JP2007097407A - Cell production method and cell culture apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell production method and a cell culture apparatus which are expected to have a higher yield. <P>SOLUTION: A culture vessel 1 is bag-like. Cells 21 are seeded in the culture vessel 1 and the culture vessel 1 is flatly placed and allowed to stand for a fixed time. When a certain time is passed, the seeded cells 21 are settled and brought into contact with an inside wall face 26 on the ground side. When another certain time is passed, the cells are bonded to the inside wall face 26. After a fixed time is passed, the culture vessel 1 is inverted (posture change process). The culture vessel is allowed to stand in an inverted state (second stationary step). When a certain time is passed, the residual cells 21 are settled, the cells are brought into contact with the inside wall face 28 on the ground side. Then, the culture vessel is maintained in a flatly positioned state and the cells bonded to the inside wall faces 26 and 28 are grown (growth step). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing animal cells by culturing animal cells, and is particularly suitable for producing cells with high adhesion dependency. The present invention also relates to a cell culture device.

Animal cell culture technology is one of the important technologies in the biotechnology field. For example, various protein drugs are produced by culturing recombinant animal cells.
On the other hand, in recent years, so-called regenerative medicine has attracted attention, and researches are being carried out daily for practical use. Here, regenerative medicine is medical care that uses cells to restore the lost function when a part of the body is killed or lost.
Culture of animal cells such as stem cells is indispensable for practical application of regenerative medicine and experimental research. Recently, it has been reported that mesenchymal stem cells having the ability to differentiate into osteoblasts, chondrocytes, adipocytes, muscle cells (myocardial / skeletal muscle), etc. are present in human bone marrow tissue, Researchers are attracting attention as to how these mesenchymal stem cells are cultured.

Here, the mesenchymal stem cells have adhesion dependency and have the property of proliferating and differentiating while attached to the wall surface or the like. Therefore, the mesenchymal stem cells can be cultured by the following method, for example.
That is, mesenchymal stem cells are separated from the bone marrow fluid removed from the human body and placed in a flask together with the medium (seeding). Although mesenchymal stem cells are commercially available, they may be collected from the provider with the consent of the provider.
The cells are then allowed to grow while attached to the bottom of the flask. The cells grow in two dimensions and cover the bottom of the flask.
When there is less room for culturing on the bottom of the flask, the cells are detached and separated by trypsin or the like, and the separated mesenchymal stem cells are seeded and grown in a new flask.
In this method, it takes time to detach the cells from the bottom of the flask and to inoculate a new flask. Also, since these operations are often performed manually, there is a risk of contamination during the operation.

Patent Documents 1 and 2 disclose a method of using a culture bag in place of the above method of using a flask.
The methods disclosed in Patent Documents 1 and 2 use a culture bag made of a resin having gas permeability. In the measures disclosed in Patent Documents 1 and 2, cells are seeded together with a medium in a culture bag, and the cells are allowed to adhere to the inner surface of the culture bag and proliferate.
JP-A-6-98756 Japanese Patent Laid-Open No. 3-160984

According to the measures disclosed in Patent Documents 1 and 2, since cells adhere to the inner surface of the culture bag and proliferate, more efficient culture can be expected compared to the method using a flask.
However, according to the experiments by the present inventors, the amount of cells collected varies depending on the site of the culture bag, and the overall yield was small.
That is, when cells are cultured with the culture bag placed vertically (suspended), the cells grow intensively on the bottom side of the culture bag, and only a few cells can be recovered from the top side.
When cells were cultured with the culture bag placed flat, there was a large difference in yield between the top wall and the ground wall. Specifically, the proliferated cells were attached to the wall on the ground side, but only a few cells were attached to the wall on the top side.

  Thus, the present invention focuses on the above-mentioned problems of the prior art, and an object of the present invention is to develop a cell production method that can expect a higher yield. In addition, an object of the present invention is to develop a cell culture apparatus that can efficiently culture cells.

  The invention according to claim 1 for solving the above-mentioned problem uses a culture container filled with a medium in which cells are seeded, and in the method for producing a cell, the cells are cultured in the culture container. Are partly or entirely made of a gas permeable resin, and the step of leaving the culture vessel in a specific posture is maintained, the step of changing the posture of the culture vessel, and the static change after the posture change. It is a cell manufacturing method characterized by having the process to place.

In the cell production method of the present invention, since part or all of the culture vessel is made of a gas permeable resin, cells can be cultured with the culture vessel sealed. Moreover, in this invention, it has the process of leaving still in the state which maintained the attitude | position of the culture container in the specific attitude | position, the cell seed | inoculated at this time sinks, and a cell adheres to the lower part side in the said attitude | position.
Furthermore, the cell production method of the present invention includes a step of changing the posture of the culture container and a step of allowing the culture vessel to stand still after the posture change. Therefore, the cells sink in the changed posture, and the cells adhere to the lower side in the posture. Therefore, in the cell production method of the present invention, cells adhere to more container surfaces, and the inner surface of the culture container can be used effectively.
The cells to be seeded in the medium are often subjected to some pretreatment, but for example, bone marrow fluid or the like may be put in the medium as it is.

