JP2011244713A - Device, system, and method for culturing cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture device which can culture cells using a scaffolding member for a long period of time, and a cell culture system and a cell culture method using the device.SOLUTION: The cell culture device 10 includes a culture chamber 300 in which cells are stored, and a culture medium inlet channel 400 and a culture medium outlet channel 500 communicating with the culture chamber 300. The cell culture device 10 also includes a porous filter 600 disposed between the culture chamber 300 and the culture medium outlet channel 500. With this structure, the scaffolding member 700 can be captured by the porous filter 600 if the scaffolding member 700 fixed to the culture chamber 300 comes off while the device is used, and therefore, blocking of the culture medium outlet channel 500 with the removed scaffolding member can be prevented and the long-time culture becomes possible.

Description

  The present invention relates to a cell culture device, and a cell culture system and a cell culture method using the same.

  Cell culture is generally performed in a state where cells and a liquid medium are contained in a container such as a petri dish. However, in recent years, with the progress of microfabrication technology in the semiconductor manufacturing field, the application of microdevices manufactured by microfabrication technology has been promoted in the medical and biotechnology research fields, and cell culture using such microdevices has been promoted. (For example, refer to Patent Document 1).

  A microdevice for cell culture (cell culture device) is formed by forming a culture chamber and a microchannel inside a flat substrate, and performs cell culture by accommodating cells and a medium in the culture chamber. The medium is exchanged using the microchannel.

  By the way, in recent years, there has been an expectation for the development of an artificial organ, and many studies have been conducted on the development of an artificial liver, which is a part of the development. However, hepatocytes are difficult to cultivate for a long period of time, and further, although liver function is expressed in the early stage of culture, there is a problem that liver function is remarkably lowered thereafter. In this regard, the present inventors can form a network on endothelial cells by using an appropriate scaffold (cell culture carrier), and can express a high liver function by co-culturing with hepatocytes. This is reported (Non-patent Document 1).

JP 2008-519598 A ([0037], FIG. 1A)

Yoichi Fujiyama, Yoichi Tagawa, and 5 others, "Analysis of liver function in co-culture with hepatocytes using tubular endothelial cell culture system", Proceedings of the 30th Annual Meeting of the Molecular Biology Society of Japan, 2007

  As described above, in cell culture, it is effective to use a scaffold according to the type of cells to be cultured. In general, a polymer gel made of collagen or the like is often used as such a scaffold. However, when such a polymer gel is coated on the surface of the culture chamber of the cell culture device as described above to form a scaffold, the gel may be peeled off during the culture and the microchannel may be clogged. . Therefore, it has been difficult to perform culture using such a scaffold for a long period of time with a conventional cell culture device.

  The present invention has been made in view of the above problems, and the object of the present invention is a cell culture device capable of performing culture using a scaffold over a long period of time, and a cell culture system using the same. And providing a cell culture method.

The cell culture device according to the present invention, which has been made to solve the above problems,
a) a culture chamber containing the cells;
b) a medium introduction channel communicating with the culture chamber;
c) a medium discharge channel communicating with the culture chamber;
d) a porous filter provided between the culture chamber and the medium discharge channel;
It is characterized by having.

  According to such a configuration, in the case where cell culture is performed by coating the scaffold in the culture chamber, even if the scaffold is detached during use, it is captured by the porous filter. Can prevent clogging of the medium discharge channel. Therefore, according to the present invention, it is possible to perform long-term culture using a scaffold material that has been difficult with conventional cell culture devices. The porous filter must be able to easily pass through the culture medium, although it cannot pass through the peeled scaffold. Therefore, it is desirable to use a porous filter having an average pore size of 0.1 to 10 μm. As such a porous filter, for example, a sheet-like porous film made of nitrocellulose or the like can be suitably used.

Further, the cell culture device according to the present invention described above,
A main body and a lid that is detachably attached to the top of the main body;
The culture chamber is formed in the main body, and the culture chamber has an opening on the upper surface of the main body;
The culture medium discharge channel is formed in the lid, and the culture medium discharge channel has an opening at a position on the lower surface of the lid facing the opening;
It is desirable that the porous filter is disposed between the main body and the lid.

