WO2011074710A1 - Contenant de séparation par centrifugation, adaptateur de maintien d'orientation pour contenant de séparation par centrifugation et instrument de séparation par centrifugation - Google Patents

Contenant de séparation par centrifugation, adaptateur de maintien d'orientation pour contenant de séparation par centrifugation et instrument de séparation par centrifugation Download PDF

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Publication number
WO2011074710A1
WO2011074710A1 PCT/JP2010/073473 JP2010073473W WO2011074710A1 WO 2011074710 A1 WO2011074710 A1 WO 2011074710A1 JP 2010073473 W JP2010073473 W JP 2010073473W WO 2011074710 A1 WO2011074710 A1 WO 2011074710A1
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Prior art keywords
container
centrifuge
accommodating
adapter
maintaining
Prior art date
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PCT/JP2010/073473
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English (en)
Japanese (ja)
Inventor
勇司 平家
吉田　孝夫
森村　孝史
Original Assignee
独立行政法人国立がん研究センター
株式会社フコク
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Application filed by 独立行政法人国立がん研究センター, 株式会社フコク filed Critical 独立行政法人国立がん研究センター
Priority to CN2010800577875A priority Critical patent/CN102712889A/zh
Publication of WO2011074710A1 publication Critical patent/WO2011074710A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/05—Means for pre-treatment of biological substances by centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B01L2300/123—Flexible; Elastomeric
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/505—Containers for the purpose of retaining a material to be analysed, e.g. test tubes flexible containers not provided for above
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
    • B04B—CENTRIFUGES
    • B04B5/00—Other centrifuges
    • B04B5/04—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers
    • B04B5/0407—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles
    • B04B2005/0435—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles with adapters for centrifuge tubes or bags

Definitions

  • the present invention relates to a centrifuge container, an adapter for maintaining the position of a centrifuge container, and a centrifuge tool, and in particular, facilitates separation and washing of cells from a cell suspension containing cultured cells.
  • the present invention relates to a centrifuge container suitable for the above, an adapter for maintaining the attitude of the centrifuge container, and a centrifuge tool.
  • cells include a method using a centrifuge, a method using a centrifuge bowl, a filtration separation method using a filter, An electrical separation method using electric charges, an affinity separation method using affinity of antigens existing on the cell surface, and the like are performed.
  • the separated and washed cells As a medical use of the separated and washed cells, it is used for treatment using cells such as hematopoietic stem cell transplantation, adoptive immunotherapy, gene therapy, etc., and it is important to aseptically separate and wash the cells. It is becoming an issue.
  • a method using a general centrifuge is known as a method capable of processing a large amount of cells at a low cost.
  • the target cell suspension or the like (hereinafter referred to as “cell suspension”) is transferred to a centrifuge cup, and the centrifuge cup is centrifuged using a centrifuge.
  • the centrifuge cup is centrifuged using a centrifuge.
  • the cells are washed by centrifuging and removing the supernatant, and can be performed with a highly versatile centrifuge and an inexpensive centrifuge cup.
  • the target product is usually divided into a plurality of stages including a step of transferring the target cell suspension to a centrifuge cup, a step of removing the supernatant, and a step of resuspension in the washing solution.
  • it is necessary to construct a sterilization system including not only the centrifuge but also the entire cell conditioning chamber, which requires a large and expensive device. It was what I needed.
  • a method using a centrifuge bowl is known as a method capable of automatically and aseptically treating a large amount of cells (Japanese Patent Application Publication No. 07-000857).
  • the method of using this centrifuge bowl introduces the cell suspension, which is the object, into a container called a centrifuge bowl that rotates about its central axis, and the centrifugal force generated in the centrifuge bowl bowl.
  • the cell and dispersion medium are further separated. Continuously, a large amount of cell suspension can be processed by introducing the cell suspension into the bowl of the centrifuge bowl and continuously removing the dispersion medium or cells. Centrifuge system. According to this method, separation / washing in a closed system is possible.
  • a sterilized disposable separation / washing kit comprising a centrifuge bowl, a plurality of bags, and a plurality of tube circuits operatively connecting them, and a centrifuge bowl
  • a dedicated centrifuge for rotation, a pump for transporting the liquid inside the cleaning kit, and a dedicated control system for controlling them are required, and the initial cost for introducing the equipment is expensive.
  • a disposable cleaning kit having a complicated structure is also expensive, which greatly increases the cost of separation and cleaning.
  • the cell suspension containing the target cell is passed through the filter member to filter out the target cell, and then the liquid is separated from the direction opposite to the cell suspension passed through the filter member.
  • the cell suspension containing target cells captured by the filter member through the filter member is removed from the filter member and recovered.
  • the filtration separation method using the filter is suitable as a method for rapidly separating a large amount of target cells from unnecessary cells and liquid components, but has a disadvantage that the recovery rate of the target cells is low. This disadvantage stems from the fact that the pore diameter of the filter member is constant during a series of processing operations.
  • the initial cost for introducing the equipment is not only high as in the case of a centrifuge bowl.
  • a disposable cleaning kit having an expensive and complicated configuration is required, which greatly increases the cost of separation and cleaning.
  • a cell separation / washing container or cell that can be processed by inexpensive or general-purpose equipment and can be separated and washed aseptically by simple and short-time work using an inexpensive disposable container. Separation and cleaning systems are desired.
