JP6192007B2 - Microdevice and bioassay system - Google Patents

Description

  The present invention relates to a microdevice and a bioassay system used for drug discovery and biotechnology research, and particularly used for culturing cells and evaluating their functions and responses.

  In recent years, development of a device for cell culture called a microchip using a microfabrication technique is progressing. The device includes a microchannel made in glass, silicon wafer, PDMS, or other resin substrate, and cultures cells while flowing a medium through the channel at a flow rate on the order of μL / min. Samples, reagents, and waste liquids can be reduced in volume, and the specific interface area of the reaction part is large, so that the cells can respond efficiently. In addition, real-time evaluation is possible by installing a measuring instrument in the culture part or in the lower part of the flow path.

  In experiments using microchips, one of the biggest problems is that some of the gas contained in the medium becomes bubbles in the microchannels, blocking the channels and peeling cultured cells from the microchips. It means that.

  In order to solve this problem, a method (Patent Document 1) in which a bubble separation unit including a plurality of holes having a water-repellent surface whose ends are open to the atmosphere is provided on the upper side of the microchannel. A method (Patent Document 2) including a deaeration unit that drops a liquid from a position higher than the liquid level of the stored liquid and removes air on the upper side of the liquid level (Patent Document 2), and a culture medium There has been proposed a method (Patent Document 3, Patent Document 4) or the like in which a chamber communicating with a supply channel is provided, and bubbles in the medium are moved upward from the chamber in the chamber.

JP 2006-223118 A JP 2011-163939 JP 2013-106595 A JP 2008-82961 A

  In the first method, it is difficult to control the contact angle of the hole surface and the size of the hole. Both the second and third methods need to store a certain amount of liquid and greatly increase the internal volume of the flow path, which not only impairs the effect of experimenting with the micro flow path, Processing is also complicated.

  Therefore, the present invention provides a microdevice for preventing the generation of bubbles in a microchannel and a bioassay system including the microdevice, which are easy to process without greatly increasing the internal volume of the channel. The purpose is to provide.

That is, the present invention is a microdevice for capturing at least a part of a gas contained in a medium or a chemical solution in cell culture in a microchip,
A culture medium or chemical solution inlet;
A culture medium or chemical solution outlet,
A microdevice comprising a porous partition wall having a hydrophobic surface provided between an inlet and an outlet of the culture medium or drug solution.

  Moreover, this invention is a bioassay system provided with the said microdevice.

  The present invention is different from the method of the prior art in that the bubbles are removed from the flow path or kept in the flow path in a state of being attached to the partition wall without separately providing a foam storage section. Since only the partition wall is introduced into the medium supply channel, processing is easy and the internal volume does not increase greatly.

It is sectional drawing which shows the one aspect | mode of the microdevice of this invention. It is sectional drawing which shows the other aspect of the microdevice of this invention. It is the schematic of a bioassay system provided with the microdevice of this invention. It is a perspective view of a bioassay system provided with the microdevice of this invention.

  Hereinafter, the present invention will be described with reference to embodiments, but the present invention is not limited to the following embodiments. Components having the same function or similar functions in the figures are given the same or similar reference numerals and description thereof is omitted. However, since the drawings are schematic, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

  FIG. 1 is a cross-sectional view perpendicular to a culture medium or chemical solution (hereinafter collectively referred to as “medium”) flow path of one embodiment of the microdevice of the present invention. The microdevice 10 includes a medium inlet 10a, a medium outlet 10b, and a porous partition wall 11 having a hydrophobic surface, and the medium flows in the direction of the arrow in the upper part of the figure. In this aspect, the diameters of the medium inlet 10a and the outlet 10b are substantially the same as the diameter of the medium flow path. For example, the device 10 can be connected to the medium flow path using a ferrule. Alternatively, the device 10 may be integrated with the bioassay system by adhering the partition wall 11 to the medium flow path. In FIG. 1, white circles schematically represent bubbles and middle black circles schematically represent some insoluble contaminants. The figure conceptually shows the state of being attached to the culture medium inlet side surface of the partition wall 11, but it is not necessarily the surface and may be inside. Surprisingly, it has been confirmed that generation of bubbles in the culture part can be prevented without removing the foam out of the flow path or providing a separate foam storage part.