  According to a second aspect of the present invention, there is provided a method for producing a cell using a culture container filled with a medium in which cells are seeded, and culturing the cells in the culture container. It is a cell manufacturing method characterized by including the attitude | position change process which is made with an adhesive resin and inverts a culture container.

  Since the cell manufacturing method of the present invention includes a posture changing step of inverting the culture container, the cells can be adhered to the wall surface of the portion located in the lower part while the culture container is inverted.

  The invention according to claim 3 is the method for producing a cell according to claim 1 or 2, wherein the culture container has a large-area front and back surface.

  In the cell culture method of the present invention, the culture container has a large and front surface. Therefore, the settled cells adhere to the inner surface of a large area.

  The invention according to claim 4 is the method for producing cells according to any one of claims 1 to 3, wherein the culture container is substantially filled with a medium and no gas phase is present.

  In the cell production method of the present invention, since the gas phase does not exist in the culture container, the adhered cells are not exposed to the gas phase when the posture of the culture container is changed or reversed.

  The invention according to claim 5 uses a culture container filled with a medium in which cells are seeded, and a method for producing a cell, wherein the cells are attached to a part or all of the inner surface of the culture container and cultured. The culture vessel is partially or entirely made of a gas permeable resin, and the culture vessel is substantially filled with a medium and there is no gas phase, and the posture of the culture vessel is changed during the culture. Thus, the cell production method is characterized in that substantially all of the inner surface to which the cells are attached is once downward.

  In the cell production method of the present invention, the posture of the culture vessel is changed during the culture so that substantially all of the inner surface to which the cells are attached is once downward. Therefore, cells can be adhered to the desired surface. In addition, since there is no gas phase in the culture vessel, the adhered cells are not exposed to the gas phase when the posture of the culture vessel is changed.

  The invention according to claim 6 uses a culture container filled with a medium in which cells are seeded, and in the method for producing cells in which cells are cultured in the culture container, part or all of the culture container is gas-permeable. The culture vessel is substantially filled with the medium and there is no gas phase. During the culture, the posture of the culture vessel is changed and almost all the inner surface of the culture vessel is temporarily lowered. It is a cell manufacturing method characterized by making it become a direction.

  In the cell production method of the present invention, the posture of the culture vessel is changed during the culture so that almost all the inner surface of the culture vessel is once downward. Therefore, cells can be adhered to almost all inner surfaces. In addition, since there is no gas phase in the culture vessel, the adhered cells are not exposed to the gas phase when the posture of the culture vessel is changed.

  The invention according to claim 7 is the method for producing cells according to any one of claims 1 to 6, wherein the culture container is always allowed to stand still.

  In the cell culture method of the present invention, the culture vessel is always left stationary. Therefore, the cultured cells are not stimulated and the cells are not damaged.

  In the invention according to claim 8, the first stationary step of maintaining one of the front and back surfaces of the culture vessel in the celestial direction and the other in the ground direction to maintain the posture is performed on the ground inner surface of the culture vessel. 8. The method according to claim 1, wherein after the cells are adhered, a posture changing step is performed to invert the front and back surfaces of the culture vessel, and further, a second stationary step is performed to maintain the posture after the top and bottom inversion. A method for producing cells according to the above.

  In the cell production method of the present invention, the first stationary step is performed in which one of the front and back surfaces of the culture container is oriented in the celestial direction and the other is arranged in the ground direction to maintain the posture. Cells can be allowed to adhere. In addition, after the cells adhere to the inner surface of the culture vessel, the posture change process is performed to invert the front and back surfaces of the culture vessel, and the posture after the inversion is maintained. Cells can be adhered to the inner surface of the cell.

  The invention according to claim 9 is the method for producing cells according to any one of claims 1 to 8, characterized in that the culture container has a bag shape.

  “Bag shape” is a concept including a bag. Since the culture container of the present invention has a bag shape, no storage space is required. Transportation is also convenient.

  The invention according to claim 10 is the method for producing cells according to any one of claims 1 to 9, wherein the inner surface of the culture vessel is hydrophilic.

  In the cell culture method of the present invention, the cells are easily adhered because the inner surface of the culture vessel is hydrophilic.

  The invention according to claim 11 is the cell manufacturing method according to any one of claims 1 to 10, wherein the cells to be seeded are adhesion-dependent cells.

  According to a twelfth aspect of the present invention, there is provided a cell manufacturing method in which a culture container filled with a medium seeded with cells is used, and the cells are cultured in the culture container. Part or all is made of gas permeable resin, the inner surface of the culture vessel is hydrophilic, the inside of the culture vessel is substantially filled with medium, there is no gas phase, and the seeded cells adhere The cell is adhered to the inner surface of the culture container in the first direction, with one of the front and back surfaces of the culture container facing upwards and the other placed in the ground direction to maintain the posture. Attach the cells to the inner surface of the culture vessel that has become the new ground by performing a posture change process to reverse the top and bottom of the culture vessel upside down and then perform a second standing step to maintain the posture after the top and bottom inversion It is a cell manufacturing method characterized by making it carry out.