  • the problem to be solved by the present invention is to be able to aseptically separate and wash cells from a cell suspension in a simple and short time operation using inexpensive disposable containers with inexpensive or general-purpose equipment.
  • a centrifuge container capable of constructing a cell separation / washing system using the container, an adapter for maintaining the posture of the centrifuge container, and a centrifuge tool. is there.
  • a centrifuge container suitable for adopting a closed system and for facilitating separation and washing of cells from a cell suspension containing cultured cells, etc., for centrifuge
  • An object of the present invention is to provide an adapter for maintaining the posture of a container and a centrifuge tool.
  • a centrifuge container is set in a centrifuge via a posture maintaining adapter,
  • a container body having a sealed internal space capable of accommodating an object to be centrifuged, and a port capable of communicating between the internal space and the outside
  • the container body has a first accommodating portion having a large volume on the upper side, and a second accommodating portion communicating with the first accommodating portion and having a relatively small volume on the lower side for accommodating the centrifugal sediment.
  • the port has an opening on the first housing portion side,
  • the boundary between the first storage portion and the second storage portion or a container wall in the vicinity thereof has flexibility, and the second container wall is brought into close contact with the boundary wall in the vicinity thereof or in the vicinity thereof.
  • a centrifuge container configured to be separable so that the housing portion and the opening of the port are in a non-communication state.
  • a tapered portion is provided on the lower side of the first accommodating portion, and the first accommodating portion is disposed via the tapered portion.
  • the boundary portion between the tapered portion and the second storage portion or a container wall in the vicinity thereof has flexibility, and the second storage portion is brought into close contact with the opposing container wall in the boundary portion or the vicinity thereof.
  • the opening of the port are separable so as to be in a non-communication state.
  • the volume of the second accommodating portion is 10% or less of the volume of the first accommodating portion.
  • the container body is formed by sealing the periphery of a superposed flexible sheet or film.
  • the container body is formed of a flexible resin by a blow molding method.
  • a fluid transfer tube having a flexibility capable of communicating with the internal space of the container body is attached to the port.
  • the transfer tube is attached so as to be capable of fusing and communicating with an aseptic joining apparatus.
  • a container body having a sealed internal space capable of accommodating an object to be centrifuged has a first accommodating portion having a large volume on the upper side, and a second accommodating portion communicating with the first accommodating portion and having a relatively small volume on the lower side for accommodating the centrifugal sediment.
  • a centrifuge container for setting a centrifuge container in which a container wall in the vicinity of or near the boundary between the first storage part and the second storage part is flexible in a bucket of a centrifuge
  • An adapter An opening for accommodating the centrifuge container on the upper surface;
  • the second housing space side includes a holding portion that holds the first housing portion,
  • An adapter for maintaining the position of the centrifuge container is provided, which includes a peripheral wall that prevents the centrifuge container from being detached during centrifugation.
  • a container body having a sealed internal space capable of accommodating an object to be centrifuged has a first accommodating portion having a large volume on the upper side, and a second accommodating portion communicating with the first accommodating portion and having a relatively small volume on the lower side for accommodating the centrifugal sediment. And consist of A tapered portion is provided on the lower side of the first accommodating portion, and the first accommodating portion communicates with the second accommodating portion via the tapered portion, An adapter for maintaining the attitude of a centrifuge container for setting a centrifuge container having a flexible container wall at or near the boundary between the taper part and the second accommodating part to a bucket of a centrifuge.
  • a first accommodation space in which the first accommodation portion is accommodated on the upper surface side and a second accommodation space in which the second accommodation portion is accommodated on the bottom surface side are provided.
  • a mortar-shaped holding portion that holds the tapered portion is provided on the second housing space side,
  • An adapter for maintaining the position of the centrifuge container is provided, which includes a peripheral wall that prevents the centrifuge container from being detached during centrifugation.
  • the inclination angle of the mortar-shaped holding part is 45 ° to 80 °.
  • the adapter for maintaining the attitude of the centrifuge container is substantially semi-dividable having a dividing surface extending in the vertical direction.
  • the divided surfaces can be closely contacted and separated.
  • the orientation maintaining adapter for the centrifuge container has transparency capable of visually confirming the setting state of the centrifuge container from the outside. is doing.
  • a container body having a sealed internal space capable of accommodating an object to be centrifuged, and a port capable of communicating the internal space with the outside are provided.
  • the container body has a first accommodating portion having a large volume on the upper side, and a second accommodating portion communicating with the first accommodating portion and having a relatively small volume on the lower side for accommodating the centrifugal sediment.
  • the port has an opening on the first housing portion side,
  • the boundary between the first storage portion and the second storage portion or a container wall in the vicinity thereof has flexibility, and the second container wall is brought into close contact with the boundary wall in the vicinity thereof or in the vicinity thereof.
  • a centrifuge container that is separable so that the container and the opening of the port are in a non-communication state; An opening for accommodating the centrifuge container on the upper surface;
  • the second housing space side includes a holding portion that holds the first housing portion,
  • An adapter for maintaining the posture of the centrifuge container, which is provided with a peripheral wall for preventing the centrifuge container from being detached during centrifugation, A centrifuge tool is provided.
