KR102364619B1 - Cell culture container and manufacturing method thereof - Google Patents

Abstract

The cell culture vessel according to an embodiment of the present invention includes a culture substrate including a base substrate and at least one deposited graphene layer disposed on the base substrate, and the deposited graphene layer may include a functional group formed on the surface.

Description

Cell culture container and manufacturing method thereof {Cell culture container and manufacturing method thereof}

The present invention relates to a cell culture vessel and a method for manufacturing the same. More particularly, the present invention relates to a cell culture vessel comprising graphene having a functional group formed on the surface and a method for manufacturing the same.

Graphene is a structure in which hexagonal shapes of 6 carbons are connected to each other to form a two-dimensional single layer. And it has a structure differentiated from graphite having a three-dimensional structure.

On the other hand, the conventional cell culture vessel was cultured and differentiated by coating it on a protein material (Extracellular matrix, ECM) substrate.

However, the cell culture vessel coated with the protein material on the substrate had low uniformity, had to be stored at a low temperature due to the nature of the protein material, and there was a large possibility of contamination during storage and coating process.

In addition, there is a problem that the uniformity of the cultured cells is also low.

The technical problem to be solved by the present invention will be described as follows.

First, the present invention is to provide a cell culture vessel with improved culture efficiency and a method for manufacturing the same.

Second, the present invention is to provide a cell culture vessel with a low possibility of contamination and convenient storage, and a method for manufacturing the same.

Third, the present invention is to provide a cell culture vessel with high uniformity of cultured cells and a method for manufacturing the same.

In addition, the present invention is to solve all problems that can be predicted or generated from the prior art in addition to the technical problems described above.

The cell culture vessel according to the present invention includes a culture substrate including a base substrate and at least one deposited graphene layer disposed on the base substrate, wherein the deposited graphene layer includes a functional group formed on the surface.

The substrate may include polystyrene (PS).

The deposited graphene layer may be a single layer or a multilayer in which a plurality of layers are stacked, and the number of the deposited graphene layers may be 1 to 5.

In the deposited graphene layer, the number of functional groups may increase from the lowest layer to the highest layer.

The functional group may include at least one of an epoxy group (COC), a hydroxyl group (-OH), a carboxyl group (-COOH), a carbonyl group (C=O), and an amino group (NH ₂ ).

The functional group may be substituted with at least a portion of carbon constituting the deposited graphene layer.

The crystal size of the graphene layer may be 5um ² to 500um ² .

Further comprising a culture substrate case including an outer wall surrounding the culture substrate, the culture substrate may be detachable from the culture substrate case.

In the method for manufacturing a cell culture vessel according to an embodiment of the present invention, at least one deposited graphene layer is disposed on a base substrate, and plasma treatment is performed on the surface of the deposited graphene layer to form a functional group on the surface of the deposited graphene layer.

Disposing the deposited graphene layer on the base substrate includes growing the deposited graphene layer, laminating the grown deposited graphene layer on a transfer film, and applying the laminated transfer film and the deposited graphene layer to the base substrate. It can be attached and heat treated.

Growing the deposited graphene layer may control a crystal size of 5um ² to 500um ² .

The deposited graphene layer disposed on the base substrate may be a single layer or a multilayer in which a plurality of layers are stacked, and the number of layers of the multilayer graphene layer may be 1 to 5.

In the multilayer graphene layer, the number of functional groups may increase from the lowest layer to the top layer.

The effect of the cell culture vessel according to the present invention configured as described above and a method for manufacturing the same will be described as follows.

In the cell culture vessel of the present invention and a method for manufacturing the same, the culturing efficiency including graphene having a functional group formed on the surface can be improved.

The cell culture vessel of the present invention and a method for manufacturing the same can have a low possibility of contamination by disposing graphene on a base substrate, convenient storage, and increased uniformity of cultured cells.