  The cell culture method of the present invention incorporates the features of each of the above-described inventions, and the cells adhere to more inner surfaces of the container, so that the inner surface of the culture container can be used effectively.

  The invention according to claim 13 relating to the production apparatus has a holding means for holding a culture container filled with a medium seeded with cells, and a posture changing means for changing the attitude of the culture container. It is a culture device.

Since the cell culture device of the present invention has posture changing means for changing the posture of the culture vessel, the cells can be adhered to many areas on the inner surface of the culture vessel.
As the posture changing means, for example, a culture vessel that can be turned upside down within a short time, a rocking device, a device that slowly rotates over a long time, a rolling device, and the like can be considered.

  The invention according to claim 14 is a cell culture device comprising holding means for holding a culture container filled with a medium in which cells are seeded, and posture inversion means for reversing the attitude of the culture container upside down. is there.

  Since the cell culture device of the present invention has the posture reversing means for reversing the posture of the culture vessel upside down, the cells can be adhered to both sides of the culture vessel inner surface.

  In the invention according to claim 15, the holding means is capable of holding a culture container having a large front and back surface, and the culture container is held with one of the front and back surfaces facing the celestial direction and the other facing the ground direction. The cell culture device according to claim 13 or 14, wherein the device is a cell culture device.

  The cell culture device of the present invention can use a culture container having a large area front and back surfaces, and can adhere cells to both sides of the culture container inner surface.

  The cell culture device according to any one of claims 13 to 15, wherein the holding means is capable of holding the culture container in a state where the front and back surfaces of the culture container can be vented. It is.

  In the cell culture device of the present invention, since the front and back surfaces of the culture container are held in a state that allows ventilation, oxygen or the like necessary for the medium can be supplied. In addition, the hydrogen ion concentration can be maintained by incorporating carbon dioxide gas.

  The invention according to claim 17 comprises a control means having a timer function, and maintains a posture with one of the front and back surfaces of the culture vessel facing upward and the other facing the ground by the control means for a certain period of time, The cell culture device according to any one of claims 14 to 16, wherein the posture of the culture vessel is inverted upside down by the posture inversion means, and the posture after further upside down is maintained for a certain time.

  In the cell culture device of the present invention, the posture in which one of the front and back surfaces of the culture vessel is directed to the top and the other is directed to the ground is maintained for a certain period of time, so that the cells can be adhered to the inner surface that has become the ground. . In addition, since the posture after turning upside down is maintained for a certain period of time, cells can be adhered to the inner surface which has become the ground direction at this time. In the present invention, since a series of these operations can be controlled by a timer, cell culture can be automated.

In the cell production method of the present invention, cells can be adhered to a wider area on the inner surface of the culture container, so that a larger amount of cells can be recovered.
In addition, the cell culture device of the present invention is effective in contributing to mechanization and automation of cell culture.

Hereinafter, embodiments of the present invention will be described.
FIG. 1 is a perspective view of a culture vessel used in the cell production method of the present invention. FIG. 2 is a perspective view showing a state where the culture container of FIG. 1 is filled with a medium and placed in a flat state. FIG. 3 is a cross-sectional view of the culture vessel in the state shown in FIG.

In the cell production method of this embodiment, a culture vessel 1 as shown in FIG. 1 is used.
The culture vessel 1 has a bag shape. The culture vessel 1 is made of a single layer or a laminated sheet described later and has a rectangular shape in plan view. That is, the culture vessel 1 is formed by overlapping two rectangular sheets and joining the periphery, and has four sides 2, 3, 4, and 5 so that each side 2, 3, 4, and 5 is airtight. Two sheets are joined to each other. Accordingly, the culture vessel 1 has a large area on the front surface 6 and the back surface 7 and a linear side surface, and has substantially no area.

  A hole 8 is provided in the side 2 which is the short side of the culture vessel 1. The holes 8 are provided so that the culture vessel 1 can be suspended when taking out the cultured cells. The hole 8 is not essential.

  Short pipes 10, 11, and 12 are attached to the side 4 facing the side 2 described above. The short tubes 10, 11, and 12 communicate with the inside and outside of the culture vessel 1. When the culture vessel 1 is filled with a culture medium, when the culture medium is replaced, or when seeded, an additive or the like is added to the culture medium. It is utilized when doing. Moreover, you may take out the cell after culture | cultivation from short tube 10,11,12.

In the present embodiment, the long sides 3 and 5 are bonded, but a culture container may be manufactured by folding a single sheet, and in this case, only one side becomes a joining side.
In addition, the culture vessel may be manufactured by extruding the sheet into a cylindrical shape, and in this case, the long sides 3 and 5 do not become the joining surface.