  • a container body having a sealed internal space capable of accommodating an object to be centrifuged, and a port capable of communicating the sealed internal space of the container body with the outside
  • the container body is composed of a first accommodating portion having a large volume on the upper side and a second accommodating portion having a relatively small volume on the lower side for accommodating the centrifugal sediment,
  • a tapered portion is provided on the lower side of the first accommodating portion, and the first accommodating portion communicates with the second accommodating portion via the tapered portion
  • the boundary portion between the tapered portion and the second storage portion or a container wall in the vicinity thereof has flexibility, and the second storage portion is brought into close contact with the opposing container wall in the boundary portion or the vicinity thereof.
  • a centrifuge container that is separable so that the opening of the port is in a non-communication state, An opening for accommodating the centrifuge container on the upper surface;
  • a first accommodation space in which the first accommodation portion is accommodated on the upper surface side and a second accommodation space in which the second accommodation portion is accommodated on the bottom surface side are provided.
  • a mortar-shaped holding portion that holds the tapered portion is provided,
  • An adapter for maintaining the posture of the centrifuge container, which is provided with a peripheral wall for preventing the centrifuge container from being detached during centrifugation, A centrifuge tool is provided.
  • the inclination angle of the mortar-shaped holding portion of the posture holding adapter of the centrifuge container is 45 ° to 80 °.
  • the adapter for maintaining the posture of the centrifuge container is substantially half-dividable having a dividing surface extending in the vertical direction, It is configured to allow close contact and separation.
  • the adapter for maintaining the position of the centrifuge container has transparency that allows visual confirmation of the setting state of the centrifuge container from the outside. Yes.
  • the first container having a large volume on the upper side and the second container having a relatively small volume for accommodating the centrifugal sediment on the lower side are provided. Therefore, after centrifugation, the precipitate is accumulated in the second accommodating portion. Then, when the supernatant is discharged after centrifugation, the second storage is provided by bringing the boundary between the first storage part and the second storage part or a flexible container wall in the vicinity thereof into close contact with each other. Since the part and the opening part of the port on the first housing part side are not in communication with each other, the supernatant can be easily discharged from the port.
  • the container main body is hermetically sealed and has a port having an opening on the first housing portion side, a series of operations such as storage of content liquid such as cell turbid liquid, centrifugation, and discharge of supernatant are performed. Work can be realized in a closed system, and cell separation and washing can be performed aseptically, simply, and inexpensively.
  • the tapered portion is provided on the lower side of the first accommodating portion, and the first accommodating portion is accommodated in the second accommodating portion via the tapered portion. Since it communicates with the portion, the precipitate is smoothly settled along the tapered portion to the second accommodation portion and does not stay in the first accommodation portion during centrifugation.
  • the centrifuge container since the volume of the second storage portion is 10% or less of the volume of the first storage portion, a centrifuged product having a high concentration of precipitate is obtained.
  • the number of repeated washings can be reduced, and the precipitate can be prevented from flowing out by repeated washing, so that the precipitate can be recovered. It can be done efficiently.
  • the container body is formed by sealing the peripheral edge of the superimposed flexible sheet or film, and thus has uniform flexibility. Volume reduction is good, that is, storage and disposal can be improved.
  • the flexible part is not limited to a part of the container body, and the whole part has flexibility, and it becomes easy to construct a closed system. Furthermore, because it is excellent in flexibility, even if the shape of the centrifuge container before centrifugation does not necessarily fit the inner structure of the housing space of the posture holding adapter, if the difference is small, Since the container wall can absorb the difference by deformation such as stretching, it fits into the inner structure of the housing space for the posture holding adapter and can avoid bag breakage.
  • the container body is formed of a resin having flexibility by a blow molding method, and therefore fits with the inner structure of the housing space of the posture holding adapter. Can be increased.
  • the flexible part is not limited to a part of the container body, and the whole part has flexibility, and it becomes easy to construct a closed system. Furthermore, because it is excellent in flexibility, even if the shape of the centrifuge container before centrifugation does not necessarily fit the inner structure of the housing space of the posture holding adapter, if the difference is small, Since the container wall can absorb the difference by deformation such as stretching, it fits into the inner structure of the housing space for the posture holding adapter and can avoid bag breakage.
  • the fluid transfer tube having flexibility that can communicate with the internal space of the container body is attached to the port, a closed system is constructed. Convenient to.
  • the transfer tube can be fused and sealed, and can be connected so as to be able to communicate with an aseptic joining device. Therefore, the cell culture bag, the medium bag, the washing liquid bag, the waste liquid Aseptically connectable and disengageable with bags for use.
  • a holding portion for holding the first receiving portion on the second receiving space side in the first receiving space, and a peripheral wall for preventing the separation of the centrifuge container during centrifugation Therefore, not only when the boundary between the first container and the second container of the centrifuge container or the container wall in the vicinity thereof has flexibility, the entire centrifuge container has flexibility. Even in the case where it is difficult to stand by itself, the far-end centrifugal container can be reliably held during centrifugation.
  • the adapter for maintaining the attitude of the centrifuge container since it has a mortar-shaped holding part that holds the tapered part provided on the lower side of the first accommodating part of the centrifuge container, A centrifuge container having such a shape can be suitably fitted.