  FIG. 2 is a cross-sectional view showing a mode in which the cross-sectional area of the partition wall 11 is larger than the cross-sectional area of the culture medium channel. Thus, by widening the cross-sectional area of the partition wall 11 and widening the flow of the culture medium, there is an effect that the pressure of the culture medium fluid is reduced and bubbles are easily released. However, since it is preferable not to increase the internal volume of the medium supply channel, the cross-sectional area of the partition wall 11 is preferably 1.1 to 100 times, more preferably 10 to 70 times the cross-sectional area of the medium supply channel. More preferred. Further, the cross-sectional shape of the partition wall 11 is not necessarily circular, and may be any shape such as a quadrangle. In FIG. 2, the distance between the culture medium inlet 10a and the partition wall 11 and the distance between the partition wall 11 and the culture medium outlet 10b are preferably the shortest distance at which the partition wall 11 can be fixed.

  The partition wall 11 is provided with a hydrophobic surface, so that at least a part of the gas present in or dissolved in the culture medium can be captured as bubbles and prevented from flowing downstream. Such hydrophobic surfaces include, for example, polyolefin resins, polyether resins, polyethersulfone resins, poly (meth) acrylate resins, polyamide resins, polyester resins, polycarbonate resins, polyimide resins, and fluorine resins. It can be composed of a hydrophobic polymer selected from the group consisting of resins and silicone resins. The entire partition wall need not be a hydrophobic polymer, and a metal such as stainless steel or titanium having a hydrophobic polymer layer formed on the surface by a hydrophobic treatment, glass, or ceramic can also be used. In view of the fact that it can be finely processed and can be disposable, it is preferably a hydrophobic such as cyclic polyolefin resin, polyetheretherketone, polytetrafluoroethylene, polyetheretherketone / polytetrafluoroethylene alloy (PAT), or polydimethylsiloxane. A porous body of a conductive polymer is used.

  The pore diameter of the partition wall 11 is preferably 0.1 μm or more, and more preferably 2 μm or more. If the pore diameter is less than the lower limit, the fluid resistance tends to increase. The upper limit of the pore diameter is not particularly limited, but is preferably 20 μm or less and more preferably 10 μm or less in terms of removing impurities that may be mixed into the medium.

  The device 10 has a tubular material that forms a medium flow path, such as PEEK (polyetheretherketone), or a partition wall 11 or a screw that sandwiches the partition wall 11 from both the medium inlet side and the outlet side. Can be made using means for pressing and fixing from both sides. Various adhesives, for example, adhesives made of UV curable resin can be used for the adhesion. As a means for pressing and fixing, for example, a commercially available filter holder or connector can be used.

  The microdevice 10 of the present invention is suitably used, for example, in the bioassay system 1 shown in FIG. In this system, a culture unit is provided with a substrate (microchip) 21 having a microchannel 21a formed on the surface thereof by a pump 33 via a medium supply channel L3, the microdevice 10, and then the channel L2. After being sent to 20, the waste liquid is collected by the waste liquid collecting section 35 through the waste liquid flow path L4. The flow rate of the medium is usually 10 μL / min or less, preferably 1 μL / min or less.

  On the other hand, a sample such as a cell is sent from the syringe 31 to the culture unit 20 via the sample supply channel L1. One of the medium supply channel L3 and the sample supply channel L1 is connected to the culture unit 20 by the channel switching valve 13. The flow path switching valve 13 includes at least three ports of a medium inlet, a sample inlet, and an outlet that have passed through the partition wall 11, and the valves are switched to connect any one of the inlet ports and the outlet ports. As long as the internal flow path is formed, any form can be used. In order to reduce the internal volume, it is preferable to use a valve having a flow path inner diameter of about 0.05 to 0.8 mm.

  The microdevice 10 may be integrated with the flow path switching valve 13. In this case, the partition wall 11 is provided between the flow path switching valve 13 and the culture medium inlet 10 a, and the culture medium outlet 10 b is connected to the switching valve 13. Connected to one port.

  FIG. 4 is a perspective view of a bioassay system including a microdevice in which a partition wall 11 and a flow path switching valve 13 are integrated. The partition wall is built in a position indicated by 11 in the figure between the channel switching valve 13 and the medium supply channel L3. The culture unit 20 disposed on the base 60 includes a substrate 21, a frame-shaped fixture 23 having an opening provided at a location corresponding to the microchannel 21 a disposed on the substrate 21, and the fixture 23. Are made up of a plurality of screws 27a... 27d attached to the base 60. A plurality of liquid supply ports 29a, 29b,... 29h connected to the microchannel 21a are provided on the cage of the fixture 23. In addition, the path | route pattern of the microchannel 21a is not restrict | limited to linear form, A various pattern can be used according to a use.

  The material of the substrate 21 is not particularly limited as long as it does not adversely affect the cell culture, and various materials can be used. In general, resins such as glass, silicon wafer, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin polymer, and polystyrene can be used. In consideration of the size of the cells, the inner diameter of the microchannel 21a is preferably 1 mm or less, and particularly preferably 100 to 500 μm.

