WO2023195043A1 - Microchannel device, micro object trapping structure, and micro object trapping method - Google Patents

Microchannel device, micro object trapping structure, and micro object trapping method Download PDF

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WO2023195043A1
WO2023195043A1 PCT/JP2022/017035 JP2022017035W WO2023195043A1 WO 2023195043 A1 WO2023195043 A1 WO 2023195043A1 JP 2022017035 W JP2022017035 W JP 2022017035W WO 2023195043 A1 WO2023195043 A1 WO 2023195043A1
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capturing
continuous
capture
microchannel device
temporary
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PCT/JP2022/017035
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French (fr)
Japanese (ja)
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伸明起 遠藤
和宏 高谷
真奈美 伊藤
壮輔 今村
佳織 高柳
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日本電信電話株式会社
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Priority to PCT/JP2022/017035 priority Critical patent/WO2023195043A1/en
Priority to JP2024513574A priority patent/JPWO2023195043A1/ja
Publication of WO2023195043A1 publication Critical patent/WO2023195043A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

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  • One aspect of the present invention relates to a microchannel device, a micro-object capture structure, and a micro-object capture method that are used to capture micro-objects such as cells.
  • Non-Patent Document 1 describes a microchannel device for isolating and culturing a single cell.
  • this microchannel device cells are first captured by the cell isolation well by flowing fluid from one direction through a microchannel in which an isolation well is disposed on the lower side and a culture well is disposed on the upper side. Then, by turning the microchannel upside down, the cells trapped by the cell isolation well are transferred to the culture well. It is designed to cultivate.
  • this microchannel device cells can be trapped and cultured using the channel, and states such as division, morphology, and phenotype of single cells can be efficiently observed.
  • Non-Patent Document 1 employs a concave hole as the structure of the culture well. For this reason, when a culture solution or the like is poured into the microchannel, cells sometimes leak out from the culture well and mix into another culture well on the downstream side, resulting in contamination.
  • This invention has been made in view of the above-mentioned circumstances, and aims to provide a technique that enables stable capture of minute objects while preventing the minute objects from leaking out of the capturing section.
  • one aspect of the microchannel device is provided with a continuous trapping section that allows fluid to flow in a state in which a microscopic object is captured in the channel, and the continuous trapping section is attached to one end. It is constructed of a cylindrical structure having an opening capable of capturing the minute object, and a gap portion set on a side surface to be smaller than the minute object and having a size that allows the fluid to flow through.
  • the continuous trapping section is a cylindrical structure having a gap in the side surface, so that fluid can smoothly flow through the gap while a micro object is captured in the cylindrical structure. can be done. Therefore, it is possible to stably maintain the captured state without leaking the micro objects and to cause the fluid to react effectively with the micro objects.
  • FIG. 1 is a diagram schematically showing an example of the structure of a microchannel device according to an embodiment of the present invention.
  • FIG. 2 is a perspective view showing a first configuration example of a continuous capture section provided in the microchannel device shown in FIG.
  • FIG. 3 is a cross-sectional view of the continuous capture section shown in FIG. 2.
  • FIG. 4 is a diagram showing an example of the size of each part of the microchannel device shown in FIG. 1.
  • FIG. 5 is a diagram showing an example of the size of each part of the continuous acquisition section shown in FIG. 3.
  • FIG. 6 is a diagram schematically showing an example of a method for capturing a minute object using the microchannel device shown in FIG. 1.
  • FIG. 7 is a cross-sectional view showing a second configuration example of the continuous capture section.
  • FIG. 8 is a cross-sectional view showing a third configuration example of the continuous capture section.
  • FIG. 9 is a cross-sectional view showing a fourth configuration example of the continuous capture unit.
  • FIG. 1 is a diagram schematically showing an example of the structure of a microchannel device according to an embodiment of the present invention.
  • cells are used as micro objects, and a device for capturing and culturing cells will be described as an example.
  • 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 placed facing each other with a void layer forming a channel 3 interposed therebetween.
  • trapping wells 5 consisting of circular recesses are arranged in a matrix at equal intervals. These trapping wells 5 function as a temporary trapping section that isolates and traps microscopic objects to be captured, in this example cells 6.
  • the size of the capture well 5 is such that when the diameter of the cell 6 to be captured is d0, in order to isolate and capture the cell 6, Depth h2; 2 ⁇ d0>h2>d0 Inner diameter d2; 2 ⁇ d0>h2>d0 is set to
  • both the depth h2 and the inner diameter d2 are set to 15 ⁇ m.
  • the size of the capture well 5 is not limited to the above value, and can be arbitrarily set depending on the size, shape, properties, etc. of the micro object to be captured.
  • a plurality of culture wells 4 are arranged in a matrix at equal intervals.
  • the culture well 4 functions as a continuous trapping section that maintains trapping of the cells 6 in order to culture the cells 6 transferred from the trapping well 5.
  • the culture well 4 has a cylindrical structure in which a plurality of arc-shaped pillars 40a are arranged with slit-shaped gaps 40b in between.
  • FIG. 3 is a diagram showing the cross-sectional shape of FIG. 2.
  • the size of the culture well 4 is such that the culture components contained in the fluid (culture solution in this example) can be transferred into the culture well 4 while continuously capturing the cells 6.
  • the height h1, the diameter d1, and the width x1 of the gap 40b are set to the diameter d0 of the cell 6 to be captured, respectively: height h1; h1>d0 Diameter d1; d1>d0 Width x1 of gap 40b; x1 ⁇ d0 It is set so that
  • the size of the culture well 4 is not limited to the above value, and can be arbitrarily set depending on the size, shape, properties, etc. of the microscopic object to be captured.
  • the number n of pillars 40a is set to n ⁇ 2 in order to ensure that the culture solution flows into and out of the culture well 4 through the gap 40b.
