JP4378514B2 - Cell culture vessel - Google Patents

Description

  The present invention relates to a cell culture vessel suitable for use in culturing cells such as animal cells under microgravity.

  When performing animal cell culture tests under microgravity such as in outer space, it is necessary to supply oxygen to the animal cells by some method. However, as is well known, under the above-mentioned microgravity, the gas-liquid is not substantially separated into two layers. I can't do it. Therefore, in general, when performing an animal cell culture test under microgravity, the culture vessel is filled with the culture solution in a full state, and a new oxygen-containing solution is continuously or intermittently added from the outside. A method of circulating and supplying the culture solution is employed.

  However, in order to maintain the oxygen concentration in the culture solution within a certain range in the culture vessel, it is necessary to replace the culture solution relatively frequently. For this purpose, a culture solution circulation pump and an oxygen supply facility are required. Since it is necessary to install it separately outside, there is a problem that the restriction on the implementation becomes large especially in the space where the execution of the test in the narrowest space is required.

  Therefore, in order to solve the above-mentioned problems, the present inventors previously formed a plate-like main body in which a space 1A that penetrates between both surfaces and is filled with a culture solution is formed as shown in FIG. 1, a culture plate 2 that is disposed on one surface of the main body 1 to block the space portion 1A and has a cell adhesion property on the surface of the space portion 1A, and the other surface of the main body 1 In addition to developing a cell culture vessel in which the transparent and oxygen-permeable membrane material 3 covering the space 1A is liquid-tightly and detachably integrated, this is disclosed in Patent Document 1. .

According to the cell culture container having the above-described configuration, since uniform oxygen is always supplied into the space 1A through the membrane material 3, no oxygen supply facility is required, and the incidental facilities can be simplified. it can.
JP 2002-153261 A

However, in the above conventional cell culture container, when the surrounding relative humidity is low, a phenomenon occurs in which bubbles are generated in the culture solution, which not only hinders the observation of cells but also the culture environment around the bubbles. There is a problem that the uniformity of the cell is impaired and the cell culture becomes an obstacle. Therefore, the conventional cell culture vessel can be used only in an environment with a high relative humidity, and when used in an environment with a low relative humidity, a separate facility for moisturizing is required. There was a point.
In addition, when acceleration is applied to the culture vessel or when the acceleration is intentionally applied, the culture fluid flows irregularly due to the density difference between the bubbles and the culture fluid, and the uniformity of the culture conditions is impaired. There was also a problem.

  The present invention has been made in view of such circumstances, and even when oxygen is supplied to a culture solution using a gas-permeable membrane material, the culture solution can be used in the culture process regardless of the relative humidity of the surroundings. It is an object of the present invention to provide a cell culture container that can prevent the generation of bubbles and thereby simplify the incidental equipment.

  As a result of intensive studies on the mechanism by which bubbles are generated in the culture solution, the present inventors have obtained the following knowledge. That is, when the water vapor in the air is not saturated, the partial pressure of the water vapor in the air is lower than the partial pressure of the water vapor in the bubble, and the partial pressure of other gas components (oxygen, nitrogen, etc.) However, since the air is higher, the other gas components are transported to the bubbles through the membrane material, and the phenomenon that the bubbles gradually increase occurs. For example, the saturated water vapor pressure at 37 ° C. is obtained from the equation of Tetens (1930) and is 6.276 kPa. That is, the pressure difference of 0.03 atm between the relative humidity of 100% and 50% is the driving force for transportation. In contrast, when the water vapor in the air is in a saturated state, the partial pressure of each gas component is almost equal in the air and in the bubbles, so that the gas transport to the bubbles as described above does not occur. Therefore, it is thought that the phenomenon of bubbles growing does not occur.

  As a result of further research focusing on such a bubble generation mechanism, the present inventors formed an aqueous solution layer on the outer surface side of the membrane material, and the gas exchange of the culture solution was dissolved in the aqueous solution. It has been found that if it is carried out between gases, bubbles can be effectively prevented from being generated in the culture solution, and the present invention has been completed.

That is, the cell culture vessel according to the present invention as set forth in claim 1, the first plate penetrate between both surfaces, hole portions inside the culture solution are filled in the culture space in which the cell culture is carried out is formed And a second plate-like member penetrating between both surfaces and filled with water or an aqueous solution to form a space filled with an aqueous solution , the outer surface side of the first plate-like member In addition, a culture plate that closes the culture space and has an inner cell adhesion property is disposed, and the culture space and the aqueous solution-filled space are separated between the opposing surfaces of the first and second plate-like members and gas A first membrane material having permeability is interposed, and a second membrane material having gas permeability is disposed on the outer surface side of the second plate member while sealing the aqueous solution filling space. It is a feature. The water includes pure water.

  According to the first aspect of the present invention, gas exchange is performed between the external air and the dissolved gas in the aqueous solution via the second membrane material, and at that time, if the water vapor partial pressure in the air is low Therefore, the gas partial pressure increases and exceeds saturation, and bubbles are generated in the aqueous solution. However, gas exchange of the culture solution is performed between the dissolved gas in the aqueous solution via the first membrane material. Since the gas partial pressure in this aqueous solution is in equilibrium, no bubbles are generated in the culture solution.

