KR20170063015A - A equipment of modifying a surface in a cell culture bag inner side and a surface modifying method therewith - Google Patents

A equipment of modifying a surface in a cell culture bag inner side and a surface modifying method therewith Download PDF

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KR20170063015A
KR20170063015A KR1020150168916A KR20150168916A KR20170063015A KR 20170063015 A KR20170063015 A KR 20170063015A KR 1020150168916 A KR1020150168916 A KR 1020150168916A KR 20150168916 A KR20150168916 A KR 20150168916A KR 20170063015 A KR20170063015 A KR 20170063015A
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cell culture
culture bag
plasma
reaction gas
surface treatment
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조용기
민성욱
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한국생산기술연구원
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/32—Gas-filled discharge tubes
    • H01J37/32009—Arrangements for generation of plasma specially adapted for examination or treatment of objects, e.g. plasma sources
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/14—Bags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/32—Gas-filled discharge tubes
    • H01J37/32431—Constructional details of the reactor
    • H01J37/3244—Gas supply means
    • H01J37/32449—Gas control, e.g. control of the gas flow

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Abstract

The present invention relates to an apparatus for surface treatment of the inner surface of a cell culture bag and a surface treatment method using the same, and more particularly, to a surface treatment apparatus for inner surface of a cell culture bag, And a reaction gas supply unit connected to the cell culture bag 100 and having a function of supplying a reaction gas into the cell culture bag 100, and a gas inlet module 200 which surrounds the cell culture bag 100 An electrode unit having a function of forming a closed space and generating plasma by discharging the reaction gas to form a plasma on the inner surface of the cell culture bag, And a plasma processing module 300 connected to the cell culture bag 100 and the plasma processing module 300. The plasma processing module 300 includes an AC power source 320, And a vacuum evacuation unit (400) for evacuating the inside and outside of the cell culture bag (100) to a vacuum state. The inside and outside of the cell culture bag (100) And a plasma discharge is selectively generated only in the inside of the cell culture bag.

Description

TECHNICAL FIELD [0001] The present invention relates to a surface treatment apparatus for treating a surface of a cell culture bag inner surface and a surface treatment method using the same,

The present invention relates to a surface treatment apparatus for treating a surface of a cell culture bag and a surface treatment method using the same. More particularly, the present invention relates to a surface treatment apparatus using a surface- And to provide a surface treatment apparatus for a surface of an inner surface of a cell culture bag capable of imparting various functions.

As the material of the cell culture bag, polyethylene material or polyamide, and a polyethylene composite material of three or more layers including an intermediate barrier layer are used as a material of a cell culture bag. Cell culture bags are medical supplies that are increasingly being used to replace petri dishes in in vivo biology experiments. Stem cell cultures, general cell cultures, bacterial cultures, tissue cultures and the like, as well as living goods and biotechnology. The cell culture bag is larger in capacity than the conventional cell culture container and has superior stability and performance, and the demand is increasing and the market is growing.

In order to improve cell / tissue regeneration by surface treatment of cell culture bag, active oxygen exposure technique using atmospheric pressure discharge is applied to polyethylene surface. The atmospheric pressure plasma method is a technology that induces reactivity on the surface inside the cell culture bag by causing the corona discharge caused by the electron emission on the surface in the atmospheric state by putting the reaction gas into the cell culture bag in the atmospheric state and applying the potential of high voltage, To form an oxide layer on the surface.

Atmospheric pressure plasma is mainly used as a method of exposing an electron stream (electron stream) or a microplasma jet, and is used in a plate type substrate material. In order to generate atmospheric pressure discharge in a three-dimensional shape such as a cell culture bag, a high voltage of 7 kV or more should be applied, but such a high voltage application method may cause surface damage due to the emission of electrons having high energy. In order to prevent damage to the surface, a plasma discharge should occur under a low voltage. However, in the case of an atmospheric pressure plasma method, there is a problem that it is difficult to treat the inner surface rather than the outer surface when a low voltage is applied.

In addition, atmospheric pressure composed of nitrogen and oxygen makes it difficult to selectively modify the surface by the reaction gas. Oxidation by oxygen gas filling is possible, but selective surface treatment of only nitrogen or barrier coating of gas tightness using hydrocarbon gas can not be performed. In other words, the atmospheric pressure plasma method is required to have a new surface treatment method which can easily form a surface that can give various functions because the composition of various surfaces is very limited.

Japanese Patent No. 4,887,222 entitled CELL CULTURE VESSEL, METHOD FOR PRODUCING THE SAME AND METHOD FOR CULTURING CELL, hereinafter referred to as Prior Art 1), is a cell culture container for culturing adherent cells, A synthetic resin sheet having a bending rigidity of 20 mm or less according to the method (A) has a container body formed in a container shape for supporting a medium required for culturing adherent cells, Wherein a culture region having a sex functional group is formed.

JP 4887222 B2

Prior Art 1 discloses a cell culture container in which a polyolefin-based resin layer on the inner surface of a cell culture container is subjected to plasma treatment. However, only an embodiment using an atmospheric pressure plasma method is described, and therefore, There is a first problem that surface damage due to application may occur, and a second problem that the configuration of various surfaces is limited.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the precise form disclosed. There will be.

