KR20140113139A

KR20140113139A - Cell spheroid culture plate

Info

Publication number: KR20140113139A
Application number: KR1020130028131A
Authority: KR
Inventors: 정석; 한세운; 신유진; 김정현; 정효은; 변재영
Original assignee: 고려대학교 산학협력단
Priority date: 2013-03-15
Filing date: 2013-03-15
Publication date: 2014-09-24

Abstract

The present invention relates to a cell spheroid culture plate comprising: a culture plate body; and a culture groove arranged on the culture plate body with a plurality of rows and lines, whereby a cell spheroid is cultured within the culture groove. The culture groove comprises: a first culture groove whereby the width of the culture groove is narrowed toward a lower portion; and a second culture groove formed beneath the first culture groove and having a lower portion with a curved surface. According to the present invention, manufacturing cost can be reduced and size of the cell spheroid culture plate can be easily increased because micromachining is performed to prepare a mold for a plastic injection by forming the culture groove to have repeated pattern.

Description

Cell spheroid culture plate < RTI ID = 0.0 >

The present invention relates to a cell spoell culture plate, and more particularly, to a cell spoell culture plate having a structure for various cell spoiloids or tissue culture.

Animal testing in new drug development is used for nonclinical / clinical experiments and initial screening. Animal experiments performed on one new drug development cost more than several hundred billion won. In addition, the ethical nature of animal experiments is getting weaker because of the strong opposition to animal experiments worldwide for ethical reasons. Thus, attempts have been made to replace animal experiments with in vitro experiments using animal or human cells.

However, there are many difficulties in predicting the actual clinical response with the conventional in vitro experiment technique. This is because most of the cells in the body mostly maintain their life activity through interaction with the surrounding cells and the extracellular matrix (ECM) three-dimensionally. However, most of the cells cultured in the in vitro experiments are two-dimensional Structure, which failed to simulate the physiological phenomenon in the body. For example, in the case of cancer, not only is it three-dimensionally present in the actual body, but also a deficiency of various nutrients and oxygen occurs at a distance of 150-200 microns or more from a blood vessel, and abnormally accumulating metabolites , It is difficult to simulate this phenomenon with existing two-dimensional in vitro experimentation techniques, resulting in different results from the results of actual animal experiments.

Therefore, in recent research groups, studies on the cultivation technology of three-dimensional cell spoiloid, which is physiologically similar to the cell tissues in the body, are under way.

Conventional traditional hanging drop methods are labor intensive and can not be mass-produced. Recently developed multi-well type Perfecta3D and Gravity PLUS products have a very small volume of droplets due to the use of a very small amount of culture medium, which causes evaporation problems, there is a problem. In addition, in the case of the 384 well type, since the number of wells to be handled is very large, it is difficult for the experimenter to directly pipethe the robot. Therefore, the robotic system may be additionally required.

Next, in the case of a single cell culture on a non-adhesive surface, since the size of the cell spoiloid formed, the number and shape of the formed cell spoil are uneven, It is difficult to apply it to a specific application (cancer-related drug development, etc.) that is essential.

In the micro-molding technique, the conventional micro-molding technique is mainly based on the semiconductor process, and thus requires special equipment for manufacturing. In addition, since the production cost is very high, it is difficult to commercialize it practically, and most of the technologies so far are available only in the laboratory. In addition, there is a disadvantage that cell spoiloid growing inside the micro-well is likely to escape during exchange of the culture medium, and cell spoiloids may be unintentionally generated in the space between the wells and the wells.

DISCLOSURE Technical Problem Therefore, an object of the present invention is to solve the problems of the prior art as described above, and it is an object of the present invention to provide a method of preventing cell spoilage from being released from the culture wall during culture medium exchange, Thereby providing a culture plate.

According to an aspect of the present invention, there is provided a method of manufacturing a microfluidic device including a culture plate body; And a culture groove which is disposed in the culture plate body in a plurality of rows and columns and in which cell spoil is cultivated, the culture groove comprising: a first culture groove which is narrowed toward the bottom; And a second culture groove formed at a lower portion of the first culture groove and having a curved lower portion.

The first culture groove may be formed with an inclined surface such that the four surfaces thereof are downwardly narrowed in width.

The inclination angle of the first culture groove may be 20 to 70 °.

The first culture groove may have a circular or polygonal cross-section.

The upper portion of the second culture groove may be formed as a parallel straight surface, and the lower portion may be formed as a semicircular shape.

The depth of the second culture groove may be at least half the width of the second culture groove.

The width of the second culture groove may be 50 to 1000 탆.