The invention described in claim 2 is characterized in that, in the cell culture container according to claim 1, the culture solution or a basic culture solution serving as a base thereof is used as the aqueous solution.

  According to the present invention, even when oxygen is supplied to a culture solution using a gas permeable membrane material, bubbles are prevented from being generated in the culture solution during the culture process, regardless of the relative humidity of the surroundings. be able to. Accordingly, it is not necessary to separately provide a moisturizing facility for preventing the generation of bubbles, and the incidental facility can be simplified. In addition, since no bubbles are generated in the culture solution, the uniformity of the culture environment and the observability can be improved, and various problems caused by the generation of bubbles can be solved.

  1 and 2 show an embodiment of a cell culture container according to the present invention. In the figure, reference numeral 10 denotes a rectangular plate shape made of a synthetic resin having a predetermined strength and corrosion resistance, such as fluororesin, polycarbonate, and acetal. It is the 1st plate-shaped member formed in. A hole 11 penetrating between both surfaces is formed at the center of the first plate member 10, and a culture space 12 for filling the culture medium and performing cell culture is formed in the hole 11. It has come to be. Here, the hole part 11 is formed in a hexagonal shape whose ridge lines are parallel to each other in the center part in the longitudinal direction and are inclined in a V shape at both end parts. The first plate-like member 10 has one end opened at one end in the longitudinal direction of the culture space 12 and the other end opened at the side of the first plate-like member 10 and one end thereof. A culture medium discharge hole (not shown) that opens to the other longitudinal end of the culture space 12, extends along the side of the first plate member 10, and similarly opens to the side surface of the first plate member 10. It is installed.

  On one surface side (outer surface side) of the first plate-like member 10, a culture plate 30 whose surface is formed in a rectangular shape by a material such as polystyrene having cell adhesion is provided, and the other surface side (inner surface side). The first film material 31 formed in a rectangular shape by a transparent and gas-permeable thin material made of, for example, fluororesin, silicone, polymethylpentene, or the like is provided on the side. The culture plate 30 and the first membrane material 31 are both set to have substantially the same outer dimensions as the first plate member 10 so as to cover the entire outer surface and the entire inner surface of the first plate member 10. That is, when both openings of the hole 11 of the first plate-like member 10 are closed by the culture plate 30 and the first membrane material 31, a sealed culture space 12 is formed. In this embodiment, the thickness dimension of the first plate member 10 is about 3 mm, the thickness dimension of the culture plate 30 is about 1.5 mm, and the thickness dimension of each membrane material 31 and 32 is about 0.1 mm. The thickness dimension of the 2nd plate-shaped member 20 mentioned later is each set to about 2 mm.

  The second plate-like member 20 is a plate-like member formed in the same planar shape from the same material as the first plate-like member 10, and the first film material 31 is narrowed between the second plate-like member 20 and the first plate-like member 10. It comes to have. In the center of the second plate-like member 20, a hole 21 penetrating between both surfaces is formed as in the first plate-like member 10, and this hole 21 is filled with water or an aqueous solution. An aqueous solution filling space 22 is formed. Various aqueous solutions can be used as the aqueous solution filled in the aqueous solution filling space 22, but the osmotic pressure difference between the aqueous solution filling space 22 and the culture space 12 is eliminated, and the culture solution in the culture space 12 In order to suppress the dilution, it is preferable to use the same culture solution as the culture space 12 or a basic culture solution as a base thereof as an aqueous solution.

  Further, on the outer surface side of the second plate-like member 20, a second film material 32 formed in a rectangular shape by a thin material similar to the transparent and gas permeable first film material 31 is provided. The outer shape of the second film member 32 is set to be substantially the same as that of the second plate member 20 so as to cover the entire outer surface of the second plate member 20. That is, when both openings of the hole portion 21 of the second plate-like member 20 are closed by the first film material 31 and the second film material 32, a sealed aqueous solution filling space 22 is formed. .

  Thus, in this embodiment, each is integrated in the state which laminated | stacked the culture plate 30, the 1st plate-shaped member 10, the 1st film | membrane material 31, the 2nd plate-shaped member 20, and the 2nd film | membrane material 32 in order. Thus, a cell culture container having the culture space 12 and the aqueous solution filling space 22 is configured.

  In the cell culture container having the above-described configuration, cell culture is performed in a state where cells are attached to the culture plate 30 and the culture space 12 is filled with the culture solution and the aqueous solution filling space 22 is filled with the aqueous solution. The cells breathe as they are cultured, but at that time they consume oxygen and generate carbon dioxide. As a result, the dissolved oxygen in the culture solution decreases and a difference in concentration occurs with the dissolved oxygen in the aqueous solution, so that passive transport in which the dissolved oxygen moves from the aqueous solution through the first membrane material 31 into the culture solution is performed. appear. Similarly, when the dissolved oxygen concentration in the aqueous solution decreases, the equilibrium with the oxygen partial pressure in the air is lost, and passive transport in which oxygen moves into the aqueous solution occurs. On the other hand, as for the carbon dioxide gas, when the dissolved concentration in the culture solution increases, passive transport occurs in which the dissolved gas in the culture solution moves to the aqueous solution and the dissolved gas in the aqueous solution is released into the air. As described above, when the passive transport occurs for each gas component, the concentration of each gas component in the culture solution is maintained almost constant.