  When coating the inside of a culture chamber with a scaffold material in a conventional cell culture device, generally, a method is used in which the liquefied scaffold material is poured into a culture chamber from a flow path for medium introduction (or medium discharge). ing. However, in this method, depending on the type of scaffolding material used, the scaffolding material may solidify in the flow path and block the flow path, and there are restrictions on the type of scaffolding material that can be used. On the other hand, according to the cell culture device of the present invention having the above-described configuration, the scaffold material can be directly introduced into the culture chamber by removing the lid from the main body, so that it is easy without clogging the flow path. A scaffold can be coated in the culture chamber. For this reason, the cell culture device according to the present invention can be used for culture using various scaffolds. In addition, the cell culture device is divided into a main body and a lid, and a porous filter is interposed between the two, so that the porous filter is easily disposed between the culture chamber and the medium discharge channel. be able to.

  Note that the contact area between the porous filter and the culture chamber should be as large as possible so that the connection between the culture chamber and the medium discharge channel is not completely blocked by the scaffold captured by the porous filter. preferable. Therefore, for example, the area of the filter in contact with the culture chamber is desirably 50% or more of the maximum area of the culture chamber in a cross section parallel to the filter.

  In the cell culture device according to the present invention, it is desirable that at least one of the contact surfaces of the main body and the lid is made of a material having self-adsorption properties.

  According to such a configuration, the lid can be stably fixed to the main body without using a special fixing means such as a screw or an adhesive, and the lid can be easily attached and detached as necessary. It becomes possible. Examples of the self-adsorbing material include silicon rubber and PDMS (polydimethylsiloxane), but are not limited thereto.

  Further, the cell culture device according to the present invention is arranged so as to close the through-hole and the sheet material having self-adsorption having a through-hole at a position corresponding to a connection portion between the culture chamber and the medium discharge channel. It is desirable that a sealing member having a porous filter is sandwiched between the main body and the lid.

  According to such a configuration, the main body and the lid can be detachably attached by the seal member, and a porous filter can be disposed between the culture chamber and the medium discharge channel. The sealing member may be detachable from both the main body and the lid, or may be fixed to the lower surface of the lid.

  In addition, a cell culture system according to the present invention includes the above cell culture device and a liquid feeding mechanism for feeding a medium to a medium introduction channel of the cell culture device.

Such a cell culture system can be suitably used for the culture of hepatocytes as described above.
That is, the cell culture method according to the present invention is a cell culture method using the cell culture system according to the present invention, wherein a coating material is coated on the culture chamber of the cell culture device, and the culture medium is applied to the culture chamber. Endothelial cells and hepatocytes are co-cultured in the culture chamber while continuously feeding.

  As described above, according to the cell culture device according to the present invention, the cell culture system and the cell culture method using the device, the cell culture using the scaffold can be performed for a long period of time.

The perspective view of the cell culture device which concerns on one Example of this invention. AA arrow sectional drawing of FIG. It is a top view of each member which constitutes a cover part in the example, and (a) shows a lid member, (b) shows the 1st sheet member, and (c) shows the 2nd sheet member. It is a top view of each member which constitutes the main part in the example, and (d) shows the 1st peripheral member, (e) shows the 2nd peripheral member, and (f) shows the bottom member. The schematic block diagram of the cell culture system containing the cell culture device of the Example. The photograph of the cell cultured using the cell culture device concerning the example. The graph which shows the liver function evaluation result of a cultured cell.

  Hereinafter, the form for implementing this invention is demonstrated using an Example. FIG. 1 is a perspective view of the cell culture device according to the present embodiment, and FIG. 2 is a cross-sectional view taken along line AA in FIG.

  The cell culture device 10 according to the present embodiment is roughly composed of a main body 100 and a cover part 200. Furthermore, the cover part 200 is comprised by the cover member 210 as shown in FIG. 3, the 1st sheet member 220, and the 2nd sheet member 230, and the main body 100 is a 1st surrounding surface member as shown in FIG. 110, a second peripheral surface member 120, and a bottom surface member 130.

