JP6550441B2 - Stir culture apparatus and medium exchange method - Google Patents

Description

  In particular, the present invention relates to a stirred culture apparatus and medium exchange method capable of continuously exchanging medium.

  In order to continuously culture a culture of cells such as iPS cells, cells of plants and animals, cells and the like at high density in a culture vessel, for example, in a three-dimensional suspension (3D suspension), the culture may While cultivating in the medium, it is necessary to exchange the fresh medium with the medium containing waste products.

  In order to withdraw the medium containing such waste products, conventionally, a discharge vessel and a discharge pump are provided in the culture vessel, and a culture such as cells does not pass through the discharge tube, and a culture solution etc. By using a culture apparatus provided with a filtration membrane (filter) that passes only the culture medium, negative pressure is applied to the discharge pipe by the discharge pump (or the inside of the culture vessel is pressurized), and the filtration is performed from the discharge pipe. By means of the membrane, only the medium other than the culture is withdrawn from the culture vessel.

  For example, as the above-mentioned stirred culture apparatus, there is Patent Document 1.

Japanese Patent Application Laid-Open No. 8-172956

  However, in the conventional culture apparatus, it is necessary to perform backwashing to prevent clogging of the filtration membrane, and a system combining operation and control of repeating filtration and backwashing (discharge and backflow) is used, and continuous The medium could not be replaced.

  In addition, it was not perfect from the viewpoint of complete clogging prevention of the filtration membrane and damage to cells.

  The present invention can be used simply because the specific gravity of the cells is surely larger than that of the mother liquor and the sedimentation is large in the culture of cells, especially after induction of differentiation in regenerative medicine that treats iPS cells and in culture using microcarriers. It is an object of the present invention to provide a culture apparatus capable of continuously discharging (extracting) only the culture medium with the above constitution.

In order to achieve the above object, the stirred culture apparatus of the present invention comprises: a culture vessel in which contents consisting of a culture and a medium are placed, and up-and- down reciprocating agitation provided in the culture vessel for stirring the contents Stopping the stirring of the means and the stirring means, and passing the desired content above the desired position at a desired time when the concentration of the culture at the desired position in the content is less than the desired concentration; It is characterized by comprising: an exhausting means for exhausting out of the culture vessel; and a supplying means for supplying a culture or a culture medium into the culture vessel.

  An operation control apparatus comprising: a stirring control unit that controls the stirring unit; a discharge control unit that controls the discharging unit; a supply control unit that controls the supplying unit; and a storage unit that stores the desired time And the control means controls the discharge means when the stored desired time has elapsed by the operation control device.

Further, in the stirred culture apparatus of the present invention, there is provided a culture vessel into which contents comprising a culture and a medium are placed, an up-and-down reciprocating stirring means for stirring the contents provided in the culture vessel, and the contents The concentration detecting device for detecting the concentration of the culture in the desired position in the inside, and stopping the stirring of the stirring means, when the concentration of the culture detected by the concentration detecting device becomes less than the desired concentration, It is characterized by comprising: a discharge means for discharging the contents above the desired position out of the culture vessel; and a supply means for supplying a culture or a medium into the culture vessel.

The operation control apparatus includes an agitation control unit that controls the agitation unit, a discharge control unit that controls the discharge unit, and a supply control unit that controls the supply unit.
By the density detecting device, when the desired concentration following concentration is detected by the operation control device, characterized in that it has a control device in which the discharge means is controlled.

Further, in the stirred culture apparatus of the present invention, a culture vessel in which contents consisting of a culture and a medium are placed , and the inside of the culture vessel provided in the culture vessel is divided into one room and the other room. towards the other from the one, or a film movable toward one from the other, the provided culture vessel, a stirring means for stirring the contents, the contents of said one of the room A discharge means for discharging the culture vessel out of the culture vessel, and a supply means for supplying a culture medium into the culture vessel, wherein the membrane does not pass the culture but passes the culture medium. Do.

  In the culture vessel, the inside of the culture vessel is divided up and down, and a sedimentation membrane capable of moving up and down is provided, and the sedimentation membrane does not pass the culture but passes the culture medium. It features.

  The medium replacement method in the culture vessel of the present invention comprises a first step of stirring the contents of the culture in the culture vessel and the contents in the culture vessel by a stirring means for a desired time, and Stopping the agitation and settling the culture in the contents for a desired time for which the concentration of the culture at the desired location in the culture vessel is less than the desired concentration; and The third step of discharging the contents in the upper culture vessel to the outside of the culture vessel by the desired amount by the discharging means, and the fourth step of supplying the desired amount of culture medium into the culture vessel by the supplying means And a fifth step of repeating the first step to the fourth step a desired number of times.

