JP2015019657A - Cell culture device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible in a closed-system cell culture device to take out one or a plurality of cell culture vessels while preventing intrusion of particles including a microorganism from outside.SOLUTION: A cell culture device includes: a cell culture vessel having an introduction port and discharge port for fluid; a supply bag that contains a liquid necessary in culture; a collection bag that collects the liquid after use; and an integrated flow channel member to which a plurality of cell culture vessels are connected in parallel, and which connects the supply bag and any cell culture vessel. The integrated flow channel member has an introduction port and a discharge port which form a flow channel by being connected with the introduction port and the discharge port of each cell culture vessel, and the cell culture vessel and the integrated flow channel member are separated from each other after being closed.

Description

  The present invention relates to a cell culture apparatus for culturing cells.

  Conventionally, cell culture work has been performed manually by skilled workers under a strict manufacturing process in a clean room that is sterilized as much as possible. Therefore, when cultivating a large amount of cells for industrialization, the burden on the worker is increased, the time and cost required to educate and nurture the worker, human error and sample mistakes, and bacteria etc. There is a possibility that contamination from humans who own the product will occur, and the countermeasures are costly. This becomes a big wall in the industrialization of cell mass culture. Therefore, it is expected to solve these problems by automating a series of cultures using an instrument. Therefore, at present, an automatic cell culture device that mimics a culture operation that is performed manually by using an arm type robot manipulator is mainly developed. Patent Document 1 provides an example in which an articulated robot manipulator is used to process a culture container transfer operation, a medium exchange, and the like. In this articulated robot manipulator, it is possible to sterilize itself.

  However, since the cell culture apparatus as described in Patent Document 1 is not a closed system, the operation of opening the lid and opening the inside of the culture container is necessary, and the inside of the cell culture apparatus in which the container is installed is required. There is a problem that the cleanliness must be maintained, and the overall system becomes larger and more costly than when working manually.

  Therefore, as in Patent Document 2, a system has been devised in which a culture vessel is sealed and a cell is cultured by supplying driving force from the outside. As a result, there is no external contamination, and the overall system cost can be reduced.

  Further, in a closed cell culture device as shown in Patent Document 2, the structure of a cell culture container that can prevent invasion of particles containing microorganisms from outside the cell culture space and liquid leakage from the cell culture space is disclosed in Patent Document 3. Is provided by.

JP 2006-149268 A JP 2011-142837 A JP 2011-10599 A

  However, in the structure of Patent Document 3, when the cell culture container is removed from the inside of the cell culture apparatus, the sealing property (closed system) on the cell culture container side is maintained, but the closed system on the side supplying the culture solution is maintained. Is not kept. For example, in a clinical trial of corneal transplantation using a cell sheet, a procedure for taking out a cell sheet from one container from the previous day's culture in a plurality of containers and inspecting it has become a procedure. In order to inspect the quality, it is conceivable that some cells are taken out and evaluated. However, in a closed cell culture apparatus to which a plurality of cell culture vessels are connected, the entire closed system is It is a problem to take out and inspect the cells as described above while keeping them.

  Accordingly, an object of the present invention is to provide a cell culture apparatus capable of taking out one or a plurality of cell culture containers while preventing invasion of particles containing microorganisms from the outside in a closed cell culture apparatus. is there.

  In order to achieve the above object, the cell culture container of the present invention comprises a cell culture container having a fluid inlet and outlet, a supply bag for storing a liquid necessary for culture, and a collection bag for recovering the used liquid. A plurality of cell culture containers connected in parallel, and comprising an integrated flow path member for connecting the supply bag and any one of the cell culture containers, Each of the cell culture containers has an introduction port and a discharge port that are connected to the introduction port and the discharge port of the cell culture container to form a flow path, and the cell culture container and the integrated flow path member are separated from each other after being closed.

  According to the present invention, in a closed system cell culture device, by providing a cell culture device capable of taking out one or a plurality of cell culture containers while preventing the entry of particles containing microorganisms from the outside, Characteristics and safety can be confirmed by conventional methods.

Schematic diagram of a closed system cell culture device. The schematic diagram of the connection part of an integrated flow path member and a cell culture container. (a) Side view of the flow path blocking structure according to the first embodiment of the present invention (b) AA sectional view. (a) Side view showing a state where the flow path is blocked by the elastic body of the flow path blocking structure according to the first embodiment of the present invention. (b) AA sectional view. The side view which shows a mode that the connection of the integrated flow path member and the cell culture container by which the flow path was interrupted | blocked by the flow path blocking structure which is the 1st Embodiment of this invention was cancelled | released. The side view which showed a mode that the flow path interruption | blocking mechanism which is the 2nd Embodiment of this invention was interrupted | blocked and a cell culture container was taken out.