According to an aspect of the present invention, there is provided a surface treatment apparatus for treating a surface of an inner surface of a cell culture bag, the apparatus comprising a cell culture bag (100) and the cell culture bag (100) A gas introducing module 200 including a reaction gas supply unit having a function of supplying a reaction gas into the inside of the cell culture bag 100 and a function of generating a plasma by enclosing the outside of the cell culture bag 100, An electrode unit having a function of discharging the reaction gas and forming a functional group on the inner surface of the cell culture bag by plasma formation, and an AC power unit 320 connected to the electrode unit to apply a voltage, Processing module 300, the cell culture bag 100 and the plasma processing module 300 so that the inside and the outside of the cell culture bag 100 are kept in a vacuum state And a vacuum exhaust unit (400) for pressing the cell culture bag (100). The pressure of the inside and the outside of the cell culture bag (100) is different from each other and a plasma discharge is selectively generated only in the cell culture bag Thereby providing a surface treatment apparatus for the inner surface of the cell culture bag.

The electrode unit may include a metal electrode 311 and a ceramic dielectric electrode 312.

The vacuum exhaust unit 400 includes a first vacuum exhaust unit 410 connected to the cell culture bag 100 to decompress the inside of the cell culture bag 100 to a vacuum state, And a second vacuum evacuation device 420 connected to the cell culture bag 100 for evacuating the outside of the cell culture bag 100 to a vacuum state.

In addition, the functional group formed from the reaction gas may be modified so that the inner surface of the cell culture bag is improved in cell adhesion or airtightness.

The functional group may be selected from the group consisting of a carboxyl group, an amine group, a hydrocarbon group, and a hydroxy group.

The reaction gas may be any one selected from the group consisting of He, Ar, O 2 , N 2 , H 2 , and hydrocarbons, or a mixed gas thereof.

In addition, the inside and outside pressures of the cell culture bag may be 50 mTorr to 50 Torr.

The pressure difference between the inside and the outside of the cell culture bag may be 1 Torr or more and 20 Torr or less.

Also, the voltage applied to the AC power source 320 may be 1 to 5 kV.

The material of the cell culture bag may be polyethylene (PE), polyamide (PA), polystyrene (PS), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyvinyl alcohol And may be any one selected from the group consisting of phthalate (PET), polypropylene (PP), polyurethane (PU), and copolymers thereof.

The plasma processing module 300 may include a driving unit having a function of moving the plasma processing module 300 up and down.

Also, the reaction gas supply unit may include a mass flow controller (MFC)

The present invention also provides a method for surface treatment using a surface treatment apparatus for an inner surface of a cell culture bag, comprising the steps of: (i) preparing a cell culture bag; (ii) decompressing the inside and outside of the cell culture bag to a vacuum state; (iii) introducing a reaction gas into the cell culture bag; (iv) applying a predetermined voltage to generate a surface plasma inside the cell culture bag; And (v) modifying the surface by forming a functional group from the reaction gas on the inner surface of the cell culture bag by the surface plasma; Wherein in step (ii), the pressure inside and outside of the cell culture bag is different, and in step (iv), a plasma discharge is selectively generated only inside the cell culture bag. A method for surface treatment of an inner surface is provided.

The present invention also provides a method of surface treatment using a surface treatment apparatus for inner surface of a cell culture bag, comprising the steps of: (a) preparing a cell culture bag; (b) depressurizing the inside and outside of the cell culture bag to a vacuum state; (c) introducing a first reaction gas into the cell culture bag; (d) applying a predetermined voltage to generate a surface plasma inside the cell culture bag; (e) forming a functional group from the first reaction gas on the inner surface of the cell culture bag by the surface plasma to modify the surface; (f) introducing a second reaction gas different from the first reaction gas in the step (c); (g) performing the steps (d) and (e); Wherein the cell culture bag has a pressure different from that of the cell culture bag in the step (b), and a plasma discharge is selectively generated only in the cell culture bag in the step (d) A method for surface treatment of an inner surface is provided.

The present invention also provides a cell culture bag having an inner surface modified using the surface treatment method according to the present invention.

The apparatus for surface treatment of the inner surface of the cell culture bag according to the present invention and the surface treatment method using the same have the first effect that the surface damage can be prevented by generating the low temperature plasma at a lower voltage than the prior art, A second effect that various functionalities can be imparted to the inner surface of the cell culture bag due to the ability to form various functional groups, and a method of regulating the pressure difference between the inside and the outside of the cell culture bag, And a cell culture bag having improved inner surface characteristics by using the surface treatment method and surface treatment apparatus of the inner surface of the cell culture bag according to the present invention have the fourth effect that the cell culture ability can be improved.