  • the adapter for maintaining the attitude of the centrifuge container since the inclination angle of the mortar-shaped holding part is 45 ° to 80 °, most of the precipitate is retained in the tapered part. And can be deposited in the second accommodating portion along the tapered portion.
  • the orientation holding adapter for the centrifuge container can be substantially divided into half parts having a dividing surface extending in the vertical direction, Since the dividing surface can be closely contacted and separated, when setting the centrifuge container, the centrifuge container can be set easily and securely by separating the dividing surface and providing a gap. Can do. And at the time of centrifugation, it can be set as the predetermined
  • the centrifuge container since the transparency of the centrifuge container can be visually confirmed from the outside, the centrifuge container is set. It is possible to visually confirm the state of being present and prevent setting errors.
  • the centrifuge device of the present invention after centrifugation, the supernatant can be easily discharged from the port, and the precipitate rises when the supernatant is discharged, and the precipitate is formed together with the supernatant. There is no risk of being discharged. Furthermore, when the supernatant is discharged, the supernatant remaining in the second storage portion can be reduced as much as possible.
  • a series of operations such as storage of content liquid such as cell turbid liquid, centrifugation, and discharge of supernatant can be realized in a closed system, and cell separation and washing are performed aseptically, easily and inexpensively. be able to.
  • the centrifugal container can be reliably held during centrifugation by the adapter for maintaining the position of the centrifugal container.
  • the precipitate is smoothly settled to the second accommodation portion along the taper portion during centrifugation and does not stay in the first accommodation portion.
  • the adapter for maintaining the position of the centrifuge container includes a mortar-shaped holding part that holds the tapered part of the centrifuge container, it can be suitably fitted to the centrifuge container having such a shape.
  • the centrifuge tool since the inclination angle of the mortar-shaped holding portion is 45 ° to 80 °, most of the precipitate is not retained in the tapered portion, Along the second container.
  • the centrifuge tool when setting the centrifuge container to the posture maintaining adapter of the centrifuge container, the centrifuge is separated by providing a gap by separating the dividing surfaces.
  • the separation container can be set easily and reliably. And at the time of centrifugation, it can be set as the predetermined
  • centrifuge tool of one embodiment of the present invention since the centrifuge container has transparency that allows visual confirmation of the setting state of the centrifuge container to the posture maintaining adapter of the centrifuge container, centrifugation is performed. The state in which the container is set can be visually confirmed, and set errors can be prevented.
  • the target suspension has been described as a cell or a cell suspension.
  • the present invention can be widely applied not only to cells or cell suspensions but also to separation or washing of dispersoids from other suspensions composed of a dispersoid and a dispersion medium, and in that case also, the scope of the present invention include.
  • the effect of the present invention is remarkable when aseptic operation is required.
  • FIG. 1 is a perspective view showing a centrifuge container according to an embodiment of the present invention.
  • FIG. 2 is a plan view showing the outline of the overall shape of the container main body when the centrifuge container of FIG. 1 is formed with a peripheral seal.
  • FIG. 3 is a plan view showing a state in which the centrifuge container of FIG. 1 is set in the posture maintaining adapter.
  • 4 is an enlarged perspective view showing the periphery of a port provided in the centrifuge container of FIG.
  • FIG. 5 is a perspective view showing an adapter for maintaining a posture of a centrifuge container according to an embodiment of the present invention.
  • 6 is a perspective view showing one of two members constituting the posture maintaining adapter of FIG. FIG.
  • FIG. 7 is a side view showing a clip for holding the container wall used when discharging the supernatant after centrifugation from the container for centrifugation shown in FIG. 8 is a perspective view showing a state in which the centrifuge container of FIG. 1 is set on the posture maintaining adapter of FIG.
  • FIG. 9 is an explanatory view schematically showing a state in which the supernatant after centrifugation is discharged from the centrifuge container of FIG.
  • the centrifuge device includes two members, a centrifuge container 1 for storing a material to be centrifuged, and a posture maintaining adapter 2 for the centrifuge container 1. It is configured. Although the details of the posture maintaining adapter 2 will be described later, the space between the object to be centrifuged and the bucket is filled in order to maintain the position and shape of the object to be centrifuged when the object to be centrifuged is stored in the bucket of the centrifuge.
  • the adapter is not necessarily a solid material, and may be deformed during centrifugation to fit the object to be centrifuged and maintain the position and shape of the object to be centrifuged.
  • the centrifuge container 1 includes a container body 10 having an inner space sealed so as to accommodate a liquid to be centrifuged, and a port 11 capable of communicating the sealed inner space of the container body 10 with the outside. , 12.
  • the container main body 10 is formed of a soft member having flexibility as a whole, and includes a first accommodating portion 10a having a large volume on the upper side and a second accommodating portion 10b having a relatively small volume on the lower side.
  • the taper part 10c which is comprised and is connected to the 2nd accommodating part 10b is provided in the lower side of the 1st accommodating part 10a.
  • At least the second housing portion 10b and the taper portion 10c of the container body 10 are configured so that the inner surfaces of the container walls 10d and 10e facing each other are brought into close contact with each other so that the second housing portion 10b is separated from the first housing portion 10a. It is configured to be segregated in a communication state.
  • the vertical position where the inner surfaces of the opposing container walls 10d and 10e are brought into close contact with each other is appropriately determined mainly by the volume of the sediment precipitated in the second accommodating portion 10b. It is.
