KR20140046661A - System for co-culturing cancer cells with feeder cells - Google Patents

Abstract

The present invention relates to a method for co-culturing cancer cells by using feeder cells. More specifically, the present invention relates to a method for co-culturing cancer cells with feeder cells by making the cancer cells and the feeder cells come in contact with each other through a membrane between them so that the growth of the cancer cells is expedited. The method for co-culturing cancer cells with feeder cells according to the present invention comprises the steps of (a) preparing an insert including a side wall forming an internal space and both ends opened, and a membrane which is installed inside the side wall to separate the internal space into a top space and a bottom space, is equipped with a first surface at the top space side and a second surface at the bottom space side, and has a plurality of pores formed through the first surface and the second surface; (b) culturing the feeder cells on the first surface of the membrane so as to make the filopodia of the feeder cells pass through the pores of the membrane to be exposed to the side of the second surface of the membrane; (c) turning over the insert so that the bottom space of the insert is disposed on the top space and the second surface faces upward; and (d) disposing cancer cells on the second surface and co-culturing the cancer cells with the feeder cells. In the method for co-culturing cancer cells with feeder cells according to the present invention, cancer cells can grow rapidly because cancer cells and feeder cells come in contact with each other directly through a membrane, having a plurality of pores, between them.

Description

[0002] System for co-culturing cancer cells with feeder cells [

The present invention relates to a system for co-culturing cancer cells using culture assisting cells. More particularly, the present invention relates to a co-culturing system capable of accelerating the growth rate of cancer cells by bringing culture assist cells and cancer cells into contact with each other through a plurality of pores.

Techniques for culturing cells and tissues are becoming increasingly important for basic or applied life sciences research. Cell culture generally refers to separating a tissue piece from an individual of a multicellular organism and culturing and propagating the tissue piece in the container

The direct interaction of a particular cell with a different type of feeder cell has a great effect on the growth, migration and differentiation of that cell. For example, in the case of cancer cells, growth with cancer-associated fibroblast (CAF) is accelerated.

Therefore, a co-culturing method of simultaneously culturing cancer cells and CAF on the same culture dish has been used. However, when the cancer cells and the CAF are cultured in the same culture dish at the same time, it is inevitable that CAF is mixed into the cultured cancer cells. Contamination of cancer cells by culture assisted cells, especially in clinical applications, can cause serious problems.

Japanese Patent Laid-Open No. 2008-0016000 discloses a method for solving the above-mentioned problem, which comprises culturing a culture cell and a stem cell into a polymer membrane having a plurality of pores, A culture method has been proposed.

However, as shown in Fig. 1, in this method, the culture-supporting cells and the stem cells are close to each other but not directly in contact with each other. Therefore, there is a problem that it is difficult to apply to co-culture of cells, which is difficult to promote growth without direct contact with CAF, like cancer cells.

Published Patent No. 2008-0016000

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a novel co-culturing system capable of culturing cancer cells in direct contact with cancer cells and culture assisting cells.

According to an aspect of the present invention, there is provided a co-culture system comprising: a sidewall defining an inner space, both open ends, and an inner space of the sidewall to distinguish the inner space from an upper space and a lower space, An insert comprising a first surface and a second surface on the lower space side, the membrane comprising a plurality of pores extending through the first surface and the second surface, A recessed bore having a receiving space formed to receive a first culture fluid for culturing a culture assisting cell attached to a first surface of the membrane and a side and a bottom surface forming an open end; And a web plate having a well in which a second culture medium for culturing cancer cells adhered to the second surface of the membrane of the insert is accommodated.

In the co-culture system described above, the insert is formed such that the first surface is facing upward in the leisure bore, and the second surface is facing upward in the well plate, and the hole of the membrane is the first culture solution. And allowing the movement of the second culture solution and the filopodia of the culture assistant cells of the first surface to be exposed to the second surface side of the membrane, and are formed to prevent the movement of the cancer cells.

Preferably, the culture-assisted cell is a cancer-associated fibroblast (CAF).

In addition, it is preferable that the film is a photoresist formed with a bio-coating layer. This is because the size, shape and distribution of the holes are uniform through the semiconductor process, and a film having a very high aperture ratio can be easily manufactured.

The thickness of the film is preferably 10 占 퐉 or less and the average diameter of the hole is preferably 1 to 10 占 퐉. When the thickness of the membrane is more than 10 탆 and the average diameter of the pores is less than 1 탆, it is difficult for the suppository cells to pass through. Further, when the average diameter exceeds 10 탆, the culture assisting cells can pass through the membrane.

The depth of the accommodation space of the leisure bore is preferably shallower than the height of the side wall of the insert. The first culture solution does not flow into the lower space beyond the side wall, but must flow into the lower space only through the membrane, so that the cancer cells are not contaminated by the culture auxiliary cells.

The cancer cell and the culture auxiliary cell co-culturing apparatus according to the present invention can grow rapidly because the cancer cell and the culture assisting cell come into direct contact with each other through a membrane having a plurality of pores formed therebetween.

