JP5592080B2 - Cell culture method and cell culture system - Google Patents

Description

  The present invention relates to a system for culturing cells under aseptic conditions, and more particularly to a cell culture method and a cell culture system suitable for culturing human cell tissue for transplantation medicine.

  An example of a method for preventing cross-contamination when a plurality of cell cultures are sequentially performed is disclosed in Patent Document 1. The aseptic culture apparatus described in Patent Document 1 includes an isolator-type work chamber, a plurality of isolator-type incubators (incubators), and a sealed transfer container that bridges the culture containers between them. The work room is equipped with equipment such as a centrifuge and a microscope. The sealed transfer container, the working chamber, and the incubator are provided with connection ports that can be contacted and separated while maintaining sterility. The raw tissue sample is pretreated in the work room, the obtained culture (cells) is stored in the culture container, the culture container is transferred to the incubator using a sealed transfer container, and the cell culture is performed in the incubator. Do. After completing the culturing operation for a certain cell, gas sterilize the working chamber, the used incubator and the sealed transfer container, and then start the culturing operation for the next cell. Prevents cross contamination.

  In addition, Patent Document 2 describes an example of a method for preventing contamination of a culture vessel. The canister described in Patent Document 2 is a box that can store a plurality of culture vessels, includes a filter for ventilation and moisture permeability, and prevents cross contamination by having a sealable structure other than that, It also has a transparent wall for cell observation.

  Furthermore, Patent Document 3 describes a method for storing sterilized consumables and the like in an area with a high degree of cleanliness. In the warehousing method described in Patent Document 3, the area is divided into a plurality of areas having gradual cleanliness so that the cleanliness increases from the outside toward the inside, and a pass box is provided at a connection portion between the areas. Is provided. When multiple wrappings are performed on sterilized products using a sterilized bag and the sterilized products are stored in an area with a high degree of cleanliness, the sterilized product packaging (sterilized bags) is discarded from the outside in each pass box. This prevents contamination from the environment adhering to the outer surface of the package applied to the sterilized product from entering an area with a high degree of cleanliness.

JP 2005-278565 A Utility Model Registration No. 3134034 JP 2008-239168 A

  The following problems can be considered in the conventional techniques represented by the above-mentioned patent documents. First, as in Patent Document 1, when a device such as a centrifuge or a microscope is installed and used inside an aseptic working chamber, a sterilizing gas (hydrogen peroxide gas having a high oxidizing power) is used when the working chamber is gas sterilized. ) Corrodes these devices and degrades their performance, causing malfunctions and shortening the service life.

  Further, as in Patent Document 2, when equipment such as an incubator or a microscope is installed outside the sterile culture chamber, a large and difficult to handle canister is required to prevent contamination of the cell sample. Moreover, this canister has the subject that it cannot respond to centrifugation.

  Further, a method of combining Patent Document 2 and Patent Document 3 and replacing the canister in Patent Document 2 with the packaging described in Patent Document 3 is also conceivable. In other words, containers such as centrifuges (centrifuge tubes) and cell culture containers are packaged in sterilized bags and then operated using the external device to keep contamination from the external device on the external surface of the package. When the container is placed inside the aseptic culture chamber, it is expected that the packaging (sterilization bag) is discarded to remove contamination from the outside together with the packaging and prevent the aseptic culture chamber from being contaminated. However, there are cases where contamination cannot always be prevented simply by combining Patent Document 2 and Patent Document 3. That is, if the centrifuged container is returned to the inside of the sterile culture chamber, even if the above-described packaging is performed, for example, the outer surface of the container is contaminated by the cell sample in the container (described later), and the sterile culture chamber is There is a problem that it may be contaminated.

  The above problem will be further examined. For example, if a centrifuge or a microscope is installed outside an aseptic area such as an aseptic culture room (hereinafter referred to as an outside of the aseptic area), these instruments are not necessarily guaranteed to be sterilized, and are therefore contaminated with microorganisms and other cells. there is a possibility. Cell samples are stored in containers such as centrifuge tubes and culture containers. When these containers are stored outside the sterile area and subjected to centrifugation or microscopic observation, microorganisms and other cells are removed from these devices. May adhere to the outer wall of the container. When this container is carried into the aseptic area, the contamination attached to the outer wall of the container is brought into the aseptic area and there is a risk of contaminating the aseptic area. When handling the container in an aseptic area in an open system (opening the lid or the like), there is a risk that the contamination further spreads inside the container, and as a result, the cell sample is contaminated. This risk is due to the above-mentioned method, that is, packaging is carried out according to Patent Document 3 when the container is carried out from the sterile area to the outside of the sterile area, and the packaging is discarded when the container is carried from outside the sterile area into the sterile area. In principle, this can be prevented.

  On the other hand, a case where contamination cannot be prevented by the above-described method alone will be examined in detail. In other words, particularly when centrifugation was performed using a centrifuge tube, it was confirmed that a very small amount of the contents of the container leaked out of the container. In this case, the contents leaking out of the container can adhere not only to the inner wall of the package but also to the outer wall of the container. When the container is brought from outside the aseptic area into the aseptic area, the inner wall of the package is decontaminated by discarding the package, but the outer wall of the container is not decontaminated. Accordingly, it is considered that the contamination attached to the outer wall of the container is brought into the aseptic region and contaminates the aseptic region. Hereinafter, such contamination originating from the cell sample itself in the container is referred to as intrinsic contamination. Also, contamination originating from outside the sterile field is called extrinsic contamination.

