KR101583159B1 - Cell culture container for producing large scale cell sheet, method for producing the container, system and method for harvesting cell sheet using the container - Google Patents

Abstract

The present invention relates to a cell culture container for producing a large scale cell sheet and a patterned sheet by radiating near infrared rays, a producing method thereof, a system for harvesting a cell sheet using the cell culture container and a harvesting method. The cell culture container can detach cells without enzyme treatment over a large area by using a conductive material as a support for cell culture, wherein the conductive material has heating characteristic when radiating near infrared rays, and can be used in a regenerative medicine field because cell sheets can be harvested without temporal and spatial constraints.

Description

Field of the Invention The present invention relates to a cell culture container for producing a large-sized cell sheet, a method for producing the same, a cell sheet harvesting system using the same, and a harvesting method }

The present invention relates to a thin film having a photothermal effect generated by irradiation with near infrared rays, specifically, a cell culture container capable of harvesting cell sheets freely from a large area to a patterned sheet by using a conductive material, , A cell sheet harvesting system using the same, and a harvesting method.

In general, cells are highly dependent on the fate of their differentiation by cell-to-extracellular matrix (ECM), including cell-to-cell and growth factors (Nakayama et al., Neurosci Res., 46, 241-249, 2003), which is known to be highly influenced by the well-known and instructive environment. Recently, bioengineering is emerging as a study on the interaction between cell environment and cell. It is not the regulation of hormones, growth factors, and serum contained in cell cultures, which are traditionally used to study or induce cell function, but the interaction of cells with cell- (Bauer S. et al., Acta Biomaterialia, 4, 1576-1582, 1985). In addition, the expression of the extracellular matrix, 2008; Guo L. et al., Biomaterials, 29, 23-32, 2008). For this, the development of biocompatibility materials and chemical surface modification are key factors.

In general, the enzyme known as trypsin is most widely used in the prior art for desorbing cells. Trypsin has a problem in that it interferes with cell interactions because it impairs chemically binding to cells attached to cell culture vessels. Therefore, when trypsin is used, the cells are desorbed into a single cell, which may result in problems of proliferation and degradation of the ability to multiply. In addition, since trypsin is treated entirely in a culture container, there are portions where it is difficult to take a desired cell partly.

As a result, many attempts have been made to obtain a cell sheet that is not a single cell in which intercellular interactions have been lost. Among the techniques that have been tried for the first time are cell sheets using thermosensitive polymers, (Paper: Teruo Okano et al, Polymers , 2012, 4, 1478-1498, Patent: US 2008-0131476-A1). The temperature-sensitive polymer (pNIPAAm), which is a core material of the technology developed by Okano's team, induces cell desorption by changing the surface characteristics of the polymer due to the phase change of the polymer according to the ambient temperature change. In the polymer, Therefore, it is mainly composed of gel, and has a structure sensitive to moisture. The biggest problem with thermo sensitive polymer based systems is that a large temperature difference (one hour or more at 4 ° C) should be applied to induce phase change, and this large temperature difference can have a negative effect on cells, Changes can be easily identified.

In order to solve the problem (concern for morphology change and detachment takes a long time) in the temperature sensitive polymer based system, copolymerization with various polymers (PEG, Alginate, polystyrene, polyurethane etc.) was attempted and the time required for desorption was shortened Concerns about low-temperature stimuli seemed to be resolved, but the preparation process for obtaining functional polymers became more complicated due to the increase in the synthesis step, which may affect the unit price rise in future prototypes (Tao Wang et al, Reactive & Functional Biosystems, Inc., Polymers , 2011, 71, 447-454, Andrzej Dworak et al., Applied Materials & Interfaces , 2013, 5, 2197-2207, Dan Liu et al., Biopolymers , 2013, 101, 58-65).

In addition, research has been conducted through electrical stimulation or ultrasonic stimulation by using a substance other than a copolymerized polymer. However, since metal materials (Au, Ag, or CNT) should be mainly used to generate electric stimulation and ultrasound, (L. Junge et al., Ultrasound) , which can be used to construct a system for generating ultrasound effects, in Med . & Amp; Biol ., 2003, 29, 1769-1776, Takao Sada et al, ACS Nano , 2011, 5, 4414-4421, Tatiana A. Kolesnikova et al, ACS Nano , 2012, 6, 9585-9595).

Therefore, it is required to develop a new technique that not only removes cells easily from a culture container, but also prevents cells from being damaged in this process, and can be desorbed only at a desired site.

Accordingly, an object of the present invention is to provide a cell culture container capable of harvesting a cell sheet without changing the morphology of cells, and harvesting a cell sheet having a free form from a large area through a developed technique, and a method for producing the cell culture container .

Another object of the present invention is to provide a cell sheet harvesting system using the cell culture container and a cell sheet harvesting method.

In order to achieve the above object, the present invention provides a substrate comprising: a substrate; And a support layer formed on the base material and containing a conductive material absorbing heat in the near infrared region to generate heat.

In the present invention, the conductive material (near-infrared heat generating component) may include at least one selected from the group consisting of a conductive polymer, a carbon nanotube, a graphene, a metal, and a metal oxide.

In the present invention, the conductive polymer may include at least one monomer selected from a heterocyclic compound represented by the following formula (1) and aniline; And a polymer or copolymer made from a composition containing at least one additive selected from a pyridine-based compound, an imidazole-based compound, and a polyethylenic compound.

