation-inducing cassette101, and the used culture medium in the culture bag18is discharged to the used culture medium bag68in the culture medium cassette20via the dummy cassette102by the activation of the first pump48. The dummy cassette102functions as a flow path which simply allows the culture medium to flow, as in the case of the dummy cassette70(FIG. 5).

In the second embodiment, as shown inFIG. 21, the CCD camera88takes images of the cells differentiated in the differentiation-inducing cassette101at predetermined time intervals. A program for carrying out the shooting is stored in a storage device of the operation control panel13. The image processing computer14carries out processing such as binarization or multithresholding on the images of the cells, and acquires the form of the cells as evaluation parameters. A program for carrying out the acquisition of the evaluation parameter is stored in the storage device of the image processing computer14. The image processing computer14determines whether the corresponding cells are differentiated or not from the change of the form of the cells with time. The operation control panel13controls the differentiation-inducing operation by the differentiation-inducing cassette101upon reception of the signal from the image processing computer14. For example, the operation control panel13continues the differentiation-inducing operation when the image processing computer14determines that the corresponding cells have a capability of differentiation. If not, the message saying so is displayed. The program for carrying out the determination of differentiation described above is stored in the storage device in the image processing computer14, and a program for carrying out the differentiation-inducing operation is stored in the storage device in the operation control panel13.

In the same manner as in the first embodiment, a program for acquiring the cell culture-related data, a program for monitoring the culture state of the cells and a program for controlling the culture operation of the cells are further stored in the storage device of the operation control panel13for the each canister16and the culture cassette17in the incubator11. The operation control panel13collects and accumulates the cell culture-related data for the each canister16and the each culture cassette17in the incubator11, monitors whether there is a trouble or not, and controls the culture operation such as the replacement of the culture medium cassette20(the change of the culture medium), and the replacement of the dummy cassette102by the differentiation-inducing cassette101in the same manner as in the first embodiment on the basis of the programs described above. The monitoring computer15receives the cell culture-related data, the culture log data, data relating to whether there is a trouble or not, and data relating to the culture operation from the operation control panel13for the each canister16and the each culture cassette17in the incubator11, and stores the same to make these data available for display (inspection) in the same manner as in the first embodiment.

Although the acquisition of the cell culture-related data is achieved in the same manner as in the first embodiment, it is different in the case of the differentiation-inducing cassette101. In other words, before the cell culture (seeFIG. 18), when adding the differentiation-inducing factor to a differentiation-inducing vessel103(FIG. 21) in the differentiation-inducing cassette101in the clean bench or the like, the operation control panel13acquires the ID of the differentiation-inducing vessel103, the ID of the differentiation-inducing factor and the operator's ID together with the date and time of operation and transmits the same to the monitoring computer15. Then, when mounting the differentiation-inducing vessel103to the differentiation-inducing cassette101, the operation control panel13acquires the ID of the differentiation-inducing vessel103, the ID of the differentiation-inducing cassette101and the operator's ID together with the date and time of operation and transmits the same to the monitoring computer15. During the cell culture (seeFIG. 19), when removing the dummy cassette102and replacing the same by the differentiation-inducing cassette101, the operation control panel13acquires the addresses of the incubator11and the canister16, the ID of the differentiation-inducing cassette101, the ID of the large tray45and the operator's ID together with the date and time of operation and transmits the same to the monitoring computer15.

Referring now toFIG. 23andFIG. 24, a differentiation-inducing intermittent perfusion culture process carried out by the cell culture apparatus100will be described.

First of all, the operator adds a culture medium and cells to the culture bag18in the clean bench or the like (S71inFIG. 23), places the culture bag18on the culture bag tray47via the platform46, and attaches the culture bag tray47to the large tray45(S72inFIG. 23). Then, the operator carries the large tray45into the single canister16illuminated by the indication lamp44, for example, in green in the incubator11.

Then, the operator mounts the culture medium cassette20to the large tray45in the canister16, and mounts the dummy cassette102(FIG. 22) (S73inFIG. 23). Then, the operator closes the canister door31of the canister16and the static culture of the cells in the culture bag18is started (S74inFIG. 23). When carrying out the static culture, in a case in which the amount of the culture suspension in the culture bag18is small, the operator activates the inclined motor76to move the elevating unit of the platform46downward, and form the liquid reservoir78(FIG. 8) in the culture bag18.

After having started the static culture, the operation control panel13activates the second pump49, and feeds the culture medium from the culture medium bag67in the culture medium cassette20to the culture bag18(S75inFIG. 23). The operation control panel13confirms whether the culture suspension in the culture bag18is increased to a level at least equal to the predetermined value ax or not from the measured value of the weight meter43(S76inFIG. 23), and when the measured value of the weight meter is increased to a value at least equal to the predetermined value α, the operation control panel13activates the inclined motor76to move the elevating unit of the platform46upward, and brings the culture bag18into the horizontal state to eliminate the liquid reservoir78(S77inFIG. 23).

Subsequently, the operation control panel13determines whether the weight of the culture suspension in the culture bag18weighted by the weight meter43is increased to a level at least equal to the predetermined value β or not (S78inFIG. 23). At a timing when a weight is increased to value at least equal to the predetermined value β, the operation control panel13activates the operating motor81to activate the shaking device80to start the shaking culture (S79inFIG. 23). Then, the operation control panel13continues to determine whether the weight of the culture suspension in the culture bag18measured by the weight meter43is increased to a value at least equal to the predetermined value χ or not (S80inFIG. 23). At a timing when a weight is increased to a value at least equal to the predetermined value χ, the operation control panel13stops the second pump49to stop feeding of the culture medium from the culture medium bag67in the culture medium cassette20to the culture bag18, stop the operating motor81and stop the shaking culture in the culture bag18(S81inFIG. 23).

The operation control panel13activates the first pump48after having settled the cells in the culture bag18to discharge the used culture medium in the culture bag18to the used culture medium bag68in the culture medium cassette20(S82inFIG. 24). Then, the operation control panel13activates the second pump49to feed the culture medium from the culture medium bag67in the culture medium cassette20to the culture bag18, activates the operating motor81to carry out the shaking culture in the culture bag18by the shaking device80(S83inFIG. 24). After having elapsed a predetermined time length, the operation control panel13stops the second pump49, stops feeding of the culture medium from the culture medium bag67in the culture medium cassette20to the culture bag18, stops the operating motor81and stops the shaking culture in the culture bag18(S84inFIG. 24).

The operation control panel13determines whether the desired culture term depending on the date and time of usage of the cells to proliferate is reached or not, or the image processing computer14determines whether the desired number of cells is reached in the culture bag18or not (S85inFIG. 24). When determining whether the desired number of cells is reached in the culture bag18or not, the operation control panel13activates the third pump50to transfer part of the cells in the culture bag18to the dummy cassette102and make the CCD camera88to take an image of the cells. The image processing computer14processes the image of the cells, determines whether the number of cells is at least equal to a specified value or not, and transmits the same to the operation panel13. When the culture term or the number of cells is not reached, the operation control panel13repeats the operation from Steps S82to S85.

The Steps S81to S85are steps of intermittent perfusion culture process in which discharge of the used culture medium in the culture bag18and supply (feed) of new culture medium into the culture bag18are carried out alternately.

In Steps S77to S85, when the operation control panel13determines that there is no more culture medium in the culture medium bag67in the culture medium cassette20from the measured value of the weight meter43, the operation control panel13prompts the operator to replace the culture medium cassette20by a new culture medium cassette20. When replacing the culture medium cassette20by the new culture medium cassette20as well, the operation control panel13stops feeding of the culture medium from the culture medium cassette20to the culture bag18and the shaking culture by the shaking device80temporarily until the replacement of the culture medium cassette20is completed.

