JP2002034551A - Method for producing cell-culturing container attached with cell and method for using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell-culturing container attached with frozen anchorage dependant animal cells, capable of being used for an experiment using objective cells just after its thawing and not peeling off the cells after the thawing. SOLUTION: This cell-culturing container is provided by culturing the anchorage dependant animal cells on the surface of the culturing container, cooling within a 0-10 deg.C range, exchanging its medium with a medium for freezing and freezing. At the beginning of a culture, it is thawed at <=10 deg.C, its medium is changed with a medium for culturing and then the culturing is started.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for freezing anchorage-dependent animal cells and a method for initiating thawing and culturing, which are used for experiments and toxicity evaluation using cells.

[0002]

2. Description of the Related Art Conventionally, anchorage-dependent animal cells have been supplied in the form of a cell suspension frozen in a container such as an ampoule or by culturing cells on the culture surface of a cell culture flask, and then culturing the culture medium. It is performed in a state where the flask is filled, the lid of the flask is closed and the flask is sealed. In each case, instead of using the cells as they are for experiments and evaluations, grow the cells once, re-seed the cells in a cell culture container suitable for experiments and evaluations, and grow the cells to some extent. It will be used for experiments and evaluations. As described above, it takes a considerable amount of time and effort to obtain cells and use them for evaluation and experiments.

For the purpose of reducing the time and labor, there is an idea that it is only necessary to freeze the cells and the culture medium in the incubator while the anchorage-dependent animal cells are cultured on the culture surface. As a method for reducing the time required for culturing until the state used in the method described above, an anchorage-dependent animal cell which has been frozen on a culture substrate together with a freezing medium in a state of being cultured on a culture substrate is disclosed in JP-A-1-16581. However, if it is attempted to freeze in a state where the anchorage-dependent animal cells are cultured, as disclosed in the above-mentioned publication, it is necessary to add a cryoprotectant to the medium, and when the culture is resumed, It is necessary to remove the freezing medium and add a culture medium. Further, in the conventional method, cells are detached during freezing and thawing. Therefore, there is a problem that the cells are sucked together with the freezing medium when the medium is replaced, or the cells are detached when the medium is dispensed.

[0004] As a countermeasure, a method has been proposed in which a culture vessel is coated in advance with a cell adhesion promoter.
The above publication discloses that it is effective to coat Celltac, fibronectin, Matrigel and the like. These cell adhesion promoting substances are expensive reagents and, when industrially viewed, increase the cost. Further, these cell adhesion promoting substances not only promote cell adhesion, but are also deeply involved in cell differentiation and expression of functions, and the state of cells may vary depending on the state of coating or the like.

[0005] It is also described that freezing in a so-called confluent state (in which individual cells are in close contact with each other and gaps between cells are almost eliminated) can suppress cell detachment. The cells may be less proliferative, thus limiting the applicable tests and experiments. Further, as another method, a method of introducing a physical hold-down for preventing cell detachment in medium exchange during thawing has been reported. Filters made of fibers are used as the retainers, but they are not transparent and may interfere with observation with a phase contrast microscope, and in the case of a 96-well plate, measure the absorbance using a plate reader. However, since there is a holder with poor transparency in the wells of the plate, it is difficult to perform measurement as it is, and the measurement must be performed by transferring the reaction solution to another 96-well plate or the like. . Further, from the viewpoint of manufacturing cost, introducing such a retainer into a large number of small wells such as a 96-well plate requires a great deal of labor and time.

[0006] For the above reasons, even if the cells are frozen in a culture state to which anchorage-dependent animal cells are adhered for the purpose of saving the labor of cell culture, the cells are detached when thawed, so that the culture state is maintained. It has been difficult to put anchorage-dependent cells frozen together with the incubator to practical use.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method in which an anchorage-dependent animal cell is frozen on a culture surface together with an incubator in a cultivated state extending on the culture surface. Because it can be used for experiments using cells, it does not detach cells when thawing, and there is no need to physically hold down to prevent cell detachment that interferes with optical measurement,
An object of the present invention is to provide an inexpensive cell culture device provided with anchorage-dependent animal cells that can be used in a wide range of experiments.

