JP2019170167A - Cell culture vessel - Google Patents

Abstract

To provide a cell culture vessel capable of facilitating filling in cell culture using clinostat, and suppressing convection flow of a culture broth generated in a culture vessel due to air bubbles or stress to cells due to the convection flow.SOLUTION: There is provided a cell culture vessel comprising: a filling port through which a culture broth is put; a culture area for culturing cells; a separation area for separating air bubbles in the culture area; and a guide/keep mechanism for guiding the air bubbles in the culture area into the separation area, and causing the separated air bubbles to stay in the separation area.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a cell culture vessel in cell culture using a clinostat.

As an apparatus for studying the gravitational response of plants and cells, a clinostat as shown in FIG. According to this apparatus, the gravity of the mounted cells can be evenly dispersed by rotating two orthogonal axes, and a microgravity environment can be generated in a simulated manner.
The above-described clinostat generally uses a culture vessel as shown in FIG. This culture vessel has a structure in which a culture solution does not spill even when the culture flask is tilted by providing the culture flask with a sealing cap.
When culturing using such a culture vessel, if air remains in the culture vessel, convection of the culture solution occurs in the vessel due to rotation or tilting of the culture vessel, which causes stress to the cells. End up.
Therefore, the culture solution in the culture vessel is preferably liquid-tight.

In other words, when the culture flask is filled with the culture solution, if the amount of the culture solution is small, air remains and does not become liquid-tight. When mounted on the clinostat in this state, the convection of the culture solution occurs due to the rotation, which may cause stress on the cells.
On the other hand, even if the culture medium is added with great care so that air does not remain for liquid-tightness, the culture liquid may overflow when the cap is closed. In that case, there is a risk of causing contamination (mold, bacterial growth, etc.).
Therefore, the work of filling the culture solution so as to be liquid-tight without leaving air requires skill.
Therefore, the present applicant has invented a container sealing structure as shown in Patent Document 3. According to the culture container of this invention, anyone can easily perform the operation of filling the culture solution without leaving air in the culture container. Therefore, even in the culture using the clinostat, the convection of the culture medium due to the residual air can be eliminated and the stress on the cells can be suppressed.

Japanese Patent Laying-Open No. 2003-9852 (FIG. 1) Japanese Patent Laid-Open No. 10-179137 (FIG. 1) JP, 2006-208920, A

However, in cell culture, there are not only bubbles generated by residual air when filling the culture solution, but also bubbles generated by changes in the temperature of the metabolites of the cells being cultured and the culture solution. In addition, there are bubbles generated in the culture vessel due to the evaporation of the culture solution. Therefore, in the container sealing structure shown in Patent Document 3, bubbles generated by residual air when filling the culture solution can be suppressed, but the bubbles generated during the culture are not considered. The remarkable effect is thin.
For this reason, in the culture using a clinostat, the stress on the cells due to the convection of the culture medium due to the bubbles cannot be completely suppressed.

  Accordingly, an object of the present invention is to provide a cell culture container that can be easily filled in a cell culture using a clinostat, and that can suppress convection of a culture solution caused by bubbles in the culture container and stress on the cells caused thereby. Is to provide.

The cell culture container of the present invention includes a filling port for containing a culture solution, a culture area for culturing cells, an isolation area for isolating bubbles in the culture area, and guiding the bubbles in the culture area to the isolation area and isolating them. And a guide fastening mechanism for retaining the formed bubbles in the isolation area.
According to this configuration, even if bubbles are generated in the culture area, the bubbles can be moved while being guided to the isolation area, and the bubbles moved to the isolation area can be kept in the isolation area.
In addition, air bubbles remaining when the culture solution is added can be moved while being guided to the isolation area, and the bubbles moved to the isolation area can be kept in the isolation area.
Therefore, the culture medium can be easily filled, and the bubbles can be moved and kept in the isolation area, so that convection due to the bubbles can be suppressed in the culture area, resulting in stress on the cells. Can be relaxed.

  According to the present invention, in a cell culture using a clinostat, a cell culture container that can be easily filled and that can suppress convection of a culture medium caused by bubbles in the culture container and thereby stress on the cells. Can be provided.

