JP7622348B2 - Method for forming a bag-shaped culture vessel - Google Patents

Description

The present invention relates to cell culture technology, and in particular to a bag-shaped culture vessel for floating cells induced from pluripotent stem cells.

In recent years, there has been a demand for efficient mass cultivation of cells, tissues, microorganisms, etc. in an artificial environment in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, etc.
When culturing a large amount of cells, it is beneficial to use a bag-shaped culture vessel (culture bag) made of a soft packaging material that allows easy production of a large-capacity culture vessel.

However, when culture experiments were carried out under various conditions on non-adherent cells using bag-shaped culture vessels, it was found that relatively high culture efficiency could not be obtained when the bag-shaped culture vessels were used.
Specifically, higher culture efficiency was achieved when a culture vessel originally suitable for culturing adherent cells, in which the culture portion of the culture vessel (the surface portion within the culture vessel where cell culture is carried out) had been hydrophilized, was used.

When culturing floating cells such as lymphocytes and dendritic cells that are cultured while suspended in a medium, it is desirable for the culture portion of the culture vessel to be hydrophobic so that the cells do not adhere to the culture portion.
In addition, when culturing floating cells, a surface treatment suitable for applying a cell adhesion inhibitor (cell low adhesion treatment agent) to the culture area is generally performed to prevent the cells from adhering to the culture area, and a culture vessel coated with this agent is used.

JP 2019-193604 A JP 2019-198288 A

Here, the floating cells used in the above culture experiments were iPS cell-derived T cells obtained by inducing differentiation of iPS cells. Therefore, the present inventors speculated that a culture vessel with different properties from ordinary culture vessels for floating cells would be suitable for culturing such floating cells.
Specifically, iPS cells are adhesive cells that can be grown by adhering to the culture section of a culture vessel. For this reason, it was speculated that the properties of a culture vessel for adhesive cells would be more suitable for iPS cell-derived T cells than for a general culture vessel for floating cells. In addition, there is also a method of culturing iPS cells in which spheres (cell aggregates) are formed without adhering the cells to the culture section of the culture vessel, and the spheres are cultured by floating them in the culture vessel.

The inventors have therefore conducted extensive research and found that when using a bag-shaped culture vessel to grow floating cells induced from pluripotent stem cells, it is preferable that the culture section of the bag-shaped culture vessel be formed from a polyethylene resin and that the static water contact angle of the culture section be 49.6° or more and 90.1° or less. It is even more preferable that the static water contact angle of this culture section be 49.6° or more and 80.0° or less.

Incidentally, the present applicant has been conducting research into appropriate conditions for manufacturing culture vessels for suspension cells and culture vessels for adherent cells, and has already proposed the culture vessel base material described in Patent Document 1 and the cell culture vessel described in Patent Document 2.
The culture vessel substrate described in Patent Document 1 is for adhesive cells, and therefore the culture vessel formed from this culture vessel substrate has a different use from that of the present invention, which is for planktonic cells. Also, the static water contact angle of the culture portion of this culture vessel is greater than 80°, which is different from the preferred range of the static water contact angle of the culture portion of the bag-shaped culture vessel of the present invention.

The cell culture vessel described in Patent Document 2 has a culture area with a static water contact angle of 80° to 95° for non-adherent cells, and a culture area with a static water contact angle of 60° to 80° for adhesive cells.
On the other hand, the bag-shaped culture vessel of the present invention is for use with floating cells induced from pluripotent stem cells, and therefore its uses are more limited compared to the cell culture vessel for floating cells described in Patent Document 2. In addition, the preferred range of the static water contact angle of the culture portion of the bag-shaped culture vessel of the present invention is different from that of the cell culture vessel for floating cells described in Patent Document 2.

The present invention was made in consideration of the above circumstances, and aims to provide a bag-shaped culture vessel capable of favorably growing floating cells induced from pluripotent stem cells.

To achieve the above object, the bag-shaped culture vessel of the present invention is a bag-shaped culture vessel for growing floating cells induced from pluripotent stem cells, the culture portion of the bag-shaped culture vessel is formed from a polyethylene resin, and the static water contact angle of the culture portion is 49.6° or more and 90.1° or less.

Furthermore, it is more preferable that the bag-shaped culture vessel of the present invention is configured so that the static water contact angle of the culture portion is 49.6° or more and 80.0° or less, and it is even more preferable that the static water contact angle of the culture portion is 74.2° or more and 80.0° or less.
Moreover, the bag-shaped culture vessel of the present invention is preferably configured such that the non-adherent cells induced from the pluripotent stem cells are iPS cell-derived T cells.

The present invention makes it possible to provide a bag-shaped culture vessel capable of favorably growing floating cells induced from pluripotent stem cells.

FIG. 2 is an explanatory diagram of a static water contact angle. FIG. 1 shows the number of cultured cells after each culture day in cell culture using various culture vessels in Test 1. FIG. 1 shows the proliferation rates after each culture day in cell culture using various culture vessels in Test 1. FIG. 1 is a graph showing the proliferation rates after each culture day in cell culture using various culture vessels in Test 1. FIG. 1 is a graph showing the proliferation rate after 11 days in cell culture using various culture vessels in Test 1.

Hereinafter, embodiments of the bag-shaped culture vessel 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 bag-shaped culture vessel of this embodiment is a bag-shaped culture vessel for growing floating cells induced from pluripotent stem cells, and is characterized in that the culture portion of the bag-shaped culture vessel is formed from a polyethylene-based resin and the static water contact angle of the culture portion is 49.6° or more and 90.1° or less.

Pluripotent stem cells are cells that can be induced to differentiate into various tissues in the body, and examples of such cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).
The floating cells are cells cultured in a culture vessel while suspended in a medium, and examples of the floating cells include lymphocytes such as T cells, dendritic cells, etc. In this embodiment, iPS cell-derived T cells can be preferably used as the floating cells induced from pluripotent stem cells.

Examples of the polyethylene-based resin that is the material of the culture section of the bag-shaped culture vessel of this embodiment include polyethylene, a copolymer of ethylene and an α-olefin, a copolymer of ethylene and vinyl acetate, and an ionomer using a copolymer of ethylene and acrylic acid or methacrylic acid and a metal ion. In this embodiment, it is particularly preferable to use polyethylene or ethylene vinyl acetate as the polyethylene-based resin.

The static water contact angle means the angle (θs) between the liquid surface and the solid surface at the point where the surface of a stationary liquid comes into contact with the surface of a solid wall, as shown in Figure 1. When the static water contact angle is large, the surface of the solid wall is relatively highly hydrophobic, and when the static water contact angle is small, the surface of the solid wall is relatively highly hydrophilic.
Specifically, it is known that when the static water contact angle in the culture portion of a culture vessel made of a polyethylene film, which is a type of polyethylene-based resin, is greater than 60° and less than 80°, adhesive cells can adhere favorably to the culture portion and be cultured efficiently.

In addition, the static water contact angle of the surface of the polyethylene film is 95° or more in an untreated state, and due to its strong hydrophobicity, it is not possible to culture adhesive cells on it. In addition, it is not possible to coat the film with a cell adhesion inhibitor, so it is not possible to culture non-adherent cells.
On the other hand, if a surface treatment is applied to the culture section of a culture vessel made of a polyethylene film to make the static water contact angle greater than 80° and less than 95°, a cell adhesion inhibitor can be coated on the culture section, making it possible to culture planktonic cells.

The static water contact angle of the culture section of the bag-shaped culture vessel of this embodiment is 49.6° or more and 90.1° or less. By having such a static water contact angle, the bag-shaped culture vessel of this embodiment is capable of favorably growing non-adherent cells induced from pluripotent stem cells.

Moreover, the static water contact angle of the culture portion of the bag-shaped culture vessel of this embodiment is more preferably 49.6° or more and 80.0° or less, and even more preferably 74.2° or more and 80.0° or less (or less).
By setting the static water contact angle of the culture section of the bag-shaped culture vessel of this embodiment within this range, it is possible to more appropriately improve the proliferation efficiency of non-adherent cells induced from pluripotent stem cells.

The mechanism behind improved proliferation under these conditions is not clear, but it is speculated that because iPS cell-derived T cells are more prone to aggregation than natural T cells, by adhering them to the culture area with a certain degree of strength, excessive aggregation is prevented, thereby preventing some of the cells from suffering from oxygen or nutrient deficiencies.
In addition, as shown in the examples below, if the static water contact angle of the culture area is too hydrophilic, the proliferation efficiency decreases. Although the reason for this is unclear, it is speculated that one of the causes is that the cells strongly adhere to the culture area, preventing them from floating up from the culture area during proliferation.

The bag-shaped culture vessel of this embodiment is characterized in that the culture section has been subjected to hydrophilization treatment by excimer treatment or corona treatment.
Excimer treatment is a surface treatment that uses an excimer lamp as a light source. Ultraviolet rays with a wavelength of 200 nm or less are called vacuum ultraviolet rays, and excimer treatment involves irradiation with this vacuum ultraviolet ray. There are excimer lamps that irradiate vacuum ultraviolet rays with various wavelengths, but xenon excimer lamps with a wavelength of 172 nm are generally used. The treatment is carried out by setting the distance between the culture unit and the lamp, the moving speed of the stage, etc. to the desired values.
When such an excimer treatment is applied to a polyethylene film, the surface properties of the polyethylene change and it becomes hydrophilic. Furthermore, the stronger the intensity of the excimer treatment, the greater the degree of hydrophilization. With the excimer treatment, it is possible to obtain a culture surface that is uniformly hydrophilized without uneven treatment.

Corona treatment is a surface treatment using corona discharge irradiation, and is carried out, for example, by using a batch-type corona treatment device and setting the output, electrode distance, stage movement speed, and the like to desired values.
When a polyethylene film is subjected to such a corona treatment, the surface properties of the polyethylene change and it becomes hydrophilic. Moreover, the stronger the corona treatment intensity, the greater the degree of hydrophilization.

An example of the cell adhesion factor that is not applied to the culture portion of the bag-shaped culture vessel of this embodiment is laminin.
In addition, examples of cell adhesion inhibitors that are not applied to the culture portion of the bag-shaped culture vessel of this embodiment include phospholipid polymers, polyvinyl alcohol derivatives, phospholipid-polymer complexes, polyhydroxyethyl methacrylate, polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, etc.

The bag-shaped culture vessel of this embodiment has one or more injection/exit ports, through which medium or cell suspension can be injected into the bag-shaped culture vessel and the medium or cell suspension can be discharged from the bag-shaped culture vessel. Tubes are connected to the injection/exit ports, and a medium supply container, cell collection container, enzyme supply container, waste container, etc. can be appropriately connected to the tubes.

As the material of the bag-shaped culture vessel (excluding the culture portion) of this embodiment, polyolefin resins such as polyethylene and polypropylene can be suitably used. For example, polyethylene, copolymers of ethylene and α-olefin, copolymers of ethylene and vinyl acetate, ionomers using ethylene, acrylic acid or methacrylic acid copolymers, and metal ions can be mentioned. In addition, polyolefins, styrene-based elastomers, polyester-based thermoplastic elastomers, silicone-based thermoplastic elastomers, silicone resins, etc. can also be used. Furthermore, silicone rubber, soft vinyl chloride resins, polybutadiene resins, ethylene-vinyl acetate copolymers, chlorinated polyethylene resins, polyurethane-based thermoplastic elastomers, polyester-based thermoplastic elastomers, silicone-based thermoplastic elastomers, styrene-based elastomers, such as SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), polyolefin resins, fluorine-based resins, etc. may also be used.

Materials that can be used for the injection port include, for example, thermoplastic resins such as polyethylene, polypropylene, polyvinyl chloride, polystyrene-based elastomers, and FEP.

The bag-shaped culture vessel of this embodiment can be formed, for example, by using a polyethylene film and subjecting at least a portion of the film to an excimer treatment or the like, thereby making the static water contact angle of the portion into a culture area of 49.6° or more and 90.1° or less, arranging the polyethylene film in a bag shape to sandwich the injection/exit port, and heat sealing the periphery.

As described above, the bag-shaped culture vessel of this embodiment makes it possible to favorably proliferate non-adherent cells such as T cells induced from pluripotent stem cells.

Below, we will explain a test in which floating cells induced from pluripotent stem cells were cultured using the bag-shaped culture vessel of this embodiment and a bag-shaped culture vessel in which the culture section has not been hydrophilized and the static water contact angle is outside the range of this embodiment.

(Test 1)
The cells used for the culture were T cells that had been induced to differentiate from iPS cells and activated using CD3 antibody and retronectin.
Specifically, the T cells were obtained by differentiating the Ff-I01s04 line provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, in accordance with a publicly known method (WO2017/221975).

The medium used for the culture was IMDM medium (Thermo Fisher Scientific) supplemented with 15% FBS (Corning), 1% penicillin-streptomycin-glutamine solution (Sigma-Aldrich), 1% insulin-transferrin-selenium solution (Thermo Fisher Scientific), 50 μg/ml L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich), 10 ng/ml IL-7 (PeproTech), and 10 ng/ml IL-15 (PeproTech).

The material of the bag-shaped culture vessel was a polyethylene film, and the culture part of the bag-shaped culture vessel was also integrally formed from the same polyethylene film.
Then, the region of the bag-shaped culture vessel that was to become the culture section was subjected to an excimer treatment or a corona treatment as a hydrophilization treatment under the following conditions.

As a device for carrying out the excimer treatment, a xenon excimer lamp device (an excimer irradiation device manufactured by MDCOM Co., Ltd.) was used, and the voltage was set to 12V.
Then, excimer treatment was performed under the following conditions of the distance between the culture area and the lamp, and the moving speed of the stage on which the polyethylene film was placed in the xenon excimer lamp device, and the static water contact angle of the culture area was measured.

When the distance between the culture portion and the lamp was 4 mm and the movement speed of the stage on which the polyethylene film was placed in the xenon excimer lamp device was 20 mm/sec, the static water contact angle of the culture portion was 90.1°.
Furthermore, when the distance was 4 mm and the stage movement speed was 5 mm/sec, the static water contact angle of the culture portion was 82.0°, when the distance was 4 mm and the stage movement speed was 2 mm/sec, the static water contact angle of the culture portion was 77.8°, and when the distance was 4 mm, the stage movement speed was 2 mm/sec, and the same treatment was performed twice, the static water contact angle of the culture portion was 74.2°.