  The medium replacement method in the culture vessel of the present invention comprises a first step of stirring the contents of the culture vessel and the culture in the culture vessel by a stirring means for a desired time, and stirring of the stirring means. Stopping the culture in the contents for a desired time at which the concentration of the culture at the desired place in the culture vessel is below the desired concentration, and And a third step of supplying a desired amount of culture medium into the culture vessel by the supply means while discharging the contents in the culture vessel of the desired amount out of the culture vessel by the discharge means. And the fourth step of repeating the third step a desired number of times.

  The medium replacement method in the culture vessel of the present invention comprises a first step of stirring the contents of the culture vessel and the culture in the culture vessel by a stirring means for a desired time, and stirring of the stirring means. Until the concentration detection device detects the concentration of the culture at the desired position in the content until the concentration of the culture at the desired position is detected to be less than or equal to the desired concentration. A second step of precipitating the medium culture, a third step of discharging the contents in the culture vessel above the desired location in a desired amount by a discharging means, and the culture step; It is characterized by comprising a fourth step of supplying a desired amount of culture medium into the container by a supplying means, and a fifth step of repeating the first step to the fourth step a desired number of times.

  The medium replacement method in the culture vessel of the present invention comprises a first step of stirring the contents of the culture vessel and the culture in the culture vessel by a stirring means for a desired time, and stirring of the stirring means. Until the concentration detection device detects the concentration of the culture at the desired position in the content until the concentration of the culture at the desired position is detected to be less than or equal to the desired concentration. A second step of precipitating the culture in the medium, discharging the contents in the culture vessel above the desired location in a desired amount by a discharge means to the outside of the culture vessel and discharging the medium into the culture vessel The method is characterized by comprising a third step of supplying a desired amount by the supply means, and a fourth step of repeating the first step to the third step a desired number of times.

  A first step of stirring the contents of the culture vessel and the culture medium in the culture vessel for a desired time by a stirring means, and the inside of the culture vessel provided with one room and the other provided in the culture vessel A second division that moves the culture to the other side by moving the membrane through which the culture does not pass but the culture medium passes from the one side to the other side; A desired amount of the contents in the culture vessel of the one chamber from the membrane, a third step of discharging the contents out of the culture vessel by the discharging means, and supplying a desired amount of culture medium into the culture vessel It is characterized by comprising a fourth step of supplying by means and a fifth step of repeating the first step to the fourth step a desired number of times.

  The medium replacement method in the culture vessel of the present invention comprises the first step of stirring the contents of the culture in the culture vessel and the culture medium in the culture vessel by a stirring means for a desired time, and in the culture vessel. The second step of settling the culture by moving downward the settling membrane through which the culture does not pass but the culture medium passes, and the inside of the culture vessel above the settling membrane A third step of discharging the contents of the container to the outside of the culture vessel by a desired amount and discharge means, a fourth step of supplying a desired amount of culture medium into the culture vessel by a supply means, and the first step It is characterized by comprising a fifth step of repeating the fourth step from the step to a desired number of times.

  The medium replacement method in the culture vessel of the present invention comprises the first step of stirring the contents of the culture in the culture vessel and the culture medium in the culture vessel by a stirring means for a desired time, and in the culture vessel. The provided culture vessel is divided into one room and the other room, the culture medium does not pass, but the culture medium is moved by moving the passing membrane from the one side to the other side. A second step of moving the culture to the other side, discharging the contents of the one chamber from the membrane by a desired amount, discharging the medium from the culture vessel by the discharging means, and The method is characterized by comprising a third step of supplying a desired amount by the supply means, and a fourth step of repeating the first step to the third step a desired number of times.

  The medium replacement method in the culture vessel of the present invention comprises the first step of stirring the contents of the culture in the culture vessel and the culture medium in the culture vessel by a stirring means for a desired time, and in the culture vessel. The second step of settling the culture by moving downward the settling membrane through which the culture does not pass but the culture medium passes, and the inside of the culture vessel above the settling membrane And a third step of supplying a desired amount of culture medium into the culture vessel by the supply means, and discharging the contents of the desired amount into the culture vessel by the desired means, and the first step to the first step. The third step is characterized by comprising a fourth step of repeating the desired number of times.

  According to the present invention, it is not necessary to provide a filtration membrane in the discharge pipe, and culture medium can be continuously exchanged.

