JP2015223154A - Culture vessel shielding device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To negate the need for frequent replacement of a culture medium by preventing evaporation of the culture medium in a culture vessel.SOLUTION: The invention provides a culture vessel shielding device 1 comprising a culture space to culture cells, and a vapor impermeable membrane 3j which is detachably attached to the culture vessel comprising a culture space ventilation part allowing ventilation to the culture space, is arranged at the position blockading the culture space ventilation part, and allows a gas required for cell culture to pass through at least one part of it and does not allows vapor to pass through.

Description

  The present invention relates to a shielding device for a culture container.

Conventionally, a culture container storage box for storing and culturing an open type culture container in a sterile state is known (for example, see Patent Document 1).
When the culture container is accommodated in this culture container storage box and further stored in an incubator for culturing, the passage of microorganisms into the culture container storage box is prohibited by the filter unit.

Japanese Patent Application Laid-Open No. 2004-154099

However, the culture container storage box of Patent Document 1 can prevent microorganisms from entering the culture container, but cannot suppress the evaporation of the medium outside the culture container, and thus requires frequent medium replacement.
The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a culture vessel shielding apparatus that can suppress evaporation of a medium in a culture container and eliminate frequent medium replacement. It is said.

In order to achieve the above object, the present invention provides the following means.
One embodiment of the present invention is detachably attached to a culture vessel that includes a culture space for culturing cells and a culture space vent that allows ventilation into the culture space, and closes the culture space vent. Provided is a shielding device for a culture vessel, which is disposed at a position and includes a water vapor impermeable membrane that passes gas necessary for cell culture and does not allow water vapor and microorganisms to pass through at least a part thereof.

  According to this aspect, since the water vapor impermeable membrane is disposed at a position where the culture space ventilation section is blocked by attaching the culture space ventilation section of the culture container to the position where the culture space ventilation section is blocked, the cells in the culture container are cultured. While the gas necessary for cell culture is supplied through the space aeration part, the water vapor in the culture vessel is maintained so as not to be discharged outside through the culture space aeration part, so that the medium in the culture space evaporates. Is suppressed, and culture can be continued without frequently changing the medium.

  In the above aspect, the apparatus includes a storage container including a storage space in which the culture container can be stored and a storage space ventilation part that allows ventilation to the storage space, and a gas necessary for cell culture is supplied to the culture space ventilation part. A microorganism-impermeable membrane that does not allow microorganisms to pass therethrough may be provided, and the water vapor-impermeable membrane may be provided in the housing space vent.

  Thus, when the culture container is accommodated in the accommodation space, the culture space in which the cells are cultured communicates with the outside of the storage container via the culture space aeration part, the accommodation space, and the accommodation space aeration part. The gas required for cell culture is supplied to the culture vessel through the microorganism-impermeable membrane and the water-vapor-impermeable membrane, while the microorganisms are prevented from entering the culture space by the microorganism-impermeable membrane, Moreover, the release of water vapor from the culture space to the outside is blocked by the water vapor impermeable membrane. Thereby, the evaporation of the culture medium in the culture space is suppressed, and the culture can be continued without frequently changing the culture medium. Further, even if the culture container is broken and the cells and culture solution in the culture container are released to the outside, they can be retained in the storage container.

Further, in the above aspect, the culture space aeration part protrudes to the side of the culture container and is configured by closing the cylindrical part that opens into the culture space with the microorganism-impermeable membrane, and the side surface of the storage container In addition, a fitting cylinder part for fitting the cylindrical part of the culture vessel may be provided, and the fitting cylinder part may be closed by the water vapor impermeable membrane.
By doing in this way, the culture space ventilation part which consists of the cylindrical part which obstruct | occluded the cylindrical part which protrudes to the side of a culture container with the microorganisms impermeable membrane is provided in the fitting cylinder part provided in the side surface of the storage container. When fitted, the microorganism-impermeable membrane and the water-vapor-impermeable membrane supply the gas necessary for cell culture from the outside through the culture space vent, and prevent the entry of microorganisms. Release can be prevented. Even in the case of using a commercially available culture vessel in which the culture space ventilation section is closed with a microorganism-impermeable membrane, a narrow closed space is formed between the microorganism-impermeable membrane and the water vapor-impermeable membrane. The water vapor evaporated in the space is retained in the closed space, and can be prevented from being released to the outside.