1 is a perspective view of a cell culture vessel according to an embodiment of the present invention.
2 is a perspective view of a culture substrate of a cell culture vessel according to an embodiment of the present invention.
3 is a conceptual view showing the graphene surface of the cell culture vessel according to the embodiment of the present invention.
4 shows the cell culture results according to Examples and Comparative Examples of the present invention.
5 shows the cell culture results according to Examples and Comparative Examples of the present invention.
6 shows the cell culture results according to Examples and Comparative Examples of the present invention.
7 is a flow chart showing a method of manufacturing a cell culture vessel in Examples of the present invention.

Advantages and features of the present invention and methods for achieving them will become apparent with reference to the following embodiments.

The present invention is defined by the scope of the claims, and if there is a separate description for the meaning of a term in the specification, the meaning of the term will be defined by the above description. Like reference numerals refer to like elements throughout.

In the cell culture vessel 100 of the present invention, the cell culture efficiency can be improved by disposing the deposited graphene layer 12 on the base substrate 11 and forming functional groups on the surface of the deposited graphene layer 12 .

In this regard, a cell culture vessel 100 according to an embodiment of the present invention will be described with reference to FIGS. 1 to 6 .

First, referring to FIGS. 1 to 3 , the cell culture vessel 100, the culture substrate 10 in which direct cell culture is made, and the culture substrate case 20 including an outer wall 21 surrounding the culture substrate 10 . may include

Specifically, the culture substrate 10 may include a base substrate 11 and at least one deposited graphene layer 12 disposed on the base substrate 11 .

The base substrate 11 may physically implement the basic structure of the culture substrate 10 and form mechanical durability.

The base substrate 11 may be at least one of a ceramic material, glass, a polymer and a polymer cured plastics.

For example, the base substrate 11 may include polystyrene (PS).

However, the constituent material of the base substrate 11 is not limited to the above-described subject, and it will be said that it includes a range that can be easily designed and changed by a person skilled in the art within an appropriate range of cell culture.

As described above, in the cell culture vessel 100 according to an embodiment of the present invention, the culture substrate 10 places the deposited graphene layer 11 on the base substrate 11, and the surface of the deposited graphene layer 11 A functional group can be formed in

Specifically, by arranging the deposited graphene layer 12 on the base substrate 11, cell culture uniformity can be improved and contamination can be minimized and easy long-term storage is possible.

Graphene of the deposited graphene layer 12 is an inorganic material composed of carbon, and since the crystal structure is regular, cell culture uniformity can be improved, contamination possibility is small, and storage is easy.

That is, since the crystal structure is regular compared to a case in which a protein coating (eg, extracellular matrix) treatment is performed on a substrate in the prior art, cell culture can be uniform. In addition, since proteins and the like are easily denatured by external environmental changes (temperature, moisture, and bacteria), the cell culture vessel 100 according to the embodiment of the present invention can facilitate long-term storage while minimizing contamination.

In addition, graphene may have a similar function to the extracellular matrix protein, which is essential as a role for adsorbing cells and supplying nutrients in cell culture, and thus may have high binding compatibility with cells.

Furthermore, the surface of the deposited graphene layer 12 included in the cell culture vessel 100 according to the embodiment of the present invention may be modified to form functional groups on the surface of the deposited graphene layer 12 .

For example, oxygen functional groups may be formed on the surface of the deposited graphene layer 12 by connecting and adding oxygen to carbon constituting the deposited graphene layer 12 or replacing carbon with oxygen.

The functional group formed on the deposited graphene layer 12 may include at least one of an epoxy group (COC), a hydroxyl group (-OH), a carboxyl group (-COOH), a carbonyl group (C=O), and an amino group (NH ₂ ).

The cell culture vessel 100 according to the embodiment of the present invention can improve adhesion or adsorption of cells to be cultured by forming functional groups on the surface of the deposited graphene layer 12 .

For example, various functional groups with high binding properties to oxygen functional groups may be disposed in cells to be cultured, and functional groups having high binding properties with oxygen functional groups are easily combined with oxygen functional groups formed on the surface of the deposited graphene layer 12 . By doing so, cells to be cultured can be effectively attached or adsorbed to the deposited graphene layer 12 . As a result, cell culture efficiency can be improved.