BRIEF DESCRIPTION OF THE DRAWINGS It is a vertical front view of the stirring culture apparatus of Example 1 of this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a vertical front view of the stirring culture apparatus of Example 1 of this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a vertical front view of the stirring culture apparatus of Example 1 of this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a vertical front view of the stirring culture apparatus of Example 1 of this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a vertical front view of the stirring culture apparatus of Example 1 of this invention. It is a longitudinal front view of the stirring culture apparatus of Example 2 of this invention. It is a longitudinal front view of the stirring culture apparatus of Example 2 of this invention. It is a longitudinal front view of the stirring culture apparatus of Example 2 of this invention. It is a longitudinal front view of the stirring culture apparatus of Example 2 of this invention. It is a longitudinal front view of the stirring culture apparatus of Example 3 of this invention. It is a longitudinal front view of the stirring culture apparatus of Example 3 of this invention. It is a longitudinal front view of the stirring culture apparatus of Example 3 of this invention.

  Examples of modes for carrying out the present invention will be shown below.

  A first embodiment of the present invention will be described with reference to FIGS.

  Reference numeral 1 denotes a spinner culture apparatus of the present invention, and 2 denotes a culture vessel of the spinner culture apparatus 1, and the culture vessel 2 includes, for example, cultures of iPS cells, cells of plants and animals, and microbial cells, Contents consisting of a medium such as a culture solution can be introduced.

  If necessary, a particulate carrier (microcarrier) having a culture surface to which cells and the like can be attached and proliferated may be contained.

  Reference numeral 3 denotes a drive shaft. The drive shaft 3 penetrates the central opening 4 of the ceiling 2 a of the culture container 2 and is vertically inserted into the culture container 2.

  Further, the drive shaft 3 is vertically movably supported by a support portion 5 such as a drive shaft seal portion or a thrust bearing portion provided in the opening 4, and the ceiling at the upper end portion of the drive shaft 3 is It was connected to a reciprocating drive 6 provided above the portion 2 a and moved up and down by driving the reciprocating drive 6.

  Reference numeral 7 denotes a stirring blade. The stirring blade 7 is formed, for example, in a circular or elliptical flat plate shape, and is provided at, for example, the lower end of the drive shaft 3 in the culture vessel 2.

  The stirring blade 7 may be provided in two or more stages other than one stage on the drive shaft 3, and the position where the stirring blade 7 is fixed to the drive shaft 3 is not the lower end but an intermediate portion There is no particular limitation.

  Further, the stirring blade 7 has various shapes including a three-dimensional shape such as an oval, a rectangle, a cross, and a cone, in addition to a circle and an ellipse. Moreover, it is good also as a bending structure which made one part or all part of this stirring blade 7 incline at arbitrary places.

  The reference numeral 8 denotes a supply pipe as a supply means for supplying contents such as a culture and a medium into the culture vessel 2, and the supply pipe 8 may be inserted into the culture vessel 2 from the outside, for example, The tip opening is inserted through the ceiling 2 a to near the bottom in the culture vessel 2.

  Further, the supply means is provided with a supply device (not shown) for supplying a culture, a culture medium and the like into the culture container 2, and the supply device is provided, for example, in the supply pipe 8. It comprises the on-off valve (not shown) and a supply pipe side pump unit (not shown) for supplying a culture, a culture medium and the like into the culture vessel 2, and the on-off valve as required. As a result, the supply pipe side pump unit supplies the culture, the culture medium, and the like into the culture vessel 2 through the supply pipe 8.

  Note that only the medium from which the culture has been removed may be supplied from the supply pipe 8 into the culture vessel 2.

  Further, 9 stops the culture medium such as cells in the contents in the culture vessel 2 by stopping the stirring blade 7 (reciprocating driving device 6), and the desired position (height of the contents) is obtained. And a drain pipe as a draining means for draining the contents hardly containing the culture above the desired position after the desired time when the culture is hardly contained in the above, and the drain pipe 9 is From the outside of the culture vessel 2 to inside, for example, penetrating the ceiling portion 2a, the tip opening thereof is almost above the desired position in the contents in the culture vessel 2 and the culture is almost It is provided at a position not included.

  The term “content hardly containing the culture” means contents whose concentration of the culture such as cells contained in the content is less than a desired concentration, and the desired concentration is the content of the culture. It is determined by the desired conditions such as the type, the type of medium, and the purpose of culture.

  In addition, the desired time is set, for example, in advance by a time obtained by experiment, simulation or the like.