  As a result of examining the cause of the leakage of the contents of the container as described above to the outside of the extremely small quantity container, the following mechanism was assumed. That is, containers such as centrifuge tubes are provided with a lid. The seal between the lid and the container body may not always be perfect. In addition, when the lid is repeatedly opened and closed and the cell sample is repeatedly attached to the lid, the cell sample gradually diffuses in the outer circumferential direction in the space between the lid and the container body, and the inside of the container formed by the container body and the lid Cell samples may leak out of space. When high acceleration is applied by a centrifuge or the like, there is a risk that the cell sample moves from the inside of the container to the outside of the container via the screw portion of the lid.

  As a result, it was confirmed that the intrinsic contamination spreads from the outer wall of the container into the aseptic region and contaminates the aseptic region (contamination in the aseptic region due to intrinsic contamination). In particular, when a plurality of cell samples are handled, it is conceivable that if the intrinsic contamination by the first cell sample spreads to the second cell sample, it may cause cross contamination (cross contamination due to intrinsic contamination).

  An object of the present invention is to provide a cell culture method and a cell culture system that can provide high-purity and safe pharmaceuticals, cells, and the like quickly and at low cost.

In order to achieve the above object, the cell culture method according to the present invention is a cell culture method in which a container for storing a cell sample is transported between an aseptic area and an outside of the aseptic area. When transporting, the container is packaged by applying a first package using a bacteria-impermeable sheet material, and when the container is transported into the aseptic area, the first package is removed, and the container contamination preventing member for preventing the contamination by a cell sample of the inner, and characterized by applying to the outer surface of the container.

A cell culture system according to the present invention that embodies the above method is, firstly, in a cell culture system that transports a container for storing a cell sample between an aseptic area and an outside of the aseptic area, When the container is transported out of the aseptic area, the first packaging using the bacteria-impermeable sheet material is applied to the container and packaged, and when the container is transported into the aseptic area, the first packaging is performed. together with the first handling means for removing the packaging are provided, the contamination preventing member for preventing the contamination by cell sample in the container, characterized in that the second handling means for performing an outer surface of the container is provided.

  Second, the first handling means includes a packaging mechanism for applying a first packaging to the container, and an unpacking mechanism for unpacking and removing the first packaging, and the second handling means includes the At least one of an addition mechanism for adding a contamination prevention member to the container and a removal mechanism for removing the contamination prevention member is provided.

  3rdly, when the said contamination prevention member conveys a container in an aseptic area | region, it is the 2nd packaging given closely to the said container outer surface using a microbe-impermeable sheet-like material. Features.

Fourthly , the contamination preventing member is applied in close contact with the outer surface of the container using a bacteria-impermeable sheet-like material when the container is transported outside the aseptic area, together with the first packaging. the packaged in a container, in transporting the container in a sterile area, wherein the first a second packaging Ru removed from the container together with packaging.

  Fifth, the contamination preventing member is a disinfectant capable of sterilizing a cell sample leaked from the container enclosed in the first package when the container is transported outside the aseptic area. To do.

Sixth, a centrifuge is provided outside the aseptic region, the container is a centrifuge tube, and the first package is configured using a fungus-impermeable material.
Seventh, an incubator is provided outside the aseptic region, the container is a cell culture container, and the first package is configured using a fungus-impermeable and gas-permeable sheet material. And

  According to the cell culturing method and the cell culturing system of the present invention, first, the endogenousness derived from the cell sample in the container is obtained by applying the first package to the container and operating the cell sample outside the sterile region. Contamination outside the aseptic area due to contamination can be prevented, and contamination in the aseptic area due to exogenous contamination from outside the container can be prevented by removing the first package when the container is transported into the aseptic area. Furthermore, by providing a contamination preventing member on the outer surface of the container, it is possible to prevent contamination in the sterile region due to intrinsic contamination derived from the cell sample in the container. From these facts, it is possible to maintain a high degree of cleanliness inside and outside the aseptic area, thereby eliminating the risk of cross-contamination in the cell culture system, and providing highly pure and safe pharmaceuticals and cells.

  In addition, since the devices and instruments that apply operations to cell samples are provided outside the sterile field, there is no need to sterilize these devices and instruments frequently, and therefore the sterilization gas of these devices and instruments is not required. Damage due to such as can be prevented. For this reason, the operating rate of the cell culture system is increased, the processing capacity is improved, and pharmaceuticals, cells, etc. can be provided quickly and at low cost.

  Second, the first handling means includes a packaging mechanism that applies the first packaging to the container, and an unpacking mechanism that unpacks and removes the first packaging, and the second handling means is a contamination preventing member for the container. By providing at least one of an addition mechanism for adding a contamination and a removal mechanism for removing a pollution prevention member, it is possible to prevent contamination caused by the worker by eliminating manual work by the worker and improve work efficiency, and to intrinsically Contamination and extrinsic contamination can be suppressed.

  Third, even if endogenous contamination derived from the cell sample is attached to the outer wall of the sample container, it can be concealed and contained by the second packaging, and the outside of the sample container provided with the second packaging. Endogenous contamination on the surface can be prevented.