Moreover, in the said aspect, the said fitting cylinder part may be comprised with the elastic material which can be expanded-contracted.
By doing in this way, even when the size of the cylindrical portion provided in the culture vessel is different, or even when the cylindrical portion is inclined with respect to the bottom surface of the culture vessel, The fitting tube portion made of an elastic material can be elastically deformed to fit the tube portion in an airtight state. Thereby, the water vapor that has passed through the microorganism-impermeable membrane can be retained in the closed space, and the release from the culture space can be suppressed.

Moreover, in the said aspect, you may provide the sealing member which obstruct | occludes the cylindrical space between the inner surface of the said fitting cylinder part, and the outer surface of the said cylindrical part.
By doing in this way, if the cylindrical part of a culture container is fitted to the fitting cylinder part of a storage container, the cylindrical space between both will be obstruct | occluded in an airtight state by a sealing member. Thereby, the water vapor that has passed through the microorganism-impermeable membrane can be retained in the closed space, and the release from the culture space can be suppressed.

Further, in the above aspect, the accommodation space ventilation portion closes a through hole formed in an extension member made of an extendable material provided in at least a part of the side surface of the storage container with the water vapor impermeable membrane. It may be constituted by doing.
By doing so, the expansion and contraction member expands following the pressing force by pressing the opening of the cylindrical portion of the culture vessel accommodated in the accommodation space against the accommodation space ventilation portion, and the culture is performed by the water vapor impermeable membrane. The space ventilation part can be sealed.

Further, in the above aspect, the apparatus includes a storage container including a storage space in which the culture container can be stored and a storage space ventilation part that allows ventilation to the storage space, and the culture space ventilation part has the water vapor impermeability. A membrane may be provided, and a microorganism-impermeable membrane that allows gas necessary for cell culture to pass and does not allow microorganisms to pass therethrough may be provided in the housing space aeration unit.

  According to the present invention, there is an effect that it is possible to suppress frequent evaporation of the medium by suppressing evaporation of the medium in the culture container.

It is a perspective view which shows the shielding apparatus for culture containers which concerns on one Embodiment of this invention. It is a perspective view which shows an example of the culture container accommodated in the shielding apparatus for culture containers of FIG. It is a longitudinal cross-sectional view which shows the culture state which accommodated the culture container of FIG. 2 in the shielding apparatus for culture containers of FIG. It is a partial longitudinal cross-sectional view which shows the 1st modification of the shielding apparatus for culture vessels of FIG. It is a partial longitudinal cross-sectional view which shows the 2nd modification of the shielding apparatus for culture vessels of FIG. It is (a) partial decomposition | disassembly longitudinal cross-sectional view which shows the 3rd modification of the shielding apparatus for culture vessels of FIG. 1, (b) It is a partial assembly longitudinal cross-sectional view. It is (a) perspective view which shows the 4th modification of the shielding apparatus for culture vessels of FIG. 1, (b) It is a partial longitudinal cross-sectional view. It is a partial longitudinal cross-sectional view which shows the 5th modification of the shielding apparatus for culture vessels of FIG. It is a longitudinal cross-sectional view which shows the 6th modification of the shielding apparatus for culture vessels of FIG. It is a longitudinal cross-sectional view which shows the 7th modification of the shielding apparatus for culture vessels of FIG.

A culture vessel shielding apparatus according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the double container shielding device 1 according to this embodiment includes a storage container 3 that houses the culture container 2 shown in FIG. 2.

