WO2019039875A2 - Culture vessel for three-dimensional cell cultivation and three-dimensional cell co-cultivation method using same - Google Patents

Abstract

The present invention relates to a culture vessel for three-dimensional cell cultivation and a three-dimensional cell co-cultivation method using the same. The culture vessel comprises a well formed by a column positioned thereon and at least one support protruding from the column within the well. In contrast to conventional techniques, the present invention allows cells to be cultured at a position spaced from a culture vessel, thus enjoying the advantage of smoothly supplying oxygen necessary for three-dimensional cell culture structures.

Description

Culture vessel for three-dimensional cell culture and three-dimensional cell culture method using the same

The present invention relates to a culture container for three-dimensional cell culture and a three-dimensional cell culture method using the same, and more particularly, to a method for culturing a three-dimensional cell The present invention relates to a culture container for three-dimensional cell culturing in which air supply necessary for a culture structure is smooth and a three-dimensional cell co-culture method using the same.

Cell culture is one of the most fundamental research methods in the field of biotechnology, and is widely used not only for the study of living organisms but also for the study of human diseases. Although more than 40 years have passed since the development and establishment of cell culture methods for eukaryotic cells, the most commonly used methods to date for adherent cell growth are polystyrene, poly Dimensional surface consisting of a substrate made of a synthetic polymer resin such as polypropylene, polyethylene, polycarbonate (PC) or glass.

However, since the cells grown by the two-dimensional cell culture method, which is a method of culturing a single cell, are attached to the surface of the cultured cell so that the cells can be adhered well, they show many differences from the cells growing in a three-dimensional biotissue environment . Therefore, the two-dimensional and three-dimensional cell cultures show general morphological differences, and the expression of the receptor, gene transcription, cell migration and apoptosis, which occur through conventional two-dimensional cell culture, Since the complex life phenomenon differs greatly from the phenomenon occurring in the actual tissue environment, the two-dimensional cell culture method has a problem that it can not accurately reflect the physiological environment of the living body in which the cells grow on the three-dimensional plane.

Indeed, in the development of therapeutic agents for metabolic diseases such as obesity, diabetes, and arteriosclerosis, drugs that have shown excellent drug efficacy in the initial in vitro test are significantly less effective in animal experiments in vivo ( in vivo ) And the like. To address this problem, an in vitro model similar to the in vivo model is needed to predict the correct efficacy and toxicity of the drug from the early stages of drug development.

Conventional cell culture vessels are difficult to apply to drug screening and toxicity tests because the supply and circulation of air is not smooth due to the spatial limitation of the three-dimensional cell culture, and the growth and tissue formation of the three-dimensional cells are difficult to be sufficiently performed.

Therefore, in order to solve such a problem, it is required to develop a technology for a cell culture container which is suitable for rapid growth of three-dimensional cells and capable of two-dimensional and three-dimensional cocurrent transfer.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems, and it is an object of the present invention to provide a method for culturing cells in a three-dimensional cell culture And to provide a three-dimensional cell co-culture method using the same.

Dimensional structure by separately forming a supporting structure for supporting the three-dimensional structure on the culture container, thereby facilitating insertion, acquisition, and removal of the three-dimensional structure.

In addition, due to the support structure optimized for the three-dimensional culture, it is possible to efficiently maintain the three-dimensional cell culture as well as two or more cells co-cultured by air circulation and smooth supply of nutrients in the culture solution.

The three-dimensional cell culture method according to the present invention can rapidly and variously regulate the growth of cells, and the three-dimensional culture differentiated cells can be effectively applied to drug screening and toxicity test by applying to an animal replacement test.

The present invention also provides a culture container for three-dimensional cell culture with high durability and a three-dimensional cell co-culture method using the same, by preventing the outer contraction phenomenon due to the supporting portion protruding from the column from being prevented by designing the support portion region in an in- .

The present invention also aims to provide a three-dimensional cell culture and differentiation or tissue culture container for restoring damaged human tissue function.