  Fourthly, even if the outer surface of the first package is contaminated due to exogenous contamination, the first packaging is discarded, so that it is possible to prevent exogenous contamination in the sterile region and the sample container, In the same way as described above, intrinsic contamination can also be prevented.

Fifth, by sealing a bactericidal agent in the first package, cells exposed to the outside of the sample container can be sterilized to prevent endogenous contamination.
Sixth, by using a centrifuge tube as a sample container, it is possible to perform centrifugation without transferring bacteria while preventing intrinsic contamination and extrinsic contamination, so that work efficiency is improved.

  Seventh, since the container is a cell culture container, and the first package is configured using a sheet material that is impermeable to bacteria and gas permeable, cell culture can be performed with the first package applied. .

BRIEF DESCRIPTION OF THE DRAWINGS It is the schematic which shows typically the whole structure of 1st Embodiment of the cell culture system which concerns on this invention. FIG. 2 is a schematic diagram schematically showing the configuration of one of the pass boxes in FIG. 1. It is the schematic which represents typically the structure of one of the pass boxes in 2nd Embodiment of the cell culture system which concerns on this invention. It is the schematic which represents typically the structure of one of the pass boxes in 3rd Embodiment of the cell culture system which concerns on this invention.

The best mode for carrying out the present invention will be described below with reference to the drawings.
The present invention relates to a cell culture method in which a container for storing a cell sample is transported between a sterile area and a non-sterile area, and when the container is transported outside the sterile area, a bacteria-impermeable sheet is used. First packaging using a material is applied to the container and packaged. When the container is transported into the aseptic area, the first packaging is removed, and contamination of the outer surface of the container by the cell sample in the container is removed. The anti-contamination member to be prevented is applied to the outer surface of the container, and is embodied by the following embodiment.

[A] First embodiment (FIGS. 1 and 2)
FIG. 1 is a schematic diagram schematically showing the overall configuration of the first embodiment of the cell culture system according to the present invention. The cell culture system 11 includes a clean room 12 in which an aseptic operation device 13, a centrifuge (hereinafter referred to as a centrifuge) 14, a carbon dioxide incubator (hereinafter referred to as an incubator) 15, and a transport device 16 are installed. Is done. The aseptic operation device 13 includes a plurality of pass boxes 17, and some of the pass boxes 17 are devices / apparatuses (hereinafter referred to as external devices) that apply operations to cell samples such as a centrifuge 14 and an incubator 15 through a transport device 16. Device). The clean room 12 and the aseptic operation device 13 include a clean air supply mechanism (not shown) using a HEPA filter, and may include an ultraviolet germicidal lamp, a gas sterilization mechanism, and the like (not shown).

  As will be described in detail later, the inside of the aseptic operation apparatus 13 is in the aseptic area, and the clean room 12, the centrifuge 14, the incubator 15, and the transport apparatus 16 are outside the aseptic area, and the sample container 18 ( 2) is transported between the inside and outside of the sterile area.

  Next, an example of a typical configuration of one or more pass boxes 17 provided in the aseptic operation device 13 will be described with reference to FIG. The pass box 17 includes a material carry-in port 19, a sample carry-out port 20, a sample carry-in port 21, a sample adjustment port 22, and a material carry-out port 23. The material carry-in port 19 includes a carry-in door 24 and carry-out doors 25 and 26, and each of the other ports 20, 21, 22, and 23 similarly includes a carry-in / out door as shown below, for example. Each port 19 to 23 of the pass box 17 includes an air cleaning mechanism (not shown) based on a HEPA filter or the like, and may include an ultraviolet germicidal lamp, a gas sterilizing mechanism, or the like.

  The material carry-in port 19 communicates with the clean room 12 through the carry-in door 24, and the material carry-out port 23 communicates with the clean room 12 through the carry-out door 27. The sample carry-out port 20 communicates with the transport device 16 through the carry-in door 28, and the sample carry-in port 21 communicates with the transport device 16, and further through the transport device 16, external devices (such as the centrifuge 14 and the incubator 15; not shown in FIG. 2) Communicate with. The sample carry-out port 20 communicates with the inside of the aseptic operation device 13 through the carry-in door 30 and the sample adjustment port 22 through the carry-out door 31. The sample carry-in port 21 communicates with the sample adjustment port 22 via the carry-in door 32.

Next, an outline of the operation of the cell culture system 11 will be described.
The clean room 12, the aseptic operation device 13, and the pass box 17 maintain predetermined cleanliness by an air cleaning mechanism, a sterilization mechanism, manual sterilization, and the like, and protect the cell samples handled in them from contamination. The pass box 17 and the aseptic operation device 13 maintain a higher degree of cleanliness than the clean room 12, and the inside of the aseptic operation device 13 is an aseptic area, and a cell sample is handled in an open system. Is possible. That is, in the clean room 12, the cleanliness level is differentiated depending on the location in consideration of the balance between the required cleanliness and the cost and labor required to maintain the cleanliness. In the present embodiment, the inside of the aseptic operation device 13 is handled within the aseptic area, and the other is handled outside the aseptic area. That is, the centrifuge 14, the incubator 15, the transport device 16, the clean room 12, and the like are outside the aseptic area.