[Chemical Formula 1]

In this formula,

X represents NH, O, S, Se or Te;

R ₁ and R ₂ are the same or as to form the other or connected to each other rings, a hydrogen _{atom, - (CH 2) ℓ -O-} (CH 2) m - (CF 2) n - (CR 7 R 8 _{) k - (CH 2) d} -Z,

-O-CH (R ₃ ) -CH (R ₄ ) -O-, or -O-CH ₂ -C (R ₅ ) (R ₆ ) -CH ₂ -O-;

R _3, R _4, R ₅ and R ₆ are the same as or different from each other, a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, a halogenated alkyl group having 1 to _{10, - (CH 2) d} -Z, - (CH 2) _{ℓ -O- (CH 2) m -} (CF 2) n - (CR 7 R 8) k - (CH 2) d -Z, or

이고;

ego;

R ₇ and R ₈ are the same or different from each other and represent hydrogen, an alkyl group having 1 to 5 carbon atoms, or - (CH ₂ ) _d -Z;

Z is a methacrylate group or an acrylate group;

m is an integer of 0 to 3, n is an integer of 0 to 5, k is an integer of 0 to 4, a is an integer of 0 to 2, b is an integer of 0 to 7, and d is an integer of 0 to 2 have.

In the present invention, the heterocyclic compound of Formula 1 may be at least one selected from the following Formulas 1a to 1m.

[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

≪ RTI ID = 0.0 &

[Formula 1e]

(1f)

[Formula 1g]

[Chemical Formula 1h]

[Formula 1i]

[Chemical Formula 1j]

[Chemical Formula 1k]

≪ EMI ID =

[Formula 1m]

In the present invention, the pyridine compound may be at least one compound selected from pyridine, halogenated pyridine, bipyridine and pyridine dicarboxylic acid, and the imidazole compound may be at least one compound selected from imidazole and alkylimidazole, The compound and the imidazole-based compound may each be contained in an amount of 0.1 to 20% by weight, independently of the total weight of the composition.

In the present invention, the polyethylene-based compound may be at least one polyethylene-based oligomer selected from polyethylene glycol oligomers, polyethylene oxide oligomers and polyethylene glycol / polypropylene glycol oligomers. The polyethylene- 0.1 to 50% by weight.

In the present invention, the weight average molecular weight of the conductive polymer may be 1,000 to 1,000,000 Da.

In the present invention, the metal of the metal and the metal oxide may be at least one selected from the group consisting of gold, silver, copper, magnesium, strontium, zinc, aluminum and arsenic.

In the present invention, the support layer may be oxidized / reduced, and the support layer may be patterned.

The substrate in the present invention may include at least one selected from polycarbonate, polypropylene, polyethylene, polystyrene, polyethylene terephthalate, polydimethylsiloxane, polylactic-coglycolic acid, polylactic acid, polycaprolactone, glass, have.

The cell culture container according to the present invention may further comprise a surface treatment layer formed on the support layer and containing an extracellular matrix material, and the extracellular matrix material may be one kind selected from collagen, gelatin, laminin and fibronectin Or more.

The cell attachment rate of the cell culture container according to the present invention may be 50-1000 cells / mm 2, and the cell desorption range may be 0.1-100 cm 2.

The present invention also provides a method for producing a cell culture container, comprising the step of forming the above-mentioned support layer on a substrate.

In the method for producing a cell culture container according to the present invention, the support layer may be formed by synthesizing a conductive polymer through a polymerization method selected from among electrochemical polymerization, solution polymerization, vapor phase polymerization, and chemical polymerization; The support layer may also be formed by coating or printing a solution for solution polymerization by a method selected from spin coating, spray coating, dip coating, roller coating, bar coating, and inkjet printing; On the other hand, a patterned support layer can be formed by arranging a morphology frame on a substrate and coating a solution polymerization solution in the morphology frame.

The present invention also relates to the aforementioned cell culture container; And a cell sheet harvesting system including a near infrared ray irradiating apparatus.

The present invention also relates to a method for culturing a cell, And irradiating the cultured cells with near-infrared light to desorb the cells in a cell sheet form.

In the cell sheet harvesting method according to the present invention, a patterned cell sheet can be harvested by placing a pattern mask in a cell culture container and irradiating near infrared rays.

According to the present invention, by using a cell culture container including a support layer containing a conductive material manufactured through a simple process and optionally surface-treated with an extracellular matrix material, it is possible to easily form various shapes Can be harvested and utilized as a cell therapy agent tailored to a damaged tissue in the field of regenerative medicine. It is expected to be a fundamental healing system for constructing a patient-tailored treatment system because it can harvest cell sheets of various shapes with short harvest time as compared with cell culture vessel systems for existing cell sheets.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing cell adhesion to a cell culture container according to the present invention and a cell culture container according to the present invention.
Fig. 2 is a photograph showing cell adhesion according to the surface modification of the support layer.
3 schematically shows a method for harvesting a cell sheet by irradiating near infrared rays according to the present invention.
Figure 4 is an optical micrograph of a cell sheet harvested in an example.
Figure 5 is a photograph showing the biological characteristics of the cell sheet harvested in the examples.
Figure 6 is a photograph of a support patterned in various shapes.

Hereinafter, the present invention will be described in detail.

Cell culture container

The cell culture container according to the present invention may comprise a substrate and a support layer.

The material of the substrate may be selected from the group consisting of polycarbonate, polypropylene, polyethylene, polystylene, polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), polylactic-co polyglycolic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL), copolymers thereof, glass, and gauze.

The shape and the shape of the substrate are not particularly limited, and for example, a petri dish, a tube, a flask, or the like can be usually used for cell culture.

The support layer may be formed on the substrate and may contain a conductive material absorbing and generating heat in the near infrared region.

The conductive material (near-infrared heat generating component) may include at least one selected from the group consisting of a conductive polymer, a carbon nanotube, a graphene, a metal, and a metal oxide, and may be formed into a thin film by singly or in combination.

The support layer may preferably be composed of a conductive polymer thin film, and may include other materials or other layers as required.