  • a container wall pressing clip 3 in order to closely contact the inner surfaces of the container walls 10d and 10e constituting the second accommodating portion 10b and the tapered portion 10c, for example, a container wall pressing clip 3 (see FIG. 7) described later can be used. .
  • a region where the inner surfaces of the opposing container walls 10d and 10e can be brought into close contact with each other will be referred to as a boundary portion 10f.
  • the inner surfaces of the container walls 10d and 10e constituting the second accommodating portion 10b and the tapered portion 10c are brought into close contact with each other so that the second accommodating portion 10b is isolated from the first accommodating portion 10a. Therefore, for example, only the supernatant can be easily discharged from the first accommodating portion 10a through the port 11 while the precipitate is accommodated in the second accommodating portion 10b.
  • the 1st accommodating part 10a contains the main-body cover part mentioned later, and the substantially rectangular parallelepiped accommodating space is formed in the upper part side. Further, the tapered portion 10c provided on the lower side of the first housing portion 10a is formed so as to gradually narrow toward the second housing portion 10b.
  • the 2nd accommodating part 10b is formed so that a tubular structure or a bag-like structure may be exhibited in a state where the content liquid is filled.
  • the second accommodating portion 10b forms a tubular structure or a bag-like structure and forms a so-called deep structure, the influence on the sediment when the supernatant is discharged can be further reduced.
  • deep pocket means that the depth is deep enough to reduce the influence on the sediment when discharging the supernatant, compared to the entrance dimension.
  • the depth is deeper than twice the diameter, and when the entrance is an ellipse, the depth is preferably deeper than one time the major diameter.
  • the container body 10 is formed, for example, by stacking two flexible sheets or films and hermetically sealing the periphery thereof, or formed from a flexible resin by a blow molding method.
  • the resin forming the container body 10 include a soft vinyl chloride resin, a low density polyethylene resin, an ethylene-vinyl acetate copolymer, a polypropylene resin, an ethylene-propylene copolymer resin, a polybutadiene resin, and a styrene-butadiene copolymer. Resins and their hydrogenated resins, polyurethane resins and the like, and mixtures of these resins.
  • low-density polyethylene includes not only general low-density polyethylene but also linear low-density polyethylene resin and metallocene catalyst-based low-density polyethylene resin.
  • the polypropylene resin also includes a stereo block polypropylene resin and a mixture of the polypropylene resin and the stereo block polypropylene resin.
  • the first accommodating portion equivalent surface 10a ′, the second accommodating portion equivalent surface 10b ′, and the tapered portion equivalent surface 10c ′ are formed as a single sheet. It can be formed from a cut sheet or film having flexibility.
  • the ports 11 and 12 are used for filling, discharging, or transferring the content liquid, and are attached to the container wall forming the first storage portion 10a at the same time as the peripheral welding by the top seals 10h and 10i on the left and right sides. And has a function of communicating the sealed internal space of the container body 10 with the outside.
  • the material for forming the ports 11 and 12 is preferably a material that is thermally bonded to the container body 10.
  • the material may be selected from polyethylene resins.
  • the ports 11 and 12 may be formed integrally with the container body 10.
  • the ports 11 and 12 are preferably provided so as to face the center direction or be easily oriented when the container main body 10 is stored in the posture maintaining adapter 2.
  • the port support pieces 13 and 14 are attached simultaneously with the peripheral welds by the top seals 10h and 10i on the left and right sides, and the ports 11 and 12 are welded to the port support pieces 13 and 14.
  • the port support pieces 13 and 14 are folded in the central direction, so that the ports 11 and 12 can be laid to face the central direction.
  • a fluid transfer tube 15 having flexibility capable of communicating with the internal space of the container body 10 can be attached to the port 11.
  • a connection adapter 16 that can be connected to an external fluid transfer tool can be attached to the port 12. Since the port support pieces 13 and 14 are folded in the center direction, the ports 11 and 12 can be laid down so as to face the center direction. Therefore, the tube for fluid transfer attached to the ports 11 and 12 or the port 11 at the time of centrifugation. 15 and the connecting adapter 16 attached to the port 12 can be accommodated in the posture holding adapter 2 described later.
  • soft vinyl chloride resin ethylene vinyl acetate copolymer, low density polyethylene resin, ethylene propylene copolymer, soft PP resin (for example, stereo block copolymer of polypropylene resin)
  • soft PP resin for example, stereo block copolymer of polypropylene resin
  • examples thereof include polybutadiene resin and styrene butadiene copolymer.
  • the transfer tube 15 is preferably provided so that it can be fused and sealed and can be connected by an aseptic joining device.
  • the fusing seal is sealed by thermally welding the tube with a commercially available tube sealer, for example, a mini tube sealer 2380 manufactured by Haemonetics, a tube sealer AC-155 manufactured by Terumo, and the tubes on both sides of the heat welded portion. It can be done by sealing so that it can be cut while maintaining the sealed state.
  • connection by the aseptic joining apparatus is such that other tubes not connected by a commercially available aseptic joining apparatus such as a sterile joining machine TSCD202 manufactured by Terumo, such as a tube of a blood storage bag, transfusion, etc. It is possible to communicate with a set tube or the like so as to communicate with each other and aseptically.
