JP3036032B2 - Cell culturing method and device - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method and an apparatus for culturing adherent cells in high density and in a large amount, and in particular, to a cell provided with a filtration facility for removing waste products metabolized by cells. The present invention relates to a culture method and a device therefor.

[Conventional technology]

In recent years, the technology of producing biological drugs such as interferon by culturing a large amount of cells in vitro has become increasingly important. In particular, since adherent cells produce many physiologically active substances, the establishment of a large-scale and high-density culture technique is essential for improving production efficiency and reducing production costs.

Incidentally, a microcarrier method and a holofiber method are generally known as methods for culturing a large amount of adherent cells at high density. Microcarrier method is several hundred μm in diameter
Is a method in which cells are attached to microcarriers and suspended and cultured in an aeration and stirring tank. This method is easy to scale up and is suitable for mass culture.

On the other hand, the holo-fiber method involves culturing cells by attaching them to a membrane in a container with a bundle of holo-fiber membranes.A fresh culture solution containing nutrients and oxygen can be supplied to the cells via the membrane, and partially the cells can be cultured to high concentrations of 10 ⁸ / ml (see, for example, JP-a-62-163687). In the holo fiber method, the culture solution is supplied and taken out by flowing the culture solution into the holo fiber and utilizing the pressure loss between the supply port and the liquid outlet.

[Problems to be solved by the invention]

However, since the microcarrier method is cultivated in a stirring tank, it is unavoidable that the microcarrier method is physically damaged by the stirring blade, and it is difficult to culture at high density. In addition, the microcarriers need to be immersed in the cell suspension for about 10 hours in order to attach the seed cells to the microcarriers before the start of culture, so they do not adhere to the microcarriers and remain in the suspension. There is a problem that many cells are killed during this time. Furthermore, when oxygen is supplied during cell culture, there are problems that the culture solution foams and that it is difficult to separate cells from microcarriers.

On the other hand, in the holo-fiber method, the pressure inside the holo-fiber is high and the pressure difference between the inside of the container and the liquid supply port is large, so the culture solution is supplied into the container through the membrane. The culture solution is taken out through the membrane because it is lower than the inside of the container. For this reason, near the center of the hollow fiber, the pressure difference between the inside of the hollow fiber and the container is small, so that the amount of the culture solution permeating the membrane is reduced. Therefore, since the supply of the culture solution becomes uneven, there is a problem that the culture at a high concentration can be performed only partially. Further, since it is difficult to detach cells from the bundle of holofiber membranes, there is a problem that the yield of cells is low, the holofibers cannot be reused, and the running cost is high.

The present invention has been made in view of such circumstances, and it is an object of the present invention to provide a cell culturing method and a device capable of culturing a large number of cells at high density by uniformly culturing cells and supplying oxygen to the cells. And

[Means for solving the problem]

The present invention, in order to achieve the above object, a plurality of perforated plates to which cells adhere to the surface, and a plurality of perforated plates are juxtaposed at predetermined intervals and a through hole communicating with the surface of the perforated plate. A filter body comprising a rotatable support shaft, a closed container for accommodating the filter body and supporting the support shaft of the filter body, and pressurizing one or more fluids including a culture solution in the closed container. And means for supplying by means of:

Further, the present invention, in order to achieve the above object, a plurality of perforated plates for attaching cells to a spindle having a through-hole, a filter body attached so as to communicate with the through-hole in the spindle, Using a culture device installed in a closed container, the culture solution is supplied to the closed container in a pressurized state, and the through-hole in the support shaft is set to the atmospheric pressure or reduced pressure to pass the culture solution containing wastes through the perforated plate and through. It is characterized in that it is taken out through a hole.

[Action]

According to the present invention, a plurality of perforated plates of a filter are arranged side by side in a closed vessel so as to communicate with the through hole, and the support shaft is provided in the closed container, and the culture solution and the like are further provided on the support shaft having the through hole formed therein. The supply means for supplying was connected to the closed vessel. Therefore, when this supply means is operated, a fresh culture solution is supplied into the closed vessel to provide nutrients and oxygen to the cells attached to the perforated plate of the filter, and at the same time to remove waste products attached to the cells. It can be separated from cells by passing through the perforated plate together with the culture solution.

