JPS63248382A - Cell culture apparatus - Google Patents

Abstract

PURPOSE:To obtain a cell culture apparatus capable of aseptically carrying out feed of culture medium, recovery of cell, etc., by putting an inner bag made of semipermeable membrane housing a cell suspension in an outer bag housing the culture medium. CONSTITUTION:The aimed cell culture apparatus is composed of a culture bag obtained by putting an inner bag 2 made of semipermeable membrane for housing a cell suspension in an outer bag 1 for housing a culture medium, a culture medium bag 50 for feeding the medium, pouring equipment 20 for pouring the cell suspension into the inner bag 1, a recovery bag 70 for recovering the cell after cultivation from the inner bag 2, a connecting tube 11 for connecting the culture bag with the medium bag 50 or recovery bag and an equipment 40 for pouring aseptic air into each bag.

Description

[Detailed Description of the Invention] [Industrial Field of Application] The present invention relates to a cell culture device, which is capable of aseptically performing operations such as injecting air into an e-condensed culture bag, exchanging the medium, and collecting cells. It has been made possible.

[Prior art] Conventionally, methods for culturing animal cells have been known, such as the static method and the roller bottle method. It is necessary to do this, but the operation is troublesome. Hollow fiber culture devices are also known, but require a device for circulating external and internal fluids, making the device complex and expensive.

For these reasons, the present applicant previously proposed a new concentration culture device that is completely different in type from the conventional one (Japanese Patent Application No. 198898/1983).

This device consists of an outer bag and an inner bag with a semipermeable membrane, and includes a cell suspension (inner solution) sealed in the inner bag with a semipermeable membrane and a culture medium J'1 in the outer bag.
! ! This system utilizes the diffusion phenomenon caused by the concentration gradient between (external liquids) and performs culture while changing the rotation of the entire container.

[Problems to be solved by the present invention] In order to use the above culture bag, the culture medium must be placed in the outer bag.
manual operation, injection of cell suspension into the semipermeable membrane inner bag,
Operations such as injecting sterile air into these bags and collecting cells after culturing are required.

The present invention aims to provide a culture device that allows such operations to be performed aseptically (although injection of the cell suspension must be performed within a clean bench).

[Means for solving the problem] Undeveloped IJI is produced by storing an inner bag 2 of a semipermeable membrane containing a cell suspension in an outer bag I containing a culture medium, and allowing cells to pass through the permeable membrane. A C-shrinking culture hauc that performs high-density culture of cells by a diffusion phenomenon caused by a concentration gradient between the suspension and the medium, a medium bag 50 that supplies the medium to the outer bag 1, and a cell suspension that is injected into the inner bag 2. An injection device 20, a collection bag 70 for collecting cultured cells in the inner bag 2, and a connecting tube 11 for connecting the concentrated culture pack and the medium bag 50 or the collection bag 70.

It consists of a device 40 for injecting sterile air into the six bags.

[Operation] After connecting the cell suspension MM injector J120 to the mouth 6 of the inner bag 2 and injecting a predetermined amount of cell suspension, connect the sV4 air injection device 40 to inject sterile air into the inner bag 2. After that, the first part 6 is sealed. Also, a culture medium bag 50 is connected to the mouth part 3 of the outer bag 1 via the connecting tube 11b. After injecting this culture medium into the outer bag 1 through the tube Ilb, sterile air is introduced in the same way as the inner bag 2 to seal the opening 3 of the culture bag. The cells in the inner bag 2 are cultured while being rotated.After culturing for a predetermined period of time, a collection haugre 0 is connected to the opening 6 of the inner bag 2, and the cultured cells are transferred to the collection bag 7.
Move to 0.

[Example] Figure 1 shows a culture bag in the present invention,
It is composed of an outer bag 1 and an inner bag 2 made of a semipermeable membrane and housed inside the outer bag 1.

The outer bag 1 is made by sealing the peripheries of two sheets of soft synthetic resin into a bag shape, and a plurality of external liquid operation ports 3 are provided at the lower end. This operation port is protected with a protector if necessary. In addition, a hole 4 or 1 is provided in a sealed portion of a corner of the outer bag 1 for attachment to a rotary agitator, which will be described later.

The inner bag 2 is made by sealing the openings at both ends of a tubular semipermeable membrane, and an internal fluid operation port 6 is provided at the upper end. This internal liquid operation port 6 protrudes to the outside of the outer bag l. This inner bag 2 is covered with a protective net 5.

The outer bag l of this example is made of polyvinyl chloride, has a sheet thickness of 0.4 sm, and has a capacity of 2,000 ml of external liquid and approximately 2.0 ml of outer liquid.
00ml of air is a total of 4,000sl. In addition, the inner bag 2 is made of regenerated cellulose, has a film thickness of 2011 m, and has a molecular weight of 10,000.6 m, which is equal to 500 m1 of internal fluid and approx.
001 air is 1,0110 ml in total.

