JPH037575A - Apparatus for cell culture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a cell culture device, and particularly to a device for culturing cells using a culture bag containing cells and a culture medium.

[Conventional technology and its problems]

For example, LAK or CTL (Cyto Toxic Lymphocytosis) is used as a treatment for cancer patients.
te) therapy is known. This treatment method involves removing lymphocytes from cancer patients, stimulating them outside the body to give them activity, obtaining lymphocytes that act specifically against cancer, and treating cancer with these lymphocytes. be.

When culturing LAK or CTL described above, a basic medium containing low molecules such as glucose and inorganic substances, serum, and IL are used.
2 and a cancer antigen. In this case, the basic medium is inexpensive, but serum and IL2 are extremely expensive. It is therefore desirable to use the latter expensive substances as efficiently as possible in LAK or CTL therapy.

Therefore, as a device used for culturing lymphocytes, the first
A batch culture apparatus as shown in FIG. 4 is already known. In FIG. 14, a culture bag stand 2 that rotates in the direction of the arrow is arranged in a culture chamber 1 whose temperature is controlled to 37°C. The culture bag stand 2 is driven by a rotation mechanism (not shown). Further, a culture bag 3 is attached to the culture bag stand 2, and as the stand 2 rotates, the liquid in the culture bag 3 is stirred. The culture bag 3 includes an outer resin bag 4,
A diaphragm bag 5 housed within a resin bag 4 is provided. A basic culture medium is stored in the resin bag 4, and the basic culture medium in the resin bag 4 can be exchanged through exchange ports 6 provided on one side of the resin bag 4. The diaphragm bag 5 contains serum and IL2 in addition to lymphocytes (T cells) or TILs. Note that the basic medium can pass through the diaphragm that constitutes the diaphragm bag 5.

In the batch type cell culture apparatus shown in FIG. 14, as the culture time passes, the basic medium changes in quality, such as glucose in the basic medium changing to lactic acid and the pH decreasing. Therefore, 3
It is necessary to temporarily stop the culture and replace the basic medium in the culture bag 3 through the replacement port 6 every ~4 days. However, during this replacement, there is a risk that germs may enter the culture bag 3. Therefore, a large number of exchange ports 6 are provided in the culture bag 3 so that the exchange ports that have been used twice are not used again, but there is still a possibility that bacteria may enter when replacing the basic medium.

An object of the present invention is to provide a cell culture device that saves the effort of replacing the culture solution and eliminates the risk of contamination during replacement.

[Means to solve the problem]

The cell culture device according to the first invention is a device for culturing cells using a culture bag containing cells and a culture solution. This device includes a culture bag stand for fixing the culture bag, a culture bag stand that is tilted and supported, and
It includes a rocking mechanism for swinging the culture bag stand around the vertical axis while preventing rotation, and a culture solution exchange mechanism connected to the culture bag and for replacing the culture solution in the culture bag.

A cell culture device according to a second invention is the cell culture device according to the first invention, in which the culture solution exchange mechanism replaces the culture solution in synchronization with the rocking by the rocking mechanism.

A cell culture device according to a third aspect of the invention is a device for culturing cells using a flexible culture bag containing cells and a culture solution. This device includes a bag fixing part for fixing the culture bag, and a roller part that rolls in pressure contact with the culture bag.

[Effect]

In the cell culture device according to the first aspect of the invention, the culture bag stand is supported in an inclined state by the swinging mechanism, and is also allowed to swing around the vertical axis without rotating. Therefore, the culture bag fixed to the culture bag stand is also similarly swung around the vertical axis. As a result, the culture solution contained in the culture bag is stirred.

In this case, since the culture bag rocks but does not rotate, the culture solution exchange mechanism can be easily connected to the culture bag. Therefore, according to the present invention, the trouble of replacing the culture solution can be saved, and the fear of contamination at the time of replacement can be eliminated.

In the second invention, the culture solution exchange mechanism replaces the culture solution in synchronization with the rocking by the rocking mechanism.