〔Example〕

Hereinafter, preferred embodiments of the cell culture method and apparatus according to the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 is an overall view showing an embodiment of the culture apparatus according to the present invention. The culture apparatus 10 includes a cylindrical closed container 12, and the closed container 12 is provided with a perforated plate unit 13. The rotating shaft 14 of the perforated plate unit 13 is rotatably supported at the center of the closed container 12 as shown in FIG. 2, and the rotating shaft 14 has a through hole 16 formed on the axis thereof. The through hole 16 is opened at the left end of the rotating shaft 14 in FIG. 2 to form an inlet 16A. .. Are attached to the rotating shaft 14, and the perforated plates 20 communicate with the through holes 16 through the communication holes 18.

Further, the closed container 12 is provided with an inlet 22 and an outlet 24. The sealed container 12 is formed in a cylindrical shape having an inner diameter of 330 mm and a length of 700 mm, and the perforated plates 20 and 20 are provided therein, so that the effective volume of the sealed container 12 is 42. A jacket 25 is attached to the outer peripheral surface of the sealed container 12 so that warm water can flow along the outer peripheral surface for temperature control.

FIG. 3 shows the details of the perforated plate 20 and the state of its attachment to the rotating shaft 14. The perforated plate 20 is a disc made of ceramic, and its inner peripheral surface communicates with the through hole 16 of the rotating shaft 14. Communication hole
It is provided in contact with 18. The perforated plate 20 has a pore diameter of 10 μm
It is formed of a porous layer 26 having a multilayer structure in which a fine porous layer 26B having a pore size of 0.01 μm to 5 μm and a large transmission resistance is provided on a porous support 26A which is coarse and has a small transmission resistance of 100 μm. For this reason, when the liquid permeates, the pressure loss on the surface of the perforated plate 20 is rate-determining, and the pressure loss when the liquid flows to the through hole 16 at any point on the perforated plate 20 is substantially equal. Therefore, the liquid is not short-passed only from the portion close to the through-hole 16 and the culture solution can be uniformly supplied and removed from any location.
13 can be used both for supplying a culture solution and for removing waste products from cells. The surface of the perforated plate 20 is polished to about 20 μm in order to facilitate cell attachment. The perforated plates 20 have a diameter of 300 mm and a thickness of 5 mm, and 50 perforated plates are arranged on the rotating shaft 14 of the sealed container 12 at intervals of 8 mm.

The sealed container 12 containing the perforated plate unit 13 is the first container.
As shown in the figure, each tank (34, 36, 3
8, 39, 40, 42, 44, 46), and a compressor 30, a vacuum pump 32, and the like, which are pressure sources for liquid feeding.

Hereinafter, these relationships will be described with reference to FIG. The inflow port 22 of the sealed container 12 communicates with a seed cell storage tank 34, a storage tank 36 for a buffer solution used for washing and the like, a storage tank 38 for an alkaline solution for washing the porous plate 20, and a culture solution storage tank 39. Each of these storage tanks 34, 36, 38, and 39 is connected to the compressor 30, so that the liquid can be pressurized by aseptic compressed air from the compressor 30 and sent to the sealed container 12. Further, a boiler 40 for generating steam for sterilization is connected to the inflow port 22.

The outlet 24 has a tube 33A for discharging the liquid in the closed container 12, and a seed cell storage tank for cells detached from the porous plate 20 after completion of the culture.
A pipe 33B for sending to 34 is connected.

A trypsin storage tank 42 for supplying trypsin for detaching cells from the perforated plate 20 after completion of the culture is connected to the outlet 16A of the rotating shaft 14 of the perforated plate unit 13. The trypsin storage tank 42 is also connected to the compressor 30 so that the liquid can be pressurized with sterile compressed air from the compressor 30 and sent to the sealed container 12. The outlet 16A has a product storage tank 4 for collecting cell products.
4. A waste storage tank 46 for collecting waste from cells is connected to the waste storage tank 46. These storage tanks 44 and 46 are connected to the vacuum pump 32 so that liquid can be collected from the sealed container 12 via the perforated plate 20.

In FIG. 1, reference numeral 48 denotes a sterile filter, reference numeral 50 denotes a switching valve for supplying a culture solution and removal of cell wastes, and reference numeral 52 denotes a hot water supply port to the jacket 25 in FIG. A hot water outlet, 56 is a motor, 58 and 60 are pulleys, and 62 is a belt.

Next, an example of production of interferon (IFN) by human fibroblasts will be described as an example of culture of the cells of the present invention using a culture device.

First, steam of 121 ° C. or higher is sent from the inlet 22 of the closed container 12 into the closed container 12, and the inside of the closed container 12 is heated at 121 ° C. for 20 minutes or more.
Empty sterilization is performed by keeping at Next, the operation supplies warm water from the warm water supply port 52, circulates warm water through the jacket 25 of the closed container 12, and stores the closed container 12 at 37 ° C.