It is the perspective view which showed typically the structure of the cell culture container in one Embodiment of this invention. It is the figure which showed the structure of the cell culture container of FIG. 1, (a) is a top view, (b) is the figure which each showed the front view. It is sectional drawing of the cell culture container shown in FIG. 2, (a) is an AA sectional view, (b) is a BB sectional view, (c) is a CC sectional view, (d) is a D- It is the figure which each showed the D line cross section. It is the figure which showed that the bubble in the culture area in the cell culture container of FIG. 2 moved to an isolation area. It is the elements on larger scale of the example which showed the operation | movement of the bubble at the time of rotation in the isolation area in the cell culture container of FIG. It is sectional drawing which showed typically the structure of the cell culture container in the modification 1 of this invention. It is sectional drawing which showed typically the structure of the cell culture container in the modification 2 of this invention. It is sectional drawing which showed typically the structure of the cell culture container in the modification 3 of this invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited to the following embodiment. Moreover, it can change suitably in the range which does not deviate from the range which has the effect of this invention.

FIG. 1 is a perspective view schematically showing a configuration of a cell culture container in one embodiment of the present invention. In FIG. 1, as an example, a lid 10 is provided on one side and a lid is not provided on the other side. However, in actual use, the lid 10 is attached to both.
As shown in FIG. 1, the cell culture vessel 100 is mounted on a clinostat (not shown) or the like that can generate a microgravity environment in a simulated manner. In that case, it is rotated by a clinostat. In addition, when mounted on a clinostat, it is possible to perform culture by adhering cells to the inner surface of the container or culturing while floating the cells in the culture solution.

  2A and 2B are diagrams showing the structure of the cell culture container of FIG. 1, wherein FIG. 2A is a top view and FIG. 2B is a front view.

  3 is a cross-sectional view of the cell culture vessel shown in FIG. 2, wherein (a) is a cross-sectional view taken along line AA, (b) is a cross-sectional view taken along line BB, and (c) is a cross-sectional view taken along line CC. Sectional drawing and (d) have each shown the DD sectional view.

In this embodiment, in the standing state of the cell culture container (for example, FIG. 2B), the ceiling is “upper”, the bottom is “lower”, the direction connecting the pair of lids is the left-right direction, and the right side of the drawing Is defined as “right” and the opposite side is defined as “left”.
In the following, what is generated by residual air, what is generated by changes in the temperature of the metabolites of the cells being cultured and the culture solution, and those that are generated due to evaporation of the culture solution are collectively referred to as “bubbles”. I'm calling.
As shown in FIGS. 2 and 3, the basic configuration of the cell culture container 100 includes a filling port 1, a culture area 2, an isolation area 3, a guide fastening mechanism 4, and the like.

  The filling port 1 has a cylindrical shape, and is provided on the upper surface of the culture area 2 as an inlet for putting the culture solution into the culture area 2. The cylindrical front end side is provided with an outer peripheral surface having a male screw thread, and the lid 10 is screwed into the male screw thread.

  The culture area 2 is an area for culturing cells, a first culture area 2a having a substantially circular cross section, and a second culture area 2b protruding outward from a part (left side) of the outer edge in a rectangular shape. It is made up of.

The isolation area 3 is an area for isolating air bubbles from the culture area 2 and has the same shape as the second culture area 2b at a position facing the second culture area 2b with respect to the first culture area 2a. Area is provided.
The isolation area 3 is provided with a filling port 6 for containing a culture medium equivalent to the filling port 1, and a lid 11 can be screwed to the upper end portion.

  The induction retaining mechanism 4 is a mechanism for guiding the bubbles in the isolation area 3 and the culture area 2 to the isolation area 3 and for keeping the isolated bubbles in the isolation area 3. A funnel-shaped cut wall 5 is provided so as to narrow the opening.

More specifically, as shown in FIGS. 3 (c) and 3 (d), the cut wall 5 is formed in the two areas from the boundary between the first culture area 2 a and the isolation area 3 toward the center line J. A pair of arc-shaped guide walls 5a are provided while blocking the vertical direction. Then, a pair of straight retaining walls 5b parallel to the center line J in the right direction are integrally formed while blocking the vertical direction from the vicinity of the center of the pair of guide walls 5a toward the isolation area 3 from the vicinity of the center. Is provided.
Although the details will be described later, the cell culture container 100 rotates while being mounted on the clinostat, but the bubbles in the container are guided toward the isolation area 3 by the guide wall 5a. However, even if it rotates, it is possible to keep air bubbles isolated in the isolation area 3 from returning to the culture area 2 by the retaining wall 5b.