On the other hand, a gas such as sterile air may be simultaneously sealed in the culture bag in order to increase the efficiency of stirring the culture solution. In this case, if the culture solution exchange mechanism is constantly operated, the culture solution exchange mechanism will discharge the gas inside the culture bag to the outside when the connection part of the culture bag to the culture solution exchange mechanism is placed upward. It will be put away. In the second invention, since the culture solution exchange mechanism is synchronized with the rocking motion by the rocking mechanism, the culture solution can be set to be replaced only when the connection part of the culture bag to the culture solution exchange mechanism is placed downward. If so, such problems will no longer occur.

In the cell culture device according to the third aspect of the invention, the roller portion rolls in pressure contact with the culture bag. As a result, the culture bag is deformed and the culture solution inside the culture bag is stirred.

Also in this case, since there is no need to rotate the culture bag, the culture solution exchange mechanism can be easily connected to the culture bag.

Therefore, according to the present invention, the trouble of replacing the culture solution can be saved, and the fear of contamination at the time of replacement can be eliminated.

〔Example〕

FIG. 1 shows an outline of the cell culture apparatus according to the present invention. This cell culture device includes a culture chamber 11, a culture medium adjustment tank 12 arranged adjacent to the culture chamber 11, a fresh medium tank 13 connected to the culture medium adjustment tank, and a culture medium tank 13 connected to the culture solution discharge side of the culture chamber 11. It mainly has a recovery medium tank 14.

The culture chamber 11 is of a lean bench type in which the temperature can be controlled, and the temperature inside the culture section 16 is set to 37° C., for example. Purified air that has passed through a filter (not shown) is introduced into the culture section 16 under a slightly high pressure state, thereby maintaining the inside of the culture section 16 in a sterile state. In addition, the culture chamber 11 is equipped with a rocking device (described in detail later).
, so that a plurality of culture bags 17 arranged within the culture section 16 can be swung.

As shown in FIGS. 2 and 3, the culture bag 17 includes a bag-shaped resin bag 18 made of flexible resin and a diaphragm bag 19 housed within the resin bag 18. One side of the resin bag 18 has a culture medium inlet 20 in its center.
and a culture medium outlet 21 are formed. Inside the bag 18, both ends of the medium inlet 20 and the medium outlet 21 are
They are bent in directions away from each other, and an opening is provided at the tip. As a result, the liquid medium introduced from the medium inlet 20 and discharged from the medium outlet 21 can be sufficiently circulated within the resin bag 18. In addition, a pair of strings 22 are fixed to both ends of the diaphragm bag 19 in the longitudinal direction, as shown in FIG. The tip of the string 22 is fixed to the resin bag 18 side, so that the diaphragm bag 19 is arranged approximately at the center within the resin bag 18. The material of the diaphragm bag 19 may be a membrane membrane depending on the diaphragm conditions.

Ultrafiltration membranes, reverse osmosis membranes, etc. can be used as appropriate.

In FIG. 1, the medium adjustment 1112 includes a medium inlet pipe 23 connected to the medium inlet 20 of each culture bag 17.
is connected. A handling pump 24 is installed in the middle of each medium introduction pipe 23 outside the culture chamber 11, and the handling pump 24 controls the medium adjustment tank 12.
The culture solution is conveyed from the culture bag 17 to the culture bag 17. A basic medium 25 to be supplied to the culture bag 17 is stored in the medium adjustment tank 12 . In order to detect the state of the basic medium 25, the medium adjustment tank 12 is provided with various sensors such as a temperature sensor, a PH sensor, a DO sensor, and a glucose sensor (not shown). A fresh culture medium tank 13 is also connected to the culture medium adjustment tank 12 via a pipe 30. The pipe 30 has a handling pump 31 in the middle thereof, so that fresh basal medium can be supplied from the fresh medium tank 13 to the medium adjustment tank 12.

The recovery medium tank 14 is connected to medium discharge vibrators 32 which are respectively connected to the medium discharge ports 21 of the culture bag 17 . The handling pump 24 is provided in the middle of the medium discharge vibrator 32, which allows the culture bag 1 to be removed.
7 to the recovery medium tank 14 .