  • the arrangement position of the culture well 4 is desirably set so that the center of the culture well 4 corresponds to the center of the capture well 5 in order to efficiently capture cells 6 from the capture well 5, which will be described later.
  • the height h3 and width w3 of the channel 3 of the microfluidic device are such that, for example, Height h3; h3>h1+d0 Width w3; w3>d1 is set to More specifically, the height h3 is set to 50 ⁇ m and the width w3 is set to 150 ⁇ m, but it is not limited to these values and can be set arbitrarily depending on the size, shape, properties, etc. of the minute object to be captured. It is possible.
  • the culture well 4 and capture well 5 described above can be produced, for example, by irradiating polydimethylsiloxane (PDMS) with an electron beam.
  • PDMS polydimethylsiloxane
  • An example of a technique for producing culture wells 4 and capture wells 5 using PDMS as a material is described, for example, in the following references.
  • the material of the microchannel device for example, glass or other silicone resin can be used, and as for the manufacturing method, for example, injection molding using photolithography can be used.
  • FIG. 6 is a flowchart schematically showing an example of the processing procedure from capturing to culturing the cells 6 using the microchannel device.
  • Step 1 the test person first installs the microchannel device so that the first substrate 1 on which the culture well 4 is formed is on the upper side and the second substrate 2 on which the capture well 5 is formed is on the lower side.
  • Step 2 the fluid containing the cells 6 to be captured is made to flow in the constant direction B through the channel 3.
  • This fluid circulation is performed, for example, by injecting fluid from a fluid inlet (not shown) using a pump. Due to the flow of the fluid, cells 6 contained in the fluid are captured one by one in the capture well 5 in Steps 3 and 4. That is, the cells 6 are isolated and captured using the capture well 5 .
  • Step 5 the test person turns the microchannel device upside down so that the second substrate 2 on which the capture well 5 is formed is on the top, and the first substrate 1 on which the culture well 4 is formed is on the bottom.
  • Step 6 the cells 6 captured in the capture well 5 fall due to gravity and are captured by the culture well 4 located directly below the capture well 5.
  • the diameter d1 of the culture well 4 is designed to be sufficiently larger than the diameter d0 of the cells 6. Therefore, the cells 6 are efficiently captured in the culture well 4 with a high probability.
  • the test person After the necessary and sufficient time has elapsed for the cells 6 to be captured by the culture well 4, the test person causes the culture solution to flow in the constant direction B through the channel 3 in Step 7.
  • This inflow of the culture solution is also performed by injecting the culture solution from a fluid inlet (not shown) using, for example, a pump.
  • the culture well 4 has a structure in which a plurality of pillars 40a are arranged in a cylindrical shape with a slit-shaped gap 40b between them. Therefore, the culture solution flows into the culture well 4 through the gap 40b, acts on the cells 6 captured in the culture well 4, and then flows out from the gap 40b.
  • the height h1 of the pillar 40a is set higher than the diameter d0 of the cell 6, and the width x1 of the gap 40b is set smaller than the diameter d0 of the cell 6. Therefore, problems such as cells 6 leaking out of the culture well 4 due to the circulation of the culture solution and contaminating the culture well 4 on the downstream side are unlikely to occur, and this can be expected to be effective in preventing the occurrence of contamination. In addition, it becomes possible to flow the culture solution at a high flow rate, thereby making it possible to cause the culture components to react with the cells 6 continuously and efficiently.
  • the microchannel device of one embodiment it is possible to continue capturing the cells 6 in the culture well 4 and prevent the occurrence of contamination, and then cause the cells 6 to perform a high-speed reaction. It becomes possible.
  • the culture well 4 has a plurality of pillars 40a whose height h1 is set higher than the diameter d0 of the cells 6, and the plurality of pillars 40a are set smaller than the diameter d0 of the cells 6. They are arranged with a gap 40b in between, and have a cylindrical structure with an inner diameter set sufficiently larger than the diameter d0 of the cell 6.
  • the cells 6 temporarily captured in the capture well 5 are efficiently transferred to the culture well 4, and the culture well 4 maintains the captured state of the cells 6 to prevent contamination. It becomes possible to cause the cells 6 to react efficiently at a high flow rate.
  • six gaps 40b are formed by combining six pillars 40a of the culture well 4. Therefore, it becomes possible to circulate the culture solution while sufficiently diffusing it in the culture well 4, thereby making it possible to cause the culture components to react with the cells 6 extremely effectively.
  • the culture well 4 may be constructed into a square cylindrical structure by arranging four pillars 42a each having an L-shaped cross section with a gap 42b in between. good.
  • the culture well 4 may be formed into a rhombic cylinder by arranging four flat pillars 43a with gaps 43b in between.
  • the arrangement shape of the pillars 43a is not limited to a diamond shape, but may be a square shape.
  • the shape of the gap may be circular, oval, elliptical, square, etc. in addition to the slit shape, and the shape of the pillar, the number of pillars and gaps, and the size of the culture well 4 with respect to the flow path 3.
  • the orientation of the arrangement can also be set arbitrarily.
  • Protrusions or the like may be formed on the inner wall surface of the pillar 40a of the culture well 4. In this way, it becomes possible to further improve the diffusivity of the culture solution within the culture well 4.
  • the present invention is not limited to the above-described embodiments as they are, but can be embodied by modifying the constituent elements at the implementation stage without departing from the spirit of the invention.
  • various inventions can be formed by appropriately combining the plurality of components disclosed in the above embodiments. For example, some components may be deleted from all the components shown in the embodiments. Furthermore, components from different embodiments may be combined as appropriate.

