Microchannel device and microobject trapping structure

Abstract

To stably capture a minute object by preventing the minute object from leaking from a capturing part.SOLUTION: There is provided a continuous capturing part for circulating a fluid in a state where a minute object is captured in a flow channel, wherein the continuous capturing part is composed of a cylindrical structure provided so as to protrude on a substrate, the cylindrical structure has an opening capable of capturing the minute object at one end, and all of gap parts provided on a side surface part are set to a size smaller than the minute object.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] One aspect of the present invention relates to a microchannel device, a micro-object capturing structure, and a micro-object capturing method used to capture a micro-object such as a cell. [Background technology]

[0002] Microfluidic devices with trapping structures are known. For example, Non-Patent Document 1 describes a microfluidic device for isolating and culturing single cells. This microfluidic device first traps cells in the cell isolation well by flowing a fluid in one direction through a microchannel with an isolation well located on the bottom and a culture well located on the top. Then, by inverting the microchannel, the cells trapped in the cell isolation well are transferred to the culture well. In this state, a culture medium or the like is pumped into the microchannel to culture the cells. This microfluidic device allows cells to be trapped and cultured using the channel, enabling efficient observation of the division, morphology, phenotype, and other aspects of single cells. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] "Microfluidic device for single cell isolation and culture," Funakoshi, published on October 27, 2021, [Retrieved March 7, 2022], Internet<https: / / www.funakoshi.co.jp / contents / 67201> Summary of the Invention [Problem to be solved by the invention]

[0004] However, the microchannel device described in Non-Patent Document 1 employs a concave hole as the structure of the culture well, which can cause contamination when cells leak from a culture well and get mixed into another culture well downstream when a culture solution or the like is flowing through the microchannel.

[0005] The present invention has been made in light of the above circumstances, and aims to provide a technique that enables stable capture of minute objects by preventing the minute objects from leaking from the capture portion. [Means for solving the problem]

[0006] In order to solve the above problems, one aspect of the microchannel device of this invention is provided with a continuous capture section that allows fluid to circulate within the channel while capturing micro-objects, the continuous capture section consisting of a cylindrical structure that protrudes from a substrate, the cylindrical structure having an opening at one end that can capture the micro-objects, and all of the gaps provided in the side section are set to a size smaller than the micro-objects.

[0007] According to one aspect of the present invention, the continuous capture section is a cylindrical structure having a gap in the side surface, which allows fluid to flow smoothly through the gap while the minute objects are captured within the cylindrical structure. This allows the fluid to react effectively with the minute objects while stably maintaining the captured state without leaking the minute objects. [Effects of the Invention]

[0008] That is, according to one aspect of the present invention, a technique can be provided that enables stable capture of minute objects by preventing the minute objects from leaking out of the capture portion. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram schematically showing an example of the structure of a microchannel device according to an embodiment of the present invention. [Figure 2] FIG. 2 is a perspective view showing a first configuration example of a continuous capture unit provided in the microchannel device shown in FIG. [Figure 3] FIG. 3 is a cross-sectional view of the continuous capture portion shown in FIG. [Figure 4] FIG. 4 is a diagram showing an example of the size of each part of the microchannel device shown in FIG. [Figure 5] FIG. 5 is a diagram illustrating an example of the size of each part of the continuous capture unit illustrated in FIG. [Figure 6] FIG. 6 is a diagram schematically showing an example of a method for capturing a minute object using the microfluidic device shown in FIG. [Figure 7] FIG. 7 is a cross-sectional view showing a second configuration example of the continuous capture section. [Figure 8] FIG. 8 is a cross-sectional view showing a third example of the configuration of the continuous capture section. [Figure 9] FIG. 9 is a cross-sectional view showing a fourth configuration example of the continuous capture section. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0011] [One embodiment] (Configuration example) 1 is a diagram schematically illustrating an example of the structure of a microfluidic device according to an embodiment of the present invention. In this embodiment, cells are used as micro-objects, and a device that captures and cultures cells will be described as an example.