  In addition, since the culture has a higher specific gravity than the medium, the concentration of the culture gradually increases from the liquid level of the contents toward the bottom. Therefore, it is preferable to provide the position of the distal end opening of the discharge pipe 9 at the desired position.

  The amount of sedimentation of the culture can be increased by lengthening the stopping time of the agitating blade 7 driven to reciprocate up and down by the reciprocating drive device 6, and thus the desired position, ie, the position of the tip opening of the discharge pipe Can be made deeper from the liquid surface of the contents to increase the discharge amount from the discharge pipe 9, but if the time for stopping the stirring blade 7 is long, the whole extraction efficiency will fall, so stirring The stopping time of the blade 7 and the depth (position) of the tip opening are determined in consideration of the entire extraction efficiency, and for example, the position of the tip opening is the amplitude of the stirring blade 7. It is provided at the position of the lower limit or the position of the amplitude center of the stirring blade 7, or the position of the middle height of the contents received, or near the liquid level of the contents received.

  Further, the discharge means is provided with a discharge device (not shown) for discharging the contents in the culture container 2 out of the culture container 2, and the discharge device is provided, for example, in the discharge pipe 9. The discharge pipe 9 is composed of an open / close valve (not shown) and a discharge pipe side pump portion (not shown) for discharging the contents in the culture container 2 out of the culture container. Etc. does not have a filtration membrane (filter) that does not pass through the culture. Then, if necessary, the on-off valve is opened, and the contents in the culture vessel 2 are extracted out of the culture vessel through the discharge pipe 9 by the discharge pipe side pump unit.

  In the above embodiment, an example of a stirring blade reciprocating up and down is given as a means for stirring the contents, but a stirring means for stirring contents of other various types such as a rotary type rotary wing etc. It may be.

  Next, the operation of the stirred culture apparatus of the present invention and the effects thereof will be described.

  In the stirred culture apparatus 1 of the present invention, first, the contents containing the culture such as cells having a specific gravity greater than that of the culture medium and the culture medium (mother liquor) such as the culture solution in the culture vessel 2 by the supply means. The substance to be stirred is allowed to be filled up to the desired height of the culture vessel 2 (the desired amount).

  That is, for example, with the on-off valve of the supply pipe 8 open, the content including the culture and the culture medium from the supply pipe 8 to the desired height of the culture container 2 by the supply pipe side pump unit The amount of

  In addition, you may make it accommodate a microcarrier as needed.

  Then, the supply means is stopped, that is, for example, the on-off valve of the supply pipe 8 is closed and the supply pipe-side pump is stopped, and as shown in FIG. Reciprocate the stirring blade 7 up and down by reciprocating it up and down by 6, etc. The contents (to-be-stirred material) in the culture vessel are stirred for a desired time by the stirring means, etc. Make things uniform (stirring step).

  Next, the agitation of the agitation means is stopped, for example, by stopping the agitation blade 7 (reciprocating driving device 6), and as shown in FIG. 2, the culture such as cells is allowed to settle for the desired time. (Settling process).

The desired time is, as described above, the time at which the concentration of the culture at the desired position is less than or equal to the desired concentration, and the desired time is the desired position, the desired time, or the desired time. It changes with conditions, such as the density | concentration of a culture medium discharge amount, etc., for example, it determines beforehand by experiment, simulation, etc.
It is determined.

  And, the specific gravity of the culture of the cells is larger than that of the mother liquor of the medium and so on, and therefore, the sedimentation of the culture of the cells and the like is relatively fast. After the lapse of time, the upper part of the culture vessel becomes only the liquid phase (medium) with almost no culture.

  Next, a desired amount of culture medium in the culture vessel 2 is extracted by the discharge means (medium extraction step). The stirring blade 7 (stirring of the stirring means) is kept stopped.

  That is, for example, while opening the on-off valve of the discharge pipe 9 and driving the discharge pipe side pump portion, as shown in FIG. 3, a desired amount of the culture medium in the culture vessel 2 is extracted Do.

  After extracting a desired amount of culture medium, the discharge means is stopped, that is, for example, the on-off valve of the discharge pipe 9 is closed and the discharge pipe side pump unit is stopped, as shown in FIG. By means, ie, for example, from the supply tube 8, a fresh medium is allowed to be filled to the desired height (the desired amount) in the culture vessel 2 (medium supply step).

