JP2010259412A - Bag for freeze preservation of cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To easily take out a freeze preserved cell in a clean state without using a large-sized apparatus such as a centrifugal separator. <P>SOLUTION: There is provided a bag 1 for freeze preservation of a cell, which includes a closed outer container 2, a mesh-formed inner container 3 placed in the outer container 2 and restricting passage of the cell but allowing passage of a liquid, a first port 4 penetrating the outer container 2 and opened in the inner container 3, and a second port 5 penetrating the outer container 2 and opened in a space A between the outer container 2 and the inner container 3. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cell cryopreservation bag.

  Conventionally, cells required for cell therapy are frozen and stored in a cryopreservation bag until they are collected from a human body and used for cell therapy. This frozen storage bag is constituted by a bag made of a laminated film of a polyimide film and a fluorinated ethylene propylene polymer film, etc., and has an outflow port and an inflow port at at least two vertices. It is known (for example, refer to Patent Document 1).

Japanese Patent No. 4034850

  However, the cryopreservation bag of Patent Document 1 is used for cryopreserving a mixed solution of cells and a preservation solution as used in cell therapy and taking out the cells in use. The bag must be centrifuged to separate the cells and the preservation solution. Therefore, there is a disadvantage that a large-scale centrifuge is required. Furthermore, since the preservation solution is attached to the cells thus separated, it is necessary to perform a washing operation in a separate process. Therefore, a separate cleaning device is required, and there is a disadvantage that the device becomes large.

  The present invention has been made in view of the above-described circumstances, and can be used for cell cryopreservation in which cryopreserved cells can be easily removed in a clean state without using a large-scale device such as a centrifuge. The purpose is to provide a bag.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a sealed outer container, a mesh-shaped inner container that is disposed in the outer container, restricts the passage of cells, and allows the passage of liquid, and penetrates the outer container, A cell cryopreservation bag comprising a first port that opens to the outside and a second port that passes through the outer container and opens into a space between the outer container and the inner container is provided.

  According to the present invention, when a mixed solution of cells and a preservative solution is supplied into the internal container via the first port, the cells in the mixed solution are held in the internal container, and the preservative solution is stored in the internal container and Housed in both external containers. In this state, the cells can be stored for a long time by freezing the mixed solution together with the cell cryopreservation bag.

  When cells are used, the mixed solution is heated and thawed together with the cell cryopreservation bag, and the second port is opened, so that the storage solution in the external container passes through the second port. To be discharged to the outside. At this time, the cells cannot leave the inner container and are retained in the inner container.

  Then, after that, the cleaning liquid is supplied into the inner container through the first port or the second port, and the cells in the inner container are cleaned by discharging the cleaned cleaning liquid from the second port. Remove the stock solution. Furthermore, a cell suspension in a form used for transplantation is obtained by supplying a transplant solution such as lactated Ringer's solution from the first port or the second port and suspending the cells in the inner container. Can do. The cell suspension thus obtained can be easily taken out of the cell cryopreservation bag through the first port.

In the said invention, the valve which opens and closes each port may be provided in the said 1st port and the said 2nd port, respectively.
By doing so, the first port and the second port can be opened and closed by operating the valve, respectively, and the storage liquid can be discharged and the cleaning liquid can be easily supplied and discharged.

Moreover, in the said invention, you may provide the 3rd port which penetrates the said external container and opens either in the said internal container or the space between this external container and the said internal container.
By doing in this way, the liquid for transplantation, such as a washing | cleaning liquid and a lactated Ringer's solution, can be supplied from a 3rd port, and the cell suspension of the form used for a transplant can be easily taken out from a 1st port.

Moreover, in the said invention, you may provide the 4th port which penetrates the said external container and opens either in the said internal container or the space between this external container and the said internal container.
By doing in this way, since the 1st-4th port can be used for the respectively independent use, and it is not necessary to switch the connection to each port, it can comprise further easily.

  According to the present invention, there is an effect that a cryopreserved cell can be easily taken out in a clean state without using a large-scale device such as a centrifuge.

