JP2019198288A - Cell culture vessel, and method for manufacturing cell culture vessel - Google Patents

Abstract

To obtain a cell culture vessel made of a polyethylene material which comprises a culture surface uniformly hydrophilized, and in which a degree of hydrophilicity is appropriately controlled corresponding to an application of the cell culture vessel.SOLUTION: A cell culture vessel made of a polyethylene material comprises a surface in which excimer treatment is applied to at least a part of the inside of the cell culture vessel as a culture surface, and a receding contact angle of the culture surface is larger than 30° and less than 78°.SELECTED DRAWING: None

Description

  The present invention relates to a cell culture technique, and in particular, to a surface treatment of a culture surface of a cell culture container.

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like, it has been required to efficiently culture a large amount of cells, tissues, microorganisms, and the like in an artificial environment.
Under such circumstances, it has been practiced to inject a large amount of cells automatically by injecting cells and a culture solution into a cell culture container.

  The cells include floating cells such as lymphocytes and dendritic cells that are suspended in the culture medium and adherent cells that are adhered to the culture surface of the cell culture vessel and cultured such as iPS cells. is there. Even suspension cells may be cultured in a specific step such as a lymphocyte activation step by adhering to a culture surface. Further, even adherent cells may be cultured by forming spheres (aggregates) in a culture solution and floating them. For this reason, in this specification, a floating cell includes a subject that is cultured while being suspended, and an adhesive cell includes a subject that is cultured while being adhered to a culture surface.

When culturing cells suspended in a culture solution, a cell adhesion inhibitor (cell low adhesion treatment agent) is generally applied (coated) to the culture surface of the cell culture container so that the cells do not adhere. There is a need.
In addition, when culturing with cells attached to the culture surface, it is necessary to improve the hydrophilicity by subjecting the culture surface to surface treatment so that the cells are sufficiently adhered to the culture surface of the cell culture container.

In particular, in a cell culture vessel made of polyethylene (PE), suspension cells cannot be appropriately cultured unless a coating layer made of such a cell adhesion inhibitor is formed on the culture surface.
Further, even when the coating layer is formed on the culture surface as described above, it is necessary to make the culture surface hydrophilic by subjecting the culture surface of the cell culture container to a surface treatment.

JP-A-6-98756

Japanese Patent No. 5315640

However, there has been a problem that it is difficult to perform highly accurate surface treatment on the culture surface of the cell culture vessel.
Specifically, it is widely practiced to make the culture surface hydrophilic by subjecting the culture surface to corona treatment. However, in corona treatment, since surface treatment is performed by discharge, uneven treatment often occurs, and it is difficult to obtain a culture surface with a uniform degree of hydrophilicity. There was a problem that it could not be performed efficiently.
For example, when iPS cells are adherently cultured in a culture vessel in which treatment unevenness has occurred, there is a problem that the cells can adhere only to the hydrophilized region on the culture surface and the culture efficiency is reduced (see FIG. 5). In FIG. 4, cells are adhered only to the colored portion.).

In addition, it is necessary to perform different degrees of hydrophilization treatment between the surface treatment on the culture surface of the culture vessel for culturing floating cells and the surface treatment on the culture surface of the culture vessel for culturing adhesive cells. .
However, the conventional method has a problem that it is difficult to perform surface treatment uniformly, and it is difficult to perform surface treatment by appropriately controlling hydrophilicity.

Here, examples of the technique related to the surface treatment of the culture surface of the cell culture container include the adhesive cell culture bag described in Patent Document 1 and the adhesive cell culture bag-shaped container described in Patent Document 2. .
In Patent Document 1, the corona discharge treatment is performed on the inner surface of the bag so that the adherent cells can be efficiently cultured. However, as described above, treatment irregularities often occur in the corona discharge treatment. There was room for it.

Moreover, in patent document 2, surface treatment is performed to the culture surface by performing ultraviolet irradiation using a low pressure mercury lamp.
However, the low-pressure mercury lamp has a problem that it is difficult to cut CC and CH bonds in polyolefin, and it is difficult to modify the culture surface (K. Tsuji, Journal of Polymer Science, 467 11 (1973)).