At the time point when the desired culture term is reached or when the desired number of cells is reached in Step S85, the operation control panel13activates the first pump48to discharge the used culture medium in the culture bag18to the used culture medium bag68in the culture medium cassette20and condense the cells until the culture suspension in the culture bag18is reduced to about ½ to ⅓ on the basis of the measured value by the weight meter43(S86inFIG. 24).

Then, after having stopped the first pump48by the operation control panel13, the operator replaces the dummy cassette102by the differentiation-inducing cassette101as shown inFIG. 21(S87inFIG. 24).

After having completed the replacement of the dummy cassette102by the differentiation-inducing cassette101, the operation control panel13activates the control unit29to feed the culture medium in the culture medium bag67in the culture medium cassette20to the culture bag18, and activates the operating motor81to carry out the shaking culture in the culture bag18by the shaking device80(S88inFIG. 24). Then, the operation control panel13activates the first pump48to transfer the culture suspension in the culture bag18to the differentiation-inducing cassette101while continuing the shaking culture in the culture bag18and feeding of the culture medium from the culture medium cassette20(S89inFIG. 24). Accordingly, the differentiation-inducing culture is started in the differentiation-inducing cassette101(S90inFIG. 24).

The operation control panel13causes the CCD camera88to take images of the cells in the differentiation-inducing cassette101during the differentiation-inducing culture at predetermined time intervals (every six hours, for example). The image processing computer14processes the picked-up image, acquires the form of the cells as evaluation parameters, and determines whether the cells are differentiated in the differentiation-inducing cassette101or not from the change of the form of the cells with time (S91inFIG. 24).

The operation control panel13stops the second pump49, the first pump48and the operating motor81to stop the differentiation-inducing culture to complete the cell culture upon reception of the signal determining that the cells are differentiated from the image processing computer14in Step S91(S92inFIG. 24). After having completed the culture, the operator collects the cells in the differentiation-inducing cassette101by the cell collecting operation in the clean bench or the like (S93inFIG. 24).

In this configuration, according to the second embodiment, the same effects as (2) to (6) and (8) in the first embodiment are achieved (provided that the cell inoculation cassette19is replaced by the differentiation-inducing cassette101) and the following effect (9) is achieved.

(9) Since the culture state of the cells (whether the cells are differentiated or not) is determined by processing the images of the cells in the differentiation-inducing cassette101and acquiring the evaluation parameters (the form of the cells) of the cell culture, and the culture operation according to the culture state (the timing of culture medium feeding from the culture medium cassette20to the culture bag18at a predetermined feeding velocity, or the cell transfer from the culture bag18to the differentiation-inducing cassette101) is carried out, the culture state of the cells is determined by a non-contact, non-invasive state. Therefore, the cells are prevented from getting damaged, the risk of contamination and the loss of the cells by sampling are avoided, and the labor of the operator is alleviated. Furthermore, since the cells of a single patient proliferate in the culture bag18in the culture cassette17and are differentiated in the differentiation-inducing cassette101which is stored in the single canister16, the cell culture is adequately carried out for the individual patients, and the cross-contamination is avoided.

According to the cell culture apparatuses10and102in the first and second embodiments, the following effect (10) is achieved.

(10) The cell culture apparatuses10and100are both adapted to transfer the cells cultured in one of the plurality of culture vessels for culturing the cells in different environments to another culture vessel on the downstream side. In other words, in the cell culture apparatus10, the cells are stimulated by the inducer for the proliferation in the cell inoculation cassette19, which is one of the culture vessels, and then the cells are transferred from the cell inoculation cassette19to the culture bag18as still another culture vessel for causing the cells to proliferate. In the cell culture apparatus100, the cells proliferate in the culture bag18, which is one of the culture vessels, and then the cells are transferred from the culture bag18to the differentiation-inducing cassette101, which is still another culture vessel for differentiating the cells. In this manner, according to the cell culture apparatuses10and100, a variety of forms of the cell culture is achieved.

Although the invention has been described on the basis of the embodiments shown above, the invention is not limited thereto.

For example, in the description of the both embodiments shown above, the image processing computer14processes the images taken by the CCD camera88and calculates the evaluation parameters, and determines the culture state of the cells (the proliferation capability of the cells, the proliferation ability of the cells) from the evaluation parameters. However, the function of the image processing computer14may be carried out by the operation control panel13.

The cell images may be taken in the culture bag18by the CCD camera88. When the shaking culture by the shaking device80is not carried out in the inducer stimulating consecutive perfusion culture process (FIG. 16andFIG. 17), it is not necessary to dispose the filter71between the second pump49and the culture bag18.

In the description of the both embodiments shown above, the shaking device80is used to stir the culture suspension for causing the cells to proliferate. However, it is also possible to shake the culture bag18in the vertical direction or to swing the same in the lateral direction to mix the culture suspension and homogenize the distribution of cells and the concentration of the components of the culture medium or to enhance the oxygen supply capacity.

In the description in the both embodiments, the operation control panel13and the image processing computer14control the individual canisters in the one incubator. However, it is also possible to prepare a plurality of the incubatores of the same type and control the individual canisters in the incubators by the operation control panel13and the image processing computer14.

In the description in the first and second embodiments, one each of the operation control panel13and the image processing computer14control the operation of the individual canisters16in the single incubator11, and one each of the operation control panel13and the monitoring computer15monitor and control the cell culture. However, as in a third embodiment shown inFIG. 25, it is also possible to install a plurality of the incubators11, arrange one each of the operation control panel13in the each incubator11, and connect the single image processing computer14and the single monitoring computer15to the each operation control panel13, so that the cell culture carried out in the each canister16of the plurality of incubators11is monitored and controlled by the monitoring computer15. With this control, the log or the like is automatically recorded and stored, and occurrence of intentional alteration or erroneous recording is prevented.

In the first and second embodiments (FIG. 1), or in the third embodiment (FIG. 25), it is possible to connect a remote monitor computer105to the single monitoring computer15using a public network104. In this case, the abnormal culture in the specific canister16of the specific incubator11is advantageously known quickly at a remote location using the remote monitor computer105.

FIG. 26is a configuration drawing showing the cell culture apparatus according to a fourth embodiment of the invention.FIG. 27is a layout of a cell culture apparatus200according to the fourth embodiment showing a configuration of a culture unit212including a single spatially independent culture chamber240and low temperature chamber230, a culture bag tray241stored in the culture chamber240, a culture medium bag tray231and waste water bag tray232stored in the low temperature chamber230.

The cell culture apparatus200shown inFIG. 26is specifically adapted to culture suspension cells used for the immune cell therapy, and includes a culture device210provided with a plurality of (three, for example) the culture units212, an operation control PLC (programmable logic controller)223for controlling the operation of the plurality of culture units212, an image processing unit221for processing cell images, a data collecting device224for collecting all the data, a touch panel222for displaying information of and entering the control of the operation control PLC223and the image processing unit221, and a monitoring computer226being connected to the operation control PLC223, the image processing unit221and a data collecting device224via a hub225for monitoring the cell culture apparatus200and the culture unit212. The operation control PLC223and the image processing unit221function as control units. The plurality of culture units212are each provided with an observation camera (CCD camera)302.