The deposited graphene layer 12 included in the cell culture vessel 100 according to the embodiment of the present invention is formed on the surface - it is possible to effectively culture the cell by the functional group.

Specifically, the delivery of nutrients is improved by the functional group - formed on the surface of the deposited graphene layer 12 , so that effective cell culture can be achieved.

The functional group is electrically negatively charged, whereas the amino acids, which are nutrients necessary for cell culture, are electrically positively charged, and the amino acids, which are nutrients, can be easily adsorbed to the functional groups formed on the surface of the deposited graphene layer 12 .

Therefore, the supply of nutrients to the cells to be cultured can be increased, thereby maximizing the cell culture and differentiation efficiency.

The deposited graphene layer 12 included in the cell culture vessel 100 according to the embodiment of the present invention may be composed of a single layer or a multi-layer in which a plurality of layers are stacked, and the number of layers of the deposited graphene layer 12 is 1 to 5 can be

In the present specification, the deposited graphene layer 12 is a concept opposite to flake-type graphene formed by pulverizing graphite, and unlike flake-type graphene formed by stacking several tens to hundreds of graphene layers, deposited graphene The pinned layer 12 may have several respective graphene layers constituting the deposited graphene layer 12, which may be significantly smaller than that of flake-type graphene.

In particular, the cell culture vessel 100 according to the embodiment of the present invention includes a multi-layer deposited graphene layer 12, thereby improving cell culture efficiency.

Specifically, as described above, the functional group included in the deposited graphene layer 12 may be formed by connecting oxygen to carbon constituting graphene and adding or replacing carbon with oxygen. Therefore, when the deposited graphene layer 12 is composed of multiple layers, functional groups can be additionally formed for each layer, so that effective cell culture can be performed as the number of spots to which cells to be cultured will be adsorbed increases.

In the multilayer deposited graphene layer 12 , the number of functional groups may increase from the lowest layer to the highest layer.

In this specification, the lowest layer means a graphene layer closest to the base substrate 11 , and the uppermost layer means a graphene layer stacked farthest from the base substrate 11 .

Since the functional group may be formed by replacing the carbon constituting the graphene due to damage to the surface of the deposited graphene layer 12 by an oxidation plasma treatment, which will be described later, the deposited graphene layer 12 having the greatest damage caused by the oxidation plasma treatment. The most functional groups are in the uppermost layer, and there may be fewer functional groups toward the lowest layer.

However, in the deposited graphene layer 12 of the cell culture vessel 100 according to the embodiment of the present invention, by controlling the number of graphene layers constituting the deposited graphene layer 12 to within 5 layers, too many graphene layers are stacked and oxidized plasma It is possible to prevent the formation of functional groups in the graphene layer adjacent to the lowest layer because the treatment effect is not realized.

The crystal size of the deposited graphene layer 12 may be about 5um ² to about 500um ²

The cell culture vessel 100 according to the embodiment of the present invention can further improve adhesion or adsorption of cells to be cultured by controlling the crystal size of the deposited graphene layer 12 .

Specifically, cells to be cultured may be more easily attached or adsorbed as the surface energy is higher.

On the other hand, the crystal size of the deposited graphene layer 12 means the size of the grain boundary (grain boundary), the smaller the size of the grain boundary, the higher the energy state (energy state).

Therefore, in the cell culture vessel 100 according to the embodiment of the present invention, by controlling the crystal size of the deposited graphene layer 12 in the above range, the surface energy of the deposited graphene layer 12 is increased to improve adhesion or adsorption properties of cultured cells. can be maximized.

The cell culture vessel 100 according to the embodiment of the present invention may include a culture substrate case 20 including an outer wall 21 surrounding the culture substrate.

The culture substrate case 20 may include a case bottom 22 that corresponds to the culture substrate 10 and on which the culture substrate 10 is placed, and an outer wall 21 formed on an edge of the case bottom 22 .

That is, the culture substrate 10 including the deposited graphene layer 12 having a functional group formed on the surface thereof is stably placed in the inner space of the culture substrate case 20, thereby protecting the culture substrate 10 from external impact. there is.