  As described above, the culture container 1 has a large area on the front surface 6 and the back surface 7, and the side surface is linear and has substantially no area. It becomes a state. That is, of the front and rear surfaces 6 and 7 of the culture vessel 1, the lower side comes into contact with the floor surface, and the upper side surface rises.

The culture container 1 of the present embodiment is made of a sheet material having gas permeability. Moreover, the inner surface of the culture vessel 1 is hydrophilic. Examples of such sheet materials include polyolefin resins and elastomers. Furthermore, examples of the polyolefin resin include polyethylene, polypropylene, and ethylene propylene copolymer. Polystyrene can also be used.
Moreover, a styrene butadiene thermoplastic resin is mentioned as an example of an elastomer. Further, the sheet material may be a polymer alloy that is a mixture of two or more kinds of polymers. Examples of the polymer alloy include a mixture of polyethylene and polypropylene, a mixture of styrene butadiene thermoplastic resin and ethylene propylene copolymer, and a mixture of styrene-ethylene-butadiene-styrene block copolymer (SEBS), vinyl acetate and polypropylene. . Furthermore, the inner surface of the culture vessel 1 may be charged so that cells can adhere more reliably.

Next, the cell production method of the present invention will be described step by step.
FIG. 4 is an explanatory view showing a state in the culture vessel in each step of the cell production method of the present invention. FIG. 5 is an explanatory view showing a state in the culture vessel in each step of the cell production method according to another embodiment of the present invention. FIG. 6 is an explanatory view showing the inside of the culture container in each step of the cell production method according to still another embodiment of the present invention.

  In the cell culture method of the present invention, the culture vessel 1 described above is used, and the culture vessel 1 is filled with the culture medium 20 as shown in FIG. The short tubes 10, 11, 12 are used for filling the medium. What should be noted here is that air does not enter the culture vessel 1. That is, the inside of the culture vessel is substantially filled with the medium 20 and no gas phase exists.

And the cell 21 is seed | inoculated to the culture container 1 like FIG.4 (b). Alternatively, cells may be dispersed in the medium 20 in advance, and the culture container 1 may be filled with this medium. Cells to be seeded are usually pretreated in some way, but for example, collected bone marrow fluid may be used as it is.
The seeded cells diffuse in the culture vessel 1. At this time, the culture vessel 1 may be shaken to assist cell diffusion.
In this state, the culture vessel 1 is laid flat and left for a certain time. In other words, the large-area front surface 6 is directed toward the top and the back surface 7 is directed toward the ground.
The floor surface 22 on which the culture container 1 is placed flat has an opening 25 as shown in FIG. Accordingly, oxygen or the like necessary for culture can be taken in from the lower surface side of the culture vessel 1. In addition, the hydrogen ion concentration can be maintained by incorporating carbon dioxide gas.

The culture vessel 1 is installed in a flat state and left in that state (first static step). In the first stationary step, the culture vessel 1 is not vibrated as much as possible.
After a while, the seeded cells 21 settle, and the cells contact the ground-side inner wall surface 26 as shown in FIG. Then, after a while, the cells adhere to the inner wall surface 26. The time during this period is about one hour to several days.

  After the predetermined time has elapsed, the culture vessel 1 is inverted upside down as shown in FIG. 4D (posture changing step). That is, the culture vessel 1 is turned over so that the large-area surface 6 faces the ground side and the back surface 7 faces the top (FIG. 4E). And it leaves still in the state which reversed the top and bottom (2nd standing process). Also in the second stationary step, the culture vessel 1 is not vibrated as much as possible.

  After a while, the remaining cells 21 settle, and the cells contact the inner wall 28 on the ground side as shown in FIG. That is, the remaining cells 21 adhere to the surface that was on the celestial side and to which the cells are not adhered. This time is also about 1 hour to several days.

After that, the flat state is maintained, and the cells adhered to the inner wall surfaces 26 and 28 are grown (growth process).
At this time, the medium is replaced as necessary. The environment is maintained so that the environment in which the culture vessel 1 is installed is suitable for cell growth. In the growth process, the culture medium in the culture vessel 1 may be moved. For example, the culture vessel 1 may be vibrated to the extent that it does not adversely affect, rotated, or tilted.
In the present embodiment, since cells adhere to substantially the entire inner surface of the culture container 1, the cells grow on the entire inner surface of the culture container 1.
When the cells in the culture vessel 1 have sufficiently proliferated, the cells are removed from the culture vessel 1 by known means.

In the embodiment described above, cells are grown in a state where the culture container 1 is placed flat, but the posture of the culture container 1 in the growth process is arbitrary.
FIG. 5 is an explanatory diagram when the cells are grown by holding the culture vessel 1 in a vertical or suspended state.