  The configuration of the cell culture device of the present invention will be described with reference to FIG.

  The cell culture device has a cell culture chamber 10, a refrigerator 11, a liquid drive unit 12, and a collection bag 15. In the cell culture chamber 10, an accumulation channel member 16, a cell culture container 17, and a cell observation device 13 for observing the state of cells are arranged. The refrigerator 11 is provided with a supply bag 14 for storing a liquid necessary for culture such as a culture solution. The supply bag 14 is connected to the accumulation channel member 16. A plurality of cell culture containers 17 can be connected in parallel to the integrated flow path member 16 that collects the flow paths for sending cells and culture solution to one or a plurality of cell culture containers. The cell culture vessel 17 includes a fluid inlet and outlet.

  Further, the collecting channel member 16 is also connected to the recovery bag 15, and the waste liquid from each cell culture container 17 can be flowed to the recovery bag 15. The flow path connecting the supply bag 14, the accumulation flow path member 16, the cell culture container 17, and the recovery bag 15 is a closed system (closed system) so as to prevent entry of microparticles containing microorganisms from the outside. The shape of the integrated flow path member 16 is shown as a hexagon in FIG. 1 as an example, but is not particularly limited.

  Next, embodiments will be described with reference to the accompanying drawings. However, it should be noted that the embodiments described below are merely examples for realizing the present invention, and do not limit the technical scope of the present invention. In each drawing, the same reference numerals are assigned to common components.

  2-5 is a figure which shows the flow-path interruption | blocking structure which is the 1st Embodiment of this invention. FIG. 2 is a schematic diagram of the connecting portion between the integrated flow path member 16 and the cell culture container 17. FIGS. 3A and 3B to FIG. A state of detaching from the road member 16 is shown.

  The integrated flow path member 16 and the cell culture vessel 17 are made of plastic having rigidity as well as plastic such as polycarbonate, polystyrene, and polypropylene. A channel 22 is formed in the cell culture container 17 and the accumulation channel member 16. The cell culture container 17 is provided with a fluid inlet, a cell culture space, and a fluid outlet. The fluid inlet and outlet are connected to the inlet and outlet of the integrated channel member 16 to form a channel. Is done.

  The integrated flow path member 16 and the cell culture container 17 are connected to each other with, for example, a pin 19 in a state where the elastic body 18 for adhesion is sandwiched between the integrated flow path member 16 and the flow path 22 of the integrated flow path member 16 and the cell culture container 17. Becomes a closed system. As shown in FIG. 3A, the integrated flow path member 16 and the cell culture container 17 have an upper end and a lower end protruding toward the cell culture container 17 side, and the cell culture container is projected to the protruding portion. A part of 17 is sandwiched and positioned. In this state, as shown in FIG. 3, the pin 19 penetrates the upper end and the lower end of the accumulation channel member 16 and the cell culture container 17 in a direction perpendicular to the channel 22, thereby collecting the accumulation channel member 16. And the cell culture vessel 17 are combined. In FIG. 3A, the lower end of the pin 19 protrudes from the integrated flow path member 16, but it is preferable that the pin 19 does not protrude.

  The material of the contact elastic body 18 is not particularly limited as long as the flow path 22 can be maintained in a closed system, but is preferably a material that is elastic, has good adhesion, and can be sterilized.

  The accumulation channel member side blocking member 20 and the cell culture container side blocking member 21 constitute a part of the contact surface between the accumulation channel member 16 and the cell culture container 17.

As shown in FIGS. 3 (a) and 3 (b), the flow path 22 is in a state where the integrated flow path member 16 and the cell culture container 17 are integrated.
When installed in the cell culture chamber 10, the integrated flow channel member side blocking member 20 and the cell culture container side blocking member 21 are accommodated in a blocking member accommodating portion located above the flow channel 22, as shown in FIG. Has been. Thus, the integrated flow path member 16 and the cell culture container 17 are connected via the flow path 22 in a state where the integrated flow path member side blocking member 20 and the cell culture container side blocking member 21 are accommodated in the blocking member accommodating portion. A state is reached (FIG. 3). Furthermore, as shown in FIG. 3, a blocking member pushing member 23 </ b> A having an L-shaped cross section is provided above the integrated flow path member side blocking member 20. Abut. Further, as shown in FIG. 3, a blocking member pushing member 23 </ b> B having an L-shaped cross section is provided above the cell culture container side blocking member 21, and the integrated channel member side blocking member 20 and the pin 19 are provided. It is in contact. Further, the blocking member pushing member 23A and the blocking member pushing member 23B are in contact with each other.