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Abstract

One aspect of this invention comprises a continuous trapping part for flowing fluid in the state where a micro object is trapped inside a channel. The continuous trapping part is composed of a cylindrical structural body that has, on one end part of the cylindrical structural body, an opening part capable of trapping the micro object and has, on a side surface part, a gap part, wherein the size of the gap part is set so as to be smaller than the micro object and to be a size allowing the fluid to flow.

Description

マイクロ流路デバイス、微小物体捕捉構造および微小物体捕捉方法Microfluidic device, micro object capture structure, and micro object capture method
　この発明の一態様は、例えば細胞などの微小物体を捕捉するために用いられる、マイクロ流路デバイス、微小物体捕捉構造および微小物体捕捉方法に関する。 One aspect of the present invention relates to a microchannel device, a micro-object capture structure, and a micro-object capture method that are used to capture micro-objects such as cells.
　トラップ構造体を有するマイクロ流路デバイスが知られている。例えば、非特許文献１には、単一の細胞を単離し培養するマイクロ流路デバイスが記載されている。このマイクロ流路デバイスは、先ず単離用ウェルを下側に配置し培養ウェルを上側に配置したマイクロ流路に一方向から流体を流すことで、上記細胞単離用ウェルにより細胞を捕捉する。そして、上記マイクロ流路の上下を反転させることにより、上記細胞単離用ウェルによりトラップされた細胞を上記培養ウェルに移し、この状態でマイクロ流路に培養液等を送液することで上記細胞を培養させるものとなっている。このマイクロ流路デバイスを用いれば、流路を利用して細胞をトラップしそのまま培養することができ、単一細胞の分裂、形態、表現型等の状態を効率良く観察することができる。 A microchannel device having a trap structure is known. For example, Non-Patent Document 1 describes a microchannel device for isolating and culturing a single cell. In this microchannel device, cells are first captured by the cell isolation well by flowing fluid from one direction through a microchannel in which an isolation well is disposed on the lower side and a culture well is disposed on the upper side. Then, by turning the microchannel upside down, the cells trapped by the cell isolation well are transferred to the culture well. It is designed to cultivate. By using this microchannel device, cells can be trapped and cultured using the channel, and states such as division, morphology, and phenotype of single cells can be efficiently observed.
　ところが、非特許文献１に記載されたマイクロ流路デバイスでは、培養ウェルの構造として凹状の穴を採用している。このため、マイクロ流路に培養液等を流したときに培養ウェルから細胞が漏れ出して下流側の別の培養ウェルに混入し、その結果コンタミネーションが発生することがあった。 However, the microchannel device described in Non-Patent Document 1 employs a concave hole as the structure of the culture well. For this reason, when a culture solution or the like is poured into the microchannel, cells sometimes leak out from the culture well and mix into another culture well on the downstream side, resulting in contamination.
　この発明は上記事情に着目してなされたもので、微小物体が捕捉部から漏出しないようにして微小物体を安定的に捕捉できるようにする技術を提供しようとするものである。 This invention has been made in view of the above-mentioned circumstances, and aims to provide a technique that enables stable capture of minute objects while preventing the minute objects from leaking out of the capturing section.
　上記課題を解決するためにこの発明に係るマイクロ流路デバイスの一態様は、流路内において微小物体を捕捉した状態で流体を流通させる継続捕捉部を備え、前記継続捕捉部を、一端部に前記微小物体を捕捉可能な開口部を有し、かつ側面部に前記微小物体より小さく前記流体を流通可能なサイズに設定された間隙部を有する筒状構造体により構成したものである。 In order to solve the above problems, one aspect of the microchannel device according to the present invention is provided with a continuous trapping section that allows fluid to flow in a state in which a microscopic object is captured in the channel, and the continuous trapping section is attached to one end. It is constructed of a cylindrical structure having an opening capable of capturing the minute object, and a gap portion set on a side surface to be smaller than the minute object and having a size that allows the fluid to flow through.
　この発明の一態様によれば、継続捕捉部を、側面部に間隙部を有する筒状構造体としたことにより、筒状構造体内に微小物体を捕捉した状態で間隙部を通して流体を円滑に流通させることができる。このため、微小物体を漏出させることなく捕捉状態を安定的に維持した上で、上記微小物体に対し流体を効果的に反応させることが可能となる。 According to one aspect of the present invention, the continuous trapping section is a cylindrical structure having a gap in the side surface, so that fluid can smoothly flow through the gap while a micro object is captured in the cylindrical structure. can be done. Therefore, it is possible to stably maintain the captured state without leaking the micro objects and to cause the fluid to react effectively with the micro objects.
　すなわちこの発明の一態様によれば、微小物体が捕捉部から漏出しないようにして微小物体を安定的に捕捉できるようにする技術を提供することができる。 That is, according to one aspect of the present invention, it is possible to provide a technique that allows a micro object to be stably captured while preventing the micro object from leaking from the capturing section.
図１は、この発明の一実施形態に係るマイクロ流路デバイスの構造の一例を模式的に示す図である。FIG. 1 is a diagram schematically showing an example of the structure of a microchannel device according to an embodiment of the present invention. 図２は、図１に示したマイクロ流路デバイスに設けられる継続捕捉部の第１の構成例を示す斜視図である。FIG. 2 is a perspective view showing a first configuration example of a continuous capture section provided in the microchannel device shown in FIG. 図３は、図２に示した継続捕捉部の横断面図である。FIG. 3 is a cross-sectional view of the continuous capture section shown in FIG. 2. 図４は、図１に示したマイクロ流路デバイスの各部位のサイズの一例を示す図である。FIG. 4 is a diagram showing an example of the size of each part of the microchannel device shown in FIG. 1. 図５は、図３に示した継続捕捉部の各部のサイズの一例を示す図である。FIG. 5 is a diagram showing an example of the size of each part of the continuous acquisition section shown in FIG. 3. In FIG. 図６は、図１に示したマイクロ流路デバイスを用いた微小物体捕捉方法の一例を模式的に示す図である。FIG. 6 is a diagram schematically showing an example of a method for capturing a minute object using the microchannel device shown in FIG. 1. 図７は、継続捕捉部の第２の構成例を示す横断面図である。FIG. 7 is a cross-sectional view showing a second configuration example of the continuous capture section. 図８は、継続捕捉部の第３の構成例を示す横断面図である。FIG. 8 is a cross-sectional view showing a third configuration example of the continuous capture section. 図９は、継続捕捉部の第４の構成例を示す横断面図である。FIG. 9 is a cross-sectional view showing a fourth configuration example of the continuous capture unit.