The known suspension cells include peripheral blood mononuclear cells, LAK cells (Lymphokine Activated killer cells), neural stem cells and ES cells. These types of suspension cells are referred simply to as “cells”, hereinafter. The cell culture apparatus200is also applicable to a case of culturing adherent cells (mesenchymal stem cells, for example) other than the suspension cells.

The cell culture apparatus200has a structure in which a plurality of (three, for example) spatially and structurally independent culture units212stacked one on top of another, and the each culture unit212is divided into the low temperature chamber230and the culture chamber240as shown inFIG. 27. The cell culture apparatus200includes a culture bag tray241, a culture medium bag tray231and a waste water bag tray232stored therein. A culture bag242as an antibody stimulating and proliferation culture vessel is placed on the culture bag tray241as described later in detail, and a culture medium bag (new culture medium bag)233as a culture medium storage unit and a waste water bag (waste culture medium bag)234as a waste water bag tray are placed on the culture medium bag tray231and the waste water bag tray232, respectively.

The culture unit212includes an openable and closable doors (not shown) for the culture chamber240and the low temperature chamber230. The culture unit212maintains the environment (temperature and CO2concentration) in the culture chamber240to an environment required for culturing the cells in the state in which the door is closed, and the environment (temperature) in the low temperature chamber230to an environment optimal for preserving the culture medium.

Therefore, the culture unit212includes temperature sensors236and243, a CO2sensor244, a door sensor (not shown) and a heater (not shown) disposed therein. A gas cylinder (not shown) installed outside is coupled to the cell culture apparatus200(the culture unit212) via a CO2supply system245. Signals from the temperature sensors236and243, the CO2sensor244and the door sensor are transmitted to the operation control PLC223. The operation control PLC223controls the heater on the basis of the temperature signals from the temperature sensors236and243, and controls the amount of CO2gas supplied from the gas cylinder into the individual culture units212on the basis of the CO2concentration signal from the CO2sensor244. The CO2in the room is discharged by a predetermined constant value by a circulation pump246.

The operation of a stirring fan247is controlled by the operation control PLC223and, when the signal indicating the fact that the door of the culture unit212is opened is transmitted from the door sensor to the operation control PLC223, the operation of the stirring fan247is stopped and the environmental change in the culture unit212is alleviated.

The cell culture apparatus200has a dry heat sterilization function, and is able to sterilize the interior of the apparatus and the individual trays in a state in which the culture bag tray, the culture medium bag tray and the waste water bag tray are set therein, so that the proliferation of bacteria in the culture unit212is prevented. The individual culture units212have an independent space structure, the each culture unit212is isolated from the cells in other culture units212and the culture bag242, the culture medium bag233and the waste water bag234stored in the culture unit212are connected in the clean bench or the like and then is installed in the culture unit212. Therefore, the culture bag242stored in the culture unit212is secured in a closed system (non-open system), and contamination that the cells in the culture bag242is contaminated by bacteria is prevented.

The culture unit212further includes the door sensor (not shown), the door lock sensor (not shown), the temperature sensors236and243, a door lock mechanism (not shown), the heater (not shown) and the stirring fan247disposed therein. The operation control PLC223controls the heater on the basis of the temperature signal from the temperature sensors236and243. The operation control PLC223controls the operation of the stirring fan247to circulate the air and CO2gas in the culture chamber240. In this manner, the interior of the culture chamber240is maintained in an environment optimal for culturing the cells.

The operation control PLC223controls the action of the door lock mechanism so that two or more doors of the culture units212are not opened simultaneously in one single cell culture apparatus200. Accordingly, erroneous installation of the cells or the culture medium among the different culture units212is prevented. The locking action of the door lock mechanism is detected by the door lock sensor and is transmitted to the operation control PLC223. The opened or closed states of the doors of the culture units212are detected by the door sensor and transmitted to the operation control PLC223.

A frame250for supporting the culture bag tray241to be stored in the culture chamber240is provided in the lower portion in the culture unit212shown inFIG. 27, and a weight meter251, which supports the frame250, is installed on the frame250above the culture unit212. The weight meter251is adapted to measure the weight of the culture bag242of the culture bag tray241stored in the culture chamber240, and actually, measures the amount of culture medium supplied from the culture medium bag233to the culture bag242and the amount of waste water discharged from the culture bag242to the waste water bag234. The measured values of the weight meter251are also transmitted to the operation control PLC223. The main body of the culture unit212is provided with an indication lamp (not shown) for indicating the presence or absence of the culture bag242in the culture unit212. The operation control PLC223illuminates the indication lamp, for example, in red, when the culture bag242is stored in the culture chamber240, and illuminates the indication lamp, for example, in green, when the culture bag242is not stored in the culture chamber240.

The culture bag tray241will be described here.

The culture bag242is mounted on the culture bag tray241as shown inFIG. 27, and the culture bag242is a culture vessel to culture the cells. The culture bag242is a function expressing culture vessel for causing the cells to express the function (for example, to cause the cells to proliferate, to cause the cells to differentiate), and is an antibody stimulating culture vessel for stimulating the cells by the antibody for proliferation in this embodiment. It also functions as a proliferation culture vessel for causing cells to proliferate stimulated by the antibody in the same culture bag242.

The culture bag242is a flexible vessel for storing the culture medium in which the cells are inoculated, and is placed on the culture bag tray241via a platform252. The culture bag242is a bag formed of, for example, oxygen permeable material.

The culture unit212will now be described.

In the culture chamber240, a supply pump261and an exhaust pump271are arranged as shown inFIG. 27. The culture bag242is connected at one end thereof to a coupling of a supply system264via the supply pump261using a tube262. The culture bag242is also connected at the other end to an exhaust system coupling274via the exhaust pump271using a tube272.

In the culture unit212, the culture bag242is connected to the coupling of a supply system264via the tube262, and is connected to the culture medium bag233via a tube265. The culture bag242is connected to the exhaust system coupling274via the tube272and is connected to the waste water bag234via a tube275.

The culture bag242is configured into a cassette structure, as shown inFIG. 27, which is immobilized with the antibody on the inner side of the part of the bottom surface, connected to the supply pump261of the culture bag242, added the culture medium in the culture bag242, inoculated the cells on the culture medium and installed on the culture bag tray241. The operation to immobilize the antibody and add the culture medium and the cells in the culture bag242is carried out under the aseptic conditions in the clean bench or the like.

The culture environment for causing the proliferation function of the cells to express, that is, by stimulating the cells by the antibody and the culture environment for causing the cells to proliferate are established in the same culture bag242by making part of immobilized antibody in the culture bag242. Therefore, by supplying the culture medium to the cells which are stimulated by the antibody in a part of the culture bag242and started to proliferate, an efficient proliferation is enabled in the same culture bag242.

The culture medium bag233as a culture medium storage vessel and the waste water bag234as a used culture medium storage vessel are placed on the culture medium bag tray231and the waste water bag tray232, respectively, and each are configured into a cassette structure. The culture medium bag233is adapted to store the culture medium to be supplied to the culture bag242. The waste water bag234is adapted to store the used culture medium (supernatant) discharged from the culture bag242. With the cassette structure of the culture medium bag tray231, the change and supply of the culture medium is enabled only by mounting the culture medium bag tray231into the low temperature chamber230in the culture unit212in a state in which the culture bag242is maintained in the culture chamber240.