  Then, the on-off valve of the supply pipe 8 is closed and the supply pipe side pump unit is stopped, and as shown in FIG. 5, the drive shaft 3 is reciprocated in the vertical direction by the reciprocating drive device 6 again. By moving the stirring blade 7 up and down by moving it, etc., the contents (to-be-stirred material) in the culture container are stirred for a desired time by the stirring means, and the contents become uniform. (Stirring step), and the settling step, the medium extracting step, and the medium feeding step are sequentially performed.

  Then, by repeating the stirring step, the settling step, the medium extracting step, and the medium supplying step a desired number of times, continuous culture without liquid level fluctuation without backwashing is performed. Will be able to do.

  In some cases, during culture, fresh medium may be supplied.

  According to the present invention, it is possible to continuously change the culture medium without providing a filtration membrane in the discharge pipe and without using a complicated culture medium separation system.

  Moreover, there is no liquid level fluctuation, and continuous culture becomes possible.

  In the second embodiment of the present invention, as shown in FIG. 6, in the stirring and culturing apparatus 1 of the first embodiment, stirring is performed to control stirring and stopping of the stirring means such as up and down reciprocation of the stirring blade 7 An operation control apparatus 10 is provided which includes a control unit 10a, a supply control unit 10b for controlling the supply means, a discharge control unit 10c for controlling the discharge means, a storage unit 10d, and an operation unit 10e for performing various calculations. .

  Further, a desired time for stirring the contents in the stirring step is stored in the storage unit 10d, and a desired time for settling the culture in the sedimentation step is stored, and the medium is removed. The desired amount of extracting the culture medium in the step is stored, and the desired amount of supplying the culture medium in the medium supplying step is stored, and the stirring step, the settling step, the medium extracting step and the medium supply are also stored. The number of times of repeating the process and the like are stored.

  In addition, the storage unit 10d includes the amplitude, the frequency, and the number of revolutions of the stirring means such as the stirring blade 7 controlled by the stirring control unit 10a, and the supply pipe side pump unit 15 controlled by the supply control unit 10b. The various control conditions such as the supply speed of the pump, the discharge speed of the discharge pipe side pump unit 16 controlled by the discharge control unit 10c, and the like are stored.

  Further, the computing unit 10e is provided with, for example, a timer (clock) and discharge operation control means for controlling the operation of the discharge means when a desired time stored by the timer has elapsed.

  According to the second embodiment of the present invention, in the stirring step in the first embodiment, the stirring control unit 10a of the operation control apparatus 10 follows the amplitude, the frequency and the stirring time stored in the storage unit 10d. The contents are stirred by a stirring means such as the stirring blade 7, and in the settling step, the culture such as cells is allowed to settle for a settling time stored in the storage unit 10d, and the medium extracting step The culture medium is extracted by the discharge pipe side pump unit according to the discharge speed and discharge amount stored in the storage unit 10d, and the supply speed and supply stored in the storage unit 10d in the culture medium supply step. According to the amount, fresh medium is supplied by the supply pipe side pump unit, and the number of times stored in the storage unit 10d is equal to the number of the stirring and the settling steps. It repeats the said medium supplying step and the medium extraction step.

  In the second embodiment of the present invention, culture and culture medium exchange can be performed automatically.

  In the settling step, the storage unit 10d stores a desired time for settling the culture, and according to this time, the stirring blade 7 (stirring of the stirring means) is stopped to shift to the culture medium extracting step. Instead, a detection device for detecting concentration information of a desired position in the culture vessel 2, for example, concentration information of a culture at a height at which the tip opening of the discharge pipe 9 is located, is used in the spinner culture apparatus 1. Provided so that concentration information from the detection device can be obtained by the calculation unit 10e of the operation control apparatus 10, and the calculation unit 10e calculates concentration if necessary from the concentration information, and the calculation unit The medium discharging step may be performed by controlling the discharging means when the concentration becomes lower than the desired concentration by the discharging operation control means provided in 10e.

  As the density detector, for example, as shown in FIG. 7, a camera 11 may be provided to detect the density from image information (density information) obtained by the camera.

  Further, as shown in FIG. 8, the concentration sensor 12 outside the tank may be provided to detect the concentration in the culture container from the outside of the culture container.

  Further, as shown in FIG. 9, an in-tank concentration sensor 13 may be provided to detect the concentration in the culture vessel.

  In the third embodiment of the present invention, in the stirred culture apparatus of the first embodiment and the second embodiment, the inside of the culture container 2 is divided into the upper and lower portions of the culture container 2 and the inside of the culture container 2 is It is possible to provide a sedimentation membrane 14 which does not pass through the culture but passes through the culture medium.