In the above-described embodiment, the first stationary step and the second stationary step are both for the purpose of adhering cells only, and are relatively short times. However, by increasing these times to some extent, Cells may be grown.
FIG. 6 shows an example in which cells are grown during the first stationary step and the second stationary step. That is, the same culture vessel 1 as in the above-described embodiment is used, the culture vessel 1 is filled with the medium 20 as shown in FIG. 6A, and the cells 21 are seeded in the culture vessel 1 as shown in FIG. 6B. To do. Then, the culture vessel 1 is placed flat and left for a certain time (first standing step). The time at this time is longer than that of the above-described embodiment.

  As a result, the seeded cells 21 settle, the cells come into contact with the ground-side inner wall surface 26 as shown in FIG. 6C, and the cells adhere to the inner wall surface 26 after a while. As time further elapses, the cells attached to the inner wall 26 on the ground side grow as shown in FIG. 6 (d).

  After the predetermined time has elapsed, the culture vessel 1 is inverted upside down as shown in FIG. 6E (posture changing step). And it leaves still in the state which reversed the top and bottom (2nd standing process). As a result, the remaining cells 21 settle, and the cells contact and adhere to the ground-side inner wall surface 28 as shown in FIG. 6 (f).

  After that, when the culture vessel 1 is allowed to stand under a certain environment, the cells adhered to the inner wall surfaces 26 and 28 grow (growth step), and a desired amount of cells can be collected.

  In the above-described embodiment, the culture vessel 1 is inverted only once and the cells are adhered to both the front and back surfaces. However, the inversion may be performed twice or more. However, if the reversal is repeated, the cells adhered at the corner may peel off, and therefore it is not preferable to repeat the reversal excessively. Although it is in the process of research, it is expected that there are cells that can be easily adhered and detached by cells. The number and speed of inversion and posture change should be changed according to the nature of the cells.

In the above-described embodiment, the culture container 1 is used only for culturing the cells. However, the cells may be induced to differentiate in the culture container 1.
In general, when cell differentiation is induced, a dedicated medium is used. Therefore, if stem cells are proliferated in the culture vessel 1 and the medium in the culture vessel 1 is replaced with a differentiation induction medium, the single culture vessel 1 can be used to proceed to differentiation induction.

  Two or more culture vessels 1 may be connected and used.

Next, the culture apparatus used for the cell manufacturing method described above will be described.
FIG. 7 is a perspective view showing a first embodiment of the culture apparatus of the present invention. FIG. 8 is a cross-sectional perspective view of the mounting table of the culture apparatus shown in FIG. 7, where (a) shows a state in which a culture vessel is installed inside, and (b) shows a state in which there is no culture vessel inside. FIG. 9 is a flowchart showing the operation of the culture apparatus shown in FIG.

  The culture apparatus 30 according to the present embodiment includes a mounting table (holding means) 31 and a rotation motor (posture reversing means) 32. The mounting table (holding means) 31 is built in an incubator (not shown) and can adjust the atmospheric temperature and the atmospheric gas temperature.

The mounting table 31 includes a plate-shaped table body 33 and a pressing member 35 that is detachably mounted on the table body 33. That is, the base body 33 has a flat plate shape, and the rotating shaft 39 is attached to both ends thereof. The rotating shaft 39 is rotatably supported by the bearing 36.
The pressing member 35 is made of a wire mesh and has a box shape. The shape of the pressing member 35 is arbitrary, and may be, for example, a plate shape. Moreover, it may be a belt or a belt shape, and may be in contact with a part of the base body 33.

An opening is provided at a part where the pressing member 35 is attached, which is a part of the base body 33, and a wire mesh 37 is provided.
The rotation motor 32 is a geared motor and rotates at an extremely low speed.
The rotation motor 32 is connected to a rotation shaft 39 provided on the base body 33. The rotation motor 32 is switched between driving and stopping by a control device (control means) 38.
The control device (control means) 38 incorporates a CPU (not shown) and has a timer function.
In the culture apparatus 30 of the present embodiment, the culture vessel 1 as shown in FIG. 1 is installed at the center of the base body 33, and the pressing member 35 is placed thereon.

Next, the function of the culture apparatus 30 of this embodiment will be described with reference to the flowchart of FIG.
In the culture apparatus 30 of the present embodiment, the culture container 1 that has been seeded is installed. The seeding operation may be performed on the culture apparatus 30.
When the seeding operation is performed on the culturing apparatus 30, the base body 33 of the culturing apparatus 30 may be oscillated and the culturing vessel 1 may be oscillated in order to assist cell diffusion. The step of swinging the base body 33 may be programmed in advance.

When the installation of the culture vessel 1 is completed, the mounting table 31 is maintained in a horizontal posture, and a timer is started (step 2). Here, the timer measures the time required for the first standing step. In the culture apparatus 30, the mounting table 31 is maintained in a horizontal posture until the timer finishes counting.
During this time, the seeded cells 21 settle, and the cells adhere to the inner wall surface 26.