Therefore, the present inventors have intensively studied and found that the surface treatment of the culture surface can be performed uniformly by hydrophilizing the culture surface using excimer treatment in a cell culture vessel made of polyethylene material.
Further, by controlling the treatment intensity by excimer treatment, a surface treatment that can obtain a culture surface suitable for culture of suspension cells, and a surface treatment that can obtain a culture surface suitable for culture of adhesive cells As a result, the present invention was completed.

  The present invention has been made in view of the above circumstances, and is a cell culture container made of a polyethylene material, which has a uniformly hydrophilized culture surface, and the degree of hydrophilization is appropriate depending on the use of the cell culture container. An object of the present invention is to provide a cell culture container controlled by the above and a method for producing the same.

  In order to achieve the above object, a cell culture vessel of the present invention is a cell culture vessel made of polyethylene material, and includes a surface subjected to excimer treatment on at least a part of the cell culture vessel as the culture surface, The receding contact angle of the culture surface is greater than 30 ° and less than 78 °.

  Further, in the method for producing a cell culture container of the present invention, the excimer treatment is performed on at least a part of the polyethylene film so that the receding contact angle of the region is greater than 30 ° and less than 78 °, and the region is a culture surface. As a method for forming a cell culture vessel made of the polyethylene film.

  Further, in the method for producing a cell culture container of the present invention, the excimer treatment is performed on at least a part of the polyethylene film so that the receding contact angle of the region is greater than 45 ° and less than 78 °. As a method for forming a suspension cell culture vessel made of the polyethylene film.

  In the method for producing a cell culture container of the present invention, an excimer treatment is performed on at least a partial region of the polyethylene film so that the receding contact angle of the region is greater than 30 ° and not greater than 45 °, and the region is a culture surface. As a method for forming an adhesive cell culture vessel made of the polyethylene film.

  According to the present invention, a cell culture vessel made of a polyethylene material, comprising a uniformly hydrophilized culture surface, the degree of hydrophilicity appropriately controlled according to the use of the cell culture vessel, And a manufacturing method thereof can be provided.

It is explanatory drawing about a static water contact angle and a receding contact angle. Average value (Avg.), Standard deviation (sd), and maximum value (Max.) Of static water contact angles at a plurality of points selected from the excimer-treated culture surface (Test 1: Examples 1 to 4) , The minimum value (Min.), And the difference between the maximum value and the minimum value (Max.-Min.), And a plurality of points selected from the culture surface subjected to corona treatment (Test 1: Comparative Examples 1 and 2) Mean water contact angle (Avg.), Standard deviation (sd), maximum value (Max.), Minimum value (Min.), And the difference between the maximum and minimum values (Max.-Min.) FIG. It is a figure showing the graph which shows the static water contact angle and receding contact angle in the culture surface (test 2) which performed the excimer process on various conditions. It is a figure showing the graph which shows the static water contact angle and receding contact angle in the culture surface (test 3) which performed the excimer process on various conditions. It is a figure which shows the result of having carried out the adhesion culture of the iPS cell using the cell culture container which has the culture surface which gave the conventional corona treatment and the process nonuniformity produced.

Hereinafter, embodiments of the cell culture container and the method for producing the cell culture container of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments and examples.
The cell culture container of the present embodiment is a cell culture container made of a polyethylene material, and includes a surface subjected to excimer treatment on at least a part of the cell culture container as a culture surface, and a receding contact angle of the culture surface is It is greater than 30 ° and less than 78 °.

First, the static water contact angle and the receding contact angle will be described with reference to FIG.
The static water contact angle means an angle (θs in FIG. 1) formed by the liquid surface and the solid surface where the surface of the stationary liquid contacts the surface of the solid wall. When the static water contact angle is large, the hydrophobicity of the surface of the solid wall is relatively strong, and when the static water contact angle is small, the hydrophilicity of the surface of the solid wall is relatively strong.
Specifically, when the static water contact angle on the culture surface of a culture vessel made of a polyethylene film which is a kind of polyolefin-based material is greater than 60 ° and 80 ° or less, the adherent cells are preferably adhered to the culture surface. It is known that it can be cultured efficiently.