In addition, each pair of culture medium introduction pipe 23 and culture medium discharge vibrator 32
A common handling pump 24 is arranged for both. Therefore, if the handling pump 24 is operated, the culture bag 1
The same amount of basal medium can be introduced and discharged at once into the cell.

Next, the culture bag rocking mechanism 3 provided in the culture chamber 11
9 will be explained.

As shown in FIG. 2, on the floor surface 4o of the culture section 16, there is a portion of the zero-rotation shaft 41 that protrudes downward from the floor surface 40, on which a rotary shaft 41 tilted at an angle θ is rotatably supported. for,
A bevel gear 42 is fixed. A bevel gear 44 fixed to the output shaft of a motor 43 meshes with the bevel gear 42 . A cam 45 as shown in FIG. 4 is fixed to the rotating shaft 41 below the bevel gear 42. As shown in FIG. cam 4
5 has an outer circumferential surface 46 formed concentrically with the rotating shaft 41. A notch 47 recessed toward the inner circumferential side is formed in a portion of the outer circumferential surface 46 . Further, a terminal 49 of a microswitch 48 is in contact with the outer circumferential surface 46, and the terminal 49 is always elastically in contact with the outer circumferential surface 46 including the notch 47.

Note that the microswitch 48 is set to be in a connected state only while the terminal 49 is disposed within the notch 47 as shown in FIG.

The upper end portion of the rotating shaft 41 is bent into a crank shape and serves as a culture bag stand support portion 50 . The center line Ot Ot of the support portion 50 is the rotation center Ox Ox of the rotating shaft 41
It is tilted by an angle θ with respect to the

A culture bag stand 52 is rotatably supported on the outer circumference of the upper end of the support part 50 via a ball bearing 51.

As shown in FIG. 3, culture bag fixing members 53 are provided at the corners of the upper end surface of the culture bag stand 52, respectively. The culture bag 17 is placed on the culture bag stand 52, and the corners of the culture bag 17 are fixed to the culture bag stand 52 by fixing members 53.

Note that the culture medium inlet 20 and culture medium outlet 21 of the culture bag 17 are arranged on the right side in FIG. 2.

On the other hand, a bracket 54 that projects downward is provided at the lower end of the right side of the culture bag stand 52.

A rotation prevention mechanism 55 for preventing rotation of the culture bag stand 520 is connected to the bracket 54. The zero rotation prevention mechanism 55 is connected to the bracket 54 via a horizontal pin 56, as shown in FIG. Lever 5 rotatably supported
7 and a pair of guide plates 58 fixed to the floor surface 40. A guide plate 5 is attached to the lower end of the lever 57.
A sliding member 59 is provided which extends in the lateral direction and is sandwiched between the parts 8 and 8. At the position corresponding to the lever 57 of the guide plate 58,
A notch 60 is provided, whereby the sliding member 5
The rotation of the lever 57 about the pivot point 9 is not blocked by the guide plate 58.

Next, how to use the above embodiment and its operation will be explained.

In the state shown in FIG. 1, there are four culture bags 17 in the culture chamber 11.
are arranged. The diaphragm bag 19 of the culture bag 17 stores, for example, T cells (lymphocytes) from a cancer patient, expensive serum, IL2, and the like. On the other hand, the medium inlet 20
A basal medium is introduced into the resin bag 18 from the base medium. The basal medium introduced into the resin bag 18 passes through the septum of the septum bag 19 and enters the bag 19, where it is used as nutrients for cell culture. Further, waste substances generated within the diaphragm bag 19 are discharged into the diaphragm bag 19 through the partition wall. The exchange of substances through this diaphragm takes place on the basis of diffusion phenomena due to concentration gradients. The basic medium in the resin bag 18 is discharged into the recovery medium tank 14 through the medium outlet 21.

