JPH0310675A - Bag for cell culture - Google Patents

Abstract

PURPOSE:To provide the subject bag composed of a gas-permeable and liquid- impermeable bag for putting a culture solution in and another liquid- impermeable bag for putting cells in accommodated in the above mentioned bag and capable of ready gas control of the culture solution without changing the culture solution. CONSTITUTION:A culture solution is initially put in the first gas-permeable and liquid-impermeable bag and cells are simultaneously put in the second liquid-permeable bag accommodated in the first bag to culture the cells using the culture solution diffused into the inside of the second bag through the liquid- permeable second bag. On the other hand, the first bag is arranged in an atmosphere of controlled gas concentration so that gas control of the culture solution may be carried out by permeation of the gas from the outside through the first gas-permeable bag and dissolution thereof into the culture solution, thus constituting the objective bag.

Description

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

[Prior art] For example, as a cell culture bag used in the production of monoclonal antibodies, lympho-in, cyto-in, interleukin, and interferon, there is an outer resin bag and a cell culture bag housed within the resin bag. A device equipped with a diaphragm bag is known.

When using this cell culture bag, the hybridoma, serum, etc. are placed in the diaphragm bag, and the culture solution is placed in the resin bag. Hybridomas are then cultured by rocking the cell culture bag at a predetermined temperature.

In the conventional cell culture bag, the resin bag is made of polyvinyl chloride. For this reason, in conventional cell culture bags, the gas inside the bag is controlled by periodically exchanging the culture medium (Japanese Patent Laid-Open No. 63-56275).

[Problem to be solved by the invention]

In the conventional cell culture bag, each time the culture bag is opened to replace the culture solution, there is a risk of bacteria entering the bag.

An object of the present invention is to provide a cell culture bag that allows easy external gas control of the culture solution within the culture bag.

[Means to solve the problem]

The cell culture bag according to the present invention is a bag for culturing cells by putting cells and a culture medium therein.

The bag includes a first gas-permeable, liquid-impermeable bag and a second liquid-permeable bag housed within the first bag.

[Effect]

In the cell culture bag according to the present invention, cells are placed in the second bag and a culture solution is placed in the first bag to culture the cells. The culture medium placed in the first bag is transferred to the liquid-permeable second bag.
It passes through the bag and into a second bag where it is utilized by the cells.

On the other hand, by placing the first bag in an atmosphere where the gas concentration is controlled, gas control of the culture solution in the culture bag is performed. In this case, the culture solution is adjusted by dissolving gas from the outside into the culture solution through the gas-permeable first bag, or by escaping from the culture solution as a gas to the outside.

[Example] FIG. 1 shows a cell culture apparatus in which an example of the present invention is adopted. This cell culture device mainly includes an incubator 1 and a culture device main body 3 disposed within a chamber 2 of the incubator 1.

The incubator 1 includes an air intake 4 provided at the lower part of the chamber 2, a blower 5 arranged downstream of the air intake 4, and a temperature adjustment means (heating means) arranged downstream of the blower 5. 6 and cooling means 7), and sterilization means 8 disposed downstream of the temperature adjustment means. The chamber 2 has an opening 9 as a working opening, which can be opened and closed freely and is provided close to the air intake port 4. Furthermore, an oxygen cylinder 10 and a carbon dioxide cylinder 11 are connected to the vicinity of the blowing means 5 via electromagnetic valves 12 and 13, respectively.

As the blowing means 5, for example, a blowing fan is used,
This makes the atmosphere inside the chamber 2 a positive pressure. As the heating means 6, for example, an electric heater is used. The cooling means 7 is composed of, for example, a heat exchanger. As the sterilization means 8, a sterilization filter made of, for example, a HEPA filter is used.

Inside the chamber 2, a temperature sensor 14 and a gas sensor 15 are installed.
is provided to detect the temperature and gas concentration inside the chamber 2.

As the culture device main body 3, a configuration as shown in FIG. 2, for example, is adopted. This culture device body 3 includes a plurality of cell culture bags 17 , a culture medium adjustment tank 26 connected to the culture solution introduction side of the culture bag 17 , a fresh medium tank 27 connected to the culture medium adjustment tank 26 , and a culture medium adjustment tank 27 connected to the culture medium adjustment tank 26 . It mainly has a recovery culture medium tank 28 connected to the liquid discharge side.

A culture medium introduction pipe 23 connected to the culture medium introduction side of each culture bag 17 is connected to the culture medium adjustment tank 26 . A handling pump 24 is disposed in the middle of the culture medium introduction vibrator 23, and the culture solution is transported from the culture medium adjustment tank 26 to the culture bag 17 by the handling pump 24.

A basic medium 25 to be supplied to the culture bag 17 is stored in the medium adjustment tank 26 . A fresh culture medium tank 27 is connected to the culture medium adjustment tank 26 via a pipe 30. The pipe 30 has a handling pump 31 in the middle thereof, so that fresh basic culture medium is supplied from the fresh culture medium tank 27 to the culture medium adjustment tank 26.