The support layer may preferably be formed on the inner surface of the substrate. The thickness of the support layer is not particularly limited, and may be, for example, 10 nm to 1 mm. If the thickness of the support layer is too small, the thin film can not be easily formed and the photothermal effect may be deteriorated. If it is too thick, the time for transferring heat due to the photothermal phenomenon may be prolonged and the cell desorption time may be prolonged.

The conductive polymer may use a conductive polymer having a photothermal effect that absorbs light in the near infrared region to generate heat. Specifically, the conductive polymer scaffold utilizes the photothermal characteristics of a conductive polymer that absorbs near-infrared rays and generates heat by radiating light energy into heat energy. When the conductive polymer scaffold is used as a support for cell culture, The cell sheet can be easily desorbed without damage to the cell wall or the cell wall protein due to the conventional trypsin treatment, and the surface on which the cells are desorbed from the conductive polymer can be maintained without changing the physical properties, There are features that can be used repeatedly in a series of processes.

The conductive polymer may be a polymer or copolymer made from a composition containing conductive monomers and additives.

As the conductive monomer, at least one selected from a heterocyclic compound represented by the following general formula (1) and aniline can be used.

[Chemical Formula 1]

In this formula,

X represents NH, O, S, Se or Te;

R ₁ and R ₂ are the same or as to form the other or connected to each other rings, a hydrogen _{atom, - (CH 2) ℓ -O-} (CH 2) m - (CF 2) n - (CR 7 R 8 _{) k - (CH 2) d} -Z,

-O-CH (R ₃ ) -CH (R ₄ ) -O-, or -O-CH ₂ -C (R ₅ ) (R ₆ ) -CH ₂ -O-;

R _3, R _4, R ₅ and R ₆ are the same as or different from each other, a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, a halogenated alkyl group having 1 to _{10, - (CH 2) d} -Z, - (CH 2) _{ℓ -O- (CH 2) m -} (CF 2) n - (CR 7 R 8) k - (CH 2) d -Z, or

이고;

ego;

R ₇ and R ₈ are the same or different from each other and represent hydrogen, an alkyl group having 1 to 5 carbon atoms, or - (CH ₂ ) _d -Z;

Z is a methacrylate group or an acrylate group;

m is an integer of 0 to 3, n is an integer of 0 to 5, k is an integer of 0 to 4, a is an integer of 0 to 2, b is an integer of 0 to 7, and d is an integer of 0 to 2 have.

Preferably, the heterocyclic compound of Formula 1 may be at least one selected from the following Formulas 1a to 1m.

[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

≪ RTI ID = 0.0 &

[Formula 1e]

(1f)

[Formula 1g]

[Chemical Formula 1h]

[Formula 1i]

[Chemical Formula 1j]

[Chemical Formula 1k]

≪ EMI ID =

[Formula 1m]

The conductive polymer may be prepared by solution polymerization, electropolymerization using electricity [Macromolecular Research, 17, 791-796, 2009], vapor polymerization [Macromolecules, 43, 2322-2327, 2010] using a heterocyclic compound and a conventional catalyst ], Solution coating polymerization [Advanced Materials, 23, 4168-4173, 2011], emulsion polymerization in water phase and the like.

The content of the conductive monomer may be 0.1 to 50% by weight, preferably 5 to 30% by weight based on the total weight of the composition including the solvent. The content of the conductive monomer affects the viscosity of the polymerization solution. If the amount is too low, the viscosity is very low and the polymerization rate is rapidly accelerated, so that it is difficult to control the reaction. If too much, the viscosity is extremely high and the polymerization temperature must be increased. It is difficult to complete the polymerization.

In addition, the conductive polymer can be produced through a polymer produced through polymerization of an aniline and / or a heterocyclic compound containing an additive. The additives that can control the photothermal properties of the conductive polymer by controlling the polymerization rate and the polymer arrangement in the polymerization of the conductive polymer include a pyridine compound, an imidazole compound, and / or a polyethylene-based compound, which is a hydrophilic substance, Polyethylene copolymer.

As the additive, at least one selected from a pyridine-based compound and a polyethylenic compound may be used, and two or more kinds of them may be used in combination. The pyridine-based compound, the imidazole-based compound, and the polyethylenic-based compound, which are additives, can be finally included in the structure of the conductive polymer, that is, they can function as the second monomer.

The pyridine compound is not particularly limited and includes, for example, halogenated pyridine such as pyridine, 4-bromopyridine, 2,2-bipyridine, pyridine-2,6-dicarboxylic acid ) Can be used. The pyridine-based compound may be contained in an amount of 0.1 to 20% by weight, preferably 1 to 5% by weight based on the total weight of the composition including the solvent. If the amount of the pyridine compound added is too small, the polymerization rate is rapidly increased, so that the arrangement of the polymerized polymer is not constant and it is very difficult to control the surface properties. If too much, the polymerization rate is remarkably lowered and polymerization with the polymer may not be completed .

The imidazole-based compound is not particularly limited, and for example, at least one selected from imidazole, alkylimidazole (2-ethyl-4-methylimidazole, etc.) can be used. The imidazole-based compound may be contained in an amount of 0.1 to 20% by weight, preferably 1 to 5% by weight, based on the total weight of the composition including (excluding) the solvent. If the addition amount of the imidazole-based compound is too small, the polymerization rate rapidly increases, so the arrangement of the polymerized polymer is not constant and it is very difficult to control the surface properties. If too much, the polymerization rate is remarkably low. have.