  The type of cell that can be used in this embodiment is not particularly limited as long as it has the ability to adhere to the microchannel.

  In addition, when administering an irritant in a drug assay or the like, solution exchange can be performed by replacing the medium supply flow path L3 with another tube and connecting a syringe containing the drug.

  In another aspect, the bioassay system may include a plurality of flow path switching valves 13 arranged in parallel. Further, the channel switching valve 13 may be further arranged in series between the culture unit 20 and the channel switching valve 13. This is because the liquid flow to the culture unit 20 can be stopped more reliably. Further, the micro device 10 may be provided also on the waste liquid flow path L4 side of the culture unit 20.

[Example]
Using the microdevice and bioassay system of the present invention, RF / 6A135 cells were cultured according to the following procedure.

(A) A bioassay system shown in FIG. 3 provided with the microdevice shown in FIG. 2 was prepared. The partition wall 11 is made of a polyether ether ketone frit (trade name: Upchurch Frit A-706), and has an inner diameter of 0.64 mm and a thickness of 0.16 mm. : 3. This was fixed to the medium supply channel by pressing with a connector.
As the flow path switching valve 13, a valve manufactured using a fluororesin was used.
As the microchip 21, a linear channel having a width of 0.3 mm, a depth of 0.1 mm, and a length of 60 mm was used, and the inside of the channel was previously coated with collagen.

(B) Next, after confirming that the channel switching valve 13 of the microdevice 10 is in a predetermined position so that the sample supply channel L1 and the channel L2 are connected, the sample supply channel L1 is 10 ^6. A cell suspension having a concentration of about 1 cell / mL was injected into the substrate 21 via the flow path switching valve 13. After the injection, the cells were allowed to stand for 2 hours to adhere the cells to the channel wall surface.

(C) After confirming that the cells adhere to the channel wall surface, it is confirmed that the flow path switching valve 13 of the microdevice 10 is in a predetermined position, and the pump 33 connected to the culture medium supply flow path L3. Was used to perfuse the medium at a flow rate of 0.1 μL / min. After 48 hours, when the microchip 21 was observed, no bubbles were observed.

[Comparative example]
RF / 6A135 cells were cultured using the same bioassay system as in the Examples except that the microdevice 10 was not provided, and the medium was perfused at a flow rate of 0.1 μL / min. After about 1 hour, bubbles were confirmed in the microchip, and peeling of the RF / 6A135 cells was observed.

  According to the present invention, there are provided a microdevice and a bioassay system that can prevent inflow of bubbles and foreign substances when a culture medium or a chemical solution is passed through a culture part in cell culture using a microchip.

1: Bioassay system 10: Micro device 11: Partition wall 13: Flow path switching valve 20: Culture unit 21: Substrate (microchip)
21a: Micro flow path 31: Syringe 33: Pump 35: Waste liquid recovery unit L1: Sample / chemical liquid supply flow path L2: Flow path L3: Medium supply flow path L4: Waste liquid flow path

Claims (6)

A bioassay system comprising a culture part having a microchip for culturing cells in the perfusion of a medium or a drug solution, and a microdevice for capturing at least part of a gas contained in the medium or drug solution,
The microdevice is
A culture medium or chemical solution inlet;
A culture medium or chemical solution outlet,
Provided between the flow inlet and outlet, provided with a porous partition wall having a hydrophobic surface,
The microdevice characterized Rukoto disposed upstream of the culture unit, bioassay system. The bioassay system according to claim 1, wherein a cross-sectional area of the partition wall is 1.1 to 100 times a cross-sectional area of the culture medium or drug solution supply channel. The hydrophobic surface of the partition is polyolefin resin, polyether resin, polyethersulfone resin, poly (meth) acrylate resin, polyamide resin, polyester resin, polycarbonate resin, polyimide resin, fluorine resin. The bioassay system according to claim 1 or 2, comprising at least one hydrophobic polymer selected from the group consisting of silicone resins. The bioassay according to claim 3, wherein the partition wall comprises at least one selected from the group consisting of cyclic polyolefin, polyetheretherketone, polydimethylsiloxane, polytetrafluoroethylene, and polyetheretherketone / polytetrafluoroethylene alloy. System . The bioassay system according to any one of claims 1 to 4, wherein a pore diameter of the partition wall is 0.1 µm to 20 µm. The bioassay system according to any one of claims 1 to 5, further comprising a flow path switching valve that connects one of the culture medium or drug solution supply flow path and the sample supply flow path to the culture unit.

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