　以下、図面を参照してこの発明に係わる実施形態を説明する。 Hereinafter, embodiments of the present invention will be described with reference to the drawings.
　［一実施形態］
　（構成例）
　図１は、この発明の一実施形態に係るマイクロ流路デバイスの構造の一例を模式的に示す図である。なお、一実施形態では、微小物体として細胞を対象とし、細胞を捕捉し培養するデバイスを例にとって説明する。 [One embodiment]
(Configuration example)
FIG. 1 is a diagram schematically showing an example of the structure of a microchannel device according to an embodiment of the present invention. In one embodiment, cells are used as micro objects, and a device for capturing and culturing cells will be described as an example.
　マイクロ流路デバイスは、培養レイヤを構成する第１基板１と、捕捉レイヤを構成する第２基板２とを、流路３を形成する空隙層を介して対向配置した構造を有する。 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 placed facing each other with a void layer forming a channel 3 interposed therebetween.
　先ず、第２基板２の面には、円形状の凹部からなる複数の捕捉ウェル５が等間隔でマトリクス状に配置されている。これらの捕捉ウェル５は、捕捉対象となる微小物体、この例では細胞６を単離捕捉する一時捕捉部として機能する。 First, on the surface of the second substrate 2, a plurality of trapping wells 5 consisting of circular recesses are arranged in a matrix at equal intervals. These trapping wells 5 function as a temporary trapping section that isolates and traps microscopic objects to be captured, in this example cells 6.
　捕捉ウェル５のサイズは、例えば図４に示すように、捕捉対象の細胞６の直径をｄ０とした場合、この細胞６を単離捕捉するために、
　　　深さｈ２；　２×ｄ０＞ｈ２＞ｄ０
　　　内径ｄ２；　２×ｄ０＞ｈ２＞ｄ０
に設定される。 For example, as shown in FIG. 4, the size of the capture well 5 is such that when the diameter of the cell 6 to be captured is d0, in order to isolate and capture the cell 6,
Depth h2; 2×d0>h2>d0
Inner diameter d2; 2×d0>h2>d0
is set to
　より具体的には、細胞の直径を１０μｍとした場合、深さｈ２および内径ｄ２のいずれも１５μｍに設定される。但し、上記捕捉ウェル５のサイズは、上記値に限定されるものではなく、捕捉対象の微小物体のサイズや形状、性質等に応じて任意に設定可能である。 More specifically, when the diameter of the cell is 10 μm, both the depth h2 and the inner diameter d2 are set to 15 μm. However, the size of the capture well 5 is not limited to the above value, and can be arbitrarily set depending on the size, shape, properties, etc. of the micro object to be captured.
　一方、第１基板１の面には、複数の培養ウェル４が等間隔でマトリクス状に配置されている。培養ウェル４は、上記捕捉ウェル５から移された細胞６を培養するために、細胞６の捕捉を維持する継続捕捉部として機能する。 On the other hand, on the surface of the first substrate 1, a plurality of culture wells 4 are arranged in a matrix at equal intervals. The culture well 4 functions as a continuous trapping section that maintains trapping of the cells 6 in order to culture the cells 6 transferred from the trapping well 5.
　培養ウェル４は、例えば図２に示すように、円弧状をなす複数のピラー４０ａを、それぞれスリット状をなす間隙部４０ｂを隔てて配置した、円筒状の構造を有する。図３は、図２の横断面形状を示す図である。 As shown in FIG. 2, for example, the culture well 4 has a cylindrical structure in which a plurality of arc-shaped pillars 40a are arranged with slit-shaped gaps 40b in between. FIG. 3 is a diagram showing the cross-sectional shape of FIG. 2.
　培養ウェル４のサイズは、例えば図４および図５に示すように、細胞６の捕捉を継続的に維持した上で、流体（この例では培養液）に含まれる培養成分を培養ウェル４内の細胞６に反応させるために、捕捉対象の細胞６の直径ｄ０に対し、高さｈ１、直径ｄ１、間隙部４０ｂの幅ｘ１がそれぞれ
　　　高さｈ１；　ｈ１＞ｄ０
　　　直径ｄ１；　ｄ１＞ｄ０
　　　間隙部４０ｂの幅ｘ１；　ｘ１＜ｄ０
となるように設定される。 For example, as shown in FIGS. 4 and 5, the size of the culture well 4 is such that the culture components contained in the fluid (culture solution in this example) can be transferred into the culture well 4 while continuously capturing the cells 6. In order to cause the cells 6 to react, the height h1, the diameter d1, and the width x1 of the gap 40b are set to the diameter d0 of the cell 6 to be captured, respectively: height h1; h1>d0
Diameter d1; d1>d0
Width x1 of gap 40b; x1<d0
It is set so that
　より具体的には、細胞６の直径ｄ０を１０μｍとした場合、高さｈ１、直径ｄ１および間隙部４０ｂの幅ｘ１はそれぞれｈ１＝３０μｍ、ｄ１＝１００μｍ、ｘ１＝８μｍに設定される。但し、培養ウェル４のサイズは上記値に限定されるものではなく、捕捉対象の微小物体のサイズや形状、性質等に応じて任意に設定可能である。 More specifically, when the diameter d0 of the cell 6 is 10 μm, the height h1, the diameter d1, and the 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 value, and can be arbitrarily set depending on the size, shape, properties, etc. of the microscopic object to be captured.