The culture bag tray241is detachably mounted to the culture unit212. At this time, the a coupling of a supply system264of the culture bag242is coupled to a joint of the culture medium bag233, and the exhaust system coupling274is coupled to a joint of the waste water bag234respectively under the aseptic conditions. In other words, the coupling of a supply system264and the joint of the culture medium bag233are coupled by, for example, inserting a rubbery joint on one of those to a needle joint on the other one of those under the aseptic conditions. Coupling between the exhaust system coupling274and the waste water bag234is also the same. The work to couple the culture bag242with the culture medium bag233and the waste water bag234is carried out in the clean bench or the like under the aseptic conditions.

With the connection among the culture bag242, the culture medium bag233and the waste water bag234as described above, a closed loop in which the culture medium in the culture medium bag233on the culture medium bag tray231is supplied to the culture bag242by the activation of the supply pump261, and the used culture medium in the culture bag242is discharged to the waste water bag234on the waste water bag tray232by the activation of the exhaust pump271is established. With the configuration of this closed loop, the system (the culture bag242, the culture medium bag233and the waste water bag234) is maintained under the aseptic conditions.

The culture (cell proliferation) in the culture bag242includes the static culture (simple feeding) in which the supply pump261is activated to supply (feed) the culture medium in the culture medium bag233on the culture medium bag tray231to the culture bag242to cause the cells to proliferate, the perfusion culture in which the supply pump261and the exhaust pump271are activated to discharge the used culture medium in the culture bag242to the waste water bag234on the waste water bag tray232and supply the culture medium in the culture medium bag233to the culture bag242to cause the cells to proliferate, and the shaking culture using a shaking device290described later. Among these, the perfusion culture includes the intermittent perfusion culture in which discharge of the used culture medium and supply of the culture medium are carried out alternately, and the consecutive perfusion culture in which the discharge of the culture medium and the supply of the culture medium are carried out simultaneously. In the consecutive perfusion culture, a filter for preventing transfer of cells is normally disposed in the tube272between the culture bag242and the exhaust pump271to prevent the cells in the culture bag242from being discharged to the waste water bag234.

After having completed the proliferation of the cells in the culture bag242, the used culture medium in the culture bag242is discharged to the waste water bag234of the waste water bag tray232, and the cells in the culture bag242is condensed. The discharge of the used culture medium is carried out by the activation of the exhaust pump271, and is carried out until the amount of the culture medium and the cells in the culture bag242is reduced to about ½ to ⅓ in quantity by the control of the operation control PLC223on the basis of the measured value by the weight meter251. By the condensation of the cells in the culture bag242, the number of times of centrifugation carried out by a centrifuge carried out later is reduced.

In the waste water bag tray232, it is also possible to replace the waste water bag234by a cell collecting bag as a cell collecting vessel which is attachable to the centrifuge after having condensed the cells in the culture bag242as described above, and then supply the culture suspension (culture medium and cells) in which the cells are condensed in the culture bag242into the cell collecting bag by activating the exhaust pump271, provided that the filter is not disposed in the tube272. Accordingly, collection of the cells in the bag which is attachable to the centrifuge may be carried out within the culture unit212, which is a space in the closed system, so that the labor for collecting the cells is saved.

In this case, as shown inFIG. 27, the culture bag tray241is stored in the culture unit212and the culture bag tray241is supported by the frame250of the culture chamber240. The platform252for directly placing the culture bag242on the culture bag tray241includes a non-elevating part252a, and a plurality of area-changing parts252band252cwhich are able to move upward and downward. The culture area of the culture bag242is controlled by the area-changing parts252band252c. Installed below the portion of the culture tray241where the area-changing parts252band252care arranged are an inclined motor (elevating mechanism280), a cam mechanism281and a positioning sensor (not shown). The inclined motor280rotates the cam mechanism281and moves upward and downward the area-changing parts252band252cof the platform252separately and independently from each other. The positions of the elevating units are detected by the positioning sensor, and transmitted to the operation control PLC223. The inclined motor280is controlled by the operation control PLC223so as to move the area-changing parts252band252cof the platform252downward in the initial stage of culture in the culture bag242. Accordingly, a liquid reservoir is formed at portions of the culture bag242corresponding to the area-changing parts252band252c. For example, the culture area of the platform252shown inFIG. 28is changed in three stages by moving the heights of the area-changing parts252band252cupward and downward respectively. Three or more of the area-changing parts may be provided, whereby the finer adjustment of the culture area is achieved.FIG. 28is a perspective view showing a configuration of the platform252inFIG. 27.

By the cells and the culture medium being stored in the liquid reservoirs in the initial stage of culture in the culture bag242, a culture condition suitable for antibody stimulation, that is, the cell density in the culture bag242is maintained to a density which is preferable for the proliferation. In the middle stage or the later stage in which the culture medium and the cells in the culture bag242are increased to a value larger than the predetermined value, the inclined motor280moves the area-changing parts252band252cof the platform252upward via the cam mechanism281, and maintains only the parts having the predetermined area of the culture bag242in the horizontal state, so that the areas of the liquid reservoirs are changed. By changing the areas of the liquid reservoirs for the cells and the culture medium according to the progress of culture, the cell density per area in the culture bag242is maintained to a density preferable for the proliferation, so that the cells efficiently proliferate in the cell proliferation stage.

For example, when the platform252is able to form two stages of liquid reservoirs as shown inFIG. 28, first of all, the culture medium and the cells are retained in a portion of the area-changing part252c, which is the lowest part, inFIG. 28to avoid lowering of the density of the cells.

When the cells has started the proliferation and hence the amount of the culture medium is increased, the height of the area-changing part252cinFIG. 28is moved upward by one stage to align the area-changing part252band the area-changing part252cat the same level, so that the area of the culture bag242which is usable for the culture is enlarged by a predetermined amount. Accordingly, the density of the cells which is preferable for the proliferation may be maintained for a certain time length again.

When the cells further proliferate and hence the amount of the culture medium is increased, the area-changing part252band the area-changing part252care moved upward by one stage to align with the part252a, so that the culture is carried out in the entire part of the culture bag242.

Furthermore, a shaking mechanism291of the shaking device290is installed above the culture bag tray241where the culture bag242is arranged as the pressing unit in the culture chamber240as shown inFIG. 27. The shaking device290includes the shaking mechanism291, an operating motor292, a cam mechanism293and the positioning sensor (not shown). As shown inFIG. 27, the shaking mechanism291includes an operating plate291aas the pressing unit disposed to the apparatus frame250via the guide rod250aso as to be capable of moving upward and downward, and a plurality of projections291bprojecting from the bottom surface of the operating plate291a. When the operating plate291ais moved upward and downward alternately on the basis of the action of the cam mechanism293by the operating motor292, the projections291bof the operating plate291apresses the culture bag242positioned below the shaking mechanism291repeatedly, that is, repeats pressing and releasing with respect to the culture bag242. Accordingly, the culture medium in the culture bag242is stirred, and the cells in the culture bag242floats and moves in the culture medium, so that the distribution of the cells and oxygen concentration distribution in the culture bag242are homogenized, and hence the proliferation of the cells is promoted.

It is also possible to use a platform352shown inFIG. 29andFIG. 30instead of the platform252described above to change the culture area in the culture bag242placed on the platform352.FIGS. 29A and 29Bare plan views showing a configuration of the platform352according to a modification of the fourth embodiment.FIG. 29Ais a drawing showing a state in which a non-elevating part353, and all area-changing parts354,355,356and357are aligned on the same plane, andFIG. 29Bshows a state in which the area-changing parts are moved downward.FIGS. 30A to 30Care partly cross-sectional views taken along the line IIIX-IIIX inFIG. 29A.FIG. 30Ais a drawing showing a state in which the area-changing parts are moved downward, and the non-elevating part353, the area-changing part355and the area-changing part357are arranged in a staircase pattern on different planes.FIG. 30Bis a drawing showing a state in which the three of the area-changing parts355,356and357from among four area-changing parts are aligned on the same plane.FIG. 30Cis a drawing showing a state in which the non-elevating part353and all the area-changing parts354,355,356and357are aligned on the same plane.