  The bottom member 130 is made of synthetic quartz, and the first circumferential member 110, the second circumferential member 120, and the lid member 210 are made of PDMS (SILPOT 184, manufactured by Toray Dow Corning). The first sheet member 220 and the second sheet member 230 are made of silicon rubber.

  Each of these members has a flat plate shape having a width of 20 mm and a length of 20 mm, and the thickness is 3 mm for the lid member 210, 0.1 mm for the first sheet member 220 and the second sheet member 230, respectively, The circumferential member 110 and the second circumferential member 120 are each 1.5 mm, and the bottom member 130 is 1 mm.

  Through holes 111, 121, and 231 having a diameter of 10 mm are provided at the centers of the first circumferential member 110, the second circumferential member 120, and the second sheet member 230, respectively. A through hole 221 having a diameter of 13.2 mm is provided in the center of the first sheet member 220. Further, a through hole 112 having a diameter of 1.5 mm is provided at a corner of the first peripheral surface member 110, and the second sheet member 230, the first sheet member 220, and the lid member 210 also correspond to the through hole 112. Through-holes 232, 222, and 211 having a diameter of 1.5 mm are provided at the positions to be operated. Further, the lid member 210 is provided with a through hole 212 having a diameter of 1.5 mm at a corner opposite to the corner where the through hole 211 is provided.

  Concave portions 213 and 113 having a depth of 0.1 mm are formed on the lower surfaces of the lid member 210 and the first peripheral surface member 110, respectively. As shown by the dotted line in FIG. 3A, the recess 213 includes a circular recess having a diameter of 11 mm provided in the center of the lid member 210, and a groove having a width of 2 mm that connects the recess and the through hole 212. Has been. Further, as shown in FIG. 4D, the recess 113 includes a circumferential groove having a width of 1 mm provided at the peripheral edge of the through hole 111 of the first peripheral surface member 110, and the groove and the through hole. It is comprised with the groove | channel of 2 mm in width which connects 112. FIG. These recesses 213 and 113 can both be formed by molding.

  The cover part 200 is formed by bonding the lid member 210, the first sheet member 220, and the second sheet member 230 in this order. Further, the main body 100 is formed by bonding the first peripheral surface member 110, the second peripheral surface member 120, and the bottom surface member 130 in this order. In addition, when joining each member, in order to obtain strong adhesiveness, it is desirable to activate and join the joint surface of each member with oxygen plasma or ultraviolet rays.

  A filter 600 made of a porous film punched into a circle having a diameter of 13 mm is fitted into the through hole 221 provided in the first sheet member 220. In this example, a commercially available nitrocellulose porous membrane (manufactured by Millipore, AAWP, pore size 0.8 μm) was used as the filter 600. Since the diameters of the through holes 231 provided in the second sheet member 230 and the recesses 213 provided in the lid member 210 are smaller than the diameters of the through holes 221 provided in the first sheet member 220, the lid is closed as described above. By bonding the member 210, the first sheet member 220, and the second sheet member 230, the peripheral portion of the filter 600 is sandwiched between the lid member 210 and the second sheet member 230 and is stably held.

  The cell culture device 10 according to the present embodiment is formed by superimposing the cover part 200 on the upper part of the main body 100 formed as described above. At this time, the culture chamber 300 is formed by the through holes 111 and 121, and the culture medium introduction channel 400 is formed by the through holes 211, 222, 232, and 112 and the recess 113. In addition, the medium discharge channel 500 is formed by the recess 213 and the through hole 212. Since the silicon rubber constituting the second sheet member 230 has a self-adsorption property, the cover part 200 can be easily attached to and detached from the main body 100. In addition, the main body 100 and the lid member 210 in the present embodiment correspond to the main body and the lid portion in the present invention, respectively, and the first sheet member 220, the second sheet member 230, and the filter 600 correspond to the seal member in the present invention.

  After sterilizing the main body 100 and the cover part 200 formed as described above with an autoclave, alcohol, or the like, the scaffold 700 is coated on the bottom surface of the culture chamber 300 with the cover part 200 removed. As the scaffold 700, for example, EHS-gel (Engelbreth-Holm-Swarm sarcoma-derived matrix) can be suitably used. EHS-gel is a basement membrane preparation isolated from EHS mouse sarcoma cells and is rich in laminin, type IV collagen and proteoglycans. Since this EHS-gel liquefies at a low temperature and becomes solid at room temperature, it can be fixed to the bottom surface of the culture chamber 300 by pouring into the culture chamber 300 in a cooled state and allowing it to stand at room temperature. Such coating with a scaffold may be performed at the manufacturing stage of the cell culture device, or may be performed by a user who has purchased the cell culture device.