Next, the operation of the cell culture container in one embodiment of the present invention will be described.
FIG. 4 is a diagram showing how bubbles in the culture area in the cell culture container of FIG. 2 move to the isolation area. (A) shows the state where the isolation area is located on the opposite side to the gravity direction, and (b) shows the state where the isolation area is located on the same direction side with respect to the gravity direction.
As shown in FIG. 4A, the bubble K generated during cell culture in the clinostat always moves in the direction opposite to the gravity due to the influence of buoyancy. Therefore, as the cell culture vessel 100 rotates, the bubble K gradually moves (invades) into the isolation area 3 along the arc-shaped guide wall 5a. At this time, the culture medium of the same amount as the invading bubbles K flows from the isolation area 3 into the culture area 2, so that the culture area 2 is always kept in a liquid-tight state. On the other hand, as shown in FIG. 4B, when the cell culture vessel 100 is rotated 180 degrees by the rotation of the clinostat from the state of FIG. 4A, the bubbles K move in the direction opposite to the gravity due to the influence of buoyancy. However, the guide wall 5a and the retaining wall 5b are fastened to the isolation area 3 so as not to return to the culture area 2.

FIG. 5 is a partially enlarged view showing the operation when the bubbles in the isolation area rotate in the cell culture container of FIG. 1, and (a) shows the position of the isolation area on the gravity direction side with respect to the position of the culture area. (B) to (h) are diagrams showing states rotated counterclockwise every 45 ° around the central axis L from the state of the previous symbol.
As shown in FIG. 5, in any state of the cell culture container 100 by the rotation of FIGS. 5A to 5H, the bubbles K in the isolation area 3 are separated from the cut wall 5 and the inner wall of the isolation area 3. It can be seen that the structure stays in the isolation area 3 and does not move to the culture area 2. 5 (a) to 5 (h) are diagrams showing movement every 45 degrees, but the same can be said at other angles.
Note that the length m and the interval n of the pair of retaining walls 5b can be appropriately determined according to the amount and size of the bubbles K, and the bubbles K are retained in the isolation area 3 as the length is increased or decreased. be able to.

  According to the cell culture container 100 in one embodiment of the present invention, bubbles in the culture area 2 can be gradually guided by the guide wall 5a during the rotation and moved into the isolation area 3. Moreover, even if the cell culture container 100 rotates, the air bubble isolated in the isolation area 3 can be retained in the isolation area 3 by the retaining wall 5b. As a result, there are no bubbles in the culture area 2, and the culture solution in the culture area 2 does not convect. Accordingly, the cells in the culture area 2 can be cultured without applying stress.

(Modification 1)
FIG. 6 is a cross-sectional view schematically showing the configuration of the cell culture container in Modification 1 of the present invention. Since the basic structure such as the filling port 1, the culture area 2, and the isolation area 3 is the same as that of the embodiment of the present invention, description of the reference numerals is omitted.

  As shown in FIG. 6, the contact portion between the bottom portion of the isolation area 3 and the retaining wall 5 b in the cell culture container 200 has an inclined portion 5 c that is inclined upward from the culture area 2 to the isolation area 3. Provided.

As described above, according to the cell culture container 200 in the first modification of the present invention, the bubbles in the culture area 2 are more easily guided into the isolation area 3 by the inclined portion 5c along the inclined portion 5c. On the other hand, since the return port becomes narrower once the bubble once enters the isolation area 3, it is difficult to return to the culture area 2 and can be retained in the isolation area 3.
In addition, at the time of loading, the cells are less likely to move into the isolation area 3, and as a result, can remain in the culture area.

(Modification 2)
FIG. 7 is a cross-sectional view schematically showing the configuration of the cell culture container in Modification 2 of the present invention. As in the first modification, the basic structure is the same as that of the embodiment of the present invention, and the description of the reference numerals is omitted. The difference from the embodiment of the present invention is the configuration of the retaining wall. Details will be described below.

  As shown in FIG. 7, the pair of retaining walls 5 d in the cell culture container 300 is configured to be inclined while approaching the center line J from the culture area 2 to the isolation area 3.