  Further, when the water vapor in the air is not saturated, the partial pressure of other gas components (oxygen, nitrogen, etc.) increases accordingly. At this time, the partial pressures of the other gas components in the bubbles generated in the aqueous solution are low because the partial pressure of water vapor is high when the atmospheric pressure is the same. Therefore, the other gas is transported to the bubbles in the aqueous solution and becomes larger. However, since the gas partial pressure in the aqueous solution is balanced, no bubbles are generated in the culture solution.

  As described above, according to the cell culture container of the present embodiment, the aqueous solution layer is formed on the outer surface side of the first membrane material 31 covering the culture solution, and the gas exchange of the culture solution is performed with the dissolved gas in the aqueous solution. Therefore, it is possible to prevent bubbles from being generated in the culture medium during the culture process regardless of the relative humidity of the surroundings. Accordingly, it is not necessary to separately provide a moisturizing facility for preventing the generation of bubbles, and the incidental facility can be simplified. In addition, since no bubbles are generated in the culture solution, the uniformity of the culture environment and the observability can be improved, and various problems caused by the generation of bubbles can be solved.

Next, the effect of the present invention will be clarified by examples.
In the example to which the present invention was applied, water was put into a 0.2 mm thick silicone film bag formed in a 3 cm × 6 cm bag shape and sealed so as not to enter air. Then, the produced silicone film bag is submerged in a culture dish filled with water, and kept in an incubator for 110 hours under the setting conditions of 22 ° C. and a relative humidity of 60%. Was observed. In this embodiment, the silicone film bag corresponds to the first membrane material of the cell culture container according to the present invention, the interior of the silicone film bag corresponds to the culture space, the inner surface of the culture dish, the outer surface of the silicone film bag, The space formed between the two corresponds to the aqueous solution filling space.

  On the other hand, in Comparative Example 1, the silicone film bag produced in the same manner as in the example was placed on a culture dish not filled with water, and the other conditions were the same as those in the example. In Comparative Example 1, when water vapor in the air is not saturated, a conventional cell culture vessel (a cell culture vessel in which an aqueous solution layer is not formed on the outer surface side of the membrane material 3 covering the culture solution) is used. It corresponds to. On the other hand, in Comparative Example 2, as in Comparative Example 1, a silicone film bag was placed on a culture dish that was not filled with water, and this was kept warm for 110 hours in an incubator under the setting conditions of 24 ° C. and relative humidity of 100%. did. This comparative example 2 corresponds to the case where a conventional cell culture vessel is used when water vapor in the air is saturated. Table 1 shows the changes before and after the test of the silicone film bags of Examples and Comparative Examples.

As can be seen from Table 1, in the examples, no bubbles were generated in the silicone film bag, and only a slight change (a decrease of about 2%) was observed in the mass of the silicone film bag before and after the test. In Comparative Example 1, about 2 ml of bubbles were generated in the silicone film bag, and the change of the mass of the silicone film bag was also larger (about 10% decrease) than the Example. On the other hand, in Comparative Example 2, as in the example, no bubbles were generated in the silicone film bag, and there was almost no change in the mass of the silicone film bag before and after the test.
As described above, in the comparative example corresponding to the conventional cell culture vessel, the generation state of bubbles greatly changes depending on the relative humidity of the surroundings, whereas in the example to which the present invention is applied, the relative humidity of the surroundings Regardless, it was confirmed that the generation of bubbles can be prevented.

It is a disassembled perspective view which shows one Embodiment of the cell culture container which concerns on this invention. It is sectional drawing of the cell culture container of FIG. It is a disassembled perspective view which shows an example of the conventional cell culture container.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 1st plate-shaped member 12 Culture space 20 2nd plate-shaped member 22 Aqueous solution filling space 30 Culture plate 31 1st film material 32 2nd film material

Claims (2)

A first plate-like member that has a hole that is a culture space in which cell culture is performed by filling the inside with a culture solution , and between both sides, and is filled with water or an aqueous solution inside And a second plate-like member having a hole formed as an aqueous solution filling space ,
A culture plate that closes the culture space and has cell adhesion on the outer surface side of the first plate member is disposed,
Between the opposing surfaces of the first and second plate-like members, a first membrane material having a gas permeability is provided between the culture space and the aqueous solution filling space ,
And above the outer surface of the second plate member, the cell culture vessel, wherein the second film material having a gas permeability as well as sealing the solution filling space is provided. 2. The cell culture vessel according to claim 1, wherein the aqueous solution is the culture solution or a basic culture solution serving as a base thereof .

Images