As shown inFIGS. 29A and 29B, the platform352includes area-changing parts357,356,355and354which are obtained by cutting a corner352aof a rectangle in plan view in sequence in the shapes of isosceles triangles each having the corner as an apex, and a non-elevating part353. The non-elevating part353is arranged on the horizontal plane. As shown inFIG. 30, the non-elevating part353is coupled to the variable area part354by a joint member353a, the area-changing part354is coupled to the area-changing part355by a joint member354a, the area-changing part355is coupled to the area-changing part356by a joint member355a, and the variable area part356is coupled to the area-changing part357by a joint member356a, so as to be rotatable with respect to each other. The joint members353a,354a,355aand356amay be, for example, hinges.

Although the platform352includes four area-changing parts, the number of the area-changing parts may be two to three, or may be five or more.

The area-changing parts355and357includes supporting legs365and367fixedly adhered on the lower surfaces thereof respectively.

As shown inFIG. 30C, the lower surface of the supporting leg365includes a plane365aand a plane365barranged in sequence from the side of the corner352aof the platform352on a horizontal plane, and an inclined plane365cwhich connects the planes365aand365b, and the plane365ais positioned at a lower level than the plane365b. On the other hand, the lower surface of the supporting leg367includes planes367a,367band367carranged in sequence from the side of the corner352aof the platform352on a horizontal plane, an inclined plane367dwhich connects the plane planes367aand367b, and an inclined plane367ewhich connects the plane367band the plane367c. The plane367ais positioned at a lower level than the plane367b, and the plane367bis positioned at a lower level than the plane367c. The distance between the plane365aand the area-changing part355is the same as the distance between the plane367aand the area-changing part357, and the distance between the plane365band the area-changing part355is the same as the distance of the plane367bfrom the area-changing part357.

A height adjusting plate370, which is movable along the line IIIX-IIIX inFIG. 29A(the direction indicated by an arrow A inFIG. 30B) by a motor380is arranged below the platform352. Elevating members375and377for moving the area-changing parts upward and downward are fixedly adhered on the height adjusting plate370.

As shown inFIG. 30C, the upper surface of the elevating member375includes a plane375band a plane375aarranged in sequence from the side of the corner352aof the platform352on a horizontal plane and an inclined surface375cwhich connects the planes375aand375b, and the plane375ais positioned at a level higher than the plane375b. On the other hand, the upper surface of the elevating member377includes planes377c,377band377aarranged in sequence from the side of the corner352aof the platform352on a horizontal plane, an inclined plane377dwhich connects the plane377aand the plane377b, and an inclined plane377ewhich connects the plane377band the plane377c. The plane377ais positioned at a level higher than the plane377b, and the plane377bis positioned at a level higher than the plane377c. The upper surface of the elevating member377has a shape corresponding to the lower surface of the supporting leg367and, when the inclined planes377dand377eare brought into abutment with the inclined planes367eand367dof the supporting leg367respectively, the planes377a,377band377ccomes into abutment with the planes367c,367band367aof the supporting leg367, respectively. The distance between the plane375aand the upper surface of the height adjusting plate370is the same as the distance of the plane377afrom the upper surface of the height adjusting plate370, and the distance of the plane375bfrom the upper surface of the height adjusting plate370is the same as the distance of the plane377bfrom the upper surface of the height adjusting plate370.

Subsequently, referring toFIGS. 30A to 30C, an example of operation to move the area-changing parts upward and downward by moving the height adjusting plate370in the direction indicated by the arrow A will be described.

As shown inFIG. 30A, when the planes367a,377band367cand the inclined planes367dand367eof the supporting leg367are brought into abutment respectively with the planes377c,377band377aof the elevating member377, the planes365aand365bof the supporting leg365and the planes375band375aof the elevating member375come into abutment with each other respectively in a state in which the inclined plane365cand the inclined plane375care apart from each other. Consequently, the area-changing part355and the area-changing part357are arranged along the horizontal direction and, simultaneously, the area-changing part354and the area-changing part356are arranged in the vertical direction, so that the platform352exhibits a staircase pattern which increase in height in sequence from the side of the corner352aof the platform352.

When the height adjusting plate370is moved by the motor380in the direction indicated by the arrow A from the state shown inFIG. 30A, the supporting leg367moves upward along the shape of the upper surface of the elevating member377(FIG. 30B). Accordingly, the planes367aand367band the inclined plane367dof the supporting leg367come into abutment respectively with the planes377band377aand the inclined plane377dof the elevating member377. On the other hand, the elevating member375moves relatively with the supporting leg365until the inclined plane365cand the inclined plane375ccome into abutment with each other in a state in which the planes365aand365bare in abutment with the planes375band375a. Consequently, as shown inFIG. 30B, the three area-changing parts355,356and357from among the four area-changing parts are positioned in the same plane.

Then, when the height adjusting plate370is moved by the motor380in the direction indicated by the arrow A from the state shown inFIG. 30B, the supporting leg367is moved upward along the shape of the upper surface of the elevating member377(FIG. 30C). Accordingly, the plane367aof the supporting leg367comes into abutment with the plane377aof the elevating member377. On the other hand, the supporting leg365moves upward along the shape of the upper surface of the elevating member375, and hence the plane365aof the supporting leg365and the plane375aof the elevating member375come into abutment with each other. Accordingly, as shown inFIG. 30C, the non-elevating part353and all the area-changing parts354,355,356and357are arranged on the same plane.

As descried above, since the area-changing parts354to357may be controlled to move upward and downward by moving the height adjusting plate370, the desired portion may be used as the liquid reservoir.

Furthermore, the shaking mechanism291of the shaking device290is installed above the culture bag tray241where the culture bag242is arranged as the pressing unit in the culture chamber240as shown inFIG. 27. The shaking device290includes the shaking mechanism291, the operating motor292, the cam mechanism293and the positioning sensor (not shown). As shown inFIG. 27, the shaking mechanism291includes the operating plate291aas the pressing unit disposed to the apparatus frame250via the guide rod250aso as to be capable of moving upward and downward, and the plurality of projections291bprojecting from the bottom surface of the operating plate291a. When the operating plate291ais moved upward and downward alternately on the basis of the action of the cam mechanism293by the operating motor292, the projections291bof the operating plate291apresses the culture bag242positioned below the shaking mechanism291repeatedly, that is, repeats pressing and releasing with respect to the culture bag242. Accordingly, the culture medium in the culture bag242is stirred, and the cells in the culture bag242floats and moves in the culture medium, so that the distribution of the cells and oxygen concentration distribution in the culture bag242are homogenized, and hence the proliferation of the cells is promoted.

As shown inFIG. 27, the position of the operating plate291ais detected by the positioning sensor, and is transmitted to the operation control PLC223, and the operating motor292is controlled by the operation control PLC223on the basis of the measured value by the weight meter251. The cell culture (shaking culture) in the culture bag242using the shaking device80described above may be carried out before the culture medium and the cells are filled in the culture bag242to a maximum level, or may be carried out after having filled therein to the maximum level. The shaking device290is controlled by the operation control PLC223on the basis of the detected position of the operating panel and the measured value of the weight meter251.