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing an apparatus for surface treatment of the inner surface of a cell culture bag according to an embodiment of the present invention. FIG.
FIG. 2 is a schematic view showing a driving part and a fixing part of the surface treatment apparatus on the inner surface of the cell culture bag according to the embodiment of the present invention.
FIG. 3 is a schematic view showing a possible electric field application method of a surface treatment apparatus on the inner surface of a cell culture bag according to an embodiment of the present invention.
4 is an SEM image of the inner surface of a cell culture bag made of polyethylene.
5 is an SEM image of the inner surface of a cell culture bag surface-treated with Ar gas according to Example 1 of the present invention.
6 is an SEM image of the inner surface of a cell culture bag in which a hydrocarbon thin film is deposited by plasma treatment using hydrocarbon gas according to Example 3 of the present invention.
FIG. 7 is an SEM image of the inner surface of a cell culture bag obtained by plasma-treating a hydrocarbon thin film using O 2 gas according to Example 4 of the present invention. FIG.
8 is an FT-IR spectrum of the inner surface of a cell culture bag surface-treated according to an embodiment of the present invention.
9 is a graph showing an XPS spectrum analysis result of a surface of a cell culture bag surface-treated according to an embodiment of the present invention.
10 is an XPS spectrum analysis result of a surface of a cell culture bag in which a hydrocarbon thin film is deposited according to Example 3 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout the specification, when a part is referred to as being "connected" (connected, connected, coupled) with another part, it is not only the case where it is "directly connected" "Is included. Also, when an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing an apparatus for surface treatment of the inner surface of a cell culture bag according to an embodiment of the present invention. FIG. 1, the present invention relates to a surface treatment apparatus for an inner surface of a cell culture bag, which comprises a cell culture bag 100 and a cell culture bag 100 connected to the cell culture bag 100, A gas inlet module 200 including a reaction gas supply unit having a function of supplying a reaction gas into the inside of the cell culture bag 100 and a function of generating a plasma by enclosing the outside of the cell culture bag 100, An electrode unit having a function of discharging the reaction gas to plasma to form a functional group on the inner surface of the cell culture bag, and an AC power supply unit 320 connected to the electrode unit to apply a voltage, A module 300 connected to the cell cultivation bag 100 and a plasma processing module 300 to connect the cell culture bag 100 and the plasma treatment module 300, Wherein the cell culture bag (100) has a pressure difference between the inside and the outside of the cell culture bag (100), and a plasma discharge is selectively generated only in the cell culture bag (100) A surface treatment apparatus for a surface is provided.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the main constituent elements of the surface treatment apparatus for the inner surface of the cell culture bag according to the present invention.

Cell culture bag is a medical consumable made of polymer material that is increasingly used in in vivo biology experiment by gradually replacing petri dish and flask. It is used for stem cell culture, general cell culture, strain culture, tissue culture etc. Is widely used in the fields of biology, medicine and household goods. The cell culture bag is larger in capacity than the conventional cell culture container and has superior stability and performance, and the demand is increasing and the market is growing.

The cell culture bag 100 may be made of polyethylene, polyamide (PA), polystyrene (PS), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyvinyl alcohol May be any one selected from the group consisting of polyethylene terephthalate (PET), polypropylene (PP), and polyurethane (PU), or copolymers thereof, but is not limited thereto.

The gas inlet module 200 may be connected to the cell culture bag 100 to supply the reaction gas into the cell culture bag 100 and may include a reaction gas supply unit. 1, a gas conduit of the gas inlet module 200 is connected to one of the ports of the cell culture bag 100, and a reactive gas is supplied into the cell culture bag 100 .

In addition, the reaction gas supply unit includes a reaction gas tank 211 for storing and supplying the reaction gas for each type, a gas flow controller (MFC) 212, a first gas supply A valve 213, and a second gas supply valve 214 for controlling whether or not the reaction gas is finally input and supplied. The reaction gas tank 211, the gas flow rate control device 212 and the first gas supply valve 213 are individually prepared according to the reaction gas to be charged, and the kind of the reaction gas is determined by the material of the cell culture bag and the function It depends on the type of machine. The kind, supply amount, mixing ratio, and the like of the reaction gas supplied from the reaction gas tank 211 by the gas flow rate control device 212, the first gas supply valve 213 and the second gas supply valve 214 are controlled, And is introduced into the culture bag.

The reaction gas may be any one selected from the group consisting of He, Ar, O 2 , N 2 , H 2 , and hydrocarbons, or a mixed gas thereof, but is not limited thereto. . The hydrocarbons may specifically be C 2 H 2 or CH 4 gases, but other hydrocarbons capable of forming hydrocarbon thin films are also possible. The type of reaction gas to be added depends on the material of the cell culture bag and the type of functional group to be produced.

In addition, the reaction gas may further include at least one of He and Ar for forming a uniform surface plasma. Preferably, helium is used. The helium is used as a gas causing plasma in the present invention as a gas effective to form a surface plasma that has a large plasma stream radius and flows along the surface. By selectively incorporating nitrogen, oxygen, or hydrogen in addition to helium, functional groups can be imparted to the inner surface of the cell culture bag as described below.

The plasma generated in the plasma processing module 300 (to be described later) causes the reactive gas to be excited and ionized to form reactive reactive species such as radicals. The reactive active species adsorbs on the inner surface of the cell culture bag while forming functional groups and modifying the surface.