  As described above, according to the cell culture device 10 of the present embodiment, the scaffold 700 can be poured directly into the culture chamber 300 with the cover part 200 removed, and medium introduction (as in the conventional cell culture device) Alternatively, since it is not necessary to introduce the scaffold material from the flow path for medium discharge), the culture chamber 300 can be coated without clogging the flow path.

  When cell culture is performed by the cell culture device 10 described above, the culture medium 300 and the cells 800 are accommodated in the culture chamber 300 with the cover part 200 removed, and then the cover part 200 is attached to the main body 100. At this time, since the cover part 200 adheres to the main body 100 due to the self-adsorption property of the silicon rubber constituting the first sheet member 220, the culture medium leaks to the outside from a portion other than the culture medium introduction channel 400 or the culture medium discharge channel 500. There is nothing. Note that the cells 800 may be accommodated in the culture chamber 300 in the form of tissue pieces, or may be accommodated in a state of being separated into individual cells.

  FIG. 5 shows an outline of a cell culture system using the cell culture device 10 according to the present embodiment. This is a combination of the cell culture device 10 and a liquid feeding mechanism for continuously feeding a medium to the cell culture device 10. The liquid feeding mechanism includes a medium storage unit 910, a medium supply pipe 920, a medium discharge pipe 930, a waste liquid storage unit 940, a liquid feed pump 950, and a control unit 960 that controls operations of the liquid feed pump 950. One end of the medium supply pipe 920 is inserted into the medium storage unit 910, and the other end is inserted into the medium introduction channel 400 of the cell culture device 10. One end of the medium discharge pipe 930 is inserted into the medium discharge channel 500 of the cell culture device 10, and the other end is inserted into the waste liquid storage unit 940.

  The medium stored in the medium storage unit 910 is sucked by the liquid feed pump 950 and sent to the cell culture device 10 through the medium supply pipe 920. The medium supplied to the cell culture device 10 passes through the medium introduction channel 400 and is introduced into the culture chamber 300 from the peripheral surface of the culture chamber 300 as indicated by an arrow in FIG. Further, with the introduction of the culture medium into the culture chamber 300, a part of the culture medium in the culture chamber 300 is discharged from the culture chamber 300 to the outside. At this time, the medium passes through a filter 600 disposed above the culture chamber 300 as shown by an arrow in FIG. 2, reaches the medium discharge channel 500, and discharges the medium connected to the medium discharge channel 500. The liquid is discharged to the waste liquid storage unit 940 through the pipe 930.

  Thus, in the cell culture device 10 according to the present embodiment, when the medium in the culture chamber 300 is discharged from the medium discharge channel 500, the peeled scaffolding material 700 is removed from the medium discharge channel 500 by passing through the filter 600. Clogging of the flow path can be prevented by preventing entry into the channel 500. In addition, as shown in FIG. 2, the medium is introduced from the peripheral surface of the culture chamber 300, and the medium is discharged from the upper part of the culture chamber 300, and the connection portion between the culture chamber 300 and each of the channels 400 and 500. As a result of the wide design, an effect of suppressing the peeling of the scaffold 700 can be obtained by preventing the generation of a strong local flow. Furthermore, since the entire upper opening of the culture chamber 300 is covered with the filter 600, the area of the filter 600 can be increased, so that the entrance of the medium discharge channel 500 is opened by the scaffold 700 captured by the filter 600. The possibility of being completely plugged can be reduced. As described above, according to the cell culture device according to the present example, it is possible to perform long-term culture using a scaffold material, which was difficult with the conventional cell culture device.