  In addition, the sedimentation membrane 14 has holes which respectively penetrate the drive shaft 3, the discharge pipe 9 and the supply pipe 8, and the discharge pipe 9 and the supply pipe 8 in the culture vessel 2. The desired portion of the lower portion is vertically extended, and the sedimentation membrane 14 is configured to be slidably movable up and down in the culture vessel 2.

  Further, between the sedimentation film 14 and the inner circumferential surface of the culture vessel 2 and between each hole of the sedimentation film 14, the drive shaft 3, the supply pipe 8, and the discharge pipe 9, Each is formed to such an extent that cultures such as cells do not pass through.

  According to the third embodiment of the present invention, as shown in FIG. 10, after the sedimentation film 14 is positioned above the liquid level of the contents and the stirring process is performed, the first embodiment is performed. As in the second embodiment, instead of waiting for the culture to settle naturally, as shown in FIG. 11, for example, the drainage together with the sedimentation membrane 14 and the stirring blade 7 is carried out. It is lowered below the tip opening of the tube 9 and the settling film 14 forcibly lowers stirring materials such as cells.

  As a result, there is almost no culture above the sedimentation membrane and only the culture medium, and only the culture medium above the sedimentation membrane 14 is discharged by the discharge pipe 9.

  According to the third embodiment of the present invention, the culture can be lowered in a short time.

  The sedimentation film 14 may be separately provided with a vertical movement device to be vertically moved.

  Further, when the settling film 14 and the stirring blade 7 can be fixed by a fixing means (not shown), for example, when the settling film 14 is moved up and down, it is located above the liquid surface The stirring blade 7 is raised to the lower end of a certain settling film 14, and both are connected and fixed by the fixing means, and then the stirring film 7 is moved downward, the settling film 14 may also be lowered.

  When the sedimentation film 14 is not moved up and down, the fixing means of the fixing means may be released.

  In the above embodiment, the sedimentation membrane 14 divides the inside of the culture vessel 2 into upper and lower parts, but for example, the inside of the culture vessel 2 is divided in the lateral direction so as to be movable in the lateral direction, etc. The inside of the culture vessel 2 may be divided into one chamber and the other chamber so that it can be moved from the one side to the other side or from the other side to the one side.

  The stirred culture apparatus of the present invention is used for a culture apparatus for medical products, food, etc.

DESCRIPTION OF SYMBOLS 1 stirring culture apparatus 2 culture container 2a ceiling part 3 driving shaft 4 opening part 5 support part 6 reciprocating drive device 7 stirring blade 8 supply pipe 9 discharge pipe 10 operation control apparatus 10a stirring control part 10b supply control part 10c discharge control part 10d memory Unit 10e Arithmetic unit 11 Camera 12 Tank outside concentration sensor 13 Tank inside concentration sensor 14 Sedimentation film 15 Supply pipe side pump part 16 Discharge pipe side pump part

Claims (3)

A culture vessel in which contents consisting of a culture and a medium are placed;
Provided in the culture container, the partition the culture vessel in one room and the other rooms, and move possible film toward the other from the one,
Stirring means provided in the culture vessel for stirring the contents;
Discharging means for discharging the contents in the one room out of the culture vessel;
And means for supplying a medium into the culture vessel,
A stirred culture apparatus characterized in that the membrane does not pass the culture but passes the culture medium. The culture vessel is divided into one room and the other room, the culture does not pass through, but the medium passes through the membrane on one side, and the contents consisting of the culture in the culture vessel and the medium are A first step of stirring by a stirring means for a desired time;
Said film by the from one side of it is moved toward the side of the other, a second step of moving the culture on the side of the other,
A third step of discharging the contents of the one chamber out of the culture vessel by a desired amount and discharging means;
Supplying a desired amount of culture medium into the culture vessel by a supplying means;
A medium replacement method in a culture vessel, comprising: a fifth step of repeating the first step to the fourth step a desired number of times. The culture vessel is divided into one room and the other room, the culture does not pass through, but the medium passes through the membrane on one side, and the contents consisting of the culture in the culture vessel and the medium are A first step of stirring by a stirring means for a desired time;
Said film by the from one side of it is moved toward the side of the other, a second step of moving the culture on the side of the other,
Desired amount of the contents of the one room, the discharging means, the discharged out of the culture vessel,
Supplying a desired amount of culture medium into the culture vessel by a supply means;
A medium replacement method in a culture vessel, comprising: a fourth step of repeating the first step to the third step a desired number of times.

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