The functional group may be selected from the group consisting of a carboxyl group, an amine group, a hydrocarbon group, and a hydroxy group, but is not limited thereto. For example, a carboxyl group or a hydroxy group is formed in an oxidizing gas atmosphere such as a carbon oxide or oxygen, and a hydrocarbon thin film may be formed in an amine group or hydrocarbon gas atmosphere in a nitriding gas atmosphere such as nitrogen.

The functional group formed from the reaction gas may be modified so that the inner surface of the cell culture bag is improved in cell adhesion or airtightness. In general, adherent cells are difficult to attach on hydrophobic surfaces, while adhesion on hydrophilic surfaces occurs well. When a carboxyl group or a hydroxy group is formed on the surface, the inner surface of the cell culture bag changes from hydrophobic to hydrophilic and can facilitate cell attachment. When the inner surface is surface-treated with hydrocarbons, the airtightness is improved and the gas permeability is reduced as the surface is hardened. In this case, the inflow of the external gas can be blocked, thereby improving the cell culture performance of the cell culture bag. In addition, when the surface is modified with a functional group formed by the low temperature plasma discharge of the present invention, lipophilicity, water repellency, dyeability and the like can be improved.

The plasma processing module 300 has a function of enclosing the outside of the cell culture bag 100 to provide a closed space and generating a plasma, and discharges the reaction gas to plasmaize the inside of the cell culture bag 100, An electrode unit having a function of forming a functional group on a surface thereof, and an alternating-current power unit 320 connected to the electrode unit and applying a voltage thereto.

A low-temperature plasma system in a vacuum state, which is constituted by a vacuum exhaust unit 400 to be described later, includes a dielectric barrier discharge (DBD), a Helicon wave plasma, a capacitively coupled plasma formed by a microwave radio frequency ; CCP) or inductively coupled plasma (ICP). But is not limited to, a dielectric barrier discharge, preferably an electrical discharge between two electrodes isolated by a dielectric barrier.

In a normal atmospheric pressure discharge (corona discharge), a current is induced with respect to an applied voltage of AC, and the induced current is mainly distributed in a range of -100 mA to 100 mA. On the other hand, when the present invention is carried out by dielectric barrier discharge, a displacement field is formed inside the dielectric with one of the electrodes (dielectric constant εr> 5) by the electric field formed by the AC power source, Inside the cell culture bag, which is in a vacuum state, a surface discharge is induced by the displacement field of the intrinsic whole to form a surface plasma.

The electrode portion has a function of causing a plasma discharge to the reaction gas to form a functional group. 1, the electrode unit includes a metal electrode 311 located outside the ceramic dielectric electrode 312 and a ceramic dielectric electrode 312 surrounding the cell culture bag 100 inside the metal electrode. . Under the dielectric barrier discharge (DBD) system, the ceramic dielectric electrode 312 has a shape and a capacity similar to that of the cell culture bag 100 and can cover the outside of the cell culture bag 100 and is formed of a ceramic having a high dielectric constant. When an AC voltage equal to or higher than the plasma discharge start voltage is applied to the metal electrode 311, a surface discharge is induced by a displacement field induced in the ceramic dielectric electrode 312 to generate a plasma.

In addition, the AC power supply unit 320 is connected to the electrode unit and performs a function of applying a voltage to the electrode unit. The voltage at this time is AC, and this AC voltage acts to induce a current of another sign by the surface discharge on the inner surface of the cell culture bag 100.

The plasma processing module 300 may include a driving unit (not shown) having a function of moving the plasma processing module 300 up and down. FIG. 2 is a schematic view showing a driving part and a fixing part of the surface treatment apparatus on the inner surface of the cell culture bag according to the embodiment of the present invention. Referring to FIG. 2, a portion including a gas inflow module 200 and a support 500 is fixed to the upper portion of the surface treatment apparatus on the inner surface of the cell culture bag according to the present invention, and a plasma treatment The portion including the module 300 can be driven up and down by the driving portion. The cell culture bag 100 is first connected to the fixing part between the supports 500 and then the lower and upper driving parts are raised to come in contact with the support 500 to enclose the cell culture bag 100, So that a vacuum state can be established outside the cell culture bag. After the surface treatment is completed, the upper and lower driving parts descend again so that the cell culture bag 100 can be detached. The driving unit may be any motor as long as it is generally used as driving means.

FIG. 3 is a schematic view showing a possible electric field application method of a surface treatment apparatus on the inner surface of a cell culture bag according to an embodiment of the present invention. 3, the AC power source 320 is connected to both sides of the metal electrode 311 located outside the ceramic dielectric electrode 312, So that a plasma discharge can be generated. 3 (b), a support 500 made of metal also acts as an electrode to connect the support 500 with the metal electrode 311 to apply a voltage thereto .

The applied voltage may be between 1 and 5 kV. If it is lower than 1 kV, it may not reach the discharge starting voltage and plasma may not be generated. If it is higher than 5 kV, there may be surface damage due to high energy electron emission caused by high voltage.