In addition, the static water contact angle on the surface of the polyethylene film is 95 ° or more in an untreated state, and since it is highly hydrophobic, it cannot be cultured with adherent cells adhered. Moreover, since a cell adhesion inhibitor cannot be coated, suspension cells cannot be cultured.
On the other hand, if the culture surface of the culture vessel made of polyethylene film is subjected to a surface treatment, and its static water contact angle is greater than 80 ° and less than 95 °, the culture surface can be coated with a cell adhesion inhibitor, It becomes possible to culture suspension cells.

Further, the receding contact angle (hereinafter sometimes simply referred to as the receding angle) means the rear contact angle (θr in FIG. 1) when a water droplet slides down the surface of the solid wall. That is, it is the rear contact angle when water drops are picked up on the surface of the horizontally supported solid wall, and the solid wall is gradually tilted, and the liquid droplet starts to fall.
The receding angle can be used as an index indicating the dynamic wettability (hydrophilicity) of the surface of the solid wall. That is, when the receding angle is large, the hydrophobicity of the surface of the solid wall is relatively strong, and when the receding angle is small, the hydrophilicity of the surface of the solid wall is relatively strong.

The excimer treatment is a surface treatment using an excimer lamp as a light source. Ultraviolet rays having a wavelength of 200 nm or less are called vacuum ultraviolet rays, and the excimer treatment is irradiated with the vacuum ultraviolet rays. Some excimer lamps irradiate vacuum ultraviolet rays of various wavelengths, but generally xenon excimer lamps having a wavelength of 172 nm are used. In the following examples, this xenon excimer lamp was used.
When such an excimer treatment is applied to a polyethylene film, the surface physical properties of the polyethylene are changed to make it hydrophilic. In addition, the stronger the excimer treatment strength, the greater the degree of hydrophilicity.

In the cell culture container of this embodiment, the receding contact angle of the culture surface of the cell culture container is greater than 30 ° and less than 78 °.
That is, the cell culture container of the present embodiment has a receding contact angle of the culture surface in a range suitable for a culture container for floating cells or a culture container for adhesive cells.
Moreover, since the cell culture container of this embodiment is formed by performing the excimer process on the culture surface, there is no treatment unevenness and the culture surface is uniformly hydrophilized.

Moreover, it is also preferable that the cell culture container of this embodiment has a receding contact angle of the culture surface of greater than 45 ° and less than 78 °, and is formed as a culture vessel for suspension cell culture.
According to such a cell culture container, the cell adhesion inhibitor can be coated on the culture surface, and a coating layer made of the cell adhesion inhibitor can be suitably formed on the culture surface. For this reason, it is possible to make it suitable for culture of suspension cells.

  Examples of cell adhesion inhibitors that can be used include phospholipid polymers, polyvinyl alcohol derivatives, phospholipid / polymer complexes, polyhydroxyethyl methacrylate, polyvinyl alcohol, agarose, chitosan, polyethylene glycol, and albumin. Moreover, you may use combining these.

Moreover, it is also preferable that the cell culture container of this embodiment has a receding contact angle of the culture surface of greater than 30 ° and 45 ° or less, and is formed as a culture vessel for adhesive cell culture.
According to such a cell culture container, the adherent cells can be sufficiently adhered to the culture surface, and can be made suitable for the culture of the adherent cells.
Moreover, from the viewpoint of making it suitable for the culture of such adhesive cells, the cell culture container of the present embodiment has a receding contact angle of the culture surface of greater than 33 ° and not greater than 45 °. It is also preferably formed as a culture container for culture.

In the cell culture container of the present embodiment, the standard deviation of the static water contact angle measured at 10 or more positions on the culture surface is preferably 5 ° or less, more preferably 4 ° or less. It is more preferably not more than 0 °, and particularly preferably not more than 2 °.
In the cell culture container of the present embodiment, the difference between the maximum value and the minimum value of the static water contact angle measured at 10 or more locations on the culture surface is preferably 10 ° or less, and 9 ° or less. More preferably, it is more preferably 8 ° or less, still more preferably 7 ° or less, still more preferably 6 ° or less, and particularly preferably 5 ° or less.
According to such a cell culture container, since the culture surface uniformly hydrophilized by the excimer treatment is provided, high culture efficiency can be obtained.