  • a commercially available aseptic joining apparatus such as a sterile joining machine TSCD202 manufactured by Terumo, such as a tube of a blood storage bag, transfusion, etc. It is possible to communicate with a set tube or the like so as to communicate with each other and aseptically.
  • soft vinyl chloride resin and ethylene vinyl acetate copolymer can be used as the resin for forming the transfer tube 15 having the above-described flexibility, fusing and sealing, and capable of being connected by a sterile joining device. It is done. However, there is no denying the possibility of adopting materials other than soft vinyl chloride resin and ethylene vinyl acetate copolymer due to the advancement of fusing sealing machines and aseptic joining devices in the future.
  • the connecting adapter 16 is formed of a soft vinyl chloride resin or the like, it is provided so as to be connected to a commercially available fluid transfer device with a bottle needle. As shown in FIG. 4, the connecting adapter 16 isolates the internal space from the outside of the container body 10 and pierces a fluid transfer device with a bottle needle, for example, a connecting tube bottle needle made by Terumo. A diaphragm 16a capable of communicating is provided in the tube 16b.
  • the posture maintaining adapter 2 has a container housing space 20 having an open top surface into which the centrifuge container 1 can be inserted, and a bottom surface to fit the bucket of the centrifuge. It is formed to have a flat outer shape.
  • the container housing space 20 has a vertical direction in which the first housing space 201 in which the first housing portion 10a of the centrifuge container 1 is housed and the second housing space 202 in which the second housing portion 10b is housed. It is configured to prepare for.
  • a mortar-shaped holding portion 203 that holds the tapered portion 10c of the centrifugal separation container 1 is formed below the first housing space 201.
  • the mortar-shaped holding portion 203 has an inclination angle of 45. By setting the angle to be greater than or equal to 0 °, most of the precipitate can be precipitated in the second accommodating portion 10b along the tapered portion 10c without staying in the tapered portion 10c of the centrifugal separation container 1.
  • the inclination angle refers to the angle at which the mortar-shaped slope rises with respect to a plane perpendicular to the centrifugal force vector during centrifugation.
  • the container housing space 20 has a cylindrical surface 20a in which the first housing portion 10a, the taper portion 10c, and the second housing portion 10b of the centrifuge container 1 can be fitted on the outer surface of the container wall during centrifuging.
  • the tapered surface 20c, tubular surface, or bag-like surface 20b of the portion 203 is formed from a peripheral wall surface continuous in the vertical direction (see FIG. 1 for the centrifugal separation container 1).
  • the term “fit” means that the container wall of the centrifuge container 1 is not damaged during centrifugation, and is not damaged, but is applied to the cylindrical surface 20a, the mortar-shaped tapered surface 20c, the tubular surface, or the bag-shaped surface 20b. This means that the deposits are efficiently accumulated in the second accommodating portion 10b along the closely adhered container wall.
  • the generation of wrinkles on the container wall is small, but even if there is a wrinkle, it is difficult to accumulate the precipitate on the wrinkle, and if it is efficiently accumulated in the second accommodating portion 10b, it is included in the fit. To do.
  • the posture holding adapter 2 is composed of two substantially half-divided members 2 ⁇ / b> A and 2 ⁇ / b> B having a dividing surface 21 extending in the vertical direction. It is set to be parallel to the centrifugal axis of the centrifuge. And, at the time of centrifugation, the two members 2A and 2B are in close contact with each other at the dividing surface 21, and when the centrifuge container 1 is accommodated in the container accommodating space 20, it can be easily accommodated in the container accommodating space 20, and In order to carry out reliably, it is comprised so that the clearance gap may be provided in the dividing surface 21 of two member 2A, 2B.
  • the two substantially divided members 2A and 2B can be integrated by tightening with bolts 24a, 24b, 25a and 25b (see FIG. 5) inserted through the through holes 22a, 22b, 23a and 23b.
  • the size of the gap of the dividing surface 21 can be adjusted by adjusting the bolts 24a, 24b, 25a, 25b.
  • Examples of the resin that forms the posture maintaining adapter 2 include a styrene resin, an acrylic resin, a polyethylene resin, a polypropylene resin, a polyamide resin, a polyester resin, a fluorine resin, a polyacetal resin, a polycarbonate resin, and an ABS resin. And hard and tough resin such as polysulfone resin.
  • a transparent acrylic resin, polycarbonate resin, polysulfone resin, transparent ABS resin, transparent polypropylene resin, polyethylene examples thereof include transparent resins such as terephthalate resin, polysulfone resin, and polymethylpentene resin.
  • the container wall pressing clip 3 opens the pressing portions 30 and 31, the handles 32 and 33, opens the handles 32 and 33, closes the pressing portions 30 and 31, and closes the boundary portion 10 f of the container body 10.
  • a spring 34 for tightly tightening see FIG. 1).
  • the holding parts 30 and 31 are set longer than the length in the width direction of the boundary part 10 d of the container body 10, and suspension holes 35 and 36 are provided at the ends of the handles 32 and 33.