The polyethylene-based compound is not particularly limited and includes, for example, at least one polyethylene selected from polyethylene glycol (PEG) oligomer, polyethylene oxide (PEO) oligomer, polyethylene glycol / polypropylene glycol (PEPG) oligomer Based oligomers can be used. The polyethylene-based compound may be contained in an amount of 0.1 to 50% by weight, preferably 5 to 30% by weight based on the total weight of the composition including (excluding) the solvent. If the addition amount of the polyethylene-based compound is too small or too large, the arrangement of the polymerized polymer is not constant, and the surface characteristics are different, so that it is difficult to control.

The conductive polymer scaffold containing the additives can control the surface characteristics of the polymer scaffold to be polymerized, thereby improving cell adhesion and proliferation. As shown in Fig. 1, it can be confirmed that the cell adhesion is significantly increased in the conductive polymer scaffold in which the additives are polymerized together, compared with the general cell culture container (TCP).

Therefore, the conductive polymer thin film having absorbance in the near infrared region is excellent in cell adhesion rate, and cell attachment and propagation are possible without forming a separate cell adhesion layer in the cell culture area.

The composition for polymerization may contain, in addition to the above monomers and additives, a polymerization initiator and a solvent. The polymerization initiator is not particularly limited, and conventional initiators can be used. The solvent is not particularly limited, and for example, butanol, isopropanol, n-Bu ₂ NClO ₄ , ethanol, methylene chloride and the like can be used.

The weight average molecular weight (Mw) of the conductive polymer is not particularly limited, but is preferably 1,000 to 1,000,000 Da. Since the molecular weight is directly related to the physical properties of the polymerized polymer, if it is too small, it is difficult to obtain a thermoelectric property because the polymerization reaction is not carried out. If it is too large, the surface properties of the polymer are changed and formation of cell culture conditions is very difficult.

In addition to the conductive polymer, carbon nanotubes (CNTs), carbon nanotubes (CNTs), and the like can be used as the near infrared ray generating component (conductive substance). Graphene; Gold, silver, copper, magnesium, strontium, zinc, aluminum, and arsenic; Metal oxides such as gold, silver, copper, magnesium, strontium, zinc, aluminum and arsenic; And / or a substance such as a near infrared absorbing compound or the like.

The support layer may be oxidized / reduced, thereby enhancing cell adhesion and proliferation.

The support layer may be patterned, so that various types of patterned cell sheets can be harvested. In order to harvest cell sheets with various shapes, for example, a patterned support based on conductive polymers for cell culture with absorbance in the near infrared region can be used. Such a patterned support for cell culture can be used for cell culture capable of forming liver, lung, heart, muscle and the like having a unique cell pattern, thereby efficiently arranging the cells. Since the patterned support for cell culture uses a polymer scaffold as a cell scaffold, it is possible to harvest cell-shaped cell clusters without special enzymes and chemical reagents when irradiated with near infrared rays. In the cell culture area where near infrared rays are not irradiated, Can be cultured.

The cell culture container according to the present invention may further include a surface treatment layer. The surface treatment layer may be formed on the support layer, specifically on the second surface opposite to the first surface of the support layer in contact with the substrate.

The surface treatment layer may include an extracellular matrix material, and may include other materials as needed. As the extracellular matrix material, at least one selected from collagen, gelatin, laminin, and fibronectin can be used. The surface treatment layer is formed by forming an electric charge on the surface of the support through oxygen plasma treatment and then immersing the support in the extracellular matrix solution for about 1 ± 0.5 hours to form a surface with the surface charge on the surface of the extracellular matrix, And then adsorbing the extracellular matrix material to the supernatant. The thickness of the surface treatment layer is not particularly limited, and may be, for example, 10 nm to 1 mm.

The surface treatment layer is a layer formed on the support layer formed on the substrate. For example, when the support is a polymer scaffold, the surface treatment layer can prevent direct contact between the cultured cells and the conjugated polymer. Specifically, the interaction between the cell and the conductive polymer is controlled so that the thermal stimulus generated through the photothermal effect is selectively concentrated on the extracellular matrix, so that the conductive polymer scaffold including the cell or the cell can be desorbed in a large sheet form Help.

Thus, a sheet having improved cell characteristics can be harvested through application of a specific protein onto a thin film of conductive material. In order to improve such characteristics, when a specific extracellular matrix that affects cell-support interactions and cell-cell interactions is used, as shown in the fourth image of FIG. 2, extracellular matrix image, The adhesion rate can be increased. In the material, various extracellular substrates such as collagen, gelatin, laminin, fibronectin and the like can be used as materials relating to cell adhesion.

The cell attachment ratio of the cell culture container according to the present invention may be 50 to 1000 cells / mm 2, preferably 150 to 1000 cells / mm 2, more preferably 200 to 1000 cells / mm 2.

The cell desorption range of the cell culture container according to the present invention may be 0.1 to 100 cm 2, preferably 0.4 to 50 cm 2, more preferably 0.7 to 10 cm 2.

As described above, when the cell culture container according to the present invention is used, a large-sized cell sheet can be harvested because a pyridine-based compound and a hydrophilic polyethylene-based compound are added when synthesizing a conductive substance, It is possible to remarkably increase the cell attachment ratio and the intercellular attraction force.

Cell culture container manufacturing method

The method for producing a cell culture container according to the present invention may include the step of forming the above-mentioned support layer on a substrate.

In the case of the conductive polymer thin film, the support layer can be formed by synthesizing a conductive polymer through a polymerization method selected from among electrochemical polymerization, solution polymerization, vapor phase polymerization, and chemical polymerization. Specifically, in order to form a conductive material support in a general cell culture container, it can be formed through electrochemical polymerization on an electrode substrate on which a conductive film is formed, and can be formed by various methods such as solution polymerization, vapor phase polymerization, Can be introduced.