  As shown in FIG. 2, the culture vessel 2 is a flask for cell culture, and includes a culture space 2a for storing a mixed solution A of cells and culture solution, and the mixed solution A in the culture space 2a. A cylindrical tubular part (culture space ventilation part) 2c provided with an opening 2b for supplying the liquid and a female screw fastened to a male screw provided on the outer periphery of the tubular part 2c, and closes the opening 2b And a cylindrical cap 2d fastened in this manner.

  The culture vessel 2 is formed in a flat box shape having a relatively wide bottom surface. When the mixed solution A is stored with the bottom surface horizontal, the mixed solution A is stored relatively shallowly and widely in the culture space 2a. It is configured so that the cells contained therein can easily grow by adhering to the bottom surface. The cylindrical portion 2c is provided on one side surface of the culture vessel 2 so as to open obliquely upward with the bottom surface thereof being horizontally disposed.

The cap 2d is provided with a through hole 2e that penetrates the end surface thereof, and a microorganism-impermeable membrane 2f that is disposed so as to close the through hole 2e. The microorganism-impermeable membrane 2f is a membrane that allows gas necessary for cell culture (CO ₂ , O ₂ , N _2, etc.) to pass but does not allow microorganisms to pass through. For example, the porous filter has a pore size smaller than that of the microorganism. And a filter made of a gas permeable plastic material. In the case of a porous filter, the pore size is preferably 0.1 to 0.45 μm, particularly preferably 0.1 to 0.2 μm.

  If the pore size is less than the lower limit value, there is a possibility that sufficient air permeability cannot be obtained, and it is not preferable, and if the pore size exceeds the upper limit value, the passage of microorganisms may not be sufficiently blocked. Examples of the filter material include polypropylene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, polyvinylidene fluoride, polyether sulfone, polyethylene, nylon, cellulose acetate, and nitrocellulose.

  The storage container 3 includes a storage space 3a in which the culture container 2 can be stored, and a storage space ventilation part 3b that opens the storage space 3a to the outside. The container 3 includes a container body 3c and a lid 3d that can open and close the upper opening of the container body 3c. The container body 3c is provided with a handle 3e for facilitating carrying, a protrusion 3f provided on the bottom surface, and an abutting portion 3g for positioning the culture container 2 in the accommodation space 3a. The abutting portion 3g is a rail-like member provided so as to move the culture vessel 2 along the abutting portion 3g in the fitting operation in which the cap 2d of the culture vessel 2 is fitted into the accommodation space ventilation portion 3b. It is.

  The lid 3d is provided with a recess 3h that can accommodate the protrusion 3f at a position corresponding to the protrusion 3f on the bottom surface of the container body 3c. Thereby, the protrusion 3f is accommodated in the recess 3h, and a plurality of storage containers 3 can be arranged in a stacked state.

  The accommodation space ventilation portion 3b is made of a flexible material such as rubber that can be expanded and contracted, is formed in a cylindrical shape having an opening 3i that connects the accommodation space 3a to the inside and outside, and is in a position to close the opening 3i. A water vapor impermeable film 3j is provided. The inner diameter dimension of the cylindrical accommodation space aeration part 3 b is configured to be slightly smaller than the outer diameter dimension of the cap 2 d of the culture vessel 2.

The water vapor impermeable membrane 3j is a polymer membrane that allows passage of gases (CO ₂ , O ₂ , N _2, etc.) necessary for cell culture but does not allow water vapor and microorganisms to pass therethrough. By changing the difference between the permeability coefficient of water vapor and the permeability coefficient of gas necessary for culture depending on the type of polymer, it is possible to control the permeation of water vapor and gas necessary for culture.

  Examples of the material of the water vapor impermeable film 3j include ethylene-vinyl acetate copolymer, polyethylene, polyvinyl chloride, polystyrene, polyolefin, styrene-based elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, and silicone rubber. be able to.