It is a front view which shows the cell cryopreservation bag which concerns on one Embodiment of this invention. It is a front view explaining supply of the liquid mixture to the cell cryopreservation bag of FIG. It is a front view which shows the state which enclosed the liquid mixture in the cell cryopreservation bag of FIG. 1, and was frozen. It is a front view explaining the state which defrosts the liquid mixture of the cell cryopreservation bag of FIG. 1, and discharges a preservation | save liquid. It is a front view explaining the state which wash | cleans the cell which remains in the inner container of the cell cryopreservation bag of FIG. 1 with a washing | cleaning liquid. FIG. 2 is a front view illustrating a state in which a transplantation liquid is supplied to the washed cells remaining in the inner container of the cell cryopreservation bag of FIG. 1. It is a front view explaining the state which discharge | releases the cell suspension produced | generated in the cell cryopreservation bag of FIG. 1 outside. It is a front view which shows the 1st modification of the cell cryopreservation bag of FIG. It is a front view which shows the 2nd modification of the cell cryopreservation bag of FIG.

A cell cryopreservation bag 1 according to an embodiment of the present invention will be described below with reference to the drawings.
A cell cryopreservation bag 1 according to this embodiment is a container that can be stored by freezing a mixture of arbitrary cells and a preservation solution, and can be thawed and removed during transplantation surgery. As shown, an outer container 2, an inner container 3 and two ports 4, 5 are provided.

  The outer container 2 is a known material similar to the conventional one, for example, a laminated film of a polyimide film and a fluorinated ethylene propylene polymer film, a copolymer film of tetrafluoroethylene and ethylene, etc. Thus, the sealed portion 2a is sealed.

The inner container 3 is a mesh container, which is smaller than the outer diameter size of the cell capable of restricting the permeation of the cell, and has a diameter that allows a free flow of the liquid, for example, 0.2 to 8 μm, preferably 1 It has a large number of through holes having ˜4 μm.
The surface of the inner container 3 is subjected to a hydrophilic treatment so that cells housed therein do not adhere.

The first port 4 extends through the seal portion 2 a at the periphery of the outer container 2 to the inside and outside of the outer container 2, and one end of the inside is opened in the inner container 3.
Similarly, the second port 5 extends through the seal portion 2 a at the periphery of the outer container 2 to the inside and outside of the outer container 2 and opens into a space A between the outer container 2 and the inner container 3. . The tip portion 5a of the second port 5 is closed in the initial state.

  The 1st port 4 and the 2nd port 5 are comprised by the tube of the material which has a certain amount of transparency and flexibility, such as a vinyl chloride resin, a silicone resin, a polyurethane resin, a polyethylene resin. These tubes can be easily welded and cut by heat, and can be cut and connected aseptically by a tube sealer.

The operation of the cell cryopreservation bag 1 according to this embodiment configured as described above will be described below.
To cryopreserve cells using the cell cryopreservation bag 1 according to the present embodiment, as shown in FIG. 2, the mixed solution B of the cells and the preservation solution is stored in the inner container via the first port 4. After being supplied into the tube 3, the tube constituting the first port 4 is welded and cut and sealed as shown in FIG. The cells C in the mixed solution B cannot pass through the through holes of the inner container 3 and are held in the inner container 3, and the preservation solution freely flows through the through holes and is stored in both the inner container 3 and the outer container 2. The
In this state, the mixed solution B is frozen together with the cell cryopreservation bag 1.

  Next, when using cells that have been cryopreserved by the cell cryopreservation bag 1 according to the present embodiment, the cell cryopreservation bag 1 is heated together with the mixture B being thawed in the second state. The tube constituting the port 5 is cut and opened. As a result, as shown in FIG. 4, the storage liquid D stored in the outer container 2 and the inner container 3 is discharged to the outside.

In this state, the cells C remain in the inner container 3 in the cell cryopreservation bag 1 and most of the preservation solution D is discharged, but a part of the preservation solution D adheres to the cells C and the like. It remains in the state.
Therefore, a cleaning liquid container (not shown) storing the cleaning liquid E is connected to the tube constituting the first port 4, and the cleaning liquid E is stored in the cell cryopreservation bag from the cleaning liquid container as shown in FIG. 1 is supplied. Thereby, the cleaning liquid E is supplied into the inner container 3, the cells C are washed, and the inner surfaces of the inner container 3 and the outer container 2 are also washed by passing through the through holes of the inner container 3. Then, the cleaning liquid E ′ after cleaning is discharged from the second port 5 to the outside.