　また、ピラー４０ａの数ｎは、培養液を間隙部４０ｂを通して培養ウェル４内に確実に流入出させるために、ｎ≧２に設定される。図２および図３では、ｎ＝６に設定した場合を例示している。ピラー数４０ａの数ｎは、多いほど、コスト高にはなるものの、培養ウェル４内における流体の拡散性を高める効果が期待できる。また、ピラー４０ａの厚さｔ１は、製作のし易さや剛性を担保するため、例えばｔ１＝２０μｍに設定されるのが望ましい。 Furthermore, the number n of pillars 40a is set to n≧2 in order to ensure that the culture solution flows into and out of the culture well 4 through the gap 40b. 2 and 3 illustrate the case where n=6 is set. The larger the number n of pillars 40a, the higher the cost, but it can be expected to have the effect of increasing the diffusivity of the fluid within the culture well 4. Further, the thickness t1 of the pillar 40a is desirably set to, for example, t1=20 μm in order to ensure ease of manufacture and rigidity.
　培養ウェル４の配置位置は、後述する捕捉ウェル５から細胞６を効率良く捕捉するために、培養ウェル４の中心が捕捉ウェル５の中心と対応するように設定されることが望ましい。 The arrangement position of the culture well 4 is desirably set so that the center of the culture well 4 corresponds to the center of the capture well 5 in order to efficiently capture cells 6 from the capture well 5, which will be described later.
　マイクロ流路デバイスの流路３の高さｈ３および幅ｗ３は、細胞６を捕捉ウェル５に運ぶために、例えば、
　　　高さｈ３；　ｈ３＞ｈ１＋ｄ０
　　　幅ｗ３；　ｗ３＞ｄ１
に設定される。
　より具体的には、高さｈ３＝５０μｍ、幅ｗ３＝１５０μｍに設定されるが、この値に限定されるものではなく、捕捉対象の微小物体のサイズや形状、性質等に応じて任意に設定可能である。 The height h3 and width w3 of the channel 3 of the microfluidic device are such that, for example,
Height h3; h3>h1+d0
Width w3; w3>d1
is set to
More specifically, the height h3 is set to 50 μm and the width w3 is set to 150 μm, but it is not limited to these values and can be set arbitrarily depending on the size, shape, properties, etc. of the minute object to be captured. It is possible.
　以上述べた培養ウェル４および捕捉ウェル５は、例えばポリジメチルシロキサン（ＰＤＭＳ）に電子線を照射することにより作製することができる。このＰＤＭＳを材料として用いる培養ウェル４および捕捉ウェル５の作製技術の一例については、例えば以下の参考文献に記載されている。 The culture well 4 and capture well 5 described above can be produced, for example, by irradiating polydimethylsiloxane (PDMS) with an electron beam. An example of a technique for producing culture wells 4 and capture wells 5 using PDMS as a material is described, for example, in the following references.
　　参考文献；「細胞をつかまえる小さな「水たまり」を開発　－細胞を１つ１つ捕捉・培養する先端医療用デバイスの実現へ－」、国立研究開発法人量子科学技術研究開発機構、2018年5月28日更新、［2022年3月29日検索］、インターネット＜ＵＲＬ；https://www.qst.go.jp/site/press/1231.html＞ References: “Developing a small “puddle” to capture cells – towards the realization of advanced medical devices that capture and culture individual cells –, National Institute for Quantum and Radiological Science and Technology, May 28, 2018 Updated on March 29, 2022, Internet <URL; https://www.qst.go.jp/site/press/1231.html>
　なお、マイクロ流路デバイスの材料については、例えばガラスやその他のシリコン樹脂を用いることができ、また製作方法については例えばフォトリソグラフィを用いた射出成型等を用いることができる。 As for the material of the microchannel device, for example, glass or other silicone resin can be used, and as for the manufacturing method, for example, injection molding using photolithography can be used.
　（動作例）
　次に、以上のように構成されたマイクロ流路デバイスの動作例を、捕捉対象の細胞６の捕捉から培養までの手順に従って説明する。 (Operation example)
Next, an example of the operation of the microchannel device configured as described above will be described according to the procedure from capturing to culturing the cells 6 to be captured.
　図６は、上記マイクロ流路デバイスを使用した細胞６の捕捉から培養までの処理手順の一例を模式的に示すフローチャートである。 FIG. 6 is a flowchart schematically showing an example of the processing procedure from capturing to culturing the cells 6 using the microchannel device.
　例えば試験担当者は、先ずStep1において、培養ウェル４が形成された第１基板１が上側、捕捉ウェル５が形成された第２基板２が下側となるようにマイクロ流路デバイスを設置する。この状態で、Step2において、捕捉対象の細胞６を含む流体を流路３に一定方向Ｂに流通させる。この流体の流通は、例えば図示しない流体注入口からポンプを用いて流体を注入することにより行われる。上記流体の流通により、Step3およびStep4において、上記流体に含まれる細胞６が捕捉ウェル５に１個ずつ捕捉される。すなわち、捕捉ウェル５により細胞６の単離捕捉が行われる。 For example, in Step 1, the test person first installs the microchannel device so that the first substrate 1 on which the culture well 4 is formed is on the upper side and the second substrate 2 on which the capture well 5 is formed is on the lower side. In this state, in Step 2, the fluid containing the cells 6 to be captured is made to flow in the constant direction B through the channel 3. This fluid circulation is performed, for example, by injecting fluid from a fluid inlet (not shown) using a pump. Due to the flow of the fluid, cells 6 contained in the fluid are captured one by one in the capture well 5 in Steps 3 and 4. That is, the cells 6 are isolated and captured using the capture well 5 .