The recovery medium tank 28 is connected to culture medium discharge pipes 32 respectively connected to the medium discharge side of the culture bag 17 . The handling pump 24 is provided in the middle of the medium discharge pipe 32, so that the basic medium can be transported from the culture bag 17 to the recovery medium tank 28.

Each culture bag 17 is attached to a culture bag swinging mechanism 39, which will be described next. As shown in FIG. 3, a rotary shaft 41 swinging at an angle θ is rotatably supported on the floor surface 40 of the chamber 2 of the incubator 1. A bevel gear 42 is fixed to a portion of the rotating shaft 41 that protrudes downward from the floor surface 40. 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. 5 is fixed to the rotating shaft 41 below the bevel gear 42. As shown in FIG. The cam 45 has an outer peripheral surface 46 formed concentrically with the rotating shaft 41 . Outer surface 46
A notch 47 that is recessed toward the inner circumferential side is formed in a portion of the hole. 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 of the rotating shaft 41 is bent into a crank shape and serves as a support portion 50 for the culture bag stand 52. The center line of the support part 50 is 0. -0. is the center of rotation of the rotating shaft 41 0□-0! It is tilted by an angle θ with respect to the

A culture bag stand 52 is rotatably supported at the upper end of the support section 50 via a ball bearing 51. As shown in FIG. 4, 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.

A bracket 54 that projects downward is provided at the lower end of the right side of the culture bag stand 52. The bracket 54 includes
A rotation prevention mechanism 55 for preventing rotation of the culture bag stand 52 is connected. As shown in FIG.
It mainly includes a lever 57 rotatably supported by a lever 57 and a pair of guide plates 58 fixed to a floor surface 40. A sliding member 59 is provided at the lower end of the lever 57 and extends laterally between the guide plates 58. A notch 60 is provided in the guide plate 58 at a position corresponding to the lever 57, so that rotation of the lever 57 about the sliding member 59 is not blocked by the guide plate 58.

The culture bag 17 includes a gas-permeable but liquid-impermeable resin bag 70 and a diaphragm bag 71 housed within the resin bag 70. The resin bag 70 is made of a gas-permeable but liquid-impermeable material such as silicone rubber or Voretex. The diaphragm bag 71 is made of a liquid-permeable material such as regenerated cellulose. As the diaphragm bag 71, a membrane membrane, an ultrafiltration membrane, a reverse osmosis membrane, etc. can be used as appropriate depending on the separation conditions.

As shown in FIG. 4, the resin bag 17 has a generally square shape, and its four corners are fixed to the culture bag stand 53 by means of fixing members 53.
It is fixed at 2. A culture medium inlet 20 and a culture medium outlet 21 are formed in the center of one side of the resin bag 18. Inside the bag 18, both ends of the culture medium inlet 20 and the culture medium outlet 21 are bent in directions away from each other, and an opening is provided at the tip thereof. Further, a medium introducing vibrator 23 and a medium discharging vibrator 32 are connected to the outer ends of the medium inlet 20 and the medium outlet 21, respectively.

As shown in FIG. 3, one end of a string 22 is fixed to the diaphragm bag 19. The other end of the string 22 is connected to the resin bag 18
, and thereby the diaphragm bag 19 is arranged approximately at the center of the resin bag 18.

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

Inside the chamber 2 of the incubator 1, sterile air is circulated while being maintained at a positive pressure state by the blowing means 5.

Temperature and oxygen concentration in chamber 2.

The carbon dioxide concentration is detected by each sensor 14, 15 and calculated by a control device (not shown). Based on the calculation result, the heating means 6. Cooling means7. The electromagnetic pulp 12.13 is controlled to a predetermined temperature (e.g. 37°C)
and a predetermined gas concentration (for example, CO□5%).

On the other hand, when the motor 43 is rotated, the rotating shaft 41 is rotated. At this time, since the support part 50 is inclined with respect to the rotation axis 41, the culture bag stand 52 swings around the intersection of the center lines of both parts. At this time, since rotation of the culture bag stand 52 is prevented by the rotation prevention mechanism 55, the culture bag stand 52 repeats only rocking.

When the rotating shaft 41 rotates, the cam 45 also rotates, so the microswitch 48 is turned ON-OFF in accordance with the rotation. When the microswitch 48 is turned on, the handling pump 24 is driven, thereby introducing fresh culture medium into the resin bag 18 and discharging the old culture medium.

When the handling pump 24 is in operation, the opening 2021 side of the culture bag stand 52 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. The basal medium introduced into the resin bag 18 passes through the partition wall of the diaphragm bag 19 and enters the bag 1.
9 and is used as nutrients for cell culture. Also,
Physiologically active substances and waste substances secreted by cells within the diaphragm bag 3 are discharged into the resin bag 18 through the diaphragm. 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 28 through the medium outlet 21.