  Thus, according to the cell culture container 300 in Modification 2 of the present invention, the air bubbles in the culture area 2 are more easily guided into the isolation area 3 by the pair of retaining walls 5d along the retaining walls 5d. Become. On the other hand, since the return port becomes narrower once the bubble once enters the isolation area 3, it is difficult to return to the culture area 2 and can be retained in the isolation area 3.

(Modification 3)
FIG. 8 is a cross-sectional view schematically showing the configuration of the cell culture container in Modification 3 of the present invention. As in the first modification, the basic structure is the same as that of the embodiment of the present invention, and the description of the reference numerals is omitted. Further, the difference between the embodiment of the present invention and the modification 2 is a structure for more tightly holding the bubbles in the isolation area 3. Details will be described below.

  Fig.8 (a) has shown the figure which added the structure to a pair of retaining wall 5b of one Embodiment of this invention. As shown in FIG. 8 (a), the pair of retaining walls 5b in the cell culture vessel 400 are integrally provided with a return portion 5e that is inclined away from the center line J from the tip to the culture area 2.

  Moreover, FIG.8 (b) has shown the figure which added the structure to a pair of retaining wall 5d of the modification 2 of this invention. As shown in FIG. 8 (b), the pair of retaining walls 5 d in the cell culture container 500 is integrally provided with a return portion 5 f that is inclined away from the center line J as it goes from the tip to the culture area 2.

Thus, according to the cell culture container 400 in the modification 3 of this invention, the air bubbles currently hold | maintained in the isolation area 3 can be kept more in the isolation area 3 by a pair of return part 5e. .
Similarly, according to the cell culture container 500 in Modification 3 of the present invention, the air bubbles retained in the isolation area 3 can be further retained in the isolation area 3 by the pair of return portions 5f.

As described above, the present invention has been described with reference to the preferred embodiments and modifications. However, this description is not a limitation, and various modifications can be made. For example, at least one of the filling ports 1 and 6 and the lids 10 and 11 may be provided.
Moreover, you may employ | adopt the sealing structure disclosed by patent document 3 of the applicant's invention instead of a filling port and a lid | cover.
Also, instead of the filling port and the lid, an injection (access) port such as a resin rubber stopper or a silicone stopper is embedded in each side surface of the second culture area 2b and the isolation area 3, and a syringe needle is embedded therein. May be directly inserted, and a culture medium may be injected by operating a pump from the outside, or air bubbles may be aspirated.
The culture area 2 is composed of a first culture area 2a having a substantially circular cross section and a second culture area 2b having a substantially rectangular shape. The second culture area 2b and the filling port 1 associated therewith. And the structure of only the 1st culture area 2a which removed the cover 10 from the structure may be sufficient.
Moreover, the external shape of the 1st culture area 2a of the culture area 2 and the 2nd culture area 2b is not restricted to this.
Furthermore, although the induction fastening mechanism 4 has been described with the funnel-shaped cut wall 5, it is sufficient that the bubbles are isolated from the culture area 2 to the isolation area 3, and the present invention is not limited to this.

1, 6 Filling port 2 Incubation area 3 Isolation area 4 Guiding mechanism 5 Cut wall 5a Guiding wall 5b, 5d Retaining wall 5c Inclined part 5e, 5f Return part J Center line K bubble 100, 200, 300, 400, 500 cells Culture vessel

Claims (6)

A filling port for containing the culture solution;
A culture area for culturing cells;
An isolation area for isolating air bubbles in the culture area;
An induction fastening mechanism that guides the bubbles in the culture area to the isolation area and causes the isolated bubbles to remain in the isolation area;
A cell culture vessel characterized by comprising:
The cell culture container according to claim 1, wherein the guide fastening mechanism includes a pair of funnel-shaped cut-out walls whose opening portions become smaller from the culture area toward the isolation area.
The cell culture container according to claim 2, wherein the cut wall comprises an arcuate guide wall and a straight retaining wall.
The cell culture according to claim 3, wherein the retaining wall is provided so as to be inclined in parallel with a center line or approaching the center line as it goes from the culture area to the isolation area. container.
The cell culture container according to claim 3 or 4, wherein the retaining wall is integrally provided with a return portion that is inclined away from the center line as it goes from the tip to the culture area.
The cell culture container according to claim 3, wherein the abutting portion between the isolation area and the retaining wall is provided with an inclined portion that is inclined upward from the culture area to the isolation area.

Images