  Prior to the use of the cell culture system 11, consumables such as the sample container 18 and cell samples and reagents (not shown) are sterilized as necessary through the loading door 24 and the unloading door 26 of the material loading port 19. It is carried into the aseptic operation device 13. The aseptic operation device 13 automatically performs operations such as medium exchange and passage on the cells. In the subculture operation, there is a step of detaching cells from the culture vessel using an enzyme solution or the like, obtaining a cell suspension, and separating the cells from the enzyme solution by centrifugation. This process will be described in detail below as an example.

  When the cell suspension is taken out to the centrifuge 14 provided outside the aseptic operation device 13, the cell suspension is first stored in the sample container 18 and sealed. Using the first packaging material 33 carried in from the material carry-in port 19, the sample container 18 is packaged in the sample carry-out port 20, and the sample container 35 is provided with the first package 34. Here, the packaging is sealed by using the first packaging material 33 that does not allow fungi to pass, thereby providing a bacteria-impermeable packaging. Then, the sample container 35 is carried out to an external device such as the centrifuge 14 via the transfer device 16 or the like.

  Since the sample container 35 is provided with the first package 34, even if the cleanliness of the transport device 16 or the centrifuge 14 is insufficient and is affected by the extrinsic contamination, the extrinsic contamination is not affected by the first packaging 34. It remains outside and the internal sample container 18 is not contaminated. On the contrary, even if endogenous contamination derived from the contents (cell sample) is generated in the sample container 18 due to an operation such as centrifugal separation or conveyance that is subjected to a large acceleration or vibration, it remains inside the first package 34. The external devices such as the external transport device 16 and the centrifuge 14, that is, the outside of the aseptic area is not contaminated by intrinsic contamination. Therefore, even when different types of cell samples are continuously cultured, cells or microorganisms derived from the first cell sample are not removed from the second cell via an external device such as the external transfer device 16 or the centrifuge 14. There is no cross contamination to cell samples. For this reason, it is not necessary to sterilize the outside of the aseptic region with a highly invasive method such as sterilization gas.

  When the sample container 35 is brought into the aseptic operation device 13 from the centrifuge 14 after the centrifugation, the first package 34 of the sample container 35 is unpacked (unpacked) at the sample carry-in port 21 to obtain the sample container 18. Waste material 36 of the first packaging 34 generated by unpacking is discharged via the material carry-out port 23. As a result, even if the outer surface of the first package 34 is contaminated by exogenous contamination, the first package 34 is discarded. No sexual contamination. The same applies to the contamination of the inner surface of the first package 34 due to intrinsic contamination.

  Next, the second packaging material 37 is closely attached to the sample container 18 and packaged by the sample adjustment port 22, and the sample container 39 is provided with the second packaging 38 and brought into the aseptic operation apparatus 13. . Here, the second packaging 38 was also a bacteria-impermeable packaging. As a result, even if intrinsic contamination derived from the cell sample is attached to the outer wall of the sample container 18, it can be shielded and contained by the second package 38, in other words, the second package 38 is applied. There is no intrinsic contamination on the outer surface of the sample container 39.

  Therefore, when the sample container 39 is transported or the like inside the aseptic operation apparatus 13, the inside of the aseptic operation apparatus 13, that is, the inside of the aseptic area is not contaminated by intrinsic contamination. For this reason, even when different types of cell samples are continuously cultured, the risk of cells or microorganisms derived from the first cell sample cross-contaminating into the second cell sample can be kept low. It is not always necessary to gas sterilize the entire aseptic area. The place where the cell sample is handled in the release system by opening the lid of the sample container 18 can be dealt with by local sterilization or the like.

Next, the packaging mechanism 40 and the unpacking mechanism 41 as the first handling means will be described.
The packaging mechanism 40 applies the first packaging 34 to the sample container 18 when the sample container 18 is carried out of the aseptic operation device 13 in the aseptic area to the outside of the aseptic area such as the centrifuge 14 and the incubator 15. The container 18 is packaged to form a sample container 35. In addition, the unpacking mechanism 41 unpacks and removes the first package 34 of the sample container 35 when the sample container 35 is carried into the aseptic area from outside the aseptic area. By using these, work efficiency can be improved and intrinsic contamination and extrinsic contamination can be suppressed. The packaging mechanism 40 is installed in the sample carry-out port 20, and the unpacking mechanism 41 is installed in the sample carry-in port 21.

  In particular, as the packaging mechanism 40, a bag-making packaging (Package, form, fill, seal) apparatus is employed. In this apparatus, a first package 34 conforming to the shape of the sample container 18 is formed by using the first packaging material 33, the sample container 18 is completely placed in the first package 34, and the first package 34 is heat sealed. By closing and sealing, the sample container 35 having the first packaging 34 is provided. As the packaging mechanism 40 of the sample carry-out port 20, various hermetic packaging devices according to the shape of the container using the blister pack method, the blow-fill-seal method, etc., in addition to the bag making packaging described above. Can be adopted.

  As the first packaging material 33, a material having no permeability with respect to microorganisms and cells, that is, a bacteria-impermeable material was used. In the case where a centrifuge tube is used as the sample container 18 in the present embodiment, the first packaging material 33 is made of polypropylene that has both bacteria impermeability and mechanical strength that can withstand centrifugal force acting on the sample container 18. A seat was adopted. The first packaging material 33 can be used after sterilizing various resin-impermeable synthetic resin materials such as polyethylene, polystyrene, and PET in addition to polypropylene. By using a centrifuge tube as the sample container 18 in this manner, centrifugation can be performed without transferring cells while preventing intrinsic contamination and extrinsic contamination, so that work efficiency is improved.