Here, a low-pressure mercury lamp is generally used in the ultraviolet treatment that may be performed on the surface treatment of the culture surface of the cell culture vessel.
The excimer treatment and the ultraviolet treatment used in the cell culture container of the present embodiment differ in the type of light source and the length of the ultraviolet wavelength.
Specifically, excimer processing using an excimer lamp (xenon excimer lamp) uses UV light with a wavelength of 172 nm, and UV processing using a low-pressure mercury lamp generally uses UV light with wavelengths of 185 nm and 254 nm. Is done.

Excimer treatment has high ability to break chemical bonds and generate active oxygen (Surface Treatment Technology Handbook, P532-538, NTS), and can modify the culture surface of cell culture vessels with various strengths. Is possible.
On the other hand, in the ultraviolet treatment, it is difficult to cleave the CC and CH bonds of the polyolefin, and it is difficult to modify the culture surface of the cell culture vessel with various strengths.
For this reason, in the ultraviolet treatment, the receding contact angle of the culture surface of the cell culture container is controlled to a range suitable for the culture of floating cells or the culture of adhesive cells, and the cell culture container suitable for each cell. It was very difficult to obtain.

The shape of the cell culture container of the present embodiment is preferably a bag shape (bag shape), but is not limited thereto. Moreover, it is also preferable to provide the cell culture container of this embodiment with an injection port for injecting and discharging cells and culture solution.
Moreover, it is also preferable that the cell culture container of the present embodiment has a plurality of wells formed on the culture surface. If a plurality of wells are formed in the cell culture container of this embodiment, cells can be collected in each well, and it is possible to increase the cell density in the initial stage of sphere culture and improve the sphere formation efficiency. Become.

In the method for producing a cell culture container according to this embodiment, an excimer treatment is performed on at least a part of a polyethylene film so that a receding contact angle of the region is greater than 30 ° and less than 78 °, and the region is used as a culture surface. A cell culture vessel made of polyethylene film is formed.
Further, in the method for producing a cell culture container of the present embodiment, at least a part of the polyethylene film is subjected to excimer treatment so that the receding contact angle of the region is greater than 45 ° and less than 78 °, and the region is cultured. It is also preferable to use a method for forming a suspension cell culture vessel made of the polyethylene film as a surface.

  Specifically, the excimer treatment conditions are such that the distance between the culture surface and the excimer lamp is 3 mm to 4 mm, and the moving speed of the culture surface relative to the lamp is 60 mm / sec (seconds) to 5 mm / sec. A culture surface with a receding contact angle greater than 45 ° and less than 78 ° can be obtained. At this time, the static water contact angle on the culture surface is approximately 80 ° to 95 °, and a coating layer made of a cell adhesion inhibitor can be appropriately formed on the culture surface.

  Further, in the method for producing a cell culture container according to this embodiment, at least a part of the polyethylene film is subjected to excimer treatment so that the receding contact angle of the region is greater than 30 ° and not more than 45 °, and the region is cultured. It is also preferable to adopt a method for forming an adhesive cell culture vessel made of the polyethylene film as a surface.

  Specifically, the excimer treatment conditions are such that the distance between the culture surface and the excimer lamp is 3 mm to 4 mm, and the movement speed of the culture surface relative to the lamp is 2 mm / sec (seconds), or the excimer under this condition By repeating the treatment twice or three times or more, a culture surface in which the receding contact angle of the culture surface is greater than 30 ° and 45 ° or less can be obtained. At this time, the static water contact angle of the culture surface is approximately 60 ° to 80 °, and adhesive cells can be suitably adhered to the culture surface.

  In the present embodiment, the excimer process conditions are not limited to the above-mentioned conditions, and it goes without saying that other process conditions may be used as long as the receding contact angle can be controlled within the above range. .

In addition, the excimer process can irradiate with the maximum output instantaneously, but the ultraviolet process takes several tens of minutes until the maximum output is obtained.
For this reason, in the manufacturing method of the cell culture container of this embodiment, it is also possible to manufacture a cell culture container more efficiently by performing an excimer process.