　試験担当者は、次にStep5において、捕捉ウェル５が形成された第２基板２が上側、培養ウェル４が形成された第１基板１が下側となるように、マイクロ流路デバイスを上下反転させる。この結果、Step6において、上記捕捉ウェル５に捕捉された細胞６が重力により落下し、上記捕捉ウェル５の直下に位置する培養ウェル４により捕捉される。このとき、培養ウェル４の径ｄ１は細胞６の直径ｄ０より十分大きなサイズに設計されている。このため、細胞６は高い確率で効率良く培養ウェル４に捕捉される。 Next, in Step 5, the test person turns the microchannel device upside down so that the second substrate 2 on which the capture well 5 is formed is on the top, and the first substrate 1 on which the culture well 4 is formed is on the bottom. let As a result, in Step 6, the cells 6 captured in the capture well 5 fall due to gravity and are 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 cells 6. Therefore, the cells 6 are efficiently captured in the culture well 4 with a high probability.
　試験担当者は、細胞６が培養ウェル４により捕捉されるまでの必要十分な時間が経過した後、Step7において、培養液を流路３に一定方向Ｂに流通させる。この培養液の流入も、図示しない流体注入口から例えばポンプを用いて培養液を注入することにより行われる。 After the necessary and sufficient time has elapsed for the cells 6 to be captured by the culture well 4, the test person causes the culture solution to flow in the constant direction B through the channel 3 in Step 7. This inflow of the culture solution is also performed by injecting the culture solution from a fluid inlet (not shown) using, for example, a pump.
　ここで、培養ウェル４は、先に述べたように複数のピラー４０ａをそれぞれスリット状の間隙部４０ｂを隔てて円筒状に配置した構造を有している。このため、培養液は上記間隙部４０ｂから培養ウェル４内に流入し、培養ウェル４内に捕捉されている細胞６に作用した後、間隙部４０ｂから流出する。 Here, as described above, the culture well 4 has a structure in which a plurality of pillars 40a are arranged in a cylindrical shape with a slit-shaped gap 40b between them. Therefore, the culture solution flows into the culture well 4 through the gap 40b, acts on the cells 6 captured in the culture well 4, and then flows out from the gap 40b.
　また、このとき上記ピラー４０ａの高さｈ１は、細胞６の直径ｄ０より高く設定され、かつ間隙部４０ｂの幅ｘ１は、細胞６の直径ｄ０より小さく設定されている。このため、上記培養液の流通により細胞６が培養ウェル４から漏出して、下流側の培養ウェル４に混入する不具合は発生し難く、これによりコンタミネーションの発生を防止する効果が期待できる。また、培養液を速い流速で流通させることが可能となり、これにより細胞６に対し培養成分を継続的かつ効率的に反応させることが可能となる。 Also, at this time, the height h1 of the pillar 40a is set higher than the diameter d0 of the cell 6, and the width x1 of the gap 40b is set smaller than the diameter d0 of the cell 6. Therefore, problems such as cells 6 leaking out of the culture well 4 due to the circulation of the culture solution and contaminating the culture well 4 on the downstream side are unlikely to occur, and this can be expected to be effective in preventing the occurrence of contamination. In addition, it becomes possible to flow the culture solution at a high flow rate, thereby making it possible to cause the culture components to react with the cells 6 continuously and efficiently.
　すなわち、一実施形態のマイクロ流路デバイスを使用すれば、培養ウェル４による細胞６の捕捉を継続してコンタミネーションの発生を防止した上で、細胞６に対し高速度の反応を行わせることが可能となる。 That is, by using the microchannel device of one embodiment, it is possible to continue capturing the cells 6 in the culture well 4 and prevent the occurrence of contamination, and then cause the cells 6 to perform a high-speed reaction. It becomes possible.
　（作用・効果）
　以上述べたように一実施形態に係るマイクロ流路デバイスでは、培養ウェル４を、高さｈ１が細胞６の直径ｄ０より高く設定された複数のピラー４０ａを、細胞６の直径ｄ０より小さく設定した間隙部４０ｂを隔てて配置し、かつ内径が細胞６の直径ｄ０より十分大きく設定された円筒状をなす構造としている。 (action/effect)
As described above, in the microchannel device according to one embodiment, the culture well 4 has a plurality of pillars 40a whose height h1 is set higher than the diameter d0 of the cells 6, and the plurality of pillars 40a are set smaller than the diameter d0 of the cells 6. They are arranged with a gap 40b in between, and have a cylindrical structure with an inner diameter set sufficiently larger than the diameter d0 of the cell 6.
　従って、捕捉ウェル５で一時的に捕捉された細胞６を効率良く培養ウェル４に移し、かつ培養ウェル４により細胞６の捕捉状態を維持してコンタミネーションを防止した上で、培養液などの流体を速い流速でかつ効率良く細胞６に反応させることが可能となる。 Therefore, the cells 6 temporarily captured in the capture well 5 are efficiently transferred to the culture well 4, and the culture well 4 maintains the captured state of the cells 6 to prevent contamination. It becomes possible to cause the cells 6 to react efficiently at a high flow rate.
　また、一実施形態では、培養ウェル４のピラー４０ａを６個組み合わせることで間隙部４０ｂを６個形成している。このため、培養ウェル４内において培養液を十分に拡散させながら流通させることが可能となり、これにより細胞６に対し培養成分を極めて効果的に反応させることができる。 Furthermore, in one embodiment, six gaps 40b are formed by combining six pillars 40a of the culture well 4. Therefore, it becomes possible to circulate the culture solution while sufficiently diffusing it in the culture well 4, thereby making it possible to cause the culture components to react with the cells 6 extremely effectively.