  The shape of the first package 34 can be arbitrarily selected according to the shape of the sample container 18, but a simple shape and the minimum necessary material are used for cost effectiveness, waste reduction, and operability improvement. The packaging used is preferred. For example, as the shape of the first package 34, a cylindrical shape having high shape compatibility with the bucket of the centrifuge was adopted. Further, by providing a structure or member such as a notch or a tape, the subsequent unpacking process can be facilitated. As a method for closing the bag, in addition to heat sealing, an adhesive tape, an adhesive, or the like can be used.

  In the present embodiment, since a material made of a sheet-like plastic material having sufficient strength is packaged as the first packaging material 33, the external dimensions and weight of the sample container 35 to which the first packaging 34 is applied can be kept small. it can. As a result, the sample container 35 provided with the first package 34 has both sufficient mechanical strength and high operability to withstand conveyance and centrifugation.

Next, the additional mechanism 42 as the second handling means will be described.
The additional mechanism 42 uses the second packaging material 37 that is impermeable to bacteria as a sample for the second packaging 38 as a contamination preventing member that prevents contamination of the outer surface of the sample container 18 by the cell sample in the sample container 18. It is applied in close contact with the outer surface of the container 18. The additional mechanism 42 is installed in the sample adjustment port 22.

  The second packaging 38 is configured such that the second packaging material 37 is in close contact with the outer wall surface of the sample container 18. Thereby, even if the intrinsic contamination derived from the sample container 18 is attached to the outer wall of the sample container 18, the outer surface of the sample container 18 is adhered and covered with the bacteria-impermeable second packaging 38. Can be prevented, and endogenous contamination can be shielded and contained. As a result, it is possible to prevent diffusion of intrinsic contamination into the aseptic operation apparatus 13, that is, the aseptic area.

  The additional mechanism 42 installed in the sample adjustment port 22 is a packaging device, and various packaging methods suitable for close-packaging can be adopted as the packaging device. For example, a shrink wrap method using a bag-like or cylindrical heat-shrinkable packaging material, a sealing method using a highly extensible sheet such as parafilm, a sealing method using an adhesive sheet, a heat sealing method, a blow-fill A packaging device using a seal method or the like can be used.

  In order to more effectively suppress the possibility of diffusion of intrinsic contamination, after unpacking the first package 34 and before applying the second package 38, the outer surface of the sample container 18 is subjected to an ultraviolet germicidal lamp or a sterilizer. It can also be decontaminated by various known methods such as an agent. When the ultraviolet sterilization method is applied to the centrifuge tube as the sample container 18, for example, a region where the pellet of the cell sample is present (the lower end of the centrifuge tube) is shielded with an ultraviolet opaque material, and only the outer surface of the sample container 18, particularly For example, ultraviolet light is intensively applied to the lid portion that is easily contaminated while rotating the centrifuge tube around the central axis. Thereby, the outer surface of the sample container 18 is decontaminated while preventing the adverse effect of ultraviolet rays on the cell sample.

  In this embodiment, the sample container 18 having a lid or the like with a septum is adopted in relation to the aspect of the second package 38. Here, the septum is a member made of an elastic material that can be punctured (pierceable) with an injection needle-like nozzle or the like, which is employed in a vacuum blood collection tube or the like. The sample container 18 provided with this septum is adopted, and the container body and lid of the sample container 18 are provided with a second packaging 38, and the second packaging material 37 of the second packaging 38 is also made of a material that can be punctured with a nozzle or the like. adopt. Thus, the contents of the sample container 39 can be handled without opening and closing the lid via the second package 38 and the septum of the sample container 18. That is, since the cell sample can be handled with the second packaging 38 applied, the generation and diffusion of endogenous contamination can be prevented to a higher level.

  As a modification of the present embodiment, the following configuration can be adopted with respect to the aspect of the second package 38. That is, instead of completely putting the container body and the lid of the sample container 18 inside the second packaging material 37, the sample container 18 is halfway (excluding the lid) in the second packaging material 37 formed in a half bag shape, for example. Put only the container body) and seal tightly by shrink wrap method. That is, the second package 38 is a semi-sealed system. As a result, the sample container 39 provided with the second package 38 opens and closes the lid and the contents in the container body while the second package 38 encloses the side surface of the container body below the lid part with the highest risk of intrinsic contamination. This has the effect of not hindering the removal of objects. In this case, no septum is required on the lid. As a form of the second packaging 38 suitable for this modified form, the adhesive sheet wrapping, the shrink wrap method, etc. can be adopted as described above, and the second packaging 38 is in close contact with the outer surface of the container body (excluding the lid) of the sample container 18. Then, a simple method of using a semi-container (container without a lid) that can be stored as packaging and storing the container body of the sample container 18 in the semi-container can also be adopted.