On the other hand, in the culture medium adjustment tank 12, the state of the basic culture medium is controlled based on detection results from various sensors. In addition, fresh basic culture medium is introduced into the culture medium adjustment tank 12 from the fresh culture medium tank 13 as needed. The basal medium controlled in a predetermined state is introduced into the culture bag 17 through the pipe 23.

In this way, the basic medium in the culture bag 17 is automatically circulated, so there is no need to manually replace the basic medium in the culture bag 17 (in addition, there is no need to manually replace the basic medium). There will be no fear.

On the other hand, since only the inexpensive basic medium is circulated and expensive IL2 and the like are confined in the diaphragm bag 19, loss of expensive substances can be prevented. Furthermore, since a large number of culture bags 17 can be used at once without providing many culture medium adjustment tanks 12, etc., culture efficiency is increased. Furthermore, by placing lymphocytes of different cancer patients in each culture bag 17 and culturing them, it is possible to treat a large number of patients at the same time.

Next, the rocking operation of the culture bag 17 by the rocking mechanism 39 within the culture chamber 11 will be explained.

When the motor 43 rotates, the rotating shaft 41 rotates.

At this time, since the support part 50 is tilted with respect to the rotation axis 41, the culture bag stand 52 swings about the intersection of the center lines of both parts. At this time, the lever 57 is driven in response to the rocking of the culture bag stand 52, but the lever 57 is maintained within the guide plate 58. As a result, even if the rotating shaft 41 rotates, the culture bag stand 52 does not rotate, so the culture bag stand 52 rotates as the rotating shaft 41 rotates.
will only repeat the oscillation.

On the other hand, when the rotating shaft 41 rotates, the cam 45 also rotates, so the micro switch 48 turns ON/OFF according to the rotation.
F. The terminal 49 of the micro switch 48 is in the notch 47
When the microswitch 48 is turned on, the handling pump 24 is driven by a control device (not shown). As a result, a fresh medium is introduced into the resin bag 18 through the pipe 23, and an old medium is discharged through the medium discharge pipe 32. At this time, as shown in FIG. 3, since the tips of the culture medium inlet 2o and the culture medium outlet 21 are bent in the direction away from each other, the culture medium can be exchanged efficiently within the resin bag 18. When this handling pump 24 operates, the culture bag stand 520 port 20.2
The first side is placed downward. Therefore, when the gas portion in the culture bag 17 is on the side of the medium inlet 20 and the medium outlet 21, the handling pump 24 will not operate. That is, the microswitch 48 is turned on.
- Since the handling pump 24 operates in synchronization with OFF, it is controlled so that only the liquid medium in the culture bag 17 circulates.

It is also possible to adopt the structure shown in FIGS. 6 and 7 as the pinwheel rotation prevention mechanism 55.

In the figure, a pair of brackets 61 and 62 are provided at the lower end of the culture bag stand 52 and at the upper end of the floor surface 40, respectively. The bracket 61 of the culture bag stand 52 is provided with a first via a bin 63 in the radial direction of the culture bag stand 52.
A link member 64 is rotatably supported. One end of the second link member 66 is rotatably supported at the lower end of the first link member 64 via a bin 65 that is perpendicular to the bin 63 in the lateral direction. At the lower end of the second link member 66,
A third link member 67 is rotatably supported via a pin 68. The lower end of the third link member 67
It is rotatably supported by a bracket 62 via a pin 69 parallel to the bracket 5.

Also with this configuration, rotation of the culture bag stand 52 can be prevented while allowing the culture bag stand 52 to swing.

S1111 Instead of the swing mechanism according to the above-described embodiment, a swing mechanism 70 shown in FIGS. 8 and 9 may be employed.

