JPH10210966A - Culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent the destruction of the agglutinated lumps formed by agglutinating many cells in order to culture the cells in the state approximate to the in-vivo state by the flowing force of a culture medium at the time of charging the culture medium and to prevent the inadvertent exertion of an adverse influence on the cells by contact by rapidly executing the exchange of the culture medium in order to minimize the temporally changes of the environment, such as the presence or absence of the culture medium, temp. and humidity and atm. components, which occur at the time of executing the exchange of the culture medium. SOLUTION: The inside surface of the culture vessel existing between a culture surface or the base of the vessel and the aperture of the culture vessel is at least partly provided with a difference in level or projecting part 1. This projecting part is so formed that the difference in level from the surface of the culture vessel 2 or the height up to the projecting part 1 attains 0.1 to 50mm, more preferably 0.5 to 5mm to decelerate the velocity at which a soln. flows or to disperse the flow of the soln. as compared to the case there are no differences in level or the projecting parts 1 when the liquid is charged along the surface.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cell culture container.

[0002]

2. Description of the Related Art Cell culture technology is a technology for living and growing cells, which are components of a living body, in vitro. It is used not only for research but also for industrial and medical applications such as production of useful substances and clinical tests. It is applied and used in the above wide fields. With the recent development of culture technology, conditions and methods for culturing cells in an environment similar to that of a living body have been devised in order to culture animal cells in a state closer to the living body. Thus, when culturing adherent cells requiring a support, in the conventional culture method, the cells were cultured in a monolayer form on the surface of the culture vessel, but the cells are also in a form closer to the living body. It is becoming possible to culture.

For example, JP-A-3-4780, JP-A-4-4780
By providing a layer of a substance similar to the intercellular matrix on the culture surface in Japanese Patent No. 4686, or applying a special surface treatment,
A method is disclosed in which a large number of cells such as hepatic parenchymal cells or spleen epithelial cells that form tissues and organs in a dimensional aggregate are aggregated to form an aggregate and cultured. Also, Japanese Patent Application Laid-Open No. 5-2369
No. 51 discloses a culture method in which hepatic parenchymal cells formed into spherical clumps are suspended in a culture solution. These methods reproduce a certain degree of tissue organization compared to tissue organs in the living body, and the level of specific differentiation function of the cells is higher than that of cells cultured in a conventional monolayer. In some cases, the level has reached the same level, and the culture period can be extended.

[0004] However, a culture environment in which cells, which are components of a living body, are cultured in vitro is an environment having many adverse effects on cells. Essentially, cells in a living body exhibit various functions of their life activities by interacting with surrounding cells, tissues, or the whole living body. Especially, the existence of cells in the living body keeps the living environment constant. Are protected and protected from various external adverse effects.
On the other hand, as for cells under conditions of culturing in vitro, various culturing methods have been developed for the functions as described above, and various functions of life activity similar to those in vivo are being reproduced. Because the cells are exposed to the outside of the living body, they are sensitive to subtle changes in the culture environment, are directly affected by various external influences, and may even die. . One of the adverse effects on cells under conditions of culturing in vitro is a temporary environmental change that occurs when performing microscopic observation or measurement analysis, especially when changing the culture medium. .

[0005] When performing normal cell culture, the culture medium, culture solution, temperature / humidity, atmospheric components, etc., which are suitable for the cell are adjusted and maintained under optimum conditions. Since nutrients and the like are consumed and secretions and wastes are discharged from the cells into the culture solution, the culture solution needs to be replaced at regular intervals. At this time, the culture vessel containing the cells must be taken out in a sterile indoor environment, and if the old culture solution that has been used before is removed before the addition of a new culture solution, the surrounding culture cells will be removed. Since the culture medium is lost, the cells will be placed in a totally unsuitable environment. For this reason, the environment surrounding the cell temporarily changes to a completely different environment, which has a great adverse effect on the cell.

[0006] In order to minimize the above-mentioned temporary changes in the environment, culture is performed in a closed system using a device capable of continuously supplying a culture solution, or when the culture solution is removed and charged. Uses a pipette or the like capable of sucking and discharging by pressurization and decompression of a pump, so that the culture solution can be quickly exchanged. However, a device that can continuously supply a culture solution must be designed for each purpose and condition of culture, and it is not very versatile for various purposes of culture or general researchers. Is also expensive. Generally, a method of culturing cells using a Petri dish, a culture flask, a multi-well plate, etc., and exchanging a culture solution in a culture container using a pipette is performed. It is necessary to repeat the above, and if the culture medium cannot be exchanged in a satisfactory manner or the operation is erroneously performed, the culture is temporarily interrupted, and the cells are placed in a completely unsuitable environment for a long time. Also, if the tip of the pipette is accidentally brought into contact with the culture surface during the culturing operation, mechanical damage is caused such that cells in the contacted portion are crushed.