1 is a view for explaining a conventional co-culture method.
FIG. 2 is a conceptual diagram showing one embodiment of a co-culture system of cancer cells and culture assisting cells according to the present invention.
Figure 3 is a perspective view of the insert shown in Figure 2;
4 is a cross-sectional view of the insert shown in Fig.
5 is a perspective view of the leisure bore shown in Fig.
6 is a perspective view of the well plate shown in Fig.
7 is a view for explaining a step of culturing a culture aid cell (feeder cell).
Fig. 8 is a diagram showing a state in which the supernatant of the culture auxiliary cell passes through the membrane. Fig.
9 is a view for explaining a step of co-culturing cancer cells and culture assisting cells.
10 is a diagram showing a state in which a supernatant of a culture auxiliary cell is in contact with cancer cells through the membrane.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are provided by way of example so that those skilled in the art can fully understand the spirit of the present invention.

FIG. 2 is a conceptual diagram showing one embodiment of a co-culture system of cancer cells and culture assisting cells according to the present invention.

Fig. 3 is a perspective view of the insert shown in Fig. 2, and Fig. 4 is a cross-sectional view of the insert shown in Fig. 3 and 4, the insert 10 includes a side wall 11 defining an inner space and both open ends, and an inner space (not shown) on the inner surface of the side wall so as to distinguish the inner space from the upper space 12 and the lower space 13. [ And a membrane (14) disposed across the membrane (14). Each insert 10 is connected in an array by a holder 20 so that all the inserts 10 can be handled at one time.

The side wall 11 is made of a biocompatible material. But not limited to, transparent biocompatible plastic such as PDMS, PMMA, PET, PC, glass, or the like.

The membrane 14 of the insert 10 is intended to keep the cancer cells and the culture assisting cells separate from each other while exchanging them. When cancer cells and culture-assisted cells exchange with each other, the growth rate of the cancer cells is accelerated. However, if the cells are not separated from each other, the membrane (14) is used because the cancer cells and the culture-assisted cells can not be separated after the completion of the cultivation of the cancer cells.

The membrane 14 has a first side 15 on the side of the upper space 12 and a second side 16 on the side of the lower space 13. Filopodia of the culture medium and the culture auxiliary cells can pass through the membrane 14, but a plurality of pores of a size that can not pass through the entire cancer cell and the culture aid cell are formed on the first surface 15 and the culture medium- And is formed so as to pass through the two surfaces (16). Thoughts are thin, transparent cell organelles of thread shape.

It is preferable that the thickness of the film 14 is 10 mu m or less and the average diameter of the holes 17 is 1 to 10 mu m. When the thickness of the membrane 14 exceeds 10 占 퐉 and the average diameter of the holes 17 is less than 1 占 퐉, it is difficult for the gastric upset of the culture auxiliary cells to pass through. Further, when the average diameter exceeds 10 탆, the culture assisting cells can pass through the membrane 14.

The membrane (14) can be made of a biocompatible polymer. For example, synthetic polymers such as poly (lactic acid) (PLA), poly (glycolic acid), poly (ethylene terephthalate) (PET), polytetrafluoroethylene (PTFE), polyurethane , Methyl methacrylic acid polymer (PMMA), polyester, polycarbonate, or the like.

Further, the film 14 may be formed of a photoresist having a bio-coating layer formed on its surface. The use of a photoresist can produce a film 14 using a semiconductor manufacturing process, so that the size, shape, and distribution of the holes 17 are uniform and the film 14 having a very high aperture ratio can be manufactured . However, since the photoresist is poor in biocompatibility, it is preferable to form a biocompatible bio-coating layer. As the bio-coating layer, a parylene coating layer can be used.

5 is a perspective view of the leisure bore shown in Fig.

5, the leisure bore 30 has a side surface 34 and a bottom surface 35 which form an internal space 32 and an open end 33. The leisure bore 30 receives the insert 10. The insert 10 is received in the recessed bore 30 such that the first side 15 faces up. The leisure bore 30 serves to accommodate the first culture medium in the step of culturing the culture auxiliary cells.

6 is a perspective view of the well plate shown in Fig.

As shown in FIG. 6, the well plate 40 includes a plurality of wells 41 recessed to receive the insert 10. The well 41 receives a second culture medium for culturing cancer cells adhering to the second side 16 of the membrane 14 of the insert 10. The insert 10 is inserted into the well 41 of the well plate 40 with the second side 16 turned upside down.

Hereinafter, a co-culture method using the co-culture system of cancer cells and culture assisting cells will be described.

First, referring to FIGS. 7 and 8, a step of culturing a culture aid cell (feeder cell) on the first side of the membrane will be described.