In the figure, a rotating shaft 7 rotatably supported on a floor surface 40
1, a bevel gear 42 and a cam 45 are fixed as in the embodiment shown in FIG. 2, and a bevel gear 44 of a motor 43 meshes with the bevel gear 42. Further, a microswitch 48 is in contact with the cam 45. Note that the rotating shaft 71
The lower end is rotatably supported by a support plate 72 via a pole bearing 73. An inclined plate 74 inclined with respect to the center line of the rotating shaft 71 is integrally provided on the upper part of the rotating shaft 71. The lower ends of four bush rods 75 extending in the vertical direction are in contact with the upper end surface of the inclined plate 74. The bushing rod 75 passes through the floor surface 40 and projects upward. Further, the bushing rod 75 is supported on the floor surface 40 so as to be freely slidable in the vertical direction. On the floor surface 40,
A culture bag stand 77 is supported via a ball joint 76 located concentrically with the rotating shaft 71 . Culture bag stand 77
The upper ends of the bushing rods 75 are in contact with the lower end surfaces of the bushing rods 75, respectively. Further, a spring 78 in a tensioned state is arranged between the lower end surface of the culture bag stand 77 and the floor surface 40. As a result, the culture bag stand 77 is always elastically biased downward.

In this case, when the motor 43 rotates, the inclined plate 74 rotates, and the bushing rod 75 moves up and down according to the inclination of the inclined plate 74. This provides a rocking motion of the culture bag stand 77. Note that the rotation of the culture bag stand 77 is as follows:
This is prevented by a spring 78. On the other hand, as in the embodiment described above, since the handling pump 24 is driven in a synchronous state depending on whether the microswitch 48 is turned on or off, only the liquid medium in the culture bag 17 is circulated.

Cliff Base N Instead of the swing mechanism according to the above-described embodiment, a swing mechanism 80 shown in FIG. 10 may be employed.

In FIG. 1O, a rotating shaft 81 has a horizontal disc 82 integrally formed at its upper end.A protrusion 83 that projects upward is integrally formed on the outer circumference of the disc 82. A roller 86 is rotatably supported on the protrusion 83 via a bottle 85 parallel to the direction of inclination of the culture bag stand 84 .

The upper end of the roller 86 is in contact with the lower end surface of the culture bag stand 84, and can roll on the lower end surface. A ball joint 87 is provided on the upper end surface of the disk 82 concentrically with the rotating shaft 81, and the center of the culture bag stand 84 is supported at the upper end of the ball joint 87. A rotation prevention mechanism 55 shown in FIGS. 2 and 5 is provided at the lower side of the culture bag stand 84.

When the rotating shaft 81 is rotated by a motor (not shown),
The roller 86 rolls on the lower surface of the culture bag stand 84, so that the culture bag stand 84 swings.

In this case, the culture bag stand 8 is rotated by the rotation prevention mechanism 55.
4 is prevented from rotating and only swinging is performed.

Further, by employing a roller mechanism 90 as shown in FIG. 11, the stirring efficiency can be improved.

In FIG. 11, the roller mechanism 90
A roller 91 disposed above and in pressure contact with the culture bag 17, a support member 92 that supports the roller 91 from above, and a drive that drives the support member 92 in the lateral direction in FIG. It has a mechanism 93.

As shown in FIG. 12, the support member 92 has a bracket 94 that rotatably supports the roller 91. The upper end center portion of the bracket 94 is rotatably supported by an upper bracket 95 via a support shaft perpendicular to the center line of the roller 91.

The bracket 95 is provided at the lower end of an elevating member 96 having a rectangular cross section. The elevating member 96 is fitted into the guide member 97 so as to be slidable only in the vertical direction.

As shown in FIG. 13, a coil spring 98 is compressed within the guide member 97, and thereby the elevating member 96 is always urged downward.

As shown in FIG. 11, a ball nut 100 is screwed into a laterally extending ball screw 99 provided on the drive mechanism 93. The ball nut 100′ is a support member 92
is fixed to the upper end of the guide member 97. One end of the ball screw 99 is connected to a bearing 10 fixed to a support frame 101.
2 is rotatably supported. The other end of the ball screw 99 is connected to the output shaft of the drive motor 103. The support frame 101 is provided with a guide groove (not shown) along the longitudinal direction of the ball screw 99.
can move in the left-right direction within the guide groove.