The culture grooves may be coated with a polymer compound, a bio-based material, or non-adhesive materials.

The non-tacky material may be selected from the group consisting of Parylene, Sigmacote, poly-HEMA, Np-vinylbenzyl-D-lactonamide (PVLA), F108, LIPIDURE, Agarose gel, bovine serum albumin (BSA), chitosan, hydroxypropyl methyl cellulose (HPMC), poly amino acid, polyvinyl alcohol polyvinyl alcohol, and titanium dioxide gel (TiO2 gel).

According to another embodiment of the present invention, A cell sphere culture plate which is detachable inside the first cell culture container and in which the first cell sphere is cultured; And a second cell culture container attached to the upper part of the cell spoil culture plate and the second cell spoil is cultured therein.

According to the present invention, it is possible to reduce the production cost by using micromachining for forming molds for plastic injection by forming culture grooves so as to have repetitive patterns, and can easily enlarge the size.

In addition, the inclined surfaces are formed in the first culture grooves so that the cells can stably enter into the culture grooves, and cells can be prevented from escaping due to unintentional flow when the cell culture liquid is replaced.

1 is a perspective view showing a culture plate of a cell sphere according to an embodiment of the present invention.
FIG. 2 is a cross-sectional view of a culture plate of a cell sphere according to an embodiment of the present invention. FIG.
3 is an exploded perspective view of a cell spoloid culture assembly in which a cell spoloid culture plate is assembled.
Figure 4 is a perspective view of a cell spoloid culture assembly in which a cell spoloid culture plate is assembled.
Figure 5 is a cross-sectional view of the cell spoloid culture assembly of Figure 4;
FIG. 6 and FIG. 7 are photographs showing results of cell spoloid formation experiments. FIG.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, an embodiment of a cell spoil culture plate according to the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 is a perspective view showing a cell spoil culture plate according to an embodiment of the present invention, and FIG. 2 is a sectional view of a cell spoil culture plate according to an embodiment of the present invention.

As shown therein, the culture plate of the present invention comprises a culture plate body 10; And a culturing groove 20 in which the culture plate body 10 is disposed in a plurality of rows and columns and in which cell spoils are cultured.

The culture plate body 10 is formed into a plastic injection molded plate shape through a mold. In order to lower the production cost and easily increase the size by using the micro-machining process for manufacturing the mold for plastic injection, the culture groove 20 has a repetitive pattern as a micro-well structure. Therefore, it is easy to mass-produce the cell spheroids, and it can be used in various sizes according to the needs of the user.

Here, the culture groove 20 has a first culture groove 22 whose width is narrowed toward the bottom; And a second culture groove (24) formed at a lower portion of the first culture groove (22) and having a curved lower portion.

In this embodiment, the first culture grooves 22 are formed at the lower portion of the second culture grooves 24, and the first culture grooves 22 have inclined surfaces that are narrower toward the lower portion. The inclined surface may be formed by four surfaces surrounding the first culture grooves 22 as shown in FIG.

Of course, the first culture grooves 22 are illustrated as having a predetermined inclined surface in this drawing, but the present invention is not limited thereto, and any shape narrowing toward the bottom can be applied.

For example, the first culture groove 22 may have a circular cross-section and may have a substantially funnel shape, or may have a polygonal cross-section such as a pentagonal or hexagonal shape. On the other hand, when the cross section of the second culture grooves 24 is circular, the first culture grooves 22 need not have a polygonal cross section.

The first culture grooves 22 are inclined as described above so that the cells introduced for culturing can enter the culture grooves 20 at an early stage. That is, the first culture grooves 22 are inclined and can easily fall into the second culture grooves 24 without entering the adjacent first culture grooves 22.

Next, if the cells introduced into the first culture grooves 22 are not cultured in the culture grooves 20 having a sufficient depth, the cells may escape from the culture grooves 20 due to unintended flow during the replacement of the cell culture liquid. Therefore, in this embodiment, the second culture grooves 24 are formed deeper than the first culture grooves 22, so that the cells can be cultured without leaving the culture grooves 20.

As shown in FIG. 2, the upper portion of the second culture groove 24 is preferably formed as a straight line, and the lower portion of the second culture groove 24 is formed as a semicircular shape. This is to allow cells to grow stably in uniform size in the form of spheroids. Of course, the shape of the second culture groove 24 is not limited to the above-described shape, and any shape can be applied if the cell spoil is formed to a sufficient depth to stably cultivate it. For example, the entire second culture groove 24 may be formed in a semicircular shape or an elliptical shape.