In addition, the plasma discharge time may be 30 seconds to 5 minutes. If the discharge time is shorter than 30 seconds, functional groups may not be formed sufficiently on the inner surface due to a short discharge time. If it is longer than 5 minutes, etching may occur depending on the polymer material. The voltage and the discharge time may be controlled depending on the kind of the reaction gas, the mixing ratio, the polymer material, and the like.

The vacuum exhaust unit 400 may be connected to the cell culture bag 100 and the plasma processing module 300 and may have a function of decompressing the inside and outside of the cell culture bag 100 to a vacuum state. The vacuum exhaust unit 400 may generate plasma discharge selectively only inside the cell culture bag 100 by differentiating the pressure inside and outside of the cell culture bag 100.

In the conventional atmospheric plasma method, a high voltage of 7 kV or more must be applied in order to plasma process the inner surface of a three-dimensional shape such as a cell culture bag. However, when such a high voltage is applied, surface damage due to the emission of electrons having high energy have. In order to prevent damage to the surface, the plasma could not be processed to the inner surface when the voltage was lowered. Accordingly, when a low-temperature plasma is generated in a reduced pressure state by using a vacuum device according to the present invention, an inner surface can be surface-treated at a lower voltage, thereby preventing surface damage due to an electron stream.

The inside and outside pressures of the cell culture bag formed in a vacuum state using the vacuum exhaust unit 400 may be 50 mTorr to 50 Torr. If it exceeds 50 Torr, the low-temperature plasma discharge start voltage can not be sufficiently lowered, and if it is lower than 50 mTorr, the cost and energy required for decompression increase unnecessarily.

In addition, the inside and outside of the cell culture bag must be decompressed to different pressures. It is an object of the present invention to modify the inner surface to have desired characteristics by the surface plasma formed inside the cell culture bag, so that the low temperature plasma is selectively generated only inside the cell culture bag, not outside. The pressure difference between the inside and the outside of the cell culture bag may be 1 Torr or more and 20 Torr or less. If the pressure difference is less than 1 Torr, a low-temperature plasma may selectively occur not only inside the cell culture bag but also outside the cell culture bag. If the pressure difference exceeds 20 Torr, the cell culture bag may be damaged or deformed due to a large pressure difference.

1, the vacuum evacuation unit 400 includes a first evacuator 410 connected to the cell culture bag 100 for evacuating the interior of the cell culture bag 100 to a vacuum state, And a second vacuum evacuator 420 connected to the plasma processing module 300 for evacuating the outside of the cell culture bag 100 to a vacuum state. A first vacuum valve 411 and a second vacuum valve 421 are provided in the first vacuum exhaust apparatus 410 and the second vacuum exhaust apparatus 420, respectively. One of the ports of the cell culture bag 100 and the first vacuum evacuation device 410 are connected to each other through a first vacuum valve 411. When the first vacuum valve 411 is opened, The evacuation device 410 is operated to depressurize the interior of the cell culture bag 100 to a vacuum state and then to close the first vacuum valve 411 to maintain the vacuum state. After the surface treatment is completed, the first vacuum valve 411 can be opened to exhaust the remaining gas. The second vacuum evacuation device 420 is connected to the plasma processing module 300 and reduces the pressure of the outside of the cell culture bag to a vacuum state through the second vacuum valve 421. Referring to FIG. 2, the second vacuum exhaust apparatus 420 may also be driven up and down in association with the up-and-down driving portion. The inside and the outside of the cell culture bag 100 can be evacuated differently by using the first vacuum evacuation device 410 and the second vacuum evacuation device 420, . However, the structure of the vacuum exhaust unit 400 is not limited thereto. For example, the pressure of the inside and the outside of the cell culture bag 100 may be different through control of a valve in one vacuum exhaust unit.

The present invention also provides a method of surface treatment using a surface treatment apparatus for inner surface of a cell culture bag according to the present invention, the method comprising the steps of: (i) preparing a cell culture bag; (ii) decompressing the inside and outside of the cell culture bag to a vacuum state; (iii) introducing a reaction gas into the cell culture bag; (iv) applying a predetermined voltage to generate a surface plasma inside the cell culture bag; And (v) modifying the surface by forming a functional group from the reaction gas on the inner surface of the cell culture bag by the surface plasma; Wherein in step (ii), the pressure inside and outside of the cell culture bag is different, and in step (iv), a plasma discharge is selectively generated only inside the cell culture bag. A method for surface treatment of an inner surface is provided.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail in each of the steps of the surface treatment method of the inner surface of a cell culture bag according to the present invention.

First, prepare a cell culture bag.

The cell culture bag may be made of a material selected from the group consisting of polyethylene (PE), polyamide (PA), polystyrene (PS), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyvinyl alcohol (PVA), polyethylene terephthalate (PET), polypropylene (PP), and polyurethane (PU), but is not limited thereto.

Second, the inside and outside of the cell culture bag are evacuated to a vacuum state.