[0008]

SUMMARY OF THE INVENTION In order to solve the above-mentioned conventional problems, the present invention is intended to reduce the factors that cause the detachment of the cell culture substrate surface which occurs when the cell is frozen and thawed while adhered to the cell substrate. As a result of the examination, it was found that the presence of the cryoprotectant added when the cells were frozen was significantly involved, and that if the temperature was high when the cells were in contact with a medium containing the cryoprotectant, the cells could be removed from the culture surface. It is found that detachment is promoted, and cells are mixed with a medium containing a cryoprotectant at a temperature close to ice-cold, and then frozen as it is, so that detachment of cells during freezing can be suppressed, and thawing It has been found that exfoliation of cells can be prevented by replacing the freezing medium with the culture medium while maintaining the low temperature in the above, and completed the present invention.

That is, the first invention of the present invention provides (1) culturing anchorage-dependent animal cells on an incubator culture surface, removing the medium,
(3) cooling to a temperature within the range of 0 to 10 ° C., (4) replacing and leaving a freezing medium containing a cryoprotectant while keeping the cooling temperature, and (5) further cooling to freeze the cells. The second invention is a method for producing a cell incubator with cells, characterized in that the cell incubator is produced by the method according to the first invention. This is a method for using a cell incubator with cells, which is characterized by thawing at a temperature of not more than 10 ° C., removing the freezing medium at a temperature of not more than 10 ° C., and then adding a culture medium to start culturing.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The anchorage-dependent animal cells that can be used in the present invention are cells that adhere to and spread on the culture surface, such as epithelial cell lines and fibroblast cell lines, and primary cultured vascular endothelial cells. HeLa, Hep G2, A-431, V
79 and Vero.

The form of the incubator used in the incubator of the present invention is not particularly limited. Examples of the form include a petri dish, a cell culture flask, a cell culture plate having a plurality of culture fractions, or a cover slip. The application is also possible to a film-like small piece for cell culture called “cell culture”. Among them, the most useful in the application of the present invention are plates such as a 96-well plate.

As for the culture surface of an incubator, a general incubator is subjected to a hydrophilization treatment for a scaffold-dependent animal cell. The degree of the hydrophilization treatment of a general culture surface is determined by a contact angle. It is about 60 degrees. In the case of cells that are easily detached, it is preferable to further hydrophilize the culture surface in order to keep the spread state of the cells after thawing better, and the degree of hydrophilization treatment is effective when the contact angle is 40 degrees or less. is there. According to the present invention, it is possible to maintain a good adhesion state of cells at the time of thawing without coating a cell adhesion factor such as fibronectin or gelatin on a culture surface, which has been conventionally required.

The procedure for freezing the incubator of the present invention will be described. First, an intended incubator is prepared, cells are seeded, and further cultured to proliferate the cells. The cell density at the time of freezing of the cell incubator depends on the purpose of the experiment to be used, but it is desirable to freeze the cell at a cell density such that about 50% of the culture surface is covered by the cells. In a so-called confluent state (in which individual cells are in close contact with each other and there are almost no gaps between cells), cells may be less proliferative and their use may be limited depending on the purpose of the experiment. Because. First, incubate the incubator
The temperature may be cooled to 0 ° C. In this case, the cooling may be performed while the medium is still contained. As for the cooling method, it may be placed in a refrigerator or placed on ice.

Next, a culture medium containing a cryoprotectant is added to the cooled incubator at a temperature of 0 to 10 ° C., and the culture is left while maintaining the cooling temperature. In the field of cell culture, a cryoprotectant is added for the purpose of suppressing cell damage during cell freezing. Various types of cryoprotectants are used, but DMSO (dimethyl sulfoxide) is inexpensive and has a high effect as a cryoprotectant. It is preferably used as a medium.

The cells acquire resistance to freezing and thawing by adding the freezing medium at low temperature and leaving them to stand.
If the temperature of the culture medium exceeds 10 ° C. during this standing, the cells will detach, but by keeping the temperature at 0 to 10 ° C., the detachment of the cells can be prevented. The time for this standing is suitably about 10 minutes to 1 hour. Finally, it is further frozen slowly by a program freezer or the like.

Next, the process from thawing to the start of culture will be described. The frozen anchorage-dependent cells are stored in a deep freezer at -80 ° C. Although the frozen culture container is quickly thawed, if it takes too long to thaw, the cells are damaged and the number of viable cells is significantly reduced.