The culture substrate 10 may be detachably coupled to the culture substrate case 20 .

Therefore, even for cultured cells of different types, there is no need to separately provide a cell culture vessel, and only the culture substrate 10 may be configured differently.

However, the coupling structure of the culture substrate 10 and the culture substrate case 20 is not limited to the above description, and it will be said that it includes a range that can be easily designed and changed by a person skilled in the art.

For example, the adhesive layer 13 may be disposed between the culture substrate 10 and the case bottom 22 so that the culture substrate 10 may be stably placed in the culture substrate case 20 .

Next, the effect of the cell culture vessel 10 according to the embodiment of the present invention will be described with reference to FIGS. 4 to 6 through specific experimental results.

First, with reference to Table 1 and Figure 4, the effect of the cell culture vessel 100 according to the embodiment of the present invention will be described.

Bare FN CVD Graphene CVD Graphene' CCK % CCK % CCK % CCK % Avg 1.33 85 1.56 100 2.02 129 1.73 111 STDEV 0.254 0.083 0.026 0.091

Table 1 shows adipose-derived mesenchymal stem cells (AdMSCs) as cultured cells, in a cell culture vessel in a bare state (only a polystyrene substrate is provided without a separate configuration), and cells in a FN-coated (Fibronectin coated on a polystyrene substrate) state. Culture vessel, CVD Graphene (graphene with crystals of about 5um ² deposited on polystyrene substrate) cell culture vessel and CVD Graphene' (graphene with crystals of about 100um ² deposited on polystyrene substrate) state Cell culture vessel - Cell culture efficiency and uniformity were determined using 10% FBS Serum (Fetal Bovine Serum) as a culture medium for cell culture vessels in the state.

Figure 4 (a) shows the cell culture results over time for each cell culture vessel.

Specifically, the photograph shown in Fig. 4(a) is a fluorescently stained image, which visually shows the distribution and amount of cultured cells. That is, the greater the amount of the stained red portion, the higher the cultivability, and the uniformity of the cell culture can be judged as the degree of distribution.

However, the contents shown in FIG. 4(a) are qualitative expressions, and cell cultivability can be determined by considering together with a quantitative value CCK (　Cell counting kit).

That is, when using a cell culture vessel in CVD Graphene state and a cell culture vessel in CVD Graphene 'state, it can be confirmed that a similar degree of cell cultivability can be implemented compared to a substrate coated with protein (FN Coated substrate).

Additionally, comparing the cell culture vessel in the CVD graphene state and the cell culture vessel in the CVD Graphene state, it can be confirmed that the smaller the crystal size of graphene, the higher the cell culture property.

Furthermore, when a cell culture vessel in CVD graphene state and a cell culture vessel in CVD graphene state are used, cells can be cultured at a more uniform or equivalent level compared to protein (FN).

In addition, referring to FIG. 4(b), FIG. 4(b) shows the primary culture result and secondary culture result (absorbance, Abs) under the above conditions, and data for 10 samples in each order was obtained.

In FIG. 4(b), it can be confirmed that the tendency of the above-described contents is maintained in all of the culture results over two times.

Next, with reference to Table 2 and Figure 5, the effect of the cell culture vessel 100 according to the embodiment of the present invention will be further described.

Bare FN CVD Graphene CVD Graphene' CVD Graphene'' CCK % CCK % CCK % CCK % CCK % Avg 0.294 51.9 0.582 100 0.32 59.7 0.617 108 0.225 35 STDEV 0.129 0.200 0.154 0.059 0.072

Table 2 shows adipose-derived mesenchymal stem cells (AdMSCs) as cultured cells, in a cell culture vessel in a bare state (only a polystyrene substrate is provided without a separate configuration), and cells in a FN-coated state (fibronectin coated on a polystyrene substrate) Culture vessel, CVD Graphene (crystalline about 5um ² graphene deposited on a polystyrene substrate) cell culture vessel, CVD Graphene' (including graphene with about 5um ² crystals on a polystyrene substrate, graphene Cell culture vessel in the state where oxygen functional groups are formed on the surface ^of Culture efficiency and uniformity were judged.