When it is confirmed in step 3 that the time required for the first stationary process has elapsed, the process proceeds to step 4 where the rotation motor 32 is rotated 180 ° and the mounting table 31 is turned over.
Here, in this embodiment, since the pressing member 35 is attached on the mounting table 31 and the culture vessel 1 is held by the pressing member 35, the culture vessel 1 falls even if the mounting table 31 is inverted. No, the culture vessel 1 is upside down.

  Subsequently, the timer starts counting again. The time counted by this timer is significantly longer than the previous timer, and the time required for the second standing step and the time required for growth are counted. Here, since the time required for the growth is significantly longer than the time required for the first standing step, in practice, it is sufficient to input the time required for the growth to the timer.

During this time, the culture container 1 is maintained in an upside down posture, and the atmosphere of the culture container 1 is maintained in an environment suitable for growth by an incubator (not shown). Moreover, in this embodiment, since the pressing member 35 and the pressing member 35 of the base main body 33 are made of a wire mesh, both the front surface side 6 and the rear surface side 7 of the culture vessel 1 are in a state in which ventilation is possible. Moreover, since the culture container 1 can permeate | transmit gas, oxygen etc. required for culture | cultivation are taken in into the culture container 1. FIG.
Therefore, in the culture vessel 1, the cells 21 settle in the inverted initial stage, and the cells contact and adhere to the inner wall 28 on the ground side as shown in FIG. 4F, and further, the adhered cells grow. .
When the timer expires, the process proceeds to step 7, where the rotation motor 32 is rotated 180 ° to return the mounting table 31 to the horizontal posture. The operator removes the culture vessel 1 from the culture device 30 and takes out the proliferated cells.
In the step of growing cells, the culture medium is replaced as necessary, but the culture apparatus 30 may automatically perform this operation. It is also recommended that cells can be removed automatically.
In the step of growing cells, the mounting table 31 may be periodically rocked.

In the embodiment described above, the culture apparatus 30 including the plate-like mounting table 31 is illustrated, but the shape of the mounting table 31 and a mechanism for rotating the mounting table 31 are arbitrary.
Hereinafter, modified examples of the culture apparatus will be described.

10 and 11 are perspective views of a culture apparatus according to another embodiment of the present invention.
The culture apparatus 40 shown in FIG. 10 employs a mounting table in which semi-cylindrical members are arranged facing each other.
That is, the culture apparatus 40 has semi-cylindrical members 42 and 43, and the culture vessel 1 is sandwiched and fixed therebetween. A spacer (not shown) is sandwiched between the culture vessel 1 and the surfaces of the semicylindrical members 42 and 43, and there is a gap between the culture vessel 1 and the surfaces of the semicylindrical members 42 and 43. is there. Therefore, the surface of the culture vessel 1 is not in close contact with the semi-columnar members 42 and 43.
A space 45 between the semi-cylindrical members 42 and 43 is a sealed space, and a gas having a desired concentration can be introduced into this space. As described above, since the surface of the culture vessel 1 is not in close contact with the semi-cylindrical members 42 and 43, the surface of the culture vessel 1 is in contact with the introduced gas.
Further, the semi-cylindrical members 42 and 43 are configured so that temperature-controlled water can pass through, and the space 45 between the semi-cylindrical members 42 and 43 can be adjusted to a desired temperature.

  The semi-columnar members 42 and 43 are supported by two rolls 46 and 47. One of the rolls 47 is rotated by a motor (not shown), and the other roll 46 is an idling roll.

  In the culture apparatus 40 of the present embodiment, when the culture vessel 1 is disposed in the space 45 between the semi-columnar members 42 and 43 and the posture is changed, the roll 47 is rotated and the semi-cylindrical shape is in contact therewith. The members 42 and 43 are rotated. As a result, the culture vessel 1 rotates with the semi-cylindrical members 42 and 43, and the top and bottom are reversed.

  Moreover, the structure which pinches | interposes the culture container 1 with the flat members 51 and 52 like the culture apparatus 50 shown in FIG. 11 is also employable. The culture vessel 1 needs to be placed in a breathable atmosphere. As a measure for that purpose, the flat members 51 and 52 are made of a material having air permeability such as a net or punching metal, or a gap is formed between the culture vessel 1 and the flat members 51 and 52. Conceivable.

In the present embodiment, a disc 53 is attached to a rotating shaft 54 protruding from a flat plate member 52, and a friction wheel 55 is engaged with the disc 53. In this embodiment, by rotating the friction wheel 55, the plate-like members 51 and 52 are rotated, and the culture vessel 1 held inside is inverted.
The disc 53 and the friction wheel 55 employed in this embodiment may be replaced with a gear train, or may be replaced with a gear and a rack, or a sprocket and a chain.

Moreover, in embodiment mentioned above, although the culture container 1 which was bag shape and was provided with the front and back of a large area was illustrated, the balloon-shaped culture container 60 as shown in FIG. 73 can also be employed.
That is, FIG. 12 shows a modified example of the culture container, (a) is a front view showing a state in which the medium is not filled, and (b) assumes a state in which the medium is filled and placed in a weightless state. (C) is the figure which assumed the state where the culture medium was filled and it was set | placed on the floor. FIG. 13 is a perspective view showing a culture container of still another shape.