  Thereby, when installing the culture container 2 in the accommodation space 3a, the lower surface of the culture container 2 is placed on the bottom surface of the container body 3c of the storage container 3 to close the cylindrical portion 2c of the culture container 2. The cap 2d is fitted to the inner opening of the accommodation space ventilation section 3b, and is slid horizontally while the side surface of the culture vessel 2 is in contact with the abutting section 3g, so that the accommodation space ventilation section 3b is pushed and expanded by the cap 2d. Can be combined. Then, after the cultivation container 2 is accommodated in the accommodation space 3a, the upper part of the container body 3c is closed by the lid 3d, whereby the preparation for cultivation is completed.

The operation of the culture vessel shielding apparatus 1 according to the present embodiment configured as described above will be described below.
In order to perform cell culture using the culture vessel shielding apparatus 1 according to the present embodiment, the culture vessel 2 in a state where the mixed solution A of cells and culture solution is stored and the cap 2d is fastened to the cylindrical portion 2c is used. Then, the lid body 3d is accommodated in the opened container body 3c, and the lower surface of the culture container 2 is placed on the bottom surface of the container body 3c.

  In this state, one side surface of the culture vessel 2 is abutted against the abutting portion 3g on the bottom surface of the vessel body 3c. Then, while maintaining this state, the cap 2d fastened to the cylindrical portion 2c of the culture vessel 2 is slid on the bottom surface so as to be close to the accommodation space ventilation portion 3b provided in the vessel main body 3c. The cap 2d is fitted while expanding the housing space vent 3b. As a result, the outer peripheral surface of the cap 2d and the inner peripheral surface of the accommodation space ventilation portion 3b are in close contact with each other, so that the microorganism impermeable membrane 2f and the water vapor impermeability are contained in the accommodation space ventilation portion 3b as shown in FIG. A closed space B sandwiched between the conductive film 3j is formed.

Then, the opening of the container body 3c is closed with a lid 3d, transported with a handle 3e, the storage container 3 containing the culture container 2 is stored in an incubator (not shown), and the inside of the incubator is suitable for culture. The culture is started while maintaining the temperature and gas concentration. If necessary, the plurality of storage containers 3 are stacked and accommodated in the incubator by fitting the protrusions 3f into the recesses 3h.
In this case, according to the shielding apparatus 1 for a culture container according to the present embodiment, the temperature in the incubator conducts heat up to the inside of the culture container 2 with the passage of time, and the cells in the culture container 2 also have a temperature suitable for culture. Become. There is no need to maintain a constant humidity in the incubator.

  Even when microorganisms are present in the incubator, the water vapor impermeable membrane 3j and the microorganism impermeable membrane 2e are prevented from entering the culture vessel 2. Further, when the temperature in the culture vessel 2 rises, the culture solution A evaporates, and a part of the culture solution A permeates the microorganism-impermeable membrane 2f of the cap 2d, but the water vapor-impermeable membrane 3j is formed outside thereof. Since it is arrange | positioned, it is prevented that water vapor | steam is discharge | released out of the accommodation space 3a.

Since the closed space B in the accommodation space vent 3d surrounded by the water vapor impermeable membrane 3j and the microorganism impermeable membrane 2f is extremely narrow, it passes through the microorganism impermeable membrane 2f and is released from the culture space 2a. The amount of water vapor is minimized.
Thus, according to the culture vessel shielding apparatus 1 according to the present embodiment, even if a commercially available culture vessel 2 having a cap 2d having a microorganism-impermeable membrane 2f that cannot block the release of water vapor is used, There is an advantage that the culture medium A is prevented from evaporating and the medium is not frequently changed.

  In the present embodiment, the cap 2d is sealed only by fitting the cap 2d to the cylindrical portion 2c provided in the storage container 2, but instead of this, as shown in FIG. The culture container 2 and the storage container 3 are more securely sealed by tightening the inner space from the outer peripheral side of the fitted storage space vent 3b to the inner peripheral surface by the annular seal member 4a or the annular member 4 having a protrusion. You may decide to connect.