In the conventional atmospheric plasma method, a high voltage of 7 kV or more must be applied in order to plasma process the inner surface of a three-dimensional shape such as a cell culture bag. However, when such a high voltage is applied, surface damage due to the emission of electrons having high energy have. In order to prevent damage to the surface, the plasma could not be processed to the inner surface when the voltage was lowered. Accordingly, when a low-temperature plasma is generated in a reduced pressure state by using a vacuum device according to the present invention, an inner surface can be surface-treated at a lower voltage, thereby preventing surface damage due to an electron stream.

Third, the reaction gas is introduced into the cell culture bag.

The reaction gas in the above step may be any one selected from the group consisting of Ar, He, O 2 , N 2 , H 2 , and hydrocarbons, or a mixed gas thereof, but is not limited thereto. Gas may be drawn in. The type of reaction gas to be added depends on the material of the cell culture bag and the type of functional group to be produced.

In addition, the reaction gas may further include at least one of He and Ar for forming a uniform surface plasma. Preferably, helium is used. The helium is used as a gas causing plasma in the present invention as a gas effective to form a surface plasma that has a large plasma stream radius and flows along the surface. By selectively incorporating nitrogen, oxygen, or hydrogen in addition to helium, functional groups can be imparted to the inner surface of the cell culture bag as described below.

Fourth, a predetermined voltage is applied to generate a surface plasma inside the cell culture bag.

In the second step, if an AC voltage equal to or higher than the plasma discharge start voltage is applied to the electrode by the AC power source while the inside and the outside of the cell culture bag are depressurized, the reaction gas introduced into the cell culture bag, Causing a plasma discharge.

Fifth, a functional group is formed from the reaction gas on the inner surface of the cell culture bag by the surface plasma to modify the surface.

By the plasma generated in the fourth step, the reaction gas is excited and ionized to form reactive reactive species such as radicals. The reactive active species adsorbs on the inner surface of the cell culture bag while forming functional groups and modifying the surface.

The functional group may be selected from the group consisting of a carboxyl group, an amine group, a hydrocarbon group, and a hydroxy group, but is not limited thereto. For example, a carboxyl group or a hydroxy group is formed in an oxidizing gas atmosphere such as a carbon oxide or oxygen, and a hydrocarbon thin film may be formed in an amine group or hydrocarbon gas atmosphere in a nitriding gas atmosphere such as nitrogen.

The functional group formed from the reaction gas may be modified so that the inner surface of the cell culture bag is improved in cell adhesion or airtightness. In general, adherent cells are difficult to attach on hydrophobic surfaces, while adhesion on hydrophilic surfaces occurs well. When a carboxyl group or a hydroxy group is formed on the surface, the inner surface of the cell culture bag changes from hydrophobic to hydrophilic and can facilitate cell attachment. When the inner surface is surface-treated with hydrocarbons, the airtightness is improved and the gas permeability is reduced as the surface is hardened. In this case, the inflow of the external gas can be blocked, thereby improving the cell culture performance of the cell culture bag. In addition, when the surface is modified with a functional group formed by the low temperature plasma discharge of the present invention, lipophilicity, water repellency, dyeability and the like can be improved.

The present invention also provides a method of surface treatment using a surface treatment apparatus for inner surface of a cell culture bag according to the present invention, comprising the steps of: (a) preparing a cell culture bag; (b) depressurizing the inside and outside of the cell culture bag to a vacuum state; (c) introducing a first reaction gas into the cell culture bag; (d) applying a predetermined voltage to generate a surface plasma inside the cell culture bag; (e) forming a functional group from the first reaction gas on the inner surface of the cell culture bag by the surface plasma to modify the surface; (f) introducing a second reaction gas different from the first reaction gas in the step (c); (g) performing the steps (d) and (e); Wherein the cell culture bag has a pressure different from that of the cell culture bag in the step (b), and a plasma discharge is selectively generated only in the cell culture bag in the step (d) A method for surface treatment of an inner surface is provided.

In the steps (f) and (g), in order to form a functional group having a performance different from that of the functional group formed by the low-temperature plasma surface treatment with the first reaction gas according to the present invention, the low- . ≪ / RTI > For example, after deposition of the hydrocarbon thin film to enhance the airtightness, an oxygen plasma surface treatment may be additionally performed to increase hydrophilicity or cell adhesion. The steps (f) to (g) may be performed a plurality of times for additional surface treatment. The description of the remaining steps of the surface treatment method is the same as the surface treatment method of the inner surface of the cell culture bag according to the present invention described above.

The present invention also provides a cell culture bag having an inner surface modified using the surface treatment method according to the present invention. The cell culture bag according to the present invention is advantageous in that it can generate a low-temperature plasma at a low voltage compared to the conventional atmospheric pressure plasma method, and can easily form a surface without damaging the surface and providing various functions.

Hereinafter, examples of the present invention will be described. However, the scope of the present invention is not limited by the following examples.