  Hereinafter, a cell culture experiment using the cell culture device according to this example will be described. FIG. 6 is a photograph showing the results of culturing hepatocytes using the cell culture device according to this example. Here, EHS-gel is used as a scaffold. First, an epithelial endothelial cell is seeded in a culture chamber coated with the EHS-gel to form an endothelial cell network, and then a liver cell is formed on the endothelial cell. Parenchymal cells were seeded and cell culture was performed. As is clear from the photograph of FIG. 6, hepatocytes are incorporated into the network of endothelial cells, and a co-culture system having a structure close to that of liver tissue in vivo is formed.

  The result of the liver function evaluation test using the hepatocytes is shown in FIG. This uses the cell culture device of this example or a general petri dish for cell culture, and the above-mentioned hippocampal endothelial cells ("GH7" in the figure) and hepatocytes ("hepatocytes" in the figure) Each of these cultured cells was cultured in a single or both co-culture system, and the urea synthesis ability (liver function of decomposing ammonia to synthesize urea) was evaluated. As is clear from the figure, the co-culture system shows a higher liver function than that in the case of culturing the liver parenchymal cells alone. In particular, cells cultured with the cell device of the present invention show higher liver function than those cultured with a petri dish. This is because the culture medium was changed at regular intervals in the culture using the petri dish, whereas the culture medium was continuously supplied (24 hours continuous feeding at a flow rate of 40 μL / min) in the culture using the cell culture device. This is probably because shear stress was applied to the tissue and the culture was performed in an environment closer to the living body.

  In addition, according to the cell culture device according to the present embodiment, the clogging of the flow path due to the peeling of the scaffolding material can be prevented as described above, so that the culture of hepatocytes in an environment close to the living body can be performed for a long time. It is possible to carry out stably over the range. For this reason, it can be used for research in various systems such as drug metabolism tests, and can be expected to be applied to an artificial liver.

  As described above, the cell culture device, the cell culture system, and the cell culture method according to the present invention have been described using the examples. However, the present invention is not limited to the above examples, and the scope of the gist of the present invention. The change is allowed as appropriate. For example, the materials and dimensions of each member constituting the cell culture device are not limited to the above, and various materials and dimensions can be adopted.

DESCRIPTION OF SYMBOLS 10 ... Cell culture device 100 ... Main body 110 ... 1st surrounding surface member 111, 112, 211, 212, 221, 231, 232 ... Through-hole 113, 213 ... Recessed portion 120 ... 2nd surrounding surface member 130 ... Bottom surface member 200 ... Cover Section 210 ... Lid member 220 ... First sheet member 230 ... Second sheet member 300 ... Culture chamber 400 ... Medium introduction channel 500 ... Medium discharge channel 600 ... Filter 700 ... Scaffold material 800 ... Cell 910 ... Medium storage unit 920 ... Medium supply pipe 930 ... Medium discharge pipe 940 ... Waste liquid storage part 950 ... Liquid feed pump 960 ... Control part

Claims (6)

a) a culture chamber containing the cells;
b) a medium introduction channel communicating with the culture chamber;
c) a medium discharge channel communicating with the culture chamber;
d) a porous filter provided between the culture chamber and the medium discharge channel;
A cell culture device comprising: A main body and a lid that is detachably attached to the top of the main body;
The culture chamber is formed in the main body, and the culture chamber has an opening on the upper surface of the main body;
The culture medium discharge channel is formed in the lid, and the culture medium discharge channel has an opening at a position on the lower surface of the lid facing the opening;
The cell culture device according to claim 1, wherein the porous filter is disposed between the main body and the lid.   The cell culture device according to claim 2, wherein at least one of the contact surfaces of the main body and the lid is made of a material having self-adsorption property.   A seal member having a self-adsorbing sheet material provided with a through hole at a position corresponding to a connection portion between the culture chamber and the medium discharge channel, and a porous filter arranged to close the through hole; The cell culture device according to claim 2, wherein the cell culture device is sandwiched between the main body and the lid.   A cell culture system comprising: the cell culture device according to any one of claims 1 to 4; and a liquid feeding mechanism for feeding a medium to a medium introduction channel of the cell culture device. A cell culture method using the cell culture system according to claim 5,
The scaffold is coated in a culture chamber of the cell culture device, and endothelial cells and hepatocytes are co-cultured in the culture chamber while continuously feeding the culture medium to the culture chamber. Cell culture method.