　［その他の実施形態］
　（１）継続捕捉部としての培養ウェル４の構造については、一実施形態で述べたもの以外に種々の変形が可能である。例えば、図７に示すように、３枚のピラー４１ａを平板状に形成してこれらのピラー４１ａをそれぞれ間隙部４１ｂを隔てて配置することにより、培養ウェル４を三角筒状の構造体に構成してもよい。 [Other embodiments]
(1) Regarding the structure of the culture well 4 as a continuous capture section, various modifications other than those described in one embodiment are possible. For example, as shown in FIG. 7, by forming three pillars 41a into a flat plate shape and arranging these pillars 41a with gaps 41b in between, the culture well 4 can be configured into a triangular cylindrical structure. You may.
　また、例えば図８に示すように、断面形状がＬ型をなす４個のピラー４２ａをそれぞれ間隙部４２ｂを隔てて配置することで、培養ウェル４を四角筒状の構造体に構成してもよい。 Alternatively, as shown in FIG. 8, for example, the culture well 4 may be constructed into a square cylindrical structure by arranging four pillars 42a each having an L-shaped cross section with a gap 42b in between. good.
　さらに、例えば図８に示すように、平板状の４枚のピラー４３ａをそれぞれ間隙部４３ｂを隔てて配置することにより、培養ウェル４を菱形形状の筒体に構成してもよい。この場合、ピラー４３ａの配置形状は菱形形状に限らず四角形状でもよい。 Furthermore, as shown in FIG. 8, for example, the culture well 4 may be formed into a rhombic cylinder by arranging four flat pillars 43a with gaps 43b in between. In this case, the arrangement shape of the pillars 43a is not limited to a diamond shape, but may be a square shape.
　その他、間隙部の形状については、スリット状以外にも円形や長円形、楕円形、角形等であってもよく、またピラーの形状、ピラーおよび間隙部の数、流路３に対する培養ウェル４の配置の向きについても、任意に設定可能である。 In addition, the shape of the gap may be circular, oval, elliptical, square, etc. in addition to the slit shape, and the shape of the pillar, the number of pillars and gaps, and the size of the culture well 4 with respect to the flow path 3. The orientation of the arrangement can also be set arbitrarily.
　（２）培養ウェル４のピラー４０ａの内壁面に突起などを形成するようにしてもよい。このようにすると、培養ウェル４内における培養液の拡散性をさらに高めることが可能となる。 (2) Protrusions or the like may be formed on the inner wall surface of the pillar 40a of the culture well 4. In this way, it becomes possible to further improve the diffusivity of the culture solution within the culture well 4.
　（３）その他、捕捉ウェルの形状やサイズ、流路３の形状やサイズ、捕捉対象となる微小物体の種類（例えば化学材料や金属材料の微粒子、ビーズ）、流体の種類（例えば水、オイル、各種培養液、各種試薬）、流体の送液手段（例えば空気圧ポンプ、シリンジポンプ、スポイト）、一時捕捉部および継続捕捉部の構造や材質、サイズ、製作方法等についても、この発明の要旨を逸脱しない範囲で種々変形して実施できる。 (3) In addition, the shape and size of the capture well, the shape and size of the flow path 3, the type of micro objects to be captured (e.g. fine particles of chemical or metal materials, beads), the type of fluid (e.g. water, oil, Various culture solutions, various reagents), fluid delivery means (e.g. pneumatic pumps, syringe pumps, droppers), structures, materials, sizes, manufacturing methods, etc. of the temporary capture section and continuous capture section also deviate from the gist of this invention. It can be implemented with various modifications within the scope.
　以上、この発明の実施形態を詳細に説明してきたが、前述までの説明はあらゆる点においてこの発明の例示に過ぎない。この発明の範囲を逸脱することなく種々の改良や変形を行うことができることは言うまでもない。つまり、この発明の実施にあたって、実施形態に応じた具体的構成が適宜採用されてもよい。 Although the embodiments of the present invention have been described above in detail, the above description is merely an illustration of the present invention in all respects. It goes without saying that various improvements and modifications can be made without departing from the scope of the invention. That is, in implementing the present invention, specific configurations depending on the embodiments may be adopted as appropriate.
　要するにこの発明は、上記実施形態そのままに限定されるものではなく、実施段階ではその要旨を逸脱しない範囲で構成要素を変形して具体化できる。また、上記実施形態に開示されている複数の構成要素の適宜な組み合せにより種々の発明を形成できる。例えば、実施形態に示される全構成要素から幾つかの構成要素を削除してもよい。さらに、異なる実施形態に亘る構成要素を適宜組み合せてもよい。 In short, the present invention is not limited to the above-described embodiments as they are, but can be embodied by modifying the constituent elements at the implementation stage without departing from the spirit of the invention. Moreover, various inventions can be formed by appropriately combining the plurality of components disclosed in the above embodiments. For example, some components may be deleted from all the components shown in the embodiments. Furthermore, components from different embodiments may be combined as appropriate.
　１…第１基板
　２…第２基板
　３…流路
　４…培養ウェル
　５…捕捉ウェル
　６…細胞
　４０ａ，４１ａ，４２ａ，４３ａ…ピラー
　４０ｂ，４１ｂ，４２ｂ，４３ｂ…間隙部
  1... First substrate 2... Second substrate 3... Channel 4... Culture well 5... Capture well 6... Cell 40a, 41a, 42a, 43a... Pillar 40b, 41b, 42b, 43b... Gap part

Claims (7)

  1. 　流路内において微小物体を捕捉した状態で流体を流通させる継続捕捉部を備えるマイクロ流路デバイスであって、
    　前記継続捕捉部が、一端部に前記微小物体を捕捉可能な開口部を有し、かつ側面部に前記微小物体より小さいサイズに設定された間隙部を有する筒状構造体により構成されるマイクロ流路デバイス。     A microchannel device comprising a continuous capture section that allows fluid to flow while capturing micro objects within the channel,
    The continuous capturing section is configured of a cylindrical structure having an opening capable of capturing the minute object at one end and a gap set to a size smaller than the minute object at the side surface. road device.