Furthermore, by performing the excimer treatment, it is possible to produce a cell culture container having a culture surface that is uniformly hydrophilized, so that high culture efficiency can be obtained.
Further, by performing excimer treatment, the receding contact angle of the culture surface can be appropriately controlled according to the use of the cell culture container. For this reason, according to the manufacturing method of the cell culture container of this embodiment, it is possible to easily manufacture the culture container for floating cells and the culture container for adhesive cells.

  As described above, according to the cell culture container and the method for producing the cell culture container of the present embodiment, the cell culture container is a cell culture container made of a polyethylene material, and has a uniformly hydrophilized culture surface. It is possible to obtain a cell culture container in which the degree of hydrophilization is appropriately controlled according to the application.

  In the following, a test comparing the uniformity of the culture surface subjected to excimer treatment and corona treatment, and the relationship between various treatment conditions and receding contact angle of excimer treatment applied to the culture surface of the cell culture vessel were confirmed. The test will be described.

(Test 1)
First, the test which compared the uniformity of the culture surface which each performed the excimer process and the corona process is demonstrated.
A polyethylene film was used as a material for the cell culture container, and the surface thereof was assumed to be a culture surface. Then, excimer treatment and corona treatment were performed on the culture surface under various conditions as surface treatments.

  As an apparatus for performing the excimer treatment, a xenon excimer lamp apparatus (an excimer irradiation apparatus manufactured by M.D. Com.) Was used, and the voltage was set to 12V. Then, the excimer treatment (Example 1) in which the distance between the culture surface and the lamp was 4 mm and the moving speed of the stage on which the polyethylene film was placed in the xenon excimer lamp apparatus was 20 mm / sec, the same distance was 4 mm, and the moving speed of the stage Excimer processing (Example 2) in which the distance is 2 mm / sec, excimer processing (Example 3) in which the distance is 3 mm and the moving speed of the stage is 20 mm / sec, and the moving speed of the stage is 3 mm. Excimer treatment (Example 4) was performed at 2 mm / sec.

  As an apparatus for performing the corona treatment, a batch type corona treatment apparatus (manufactured by Kasuga Electric Co., Ltd.) was used. Then, the output is 280 W, the distance between the electrodes is 5 mm, and the batch type corona treatment apparatus has a corona treatment (Comparative Example 1) in which the moving speed of the stage on which the polyethylene film is disposed is 50 mm / sec, the output is 520 W, the distance and the stage The corona treatment (Comparative Example 2) was performed under the same conditions as in Comparative Example 1.

  Then, arbitrarily select 10 points from the film surface and measure the static water contact angle at each point, and average (Avg.), Standard deviation (sd), maximum value (Max.), And minimum value. (Min.) And the difference between the maximum value and the minimum value (Max.-Min.) Were calculated. The result is shown in FIG.

As shown in the figure, the standard deviations (sd) of the static water contact angles of Examples 1 to 4 were 1.7 °, 0.5 °, 0.7 °, and 0.5 °, respectively. . On the other hand, the standard deviations of the static water contact angles of Comparative Examples 1 and 2 were 6.5 ° and 6.3 °, respectively.
From this, it can be seen that in the surface treatment of the culture surface by excimer treatment, the uniformity of the surface treatment is greatly improved as compared with the corona treatment.

Moreover, as shown in the figure, the difference between the maximum value and the minimum value (Max.-Min.) Of the static water contact angles in Examples 1 to 4 is 4.8 °, 1.6 °, 2 °, respectively. .5 ° and 1.4 °. On the other hand, the difference between the maximum value and the minimum value of the static water contact angles in Comparative Examples 1 and 2 was 11.9 ° and 14.9 °, respectively.
This also revealed that the surface treatment uniformity by the excimer treatment greatly improves the surface treatment uniformity compared to the corona treatment.

(Test 2)
Next, the test which confirmed the relationship between the various process conditions of the excimer process performed with respect to the culture surface of a cell culture container and receding contact angle is demonstrated.
A polyethylene film was used as a material for the cell culture container, and the surface thereof was assumed to be a culture surface. And as a surface treatment for this culture surface, excimer treatment was performed under various conditions.