  As shown in FIG. 3, a part of the blocking member pushing member 23 </ b> A is located at the uppermost portion, and the pin 19 and the blocking member pushing member 23 </ b> B are in contact with the lower surface portion thereof. Furthermore, there is a gap between a part of the blocking member pushing member 23A described above and the accumulation flow path member 16, and the pin 19 is located in this portion. Further, the integrated flow path member 16 has a through hole penetrating from the upper part to the blocking member accommodating portion, and the blocking member pushing member 23A is provided in the through hole so as to be movable in the vertical direction of FIG. Similarly, the cell culture container 17 has a through-hole penetrating from the upper part to the blocking member accommodating portion, and the blocking member pushing member 23B is provided in this through-hole so as to be movable in the vertical direction of FIG.

  In this state, a force is applied from above to the blocking member pushing member 23A. Since the accumulation channel member 16 and the cell culture container 17 are coupled by the pin 19, the accumulation channel member side blocking member is in a state where the accumulation channel member side blocking member 20 and the cell culture container side blocking member 21 are pressed against each other. 20 and the cell culture container side blocking member 21 are pushed into the flow path 22.

  At this time, since the accumulation channel member 16 and the cell culture container 17 are coupled by the pin 19, the accumulation channel member side blocking member 20 and the cell culture container side blocking member 21 are pressed against each other. A force is exerted so as to seal each of the flow path 22A and the cell culture container flow path 22B. FIG. 4 shows the flow of force exerted by the accumulation channel member side blocking member 20 and the cell culture container side blocking member 21 respectively. Thereby, the flow path 22B of the cell culture container and the flow path 22A of the integrated flow path member connected to the cell culture container 17 are blocked, and the cell culture container 17 and the integrated flow path member 16 are independently closed systems. The cell culture container 17 can be aseptically removed from the system.

  The diameter of each blocking member 20, 21 is required to be larger than the diameter of the flow path. Since the diameter of each blocking member 20 and 21 is larger than the diameter of the flow path, the flow path can be sealed.

  The material of each blocking member 20, 21 is preferably a material that is elastic, has good adhesion, and can be sterilized, but is not particularly limited as long as the flow path 22 can be sealed. The flow path 22 may be blocked by using the blocking members 20 and 21 and the blocking member pushing members 23A and 23B as magnetic bodies and moving the blocking members by magnetic force.

  When the blocking member push-in members 23A and 23B push in the respective blocking members, the pins 19 that connect the accumulation channel member 16 and the cell culture vessel 17 are simultaneously pressed. Can be extracted. That is, the pin 19 cannot be pulled out until each blocking member 20, 21 is pushed into the flow path 22. Until the pin 19 is withdrawn, the cell culture vessel 17 cannot be withdrawn from the accumulation channel member 16.

  Accordingly, since the cell culture container 17 cannot be extracted from the integrated flow path member 16 unless the flow path 22 is blocked, the cell culture container 17 may be mistakenly extracted without the flow path 22 being blocked. It is possible to prevent the closed system from collapsing.

  Further, the blocking member pushing members 23A and 23B are divided into two parts. As a result, as shown in FIG. 5, the blocking portion pushing member is not removed when the collecting channel member 16 and the cell culture container 17 are separated. Can be separated. However, the blocking member pushing members 23A and 23B may be a single component. In that case, it is necessary to remove the blocking member push-out members 23A and 23B when separating the integrated flow path member 16 and the cell culture vessel 17. There is no particular limitation on the number of parts of the blocking member pushing member.

  In FIG. 5, the contact elastic body is attached to the cell culture container, but it may be attached to the accumulation channel member side, or may not be attached to either.

  2 to 5, the flow path blocking structure related to one cell culture container 17 is representatively shown. However, this flow path blocking structure is applicable to all the cell culture containers 17 connected to the integrated flow path member 16. Yes, it is not limited by the position of the cell culture vessel 17 or the like. Therefore, when the cell culture container 17 is taken out, any cell culture container 17 can be taken out from the plurality of cell culture containers 17.