In the culture chamber240, as shown inFIG. 27, an illuminating lamp301is installed at a position above the culture bag242where the antibody is immobilized and a CCD camera302as an image acquiring unit is installed below the same. The CCD camera302is added with optical equipment such as a lens, a prism, a barrel according to the observing mode or the like. The illuminating lamp301is adapted to illuminate the culture bag242from above. The CCD camera302is adapted to take images of the cells in the culture bag242from below and acquires the images thereof. The illuminating action of the illuminating lamp301and the shooting action of the CCD camera302are controlled by the operation control PLC223, and the images of the cells in the culture bag242are acquired at predetermined time intervals (six hours, for example). The cell images taken at the predetermined time intervals are stored in an image memory circuit (not shown) of the image processing unit221.

The image processing unit221carries out image processing, for example, binarization or multithresholding, for the cell images taken at the predetermined time intervals stored in the image memory circuit of the image processing unit221, so that an average value of the projected areas of the single cells and the increasing rate of the non-single cell, which is a cell aggregate formed of a plurality of single cells are calculated as evaluation parameters of the cell culture. The average value of the projected areas of the single cells (the average projected area of the single cell) is calculated from the cell image taken when 24 hours, for example, has elapsed from the moment when the culture is started after having mounted the culture bag242on culture bag tray241and stored culture bag tray241in culture chamber240.

Whether the above-described cells are the non-single cell or not is determined with reference to the projected area of 100 μm2in such a manner that the cells having a projected area at least equal to 100 μm2are determined as the non-single cells and the cells having projected are less than 100 μm2are determined as the single cells. It is because all the cells had projected areas less than 100 μm2when the projected areas of the single cells in the initial stage of culture were measured. The change of the ratio of the non-single cells with respect to all the cells is computed from follow-up images of the cells (for example, images of the cells after having elapsed 24 hours, 48 hours and 72 hours from the start of culture) to calculate the increasing rate of the non-single cells. The increasing rate of the non-single cells and the average projected area of the single cells are outputted from the image processing unit221to the operation control PLC223.

The operation control PLC223calculates a lag time from the average projected area of a single cell and estimates the timing when the corresponding cell has started proliferation. In this specification, the lag time means a time length of an induction phase required from inoculation of cells in the portion of the immobilized antibody in the culture bag tray241until the proliferation is started. The operation control PLC223determines whether the culture state of the cells, that is, whether the cell has a capability to proliferate by the stimulation from the antibody or not, from the timing when the cells has started to proliferate, and evaluates the cells. The operation control PLC223stops the culture in the cell culture apparatus200for the cells which having a very low proliferation capability on the basis of the evaluation.

The operation control PLC223calculates a minimum doubling time of the cells from the increasing rate of the non-single cell. The minimum doubling time in this specification means a minimum time period required for the number of cells at a certain time instant to be increased to two times the number of cells. The operation control PLC223determines the culture state of the cells, that is, the proliferation ability of the cells from the minimum doubling time and decide the timing or the velocity of the feeding of the culture medium to the culture bag242.

The operation control PLC223and the image processing unit221functioning as control units include, although not shown, a CPU for executing computation or control, a storage device (memory) for storing a processing program or data, and a input/output circuit for the connection with the input devices such as a keyboard, a mouse or a touch panel for supplying data or commands and the output devices such as the monitor. The image processing unit221further includes the image memory circuit for storing image data from the CCD camera302.

The storage device of the image processing unit221stores a program for processing (for example, binarization of multithresholding) the images of the cells in the culture bag242shot at predetermined time intervals by the CCD camera302and calculating the evaluation parameters for the cell culture (the average projected area of the single cell, the increasing rate of the non-single cell).

The storage device of the operation control PLC223also stores a program for determining the culture state of the cells (the proliferation capability of the cells, the proliferation ability of the cells) from the evaluation parameters of the cell culture and a program for controlling the equipment relating to the cell culture apparatus200and the culture unit212(for example, the supply pump261and the exhaust pump271) according to the culture state of the cell and executing the culture operation. The storage device of the operation control PLC223further stores an equipment controlling program for controlling the equipment relating to the culture device210and the culture unit212on the basis of the signals from various sensors of the cell culture apparatus200and the culture unit212. The storage device of the image processing unit221also stores an equipment controlling program for controlling the CCD camera302at predetermined time intervals and acquiring the cell images.

Referring now to flowcharts shown in the process drawings inFIG. 31toFIG. 40, a process of culturing cells carried out by the operation control PLC223and the image processing unit221by executing the above-described programs will be described.

FIG. 31andFIG. 32show an antibody stimulating intermittent perfusion culture process in a case of collecting the cells in the culture bag242.

As shown inFIG. 31, the operator immobilizes an antibody in the culture bag242in the clean bench or the like (W01inFIG. 31), adds the culture medium, and mounts the culture bag242having inoculated the cells on the culture medium bag tray231. Then, the operator mounts the culture medium bag233in which the culture medium is added in the clean bench or the like onto the culture medium bag tray231, mounts the waste water bag234on the waste water bag tray232, and couples the culture bag242with the culture medium bag233and the waste water bag234using the coupling of a supply system264and the exhaust system coupling274, respectively (W02inFIG. 31).

Then, the operator carries the culture bag tray241, the culture medium bag tray231and the waste water bag tray232in the single culture unit212of the cell culture apparatus200, illuminated by an illumination lamp, for example, in green, causes the culture bag tray241to be supported by the frame250of the culture chamber240and installs the culture medium bag tray231and the waste water bag tray232in the low temperature chamber230. Then, the operator connects the pump tubes262and272of the culture bag242to the supply pump261and the exhaust pump271, respectively, (W03inFIG. 31).

Then, the operator confirms images in the culture bag242stored in the culture unit212, which are outputted from the CCD camera302(W05inFIG. 31). Before confirming the outputted images, the operator activates the inclined motor280of the culture unit212to move the area-changing parts252band252cof the platform252downward to form the liquid reservoirs in the culture bag242(W04inFIG. 31). The operator then measures the weight of the installed culture bag242by the weight meter251of the culture unit212.

Then, the operator closes the door of the culture unit and starts the cell culture in the culture unit212(W06inFIG. 31). Accordingly, the cells are stimulated by the antibody for the proliferation in the liquid reservoirs in the culture bag242(W07inFIG. 31). The CCD camera302of the culture unit212takes the images of the cells in the liquid reservoirs in the culture bag242at predetermined time intervals (every six hours, for example), the image processing unit221calculates the evaluation parameters of the cell culture from the picked-up image. The operation control PLC223calculates the lag time from the evaluation parameter, determines whether the cells has a capability to proliferate upon reception of stimulation from the antibody or not, calculates the minimum doubling time, and determines the proliferation ability of the cells (W08inFIG. 31).

When the capability of proliferation cannot be seen even when a predetermined time period (24 hours, for example) has elapsed since the cells has stimulated by the antibody in the culture bag242, the operation control PLC223cancels the culture of the cell in the cell culture apparatus200(W08′ inFIG. 31). When it is determined that the cells in the culture bag242has a capability of proliferation, the operation control PLC223decides the velocity and timing of feeding of the culture medium into the culture bag242on the basis of the proliferation ability of the cells. The operation control PLC223activates the supply pump261on the basis of this decision, and feeds the culture medium in the culture medium bag233on the culture medium bag tray231to the culture bag242(W09inFIG. 31).