 The support layer may be formed by coating or printing a solution of a solution polymerization solution in a method selected from a spin coating, a spray coating, a dip coating, a roller coating, a bar coating and an ink jet printing method in the case of the conductive polymer thin film have. That is, since the solution process can be performed through solution polymerization, it can be applied to spin coating, spray coating, immersion coating, roller coating, bar coating, inkjet printing and the like, and thus it is easy to harvest a cell sheet having various patterns.

When the support layer is composed of carbon nanotubes (CNT), graphene, a single metal, a metal oxide, or other near-infrared absorbing compound in addition to the conductive polymer, it can be formed by a conventional deposition or coating method.

In addition, the support layer can be oxidized / reduced, thereby improving cell attachment and proliferation as compared to a conventional cell culture container. The coral / reduction treatment method aims at controlling the doping state of the conductive thin film. First, the conductive thin film is put into the electrolyte solution and the voltage in the range of 1.5 V to -1.5 V is applied to the anode 10 times, and when the conductive thin film is in the reduced state, it is stopped for 2 seconds and 10 seconds, then the power is removed, and the resultant is washed with pure solvent and dried. In the above method, as the applied voltage is closer to -1.5 V, the doping state of the conductive thin film is transferred to the reduced state, so that the adhesion and proliferation of cells are increased like the oxidation / reduction image shown in the third photograph of FIG. Lt; RTI ID = 0.0 > cell < / RTI > Cells that have been fully cultured in a reduced state conductive thin film support can be harvested into cell clusters in a sheet form and the entirely desorbed cell sheet can be present in the culture while retaining the sheet shape after delivery to a new culture vessel.

In addition, a patterned support layer can be formed by placing a morphology frame on a substrate and coating the conductive thin film within the morphology frame. In order to obtain cell sheets of various shapes, the conductive thin film support layer may be formed on the surface of the culture container substrate together with a shape frame of a desired shape. For example, in the case of the solution coating polymerization method, the coating step can be carried out after fixing the desired shape of the mold frame on the surface of the container and pouring the solution before injecting the solution for coating into the container. The morphology frame is formed by embossing a thin film having a thickness of less than 200 탆 in a desired shape and applying it in a container, and optionally, a material (collagen, gelatin, laminin, fibronectin, etc. extracellular matrix) Is applied one more time, and the used pattern frame is removed, a desired pattern can be formed as shown in FIG. Since all the work for pattern formation can be carried out within 30 minutes, it can be applied quickly and easily compared with the photolithography generally used for patterning the existing polymer.

Cell sheet harvesting system

The cell sheet harvesting system according to the present invention comprises the above cell culture container; And a near infrared ray irradiation apparatus.

The cell culture container is a conductive thin film support layer formed on the inner surface of a general cell culture container and having a photothermal effect; And optionally a surface treatment layer formed on the support layer and for enhancing the biological properties of the cells. The cell culture container is a cell culture container prior to harvesting the cell sheet, and may have the same characteristics as a general cell culture container that induces cell proliferation and differentiation.

The near-infrared ray irradiation apparatus is a device for irradiating near-infrared rays to generate a photothermal effect of a conductive material, and a conventional near-infrared laser irradiation apparatus having a light source, a laser controller, a collimator, a shutter, .

The near-infrared rays may have a wavelength range of 700 nm to 3 占 퐉. Near infrared rays are preferably laser beams. The beam irradiation range is not particularly limited, and may be, for example, 0.5 to 100 cm 2, preferably 0.79 to 79 cm 2. The irradiation time is not particularly limited and can be, for example, from 1 minute to 1 hour. The irradiation intensity is not particularly limited, and may be, for example, 0.1 to 10 W. [ The irradiation dose is not particularly limited and may be, for example, 1 to 1000 J (or W · s). Under these conditions, a cell sheet having a minimum diameter of 10 mu m or more can be harvested.

The near-infrared rays can be irradiated freely from above and below the conductive thin film support on which the cells have been cultured, and preferably from the bottom.

The temperature difference occurring before and after the near-infrared irradiation may be 0 to 40 占 폚, preferably 1 to 20 占 폚 for 1 to 10 minutes.

The cells that can be cultured and harvested in the cell harvesting system according to the present invention are not particularly limited and may include cells derived from various tissues / organs such as fat, bone marrow, breast, cord blood, blood, liver, Stem cells or adult tissue cells.

The cell harvesting system according to the present invention can harvest cell sheets of various shapes with short harvest time as compared with the cell culture vessel system for existing cell sheets, so that it can be utilized as a fundamental healing system necessary for constructing a customized treatment system for damaged areas It is possible.

Cell sheet harvesting method

The method for harvesting a cell sheet according to the present invention comprises the steps of: culturing cells in the cell culture container; And irradiating the cultured cells with near-infrared light to desorb the cells in a cell sheet form.

After cells are cultured in a cell culture container, cells in culture can be harvested in a sheet form by irradiating the cell culture container with a near-infrared light ray. The principle of manufacturing the cell sheet through the conductive thin film support is basically a The temperature difference due to the photothermal effect generated from the conductive substance hydrolyzes the extracellular matrix existing between the cell and the surface of the supporter, and thus the interaction between the cell and the supporter surface is weakened and the cell sheet can be desorbed naturally.

In addition, the patterned cell sheet can be harvested by placing a pattern mask in a cell culture container and irradiating near infrared rays. In addition to the method of patterning the conductive thin film support as described above, variously patterned cell sheets can be produced according to the type and condition of near infrared irradiation. Specifically, when a metal mask patterned in a desired shape is placed on a container before irradiating the conductive thin film support on which the cells have been cultivated and near infrared rays are irradiated, the same cell sheet as the square pattern of FIG. 5 is obtained have. A metal mask patterned in various shapes such as circles, squares, triangles, etc. in the beam diameter range described above can be introduced, and the size of one pattern can be, for example, 10 μm to 50 cm.