The thawing method is as follows:
By immersing the incubator in running water of not more than 10 ° C., quick thawing can be easily performed while maintaining the temperature at not more than 10 ° C.
Alternatively, the incubator is immersed in water at about room temperature, and when about half of the medium in the well has melted, the medium is transferred to ice cooling. next,
Remove the freezing medium at 10 ° C. or lower, and immediately add the culturing medium. At this time, the thawed cells do not detach. After the culture medium is added, the cells are placed in a carbon dioxide incubator at 37 ° C. to start culture.

[0018]

The present invention will be specifically described below with reference to examples. (Example 1) HeLa cells were seeded in a 96-well multiwell plate for cell culture (product number MS-3096F) manufactured by Sumitomo Bakelite Co., Ltd. The medium used was a MEM medium supplemented with 5% calf serum. About 10,000 cells were seeded per well and cultured in a carbon dioxide incubator. When the cell density reached about 50% of the culture surface by microscopic observation, the cells and the incubator were frozen together with the cells. The freezing procedure was performed as follows. The cultivated medium was removed, covered with a lid to prevent the culture surface from drying, and left in a refrigerator (4 ° C.) for 1 hour. At the same time, DMSO was added to the above culture medium at a concentration of 10% to prepare a freezing medium, which was cooled with ice.

The ice-cooled freezing medium was dispensed in a volume of 100 μl into each well of the plate cooled in the refrigerator, and left again in the refrigerator for 30 minutes. Thereafter, the mixture was cooled to -80 ° C at a rate of 1 ° C per minute in a deep freezer, and the whole plate was frozen and stored at -80 ° C. After storage, the plate is taken out, the outer bottom surface of the plate is immersed in running water at 10 ° C. to thaw, the medium for freezing is removed by suction, 100 μl of medium for culture is added per well, and the plate is placed in a carbon dioxide incubator at 37 ° C. To start the culture.

(Comparative Example 1) HeLa was placed in a 96-well multiwell plate for cell culture (product number MS-3096F) manufactured by Sumitomo Bakelite Co., Ltd.
Cells were seeded. The medium used was a MEM medium supplemented with 5% calf serum. About 10,000 cells were seeded per well and cultured in a carbon dioxide incubator. When the cell density reached about 50% of the culture surface by microscopic observation, the cells and the incubator were frozen together with the cells. The freezing procedure was performed as follows.

Remove the culture medium and add DMSO to 10%
, And 100 μl of a freezing medium was dispensed into each well, immediately cooled in a deep freezer at a rate of 1 ° C. per minute to −80 ° C., and the whole plate was frozen to −80 ° C. Stored at ° C. After storage, the plate was taken out, the outer bottom surface of the plate was immersed in warm water at 37 ° C. to completely thaw, the culture medium for freezing was removed by suction, 100 μl of culture medium was added per well, and carbon dioxide gas was added at 37 ° C. Culture was started in the incubator.

Example 2 For Hep G2 cells, a modified Dulbecco's MEM medium supplemented with 10% fetal calf serum was used as a medium. Thereafter, freezing and thawing were performed under the same conditions as in Example 1.

(Comparative Example 2) Hep G2 cells were frozen and thawed under the same conditions as in Comparative Example 1 using Dulbecco's modified MEM medium supplemented with 10% fetal bovine serum as the medium.

Example 3 HeLa cells were seeded on a 60 mm Petri dish for cell culture (product number MS-10600) manufactured by Sumitomo Bakelite Co., Ltd. The medium used was a MEM medium supplemented with 5% calf serum. About 500,000 cells were seeded per petri dish, and frozen and thawed in the same manner as in Example 1 below.

Comparative Example 3 HeLa cells were seeded on a 60 mm Petri dish for cell culture (product number MS-10600) manufactured by Sumitomo Bakelite Co., Ltd. The medium used was a MEM medium supplemented with 5% calf serum. About 500,000 cells were seeded per petri dish, and frozen and thawed in the same manner as in Comparative Example 1 below.

Example 4 Hep G2 cells were frozen and thawed under the same conditions as in Example 3 using a Dulbecco's modified MEM medium supplemented with 10% fetal bovine serum as a medium.

Comparative Example 4 Hep G2 cells were frozen and thawed under the same conditions as in Comparative Example 3, using Dulbecco's modified MEM medium supplemented with 10% fetal bovine serum as the medium.

