JP3175393U - Cell culture system - Google Patents

Abstract

A cell culture system in a sealed state capable of cultivating floating cells and continuously changing air while maintaining aseptic conditions.
A closed floating cell culture system capable of culturing floating cells of an additive includes a sterile culture pack, a connector device, a sterility maintenance device, and a connection device. The aseptic culture pack 10 is used for accommodating cultured cells and has a sealable opening 12. One end of the connector device 20 is inserted into the sealable opening 12 of the sterile culture pack 10. The sterility maintenance device 30 is attached to the end of the culture system. The connection device 40 is connected between the connector device 20 and the sterility maintenance device 30 to form a sealed cell culture space.
[Selection] Figure 1

Description

  The present invention relates to a cell culture system, and more particularly to a hermetically sealed cell culture system capable of culturing floating cells of additives.

  At the present time when regenerative medicine has been actively developed, various researches are ongoing, particularly for searching for special stem cells or self-renewing cells of organs in living organisms. In research related to self-replicating cells, research related to hematopoietic stem cells has received the most attention. That's because hematopoietic stem cell transplantation is a new treatment option, from cancer to metabolic disorders to immune deficiencies.

Hematopoietic stem cells are generally collected from bone marrow, surrounding blood and umbilical cord blood. Currently, human hematopoietic stem cells from adult bone marrow and posterior peripheral blood and umbilical cord blood transplants are not only used to treat clinical blood cancer (leukemia and malignant lymphoma), but also high-dose chemistry for non-hematologic cancer Helps restore the immune system through treatment.
However, in order to proceed with transplantation effectively, a large amount of hematopoietic stem cells from different collection sources are required. If the amount of transplantation of hematopoietic stem cells is sufficient for rapid recovery, it is possible to keep generational changes of blood and immune cells uniform and to greatly improve the success rate of transplantation. Generally speaking, to effectively transplant hematopoietic stem cells, the number of mononuclear cells (MNC) reaches 8 × 10 ⁶ cells / kg, and the number of CD34 hematopoietic stem cells reaches 2 × 10 ⁴ cells / kg. It is necessary to reach kg.

  One unit (pack) of hematopoietic stem cells collected from umbilical cord blood, peripheral blood, or bone marrow has a limited number of cells and can only be used for people weighing 20-30 kg, so it can be applied to transplantation for newborns or infants However, it is clear that there is not enough transplant for adults. Therefore, to reach an effective transplant amount, it is necessary to proliferate limited hematopoietic stem cells in an in vitro culture system.

The in vitro culture methods currently used are mainly cultured using a culture tray or a culture bottle. This method is a semi-open culture method. When replenishing or transferring cultured cells, the lid or stopper of the culture tray or culture bottle must be opened, so that microorganisms in the air can enter the culture tray or culture bottle. And may contaminate cells or culture media in the culture tray or bottle.
On the other hand, since the culture environment is hermetically sealed in the method of growing cells in vitro using a commercially available blood pack, it is possible to avoid secondary contamination that occurs when the cultured cells are replenished or transferred. However, since the air inside and outside the blood pack cannot be exchanged, it affects the cell proliferation effect.

In view of the problems of the in vitro culture method described above, the present invention provides a closed culture system capable of culturing additives based on research and development spirit and expertise, thereby causing secondary contamination problems in a semi-open culture apparatus. To achieve a relatively good cell proliferation effect.
Specifically, the main object is to provide a sealed cell culture system capable of cultivating floating cells (for example, hematopoietic stem cells) and maintaining the sterility and continuously changing air. .

  In order to achieve the above-described object, a sealed cell culture system capable of culturing floating cells of an additive according to the present invention includes a sterile culture pack, a connector device, a sterile maintenance device, and a connection device. The aseptic culture pack is used for accommodating cultured cells and has a sealable opening. One end of the connector device is inserted into the sealable opening of the sterile culture pack. A sterile maintenance device is attached to the end of the culture system. The connection device is connected between the connector device and the sterility maintenance device to form a sealed cell culture space.

  In one embodiment of the present invention, the sterile culture pack is constructed from a flexible material. The sterility maintenance device may have a wet film or a sterility filter.

  In one embodiment of the present invention, the culture system further comprises a flow guide device. The flow guide device is mounted between the connector device and the sterility maintenance device, and is used for connection to the replenishment device. The replenishing device can inject a cell culture additive with a flow guide device and introduce the cultured cells into the sterile culture pack or derive the cultured cells from the sterile culture pack.