The resin bag 18 is gas permeable. Therefore, the gas in the chamber 2 passes through the resin bag 18 and the resin bag 1
8 or from inside the resin bag 18.
Go out. In this way, since gas can pass through the resin bag 18, the gas inside the resin bag 18 can be easily controlled. That is, by simply changing the gas concentration in the chamber 2, for example, the dissolved oxygen concentration or pH of the culture solution in the resin bag 18 can be changed.
adjustments can be made.

In this embodiment, the incubator is configured to maintain sterility, but it does not necessarily have to be a sterile chamber as long as care is taken in aseptic operation when connecting tubes.

[Other Examples]

(a) When using a normal incubator without using an incubator that can adjust the oxygen concentration and carbon dioxide concentration, a culture bag 70 shown in FIG. 7 is used. Note that the incubator and culture device main body to be used can be the same as those in the above-mentioned embodiments except for the gas concentration adjustment section.

The culture bag 70 shown in FIG.
In addition to this structure, a gas bang 71 made of flexible resin (eg, vinyl chloride) is further provided on the outside.

The resin bag 72 of the culture bag 70 corresponds to resin bag 1 of the above-described embodiment, and the diaphragm bag 73 corresponds to 19 of the above-described embodiment. Furthermore, the culture medium introduction lower 5, medium discharge lower and string 77 of the culture bag 70 are respectively the medium introduction lower 5, medium discharge lower and string 2 of the above-mentioned embodiment.
It corresponds to 2.

Furthermore, in the culture bag 70, strings 74 are fixed to the four corners of the resin bag 72 in order to position the resin bag 72. The tip of the string 74 is fixed to the gas bag 71 side.

An in/out boat 78 is connected to the diaphragm bag 73.

The in/out boat 78 projects from the diaphragm bag 73 to the outside through the resin bag 72 and the gas bag 71. Note that this in/out boat 78 is sealed in a liquid-tight state. Furthermore, inside the resin bag 72, a pH sensor 7 is provided.
9, O, i The tip of a sensor unit 82 including a 4 degree sensor 80 and a temperature sensor 81 is arranged. The sensor unit 82 includes a resin bag 72 and a gas bag 71.
The base portion thereof protrudes to the outside through the protrusion, and a lead wire 83 extends from the protrusion portion. In the gas bag 71,
A pair of gas inlet 84 and gas outlet 85 are provided. The gas inlet 84 and the gas outlet 85 are connected to a gas concentration control unit (not shown). In the gas concentration control unit, gas whose oxygen concentration and carbon dioxide concentration are controlled is introduced into the gas bag 71 from the gas inlet 84 and discharged from the gas outlet 85.

In this case, a gas with controlled concentrations of oxygen and carbon dioxide is introduced into the gas bag 71, and the culture solution in the resin bag 72 is gas-controlled. In addition, a resin bag'! The state of the culture solution in the culture medium 2 is detected by each sensor 79, 80, 81, and the gas supply and storage are controlled based on the detection results.

Φ) The present invention can be similarly applied to a culture bag used in a punch type culture device as shown in FIG.

In FIG. 8, a culture bag stand 92 that rotates in the direction of the arrow is arranged in a temperature-controlled culture chamber 91. The culture bag stand 92 is driven by a rotation mechanism (not shown). In addition, on the culture bag stand 92,
A culture bag 93 is attached, and by rotating the table 92, the liquid in the culture bag 93 is stirred. The culture bag 93 has an outer resin bag 94
and a diaphragm bag 95 housed within a resin bag 94. The resin bag 94 is provided with a large number of exchange ports 96 for exchanging the culture solution. The materials of the resin bag 94 and the diaphragm bag 95 are the same as those in the above embodiment.

In this case as well, by making the culture chamber 91 capable of controlling the gas concentration as shown in FIG. 1, the gas of the culture solution in the resin bag 94 can be controlled.

〔Effect of the invention〕

According to the cell culture bag according to the present invention, since the first bag is gas-permeable and liquid-impermeable, it is possible to control the pH and Do of the culture solution without replacing the culture solution. Become.

[Brief explanation of drawings]

FIG. 1 is a schematic vertical cross-sectional view of an incubator in which an embodiment of the present invention is adopted, FIG. 2 is a schematic configuration diagram of the main body of the culture device, and FIG. 3 is a partially cutaway side view of the rocking mechanism. Fig. 4 is a view in the direction of the ■ arrow in Fig. 3, Fig. 5 is a sectional view taken along the line V-■ in Fig. 3,
6 is a partial view of FIG. 4 in the direction of the ■ arrow, FIG. 7 is a plan view of another embodiment, and FIG. 8 is a schematic vertical cross-sectional view of a cell culture apparatus in which still another embodiment is adopted. 17.70.93...Culture bag, 18,72°94
...Resin bag, 19,73.95...Diaphragm bag.

Claims (1)

[Claims] (1) A cell culture bag for culturing cells by placing cells and a culture medium, the bag comprising: a gas-permeable but liquid-impermeable first bag; and a liquid-permeable bag housed within the first bag. a second bag, and a cell culture bag comprising: