JP6439979B2 - Cell culture equipment and cell culture method - Google Patents

Description

  The present invention relates to a cell culture facility and a cell culture method.

  In recent years, there is a field of regenerative medicine that has made significant progress as a new medical field dealing with cells. In order to provide patients with regenerative medicine technology safely, in addition to conventional management, chemical substances, viruses And management of mycoplasma is required. For example, when cells are contaminated with mycoplasma, it is considered to have various effects such as gene expression and abnormal immune response. Therefore, cells used for treatment are required to be free from contamination during the culture process. In facilities handling regenerative medical products, it is required to confirm the presence or absence of contamination such as mycoplasma from the viewpoint of safety.

  Mycoplasma is difficult to confirm by normal observation during culture and is determined by a mycoplasma negative test, but it is very difficult to reliably prove that the cultured cells are free of contamination. For this reason, from the viewpoint of preventing contamination, the cell culture facility itself is required to be a clean environment free from mycoplasma. Conventional cell culture facilities provide an environment in which air that keeps fine particles and microorganisms below a predetermined value using filters such as HEPA (High Efficiency Particulate Air) and ULPA (Ultra Low Penetration Air) is provided. (For example, patent document 1).

JP 2009-219382 A

  However, there has been a problem that viruses and mycoplasmas may remain in the air that has passed through the HEPA filter and the ULPA filter.

  The present invention has been made in view of the above circumstances, and is supplied with clean air from which viruses and mycoplasma have been removed, and a cell culture facility as a clean environment suitable for cell culture, and under the clean environment A cell culture method for culturing cells is provided.

[1] A cell culture facility comprising a cell culture environment in which clean air that has passed at least once through a virus / mycoplasma treatment unit, a chemical filter, and a HEPA filter is introduced, and the clean air is filled. .
[2] A cell manipulation chamber and at least one of a cell manipulation unit and a cell culture unit installed in the cell manipulation chamber are provided, and the clean air is supplied to the cell manipulation unit and the cell culture unit. The cell culture facility according to [1], wherein the cell culture facility can be introduced into at least one of them.
[3] In the above [2], at least one of the virus / mycoplasma treatment unit, the chemical filter, and the HEPA filter is provided in an air introduction path of the cell operation chamber. Cell culture equipment.
[4] The cell culture facility according to [2] or [3], wherein the virus / mycoplasma treatment unit is provided in at least one of the cell operation unit and the cell culture unit. .
[5] A HEPA filter is provided in an air introduction path of the cell operation chamber, and the virus / mycoplasma treatment unit, the chemical filter, and the HEPA filter are provided in the cell operation unit and the cell culture unit. The cell culture facility according to any one of [2] to [4] above, wherein:
[6] The HEPA filter and the chemical filter are provided in the air introduction path of the cell operation chamber, and the virus / mycoplasma treatment unit and the HEPA filter are provided in the cell operation unit and the cell culture unit. The cell culture facility according to any one of [2] to [4] above, wherein:
[7] A step of generating clean air by passing air at least once through a virus / mycoplasma treatment unit, a chemical filter, and a HEPA filter, and in an environment in which the clean air is introduced and the clean air is filled And a step of culturing the cells.

According to the cell culture facility of the present invention, it is possible to provide an environment for safely culturing cells from which impurities such as dust, chemical substances that can adversely affect cells, viruses and mycoplasma are removed, and the risk of contamination is extremely low. be able to.
According to the cell culture method of the present invention, cells can be safely cultured in an environment in which impurities such as dust, chemical substances that can adversely affect cells, viruses and mycoplasma are removed, and the risk of contamination is extremely low. it can.
According to the present invention, productivity of cells used for applications such as regenerative medicine can be increased.

It is a schematic diagram which shows the cell culture equipment of 1st embodiment of this invention. It is a schematic diagram which shows the modification of the cell culture equipment of 1st embodiment of this invention. It is a schematic diagram which shows the cell culture equipment of 2nd embodiment of this invention. It is a schematic diagram of the safety cabinet as an example of the cell operation part installed in the cell culture equipment of 2nd embodiment of this invention. It is a schematic diagram which shows the cell culture equipment of 3rd embodiment of this invention. It is a schematic diagram of the safety cabinet as an example of the cell operation part installed in the cell culture equipment of 3rd embodiment of this invention.

<Cell culture equipment>
Each embodiment of the cell culture equipment according to the present invention has a cell culture environment in which clean air that has passed at least once through a virus / mycoplasma treatment unit, a chemical filter, and a HEPA filter is introduced and is filled with the clean air. Is provided.
In this specification, “clean air” refers to air from which impurities have been removed by passing at least once through a virus / mycoplasma treatment unit, a chemical filter, and a HEPA filter. The “cell culture environment filled with clean air” is an environment (space) suitable for cell culture that is filled with clean air in a state where it is difficult for air containing impurities from flowing in from the outside.

The virus / mycoplasma processing unit (hereinafter referred to as VM processing unit) provided in the cell culture environment of each embodiment includes a processing apparatus capable of removing or inactivating at least one of the virus and mycoplasma. Means a unit.
As the processing apparatus, for example, ions capable of decomposing or inactivating at least one of virus and mycoplasma are generated, and the ions are applied to the virus and mycoplasma present in the air passing through the VM processing unit. An ion generator that can be brought into contact can be mentioned.

  The ionization method of the ion generator is not particularly limited, and examples thereof include a known discharge method, irradiation method, and electric field method. Among these, a discharge system that allows easy contact of the ions with viruses and mycoplasma is preferable. Specifically, air corona discharge, plasma discharge, arc discharge, underwater corona discharge and the like are preferable.

As an example of the ion generator, an AC high voltage is applied between the electrodes to cause plasma discharge, thereby ionizing oxygen and water vapor in the air, thereby positive ions H ⁺ (H ₂ O) _n and negative ions. An apparatus that generates O ₂ ⁻ (H ₂ O) _n that is an ion is known (for example, Japanese Patent No. 3680121). These ions can surround and remove microorganisms such as airborne bacteria, viruses, and mycoplasmas. As the mechanism, H ⁺ (H ₂ O) _n and O ₂ ⁻ (H ₂ O) _n adhere to the surface of the microorganism, and active hydrogen peroxide or hydroxy radical is generated. It is believed to inactivate the microorganism.

  Further, as the processing apparatus, for example, a virus that generates light capable of directly or indirectly decomposing or inactivating at least one of a virus and a mycoplasma and exists in the air passing through the VM processing unit. And a light generator capable of irradiating the mycoplasma with the light.

  The configuration of the light generator is not particularly limited. For example, the device may be a device configuration in which the irradiation light directly inactivates viruses and mycoplasmas, or the irradiation light releases radical species from the irradiated material. The device configuration may be such that the virus and mycoplasma are indirectly inactivated by the radical chemical species.

  An example of the light generator is an apparatus that irradiates the air flowing through the flow path with ultraviolet rays and inactivates viruses and mycoplasma in the air. As such an apparatus, for example, an ultraviolet ray processing apparatus disclosed in JP2013-136031A can be cited. Although the said ultraviolet treatment apparatus is an apparatus which irradiates an ultraviolet-ray to the to-be-processed water which flows through a pipe line, it should just use air instead of to-be-processed water as an to-be-irradiated body in this embodiment.

  As another example of the light generation device, the air that has been sent and a liquid containing a photosensitizing dye are brought into gas-liquid contact, and light that generates singlet oxygen by exciting the photosensitizing dye is irradiated. In addition, a device that inactivates viruses and mycoplasma in the air by singlet oxygen can be used. As such an apparatus, for example, a virus inactivation apparatus disclosed in JP2013-78650A can be cited.

  As another example of the light generation device, a photocatalytic filter that captures viruses, bacteria, fine particles, and the like contained in the supplied air is provided, and the virus and mycoplasma captured by irradiating the photocatalytic filter with light are excluded. A device to be activated is mentioned. As such an apparatus, for example, an air conditioner disclosed in Japanese Patent Laid-Open No. 2005-160494 can be cited.

  In addition, as the processing apparatus, for example, a contact portion including a substance that can inactivate at least one of a virus and a mycoplasma by physical contact has a configuration capable of contacting the supplied air. Apparatus. As an example of such a substance and contact portion, for example, a monovalent copper compound such as cuprous oxide, cuprous sulfide, cuprous iodide, cuprous chloride disclosed in JP2011-153163A is used. Examples include a virus inactivating agent contained as an active ingredient, and a contact portion comprising a base material containing the virus inactivating agent.

  In the cell culture facility of each embodiment, supplying clean air obtained by inactivating viruses and mycoplasma that have been difficult to remove with conventional HEPA filters and chemical filters in a VM processing unit to an environment in which cell culture is performed. Can do. Thereby, it can prevent that a virus and mycoplasma mix with a cultured cell, and contaminate a cultured cell (contamination).

The HEPA filter provided in the cell culture environment of each embodiment is the same as a known HEPA filter, and it captures 99.97% or more of particles that satisfy the standard of JIS Z8122 and have an average particle size of 0.3 μm with a rated air volume. It is an air filter having the performance of the collection pressure and the initial pressure loss of 245 Pa or less.
Therefore, even if the air before cleaning contains impure particles such as dust, dust, bacteria, and mold, these impure particles can be removed by the HEPA filter.

  The chemical filter provided in the cell culture environment of each embodiment is the same as a known chemical filter, and chemisorbs or absorbs chemical substances (hereinafter referred to as molecular contaminants) that may adversely affect the function expression or proliferation of cells. It is a filter that can be removed by physical adsorption. Examples of the adsorbent constituting the chemical filter include activated carbon; inorganic porous materials such as silica gel and activated alumina; and ion exchange resins.

Specific chemical filters include, for example, cationic filters that remove basic gases such as ammonia and amines; hydrofluoric acid, hydrogen chloride, sulfuric acid, sulfur oxide (SO _X ), nitric acid, nitrogen oxide (NO _X ), Anionic filters that remove acidic gases such as ozone and hydrogen peroxide; organic filters that remove organic gases such as dioctyl phthalate (DOP), dibutyl phthalate (DBP), siloxane, and phosphate esters; Can be mentioned.
Therefore, even if molecular contaminants are contained in the air before being cleaned, the molecular substances can be removed by the chemical filter.

  When there are a plurality of types of molecular contaminants, the cell culture equipment of each embodiment is preferably provided with a plurality of types of chemical filters capable of removing each molecular contaminant. In the present specification, unless otherwise specified, a single type of chemical filter and a plurality of types of chemical filters are not distinguished from each other, and are simply referred to as chemical filters in a sense including both.

The cell culture environment provided in the cell culture facility of each embodiment is a space configured so that cells can be handled and the incubator containing the cells can be held at a predetermined temperature.
The scale of the cell culture environment is not particularly limited, and may be an entire room configured semi-closed as a clean room, or may be a predetermined space of a cell culture related apparatus such as a safety cabinet or an incubator. Here, as a specific example of the safety cabinet, Class II safety cabinet 1300 series 1320 A2 manufactured by Thermo Scientific Co., Ltd. can be exemplified.

  In the cell culture environment provided in the cell culture facility of each embodiment, since clean air that has passed through the VM treatment unit, the chemical filter, and the HEPA filter is introduced at least once, and the clean air is filled, viruses, Contamination of cultured cells by mycoplasma can be prevented.

<Cell culture method>
A first embodiment of the cell culture method according to the present invention includes a step of generating clean air by passing outside air through a VM processing unit, a chemical filter, and a HEPA filter at least once, and introducing the clean air and And culturing the cells in an environment filled with clean air. The cell culturing operation in this embodiment is performed by a conventional method.

  The cell culture method of this embodiment may include a step of performing an operation other than the above. For example, you may have the process of introduce | transducing the said clean air in at least any one among the cell operation part installed in the cell operation room mentioned later and a cell culture part.

  The cell culture method of this embodiment can be carried out using the cell culture facility according to the present invention, and is preferably performed using, for example, the cell culture facility 1 described below.

  Hereinafter, each embodiment of the cell culture equipment according to the present invention will be described with reference to the drawings. Arrows G1 to G5 shown in each figure indicate the direction of air flow.

(First embodiment)
A cell culture facility 1A (1) shown in FIG. 1 includes a cell operation chamber 10 configured semi-closed as a clean room. The cell operation chamber 10 includes a cell operation unit 11 provided with a table for handling cells and containers to be cultured, and a cell culture unit 12 functioning as an incubator that can be set to a predetermined temperature and CO ₂ concentration. It has been.

An air introduction path 10 a for taking in air (outside air) G <b> 1 is provided on the ceiling of the cell operation chamber 10. On the back side of the ceiling, a chemical filter 3, a HEPA filter 2, and a VM processing unit 4 are arranged in this order in the air introduction path 10a as viewed from the air G1 introduction side.
The air G <b> 1 is introduced into the chemical filter 3 through an air supply facility (not shown) such as an air supply fan and a duct disposed on the ceiling of the cell operation chamber 10. When the air G1 passes through the chemical filter 3, air (molecular contaminant removal gas) G3 from which most of the molecular contaminants in the air G1 are removed is generated. Next, the air G3 passes through the HEPA filter 2 to generate air (impure particle-removed air) G2 from which the impure particles existing in the air G3 are removed. Subsequently, when the air G2 passes through the VM processing unit 4, the air (VM-removed air) G4 from which viruses and mycoplasma present in the air G2 are removed is converted into clean air G5 from which various impurities are removed. Generated. Clean air G5 is introduced into the cell operation chamber 10 from the air introduction path 10a.

The order of installing the chemical filter 3, the HEPA filter 2, and the VM processing unit 4 installed in the air introduction path 10a of the present embodiment is not particularly limited, and can be installed in any order. From the viewpoint of reducing the risk that ions generated in the VM processing unit 4 come into direct contact with the cultured cells, it is preferable to install at least one of the HEPA filter 2 and the chemical filter 3 on the downstream side of the VM processing unit 4. With this preferred configuration, ions flowing out from the VM processing unit 4 can be removed by the HEPA filter 2 or the chemical filter 3.
In the cell culture facility 1, the VM processing unit 4 and the filters 2 and 3 may be installed singly or plurally.

Air introduction paths 11a and 12a for taking in clean air G5 are provided on the ceiling or wall surfaces of the cell operation unit 11 and the cell culture unit 12, respectively.
The clean air G5 is introduced into the cell operation unit 11 from the air introduction path 11a and is introduced into the cell culture unit 12 from the air introduction path 12a.
In the present embodiment, the air introduction paths 11a and 12a are configured by simple openings. The cell operation unit 11 and the cell culture unit 12 are provided with an exhaust fan (not shown) that exhausts the introduced clean air G5 to the outside.

  In the present embodiment, since clean air G5 is introduced into the entire chamber 10 and the clean air G5 is filled, not only the inside of the cell operation unit 11 and the cell culture unit 12, It can be handled without contaminating cells at any place in the room. Therefore, the cell operation unit 11 is not necessarily a safety cabinet, and may be a simple experimental table.

(Modification of the first embodiment)
In the cell culture facility 1A illustrated in FIG. 1, the air introduction path 10a is provided at one place on the ceiling of the cell operation chamber 10, but the position where the air introduction path 10a is arranged is not particularly limited.
For example, like the cell culture facility 1A ′ shown in FIG. 2, the air introduction path 10a may be opened in the entire ceiling of the cell operation chamber 10. Moreover, the shape of the cell operation chamber 10 is not limited to the shape shown in FIG. 1. For example, the ceiling of the cell operation chamber 10 may be formed so as to increase from the outer peripheral portion toward the central portion. In that case, it is preferable that the air introduction path 10 a is opened at the top of the ceiling of the cell operation chamber 10.
Moreover, the position where the air introduction paths 11a and 12a of the cell operation unit 11 and the cell culture unit 12 are disposed and the position where the air introduction path is opened are not particularly limited. .

(Second embodiment)
A cell culture facility 1B (1) of the second embodiment of the present invention is shown in FIG. Among the components of the cell culture facility 1B, the same components as those of the cell culture facility 1A shown in FIG.

The cell culture facility 1B of the present embodiment is provided with a cell operation chamber 10, and an HEPA filter 2 is provided in an air introduction path 10a of the cell operation chamber 10.
Accordingly, the air G1 passes through the HEPA filter 2 through the blower facility (not shown) of the cell operation chamber 10 to become air G2 from which the impure particles in the air G1 are removed, and the air G1 enters the cell operation chamber 10 from the air introduction path 10a. It is introduced indoors.

The cell operation unit 11 and the cell culture unit 12 are configured to be semi-closed, and are kept clean by introducing clean air G5 into the internal space for handling cells and filling the air G5.
The air G5 is introduced by the air G2 introduced into the cell operation chamber 10 through the VM processing unit 4, the chemical filter 3, and the HEPA filter 2 installed in this order in the cell operation unit 11 and the cell culture unit 12. Generated.
The air G2 passes through the VM processing unit 4 and becomes air G4, and then passes through the chemical filter 3 and becomes air G3 as clean air G5. The air G3 (G5) further passes through the HEPA filter 2 to generate air G2 ′ (G5) as extremely clean air G5.

  In the cell operation part 11 and the cell culture part 12, the place which installs VM processing unit 4, the chemical filter 3, and the HEPA filter 2 is not specifically limited. For example, when the cell operation part 11 is the well-known safety cabinet 11, it can install above the cell handling space part 11b which the safety cabinet 11 shown in FIG. 4 has.

  In the configuration of FIG. 4, first, air G2 is sent from the intake port on the front surface of the safety cabinet 11 to the ventilation duct 11e on the rear surface side by the exhaust blower 11c and the circulation blower 11d. Next, a part of the air G2 introduced into the safety cabinet 11 is sent by the circulation blower 11d so as to pass through the VM processing unit 4, the chemical filter 3, and the HEPA filter 2 in order. The extremely clean air G2 ′ (G5) that has passed through the last HEPA filter 2 is introduced into the cell handling space 11b, fills the cell handling space 11b, and is then drawn back into the ventilation duct 11e on the back side and exhausted. After passing through the HEPA filter 2 by the blower 11c, the air is exhausted out of the safety cabinet 11.

  The semi-closed cell handling space portion 11b into which extremely clean air G2 'is introduced and filled with air G2' is a clean space free from viruses and mycoplasmas and suitable for cell culture. It is space.

(Third embodiment)
A cell culture facility 1C (1) of the third embodiment of the present invention is shown in FIG. Among the components of the cell culture facility 1C, the same components as those of the cell culture facility 1A shown in FIG.

The cell culture facility 1C of the present embodiment is provided with a cell operation chamber 10, and a chemical filter 3 and a HEPA filter 2 are provided in the air introduction path 10a of the cell operation chamber 10 in this order as viewed from the air G1 introduction side. It has been.
Therefore, the air G1 passes through the chemical filter 3 through the blower facility (not shown) of the cell operation chamber 10 to become air G3 from which molecular contaminants in the air G1 have been removed, and passes through the HEPA filter 2 and passes through the air. Air G2 from which the impure particles in G3 are removed is introduced into the cell operation chamber 10 from the air introduction path 10a.

The cell operation unit 11 and the cell culture unit 12 are configured to be semi-closed, and are kept clean by introducing and filling clean air G5 into the internal space for handling cells.
The air G5 is generated when the air G2 introduced into the cell operation chamber 10 passes through the VM processing unit 4 and the HEPA filter 2 installed in the cell operation unit 11 and the cell culture unit 12 in this order.
The air G2 passes through the VM processing unit 4 to become air G4 as clean air G5, and further passes through the HEPA filter 2 to generate air G2 ′ (G5) as extremely clean air G5.

  In the cell operation part 11 and the cell culture part 12, the place which installs VM processing unit 4 and the HEPA filter 2 is not specifically limited. For example, when the cell operation part 11 is the well-known safety cabinet 11, it can install above the cell handling space part 11b which the safety cabinet 11 shown in FIG. 6 has.

  In the configuration of FIG. 6, first, the air G2 is sent from the intake port on the front surface of the safety cabinet 11 to the ventilation duct 11e on the rear side by the exhaust blower 11c and the circulation blower 11d. Next, a part of the air G2 introduced into the safety cabinet 11 is sent by the circulation blower 11d so as to pass through the VM processing unit 4 and the HEPA filter 2 in order. The extremely clean air G2 ′ (G5) thus generated is introduced into the cell handling space 11b, fills the cell handling space 11b, and is then drawn back into the back side ventilation duct 11e and is exhausted by the exhaust blower 11c. After passing through the HEPA filter 2, it is exhausted out of the safety cabinet 11.

  The semi-closed cell handling space portion 11b into which extremely clean air G2 'is introduced and filled is a clean space free from viruses and mycoplasma, and is a space suitable for cell culture.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention.

DESCRIPTION OF SYMBOLS 1,1A, 1B, 1C ... Cell culture equipment, 2 ... HEPA filter, 3 ... Chemical filter, 4 ... Virus mycoplasma processing unit (VM processing unit), 10 ... Cell operation room, 11 ... Cell operation part, 11a ... Air Introducing path, 11b ... cell handling space, 11c ... exhaust blower, 11d ... circulating blower, 11e ... venting duct, 12 ... cell culture part, 12a ... air introducing path,
G1 ... Air (outside air),
G2: Air that has passed through the HEPA filter,
G3: Air that has passed through the chemical filter,
G4: Air passing through the virus / mycoplasma treatment unit,
G5: Clean air that has passed through the HEPA filter, chemical filter, and virus / mycoplasma treatment unit

Claims (7)

  A cell culture facility comprising a cell culture environment in which clean air that has passed at least once through a virus / mycoplasma treatment unit, a chemical filter, and a HEPA filter is introduced, and is filled with the clean air. A cell manipulation room, and a cell manipulation unit installed in the cell manipulation chamber and at least one of a cell culture unit, and
The cell culture equipment according to claim 1, wherein the clean air can be introduced into at least one of the cell operation unit and the cell culture unit.   The cell culture facility according to claim 2, wherein at least one of the virus / mycoplasma processing unit, the chemical filter, and the HEPA filter is provided in an air introduction path of the cell operation chamber. .   The cell culture facility according to claim 2 or 3, wherein the virus / mycoplasma treatment unit is provided in at least one of the cell operation unit and the cell culture unit. A HEPA filter is provided in the air introduction path of the cell manipulation chamber;
The cell culture according to any one of claims 2 to 4, wherein the cell manipulation unit and the cell culture unit are provided with the virus / mycoplasma treatment unit, the chemical filter, and the HEPA filter. Facility. An HEPA filter and the chemical filter are provided in an air introduction path of the cell manipulation chamber;
The cell culture facility according to any one of claims 2 to 4, wherein the cell manipulation unit and the cell culture unit are provided with the virus / mycoplasma treatment unit and the HEPA filter. Passing air through the virus and mycoplasma treatment unit, chemical filter and HEPA filter at least once to produce clean air;
Introducing the clean air and culturing cells in an environment filled with the clean air;
A cell culture method comprising:

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