  FIG. 6 is a diagram showing a second embodiment of the present invention. In the second embodiment, a heat flow path blocking mechanism 26 is provided, the tube 27 connecting the cell culture container 17 and the accumulation flow path member 16 is deformed by heat to block the flow path 22 and then the blocking section is cut. By doing so, the cell culture container 17 and the accumulation channel member 16 are each independently closed. Further, by using a material that can be deformed by heat for a part of the cell culture vessel 17 and a part of the accumulation channel member 16 without using the tube 27, the part is deformed to block the channel. You may do it. In this embodiment, in order to prevent the closed system of the flow path 22 from collapsing by mistakenly withdrawing the cell culture container 17 in a state where the flow path 22 is not blocked, the integrated flow path member 16 and the cell culture container 17 are connected. It is desirable that the connecting tube 27 is fixed to each of the accumulation channel member 16 and the cell culture container 17 so that the user cannot easily remove it.

  One heat channel blocking mechanism 26 may be provided for each cell culture vessel 17 or one cell culture device may be provided. Alternatively, a flow path blocking mechanism that the cell culture apparatus does not have may be brought into the apparatus and used, or the entire flow path from the supply bag 14 to the collection bag 15 is taken out of the apparatus and the flow path 22 is set outside the apparatus. You may block it.

DESCRIPTION OF SYMBOLS 10 ... Cell culture room 11 ... Refrigerator 12 ... Liquid drive part 13 ... Cell observation equipment 14 ... Supply bag 15 ... Collection bag 16 ... Accumulation channel member 17 ... Cell culture container 18 ... Container contact | adherence elastic body 19 ... Pin 20 ... Accumulation channel member side blocking member 21... Cell culture container side blocking member 22... Channel 22A... Accumulation channel member channel 22B... Cell culture container channels 23A and 23B. A flow of force exerted by the blocking member on the blocking member on the integrated flow channel member 25... A flow of force applied by the blocking member on the integrated flow channel member side on the blocking member on the cell culture container 26.

Claims (6)

A cell culture device having a cell culture container having a fluid inlet and a fluid outlet, a supply bag for storing a liquid necessary for culture, and a recovery bag for recovering the liquid after use,
A plurality of cell culture containers are connected in parallel, and include an integrated flow path member that connects the supply bag and any of the cell culture containers,
The integrated channel member has an inlet and an outlet that are connected to the inlet and outlet of each cell culture container to form a channel,
A cell culture apparatus, wherein the cell culture container and the collecting channel member are separated from each other after being closed. In claim 1,
The cell culture container and the collection channel are arranged so that the cell culture container and the collection channel member cannot be separated from each other until the introduction port and the discharge port in the cell culture container and the introduction port and the discharge port in the collection channel member are sealed. A cell culture apparatus, wherein members are connected. In claim 2,
A first sealing member that seals the inlet of the cell culture container, a second sealing member that seals the outlet of the cell culture container, and a third that seals the inlet of the accumulation channel member A sealing member and a fourth sealing member for sealing the discharge port of the integrated flow path member;
The cell culture container includes a first accommodating portion that accommodates the first sealing member before the first sealing member seals the inlet, and a second sealing member before the outlet is sealed. And a second housing portion for housing the second sealing member,
The integrated flow path member includes a third housing portion that houses the third sealing member before the third sealing member seals the introduction port, and a fourth sealing member that seals the discharge port. A cell culture apparatus comprising: a fourth storage portion that previously stores a fourth sealing member. In claim 3,
A fixing member that fixes the cell culture container and the accumulation channel member by penetrating in a direction perpendicular to a direction in which the liquid flows; the first sealing member seals the inlet of the cell culture container; The second sealing member seals the outlet of the cell culture container, the third sealing member seals the inlet of the integrated channel member, and the fourth sealing member discharges the outlet of the integrated channel member The cell culture device, wherein the fixing member does not come off until the cell is sealed. In claim 1,
A first connection channel member that connects the introduction port in the cell culture container and the introduction port in the accumulation channel member; a second connection channel member that connects the discharge port in the cell culture container and the discharge port in the accumulation channel member; Prepared,
A cell culture apparatus characterized in that these connection flow path members are deformed and cut in a state where the flow path is blocked. In claim 5,
A cell culture apparatus comprising a blocking mechanism for blocking the connection flow path member.