In order to achieve the above object, a culture container for three-dimensional cell culture according to the present invention comprises a well formed by a column located on a bottom surface of the culture container, And at least one support portion.

The support may be located at a height of 20 to 60% of the column apart from the bottom surface of the culture vessel.

Alternatively, the supporting portion may be in contact with the bottom surface of the culture container, and a supporting portion may be formed along the side surface of the column. Here, the uppermost end of the support portion may be located at a height of 20 to 60% of the column from the culture container.

In the culture vessel for three-dimensional cell culture according to the present invention, the distance from the column to the end of the support may be 15 to 30% of the diameter of the well, with respect to the cross section of the well.

In addition, at least a part of the column area in contact with the support part and the support part area in contact with the column area can be removed.

In addition, a culture container for three-dimensional cell culture according to the present invention is characterized in that a well is formed by a column located on a bottom surface of the culture container, And at least a part of the upper surface of the support portion may be formed in a concave shape. Wherein the upper surface of the support portion is divided into an edge region and a central region, the central region is concave and the edge region is flat or convexly protruding in the protruding direction, and at the upper surface of the support, The area may occupy 10 to 30%, and the central area may occupy 70 to 90% area.

The top of the support may be curved and the top of the support may be 20 to 60% of the height of the column.

A three-dimensional cell co-culturing method according to the present invention is a three-dimensional cell co-culturing method comprising the steps of: forming a three-dimensional cell co- Preparing a culture container for cell culture; An inoculation step of inoculating the first cell into the well; An exchange step of removing the supernatant and at least partially exchanging the culture medium after the first cell is cultured; And a co-culturing step of co-culturing the three-dimensional structure inoculated with the second cell on the support.

And a replacement step of replacing the culture medium at intervals of 1 to 5 days after the co-cultivation step.

According to the culture container for the three-dimensional cell culture of the present invention and the three-dimensional cell culture method using the same, the cells are cultured at a position apart from the bottom surface of the culture container, There is a good advantage of supply.

By embodying a support structure for separately supporting a three-dimensional structure on a culture container, it is easy to insert, acquire and remove the three-dimensional structure.

In addition, due to the support structure optimized for three-dimensional culture, there is an advantage that two or more types of cell co-culture can be efficiently performed as well as three-dimensional cell culture.

The three-dimensional cell culture method according to the present invention is capable of rapid cell growth and various size control, and is more effective for drug screening, drug efficacy and toxicity test by a biomimetic model or an animal replacement test method.

In addition, since the support portion region is designed to be inwardly recessed, it is possible to prevent the outer contraction due to the support portion protruding from the column, thereby providing high durability.

In addition, there is an advantage that it can be utilized as a three-dimensional cell culture and tissue culture container for restoring damaged human tissue function.

1 is a perspective view showing a first embodiment of a culture container for three-dimensional cell culture of the present invention

2 is a plan view showing a first embodiment of a culture container for three-dimensional cell culture of the present invention

3 is a perspective view showing a second embodiment of a culture container for three-dimensional cell culture of the present invention

FIG. 4 is a perspective view showing a 16-well form of a culture container for three-dimensional cell culture of the present invention

5 is a perspective view showing a third embodiment of the culture container for three-dimensional cell culture of the present invention

6 is an external view showing a third embodiment of the culture container for three-dimensional cell culture of the present invention

7 is a perspective view showing a fourth embodiment of the culture container for three-dimensional cell culture of the present invention

8 is a plan view showing a fourth embodiment of the culture container for three-dimensional cell culture of the present invention

9 is a plan view showing a fifth embodiment of the culture container for three-dimensional cell culture of the present invention

10 is a plan view showing a sixth embodiment of the culture container for three-dimensional cell culture of the present invention

11 is a graph showing experimental results of three-dimensional cell culture with a culture container for three-dimensional cell culture of the present invention

FIG. 12 is an embodiment of a culture container for three-dimensional cell culture according to the present invention, wherein (a) is an image for culturing beads containing cells in the culture container in which the culture is carried out, and (b) Is obtained in a culture vessel.

Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings on a culture container for three-dimensional cell culture according to the present invention and a three-dimensional cell culture method using the same. The present invention may be better understood by the following examples, which are for the purpose of illustrating the present invention and are not intended to limit the scope of protection defined by the appended claims.

1 and 2, a first embodiment of a culture container 100 for three-dimensional cell culture of the present invention is a cell culture container 100 for culturing three-dimensional cells in a well (not shown) by a column 120 placed on a bottom surface 110 of a culture container and at least one supporting part 130 protruding from the column 120 may be formed in the well 140. In addition,

First, the bottom surface 110 of the culture container is a surface on which a base is formed, and is made of any material applied to cell culture such as polystyrene (PS), polypropylene (PP), polyethylene (PE), polycarbonate It may be configured.

The column 120 is formed in contact with the bottom surface 110 of the culture container and is preferably made of the same material as the bottom surface 11 and can be integrally injected. The pillars 120 may be located on the bottom surface 110 of one culture container in plural and the wells 140 may be formed for each pillars 120. As shown in Figure 4, it can be configured to have various numbers of wells, such as 16 wells, 96 wells.

The cross-sectional shape of the column 120 can be circular or polygonal, and can be preferably elliptical or circular, and most preferably circular.

It is preferable that the support portion 130 is located at a height of 20 to 60% of the column 120 away from the bottom surface 110 of the culture container and more preferably 25 To 50%, and most preferably at a height of 30 to 35%. If it is located at a height of less than 20%, it is difficult to smoothly circulate and supply air to the cells in the lower part. If it is located at a height of more than 60%, the three-dimensional cell structure and the well 140) is difficult to co-cultivate with other cells.

2, the distance from the column 120 to the end of the support part 130 is preferably 15 to 30% of the diameter of the well 140 with respect to the cross section of the well 140, , More preferably 20 to 25%, and most preferably 22 to 23%. If it is less than 15%, it is difficult to stably support the three-dimensional cell structure. If it exceeds 30%, the separation effect from the bottom surface 110 of the support part 130 is deteriorated. That is, There is a problem that adversely affects growth.

 The number of the support portions 130 is preferably 2 to 5, more preferably 3 to 4, and most preferably 3. If less than two, the three-dimensional cell structure is virtually impossible to support. If the number exceeds 5, the open space between the support portion 130 and the bottom surface 110 of the culture container is excessively narrow, There is a problem that efficiency of cell co-culture or the like is significantly lowered.

As shown in FIG. 3, in the second embodiment of the culture container 100 for three-dimensional cell culture of the present invention, the support part 130 is in contact with the bottom surface 110 of the culture container, The support portion 130 may be formed along the side surface of the support member 120. [

Since the supporting part 130 is formed along the side surface of the column 120 from the bottom surface 110 of the culture container, the injection can be easily performed integrally, which simplifies the manufacturing process and increases the economic efficiency. In addition, since the support portion 130 is formed in a columnar shape along the side surface of the column 120, it has higher stability and durability.

It is preferable that the uppermost end of the support part 130 is located at a height of 20 to 60% of the column 120 from the bottom surface 110 of the culture container, more preferably 25 to 50% , And most preferably at a height of 30 to 35%. If the height is less than 20%, it is difficult to smoothly supply oxygen to the cells in the lower part. If the height is more than 60%, the three-dimensional cell structure and the well 140, There is a problem that it is difficult to co-cultivate other cells in the cells.

5 and 6, the third embodiment of the culture container 100 for three-dimensional cell culture according to the present invention is characterized in that at least a part of the area of the column 120 in contact with the support part 130, At least a part of the region of the support 130 contacting the region 120 may be removed.