  By the way, in addition to the case where the centrifuge 14 is used as an external device, when the incubator 15 is applied as an external device, a cell culture container is used as the sample container 18. In this case, as a characteristic of the first packaging material 33, Gas permeability is required in addition to bacteria impermeability. Materials that have both properties include Teflon (registered trademark), cellulose, polyolefin, polyacetal, polyamide, polyester, polysulfide, ultrahigh molecular weight polyethylene porous film, sterilized paper, glassine paper, nonwoven fabric, high density polyethylene fiber sheet, There are microporous materials such as Tyvek®, fine porous film and membrane filter. The sheet made of this microporous material is used alone or in combination with another fungus-impermeable material and sterilized as necessary, and used as the first packaging material 33. Further, for example, various composite materials such as a material having a high virus barrier property obtained by coating a nonwoven fabric with a gas permeable polyethylene film can be selected as the first packaging material 33.

  By adopting a microporous material that is both impermeable to bacteria and gas permeable, oxygen, carbon dioxide, etc. can be permeated, so that cell culture can be performed with the first packaging 34 applied. Furthermore, by selecting or using a material with high transparency, it becomes possible to observe cells in the sample container 18 with the first packaging 34 applied.

From the above, according to the present embodiment, the following effects (1) and (2) are obtained.
(1) By applying the first package 34 to the sample container 18 and manipulating the cell sample outside the aseptic area (for example, centrifugation), the sample container 18 is outside the aseptic area due to intrinsic contamination derived from the cell sample in the sample container 18. Contamination can be prevented. Further, by removing the first package 34 when the sample container 18 is carried into the aseptic area (in the aseptic operation device 13), contamination in the aseptic area due to exogenous contamination originating from outside the sample container 18 can be prevented. Further, by applying the second package 38 to the outer surface of the sample container 18, contamination in the sterile region due to intrinsic contamination derived from the cell sample in the sample container 18 can be prevented. From these facts, it is possible to maintain a high degree of cleanliness inside and outside the sterile region, so that the risk of cross-contamination in the cell culture system 11 can be eliminated, and highly pure and safe pharmaceuticals and cells can be provided.

  (2) Since the devices / equipment for applying the operation to the cell sample are provided as external devices outside the aseptic area, it is not necessary to sterilize these devices / equipment at a high frequency. Can prevent damage caused by sterilization gas. For this reason, the operating rate of the cell culture system 11 is increased, the processing capacity is improved, and pharmaceuticals, cells, and the like can be provided quickly and at low cost.

[B] Second embodiment (FIG. 3)
FIG. 3 is a schematic view schematically showing the configuration of one of the pass boxes in the second embodiment of the cell culture system according to the present invention. In the second embodiment, the same parts as those in the first embodiment are denoted by the same reference numerals, and the description is simplified or omitted.

The cell culture system 51 of the present embodiment is different from the above embodiment in the configuration of the sample adjustment port 53 of the pass box 52 and the sample container 55.
Specifically, the second package 54 for preventing contamination of the outer surface of the sample container 18 by the cell sample in the sample container 18 is in close contact with the outer surface of the sample container 18 using a bacteria-impermeable sheet material. The sample container 18 is in the form of a sample container 55 with a second package 54 applied thereto. Further, the additional mechanism 42 is not installed in the sample adjustment port 53 (therefore, the second packaging material 37 is not used), and the removal mechanism 56 for removing the second packaging 54 of the sample container 55 serves as the second handling means. is set up. By providing the addition mechanism 42 and the removal mechanism 56, it is possible to improve work efficiency and suppress intrinsic contamination and extrinsic contamination.

  The sample container 55 provided with the second package 54 is packaged by the first package 34 when being transported out of the sterile region such as the centrifuge 14 by the packaging mechanism 40 in the sample transport port 20 of the pass box 52. The sample container 57 is formed. When the sample container 57 is carried from outside the aseptic area into the aseptic area (in the aseptic operation device 13), first, the first package 34 is unpacked by the unpacking mechanism 41 at the sample loading port 21 of the pass box 52. The second package 54 is unpacked and removed by the removal mechanism 56 at the sample adjustment port 53, and only the sample container 18 is carried into the aseptic operation device 13.

  The operation of the cell culture system 51 of the present embodiment is the same as the modification of the first embodiment in that the container body of the sample container 18 is packaged, but the following points are different. That is, in the form of a sample container 55 in which the second package 54 is closely attached to the sample container 18, after sterilization, the sample is carried into the aseptic operation device 13 through the material carry-in port 19. Then, after putting the sample in the sample container 55 and sealing it, the sample delivery port 20 performs hermetically packaging using the first packaging 34 by the packaging mechanism 40. Specifically, the sample package 57 is formed in the first package 34 on the second package 54, and is then carried out of the aseptic operation device 13.

  When the sample container 57 is brought into the aseptic operation device 13 from outside the aseptic region after the processing in the external device (outside the aseptic region) such as the centrifuge 14, the first packaging is first performed by the unpacking mechanism 41 at the sample carry-in port 21. 34 is unpacked and the sample container 55 which gave the 2nd package 54 is obtained. Next, the second package 54 is unpacked by the removal mechanism 56 at the sample adjustment port 53 and brought into the aseptic operation apparatus 13 in a state where only the sample container 18 is provided. The waste material 58 of the second package 54 and the waste material 36 of the first package 34 generated by unpacking are discharged from the material carry-out port 23.