[0012] The microchannel device has a structure in which a first substrate 1 constituting a culture layer and a second substrate 2 constituting a capture layer are arranged opposite each other with a void layer forming a channel 3 interposed therebetween.

[0013] First, a plurality of trapping wells 5, each consisting of a circular recess, are arranged in a matrix at equal intervals on the surface of the second substrate 2. These trapping wells 5 function as temporary trapping sections that isolate and trap minute objects to be trapped, in this example, cells 6.

[0014] For example, as shown in FIG. 4, when the diameter of a cell 6 to be captured is d0, the size of the capture well 5 is set as follows: Depth h2; 2×d0>h2>d0 Inner diameter d2; 2×d0>h2>d0 is set to

[0015] More specifically, if the diameter of a cell is 10 μm, the depth h2 and inner diameter d2 are both set to 15 μm. However, the size of the trapping well 5 is not limited to the above values ​​and can be set arbitrarily depending on the size, shape, properties, etc. of the minute object to be trapped.

[0016] On the other hand, a plurality of culture wells 4 are arranged at equal intervals in a matrix on the surface of the first substrate 1. The culture wells 4 function as continuous capture units that maintain the capture of the cells 6 transferred from the capture wells 5 in order to culture the cells 6.

[0017] The culture well 4 has a cylindrical structure in which a plurality of arc-shaped pillars 40a are arranged with slit-shaped gaps 40b between them, as shown in Fig. 2. Fig. 3 is a diagram showing the cross-sectional shape of the well 4 in Fig. 2.

[0018] As shown in FIGS. 4 and 5, the size of the culture well 4 is determined such that, with respect to the diameter d0 of the cell 6 to be trapped, the height h1, diameter d1, and width x1 of the gap 40b are set to be equal to or smaller than the diameter d0 of the cell 6 to be trapped, so that the cell 6 in the culture well 4 can be reacted with the culture components contained in the fluid (culture solution in this example) while continuously trapping the cell 6. Height h1; h1>d0 Diameter d1; d1>d0 Width of gap 40b x1; x1 <d0 It is set so that:

[0019] More specifically, if the diameter d0 of the cell 6 is 10 μm, the height h1, diameter d1, and width x1 of the gap 40b are set to h1=30 μm, d1=100 μm, and x1=8 μm, respectively. However, the size of the culture well 4 is not limited to the above values ​​and can be set arbitrarily depending on the size, shape, properties, etc. of the micro-object to be captured.

[0020] The number n of pillars 40a is set to n≧2 to ensure that the culture solution flows into and out of the culture well 4 through the gaps 40b. FIGS. 2 and 3 show an example where n=6. The larger the number n of pillars 40a, the higher the cost, but the more effective it is to increase the diffusibility of the fluid in the culture well 4. The thickness t1 of pillars 40a is desirably set to, for example, t1=20 μm to ensure ease of manufacturing and rigidity.

[0021] The position of the culture well 4 is preferably set so that the center of the culture well 4 corresponds to the center of the capture well 5 in order to efficiently capture the cells 6 from the capture well 5 described below.

[0022] The height h3 and width w3 of the channel 3 of the microfluidic device are set to, for example, Height h3; h3>h1+d0 Width w3; w3>d1 is set to

[0023] More specifically, the height h3 is set to 50 μm and the width w3 to 150 μm, but these values ​​are not limited to these and can be set arbitrarily depending on the size, shape, properties, etc. of the minute object to be captured.

[0024] The culture wells 4 and capture wells 5 described above can be fabricated, for example, by irradiating polydimethylsiloxane (PDMS) with an electron beam. An example of a fabrication technique for the culture wells 4 and capture wells 5 using PDMS as a material is described in, for example, the following references:

[0025] References: "Development of a small 'puddle' that captures cells - Toward the realization of an advanced medical device that captures and cultures cells one by one", National Institutes for Quantum and Radiological Science and Technology, updated May 28, 2018, [Retrieved March 29, 2022], Internet<URL;https: / / www.qst.go.jp / site / press / 1231.html> The microchannel device may be made of a material such as glass or other silicon resins, and may be manufactured by injection molding using photolithography, for example.