Some of the removed regions of the support 130 and the removed portions of the column 120 in contact with the support 130 are the undercut regions 150 shown in Figures 5 and 6 and are formed in the well 140 It is preferable that all the remaining areas except the surface of the support part 130 are removed.

7 and 8, the fourth embodiment of the culture container 100 for the three-dimensional cell culture of the present invention is characterized in that the column 120 is placed on the bottom surface 110 of the culture container, And a well 140 may be formed in the well 140. The well 140 may include at least one support 130 protruding from the bottom surface 110 of the culture container. This serves to separate the three-dimensional cell structure from the bottom surface 110 of the culture container by protruding the support portion 130 on the bottom surface 110 of the culture container rather than the column 120.

The uppermost portion of the support portion 130 is preferably located at a height of 20 to 60% of the column 120 from the bottom surface 110 of the culture container, more preferably at a height of 25 to 50% , And most preferably at a height of 30 to 35%. If the height is less than 20%, it is difficult to smoothly supply oxygen to the cells in the lower part. If the height is more than 60%, the three-dimensional cell structure and the well 140, There is a problem that it is difficult to co-cultivate other cells in the cells.

8, the diameter of the support 130 is preferably 2 to 10% of the diameter of the well 140, more preferably 4 to 8 times the diameter of the well 140, %, And most preferably 5 to 6%. If it is less than 2%, it is difficult to stably support the three-dimensional cell structure. If it exceeds 10%, the effect of separating the supporting part 130 from the bottom surface 110 of the culture container is deteriorated, There is a problem that the supply of the components is difficult and adversely affects the cell growth.

9, in the fifth embodiment of the culture container 100 for three-dimensional cell culture according to the present invention, the upper end of the support part 130 may be configured as a curved surface shape 131. FIG. As a result, it is possible to prevent the cell damage due to the angled region and also to increase the survival rate because the cell-inoculated three-dimensional structure can be supported more stably.

10, in the sixth embodiment of the culture container 100 for three-dimensional cell culture of the present invention, at least a part of the upper surface of the support part 130 may be formed in a concave shape. Since the central portion at the upper end of the support portion 130 is recessed most deeply, cell support is more stable, and the co-culture efficiency and survival rate can be remarkably improved. In addition, the surface where cells contact with the support 130 is minimized, and the concave surface is effective for cell growth since it can maximize the smooth air circulation effect.

The upper surface of the support part 130 is divided into an edge area and a center area 132. The center area 132 is concave and the edge area may be flat or convexly protruding in the protruding direction. have. The cell is brought into contact with only the edge region, thereby minimizing the damage, and the survival rate is greatly increased by the smooth air circulation.

In the upper surface of the support portion 130, the edge region may occupy 10 to 30% and the central region may occupy 70 to 90%. More preferably, the edge region may be 15 to 20% Is effective to occupy an area of 80 to 85%. This is an optimized area range through several experiments to maximize cell growth and survival while providing optimal area for stable cell support.

Next, the three-dimensional cell culture method of the present invention comprises a preparing step (S10); And a culture step (S11). This is a method for effectively culturing cells using the culture container structure for three-dimensional cell culture of the present invention.

First, the preparation step S10 includes at least one support protruding from the column or at least one support protruding from the bottom surface of the culture container in a well formed by a column located on a bottom surface of the culture container And a culture vessel for three-dimensional cell culture including the culture medium. In the preparation step (S10), the uppermost end of the support part may be located at a height of 20 to 60% of the column from the bottom surface of the column or the culture container.

The culturing step (S11) is a step of placing the cell-inoculated three-dimensional structure on the supporting part and culturing it. Due to the support structure optimized for the three - dimensional culture, three - dimensional cell culture proceeds efficiently.

Next, the three-dimensional cell co-culturing method of the present invention comprises preparing (S20); Inoculation step S21; Exchange step S22; And a co-culture step (S23). This is a method by which the first cell and the second cell can be effectively co-cultured using the culture container structure for the three-dimensional cell culture of the present invention.