  Even if the intrinsic contamination derived from the contents (cell sample) of the sample container 55 occurs in the process of the external device such as the centrifuge 14 by the above-described operation, it is only the contamination of the second package 54. If the second package 54 is removed as the waste material 58 at the sample adjustment port 53 and is discharged from the material carry-out port 23, the sample container 18 is not contaminated, so that no contamination is mixed inside the aseptic operation device 13. Therefore, even if the sample container 18 is transported inside the aseptic operation device 13, the inside of the aseptic operation device 13, that is, the aseptic region, is not contaminated by intrinsic contamination.

  As the second package 54 in the sample container 55, for example, an adhesive sheet may be used, and a state in which the sample container 18 is tightly wound around the sample container 18 may be employed. More preferably, the adhesive sheet with perforation for cutting is wound a plurality of times around the circumference, and the adhesive sheet is removed by the length of the perforation interval when it is carried into the aseptic area. Is possible. With the latter configuration, the sample container 55 with the second package 54 can be unpacked a plurality of times, and the clean surface can be repeatedly exposed on the surface of one sample container 55. In this case, one sample container 55 Can be carried in and out repeatedly. Further, by setting the interval between the perforations to be equal to or greater than the circumference, the perforation for the next cutting is not exposed on the outer peripheral surface, so that the packaging integrity of the second packaging 54 can be maintained high. As a method for facilitating the cutting, various known methods such as a method of adhering a string or a tape in addition to perforation, a method of reducing the thickness of the sheet of the cut portion, and the like can be adopted.

From the above, according to the present embodiment, in addition to the same effects as the effects (1) and (2) of the first embodiment, the following effect (3) is achieved.
(3) By applying the second package 54 in close contact with the sample container 18 in advance using a second packaging material (not shown), the removal mechanism 56 allows the second package 54 to be attached to the sample adjustment port 53. Since unpacking is sufficient and the packaging work is not necessary, the operation is simple and the process time can be shortened.

[C] Third embodiment (FIG. 4)
FIG. 4 is a schematic view schematically showing the configuration of one of the pass boxes in the third embodiment of the cell culture system according to the present invention. In the third embodiment, the same parts as those in the first embodiment are denoted by the same reference numerals, and the description is simplified or omitted.

  The cell culture system 61 of the present embodiment is different from the first embodiment in that a sample carrying port 63 in the pass box 62 is provided with a sterilizing agent enclosure mechanism 64 as an additional mechanism together with the packaging mechanism 40, and In addition to the unpacking mechanism 41, the sample carry-in port 65 is provided with a sterilizing agent removing mechanism 66 as a removing mechanism, and the pass box 62 is not provided with the sample adjusting port 22.

  The sterilizing agent sealing mechanism 64 is disposed between the first packaging 34 applied to the sample container 18 by the packaging mechanism 40 and the sample container 18 when the sample container 18 is carried out of the aseptic area such as the centrifuge 14. 67 is enclosed. The sterilizing agent removing mechanism 66 removes the sterilizing agent 67 from between the first package 34 and the sample container 18 when the sample container 18 is carried into the aseptic region (in the aseptic operation apparatus 13). . The sterilizing agent sterilizes the cell sample leaked to the outside from the sample container 18.

  The operation of the cell culture system 61 of this embodiment is basically the same as that of the first embodiment, except for the following points. That is, the sample container 18 is carried into the aseptic operation device 13 through the material carry-in port 19 as in the first embodiment. After the cell sample is put in the sample container 18 and sealed, the sample container 18 is hermetically packaged with the first package 34 together with the sterilizing agent 67 using the packaging mechanism 40 and the sterilizing agent sealing mechanism 64 at the sample carrying-out port 63. Specifically, first, the sample container 18 is packaged by the packaging mechanism 40 at the sample carry-out port 63 using the first packaging material 33 while leaving a part of the opening. Next, a bactericide 67 (70% ethanol aqueous solution mist in this embodiment) is blown into the first package 34 through the opening by the bactericide enclosure mechanism 64, and then the opening is sealed and sealed. In this way, the first package 34 enclosing the bactericidal agent 67 is applied to the sample container 18 to form the sample container 68 and then carried out of the aseptic operation apparatus 13.

  When the sample container 68 is brought into the aseptic operation device 13 from outside the aseptic region after the processing in the external device (outside the aseptic region) such as the centrifuge 14, the first package 34 is unpacked by the unpacking mechanism 41 at the sample carry-in port 65. The sample container 18 is obtained by unpacking and further removing the ethanol as the sterilizing agent 67 by air-drying or wiping, or by removing it by the sterilizing agent removing mechanism 66. The waste material 36 of the first package 34 generated by unpacking, air containing ethanol, or a cloth is discharged from the material carry-out port 23.

  Even if the cell sample which is the contents of the sample container 18 leaks out in the course of processing in an external device such as the centrifuge 14 due to the above-described operation, intrinsic contamination occurs on the outer surface of the sample container 18. Since it is sterilized and decontaminated by the action of ethanol which is the sterilizing agent 67, no contamination is mixed inside the aseptic operation device 13. Therefore, even if the sample container 18 is transported inside the aseptic operation device 13, the inside of the aseptic operation device 13, that is, the aseptic region, is not contaminated by intrinsic contamination.