The culture container 60 shown in FIG. 12 has a balloon shape, and becomes spherical when filled with a medium (there is no gravity and it actually hangs downward). Therefore, there is no distinction between the front and back surfaces of the culture vessel 60.
However, when the culture vessel 60 is placed on the floor surface 61, the installation surface with the floor surface 61 becomes flat due to the weight of the internal culture medium.

  When the culture container 60 as shown in FIG. 12 is used, after the cells are seeded, the culture container 60 is rolled to change the posture of the culture container 60, and almost all the inner surface of the culture container 60 is once lowered. Try to be in the direction. As a result, the cells adhere to the entire inner surface of the culture vessel 60. After that, the culture vessel 60 is left in a predetermined posture to grow the adhered cells.

In addition to the examples described above, culture vessels 70 to 73 having a shape as shown in FIG. A culture container 70 shown in FIG. 13A has a shape like a winged envelope, and a thickness forming portion 75 is provided on a side surface portion. The thickness forming part 75 is normally folded by a crease 76.
The culture container 71 shown in FIG. 13B is box-shaped. The culture vessel 72 shown in FIG. 13 (c) is cylindrical. The culture vessel 73 shown in FIG. 13 (d) has a polygonal column shape.

  Among the culture vessels 70 to 73 shown in FIG. 13, for a shape having a clear front and back surface, such as the culture vessels 70 and 71, a method of changing the posture by inverting the culture vessels 70 and 71 is recommended. On the other hand, for the culture vessels 72 and 73 having no clear front and back surfaces, the surface to which the cells are to be adhered is once downward by rotating or shaking the culture vessels 72 and 73. Change posture.

  In the above-described culture apparatuses 30, 40, and 50, the posture of the culture vessel 1 was changed by rotation. A method of changing the posture of the culture vessel 1 by rotation is recommended in that the inlet / outlet of the culture medium can be moved to any optimum position, but even if the posture of the culture vessel 1 is changed by a method other than rotation, Good.

In the above-described embodiment, the culture vessel 1 and the like are allowed to stand at the time of culture (during the propagation process), but the posture change such as rotation or swinging of the culture vessel 1 or the like is performed so as not to adversely affect the culture. The cells may be allowed to grow while performing.
For example, when using the culture vessels 72 and 73 having no clear front and back surfaces such as the culture vessels 72 and 73 shown in FIGS. 13 (c) and 13 (d), the rotation speed is always slow without being left still. You can continue to rotate and grow the cells.

The somatic stem cell culture experiment by the method of the present invention was carried out by the following procedure. The CO ₂ incubator was set to a general setting of temperature 37 ° C., relative humidity 95%, and CO ₂ gas 5%.

A gas permeable bag having a capacity of 200 mL was filled with about 150 mL of Dulbecco's modified Eagle medium (hereinafter referred to as “FCS-DMEM”) containing 10% calf serum, and after sealing, pre-incubated in a CO ₂ incubator. Meanwhile, cryopreserved somatic stem cells were thawed, washed with FCS-DMEM, and then suspended in several mL of FCS-DMEM. An amount of cell suspension corresponding to the number of 100 cells was added to the Eagle medium in the pre-incubated bag. After slowly shaking the bag so that the cells were uniformly dispersed in the bag, the bag was allowed to stand in a CO ₂ incubator for 1-2 hours to allow the cells to adhere to one inner surface of the bag. Next, the bag was turned upside down and allowed to stand for an additional 1 to 2 hours to allow cells to adhere to the other inner surface of the bag. Thereby, the cell adhered to the inner surface of both surfaces of the bag. Thereafter, the bag was hung vertically and allowed to stand for about 1 month in a CO ₂ incubator.

  When the bag was visually observed after standing, cells grown on both inner surfaces of the bag were attached to the entire surface. The medium was extracted from the bag, and the cells attached to the inner surface of the bag were collected by trypsin treatment. The number of collected cells was counted, and the obtained cell number was divided by the surface area of the bag inner surface to calculate the cell density. As a result, a cell density of 3 × 1000 cells / square centimeter was realized. This cell density was sufficient for the purpose of culturing stem cells such as embryonic stem cells.