According to another embodiment of the present invention, not only the desorption of the cell sheet composed of cells only in the conductive thin film support in which the cells are cultured, but also the separation of the support and the cells together according to the material and / or the shape of the container substrate and / . The conductive thin film support may have various forms depending on the type of the container substrate. (PDMS), a biocompatible polyethylene coglycol-based compound (PLGA, PLA, PCL, etc.), a wound healing gauze (cotton, rayon , Synthetic fibers, etc.), it is possible to produce various types of cell sheets cultured on a conductive thin film support formed on a flexible substrate, and to harvest not only cells but also sheets containing cells together with a support. The reason why the cells and the supporter are harvested together is that the supporter formed in the flexible substrate is cultured together with the cells in a general culture vessel, so that the suppository can be taken out in a patch form together with the formed cell sheet and used for wound. Thus, when the supporter and the cell are harvested together, the skin-like tissue is exposed to the air, so that when the wound healing is carried out with the healing system covered with the sealable material such as a cane, The supporter maintains the cell sheet and simultaneously sutures the wound, so that it is possible to prevent the secondary infection from the outside.

According to another embodiment of the present invention, in addition to the cell sheet harvesting method described above, the cell sheet can be harvested utilizing an extracellular matrix-based stamp (a tool for delivering the harvested cell sheet). The stamp utilizes the interaction between the support and the cell, and it is possible to manufacture various cell sheets by controlling the interaction between the cell and the stamp surface comprising the extracellular matrix according to the photothermal effect by the near-infrared rays and the application time of the stamp. As the extracellular matrix of the stamp, various extracellular matrices such as collagen, gelatin, laminin, and fibronectin can be used, and there is no particular limitation.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are for illustrating the present invention only, and the scope of the present invention is not limited thereto.

The reagents and solvents used in the following examples were purchased and used from Aldrich (USA) and Invitrogen (USA) unless otherwise noted.

Example  1: Human adipose-derived adult stem cell culture

Human fat was obtained from the consensus of patients approved by the Severance Hospital Clinical Trials Board (IRB) and this study was approved by the Severance Hospital Bioethics Review Board. The tissues obtained from human fat were solidified and cut or cut through surgical scissors, diluted 1: 5 in PBS (phosphate buffer saline) buffer solution, transferred to a 50 mL centrifuge tube and centrifuged at rpm for 10 minutes. After centrifugation, the supernatant was discarded, and fresh PBS buffer solution was poured onto the precipitated tissue on a filter for 100 쨉 m to filter only the stem cells. The filtered cells were centrifuged one more time and cells were harvested and suspended in culture medium (DMEM (low glucose) + 1% P / S + 10% FBS) and then cultured in 100 mm cell culture dishes. After the cells were cultured in a CO ₂ incubator for 1 day, the supernatant was removed, and the cells adhered to the bottom of the culture container were replenished with the same culture medium as that used for the initial culture, and were allowed to proliferate for one week. Adult-derived adult stem cells were cultured and maintained in the above manner.

Example  2: Fabrication of Conductive Polymer Support for Cell Sheet Culture

Impurities were removed from the surface of the cell culture container made of polystyrene by using a nitrogen gun, and a conductive polymer scaffold layer having absorbance in the near infrared region was formed on the surface of the container. Table 1 shows the composition and characteristics of the solution for the formation of the thin film of the conductive polymer scaffold. In Table 1, Constant potential (1.1 V / 0.02 C cm ^-2 ) was used in Examples 1-13, and solution coating polymerization was used in all of the other Examples. The electroconductive polymer thin film formed on the substrate confirmed the degree of cell adhesion according to the embodiment of Table 2. Table 2 shows the cell adhesion rate according to the thin film of the polymer scaffold The polymerization and the surface modification were performed singly or simultaneously. In order to remove unreacted substances and impurities remaining in the polymer scaffold, the scaffold was rinsed with an anhydrous alcohol solution and then subjected to alcohol treatment repeatedly for sterilization treatment. The prepared conductive polymer scaffold was sterilized, The cell adhesion ratio of the conductive polymer scaffold prepared in Example 2-7 was highest in the polymerization coating conditions It was confirmed through analysis.

Example Monomer Additive (wt%) Thickness (nm) Conductivity (S / cm) 1-1 Formula 1a Pyridine (1.34) 230 357 1-2 Formula 1a Pyridine (2.67) 160 1060 1-3 Formula 1a PEO (9.1) 252 568 1-4 Formula 1a PEPG (16.6) 225 874 1-5 Formula 1a PEPG (23.1) 183 1015 1-6 Formula 1a Pyridine (1.22) /
PEPG (9.0) 193 1031 1-7 Formula 1a Pyridine (1.12) /
PEPG (16.5) 122 1362 1-8 Formula 1a Pyridine (1.03) /
PEPG (22.8) 88 1253 1-9 Formula 1a Imidazole (2) 118 154 1-10 Formula 1a 2-ethyl-4-methyl
Imidazole (2) 120 420 1-11 1d PEG (16) 150 12 1-12 1 g Pyridine (2.26) 120 1.5 x 10 ^-3 1-13 1l - 130 335 1-14 1m Pyridine (2.26) 130 5.6 x 10 ^-5