(Comparison of Variation in the Number of Viable Cells in Wells in a 96-Well Plate) Examples 1 and 2 and Comparative Examples 1 and 2 were used. 100 μl of each well was added and cultured for one day. The operation with the dispenser was performed by aligning the tip of the tip of the dispenser with the bottom end of each well. After that, WST-8
10 μl was added to each well, and the amount of formazan produced in accordance with the number of living cells was measured for absorbance at a wavelength of 450 nm using a plate reader. The variation in the number of living cells in the plate was calculated from the absorbance of each of the 96 wells per plate. The variability was compared by dividing (maximum value-minimum value) by the average value of the entire plate and the coefficient of variation (cv value). The results are shown in Tables 1 and 2.

(Comparison of cell viability upon thawing) Examples 3 and 4
Comparative Examples 3 and 4 were used. When the freezing medium was removed after thawing, this medium was recovered and DMSO was removed by centrifugation to prepare a cell suspension. The petri dish was subjected to trypsin treatment, the cells adhered to the culture surface were peeled off and collected, and trypsin was removed by centrifugation to prepare a cell suspension. For each of the above cell suspensions, the number of cells and the number of viable cells were measured by the trypan blue exclusion method, and the viable cell ratio was measured.
Table 3 shows the results.

(Comparison of Cell Detachability at Thawing) Examples 3 and 4 Comparative Examples 3 and 4 were used. When removing the freezing medium, the medium was recovered and DMSO was removed by centrifugation to prepare a cell suspension. After adding 5 ml of each cell culture medium per petri dish and shaking horizontally for 30 seconds at a stroke of 5 cm and once per second for 30 seconds, the culture medium is removed from the petri dish and suspended in the medium. The Petri dish with the collected cells was subjected to trypsin treatment, the cells adhered to the culture surface were peeled off and collected, and trypsin was removed by centrifugation to prepare a cell suspension. For each cell suspension, only the number of viable cells was measured by the trypan blue exclusion method, and after shaking, the number of viable cells adhered and remaining in the Petri dish was divided by the total number of viable cells to calculate the residual adhesion rate. As an index of the degree of peeling. Table 4 shows the results.

[0031]

[Table 1]

[0032]

[Table 2]

[0033]

[Table 3]

[0034]

[Table 4]

[0035]

According to the method for freezing and thawing cells of the present invention, there is no detachment of cells at the time of thawing cells, no loss of cells at the time of medium exchange, and in a 96-well plate, the variation in the number of cells between wells is small. Furthermore, the cell viability is high, and it is useful as a method for producing and using a cell incubator with anchorage-dependent animal cells while maintaining the culture state.

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[Procedure amendment]

[Submission date] October 9, 2001 (2001.10.
9)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction contents]

It is also described that freezing in a so-called confluent state (in which individual cells are in close contact with each other and gaps between cells are almost eliminated) can suppress cell detachment. The cells may be less proliferative, thus limiting the applicable tests and experiments. Further, as another method, a method of introducing a physical hold-down for preventing cell detachment has been reported. The introduction of the pusher hinders observation with a phase contrast microscope. Further, in the case of a 96-well plate, measurement such as absorbance by a plate reader is performed. However, if a well is present in the plate, it becomes difficult to measure the plate as it is. From a manufacturing cost perspective,
Attempting to introduce such a hold-down into a large number of small wells, such as a 96-well plate, would require a great deal of labor and time.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction contents]

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method in which an anchorage-dependent animal cell is frozen on a culture surface together with an incubator in a cultivated state extending on the culture surface. A cell incubator equipped with anchorage-dependent animal cells that can be used for experiments with cells, does not peel off when thawing, and does not interfere with optical measurements. It is to provide at low cost.

Claims (3)

[Claims] (1) culturing an anchorage-dependent animal cell on an incubator culture surface, (2) removing a medium, (1) cooling to a temperature within a range of 0 to 10 ° C., and (2) controlling a cooling temperature. A method for producing a cell incubator with cells, comprising at least the steps of exchanging and leaving a cryoprotectant-containing cryoprotectant while keeping the temperature, and (3) further cooling and freezing the cells. 2. The method according to claim 1, wherein the cryoprotectant is dimethyl sulfoxide. 3. The cell incubator according to claim 1 or 2, wherein the culture is started at a temperature of 10 ° C. or lower when starting the culture.
A method for using a cell incubator with cells, further comprising removing a freezing medium at a temperature of 10 ° C. or lower, and then adding a culture medium to start culturing.