[Example 1]

A cell culture bag made of polyethylene was prepared and the inner surface of the cell culture bag was modified using a surface treatment apparatus for the inner surface of the cell culture bag according to the present invention. The cell culture bag was connected to the plasma treatment module of the surface treatment apparatus, and then the inside and the outside of the cell culture bag were respectively evacuated to a vacuum state by using two vacuum evacuation apparatuses. The internal and external pressures were maintained at 300 mTorr and 20 Torr, respectively, so that the pressure difference was 20 Torr. Argon (Ar) gas was supplied as a reaction gas into the cell culture bag through a gas inlet module. The surface plasma discharge was performed by applying AC power of 40 kHz at a voltage of 3.0 kV for 30 seconds to form a surface plasma in the cell culture bag.

[Example 2]

The surface of the inner surface of the cell culture bag was treated in the same manner as in Example 1, except that the reaction gas was injected at a ratio of 1: 1 as a mixed gas of argon and oxygen.

[Example 3]

Except that the reaction gas was injected at a ratio of 1: 1 as a mixed gas of argon and hydrocarbon, and the surface plasma discharge was performed for 60 seconds. The procedure of Example 1 was repeated to prepare a hydrocarbon thin film Respectively.

[Example 4]

Example 3 was carried out under the same conditions as in Example 1 except that a cell culture bag having a hydrocarbon thin film formed on its inner surface was injected with a mixed gas of argon and oxygen at a ratio of 1: And the inner surface of the cell culture bag was surface-treated.

[Example 5]

Example 3 was carried out under the same conditions as in Example 1 except that a cell culture bag having a hydrocarbon thin film formed on its inner surface was injected with a mixed gas of argon and hydrogen at a ratio of 1: And the inner surface of the cell culture bag was surface-treated.

[Experimental Example 1]

The inner surface shape of the cell culture bag was observed using a scanning electron microscope (SEM) at a magnification of 50,000 times.

4 is an SEM image of the inner surface of a cell culture bag made of polyethylene without surface treatment.

5 to 7 are SEM images of inner surfaces of a cell culture bag subjected to a plasma surface treatment according to an embodiment of the present invention. FIG. 5 is a SEM image of the inner surface of a cell culture bag obtained by plasma-treating the surface of the cell using Ar gas according to Example 1 of the present invention. Compared with FIG. 4, it can be seen that the surface did not change significantly by the Ar plasma.

6 is an SEM image of the inner surface of a cell culture bag in which a hydrocarbon thin film is deposited by plasma treatment using hydrocarbon gas according to Example 3 of the present invention. Referring to FIG. 6, it can be seen that the roughness of the surface is better than that of FIG. 4, and it can be seen that a hydrocarbon thin film is formed.

FIG. 7 is a SEM image of the inner surface of a cell culture bag obtained by plasma-treating a hydrocarbon thin film according to Example 4 of the present invention using O 2 gas. Referring to FIG. 7, it is confirmed that a hydrocarbon functional group is formed by oxygen plasma while the hydrocarbon thin film is maintained.

[Experimental Example 2]

Fourier Transform Infrared Spectroscopy (FT-IR) analysis was performed to confirm the functionalization and chemical bonding of the inner surface of the cell culture bag made of polyethylene to the plasma surface treatment. The results are shown in Fig. 8 is an FT-IR spectrum of the inner surface of a cell culture bag surface-treated according to an embodiment of the present invention. Referring to Fig. 8, in the case of Example 1 in which the Ar plasma was treated, no carboxyl group (-COOH) and hydroxyl group (-OH) appeared on the surface. (-CH), a carboxyl group (-COOH) and a hydroxyl group (-OH) were formed in the case of Example 3, Example 4 and Example 5 after the hydrocarbon thin film was deposited.

[Experimental Example 3]

The X-ray photoelectron spectroscopy (XPS) surface analysis method was used to analyze the embodiments of the present invention to identify changes in the surface binding structure of the inner surface of the cell culture bag. 9 is a graph showing an XPS spectrum analysis result of the inner surface of a cell culture bag surface-treated according to an embodiment of the present invention. Referring to FIG. 9, it can be seen that the surface bonding structure varied in various ways depending on the kind of the plasma surface treatment.

10 is an XPS spectrum analysis result of a surface of a cell culture bag in which a hydrocarbon thin film is deposited according to Example 3 of the present invention. Referring to FIG. 10, the C-C bond and the C-H bond appeared in the polyethylene backbone, but the fraction of the C-H bond was increased by the deposition of the hydrocarbon thin film. And C-O and C = O bonds can be said to form a carboxyl group and a hydroxyl group on the surface.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

100: Cell culture bag
200: gas inlet module
The reaction gas supply part
211: Reaction gas tank
212: Gas flow control device (MFC)
213: first gas supply valve
214: second gas supply valve
300: plasma processing module
The electrode portion
311: metal electrode
312: Ceramic dielectric electrode
320: AC power source
400: Vacuum exhaust unit
410: First vacuum exhaust device
411: First vacuum valve
420: Second vacuum exhaust device
421: Second vacuum valve
500: Support

Claims (15)