Next, various equipment used for such culture bags,
This will be explained along with how to handle it. In addition, in the following examples, the culture of human lymphocytes will be explained as an example.
The present invention is also effective for high-density culture of blood-derived cell lines, such as mouse hybridomas.

(I) Cleaning of the culture bag The inner bag 2 is coated with chrycerin to prevent drying. Therefore, it is necessary to wash with physiological saline before use.

First, as shown in FIG. 2, the connecting tube 11 is connected to the bag 10 containing physiological saline, and the other end is connected to the internal fluid operation port 6. This connecting tube 11 is provided with infusion needles 12 and 13 at both ends of the tube, and a clamp 14 is attached to the middle portion of the tube. This clamp 14 is initially kept closed, and after connecting the operating port 6 and the bag 1O, it is opened to inject approximately 500 cm of physiological saline by a drop.

Next, close the clamp 14 and connect the connecting tube 11 to the bag 1.
After removing it from 0, tightly tie and seal the two places near the operation port 6, and cut out the unnecessary part.

After checking for leaks in the inner bag, pour approximately 200 ml of physiological saline into the outer bag in the same manner as described above.

FIG. 3 shows a state in which after saline is injected into the outer bag 1, the connecting tube lla is tightly tied and sealed 15.15a. Note that the external liquid operation port 3 is used without a protector.

Subsequently, the culture bag was attached to a rotary agitator (described later) and rotated at 4 to 5 rpm for about 15 minutes.

(TI) Lymphocyte suspension (internal solution) and culture medium (external solution)
Injection (1) Discharge of washing solution After removing the culture bag from the rotary agitator, pull out the connecting tube 11a from the external liquid operation port 3, and connect a new connecting tube (clamp closed). After opening the clamp of this connecting tube and discharging the cleaning liquid in the outer bag 1 by a drop, the connecting tube is tightly tied twice to seal it, and the unnecessary portion is cut off.

Thereafter, after checking whether there is a leak in the inner bag 2, the cleaning liquid inside the inner bag 2 is drained in the same manner as described above, and the connecting tube is sealed and cut off.

(2) Injection of internal fluid Next, inject the lymphocyte suspension into the inner bag 2, or perform this operation in a clean bench.

The lymphocyte suspension is injected into the inner bag 2 by the following procedure.

The connecting tube 11 is pulled out from the internal fluid operation port 6, and a cell suspension injection device 20 as shown in FIG. 4 is connected. This injection device 20 has a funnel 21 connected to a guide tube 22, and an infusion needle 23 attached to the tube.

After inserting the infusion needle 23 into the internal fluid operation port 6, the lymphocyte suspension is injected into the inner bag 2 from the funnel 21 by a drop.

After injecting the lymphocyte suspension, the injection device 20 is pulled out and an operating adapter 30 as shown in FIG. 5 is connected. This operation adapter 30 has a cap 32 with a rubber stopper fitted to the rear end of an infusion needle 31.

Next, a sterile air injection device 40 as shown in FIG. 6 is connected to this operating adapter 30. This syringe J440 has a TE stopcock 43, a sterilizing filter 42, and a disposable 3i41 connected in sequence to the tip of a disposable syringe 44.

After inserting the disposable needle 41 into the rubber stopper portion of the operating adapter 30, sterile air is injected by interlocking the screws 1 to 43 of the syringe 44 and switching the three-way stopcock 43 to tension the inner bag 2. Strict settings are not required for sterile air injection-1. After sterile air is injected in this manner, the injection device 40 is removed.

■Injection of external solution As shown in Fig. 7, a connecting tube llb with the same configuration as above is inserted into the culture medium bag 50 prepared in advance.
After connecting one of the infusion needles 12b, the connecting tube connected to the external liquid operation port 3 of the outer bag is removed, and this operation [1
3 to the other infusion needle 13b. Thereafter, the clamp 14b is opened and external liquid is injected by a drop.

After injection, remove the connecting tube 11b from the external liquid operation port 3,
An operation adapter 30 having the same configuration as above is connected.

Next, replace the sterile filter 42 and disposable needle 41 of the sterile air injection device 40 used for injecting the internal fluid with new ones, and inject sterile air into the outer bag l by injecting into the inner bag 2 and turning it. Give tension to the bag. Inner bag 2 for injection amount
Similarly, no strict settings are required.

(III) Start of culture Using a rotary agitator as shown in Figure 8,
Place in a constant temperature room or incubator set at ℃, and set the above culture bag. This agitator is provided with a rotating plate 60 that is appropriately inclined in a box 61, and the rotating plate 60 is rotatable by a drive mechanism built into the box 61. In addition, first holes fJ: 62 are provided at the four corners of the rotary plate 60 for fixing the culture bag described in -, and the outer bag l can be attached to the rotary plate by fixing the hole 4 with the first screw 62. It can be fixed at 60. After the culture bag is placed on the rotary plate 60, the plate is rotated once at 4 to 5 rpm to start culturing.

(1’V) Culture J1! ! (External fluid) replacement The time for replacement is determined by the yellowing of the external fluid.