  In one embodiment of the present invention, the sterile culture pack has a sealable first opening and a sealable second opening. The connection device has a first pipeline and a second pipeline. The connector device has a first connector and a second connector. One end of the first connector is inserted into the sealable first opening, and the other end is connected to the front end of the first pipe line. One end of the second connector is inserted into the sealable second opening, and the other end is connected to the front end of the second pipe line.

Furthermore, the sterility maintenance device is connected to the end of the first conduit, and the second conduit is connected to the replenishment device at the end, and is used for supplying culture additives or introducing cultured cells into a sterile culture pack. Cultured cells may be derived from a sterile culture pack.
In addition, the sterile culture pack is connected in series to the storage device by the second conduit, so that the cultured cells in the sterile culture pack can be transferred to the storage device.

  In order to clarify the above-described objects and features of the present invention, detailed description will be made based on the following embodiments and drawings.

1 is a schematic diagram showing a configuration of a cell culture system according to a first embodiment of the present invention. It is a schematic diagram which shows the structure of the cell culture system by 2nd Embodiment of this invention. It is a schematic diagram which shows the structure of the cell culture system by 3rd Embodiment of this invention. It is a schematic diagram which shows the structure of the cell culture system by 4th Embodiment of this invention.

  Hereinafter, the present invention will be described with reference to a plurality of embodiments. The contents described in the drawings and specification, i.e., the relatively preferred embodiments, are merely examples for explaining the present invention. The present invention is not limited to the specific embodiments described in the drawings and specification.

Hereinafter, a closed floating cell culture system 1 according to the present invention will be described with reference to the drawings.
(First embodiment)
FIG. 1 is a schematic diagram showing the first embodiment. The closed floating cell culture system 1 according to the first embodiment includes a sterile culture pack 10, a connector device 20, a sterility maintenance device 30, and a connection device 40.

  The aseptic culture pack 10 is used for accommodating culture objects such as floating cells and culture media, and has a sealable opening 12. One end of the connector device 20 is inserted into the sealable opening 12 of the sterile culture pack 10. The sterility maintenance device 30 is attached to the end of the culture system 1. The connection device 40 is connected between the connector device 20 and the sterility maintenance device 30 to form a sealed cell culture space.

In the sealed floating cell culture system 1 according to the present invention, the volume of the sterile culture pack 10 varies depending on the number of cells accommodated. The aseptic culture pack 10 is relatively preferably composed of a flexible material such as a commercially available blood pack.
The sterility maintenance device 30 has a wet film. The air inside and outside the culture system 1 is exchanged through the wet film by the osmotic diffusion action. On the other hand, the liquid is still contained in the culture system 1.
It is relatively preferable that the sterility maintenance device 30 further includes a sterility filter. The sterile filter has a pore diameter of 0.22 μm, and can effectively block microorganisms from entering the culture system 1 and maintain a sterile culture state.

(Second Embodiment)
FIG. 2 is a schematic diagram showing the second embodiment. Differences from the first embodiment are as follows.
The sealed floating cell culture system 1 according to the second embodiment further includes a flow guide device 50. The flow guide device 50 is mounted between the connector device 20 and the sterility maintenance device 30. The flow guide device 50 may employ a three-way valve. The connector device 20 and the sterility maintenance device 30 can be connected via the connection device 40. Moreover, the diversion apparatus 50 may be comprised from the Y-shaped connection apparatus 40 (not shown in the figure).

The closed floating cell culture system 1 further includes a supply device 60. The replenishing device 60 is connected to the flow guide device 50. The culture additive or cultured cells are introduced into the aseptic culture pack 10 by the replenishing device 60 via the flow guide device 50 or are derived from the aseptic culture pack 10.
The replenishing device 60 may be composed of a peristaltic pump or a syringe.

(Third embodiment)
FIG. 3 is a schematic diagram showing the third embodiment. Differences from the above-described embodiment are as follows.
The sealable opening 12 of the sterile culture pack 10 has a sealable first opening 12a and a sealable second opening 12b.
The connector device 20 includes a first connector 22 and a second connector 24. The first connector 22 is inserted into the first opening 12a that can be sealed at one end. The second connector 24 is inserted into the second opening 12b whose one end can be sealed.
The connection device 40 has a first pipeline 42 and a second pipeline 44. The front end of the first conduit 42 is connected to the other end of the first connector 22. The front end of the second conduit 44 is connected to the other end of the second connector 24.

  The replenishing device 60 is connected to the end of the second conduit 44 and can introduce cultured cells into the aseptic culture pack 10 or derive the cultured cells from the aseptic culture pack 10. This embodiment can increase the number of sealable openings 12a and 12b, connectors 22 and 24, and conduits 42 and 44 according to the culture demand.