It is a perspective view of the culture container used with the cell manufacturing method of this invention. FIG. 2 is a perspective view showing a state in which the culture container of FIG. 1 is filled with a medium and placed in a flat state. It is sectional drawing of the culture container in the state shown in FIG. It is explanatory drawing which shows the mode in the culture container in each process of the cell manufacturing method of this invention. It is explanatory drawing which shows the mode in the culture container in each process of the cell manufacturing method in other embodiment of this invention. It is explanatory drawing which shows the mode in the culture container in each process of the cell manufacturing method in other embodiment of this invention. It is a perspective view which shows 1st embodiment of the culture apparatus of this invention. It is a cross-sectional perspective view of the mounting table of the culture apparatus shown in FIG. 7, (a) shows the state where the culture vessel is installed inside, and (b) shows the state where there is no culture vessel inside. It is a flowchart which shows operation | movement of the culture apparatus shown in FIG. It is a perspective view of the culture apparatus of other embodiment of this invention. It is a perspective view of the culture apparatus of further another embodiment of this invention. The modification of a culture container is shown, (a) is a front view which shows the state which is not filled with a culture medium, (b) is a front view which assumed the state with which the culture medium was filled and it was set to the weightless state. (C) is the figure supposing that the culture medium was filled and it was set | placed on the floor. It is a perspective view which shows the culture container of another shape.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Culture container 6 Front surface 7 Back surface 20 Medium 26,28 Inner wall surface 30 Culture apparatus 31 Mounting stand (holding means)
32 Rotating motor (posture reversing means)
33 Main body 35 Holding member 40 Culture device 50 Culture device 60 Culture vessel 70-73 Culture vessel

Claims (17)

  In the method for producing a cell using a culture container filled with a medium seeded with cells and culturing the cells in the culture container, part or all of the culture container is made of a gas permeable resin, A cell production method comprising: a stationary step of allowing the container to remain in a specific posture, a posture changing step of changing the posture of the culture vessel, and a step of further standing after the posture change .   In the method for producing a cell using a culture container filled with a medium seeded with cells and culturing the cells in the culture container, part or all of the culture container is made of a gas permeable resin, A cell production method comprising a posture changing step of inverting a container.   The method for producing a cell according to claim 1 or 2, wherein the culture container has a large-area front and back surface.   The method for producing cells according to any one of claims 1 to 3, wherein the culture vessel is substantially filled with a medium and no gas phase is present.   In a method for producing a cell using a culture container filled with a medium in which cells are seeded and attaching the cells to a part or all of the inner surface of the culture container, the part or all of the culture container Is made of gas permeable resin, the inside of the culture vessel is substantially filled with medium and there is no gas phase, and the inner surface of the culture vessel is attached by changing the posture of the culture vessel during culture. A method for producing cells, characterized in that the whole is once downward.   In the method for producing a cell using a culture container filled with a medium seeded with cells and culturing the cells in the culture container, part or all of the culture container is made of a gas permeable resin, The inside of the container is substantially filled with a medium and there is no gas phase, and the posture of the culture container is changed during culture so that almost all the inner surface of the culture container is once downward. A cell production method.   The method for producing cells according to any one of claims 1 to 6, wherein the culture container is always allowed to stand.   Implement the first stationary process to keep the posture by placing one of the front and back surfaces of the culture vessel facing the sky and the other in the ground direction, and the posture changing step after the cells adhere to the inner surface of the culture vessel The cell production method according to any one of claims 1 to 7, wherein a second stationary step is carried out, wherein the second stationary step is carried out by reversing the front and back surfaces of the culture vessel and maintaining the posture after the reversal of the top and bottom.   The method for producing cells according to any one of claims 1 to 8, wherein the culture container has a bag shape.   The method for producing cells according to any one of claims 1 to 9, wherein the inner surface of the culture vessel is hydrophilic.   The cell production method according to any one of claims 1 to 10, wherein the cells to be seeded are adhesion-dependent cells.   In the method for producing a cell, wherein a culture container filled with a medium in which cells are seeded is used, and the cells are cultured in the culture container, the culture container is in a bag shape, and part or all of the culture container is made of a gas permeable resin. The inner surface of the culture vessel is hydrophilic, the inside of the culture vessel is substantially filled with a medium and there is no gas phase, and the cells to be seeded are adherent cells. Place the back side in the celestial direction and place the other side in the ground direction to perform the first stationary process, and after the cells adhere to the grounded inner surface of the culture vessel, perform the posture change process and culture A cell production method characterized in that cells are adhered to the inner surface of a culture container that is newly ground by performing a second stationary step for reversing the front and back surfaces of the container and maintaining the posture after the reversal of the top and bottom .   A cell culture apparatus comprising: holding means for holding a culture container filled with a medium seeded with cells; and posture changing means for changing the posture of the culture container.   A cell culture apparatus comprising: holding means for holding a culture container filled with a medium seeded with cells; and posture inversion means for reversing the orientation of the culture container.   The holding means is capable of holding a culture container having a large area front and back surfaces, and the culture container is held with one of the front and back surfaces facing upwards and the other facing the ground direction. Or the cell culture apparatus of 14.   The cell culture device according to any one of claims 13 to 15, wherein the holding means is capable of holding the culture container in a state where the front and back surfaces of the culture container can be vented.   A control means having a timer function is provided, and the control means maintains a posture in which one of the front and back surfaces of the culture vessel is directed to the celestial direction and the other is directed to the ground direction for a certain period of time. The cell culture device according to any one of claims 14 to 16, wherein the cell culture device is turned upside down and maintains the posture after further upside down for a certain period of time.

Images