[0026] (Example of operation) Next, an example of the operation of the microfluidic device configured as above will be described according to the procedure from capturing the target cell 6 to culturing it.

[0027] FIG. 6 is a flow chart showing a schematic example of a procedure from capturing to culturing cells 6 using the microfluidic device.

[0028] For example, in Step 1, a tester first sets up the microfluidic device so that the first substrate 1 on which the culture wells 4 are formed is on the upper side and the second substrate 2 on which the capture wells 5 are formed is on the lower side. In this state, in Step 2, a fluid containing cells 6 to be captured is circulated through the channel 3 in a certain direction B. This fluid circulation is achieved, for example, by injecting the fluid using a pump from a fluid inlet (not shown). As a result of the fluid circulation, in Steps 3 and 4, the cells 6 contained in the fluid are captured one by one in the capture wells 5. In other words, the cells 6 are isolated and captured by the capture wells 5.

[0029] Next, in Step 5, the tester turns the microfluidic device upside down so that the second substrate 2 on which the capture well 5 is formed is on the upper side and the first substrate 1 on which the culture well 4 is formed is on the lower side. As a result, in Step 6, the cell 6 captured in the capture well 5 falls due to gravity and is captured by the culture well 4 located directly below the capture well 5. At this time, the diameter d1 of the culture well 4 is designed to be sufficiently larger than the diameter d0 of the cell 6. Therefore, the cell 6 is captured in the culture well 4 efficiently with a high probability.

[0030] After a sufficient time has passed for the cells 6 to be captured by the culture well 4, the tester then, in Step 7, causes the culture medium to flow through the flow channel 3 in a fixed direction B. This inflow of the culture medium is also achieved by injecting the culture medium through a fluid inlet (not shown) using, for example, a pump.

[0031] As described above, the culture well 4 has a structure in which multiple pillars 40a are arranged in a cylindrical shape with slit-like gaps 40b between them. Therefore, the culture solution flows into the culture well 4 through the gaps 40b, acts on the cells 6 trapped in the culture well 4, and then flows out of the gaps 40b.

[0032] Furthermore, the height h1 of the pillars 40a is set to be higher than the diameter d0 of the cells 6, and the width x1 of the gaps 40b is set to be smaller than the diameter d0 of the cells 6. Therefore, the flow of the culture medium is unlikely to cause the cells 6 to leak out of the culture wells 4 and mix with the downstream culture wells 4, which is expected to be effective in preventing contamination. Furthermore, the culture medium can be circulated at a high flow rate, which allows the culture components to react with the cells 6 continuously and efficiently.

[0033] That is, by using the microfluidic device of one embodiment, it is possible to cause a high-speed reaction on the cells 6 while preventing contamination by continuing to capture the cells 6 in the culture well 4.

[0034] (Actions and Effects) As described above, in one embodiment of the microchannel device, the culture well 4 has a cylindrical structure in which multiple pillars 40a, each with a height h1 set higher than the diameter d0 of the cell 6, are arranged across gaps 40b set smaller than the diameter d0 of the cell 6, and the inner diameter is set sufficiently larger than the diameter d0 of the cell 6.

[0035] Therefore, the cells 6 temporarily captured in the capture well 5 can be efficiently transferred to the culture well 4, and the captured state of the cells 6 can be maintained by the culture well 4 to prevent contamination, and then a fluid such as a culture medium can be reacted with the cells 6 at a high flow rate and efficiently.

[0036] In one embodiment, six gaps 40b are formed by combining six pillars 40a of the culture well 4. This allows the culture solution to circulate while being sufficiently diffused within the culture well 4, thereby allowing the culture components to react with the cells 6 extremely effectively.