As an apparatus for performing excimer treatment, a xenon excimer lamp apparatus (an excimer irradiation apparatus manufactured by M.D. Com.) Was used, and the voltage was 12 V, and the distance between the culture surface and the lamp was 4 mm.
In the xenon excimer lamp apparatus, excimer treatments having different intensities were performed on different film surfaces by setting various conditions for the moving speed of the stage on which the polyethylene film was placed and the number of excimer treatments. .

  Specifically, the polyethylene film that was not subjected to excimer treatment (untreated) and the stage moving speed were 60 mm / sec, 40 mm / sec, 20 mm / sec, 10 mm / sec, 5 mm / sec, 2 mm / sec, respectively. A polyethylene film subjected to one excimer treatment was prepared. The slower the stage moving speed, the longer the excimer processing time and the higher the processing intensity.

  Further, since the minimum moving speed of the stage of the apparatus used was 2 mm / sec, as an excimer treatment with a higher strength, a polyethylene film subjected to excimer treatment twice at 2 mm / sec and 3 mm at 2 mm / sec. A polyethylene film subjected to the excimer treatment was prepared.

For measurement of the static water contact angle and receding contact angle, a solid-liquid interface analysis system DropMaster 700 (manufactured by Kyowa Interface Science Co., Ltd.) was used.
Specifically, the static water contact angle (θs) was measured by dropping 3 μl of pure water on the film. The receding contact angle (θr) was obtained by dropping 30 μl of pure water on the film, tilting the measuring table by 1 ° every second, and calculating the receding contact angle (θr) during sliding by the tangent method. . The result is shown in FIG.

As shown in the figure, the static water contact angle of the culture surface not subjected to the excimer treatment was 100.9 °, and the receding contact angle was 90.2 °.
Further, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 60 mm / sec were 95.0 ° and the receding contact angle was 77.2 °.
Further, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 40 mm / sec were 93.1 ° and the receding contact angle were 68.3 °.

In addition, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 20 mm / sec were 90.1 °.
In addition, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 10 mm / sec were 87.5 ° and the receding contact angle were 54.3 °.
In addition, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 5 mm / sec were 82.0 °.

Further, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 2 mm / sec were 77.8 ° and 42.4 °, respectively.
Further, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment twice at a stage moving speed of 2 mm / sec were 74.2 °.
Further, the static water contact angle and the receding contact angle of the culture surface after excimer treatment three times at a stage moving speed of 2 mm / sec were 72.4 °.

From the above results, it became clear that the receding contact angle can be adjusted appropriately by controlling the strength of the excimer treatment on the polyethylene film.
Specifically, the excimer treatment conditions are such that the distance between the culture surface and the lamp is 4 mm, and the stage moving speed is 60 mm / sec to 5 mm / sec, so that the receding contact angle is greater than 45 ° and less than 78 °. It turned out that it can control to become.
At this time, the static water contact angle is in the range of 80 ° to 95 °, and the surface treatment suitable for the coating of the cell adhesion inhibitor is performed, that is, suitable for the production of the culture vessel for floating cells. I understand that

Further, the excimer treatment conditions are such that the distance between the film surface and the lamp is 4 mm and the moving speed of the stage is 2 mm / sec or more, so that the receding contact angle is greater than 30 ° and 45 ° or less. It turns out that it can be controlled.
At this time, the static water contact angle is in the range of 60 ° to 80 °, and the surface treatment suitable for adhesion of the adherent cells is performed, that is, suitable for the production of the culture vessel for adherent cells. I found out.

(Test 3)
Next, the test 2 and the test confirmed by changing the conditions will be described with respect to the relationship between various treatment conditions of the excimer treatment applied to the culture surface of the cell culture container and the receding contact angle.
In this test, the same excimer irradiation apparatus as in Test 2 was used, and the distance between the film surface and the lamp was set to 3 mm at the same voltage. Other various conditions were the same as those in Test 2. The result is shown in FIG.