  In addition, although the example which inject | pours the inside of the 1st package 34 by using the bactericidal agent 67 as mist was demonstrated, the introduction method of the bactericidal agent 67 is not restricted to this. For example, it is possible to directly inject an ethanol aqueous solution into the gap between the sample container 18 and the first package 34 and use the sterilizing action of ethanol vapor generated therefrom, or the nonwoven fabric is impregnated with the sterilizing agent 67. Things can also be sandwiched between the sample container 18 and the first package 34.

  Also, the type of the bactericidal agent 67 is not limited to the ethanol aqueous solution, and various known disinfecting liquids such as sodium percarbonate liquid (oxygen-based disinfecting liquid), popidone iodine liquid, and chlorine-based disinfecting liquid can also be used. Further, various sterilization gases such as ozone and hydrogen peroxide can be preferably used at a necessary minimum concentration. When using this sterilization gas, it is preferable to provide the sample carry-in port 65 with a sterilization gas decomposition mechanism. On the other hand, a film containing a sterilizing solid material such as copper or silver as the sterilizing agent 67 can be sandwiched between the sample container 18 and the first package 34. Furthermore, by using a film having a bactericidal action as the first packaging material 33, the first packaging 34 and the bactericidal agent 67 may be combined.

From the above, according to the present embodiment, in addition to the same effects as the effects (1) and (2) of the first embodiment, the following effect (4) is achieved.
(4) In the present embodiment, the second packaging material 37 and the sample adjustment port 22 are unnecessary, and the sample container 18 can be used in an unwrapped state in the aseptic operation device 13. The configuration is simple and the operation in the aseptic operation device 13 is simple.

  As mentioned above, although this invention was demonstrated based on said each embodiment, this invention is not limited to this. For example, in each embodiment, as an aseptic operation device 13, an automatic device that automatically performs operations such as medium exchange and passage in cell culture has been described as an example. Not limited to cell culture, but also for automated devices that automatically perform aseptic operations to research, develop, and produce various biological products such as transplanted cell tissues, animal and plant tissues, antibodies, vaccines, serum, etc. It is applicable to. Moreover, it is applicable also to the automation apparatus which requires aseptic operation, such as research and development and production of a general chemical synthetic drug and food. Of course, the present invention can be applied not only to the above-described automation apparatus for various purposes but also to a semi-automatic or manual apparatus.

DESCRIPTION OF SYMBOLS 11 ......... Cell culture system, 13 ......... Aseptic operation device (in sterile field), 14 ......... Centrifuge (outside sterile field), 15 ...... Incubator (outside sterile field), 16 ......... Transfer device (Outside aseptic area), 17 ... pass box (outside aseptic area), 18 ... sample container, 34 ... first packaging, 38 ... second packaging (contamination prevention member), 40 ......... Packaging mechanism (first handling means), 41 ......... Unpacking mechanism (first handling means), 42 ......... Addition mechanism (second handling means), 51 ......... Cell culture system, 53 ......... Sample preparation Port, 55 ......... Sample container, 54 ......... Second packaging (contamination prevention member), 56 ......... Removal mechanism (second handling means), 61 ......... Cell culture system, 64 ......... Bactericidal agent enclosed Mechanism (addition mechanism, second handling means), 66... Disinfectant removal mechanism (removal mechanism, second handling means) ).

Claims (8)

In a cell culture method for transporting a container for storing a cell sample between inside and outside a sterile area,
When the container is transported out of the sterilization area, a first package using a bacteria-impermeable sheet-like material is applied to the container and packaged, and when the container is transported into the sterilization area, the first packaging is performed. 1 Remove the packaging and
Cell culture method characterized by the contamination preventing member for preventing the contamination by cell sample in the container, it applied to the outer surface of the container. In a cell culture system that transports a container for storing a cell sample between inside and outside a sterile area,
When the container is transported out of the sterilization area, a first package using a bacteria-impermeable sheet-like material is applied to the container and packaged, and when the container is transported into the sterilization area, the first packaging is performed. A first handling means for removing one package is provided;
Cell culture system, wherein a contamination preventing member for preventing the contamination by cell sample, the second handling means for performing an outer surface of said container is provided in the vessel. The first handling means includes a packaging mechanism that performs first packaging on the container, and an unpacking mechanism that unpacks and removes the first packaging,
The cell culture according to claim 2, wherein the second handling means includes at least one of an addition mechanism for adding a contamination prevention member to the container and a removal mechanism for removing the contamination prevention member. system.   The anti-contamination member is a second package that is applied in close contact with the outer surface of the container by using a fungus-impermeable sheet material when the container is transported into a sterile region. 4. The cell culture system according to 2 or 3. When the container is transported out of the sterile field, the contamination prevention member is applied in close contact with the outer surface of the container using a bacteria-impermeable sheet material and is packaged in the container together with the first package. the cell culture system according to claim 2 or 3, characterized in that during the transport of the container in a sterile area, which is the second packaging Ru removed from the container together with the first wrapping.   The antifouling member is a disinfectant capable of sterilizing a cell sample that is enclosed in the first package and leaks from the container when the container is transported outside the aseptic region. The cell culture system described.   The centrifuge is provided outside the aseptic region, the container is a centrifuge tube, and the first package is configured using a bacteria-impermeable material. 2. The cell culture system according to item 1.   The incubator is provided outside the aseptic area, the container is a cell culture container, and the first package is configured using a fungus-impermeable and gas-permeable sheet material. The cell culture system of any one of thru | or 6.