  In addition, the cap 2d of the culture vessel 2 is fitted into the flexible accommodation space ventilation portion 3b. Instead, as shown in FIG. 5, the inside of the accommodation space ventilation portion 3b made of a rigid body is used. A sealing member 5 made of an O-ring or the like may be disposed on the peripheral surface, and the cylindrical gap between the accommodation space ventilation portion 3b and the cap 2d may be closed by the sealing member 5.

  In addition, the storage space ventilation part 3b is formed in the container main body 3c of the storage container 3, but instead of this, as shown in FIG. 6A, the cylinder main body 3c and the lid 3d are divided into two cylinders. A bag-shaped water vapor impermeable membrane 6 is formed between the combined accommodation space ventilation portion 3b and the cap 2d. You may decide. That is, as shown in FIG. 6 (b), the container body 3c is closed by the lid 3d with the seal member 6b fitted on the outer periphery of the cap 2d. It is possible to seal the gap between the two by compressing the sealing member 6b between the outer peripheral surface and the outer peripheral surface of the member. Reference numeral 6c denotes an O-ring groove formed by combining the container body 3c and the lid 3d.

  In addition, the cap 2d is fitted into the housing space ventilation portion 3b whose opening is closed by the water vapor impermeable membrane 3j. Instead of this, as shown in FIG. A part of the side wall of the container body 3c may be constituted by a stretchable silicon film 7a, and a hole provided in a part of the silicon film 7a may be closed by the water vapor impermeable film 7b. Accordingly, as shown in FIG. 7B, the end surface of the cap 2d is inclined by pressing the end surface of the cap 2d of the culture vessel 2 against the water vapor impermeable membrane 7b to deform the silicon film 7a. Alternatively, even if the cap 2d is different in size, the end face of the cap 2d is brought into close contact with the water vapor impermeable film 7b, and the opening 2e at the end face of the cap 2d is formed by the deformation of the silicon film 7a. It can be occluded by 7b.

  In the present embodiment, as shown in FIG. 8, a culture vessel 8 in which the opening of the cap 2d is closed with a water vapor impermeable membrane 8a may be employed. In this case, the storage container 3 may be one in which the opening 3i is closed with the microorganism-impermeable membrane 8b. Thereby, since water vapor | steam is interrupted | released from the culture container 8, it does not need to perform frequent culture medium exchange.

In the present embodiment, as shown in FIG. 9, a bag-shaped water vapor impermeable membrane 6 in which the opening 6a can be expanded and contracted by a sealing member 6b such as a rubber band may be employed. In this case, not only the culture vessel 2 composed of the above-described cell culture flask but also a vent formed by a gap formed between the vessel main body 9a and the lid 9b, such as a petri dish as shown in FIG. In the case of the culture vessel 9 that performs the above, the evaporation of the culture solution A in the culture vessel 9 can be suppressed by covering the gap between the vessel main body 9a and the lid body 9b.
Further, the present invention is not limited to the bag shape, and as shown in FIG. 10, a member in which seal members 10b and 10c such as rubber bands are arranged at both ends of the tubular water vapor impermeable film 10a may be adopted.

Next, the relationship between the closed space B closed by the water vapor impermeable membranes 3j, 6, 8a, and 10a and the culture space 2a will be described as follows.
Since the humidity in the sealed space B is about 50 to 60% equivalent to the work space at the time of assembly, the water vapor of the culture solution A moves from the culture space 2a to the closed space B during the culture. Therefore, the closed space B is preferably as narrow as possible.

As a result of arranging the culture vessel 2 alone in the incubator and measuring the evaporation amount, it was found that about 1% of the culture solution A evaporates in one day.
Although the frequency of medium exchange varies depending on the cell type, assuming that it is at least 1 day and 7 days at the longest, the amount is about 1 to 7% of the culture medium A.