[0037] [Other embodiments] (1) The structure of the culture well 4 as the continuous capture unit can be modified in various ways in addition to the one described in the embodiment. For example, as shown in Fig. 7, the culture well 4 may be configured as a triangular cylindrical structure by forming three pillars 41a into flat plates and arranging these pillars 41a with gaps 41b between them.

[0038] Furthermore, as shown in FIG. 8, for example, the culture well 4 may be configured as a rectangular cylindrical structure by arranging four pillars 42a each having an L-shaped cross section with gaps 42b between them.

[0039] 8, the culture well 4 may be configured as a diamond-shaped cylinder by arranging four flat pillars 43a with gaps 43b between them. In this case, the arrangement of the pillars 43a is not limited to a diamond shape and may be a square shape.

[0040] In addition, the shape of the gaps may be circular, oval, elliptical, rectangular, etc., other than slit-like, and the shape of the pillars, the number of pillars and gaps, and the orientation of the culture well 4 relative to the flow path 3 can also be set arbitrarily.

[0041] (2) Protrusions or the like may be formed on the inner wall surface of the pillars 40a of the culture well 4. In this way, the diffusibility of the culture solution in the culture well 4 can be further improved.

[0042] (3) In addition, various modifications can be made to the shape and size of the capture well, the shape and size of the flow path 3, the type of micro-object to be captured (e.g., microparticles of chemical or metallic materials, beads), the type of fluid (e.g., water, oil, various culture media, various reagents), the means for delivering the fluid (e.g., air pressure pump, syringe pump, dropper), the structure, material, size, and manufacturing method of the temporary capture section and the continuous capture section, etc., without departing from the spirit of this invention.

[0043] Although the embodiments of the present invention have been described in detail above, the above description is merely an example of the present invention in every respect. It goes without saying that various improvements and modifications can be made without departing from the scope of the present invention. In other words, when implementing the present invention, specific configurations according to the embodiments may be appropriately adopted.

[0044] In short, this invention is not limited to the above-described embodiments, and in the implementation stage, the components can be modified and embodied without departing from the spirit of the invention. Furthermore, various inventions can be formed by appropriately combining multiple components disclosed in the above-described embodiments. For example, some components may be omitted from all the components shown in the embodiments. Furthermore, components from different embodiments may be appropriately combined. [Explanation of symbols]

[0045] 1...First board 2...Second board 3...Flow path 4...Culture well 5...Capture well 6...Cell 40a, 41a, 42a, 43a...pillars 40b, 41b, 42b, 43b...Gap part

Claims

1. A microchannel device having a continuous capture unit that allows a fluid to flow in a channel while capturing a micro object, A microchannel device in which the continuous capture section consists of a cylindrical structure protruding from a substrate, the cylindrical structure having an opening at one end capable of capturing the micro-object, and all of the gaps provided on the side section are set to a size smaller than the micro-object.

2. a continuous capture unit that circulates fluid in a state in which minute objects are captured within the flow channel; a temporary capture unit that isolates and captures the minute objects in the flow channel and transfers the captured minute objects to the continuous capture unit; Equipped with The continuous capture unit is configured as a cylindrical structure having an opening at one end capable of capturing the minute object and a gap on the side surface. Microfluidic device.

3. 3. The microchannel device according to claim 1, wherein the tubular structure is a cylinder having the opening at an upper portion and the slit-shaped gap formed in the side surface.

4. 3. The microchannel device according to claim 1, wherein the cylindrical structure has a plurality of gaps formed in a side surface thereof at positions corresponding to a direction of flow of the fluid.

5. The microfluidic device according to claim 2 , wherein the continuous trapping section and the temporary trapping section are arranged such that their openings face each other across the channel.

6. A micro-object capturing structure that is provided in a flow path and allows a fluid to flow while capturing a micro-object, A micro-object capture structure comprising a cylindrical structure arranged to protrude from a substrate, the cylindrical structure having an opening at one end capable of capturing the micro-object, and all of the gaps provided in the side portion being set to a size smaller than the micro-object.

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