It is convenient to prepare the culture medium on Mars and store it in small quantities in culture medium bags.

When replacing the medium, first stop the rotation of the rotary agitator and remove the culture bag. Next, after connecting the connecting tube (clamp closed) with the same configuration as shown in Fig. 2 to the unused external liquid operation device 3, open the clamp and drain the external liquid using the head.

After draining the external fluid, close the clamp and connect another connecting tube (clamp closed) to the pre-prepared culture medium bag. Thereafter, the connecting tube connected to the external liquid operation port 3 is removed, the connecting tube of the culture medium bag is connected, and the clamp is opened to inject external liquid using a drop.

After the injection of the external fluid is completed, the clamp is closed, the connecting tube is removed from the culture medium bag, the connecting tube is tightly tied twice to seal, and the unnecessary portion is cut out. At this time, the outer bag l
If there is no tension, inject sterile air using the same method as above.

Thereafter, the culture bag is fixed to a rotary agitator and culture is restarted.

(V) Completion of culture and collection of lymphocyte suspension (inner fluid) After completion of culture, remove the culture bag from the rotary agitator, and place the collection bag 70 as shown in Figure 9.
Prepare.

A guide tube 71 is connected to the collection bag 70, and an infusion solution t172 is attached to the tip thereof. The guide tube 71 is tied loosely at the part near the storm bag, as shown in the figure, to form a loop 73.

Next, the operation adapter connected to the internal fluid operation port 6 is removed, the infusion needle 72 of the collection bag 70 is connected, and the internal fluid is collected into the collection bag 70 via the guide tube 71 due to the drop.

After collection, the loop 73 of the guide tube 71 is tightly tied and sealed, and then the infusion needle 72 is removed from the internal fluid operation r-+ 6. Tie another similar knot right next to the knot, seal it, and cut off the unnecessary part.

In addition, the culture bag, medium bag, collection bag,
Connection tubes, operating adapters, cell injection devices, sterile air injection devices, etc. are disposable products with plastic covers.

Table F shows data when various cells were cultured using the apparatus of the present invention.

[Effects] According to the present invention, the following excellent effects can be achieved.

(1) Compared to conventional rotary culture using roller bottles, it requires less space and equipment for culturing.Furthermore, it does not require a complicated system like cell culture using empty fibers. It is close to Tenba concentration culture or 1+ (Moat).

■Cultured cells and culture medium are enclosed in different ensembles, and cell injection/
Collection, culture medium exchange, and sterile air injector can be performed by connecting the respective operating instruments with a single touch.
It is either handled or shipped in a tube.

(:j) ,,h g"By using each instrument,
Cell collection and medium exchange can be performed aseptically without using a clean bench.

(4) Compared to the conventional rotary culture method using roller bottles,
Since nutrients permeate through the semipermeable membrane to the cell side, cells can be cultured at high density within the encapsulated semipermeable membrane by exchanging the medium at appropriate times.

■Since the cells and culture medium are gently agitated by the rotary agitator, the culture j'l! ! The components are uniformly dispersed, and the cells are less likely to adhere to the container wall or aggregate, making it possible to obtain a high recovery rate.

[BRIEF DESCRIPTION OF THE DRAWINGS] FIG. 1 is a schematic diagram of the concentrated culture bag according to the present invention [4, 2
The figure is a schematic diagram for explaining the method of cleaning the concentrated culture bag, and FIG. 3 is a schematic diagram of the connecting tube sealed after cleaning the outer bag. Figure 4 is a schematic diagram showing the cell suspension injection device connected to the inner bag, Figure 5 is a schematic diagram of the operating adapter used in the present invention, and Figure 6 is a sterile air injection device connected to the inner bag. FIG. 7 is a schematic diagram showing the state in which the culture medium pack is connected to the outer bag. FIG. 8 is a schematic diagram of the rotary agitator used in the present invention. FIG. 3 is a schematic diagram showing a state in which a collection bag is connected. In the figure, l is an outer bag, 2 is an inner bag, 3 is an external fluid operation port, 5 is an inner bag protection net, 6 is an internal fluid operation port, 10 is a saline bag, 11 is a connecting tube, and 20 is a cell A floating liquid injection device, 30 is an operation adapter, 40 is a sterile air injection device, 5
0 is a medium bag, and 70 is a cell collection bag. Patent applicant: Kawasumi Chemical Industry Co., Ltd. Representative: Shigemi Nishino, patent attorney Figure 3 Figure 5 Figure 4 Figure 9

Claims (1)

[Claims] An inner bag containing a semipermeable membrane containing a cell suspension is housed in an outer bag containing a medium, and cells are cultured at high density through the semipermeable membrane by a diffusion phenomenon due to a concentration gradient between the cell suspension and the medium. a culture medium bag for supplying a culture medium to the outer bag, an injection device for injecting a cell suspension into the inner bag, and a collection bag for collecting cells after culturing in the inner bag;
A cell culture device comprising a connecting tube connecting the concentrated culture bag and a medium bag or a collection bag, and a device for injecting sterile air into each bag.