With the activation of the supply pump261, the static culture of the cells is started in the liquid reservoir having a predetermined area in the culture bag242(W10inFIG. 31). The operation control PLC223determines whether the weight of the culture medium and the cells in the culture bag242measured by the weight meter251is increased to a value at least equal to the predetermined value α or not (W11inFIG. 31). At a timing when the value at least equal to the predetermined value α is reached, the operation control PLC223activates the inclined motor280, moves the area-changing parts252band252cof the platform252upward via the cam mechanism281and brings a predetermined area of the culture bag242into a horizontal state to change the liquid reservoir (W12inFIG. 31). This process is repeated until all the steps formed on the platform252are eliminated and the culture bag242is brought into a horizontal state (W13inFIG. 31).

Then, the operation control PLC223determines whether the weight of the culture medium and the cells in the culture bag242measured by the weight meter251is increased to a value at least equal to the predetermined value β or not (W14inFIG. 31). At the timing when the value at least equal to the predetermined value β is reached, the operation control PLC223activates the operating motor292. Accordingly, the shaking device290is activated, and the shaking culture in which the shaking mechanism291of the shaking device290presses the culture bag242repeatedly is started (W15inFIG. 31). The operation control PLC223continues to determine whether the weight of the culture medium and the cells in the culture bag242measured by the weight meter251is increased to a value at least equal to the predetermined value χ or not (W16inFIG. 32). At a timing when the value at least equal to the predetermined value χ is reached, the operation control PLC223stops the supply pump261to stop the feeding of the culture medium from the culture medium bag233on the culture medium bag tray231to the culture bag242(W17inFIG. 32), stops the operating motor292to stop the shaking culture in the culture bag242(W18inFIG. 32).

The operation control PLC223activates the exhaust pump271after having settled the cells in the culture bag242to discharge the used culture medium (supernatant in the culture bag242) in the culture bag242to the used waste water bag234on the waste water bag tray232(W19inFIG. 32). Then, the operation control PLC223determines whether the weight of the culture medium and the cells in the culture bag242measured by the weight meter251is reduced to a value at most equal to a predetermined value δ or not (W20inFIG. 32). At a timing when the value at most equal to the predetermined value δ is reached, the operation control PLC223stops the exhaust pump271to stop the discharge of the used culture medium from the culture bag242(W21inFIG. 32). Then, the operation control PLC223activates the supply pump261to feed the culture medium from the culture medium bag233on the culture medium bag tray231to the culture bag242, and activates the operating motor292to carry out the shaking culture in the culture bag242by the shaking device290(W22inFIG. 32). The operation control PLC223stops the supply pump261after having elapsed a predetermined time to stop the feeding of the culture medium from the culture medium bag233on the culture medium bag tray231to the culture bag242and continue the shaking culture in a state in which the operating motor292is activated (W23inFIG. 32).

The operation control PLC223determined whether the desired culture term depending on the date and time of usage of the cells to proliferate is reached or not, or the image processing unit221determines whether the desired number of cells is reached in the culture bag242or not (W24inFIG. 32). When the desired culture term or the desired number of cells is not reached, the process steps in Steps W18to W23are repeated. When counting the number of cells, the shaking device290is stopped once to wait until the cells are settled. Then, the images of the interior of the culture bag242are taken by the CCD camera302. The image processing unit221estimates and calculates the number of cells existing in the bag from the acquired images.

Steps W18to W23are steps of intermittent perfusion culture in which discharge of the used culture medium in the culture bag242and supply (feed) of new culture medium into the culture bag242are carried out alternately.

At the time point where the desired culture term or the number of cells are reached in Step W24, the operation control PLC223stops the operating motor292as shown inFIG. 32to stop the shaking culture in the culture bag242(W25inFIG. 32). Then, the operation control PLC223activates the exhaust pump271after having settled the cells in the culture bag242to discharge the used culture medium in the culture bag242to the waste water bag234on the waste water bag tray232, and condense the cells until the culture medium and the cells in the culture bag242are reduced to about ½ to ⅓ on the basis of the measured value by the weight meter251(W26inFIG. 32)

The operation control PLC223then stops the exhaust pump271to complete the cell culture (W27inFIG. 32). After having completed the culture, the operator transfers the cells in the culture bag242to a vessel for the centrifugation in the clean bench or the like, and then, the cells are collected by the centrifugation (W28inFIG. 32).

In the similar antibody stimulating intermittent perfusion culture process, the case in which the process of collecting the cells by the cell collecting bag is included is shown inFIG. 33andFIG. 34. Therefore, since Steps W31to W56of the process shown inFIG. 33andFIG. 34are the same as Steps W01to W26shown inFIG. 31andFIG. 32, description is omitted.

In Step W56shown inFIG. 34, after having condensed the cells in the culture bag242by the activation of the exhaust pump271, the operation control PLC223stops the exhaust pump271and prompts the operator to replace the waste water bag234on the waste water bag tray232by the cell collecting bag (W57inFIG. 34). The cell collecting bag is a bag to be mounted to the centrifuge and used for the centrifugation.

After having replaced the waste water bag234on the waste water bag tray232by the cell collecting bag, the operation control PLC223activates the exhaust pump271and the operating motor292. Then, the operation control PLC223shakes the interior of the culture bag242by the shaking device290to transfer the cells in the culture bag242to the cell collecting bag mounted on the waste water bag tray232together with the culture medium (W58inFIG. 34). Then, the operation control PLC223stops the exhaust pump271and the operating motor292to stop the collection of the cells from the culture bag242to complete the cell culture (W59inFIG. 34). After having completed the cell collection, the operator mounts the cell collecting bag to the centrifuge, so that the cells are collected by the centrifugation (W60inFIG. 34).

Referring now toFIG. 35andFIG. 36, process steps in an antibody stimulating consecutive perfusion culture process will be described. Since Steps X01to X15in the antibody stimulating consecutive perfusion culture process shown inFIG. 35andFIG. 36are the same as Steps W01to W15in the antibody stimulating intermittent perfusion culture process shown inFIG. 31andFIG. 32, description is omitted.

In the antibody stimulating consecutive perfusion culture process, a filter (not shown) is disposed between the culture bag242and the exhaust pump271.

The operation control PLC223activates the exhaust pump271at a timing when the weight of the culture medium and the cells in the culture bag242is increased to a value at least equal to the predetermined value χ (X16inFIG. 36) to discharge the used culture medium in the culture bag242to the waste water bag234on the waste water bag tray232while the culture medium is fed from the culture medium bag233on the culture medium bag tray231into the culture bag242and the shaking culture by the shaking device290is carried out in the culture bag242. Accordingly, the consecutive perfusion culture in which the feeding of the culture medium to the culture bag242and the discharge of the culture medium from the culture bag242are carried out simultaneously is started in the culture bag242(X17inFIG. 36). At this time, the cells in the culture bag242are prevented from flowing by the filter and hence do not flow into the waste water bag234. During the consecutive perfusion culture, the shaking culture by the shaking device290is simultaneously carried out.

The operation control PLC223determines whether a desired culture term depending on the date and time of usage of the cells to proliferate is reached or not, or the image processing unit221determines whether the desired number of cells is reached in the culture bag242or not (X18inFIG. 36). When the desired culture term or the desired number of cells is not reached, the consecutive perfusion culture in Step X17is repeated. When counting the number of cells, the shaking device290is stopped once to wait until the cells are settled. Then, the images of the interior of the culture bag242are taken by the CCD camera302. The image processing unit221estimates and calculates the number of cells existing in the bag from the acquired images.