First, the preparing step S20 includes at least one support protruding from the column or at least one support protruding from the bottom surface of the culture container in a well formed by a column located on the culture container. And preparing a culture container for the 3D cell culture. In the preparation step (S20), the uppermost end of the support part may be located at a height of 20 to 60% of the column from the bottom surface of the column or the culture container.

Inoculation step S21 is a step of inoculating the first cells into the wells. That is, the first cells and the culture liquid can be inoculated into the wells.

Herein, the first cell may be any cell capable of co-culturing with the second cell, but may be an adherent cell, and may be a mesenchymal stem cell, a mesenchymal stem cell, a fat precursor cell, a smooth muscle cell (Smooth Muscle Cell, SMC) Or a macrophage.

The exchange step S22 is a step of culturing the first cells, removing the supernatant, and at least partially exchanging the culture liquid. The co-culture efficiency can be increased by removing the supernatant resulting from the first cell culture in the culture medium in the well and replacing part or all of the culture medium.

The co-culture step (S23) is a step of co-culturing the three-dimensional structure inoculated with the second cells on the support. Here, the three-dimensional structure is preferably in the form of a bead or a support.

Herein, the second cell may be a culturable eukaryotic cell, and more particularly, an epithelial cell, a fibroblast, an osteoblast, a cartilage cell, a hepatocyte, a umbilical cord cell, a umbilical cord mesenchymal stem cell (UCMSC ), Adipose-derived mesenchymal stem cells (ADMSC), or bone marrow-derived mesenchymal stem cells (BMMSC).

Finally, the replacing step S24 is a step of replacing the culture solution every 1 to 5 days after the co-cultivation step S23. More preferably, it is effective to replace it every two to three days. This is to supply the necessary nutrients to the cell culture and to remove the cell-viability inhibitor that is generated during the cultivation, so that the cells or tissues are continuously grown.

Example 1) Culture vessel preparation and sterilization

As an embodiment of the present invention, a culture container is manufactured by using a 3D printing technique that forms a layer by stacking one layer by using a solid freeform fabrication (SFF) method using rapid prototyping (RP) equipment, Was performed using an electron beam accelerator.

Example 2) First cell culture

Experiments were conducted under the following conditions to measure the effect of the three-dimensional cell culture using the culture container for the three-dimensional cell culture of the present invention.

Cells: 3T3-L1 (lipocal precursor cells) (2.45x10 ⁶ cells / 5mL / Tube), adipose-derived mesenchymal stem cells (ADMSC)

- Culture period: Culture for 7 days

- Cell count: 0, 3, 7 days Cell count

- Alginate mixture: 1.3% concentration

Specifically, the frozen first cells were thawed in a constant-temperature water bath at 37 ° C and transferred to a 15 ml tube, followed by addition of a basic medium (DMEM + 1% antibiotics) to 1,500 The supernatant was removed by centrifugation for 5 min at RPM and the supernatant was added again. The above procedure was repeated to completely remove the frozen storage component. The cells were then transferred to a 100 mm culture dish with DMEM + 1% antibiotics + 10% The cells were inoculated and cultured in a 5% carbon dioxide incubator at 37 ° C. After 4 to 5 days, the cells were sufficiently proliferated. Then, 0.05% trypsin-EDTA was added thereto to inoculate cells per well in the culture vessel at a concentration of 1 × 10 ³ .

Example 3) Production of beads containing second cells

The second cell was adipose-derived mesenchymal stem cell (ADMSC), and the 3-dimensional structure inoculated with the second cell was prepared in bead form.

The beads include a melting step in which a culture solution, alginate and gelatin are put into a tube and melted at 65 캜; The temperature of the tube was lowered to 37 DEG C, the second cell (2.45x10 < ⁶ > cells / 5mL / Tube) was added to the tube and stirred at 500RPM for 2 minutes; And a manufacturing step of making the mixture in the tube into a bead form and crosslinking it in a calcium chloride solution.