In this state, the cells are attached to the surfaces of the perforated plates 20, 20,. The adhesion to the surface is achieved by the compressor 30 and the vacuum pump 32
Fulfills the sealed container 12 and the cells operated from seed cells tank 34, concentration 10 ⁵ cells / ml were suspended in culture medium (fetal calf serum 5
% Of the MES medium) from the culture solution storage tank 39 to the closed container 12.
After filling the buffer, the sterilized and dissolved oxygen-saturated buffer solution (PBS solution) is pressurized to 0.5 kgf / cm ² and sent from the buffer solution storage tank 36 to the closed container 12 via the inlet 22, Perforated plates 20, 20 ...
This is performed by taking out the culture solution from the through-hole 16 of the rotating shaft 14 through the above.

As a result, the cells flow in the direction of the perforated plates 20, 20,... Together with the liquid, but cannot permeate through the perforated plates 20, 20,. Whereas the time required for cell attachment by only immersion, which is normally performed, requires 4 to 14 hours, the method of the present invention involving filtration can be completed in about 30 minutes. For this reason, the possibility of bacterial contamination can be reduced, and the bacterial adhesion rate and survival rate can be made almost 100%.

Next, a fresh culture solution containing nutrients and oxygen is supplied,
The culture solution containing waste products is taken out, and the cells attached to the perforated plates 20, 20,. This growth occurs in closed containers 12
From the inlet 22, 0.01~1.0kgf / cm ² pressurized fresh culture medium was pressed into the through hole 16 atmospheric pressure or -0.05kgf / cm ²
This is performed by taking out the culture solution through the outlet 16A under a reduced pressure. At this time, the pressurized fresh culture solution provides nutrients and oxygen to the cells adhering to the surface of the perforated plate 20 and contains waste products metabolized by the cells.
Is taken out of the through hole 16 through the inner surface of the hole. In a culture method involving the removal of such wastes, perforated plates 20, 20
… High-density cells adhere on top.

Subsequently, an IFN inducer is added to the culture solution to be supplied, and IFN production is started. IFN is taken out together with the culture solution from the outlet 16A of the through-hole 16.

Next, the cells for which IFN production has been completed are detached from the perforated plates 20, 20,..., And the perforated plates 20, 20,. This can be performed by exchanging the culture solution in the sealed container 12 with a buffer solution, and then exuding the trypsin solution from the inner surfaces of the porous plates 20, 20,. According to this method, the perforated plate 20,
Since the attachment surface of the cells attached to 20 ... can be directly treated with trypsin, the detachment is completed in 2 minutes. This time corresponds to about 1/10 of the conventional method of only stirring in the trypsin solution.

At this time, when the motor 56 is driven to rotate the perforated plates 20, 20,..., The cells can be detached in a shorter time. The detached cells are taken out together with the buffer when the inside of the closed container 12 is washed with the buffer, and a part thereof is stored in the seed cell storage tank for use as seed cells for the next culture.

After the cells have been discharged, in order to wash the perforated plates 20, 20,..., The closed container 12 is filled with an alkali solution (0.5% sodium hydroxide solution containing a surfactant), and the perforated plates 20, 20,.
And rotate for about 1 hour. By this operation, the perforated plate 20,
Since 20 ... can remove deposits and residues on the surface thereof, they can be used for repeated culture.

In the above embodiment, as a method of supplying oxygen during the culture, a fresh medium containing oxygen was supplied to the closed container 12, but in the case of cells requiring a large amount of oxygen, oxygen is supplied by the following method. Can be. First, adjust the volume of the culture solution to approximately
The half-hole of the perforated plates 20, 20,... Is rotated from 30 to 50 rpm after the perforated plates 20, 20,. When a gas containing oxygen (such as 5% carbon dioxide and sterile air) is sent from the inflow port 22 in this state, the efficiency of supplying oxygen to the cells is greatly improved.

Further, in the above-described embodiment, as a method of recovering the product, a fresh culture solution was sent to recover the product in which the cells were metabolized.
An inexpensive buffer may be used instead of the culture solution.

Further, in the above embodiment, the rotating shaft 14 of the perforated plate unit 13
However, the present invention is not limited to this, and in the case of increasing the culture volume, two sets of perforated plate units may be used using two rotating shafts as shown in FIG. FIG. 5 shows an overall view of the cell culture apparatus in this case. In FIG. 5, reference numeral 60 denotes a closed container.
In the drawing, the same members as those in the above-described embodiment are denoted by the same reference numerals, and description thereof is omitted.