Figure 5 (a) shows the cell culture results over time for each cell culture vessel.

Specifically, the photograph shown in Fig. 5(a) is a fluorescence staining image, which visually shows the distribution and amount of cultured cells. That is, the greater the amount of the stained red portion, the higher the cultivability, and the uniformity of the cell culture can be judged as the degree of distribution.

However, the contents shown in FIG. 5(a) are qualitative expressions, and cell cultivability can be determined by considering together with the quantitative value CCK (　Cell counting kit).

That is, when using a cell culture vessel in the CVD Graphene' state, cell adsorption is improved by the oxygen functional group formed on the deposited graphene layer 12, so that the improved cell culture performance compared to the FN Coated substrate. At the same time, it is possible to implement improved cell culture uniformity over a protein-coated substrate.

Additionally, compared to the cell culture vessel in the CVD Graphene state and the cell culture vessel in the CVD Graphene state, the cell culture vessel in the CVD Graphene state and the cell culture vessel in the CVD Graphene state do not have FBS that supplies nutrients to the cells, so the culturability This can be reduced, and when a cell culture vessel in CVD Graphene' state is used, even if there is no FBS that supplies nutrients during cell culture, it can be confirmed that the function as a culture substrate can be realized by the oxygen functional group of CVD Graphene'. .

In addition, referring to FIG. 5(b), 5(b) shows the culture results (absorbance, Abs) according to time under the above conditions.

As can be seen in FIG. 5(b), it can be confirmed that the cell culture vessel in the CVD graphene' state has excellent cell cultivability in a culture medium environment without serum -- due to the oxygen functional group formed on the deposited graphene layer 12. there is.

Further, FIG. 6 shows the degree of differentiation by adding serum as a culture medium to the experimental examples of FIG. 5 .

Specifically, FIG. 6(a) shows the degree of differentiation of RNA markers expressed in the early-mid-late stage in the cell differentiation process, and FIG. 6(b) is a photograph showing the differentiation state of the experimental examples.

Referring to FIG. 6( a ), it can be confirmed that all of the RNA markers have excellent cell differentiation efficiency of CVD Graphene', and even referring to FIG. 6( b ), the calcium component of bone stained in red is It can be confirmed that many CVD Graphene' have the best cell differentiation properties.

Next, a method for manufacturing a cell culture vessel according to an embodiment of the present invention will be described with reference to FIG. 7 .

In the method for manufacturing a cell culture vessel according to an embodiment of the present invention, at least one deposited graphene layer 12 is disposed on a base substrate 10, and plasma treatment is performed on the surface of the deposited graphene layer 12 to deposit the deposited graphene layer 12 ) can form a functional group on the surface.

Specifically, deposited graphene may be grown to dispose the deposited graphene layer 12 on the base substrate 11 (S10).

Growing deposited graphene may grow graphene on the metal foil through a chemical vapor deposition process on the metal foil.

For example, the deposited graphene layer 12 may be generated and grown on the copper foil while the copper foil passes through the chemical vapor deposition apparatus.

The crystal size of the deposited graphene layer 12 may be about 5um ² to 500um ² as described above, and the crystal size as described above may be achieved by controlling the deposition environment in the chemical vapor deposition process.

The deposited graphene layer 12 thus completed may be laminated by laminating it to a thermal transfer film together with the metal foil. (S20)

After lamination, the metal foil may be etched and removed using an etching solution, for example, an aluminum sulfate solution. (S30)

After being removed from the metal foil, the thermal transfer film and the deposited graphene layer 12 may remain.

After the metal foil is etched and removed, it can be cut to correspond to the size of the cell culture vessel 100 to be used. (S40)

For example, it may be cut to correspond to the area of the base substrate 11 .

The cut thermal transfer film and the deposited graphene layer 12 are directly attached to the base substrate 11, and the thermal transfer film may be removed through a heat treatment process (S50, S60).