  • the centrifugal separation container 1 As described above, according to the centrifugal separation container 1 according to the present embodiment, the first container 10a having a large volume on the upper side and the relatively small volume for accommodating the centrifugal sediment on the lower side. Therefore, after centrifugation, the sediment is accumulated in the second storage portion 10b. Then, when the supernatant is discharged after centrifugation, the boundary wall 10d between the first container 10a and the second container 10b or the flexible container walls 10d and 10e in the vicinity thereof are brought into close contact with each other. As a result, the second accommodating portion 10b and the opening of the port 11 on the first accommodating portion 10a side are in a non-communication state, so that the supernatant can be easily discharged from the port 11.
  • the sediment rises when the supernatant is discharged, and together with the supernatant There is no risk of sediment being discharged. Further, when discharging the supernatant, the place where the container walls 10d and 10e are in close contact is directly above the centrifugal sediment accumulated in the second storage portion 10b, so that the supernatant remaining in the second storage portion 10b is obtained. Can be reduced as much as possible.
  • the container main body 10 is sealed and the port 11 having an opening is provided on the first accommodating portion 10a side, the content liquid such as a cell turbid liquid is accommodated, centrifuged and the supernatant is discharged.
  • the content liquid such as a cell turbid liquid
  • centrifuged is accommodated, centrifuged and the supernatant is discharged.
  • a tapered portion 10c is provided on the lower side of the first accommodating portion 10a, and the first accommodating portion 10a communicates with the second accommodating portion 10b via the tapered portion 10c. Sometimes the sediment smoothly settles along the tapered portion 10c to the second accommodating portion 10b and does not stay in the first accommodating portion 10a.
  • the first accommodation space 201 in which the first accommodation portion 10a is accommodated on the upper surface side, and the second accommodation portion 10b on the bottom surface side. While having the 2nd accommodation space 202 accommodated, the mortar-shaped holding part 203 holding the taper part 10c of the 1st accommodation part 10a is provided in the 2nd accommodation space 202 side in the 1st accommodation space 201.
  • the centrifuge container 1 used here has two ports 11 and 12 at the top of the container body 10, and a fluid transfer tube 15 or a connection adapter 16 is connected to each of the ports 11 and 12.
  • a second housing portion 10b having a deep bag-like structure and a tapered portion 10c that gradually narrows in a tapered shape toward the second housing portion 10b are formed.
  • the container body 10 is made by superposing flexible sheets 10d and 10e obtained by inflation molding an ethylene vinyl acetate copolymer having a vinyl acetate content of 15% by weight, and sealing the periphery with a high frequency seal, followed by injection molding.
  • the medium-density polyethylene resin port 11 formed by the method was heat-sealed to the port support piece 14 to form the entire container wall from a flexible sheet.
  • the taper portion 10c of the container body 10 was set to 90 ° at the time of peripheral sealing so that the taper (angle formed by the facing ridge line) when the inner solution was filled and three-dimensionalized was close to 60 °.
  • the volume ratio of the space on the narrow side to the space on the port side when the container walls 10d and 10e facing each other with the boundary wall 10f sandwiched by the container wall pressing clip 3 was set to about 1: 150.
  • the fluid transfer tube 15 was provided with a flexible vinyl chloride resin having an inner diameter of 4.7 mm and an outer diameter of 6.7 mm.
  • the adapter 16 for connection is equipped with a flexible polyvinyl chloride resin with an inner diameter of 4.4 mm so that the bottle needle of the fluid transfer device with a bottle needle can be easily pierced and connected, and not easily removed. It was.
  • the posture maintaining adapter 2 was manufactured by cutting a transparent acrylic resin block, and was equipped with a transparent inner surface that was made visible by polishing the cut surface.
  • the inclination angle of the tapered surface of the container housing portion the inclination angle of the portion where the inclination angle becomes maximum is set to 63 ° so that the taper portion of the container body fits, and the inclination angle of the portion where the inclination angle becomes minimum is set. Set to 57 °.
  • the fluid transfer tube 15 After transferring a cell suspension composed of 255 ml medium (KBM400: manufactured by Kojin Bio Inc.) containing 8.8 ⁇ 10 4 cells / ml of K562 cells to the centrifuge container 1 having a capacity of 300 ml, the fluid transfer tube 15 was fused and sealed with a Terumo tube sealer (Terflex AC-157), and the container was sealed.
  • KBM400 manufactured by Kojin Bio Inc.
  • the centrifuge container 1 filled with the culture solution is visually observed.
  • the bolts 24a, 24b, 25a, and 25b were tightened so that the divided surfaces 21 were brought into close contact with each other while being checked.
  • the port 11 and the fluid transfer tube 15 are temporarily accommodated along the central top seal 10g, and the portion exceeding the other cylindrical surface 20a facing each other constituting the first accommodation space 201 of the posture maintaining adapter 2 will be described. Then, it was bent near the other inner wall and stored along the inner wall.
  • the connecting adapter 16 was also accommodated along the center top seal 10g.
  • centrifuge container 1 Along with the posture maintaining adapter 2 in which another centrifuge container 1 produced in the same manner is inserted, each is placed on a diagonal bucket of a centrifuge (a large capacity cooling centrifuge 9810 manufactured by Kubota Corporation), respectively. Centrifuge for 20 minutes at 400G.
  • a centrifuge a large capacity cooling centrifuge 9810 manufactured by Kubota Corporation
  • Centrifuge for 20 minutes at 400G the two centrifuge containers 1 are referred to as a first container and a second container.