Further, in the above-mentioned method of culturing a large number of cells forming tissues and organs in a living body in a three-dimensional aggregate to form an aggregate, the cells adhere to surrounding cells. Agglomerates tend to be broken when subjected to shearing force because they are not strongly fixed to the culture vessel compared to the conventional method in which cells are adhered to the culture surface and cultured in a single layer. . Therefore, when the culture solution is introduced, it must be performed carefully so as not to destroy the cell aggregates due to the shear force due to the flow, and the culture solution cannot be exchanged quickly. In particular, when the opening of the container is narrow like a culture flask, or when the culture operation is performed with the container in a fixed direction like a multi-well plate, the location and flow of the culture solution to be charged are always changed. It becomes almost constant by exchanging the liquid, and concentrates on cells at a specific site, causing damage by shearing force. Affected cells generally die without repair, and at that time, enzymes released from the dead cells are known to affect normal cells that are not further affected by surrounding cells. I have. In the case of performing such a culture method, the culture is performed for a long period of time, so that it is necessary to exchange the culture medium many times. This results in repeated and prolonged periods of time in totally unsuitable environments. Thus, researchers who use cells for research and development by culturing cells in a state closer to the living body need particularly excellent culture operation techniques.

[0008]

That is, an object of the present invention is to provide a method for minimizing temporary changes in the environment such as the presence or absence of a culture medium, temperature, humidity, and atmospheric components that occur when a culture medium is exchanged. The culture medium can be exchanged at a time, and agglomerated masses of cells to be cultured in a state closer to the living body are not destroyed by the shear force due to the flow when the culture medium is injected. Another object of the present invention is to prevent cells from being mechanically damaged by contact. The shearing force due to the liquid at the time of dispensing the culture solution is determined by the amount of solution discharged and the range of discharge. The larger the amount of discharge and the narrower the range of discharge, the greater the flow rate and the greater the shearing force. Therefore, reducing the discharge amount and increasing the discharge range are methods for reducing the shearing force. But,
Since lowering the discharge volume cannot be freely controlled in the sense of the original exchange of the culture solution, expanding the discharge range is the only method that can reduce the shearing force. The easiest way to widen the discharge range is to make the discharge port larger, but in such a case, the amount of the solution cannot be accurately measured with a head or the like. Therefore, not a dispensing device,
A device for expanding the discharge range on the incubator side is required.

[0009]

The present invention relates to a culture vessel characterized in that a step or a projection is provided on at least a part of the culture surface or the inner surface of the vessel between the bottom of the vessel and the opening of the culture vessel. .

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A detailed description will be given below with reference to FIGS. The main culture method to be used by the present invention is
This is a method in which cell lines such as HeLa cells and PC12 cells and adherent animal cells such as hepatocytes, vascular endothelial cells, and fibroblasts are fixed as cells to be cultured and cultured in a culture vessel. Furthermore, a method in which adherent cells are fixed to microcarriers such as beads and suspended in a culture solution, a method in which a large number of cells alone are aggregated to form aggregates and suspended in the culture solution, or a method in which floating cells such as red blood cells are cultured in a culture solution It is also effective in cultivation such as a method of suspending in a medium. As the culture vessel, a conventionally known one can be used without any particular limitation. In the present invention, there is no particular limitation on the member constituting the step or the projection (1), and there is no particular limitation on the means for attaching the step or the projection (1), but it is preferably the same as the culture vessel (2). Using materials,
When the culture vessel (2) is provided at the same time as the production, the labor for processing can be simplified. For example, the disposable culture vessel (2) made of polystyrene is produced by injection molding using a mold, but can be produced by performing injection molding using a mold provided with a step or a convex portion (1). It is possible. Further, there is no particular limitation on the position where the step or the convex portion (1) is provided, and the size and shape such as height and width.

However, if the height (3) from the surface of the culture vessel (2) to the step or the projection (1) is too low, the effect of reducing the flow speed of the solution or dispersing the flow of the solution is obtained. The height (3) is preferably 0.1 to 20 mm, since it is difficult to leave the culture solution on the step or the projection (1) or to perform the culturing operation if it is too high. Preferably, it is 0.5 to 5 mm. As for the width (4) with respect to the direction in which the culture solution flows, the larger the width is, the more the liquid can be dispersed, and the area where the culture solution can be introduced with the effect of the present invention is widened and the culture operability is improved. Therefore, it is appropriate to increase the size as much as possible according to the size of the culture vessel (2). However, as shown in FIG.
Is small, it is possible to sufficiently disperse the flow of the solution by combining a plurality of steps or projections (1). From this, it is preferable to select the size of the step or the width (4) of the projection (1) according to the type and size of each culture container and the manufacturing method.