Example Pyridine series
Compound (wt%) Polyethylene system
Compound (wt%) thickness
(nm) conductivity
(S / cm) Surface modification Cell adhesion rate
(cells / mm 2) 2-1 Pyridine (1.34) PEPG (0) 230 357 No treatment 55 2-2 Pyridine (2.67) PEPG (0) 160 1060 No treatment 60 2-3 Pyridine (0) PEO (16.6) 252 568 No treatment 75 2-4 Pyridine (0) PEPG (16.6) 225 874 No treatment 150 2-5 Pyridine (0) PEPG (23.1) 183 1015 Oxidation / reduction
process 175 2-6 Pyridine (1.22) PEPG (9.0) 193 1031 Extracellular matrix
Processing (ECM) 200 2-7 Pyridine (1.12) PEPG (16.5) 122 1362 Extracellular matrix
Processing (ECM) 350 2-8 Pyridine (1.03) PEPG (22.8) 88 1253 Extracellular matrix
Processing (ECM) 300

Example  3: Cell sheet harvest from conductive polymer scaffold

Human adult skin derived adult stem cells were cultured in a conductive polymer scaffold. Three days after the cell culture, temperature changes accompanying and desorbed cell desorption range were confirmed by changing irradiation conditions of the near-infrared light ray according to the conditions described in Table 3. [ Also, under the same irradiation range conditions, the kind of the container substrate was changed as in Example 3-4, and the temperature of the irradiated region and the area of the desorbed cell sheet were confirmed. Table 3 shows the cell sheet harvesting conditions and results according to the near-infrared irradiation conditions.

Example Coated substrate Survey strength
(W) Dose
(J or W · s) Temperature change
(° C) Cell desorption
Range (㎠) cell
Survival rate (%) 3-1 Petri dish
(polystyrene) 1.0 300 34 0.13 89 3-2 Petri dish
(polystyrene) 1.5 450 37 0.95 96 3-3 Petri dish
(polystyrene) 2.0 600 40 1.33 95 3-4 PET film 1.5 450 34 0.89 97

Example  4: Cell sheet harvesting by supporter type

A cell culture container made of polystyrene was used to remove impurities on the surface by using a nitrogen gun, and a graphene thin film, a gold thin film, a single-walled carbon nanotube (SWNTs) support layer having absorbance in the near- . Human adult stem cells were cultured in a conductive thin film support. Three days after the cell culture, the range of desorption of the cell sheets harvested by irradiation under the same conditions of near infrared rays under the conditions described in Table 4, and the temperature changes occurred were confirmed.

Example Support type Thin film thickness
(nm) Surface roughness
(nm) Temperature change
(° C) Cell desorption
Range (㎠) cell
Survival rate (%) 4-1 Graphene thin film 300 107 47 1.21 63 4-2 Gold thin film 20 2.7 55 1.45 17 4-3 conductivity
Polymer thin film 230 38.4 40 1.33 95

Example  5: Extracellular matrix  Treated cell sheet harvest

The surface of the conductive polymer scaffold of Example 3 was treated with an extracellular matrix to improve the cell characteristics, and human skin-derived adult cells were cultured. Three days after the cell culture, cell sheet desorption experiments were carried out according to the conditions shown in Table 5. [ Table 5 shows cell sheet harvesting conditions and results according to extracellular matrix treatment. In Example 5-1, cell desorption could not be induced because the conductive polymer scaffold was not present. In Examples 5-2 to 5-4, different types of cell sheets could be harvested. In Example 5-2 in which the extracellular matrix was not treated, the cell shape in the harvested cell sheet was desorbed, Were observed. On the other hand, in Examples 5-3 and 5-4, the cell sheet was harvested as it was in the cultured cell type, and the cell survival rate of each cell was found to be 95% or more, Respectively.

Example coating
Condition Cell culture photograph Desorbed cell sheet Cell morphology,
Survival rate (%) 5-1 Normal
culture
Vessel
(TCP)

Detachable radish 5-2 conductivity
Polymer
Support

rectangle
(96) 5-3 Collagen
Treated
conductivity
Polymer
Support

Fiber type
(95) 5-4 Laminin
Treated
conductivity
Polymer
Support

Fiber type
(95)

Example  6: Cell sheet harvesting in various other forms

Human skin-derived adult cells were cultured in a conductive polymer scaffold. Three days after the cell culture, various types of cell sheets were prepared by irradiating near infrared rays according to the conditions shown in Table 6. Under the same irradiation range conditions, the shape of the container substrate was varied to determine the temperature change in the irradiated area and the area of the cell sheet to be desorbed. Table 6 shows various cell sheet harvesting conditions and results.

Example Coated substrate Research
century
(W) Dose
(J or
W · s) Temperature
change
(° C) Cell desorption
range
(㎠) Cell sheet
Harvest method cell
Survival rate
(%) 6-1 Petri dish
(polystyrene) 1.5 450 37 0.95 Cells only
harvesting 96 6-2 PLGA film 1.7 510 39 0.99 The support
Harvest together 96 6-3 gauze 1.7 510 35 0.93 The support
Harvest together 95 6-4 PET film 1.5 450 34 0.89 The support
Harvest together 97

Example  7: Cell sheet analysis

The moment of harvesting the cell sheet through Example 3 was photographed in real time through an optical microscope. When the near infrared ray was irradiated, the end of the cell sheet was gradually desorbed from one corner of the container to the other, and some desorbed cell sheets floated in the culture vessel, and almost all of the sheets were harvested at the completion of the irradiation , And the shape still after the transfer to the new cell culture container was confirmed through Fig. In FIG. 5, when the cell morphology was observed at high magnification through an optical microscope, the morphology of the cells observed on the first day of cell culture in the prepared culture container maintained the inherent morphology after harvesting, and the cell survival rate .