As shown in the figure, the static water contact angle of the culture surface not subjected to the excimer treatment was 100.9 °, and the receding contact angle was 90.2 °.
In addition, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 60 mm / sec were 92.9 ° and 71.8 °, respectively.
Further, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 40 mm / sec were 90.6 ° and 67.1 °, respectively.

In addition, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 20 mm / sec were 87.7 °.
Further, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 10 mm / sec were 85.1 ° and the receding contact angle were 53.4 °.
In addition, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 5 mm / sec were 80.4 °.

In addition, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment at a stage moving speed of 2 mm / sec were 75.3 ° and the receding contact angle were 41.5 °.
Further, the static water contact angle and the receding contact angle of the culture surface subjected to excimer treatment twice at a stage moving speed of 2 mm / sec were 71.7 °.
In addition, the static water contact angle and the receding contact angle were 70.0 ° and 33.4 °, respectively, on the culture surface subjected to excimer treatment 3 times at a stage moving speed of 2 mm / sec.

From the above results, it became clear that the receding contact angle can be adjusted appropriately by controlling the strength of the excimer treatment on the polyethylene film.
Specifically, the excimer treatment conditions are such that the distance between the culture surface and the lamp is 3 mm, and the stage moving speed is 60 mm / sec to 5 mm / sec, whereby the receding contact angle is greater than 45 ° and less than 78 °. It turned out that it can control to become.
At this time, the static water contact angle is in the range of 80 ° to 95 °, and the surface treatment suitable for the coating of the cell adhesion inhibitor is performed, that is, suitable for the production of the culture vessel for floating cells. I understand that

In addition, the excimer treatment conditions are such that the distance between the culture surface and the lamp is 3 mm, and the stage moving speed is 2 mm / sec or more, so that the receding contact angle is greater than 30 ° and 45 ° or less. It turns out that it can be controlled.
At this time, the static water contact angle is in the range of 60 ° to 80 °, and the surface treatment suitable for adhesion of the adherent cells is performed, that is, suitable for the production of the culture vessel for adherent cells. I found out.

The present invention is not limited to the above embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention.
For example, in the above-described embodiments and examples, specific conditions are shown as conditions for excimer treatment, but the present invention is not limited to these conditions, and the receding contact angle of the culture surface of the cell culture container is within the scope of the present invention. As long as it is a condition that can be processed as described above, it can be changed as appropriate.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used when producing a culture vessel having a uniformly hydrophilized culture surface used for suspension cells or adhesive cells.

Claims (8)

A cell culture vessel made of polyethylene material,
A cell culture vessel comprising a surface on which at least a part of the cell culture vessel is subjected to excimer treatment as a culture surface, and a receding contact angle of the culture surface being greater than 30 ° and less than 78 °. The cell culture container according to claim 1, wherein a receding contact angle of the culture surface is greater than 45 ° and less than 78 °, and is formed as a culture vessel for suspension cell culture. The cell culture container according to claim 1, wherein a receding contact angle of the culture surface is greater than 30 ° and 45 ° or less, and is formed as a culture vessel for culturing adhesive cells. The cell culture container according to any one of claims 1 to 3, wherein a standard deviation of static water contact angles measured at 10 or more locations on the culture surface is 2 ° or less. The cell culture container according to any one of claims 1 to 3, wherein a difference between a maximum value and a minimum value of static water contact angles measured at 10 or more locations on the culture surface is 5 ° or less. . Excimer treatment is performed on at least a part of the polyethylene film so that the receding contact angle of the region is greater than 30 ° and less than 78 °, and the cell culture vessel made of the polyethylene film is formed using the region as a culture surface. A method for producing a cell culture container. An excimer treatment is applied to at least a part of the polyethylene film so that the receding contact angle of the region is greater than 45 ° and less than 78 °, and the region is used as a culture surface, and the culture vessel for floating cells is made of the polyethylene film. Forming a cell culture container. An excimer treatment is applied to at least a part of the polyethylene film so that the receding contact angle of the region is greater than 30 ° and 45 ° or less, and the region is used as a culture surface, and the culture cell for adhesive cells comprises the polyethylene film. Forming a cell culture container.