Assuming that the volume of the culture space 2a of the culture vessel 2 is vcm ³ , the culture solution A is stored in about 5% of the culture space 2a. <W <7/100 × 5/100 × v (1)
It becomes.

Next, the evaporation amount W when the closed space B is formed is calculated.
Assuming that the temperature is 37 ° C., the humidity of the enclosed space B is 50%, and the volume of the enclosed space B is V, the saturated water vapor amount at the temperature of 37 ° C. is 43.9 g / m ³ .
Evaporation amount W = saturated water vapor amount of volume V−water vapor amount already contained in volume V
= 43.9 × V / 10 ⁶ −43.9 × 50/100 × V / 10 ⁶
= 21.95 × V / 10 ⁶ (2)
It becomes.

The volume V of the closed space B when the evaporation amount w and the evaporation amount W are equal is obtained from the above equations (1) and (2).
5 × 10 ⁻⁴ × v <21.95 × V / 10 ⁶ <3.5 × 10 ⁻³ × v
That is,
22.8v <V <159.6v
It becomes.

  Therefore, if the volume V of the closed space B is about 160 times or less the volume v of the culture space 2a of the culture vessel 2, the culture solution A is evaporated more than when the culture vessel 2 is contained in the incubator and cultured. The amount can be kept lower than 7 days, and if it is about 23 times or less, it can be kept lower than 1 day.

DESCRIPTION OF SYMBOLS 1 Shielding device for culture vessels 2a Culture space 2c Cylindrical part (culture space ventilation part)
2f Microorganism-impermeable membrane 3 Storage container 3a Storage space 3b Storage space ventilation part (fitting cylinder part)
3j, 6, 8a, 10a Water vapor impermeable membrane 5 Seal member 7a Silicon film (extension member)

Claims (7)

  A culture space provided with a culture space for culturing cells and a culture space aeration unit allowing ventilation into the culture space, and detachably attached to the culture space, and disposed at a position for closing the culture space aeration unit. A shielding device for a culture vessel, which is partially provided with a water vapor impermeable membrane that allows gas necessary for cell culture to pass and does not allow water vapor and microorganisms to pass through. A storage container including a storage space in which the culture container can be stored, and a storage space ventilation section that allows ventilation to the storage space;
The microorganism-impermeable membrane that allows gas necessary for cell culture to pass and does not allow microorganisms to pass therethrough is provided in the culture space aeration portion, and the water vapor impermeable membrane is provided in the accommodation space aeration portion. The shielding apparatus for culture containers as described. The culture space aeration part protrudes to the side of the culture vessel, and is configured by closing the cylindrical part opening to the culture space with the microorganism-impermeable membrane,
On the side surface of the storage container, a fitting cylinder part for fitting the cylindrical part of the culture container is provided,
The shielding device for a culture vessel according to claim 2, wherein the fitting tube portion is closed by the water vapor impermeable membrane.   The culture vessel shielding device according to claim 3, wherein the fitting tube portion is made of an elastic material capable of expanding and contracting.   The culture vessel shielding apparatus according to claim 3, further comprising a seal member that closes a cylindrical space between an inner surface of the fitting cylindrical portion and an outer surface of the cylindrical portion.   The housing space ventilation portion is configured by closing a through hole formed in an extending member made of an extendable material provided on at least a part of a side surface of the storage container with the water vapor impermeable film. The shielding apparatus for culture vessels according to claim 2. A storage container including a storage space in which the culture container can be stored, and a storage space ventilation section that allows ventilation to the storage space;
The said water vapor impermeable membrane is provided in the said culture space ventilation | gas_flowing part, The microorganisms impermeable film which allows the gas required for cell culture to pass through and does not allow microorganisms to pass through is provided in the said accommodation space ventilation | gas_flowing part. The shielding apparatus for culture containers as described.

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