  2. 　前記筒状構造体は、上部に前記開口部を有し、かつ前記側面部にスリット状をなす前記間隙部が形成された円筒体からなる、請求項１に記載のマイクロ流路デバイス。 The microchannel device according to claim 1, wherein the cylindrical structure is a cylindrical body having the opening at the top and the slit-shaped gap formed at the side surface.
  3. 　前記筒状構造体は、側面部の前記流体の流通方向に対応する部位に前記間隙部を複数個形成してなる、請求項１に記載のマイクロ流路デバイス。 2. The microchannel device according to claim 1, wherein the cylindrical structure has a plurality of gaps formed in a side surface thereof at a portion corresponding to the flow direction of the fluid.
  4. 　前記流路内において前記微小物体を単離捕捉し、捕捉した前記微小物体を前記継続捕捉部に転移させる一時捕捉部を、さらに備える、請求項１に記載のマイクロ流路デバイス。 The microchannel device according to claim 1, further comprising a temporary capture section that isolates and captures the microscopic object within the flow channel and transfers the captured microscopic object to the continuous capture section.
  5. 　前記継続捕捉部および前記一時捕捉部は、それぞれの開口部が前記流路を隔てて相互に対向する位置に配置される、請求項４に記載のマイクロ流路デバイス。 The microchannel device according to claim 4, wherein the continuous capture section and the temporary capture section are arranged at positions where respective openings face each other across the channel.
  6. 　流路内に設けられ、微小物体を捕捉した状態で流体を流通させる微小物体捕捉構造であって、
    　一端部に前記微小物体を捕捉可能な開口部を有し、かつ側面部に前記微小物体より小さく前記流体を流通可能なサイズに設定された間隙部を有する筒状構造体により構成される、微小物体捕捉構造。     A micro object capturing structure that is provided in a flow path and allows fluid to flow while capturing micro objects, the structure comprising:
    A microscopic structure consisting of a cylindrical structure having an opening at one end that can capture the microscopic object, and a cylindrical structure having a gap smaller than the microscopic object and sized to allow the fluid to flow through the side surface. Object capture structure.
  7. 　流路内に一時捕捉部および継続捕捉部が配置され、前記一時捕捉部が凹状部位を有し、前記継続捕捉部が一端部に開口部を有しかつ側面部に捕捉対象の微小物体より小さいサイズに設定された間隙部を有する筒状構造体からなるマイクロ流路デバイスを用いて、前記微小物体を捕捉する微小物体捕捉方法であって、
    　前記マイクロ流路デバイスを、前記一時捕捉部が下側でかつ前記継続捕捉部が上側となるように配置し、この状態で前記流路内に一定方向に前記微小物体を含む第１の流体を流通させることにより、前記一時捕捉部により前記微小物体を捕捉させる第１の過程と、
    　前記一時捕捉部により前記微小物体が捕捉された状態で、前記マイクロ流路デバイスを、前記継続捕捉部が下側でかつ前記一時捕捉部が上側となるように上下を反転させ、この状態で、前記一時捕捉部に捕捉されている前記微小物体を前記継続捕捉部に移動させる第２の過程と、
    　前記微小物体が前記継続捕捉部に移動した状態で、前記流路内に所定の成分を含有する第２の流体を流通させることにより、前記微小物体に対し前記成分を反応させる第３の過程と
    　を備える微小物体捕捉方法。     A temporary trapping part and a continuous trapping part are arranged in the flow path, the temporary trapping part has a concave part, the continuous trapping part has an opening at one end, and a side part smaller than the micro object to be captured. A method for capturing a minute object, the method comprising capturing the minute object using a microchannel device comprising a cylindrical structure having a gap set to a certain size.
    The microchannel device is arranged such that the temporary trapping section is on the lower side and the continuous trapping section is on the upper side, and in this state, the first fluid containing the microscopic object is introduced into the channel in a certain direction. A first step of causing the temporary capturing unit to capture the minute object by circulating it;
    In a state where the micro object is captured by the temporary capture section, the microchannel device is turned upside down so that the continuous capture section is on the bottom side and the temporary capture section is on the top side, and in this state, a second step of moving the minute object captured by the temporary capturing unit to the continuous capturing unit;
    a third step of causing the component to react with the micro object by flowing a second fluid containing a predetermined component in the flow path while the micro object has moved to the continuous trapping section; A method for capturing a minute object.
PCT/JP2022/017035 2022-04-04 2022-04-04 Microchannel device, micro object trapping structure, and micro object trapping method WO2023195043A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009219480A (en) * 2008-02-21 2009-10-01 Toyama Prefecture Microwell array chip
JP2013029391A (en) * 2011-07-28 2013-02-07 Toyama Prefecture Microchip for capturing floating cancer cell in blood
CN108823064A (en) * 2018-07-04 2018-11-16 李劲松 A kind of cell capture device suitable for medical test
US20210071125A1 (en) * 2019-09-05 2021-03-11 Charles Stark Draper Laboratory, Inc. Systems and methods for seeding cell cultures in a microfluidic device

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009219480A (en) * 2008-02-21 2009-10-01 Toyama Prefecture Microwell array chip
JP2013029391A (en) * 2011-07-28 2013-02-07 Toyama Prefecture Microchip for capturing floating cancer cell in blood
CN108823064A (en) * 2018-07-04 2018-11-16 李劲松 A kind of cell capture device suitable for medical test
US20210071125A1 (en) * 2019-09-05 2021-03-11 Charles Stark Draper Laboratory, Inc. Systems and methods for seeding cell cultures in a microfluidic device

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