Example  8: Fabrication of cell sheet pattern

In order to obtain cell sheets of various shapes, the shape of the desired shape was applied together with the step of coating the polymer on the surface of the culture container. Before the solution for coating was injected into the container, the shape of the desired shape was fixed to the surface of the container, and the solution was poured, followed by the coating step. The shape frame is formed by patterning a thin PET film of less than 200 μm in a desired shape and applying it to the container, and then adding a substance (collagen, gelatin, laminin, fibronectin or other extracellular matrix) After further application of the pattern, the used pattern frame was removed, and the desired pattern was formed as shown in FIG. All the work for pattern formation was possible within 30 minutes, and thus it was possible to apply it remarkably quickly and easily compared with the photolithography generally used for patterning the existing polymer. In addition to the method of patterning the conductive polymer scaffold, patterned cell sheets could be prepared according to the near infrared ray irradiation conditions. A cell sheet having the same shape as the square pattern of FIG. 5 was prepared by arranging and irradiating a pattern mask of a desired shape in the container before applying the near infrared rays to the conductive polymer scaffold on which the cells were cultured.

Claims (25)

materials;
A support layer formed on the substrate and containing a conductive material that absorbs light in the near infrared region to generate heat; And
A surface treatment layer formed on the support layer and containing extracellular matrix material,
The conductive material is a conductive polymer, and the conductive polymer is at least one monomer selected from a heterocyclic compound represented by the following formula (1) and aniline; And a polymer or copolymer made from a composition containing at least one additive selected from a pyridine-based compound, an imidazole-based compound and a polyethylenic compound:
[Chemical Formula 1]

In this formula,
X represents NH, O, S, Se or Te;
R ₁ and R ₂ are the same or as to form the other or connected to each other rings, a hydrogen _{atom, - (CH 2) ℓ -O-} (CH 2) m - (CF 2) n - (CR 7 R 8 _{) k - (CH 2) d} -Z,

-O-CH (R ₃ ) -CH (R ₄ ) -O-, or -O-CH ₂ -C (R ₅ ) (R ₆ ) -CH ₂ -O-;
R _3, R _4, R ₅ and R ₆ are the same as or different from each other, a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, a halogenated alkyl group having 1 to _{10, - (CH 2) d} -Z, - (CH 2) _{ℓ -O- (CH 2) m -} (CF 2) n - (CR 7 R 8) k - (CH 2) d -Z, or

ego;
R ₇ and R ₈ are the same or different from each other and represent hydrogen, an alkyl group having 1 to 5 carbon atoms, or - (CH ₂ ) _d -Z;
Z is a methacrylate group or an acrylate group;
m is an integer of 0 to 3, n is an integer of 0 to 5, k is an integer of 0 to 4, a is an integer of 0 to 2, b is an integer of 0 to 7, and d is an integer of 0 to 2 .
delete delete The method according to claim 1,
Wherein the heterocyclic compound of Formula 1 is at least one selected from the following Formulas 1a to 1m.
[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

≪ RTI ID = 0.0 &

[Formula 1e]

(1f)

[Formula 1g]

[Chemical Formula 1h]

[Formula 1i]

[Chemical Formula 1j]

[Chemical Formula 1k]

≪ EMI ID =

[Formula 1m]

The method according to claim 1,
Wherein the pyridine-based compound is at least one selected from pyridine, halogenated pyridine, bipyridine, and pyridine dicarboxylic acid.
The method according to claim 1,
Wherein the imidazole-based compound is at least one selected from imidazole and alkylimidazole.
The method according to claim 1,
Wherein the pyridine-based compound and the imidazole-based compound are each independently contained in an amount of 0.1 to 20% by weight based on the total weight of the composition.
The method according to claim 1,
Wherein the polyethylene-based compound is at least one polyethylene-based oligomer selected from the group consisting of polyethylene glycol oligomer, polyethylene oxide oligomer, and polyethylene glycol / polypropylene glycol oligomer.
The method according to claim 1,
Wherein the polyethylene-based compound is contained in an amount of 0.1 to 50% by weight based on the total weight of the composition.
The method according to claim 1,
And the weight average molecular weight of the conductive polymer is 1,000 to 1,000,000 Da.
delete The method according to claim 1,
Wherein the support layer is oxidized and reduced.
The method according to claim 1,
Wherein the support layer is patterned.
The method according to claim 1,
The base material is characterized in that it comprises at least one selected from polycarbonate, polypropylene, polyethylene, polystyrene, polyethylene terephthalate, polydimethylsiloxane, polylactic-coglycolic acid, polylactic acid, polycaprolactone, glass, Cell culture container.
delete The method according to claim 1,
Wherein the extracellular matrix material is at least one selected from the group consisting of collagen, gelatin, laminin, and fibronectin.
The method according to claim 1,
Wherein the cell adhesion rate is 50 to 1000 cells / mm < 2 >.
The method according to claim 1,
Wherein the cell desorption range is 0.1 to 100 cm < 2 >.
A method for manufacturing a cell culture container, comprising: forming a support layer according to claim 1 on a substrate.
20. The method of claim 19,
Wherein the support layer is formed by synthesizing a conductive polymer through a polymerization method selected from electropolymerization, solution polymerization, vapor phase polymerization, and chemical polymerization.
20. The method of claim 19,
Wherein the support layer is formed by coating or printing a solution polymerization solution by a method selected from spin coating, spray coating, immersion coating, roller coating, bar coating, and inkjet printing.
20. The method of claim 19,
A method for producing a cell culture container, comprising: forming a patterned support layer by disposing a morphology frame on a substrate; and coating a solution polymerization solution in the morphology frame.
A cell culture container according to claim 1; And
A cell sheet harvesting system comprising a near-infrared irradiator.
Culturing the cells in the cell culture container according to claim 1; And
Irradiating the cultured cells with near-infrared light to desorb the cells in a cell sheet form.
25. The method of claim 24,
A cell sheet harvesting method characterized by placing a pattern mask in a cell culture container and irradiating near infrared rays to harvest the patterned cell sheet.

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