The insert 10 is placed inside a reservoir 30 and then a first culture liquid 31 suitable for culture assisting cell culture is introduced into the reservoir 30. It is preferable that the first culture liquid 31 is injected so as not to exceed the side wall 11 of the insert 10. When the first culture fluid 31 containing the culture auxiliary cells flows over the side wall 11 of the insert 10, the lower space 13 of the insert 10 in which the cancer cells are cultured may be contaminated by the culture auxiliary cells It is because.

Next, the culture assisting cell (A) is put on the first side 15 of the membrane 14 of the insert 10. Then, the culture medium (A) is cultured by injecting the leisure bubble (30) into an incubator. As shown in FIG. 6, during the incubation of the culture assisting cell (A), the supernatant (C) of the culture assisting cell (A) passes through the holes (17) of the membrane (14) of the insert Is exposed on the two sides (16). Then, the culture assisting cell (A) adheres to the first surface (15) and is fixed.

Next, the step of reversing the insert will be described.

The culture assisting cell A is sufficiently grown and the supernatant stomach C of the culture assisting cell A is exposed on the second surface 16 and the culture assisting cell A is attached to the second surface 16 Once fixed, the insert 10 is removed from the recessed bore 30. Next, a second culture liquid 42 suitable for culturing cancer cells is injected into a well plate (40) having a plurality of wells (41) into which the insert (10) can be inserted. It is preferable that the second culture liquid 42 is injected so as not to exceed the side wall 11 of the insert 10. When the second culture medium 42 containing the culture assisting cells A flows over the side wall 11 of the insert 10 during co-culturing of the cancer cells B and the culture assisting cells A, Because the lower space 13 of the insert 10 where it is cultured can be contaminated by the culture assisting cell (A) after it is turned upside down. Then, the insert 10 is turned upside down and inserted into the well plate 40 (see Fig. 9).

Finally, referring to Figs. 9 and 10, a step of arranging cancer cells on the second surface of the membrane and co-culturing with the culture assisting cells will be described.

The cancer cells B are placed on the second surface 16 of the membrane 14 of the insert 10 which is inserted into the well plate 40 in turn. The well plate 40 is put into an incubator to co-culture the cancer cells B and the culture assisting cells (A). At this time, since the supernatant stomach (C) of the culture assisting cell (A) is exposed to the second surface (16) side, the cancer cells (B) are in direct contact with the supernatant stomach (C) (see Fig. The culture aid cell (A) promotes the growth of the cancer cell (B) by supplying the cancer cell (B) with the nutrients necessary for the growth of the cancer cell (B) through the upper gastrointestinal (C).

When the growth of the cancer cells (B) is completed, the cancer cells (B) are separated from the membrane (14) of the insert (10). In this process, it is known that the supernatant (C) of the culture aid cell (A) can be attached to the cancer cell (B), but the supernatant (C) does not contaminate the cancer cell (B). Therefore, it is possible to prevent the contamination by the culture auxiliary cell (A) while rapidly growing the cancer cell (B).

The embodiments described above are merely illustrative of the preferred embodiments of the present invention, the scope of the present invention is not limited to the described embodiments, those skilled in the art within the spirit and claims of the present invention It will be understood that various changes, modifications, or substitutions may be made thereto, and such embodiments are to be understood as being within the scope of the present invention.

10: insert 11: side wall
12: upper space 13: lower space
14: membrane 15: first side
16: second side 17: hole
20: Holder 30: Leisure Boya
31: First culture medium 40: Well plate
41: well 42: second culture solution

Claims (5)

A side wall defining an inner space and both ends open; an inner surface of the side wall so as to distinguish the inner space from an upper space and a lower space, the upper surface having a first surface on the upper space side and a second surface on the lower space side, An insert comprising a membrane formed with a plurality of pores extending through the first and second surfaces,
A recessed bore having a receiving space formed to receive a first culture fluid for culturing the culture assisting cells attached to the first surface of the insert and the membrane of the insert, and side and bottom surfaces forming an open end,
And a web plate having a well accommodated in a second culture liquid for culturing cancer cells adhered to a second surface of the membrane of the insert, the web plate being recessed to receive the insert,
The insert is formed so that the first surface is facing upward in the leisure bore, and the second surface is received upward in the well plate, and the hole of the membrane is the movement of the first culture liquid and the second culture liquid. And co-culture system of cancer cells and culture assistant cells formed so as to prevent the migration of the cancer cells from the filamentous foot (Filopodia) of the culture assistant cells of the first side. The method of claim 1,
The culture-assisted cells are co-cultured with cancer-associated fibroblasts (CAF), cancer cells and culture-assisted cells. The method of claim 1,
Wherein the membrane is a co-culture system of a cancer cell and a culture aid cell, which are photoresists formed with a bio-coating layer. The method of claim 1,
Wherein the membrane has a thickness of 10 mu m or less and an average diameter of the hole is 1 to 10 mu m. The method of claim 1,
The depth of the receiving space of the leisure bore is set so that the depth of the accommodating space of the leisure bore is shallower than the height of the sidewall of the insert, .

Images