In order to efficiently disperse the flow of the culture solution, the step or the projection (1) is angled as shown in FIG. 6 to guide a part of the solution to change the flow of the solution in an arbitrary direction. Is also good. Further, in order to sufficiently slow down the flow of the culture solution, two or more steps or projections (1) may be provided in the direction in which the solution flows as shown in FIG. 7, and the solution may be repeatedly slowed down. It is preferable to make an appropriate selection according to the type, size and production method of the culture vessel. Further, according to the present invention, the step or the projection (1) on the inner surface of the container becomes caught by the pipette. This makes it possible to reduce damage to a layer of a substance similar to cells or an intercellular matrix on the culture surface and damage to a special surface treatment by bringing the tip of the pipette into contact with the culture surface during the culture operation. Hereinafter, the present invention will be described with reference to examples.

[0013]

EXAMPLE A trial mold for a disposable, 6-hole polystyrene multiwell plate was machined so that the cylindrical diameter of the mold for modeling the inner surface of the well was two steps of 35φ and 33φ. Next, polystyrene was injection-molded using this mold, subjected to surface treatment and sterilization, to obtain a culture vessel according to the present invention. Using the multiwell plate obtained by the above method, liver parenchymal cells were formed into aggregates on the collagen gel layer according to the prescription of JP-A-3-4780, and three-dimensional culture was performed. When the culture medium was exchanged, the culture medium was dispersed right and left along the step, and was gently poured onto the culture surface. Moreover, after the cells formed aggregates, it was observed that the aggregates were cultured without being destroyed for at least two weeks as shown in FIG. On the other hand, when a normal multi-well plate is used, when the culture medium is exchanged, the culture medium is poured directly into the culture surface as shown in FIG. Was taken out into the indoor environment for a long time as compared with the case of using. Further, several days after the formation of the aggregates, it was observed as shown in FIG. 10 that the aggregates of the cells were destroyed from the portion where the culture solution was charged.

[0014]

According to the present invention, the culture medium can be quickly exchanged to minimize the temporary changes in the environment such as the presence or absence of the culture medium, temperature, humidity, and atmospheric components that occur when the culture medium is exchanged. It can be performed, without being destroyed by the flowing force of the culture solution when the culture solution is thrown into the aggregate where a large number of cells are aggregated in order to culture the cells in a state closer to the living body,
In addition, it is possible to prevent cells from being damaged by contact accidentally.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of a culture vessel according to one embodiment of the present invention.

FIG. 2 is a view showing a use state of a culture vessel according to one embodiment of the present invention.

FIG. 3 is a diagram showing a use state of a culture vessel according to one embodiment of the present invention.

FIG. 4 is a partially enlarged view showing the configuration of a culture vessel according to one embodiment of the present invention.

FIG. 5 is a diagram showing a configuration and a use state of a culture vessel according to one embodiment of the present invention.

FIG. 6 is a diagram showing a configuration and a use state of a culture vessel according to one embodiment of the present invention.

FIG. 7 is a diagram showing a configuration and a use state of a culture vessel according to one embodiment of the present invention.

FIG. 8 is a distribution diagram showing a state of an aggregate when culturing is performed in a culture vessel according to an embodiment of the present invention.

FIG. 9 is a diagram showing a use state of a culture vessel as a comparative example of the present invention.

FIG. 10 is a distribution diagram showing a state of an aggregate when cultured in a culture vessel as a comparative example of the present invention.

[Explanation of symbols]

1. Steps or protrusions 2. Culture vessel 3. Height 4. Width 5. 5. A portion composed of a plurality of steps or projections 9. Site composed of multiple steps or projections Pipette 11. Culture solution and flow of culture solution 20. 21. Plan view of well of multi-well plate The range in which the aggregate was maintained on day 14 after the formation of the aggregate. 22. Range in which aggregate was broken on day 7 after aggregate formation After the formation of the aggregate, the area where the aggregate was broken on the 14th day

Claims (2)

[Claims] 1. A culture vessel characterized in that a step or a projection is provided on at least a part of the culture vessel or the inner surface of the culture vessel between the vessel bottom and the culture vessel opening. 2. The culture vessel according to claim 1, wherein the height of the step or the projection from the surface of the culture vessel is 0.1 to 50 mm.