The heat treatment process may be performed at a temperature of about 90 degrees to about 120 degrees.

Further, after forming the deposited graphene layer 12 on the base substrate 11, the thermal transfer film and the new deposited graphene layer 12 laminated on the deposited graphene layer 12 are subsequently attached and additional heat treatment is performed to By thermal transfer, the number of layers of the deposited graphene layer 12 can be formed as a single layer or a multi-layer in which a plurality of layers are stacked.

For example, if the above steps are repeated three times, the number of layers of the deposited graphene layer 12 may be three.

Subsequently, a plasma treatment ( S70 ) may be performed on the deposited graphene layer 12 .

Plasma treatment ( S70 ) may use oxygen plasma, and through oxygen plasma treatment, damage to the deposited graphene layer 12 may be caused and surface energy may be increased so that cells to be cultured may be well attached.

Furthermore, during oxygen plasma treatment, oxygen functional groups may be formed on the surface of the deposited graphene layer 12 by adding or replacing oxygen to carbon constituting graphene.

However, since the oxygen plasma treatment decreases the power transmitted toward the inside of the deposited graphene layer 12, when oxygen plasma treatment is performed on the multilayer deposited graphene layer 12, the number of oxygen functional groups increases from the lowest layer to the top layer. can do.

However, the plasma treatment ( S70 ) is not limited to only the oxygen plasma treatment, and for example, a nitrogen plasma treatment may be used. In the case of using nitrogen plasma treatment, a functional group including an amino group (NH ₂ ) and the like may be formed on the deposited graphene layer 12 .

As mentioned above, although the embodiments of the present invention have been illustrated and described, the present invention is not limited to the specific embodiments described above, and it is common in the technical field to which the present invention pertains without departing from the gist of the present invention as claimed in the claims. Various modifications may be made by those having the knowledge, and these modifications should not be individually understood from the technical spirit or perspective of the present invention.

Claims (13)

base substrate; and
Containing a culture substrate comprising; at least one deposited graphene layer disposed on the base substrate,
The deposited graphene layer includes a functional group formed on the surface,
The deposited graphene layer is a multilayer in which a plurality of layers are stacked,
The multilayer deposited graphene layer is a cell culture vessel in which the number of functional groups increases from the lowest layer to the top layer. According to claim 1,
The substrate is a cell culture vessel comprising polystyrene (PS). According to claim 1,
The number of layers of the deposited graphene layer is 2 to 5 cell culture vessel. delete According to claim 1,
The functional group comprises at least one of an epoxy group (COC), a hydroxyl group (-OH), a carboxyl group (-COOH), a carbonyl group (C=O), and an amino group (NH ₂ ). According to claim 1,
The functional group is a cell culture vessel in which at least a portion of the carbon constituting the deposited graphene layer is substituted. According to claim 1,
A cell culture vessel having a crystal size of the graphene layer of 5um ² to 500um ² . According to claim 1,
Further comprising a culture substrate case comprising an outer wall surrounding the culture substrate,
The culture substrate is a cell culture vessel detachable from the culture substrate case. disposing at least one deposited graphene layer on the base substrate;
Plasma treatment on the surface of the deposited graphene layer,
Forming a functional group on the surface of the deposited graphene layer,
The deposited graphene layer is a multilayer in which a plurality of layers are stacked,
The multilayer deposited graphene layer is a cell culture vessel manufacturing method in which the number of functional groups increases from the lowest layer to the uppermost layer. 10. The method of claim 9,
Disposing the deposited graphene layer on the base substrate,
Growing the deposited graphene layer,
Laminating the grown deposited graphene layer on a transfer film,
A method of manufacturing a cell culture vessel by attaching the laminated transfer film and the deposited graphene layer to the base substrate and performing heat treatment. 11. The method of claim 10,
Growing the deposited graphene layer is a cell culture vessel manufacturing method to control the crystal size to 5um ² to 500um ² . 10. The method of claim 9,
The method of manufacturing a cell culture vessel wherein the number of layers of the multi-layer deposited graphene layer is 2 to 5.

delete

Images