In the embodiment shown in FIG. 4, the perforated plates of the two sets of perforated plate units are provided so as not to be adjacent to each other, but may be provided so that the two sets of perforated plates are engaged with each other. Note that the number of perforated plate units is not limited to two, and a plurality of sets may be provided.

〔The invention's effect〕

According to the culture method and the apparatus according to the present invention, the cells and the culture solution can be uniformly separated over the entire area of the perforated plate of the filter, so that the nutrients and oxygen contained in the culture solution in the cells attached to the perforated plate Can be supplied uniformly. Therefore, a large amount of cells can be cultured at a high density, so that efficient production can be performed.

Further, by filtering the cell suspension, the cells can be reliably and quickly attached to the porous plate of the filter before the start of the culture.

Furthermore, since the cells can be easily detached from the perforated plate of the filter after completion of the culture, the perforated plate can be regenerated.

In addition, by forming the porous plate of the filter into a multilayer structure in which a porous layer having a small pore size is provided on the outer periphery of a porous support having a large pore size, culture can be performed uniformly from any location on the surface of the porous plate. The solution can be supplied and taken out, and the cells can be easily attached.

[Brief description of the drawings]

1 is an overall view showing a cell culture apparatus according to the present invention, FIG. 2 is a cross-sectional view showing the structure of a perforated plate unit, FIG. 3 is an enlarged view of a perforated plate, and FIG. Sectional view of a perforated plate unit used in another embodiment of the culture apparatus of FIG.
The figure shows a cell culture device using the perforated plate unit of FIG. 10 …………………………………………………………………………………………………………………………………………………. Cell storage, 39 …… Culture storage tank.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shigemi Suzuki 2-81-1, Honmura, Chigasaki-shi, Kanagawa Prefecture Tochiki Kiki Co., Ltd. Chigasaki Plant (72) Inventor Shigeru Yamano 2-81-1, Honmura, Chigasaki-shi, Kanagawa Prefecture No. 72 in Toga Equipment Co., Ltd. Chigasaki Factory (72) Inventor Nobuyuki Maebashi 2-8-1, Honmura Chigasaki-shi, Kanagawa Prefecture Totoki Equipment Co., Ltd. Chigasaki Factory (56) References JP-A-60-99881 (JP, A JP, 2-200176 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12M 1/00-3/08

Claims (8)

(57) [Claims] 1. A plurality of perforated plates to which cells adhere to the surface,
A filter body comprising a plurality of perforated plates arranged side by side at predetermined intervals and a rotatable support shaft having a through hole communicating with the surface of the perforated plate; and a filter housing for accommodating the filter body and supporting the filter body. An apparatus for culturing cells, comprising: a sealed container that supports a shaft; and a unit that supplies one or more fluids including a culture solution to the sealed container in a pressurized state. 2. The method according to claim 1, further comprising: a means for supplying a fluid for separating cells from the perforated plate in a pressurized state in a through hole of a support shaft of the filter. Cell culture equipment. 3. The porous plate of the filter has a pore size of 10 μm to 100 μm.
The cell culture device according to claim 1 or 2, wherein a porous layer having a pore size of 0.01 µm to 5 µm is provided on the outer periphery of the porous support to form a multilayer structure. 4. A culturing apparatus in which a filter provided by mounting a plurality of perforated plates for attaching cells to a spindle having a through hole so as to communicate with the through hole in the spindle is provided in a closed container. Using, while supplying the culture solution to the closed container in a pressurized state, the through-hole in the support shaft is set to atmospheric pressure or reduced pressure, and the culture solution containing waste products is taken out through the perforated plate and the through-hole. Cell culture method. 5. A seed cell suspension is supplied into a closed container, and half or all of the perforated plate is immersed in the seed cell suspension. 5. The method for culturing cells according to claim 4, wherein the cells are filtered and the cells are attached to a perforated plate. 6. A culture solution is supplied into a closed container, a part of the perforated plate is immersed in the culture solution, and compressed air is supplied into the closed container to contact the perforated plate with a sterile gas containing oxygen. The method for culturing cells according to claim 4 or 5, wherein oxygen is supplied to the cells by rotating the perforated plate. 7. A fresh culture solution or buffer solution is pressurized and supplied into the closed container, and then the inducer is pressurized and supplied into the closed container, and the culture solution containing the produced product is supplied to the perforated plate and (4) The liquid is taken out through the through hole.
The method for culturing cells according to (5) or (6). 8. The cell according to claim 4, wherein the liquid for exfoliation is leached from the perforated plate through the through-hole, and the cells attached to the perforated plate are separated from the perforated plate. ) Or (7).