The corona treatment was performed using a batch-type corona treatment device (manufactured by Kasuga Electric Co., Ltd.) with an output of 520 W, an interelectrode distance of 5 mm, and a moving speed of the stage on which the polyethylene film was placed in the batch-type corona treatment device of 50 mm/sec. This was repeated three times, and the static water contact angle of the culture section was 49.6°.
These static water contact angles were obtained by measuring the static water contact angles at 10 randomly selected points on the film surface and calculating the average value.

The bag-shaped culture vessels were formed by sealing each of the polyethylene films treated as described above with an impulse sealer so that the area of the culture section was 10 cm x 10 cm. At this time, a polyethylene port was sandwiched and sealed to attach an injection and exit port.

The cells were cultured according to the following culture protocol.
First, 1.5×10 ⁶ cells were suspended in 30 ml of medium and placed in each bag-shaped culture vessel.
Next, 3 days after the start of the culture, the number of cells was counted, 15 ml of the suspension in each bag-like culture vessel was removed, and 30 ml of fresh medium was added.

Five days after the start of the culture, the number of cells was counted, 35 ml of the suspension in each bag-like culture vessel was removed, and 30 ml of fresh medium was added.
Seven days after the start of the culture, the number of cells was counted, 30 ml of the suspension in each bag-like culture vessel was removed, and 30 ml of fresh medium was added.

The final cell number was counted 11 days after the start of culture, and the results are shown in FIG.
In the above culture protocol, the suspension is discarded midway because if the cell density becomes too high, the growth stops, and the effect of the static water contact angle of the culture area cannot be properly evaluated. Also, the number of seeded cells on each day shown in Figure 2 indicates the number of cells remaining in the bag-shaped culture vessel after the suspension is discarded.

The fold increase in cell number was calculated for each day interval in which the cells were counted, and the results were multiplied to determine the total proliferation fold.
Specifically, as shown in FIG. 3 , the proliferation fold of the number of cells on day 3 relative to the number of cells on day 0, the proliferation fold of the number of cells on day 5 relative to the number of cells seeded on day 3, the proliferation fold of the number of cells on day 7 relative to the number of cells seeded on day 5, and the proliferation fold of the number of cells on day 11 relative to the number of cells seeded on day 7 were calculated, and these were multiplied together to calculate the total proliferation fold.

The proliferation rate on each day was calculated in the same manner by multiplying the rates calculated for each of the previous days. The results are shown in a graph in Figure 4. Furthermore, the proliferation rate on the 11th day is shown in a graph in Figure 5.

As shown in these figures, when the static water contact angle of the culture area of the bag-shaped culture vessel is 49.6° or more and 90.1° or less, the proliferation rate of the non-adherent cells induced from pluripotent stem cells is significantly improved compared to a bag-shaped culture vessel in which the culture area has not been subjected to hydrophilization treatment.
In particular, excellent proliferation efficiency is shown when the static water contact angle of the culture portion is 74.2° or more and at least 80.0° or less.

The present invention is not limited to the above-described 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 embodiments and examples, specific conditions were shown as the conditions for the excimer treatment, but these are not limited to these, and the surface treatment method and treatment conditions can be changed as appropriate as long as they are capable of treating the culture portion of the bag-shaped culture vessel so that the static water contact angle falls within the range of the present invention.

The present invention can be suitably used when efficiently producing large amounts of iPS cell-derived T cells, etc.

Claims (6)

A method for forming a bag-shaped culture vessel for growing floating cells induced from pluripotent stem cells, comprising forming a culture portion of the bag-shaped culture vessel from a polyethylene-based resin, and setting a static water contact angle of the culture portion to 74.2° or more and 77.8° or less ,
The cell adhesion inhibitor is not applied to the culture area.
A method for forming a bag-shaped culture vessel comprising the steps of: The method for forming a bag-shaped culture vessel according to claim 1, characterized in that the floating cells induced from the pluripotent stem cells are iPS cell-derived T cells. The method for forming a bag-shaped culture vessel according to claim 1 or 2, characterized in that the polyethylene resin is polyethylene or ethylene vinyl acetate. The method for forming a bag-shaped culture vessel according to any one of claims 1 to 3, characterized in that the culture section is hydrophilized by excimer treatment or corona treatment. The method for forming a bag-shaped culture vessel according to any one of claims 1 to 4, characterized in that neither a cell adhesion factor nor a cell adhesion inhibitor is applied to the culture portion. The method for forming a bag-shaped culture vessel according to any one of claims 1 to 5, characterized in that the bag-shaped culture vessel is provided with one or more injection/exit ports.

Images