In this embodiment, in addition to the rocking motion of the culture bag 17 by the bag rocking mechanism 39, the stirring motion by the roller mechanism 92 is added. When the ball screw 99 is rotated by the drive motor 103, the roller 91 rolls on the culture bag 17 in a pressed state.

This rolling action of the roller 91 stirs the culture medium within the culture bag 17. The stirring effect caused by the rolling of the roller 91 is particularly effective for stirring the culture medium in the diaphragm bag 19 which does not have an air layer.

In addition, the bracket 94 changes to the bracket 95 according to the change in the rocking posture of the culture bag 17 by the bag rocking mechanism 39.
The elevating member 96 also moves up and down relative to the guide member 97. Thereby, the state in which the roller 91 is in pressure contact with the culture bag 17 is maintained at all times, and stirring is performed reliably.

Note that the driving by the drive motor 103 is performed based on timing detection by a combination of a cam 45 and a microswitch 48. The reciprocating motion of the roller 91 is timing-controlled so that the roller suppresses the portion of the diaphragm bag 19 exposed to the air layer within the resin bag 18.

Backyard Team ■ The roller mechanism 90 described above can be similarly employed in other bag swinging mechanisms (eg, bag swinging mechanisms 70, 80). In addition, the bag swinging mechanism is not used, and the roller mechanism 9 is simply
The culture bag 17 may be stirred using only 0.

〔Effect of the invention〕

According to the first invention, since the culture bag stand, the rocking mechanism, and the culture solution exchange mechanism are provided as described above, the effort of replacing the culture solution can be saved and there is no fear of contamination at the time of exchange.

According to the second aspect of the invention, the culture solution exchange mechanism operates in synchronization with the swinging motion by the swinging mechanism, so that the culture solution in the culture bag stand can be efficiently and smoothly circulated.

According to the third aspect of the invention, since the roller portion is provided that rolls in pressure contact with the culture bag, the effort of replacing the culture solution can be saved and the fear of contamination at the time of replacement can be eliminated.

[Brief explanation of drawings]

FIG. 1 is a schematic configuration diagram of an embodiment of the present invention, FIG. 2 is a partially cutaway side view of an embodiment of the swing mechanism, and FIG.
Figure 4 is a cross-sectional view of ■-IV in Figure 2.
The figure is a partial view in the direction of the ■ arrow in Figure 2, Figures 6 and 7 are front and side views of a modified example of the rotation prevention mechanism, and Figure 8 is a partial view of Figure 2 of another embodiment of the swinging mechanism. Corresponding figures, FIG. 9 is a sectional view taken along line IX-IX in FIG. 8, and FIG.
FIG. 11 is a diagram corresponding to FIG. 2 of another embodiment, FIG. 12 is a perspective partial view of the roller mechanism, FIG. 13 is a vertical cross-sectional partial view of the support member, and FIG. 14 1 is a schematic vertical cross-sectional view of a conventional cell culture device. 17... Culture bag, 24... Handling pump, 397
0.80... Rocking mechanism, 45... Cam, 48...
Micro switch, 52, 77.84... Culture bag stand, 90... Roller mechanism, 91... Roller.

Claims (3)

[Claims] (1) A cell culture device for culturing cells using a culture bag containing cells and a culture solution, comprising: a culture bag stand for fixing the culture bag; and an inclined culture bag stand. a rocking mechanism for supporting the culture bag stand and swinging it around a vertical axis while preventing rotation; and a culture solution exchange mechanism connected to the culture bag and for replacing the culture solution in the culture bag. Cell culture equipment equipped with (2) The cell culture device according to claim 1, wherein the culture solution exchange mechanism replaces the culture solution in the culture bag in synchronization with the rocking by the rocking mechanism. (3) A cell culture device for culturing cells using a flexible culture bag containing cells and a culture solution, comprising: a bag fixing part for fixing the culture bag; and a bag fixing part for fixing the culture bag; A cell culture device comprising: a roller portion that rolls in pressure contact with a roller portion;