After stirring, the bubbles are removed by centrifugation at 1500 RPM. In the preparation step, the concentration of the calcium chloride solution was 5%.

The crosslinked beads were washed with phosphate buffered saline (PBS), and cultured in a culture medium containing 10% fetal bovine serum at 37 ° C in a 5% carbon dioxide incubator. As a result, (Fig. 10 (b)).

Example 4) Supporting and Sterilization Process

The support was made using polycaprolactone. First, polycaprolactone was introduced into a tube and a pressure of 650 to 730 kPa was applied at 90 ° C. Then, a three-dimensional support was prepared using the suction function of the air controller, and the sterilization process was performed using electron beam acceleration.

Example 5) Three-dimensional cell culture using a support containing a second cell

In the three-dimensional cell culture using the supernatant containing the second cells, the second cells were inoculated on 4 drop supports on a 4-drop support, cultured for 1 hour, and cultured in 1 ml medium at 37 ° C under 5% A three-dimensional structure of the support type in which the second cells were inoculated was prepared.

As shown in FIG. 11 and FIG. 12, the three-dimensional cell culture using the culture container according to the present invention showed that the cell number did not decrease but increased as the culture time passed, unlike the two-dimensional cell culture using the general culture container Able to know. That is, it has been confirmed in actual experiments that the three-dimensional cell culture can be more continuously performed with the culture container of the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is clear that the present invention can be suitably modified and applied in the same manner. Therefore, the above description does not limit the scope of the present invention, which is defined by the limitations of the following claims.

The present invention relates to a culture container for three-dimensional cell culture and a three-dimensional cell co-culture method using the same, and is industrially applicable.

Claims (11)

1. In a culture container for three-dimensional cell culture,

   A well is formed by a column located on a bottom surface of the culture container,

   And at least one support protruding from the column in the well.
2. The method according to claim 1,

   Wherein the support portion is located at a height of 20 to 60% of the column, spaced from the bottom surface of the culture container.
3. The method according to claim 1,

   Wherein the supporting portion is in contact with a bottom surface of the culture container, and a supporting portion is formed along a side surface of the column.
4. The method of claim 3,

   And the uppermost end of the support is located at a height of 20 to 60% of the column from the bottom surface of the culture container.
5. The method according to claim 1,

   Wherein the distance from the column to the end of the support is 15 to 30% of the diameter of the well, with respect to the cross section of the well.
6. The method of claim 3,

   Wherein at least a part of the column area contacting the support part and at least a part of the support part area contacting the column area are removed.
7. In a culture container for three-dimensional cell culture,

   A well is formed by a column located on a bottom surface of the culture container,

   And at least one support protruding from a bottom surface of the culture container, wherein at least a part of the upper surface of the support is concave.
8. 8. The method of claim 7,

   The upper surface of the support portion is divided into an edge region and a central region. The central region is concave and the edge region is flat or convexly protruding in a protruding direction.

   Wherein the edge region occupies an area of 10 to 30% and the central region occupies an area of 70 to 90% on the upper surface of the support.
9. 8. The method of claim 7,

   The upper end of the support portion is formed in a curved shape,

   Wherein the top of the support is located at a height of 20 to 60% of the column.
10. Preparing a culture container for three-dimensional cell culture comprising a column or at least one support protruding from the bottom in a well formed by a column located on a bottom surface of the culture container;

    An inoculation step of inoculating the first cell into the well;

    An exchange step of removing the supernatant and at least partially exchanging the culture medium after the first cell is cultured; And

    And co-culturing the three-dimensional structure inoculated with the second cells on the support,

    Wherein the uppermost end of the support is located at a height of 20 to 60% of the column from the bottom surface of the column or the culture container in the preparing step.
11. 11. The method of claim 10,

    After the co-culture step,

    And replacing the culture medium at intervals of 1 to 5 days.

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