  • the retaining clip 3 directly contacts the container walls 10d and 10e facing each other immediately above the interface, that is, in the vicinity of the boundary between the second accommodating portion 10b and the taper portion 10c so as not to disturb the precipitate interface.
  • the second accommodating portion 10c is isolated from the first accommodating portion 10a on the port 11 side.
  • the supernatant liquid was stirred so that the container wall on the supernatant side of the centrifuge container 1 was grasped by hand, and the cells precipitated or adhered to the wall surface of the centrifuge container 1 were dispersed in the supernatant liquid.
  • the container wall pressing clip 3 is hung on the hanger through the suspension holes 35 and 36 so that the port 11 is lowered, and the space on the port 11 side, that is, the first accommodating portion 10a.
  • the medium existing inside, that is, the supernatant is discharged from the fluid transfer tube 15, the amount of the supernatant and the number of cells floating in the supernatant are measured, and the total number of cells floating in the supernatant is measured.
  • the ratio between the total number of cells remaining in the centrifuge container 1 and the initial total number of cells was calculated and used as the recovery rate.
  • the clip 3 for holding the container wall was removed and inserted into the posture maintaining adapter 2 in the same manner as the first centrifugation, and the second centrifugation for 20 minutes was performed at 400G. Later, in the same manner as in the first container, the total number of cells suspended in the supernatant and the recovery rate were calculated.
  • the centrifuge container, the centrifuge container orientation maintaining adapter, and the centrifuge tool according to the present invention can efficiently separate cells when used for cell separation and washing after cell culture.
  • a means for collecting or washing cells antibodies, proteins used for immunotherapy, as a means for collecting or washing cells for use in regenerative medicine, and as a means for pharmaceutical research and development by cell culture
  • it is extremely suitable as a means for collecting or washing fine particles other than cells.

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Abstract

L'invention porte sur un contenant de séparation par centrifugation, sur un adaptateur de maintien d'orientation destiné au contenant de séparation par centrifugation et sur un instrument de séparation par centrifugation qui peut être utilisé pour séparer ou nettoyer de manière aseptique des cellules dans une suspension cellulaire à l'aide de contenants jetables génériques bon marché, qui sont aptes à adopter un système fermé. Le contenant de séparation par centrifugation précité, qui est installé dans une centrifugeuse au moyen de l'adaptateur de maintien d'orientation, est muni d'un corps de contenant comprenant un espace interne hermétique pouvant contenir un article à centrifuger, et d'un orifice qui relie l'espace interne à l'extérieur du corps. Le corps de contenant comprend une première partie de confinement de grand volume et un seconde partie de confinement d'un volume relativement petit, qui est reliée à la première partie de confinement et qui contient le sédiment centrifugé. L'orifice est muni d'une ouverture sur le côté de la première partie de confinement. Les parties limitrophes de la première et de la seconde partie de confinement ou les parois du contenant à proximité des parties limitrophes sont souples et les parties limitrophes opposées ou les parois de contenant à proximité des parties limitrophes sont unies de sorte que la seconde partie de confinement et l'ouverture de l'orifice peuvent être séparées de manière à ne pas être reliées.
PCT/JP2010/073473 2009-12-18 2010-12-17 Contenant de séparation par centrifugation, adaptateur de maintien d'orientation pour contenant de séparation par centrifugation et instrument de séparation par centrifugation WO2011074710A1 (fr)

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CN2010800577875A CN102712889A (zh) 2009-12-18 2010-12-17 离心分离用容器、离心分离用容器的姿态保持用适配器和离心分离用用具

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JP2009-288141 2009-12-18
JP2009288141A JP2011125813A (ja) 2009-12-18 2009-12-18 遠心分離用容器、遠心分離用容器の姿勢保持用アダプター及び遠心分離用用具

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US9296984B2 (en) 2010-07-09 2016-03-29 The Gid Group, Inc. Tissue processing apparatus and method for processing adipose tissue
US9206387B2 (en) 2010-07-09 2015-12-08 The Gid Group, Inc. Method and apparatus for processing adipose tissue
WO2013106655A1 (fr) 2012-01-11 2013-07-18 The Gid Group, Inc. Procédé de traitement de tissu adipeux et appareil de traitement
BR112013000605B1 (pt) 2010-07-09 2021-11-09 The Gid Group, Inc. Aparelho utilizável para a coleta de material biológico humano e processamento póscoleta do material coletado, conjunto e método para processar material biológico humano
BR112015004003B1 (pt) 2012-09-06 2020-05-19 The Gid Group Inc aparelho para processar material biológico humano contendo tecido fibroso e método para processar tecido adiposo
EP3038629B1 (fr) 2013-09-05 2020-11-18 GID BIO, Inc. Procédé de traitement de tissu adipeux
JPWO2015037120A1 (ja) 2013-09-13 2017-03-02 株式会社メディネット 細胞懸濁液ろ過器具及び細胞懸濁液を含む点滴用溶液の調製方法
JP6431438B2 (ja) * 2015-04-28 2018-11-28 株式会社フコク 遠心分離用容器の姿勢保持用アダプタ
WO2018044791A1 (fr) 2016-08-30 2018-03-08 Lifecell Corporation Systèmes et procédés de commande d'un dispositif médical
USD851777S1 (en) 2017-01-30 2019-06-18 Lifecell Corporation Canister-type device for tissue processing
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