(Fourth embodiment)
FIG. 4 is a schematic diagram showing the fourth embodiment. Differences from the above-described embodiment are as follows.
The aseptic culture pack 10 and the storage device 70 can be connected in series by the second conduit 44 or the flow guide device 50. The storage device 70 may be another sterile culture pack. The hematopoietic hepatocytes that have been proliferated and cultured move from the sterile culture pack 10 to the storage device 70 via the second conduit 44, and can respond to hematopoietic stem cell transplantation. The storage device 70 may be expanded by a plurality of serial connections according to the demands of a culture engineer.

Subsequently, cell culture is carried out using different culture lines, and the results are compared as follows.
First, a frozen blood sample of umbilical cord blood is thawed in a 37 ° C. water bath. Subsequently, erythrocytes in the sample are lysed and removed with cord blood erythrocyte lysate (RBC lysis buffer) containing 0.83% ammonium chloride (pH 7.0) containing 0.1% sodium bicarbonate. Subsequently, the amount of 300 g is centrifuged 2 to 3 times (adjusted according to the transparency of the liquid) for 5 minutes, and mononuclear cells (MNC) are collected. Alternatively, CD34 stem cells are collected by a method of separating a specific target with magnetic beads.

  Harvested mononuclear cells and CD34 stem cells were suspended in IMDM medium containing 10% FBS, followed by addition of cytokines such as 5 ng / ml IL-3, 10 ng / ml IL and 15 ng / ml SCF. Subsequently, they are stored in a culture tray, a commercial culture pack, and a culture system according to the present invention for cell culture.

Table 1 shows the results of proliferation culture of mononuclear cells and CD34 stem cells using different culture apparatuses. According to the experimental results, the number of mononuclear cells cultured in the culture system according to the present invention increased from 15.20 × 10 ³ μl to 30.29 × 10 ³ μl after culturing for 3 days, and the number of CD34 stem cells Was found to increase from 211 to 1053. Accordingly, the present invention not only has a proliferation effect superior to a commercially available culture pack, but also has a cell proliferation number close to that obtained by culturing with a culture medium.

  As described above, based on the culture results of mononuclear cells and CD34 stem cells, the floating cell culture system according to the present invention achieves the sealing required for the culture environment without affecting the cell proliferation effect, and prevents secondary contamination. It turns out that it can be avoided.

  As mentioned above, this invention is not limited to the said embodiment at all, In the range which does not deviate from the meaning of invention, it can be implemented with a various form.

1: Sealed floating cell culture system,
10: Aseptic culture pack,
12: Sealable opening,
12a: Sealable first opening,
12b: a second opening that can be sealed;
20: Connector device,
22: first connector,
24: second connector,
30: Aseptic maintenance device,
40: connection device,
42: first pipeline,
44: Second pipeline,
50: Current guide device,
60: Supply device,
70: Storage device.

Claims (10)

A closed cell culture system capable of culturing floating cells of additives,
A sterile culture pack used for accommodating cultured cells and having a sealable opening;
A connector device having one end inserted into a sealable opening of a sterile culture pack;
A sterile maintenance device attached to the end of the cell culture system;
A connection device that forms a sealed cell culture space by being connected between the connector device and the sterility maintenance device;
A cell culture system comprising:   The cell culture system according to claim 1, wherein the aseptic culture pack is made of a flexible material.   The cell culture system according to claim 1, wherein the sterility maintenance device has a wet film.   The cell culture system according to claim 3, wherein the sterility maintenance device further includes a sterility filter.   The cell culture system according to claim 1, further comprising a flow guide device mounted between the connector device and the sterility maintenance device.   6. The apparatus according to claim 5, further comprising a replenishing device connected to the flow guide device, for introducing the culture additive and the cultured cells into the sterile culture pack via the flow guide device, or for leading out the sterile culture pack. Cell culture system. The sealable opening has a sealable first opening and a sealable second opening;
The connecting device has a first pipeline and a second pipeline,
The connector device has a first connector and a second connector,
One end of the first connector is inserted into the first opening that can be sealed, and the other end is connected to the front end of the first pipeline,
The cell culture system according to claim 1, wherein one end of the second connector is inserted into a second opening that can be sealed, and the other end is connected to a front end of the second conduit. The sterility maintenance device is connected to the end of the first conduit,
The cell according to claim 7, further comprising a replenishing device connected to the end of the second conduit, for introducing the culture additive and the cultured cell into or out of the sterile culture pack. Culture system.   The cell culturing system according to claim 6 or 8, wherein the replenishing device comprises a peristaltic pump or a syringe.   The cell culture system according to claim 7, wherein the sterile culture pack is connected in series to the storage device by a second conduit, thereby transferring the cultured cells in the sterile culture pack to the storage device.

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