At the time point where the desired culture term or the number of cells are reached in the Step X18, the operation control PLC223stops the supply pump261, the exhaust pump271and the operating motor292to stop the perfusion culture and the shaking culture (X19inFIG. 36). Then, the operation control PLC223activates the exhaust pump271after having settled the cells in the culture bag242to discharge the used culture medium in the culture bag242to the waste water bag234on the waste water bag tray232and condense the cells until the culture medium and the cells in the culture bag242are reduced to about ½ to ⅓ on the basis of the measured value by the weight meter251(X20inFIG. 36). To stop the shaking device in the condensing process is to prevent a large amount of cells from flowing into the tube and from clogging the filter.

The operation control PLC223then stops the exhaust pump271to complete the cell culture (X21inFIG. 36). After having completed the culture, the operator transfers the cells in the culture bag242to a vessel for the centrifuge in the clean bench or the like, and then, the cells are collected by the centrifugation (X22inFIG. 36).

Referring now toFIG. 37andFIG. 38, processing steps in an antibody stimulating simple feeding culture process will be described. Since Steps Y01to Y15in the antibody stimulating simple feeding culture process inFIG. 37andFIG. 38are the same as Steps in W01to W15in the antibody stimulating intermittent perfusion culture process inFIG. 31andFIG. 32, description is omitted.

The operation control PLC223determines whether the weight of the culture medium and the cells in the culture bag242measured by the weight meter251is increased to a value at least equal to the predetermined value χ or not (Y16inFIG. 38). At a timing when a value at least equal to or larger than the predetermined value χ is reached, the operation control PLC223stops the supply pump261to stop the feeding of the culture medium from the culture medium bag233on the culture medium bag tray231to the culture bag242and to continue the shaking culture while activating the operating motor292(Y17inFIG. 38).

The operation control PLC223determines whether a desired culture term depending on the date and time of usage of the cells to proliferate is reached or not, or the image processing unit221determines whether the desired number of cells is reached in the culture bag242or not (Y18inFIG. 38). When the desired culture term or the desired number of cells is not reached, the shaking culture is continued (No in Y18).

When counting the number of cells, the shaking device290is stopped once to wait until the cells are settled. Then, the images of the interior of the culture bag242are taken by the CCD camera302. The image processing unit221estimates and calculates the number of cells existing in the bag from the acquired images.

At a timing when the desired culture term or the number of cells is reached in Step Y18, the operation control PLC223stops the operating motor292as shown inFIG. 38to stop the shaking culture in the culture bag242(Y19inFIG. 38). After having settled the cells in the culture bag242, the exhaust pump271is activated to discharge the used culture medium in the culture bag242to the waste water bag234on the waste water bag tray232and condense the cells until the culture medium and the cells in the culture bag242are reduced to about ½ to ⅓ on the basis by the measured value of the weight meter251(Y20inFIG. 38).

The operation control PLC223then stops the exhaust pump271to complete the cell culture (Y21inFIG. 38). After having completed the culture, the operator transfers the cells in the culture bag242to the vessel for the centrifuge in the clean bench or the like, and then the cells are collected by the centrifugation (Y22inFIG. 38).

Subsequently, in the similar antibody stimulating simple feeding culture process, the case in which the step of collecting the cells by the cell collecting bag is included is shown inFIG. 39andFIG. 40. Therefore, since Steps Y31to Y50in the process shown inFIG. 39andFIG. 40are the same as Steps Y01to Y20inFIG. 37andFIG. 38, description is omitted.

In Step Y50shown inFIG. 40, after having condensed the cells in the culture bag242by the activation of the exhaust pump271, the operation control PLC223stops the exhaust pump271and prompts the operator to replace the waste water bag234on the waste water bag tray232by the cell collecting bag (Y51inFIG. 40). The cell collecting bag is a bag to be mounted to the centrifuge and used for the centrifugation.

After having replaced the waste water bag234on the waste water bag tray232by the cell collecting bag, the operation control PLC223activates the exhaust pump271and the operating motor292. Then, the shaking device290shakes the interior of the culture bag242to transfer the cells in the culture bag242to the cell collecting bag mounted on the waste water bag tray232together with the culture medium (Y52inFIG. 40). The operation control PLC223then stops the exhaust pump271and the operating motor292to stop the collection of the cells from the culture bag242and complete the cell culture (Y53inFIG. 40). After having completed the cell collection, the operator mounts the cell collecting bag to the centrifuge, so that the cells are collected by the centrifugation (Y54inFIG. 40).

In this configuration, according to the embodiments shown above, the following effects (1) to (7) are achieved.

(1) The image processing unit221processes the images of the cells in the culture bag242taken by the CCD camera302to acquire the evaluation parameters of the cell culture (the average projected area of the single-cell, the increasing rate of the non-single cell), the operation control PLC223determines and evaluates the culture state of the cells (the proliferation capability and the proliferation ability) to carry out the culture operation according to the culture state (the culture medium feeding at the predetermined feeding velocity or the timing from the culture medium bag233to the culture bag242). Consequently, since the culture state of the cells is determined in a non-contact state, the cells are prevented from getting damaged, and the operator needs not carry out the culture operation one by one, the labor of the operator is alleviated. Since the cells of one patient may be inoculated to the single culture bag242stored in the culture unit212, and the culture operation according to the culture state of the cells may be carried out on the individual basis, so that the adequate culture operation is achieved. Since the culture operation suitable for the culture state of the cells is achieved, the culture operation by the hour is enabled, and the culture is accelerated to shorten the culture term.

(2) The culture medium bag233, the waste water bag234and the culture bag242are coupled in the clean bench or the like and are configured into a closed loop to install in the culture unit212. Therefore, aseptic conditions in a completely closed system are maintained.

(3) The culture process is carried out automatically since the culture bag tray241, the culture medium bag tray231and the waste water bag tray232are installed in the culture unit212at the time of starting the culture until the culture is completed. Therefore, the cells in the culture bag242is prevented from getting damaged due to the change of environment, and the aseptic operation for supplying the culture medium to the culture bag242in the clean bench or the like may be omitted.

(4) The antibody stimulation and the cell proliferation in the initial state of culture in culture bag242may be carried out in the same culture bag242, and the liquid reservoirs where the cells and the culture medium are stored in the culture bag242may be changed into the predetermined areas. Therefore, the cell density per area during the culture is maintained at a density preferable to the proliferation, so that the cells proliferate efficiently.

(5) The used culture medium in the culture bag242is discharged and stored in the waste water bag234on the waste water bag tray232, the cell density in the culture bag242may be increased and hence condensed. Therefore, the number of times of operation of the centrifugation for collecting the cells is reduced. Consequently, the labor for collecting the cells is saved, and the damage of the cells in association with the centrifugation is reduced.

(6) When all the cells condensed in the culture bag242are to be collected into the cell collecting bag mounted to the waste water bag tray232, the cells may be collected by mounting the cell collecting bag directly to the centrifuge. Therefore, the labor for collecting the cells is saved.

(7) The culture medium in the culture bag242is stirred by repeatedly pressing the flexible culture bag242having the culture medium to which the cells are inoculated stored therein by the projections291bof the operating plate291ain the shaking mechanism291of the shaking device290. Therefore, the distribution of cells and the oxygen concentration distribution in the culture bag242are homogenized, so that the proliferation of the cells is accelerated and the efficiency of the cell culture is improved.

The cells only float in the culture medium stirred by being pressed repeatedly by the operating plate291aof the shaking device290. Therefore, the cells are prevented from getting damaged.