In a surface treatment apparatus for an inner surface of a cell culture bag,
A cell culture bag 100;
A gas inlet module 200 connected to the cell culture bag 100 and having a function of supplying a reaction gas into the cell culture bag 100;
And a function of forming a functional group on the inner surface of the cell culture bag by discharging the reaction gas and plasmaizing the cell culture bag 100 with a function of enclosing the outside of the cell culture bag 100 to provide a closed space and generating plasma, A plasma processing module 300 including an electrode unit and an AC power unit 320 connected to the electrode unit and applying a voltage thereto;
A vacuum exhaust unit 400 connected to the cell culture bag 100 and the plasma processing module 300 to decompress the inside and outside of the cell culture bag 100 to a vacuum state; , ≪ / RTI >
Wherein the inner and outer pressures of the cell culture bag (100) are different from each other, so that a plasma discharge is selectively generated only in the cell culture bag (100).
The method according to claim 1,
Wherein the electrode unit comprises a metal electrode (311) and a ceramic dielectric electrode (312).
The method according to claim 1,
The vacuum exhaust unit 400 includes a first vacuum exhaust unit 410 connected to the cell culture bag 100 to decompress the inside of the cell culture bag 100 to a vacuum state, And
A second vacuum exhaust device 420 connected to the plasma processing module 300 to decompress the outside of the cell culture bag 100 to a vacuum state; Wherein the surface of the inner surface of the cell culture bag is treated with the surface treatment agent.
The method according to claim 1,
Wherein the functional group formed from the reaction gas is modified so that the inner surface of the cell culture bag is improved in cell adhesiveness or airtightness.
The method according to claim 1,
Wherein the functional group is any one or more selected from the group consisting of a carboxyl group, an amine group, a hydrocarbon group, and a hydroxy group.
The method according to claim 1,
Wherein the reaction gas is any one selected from the group consisting of He, Ar, O 2 , N 2 , H 2 , and hydrocarbons or a mixed gas thereof.
The method according to claim 1,
Wherein the inner and outer pressure of the cell culture bag is 50 mTorr to 50 Torr.
The method according to claim 1,
Wherein the pressure difference between the inside and the outside of the cell culture bag is 1 Torr or more and 20 Torr or less.
The method according to claim 1,
And the voltage applied from the AC power supply unit (320) is 1 to 5 kV.
The method according to claim 1,
The cell culture bag may be made of a material selected from the group consisting of polyethylene (PE), polyamide (PA), polystyrene (PS), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyvinyl alcohol (PVA), polyethylene terephthalate (PET), polypropylene (PP), polyurethane (PU), or a copolymer thereof.
. The method according to claim 1,
And a driving unit having a function of moving the plasma processing module 300 up and down.
The method according to claim 1,
Wherein the reaction gas supply unit comprises a gas flow rate controller (MFC) (212).
A surface treatment method using a surface treatment apparatus on the inner surface of a cell culture bag according to claim 1,
(i) preparing a cell culture bag;
(ii) decompressing the inside and outside of the cell culture bag to a vacuum state;
(iii) introducing a reaction gas into the cell culture bag;
(iv) applying a predetermined voltage to generate a surface plasma inside the cell culture bag;
(v) forming a functional group from the reaction gas on the inner surface of the cell culture bag by the surface plasma to modify the surface; , ≪ / RTI >
Wherein the inner and outer pressures of the cell culture bag are differentiated in the step (ii), and a plasma discharge is selectively generated only in the cell culture bag in the step (iv) .
A surface treatment method using a surface treatment apparatus on the inner surface of a cell culture bag according to claim 1,
(a) preparing a cell culture bag;
(b) depressurizing the inside and outside of the cell culture bag to a vacuum state;
(c) introducing a first reaction gas into the cell culture bag;
(d) applying a predetermined voltage to generate a surface plasma inside the cell culture bag;
(e) forming a functional group from the first reaction gas on the inner surface of the cell culture bag by the surface plasma to modify the surface;
(f) introducing a second reaction gas different from the first reaction gas in the step (c);
(g) performing the steps (d) and (e); , ≪ / RTI >
Wherein the inner and outer pressures of the cell culture bag are different from each other in step (b), and a plasma discharge is selectively generated only in the cell culture bag in step (d) .
The cell culture bag according to claim 13 or 14, wherein the inner surface is modified using the surface treatment method of the inner surface of the cell culture bag.
KR1020150168916A 2015-11-30 2015-11-30 A equipment of modifying a surface in a cell culture bag inner side and a surface modifying method therewith KR20170063015A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110016436A (en) * 2019-04-26 2019-07-16 北京康源嘉成生物科技有限公司 A kind of electrical environment biological culture system
WO2021256768A1 (en) * 2020-06-19 2021-12-23 주식회사 아모그린텍 Cell culture device
WO2021256769A1 (en) * 2020-06-19 2021-12-23 주식회사 아모그린텍 Cell culture device

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110016436A (en) * 2019-04-26 2019-07-16 北京康源嘉成生物科技有限公司 A kind of electrical environment biological culture system
WO2021256768A1 (en) * 2020-06-19 2021-12-23 주식회사 아모그린텍 Cell culture device
WO2021256769A1 (en) * 2020-06-19 2021-12-23 주식회사 아모그린텍 Cell culture device

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