Next, the culture grooves 20 described above are preferably designed as follows. First, in order to prevent the cells from escaping due to an unintentional flow when the cell culture medium is replaced as described above, the depth of the second culture groove 24 is preferably at least half the width of the second culture groove 24 Do. It is preferable that the cells of the first culture groove 22 have an inclination angle of 20 to 70 degrees and the width of the second culture groove 22 is 50 to 1000 占 퐉.

Meanwhile, the culture groove 20 may be coated with a polymer compound, a bio-based material, or a non-adhesive material. Particularly, when the non-sticky substance harmless to the cells is coated on the culture groove 20, the cells can be stably grown in a uniform size in the form of spoiloid, and the cells can be easily obtained when the formed cell spoloids are collected .

Examples of non-tacky materials include, but are not limited to, parylene, Sigmacote, poly-HEMA, polyvinylbenzyl-D-lactonamide (PVLA), F108, LIPIDURE, Agarose gel, bovine serum albumin (BSA), chitosan, hydroxypropyl methyl cellulose (HPMC), poly amino acid, polyvinyl alcohol, Alcohol, polyvinyl alcohol, titanium dioxide gel (TiO2 gel), and the like.

3 is an exploded perspective view of a cell spoloid culture assembly in which a cell spoell culture plate is assembled, and FIG. 4 is an exploded perspective view of a cell spoloid culture plate in which a cell spoloid culture plate is assembled Figure 5 is a cross-sectional view of the cell spoloid culture assembly of Figure 4;

As shown in the figure, the above-described cell spoil culture plate is detachable inside the first cell culture container 30. In this figure, the cell culture medium plate is shown attached to the first cell culture container 30, but the present invention is not limited thereto and may be integrally formed with the first cell culture container 30.

In the cell spoil culture plate attached to the inside of the first cell culture container 30, the first cell spoloid 50 is cultured. The first cell spoloid 50 can be stably placed in the above-described culture groove 20.

The second cell culture container 40 in which the second cell spoloid 52 is cultured is attached to the upper part of the cell spoil culture plate. The second cell culture container 40 may be attached to the upper surface of the first cell culture container 30 so as to surround the cell spoal culture plate. When the culture solution flows into the first and second cell culture vessels 30 and 40 in the attached state as described above, the first and second cell spoils 50 and 52 can be cultured.

In FIG. 6, the experimental results of cell spleod formation of mouse neural stem cells from days 1 to 11 were compared with those of non-tacky material coated and not coated, The results of the cell spoloid formation experiments of HepG2 hepatocarcinoma cell line from days 11 to 11 were compared with those of non-tacky substance-coated and non-coated substance.

The scope of the present invention is not limited to the embodiments described above, but may be defined by the scope of the claims, and those skilled in the art may make various modifications and alterations within the scope of the claims It is self-evident.

10: culture plate body 20: culture groove
22: first culture groove 24: second culture groove
30: First cell culture container 40: Second cell culture container
50: first cell spheroid 52: second cell spheroid

Claims

Culture plate body; And
And a culture groove which is arranged in a plurality of rows and columns in the culture plate body and in which cell spoil is cultivated,
Wherein the culture-
A first culture groove which is narrowed toward the bottom; And
And a second culture groove formed at a lower portion of the first culture groove and having a curved lower portion.

The method according to claim 1,
Wherein the first culture groove has an inclined surface formed so that its four faces are downwardly narrowed.

3. The method of claim 2,
Wherein the first culture groove has an inclination angle of 20 to 70 °.

The method according to claim 1,
Wherein the first culture groove has a circular or polygonal cross-section.

The method according to claim 1,
Wherein the upper portion of the second culture groove is formed as a parallel straight surface and the lower portion is formed as a semicircular shape.

The method according to claim 1,
Wherein the depth of the second culture groove is at least half the width of the second culture groove.

The method according to claim 1,
And the width of the second culture groove is 50 to 1000 占 퐉.

The method according to claim 1,
Wherein the culture groove is coated with a polymer compound, a bio-based material, or non-adhesive materials.

9. The method of claim 8, wherein the non-
Poly-HEMA, polyvinylbenzyl-D-lactonamide (PVLA), F108, LIPIDURE, agarose gel, Bovine serum albumin (BSA), chitosan, hydroxypropyl methyl cellulose (HPMC), poly amino acid, polyvinyl alcohol, titanium dioxide And a material of gel (TiO2 gel).

A first cell culture container;
The cell spleod culture plate according to any one of claims 1 to 8, which is detachable inside the first cell culture container and in which the first cell spoloid is cultured; And
And a second cell culture container attached to the upper part of the cell spoil culture plate and the second cell spoil is cultured therein.