  In the state where the cleaning liquid E ′ after the cleaning has been discharged, as shown in FIG. 6, the second port 5 is closed by welding, and the first port 4 is placed in a lactating Ringer solution container (not shown) for transplantation. Reconnect and supply the lactated Ringer's solution F into the cell cryopreservation bag 1. Thereby, since the cell C is suspended by the lactate Ringer solution F to form the cell suspension G, the lactate Ringer solution container is separated from the first port 4 as shown in FIG. By inverting 1, the cell suspension G in the cell cryopreservation bag 1 can be discharged to the outside through the first port 4.

  As described above, according to the cell cryopreservation bag 1 according to the present embodiment, when only the cells C are taken out from the thawed mixed solution B, a large-scale device such as a centrifuge is not necessary. There is an advantage that it can be easily taken out. Further, the cell suspension G containing clean cells C from which the preservation solution D has been removed can be taken out without using a special device such as a washing device, and can be used for transplantation as it is.

  In this embodiment, the case of having two ports 4 and 5 has been described, but instead of this, as shown in FIG. 8, three ports 4, 5, 6 or as shown in FIG. You may decide to have four ports 4, 5, 6, and 10.

  A third port 6 shown in FIG. 8 opens into the inner container 3. And the tubes 6a and 6b which comprise the 3rd port 6 branch into two, and the valves 7 and 8 are provided, respectively. The second port 5 is also provided with a valve 9.

One tube 6a constituting the third port 6 is alternatively provided with a cleaning liquid container (not shown) storing the cleaning liquid E or a transplanting liquid container (not shown) storing the lactated Ringer's solution F used for transplantation. Connected.
In this way, after supplying the mixed solution B of the cells C and the preservation solution D from the first port 4, the first port 4 is welded and closed, and the subsequent washing solution E and lactated Ringer solution F Can be performed from one tube 6a and the cell suspension G can be released from the other tube 6b by opening and closing the valves 7 and 8 of the third port 6. Further, the storage liquid D can be discharged and the cleaning liquid E ′ after cleaning can be discharged by opening and closing the valve 9 of the second port 5.

  A fourth port 10 shown in FIG. 9 is provided for assigning ports to the two tubes 6 a and 6 b of the third port 6, and is provided with a valve 11. That is, a cleaning liquid container (not shown) storing the cleaning liquid E is connected to one tube 6a constituting the third port 6, and a transplanting liquid storing the lactated Ringer's solution F used for transplantation is connected to the other tube 6b. A container (not shown) is connected. The cell suspension G can be released from the fourth port 10. Thereby, there is an advantage that it is not necessary to switch between the supply of the cleaning liquid E or the lactated Ringer's solution F and the release of the cell suspension G, and the configuration can be simplified.

  When the fourth port 10 for releasing the cell suspension G is provided, the third port 6 for supplying the washing liquid E or the lactated Ringer's solution F into the cell cryopreservation bag 1 is connected to the outer container 2 and the inner part. You may open to the space A between the containers 3. FIG.

A Space D Storage solution (liquid)
E Cleaning liquid (liquid)
E 'Cleaning liquid (liquid)
F Lactated Ringer's solution (liquid)
DESCRIPTION OF SYMBOLS 1 Cell cryopreservation bag 2 External container 3 Internal container 4 1st port 5 2nd port 6 3rd port 7, 8 Valve 10 4th port

Claims (4)

A sealed outer container;
A mesh-like inner container that is disposed in the outer container and restricts the passage of cells and allows the passage of liquid;
A first port penetrating the outer container and opening into the inner container;
A cell cryopreservation bag comprising a second port that penetrates through the outer container and opens into a space between the outer container and the inner container.   The cell cryopreservation bag according to claim 1, wherein the first port and the second port are each provided with a valve for opening and closing each port.   The cell cryopreservation according to claim 1 or 2, further comprising a third port that penetrates through the outer container and opens in either the inner container or a space between the outer container and the inner container. bag.   The cell cryopreservation bag according to claim 3, further comprising a fourth port that penetrates through the outer container and opens in either the inner container or a space between the outer container and the inner container.

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