Method for forming artificial cell membrane chip, and artificial cell membrane chip

Abstract

Provided is a method for forming an artificial cell membrane chip, the method making it possible to efficiently create a plurality of wells. Specifically, the method for forming an artificial cell membrane chip is for forming an artificial cell membrane chip provided with a plurality of wells that form an artificial cell membrane, the method including: a step for preparing a substrate in which a plurality of recesses are provided in a row in a plate-shaped substrate, and a groove for connecting the recesses to one another is formed between adjacent recesses in the row; and an insertion step for inserting band-shaped sheets into the recesses and the groove. In the insertion step, one of the sheets is inserted into at least two of the recesses and the groove between said recesses, and the sheet separates the recesses into two portions and is provided with a through hole for communicating the two portions. The wells comprise the sheet and the recesses separated into two portions by the sheet.

Description

Method for forming an artificial cell membrane chip and artificial cell membrane chip

[0001] The present invention relates to a method for forming an artificial cell membrane chip and an artificial cell membrane chip.

[0002] Cells that make up an organism, and mitochondria, Golgi bodies, endoplasmic reticulum, etc. that exist inside cells are covered on the outside by a biological membrane, and this biological membrane is basically composed of a lipid bilayer. And various proteins with physiological activity are held on the lipid bilayer in a form that penetrates this lipid bilayer, and such proteins are called transmembrane proteins. These transmembrane proteins play important roles in vivo.

[0003] One type of such transmembrane protein is an ion channel. An ion channel is a general term for proteins that passively allow ions to permeate. In addition to maintaining and changing the membrane potential of cells, it also allows the inflow and outflow of ions in cells. It is involved in the generation of action potentials in electrically excitable cells such as nerve cells, the generation of receptor potentials in sensory cells, and the maintenance of the resting membrane potential in cells.

[0004] When an ion channel opens, ions permeate, and when it closes, the permeation of ions stops. Therefore, the opening and closing of the ion channel changes the flow of ions passing through the lipid bilayer, and the ion current changes.

[0005] In recent years, as described in Patent Documents 1 to 3, an artificial lipid bilayer is formed, an ion channel is introduced therein, and the effects of various pharmaceuticals are confirmed to develop pharmaceuticals.

[0006] Japanese Patent Publication No. 2010-513332, Japanese Unexamined Patent Application Publication No. 2012-81405, Japanese Unexamined Patent Application Publication No. 2017-158464

[0007] As described in Patent Document 2, the artificial cell membrane is created by adding a lipid bilayer-forming lipid solution to two recesses separated by a septum having through-pores. Specifically, it is created by providing two adjacent and connected recesses on a substrate (for example, a plastic plate) and inserting a septum into the connected portion. A single well is formed by these two recesses and the septum, and the lipid bilayer-forming lipid solution is added to it to create the artificial cell membrane.

[0008] On the other hand, when measuring ion currents flowing through ion channels for drug development, multiple artificial cell membranes are used simultaneously to measure ion currents in order to enable rapid development. In this case, forming each well on a separate substrate and using them individually would be time-consuming and laborious for both well creation and ion current measurement. Therefore, it is conceivable to form multiple wells on a single substrate and measure ion currents using a substrate (artificial cell membrane chip) with multiple wells.

[0009] However, creating an artificial cell membrane chip involved the time-consuming and laborious process of inserting each septum into the recesses to create a single well.

[0010] The present invention has been made in view of the above, and its objective is to provide a method for forming an artificial cell membrane chip that can efficiently create multiple wells.

[0011] The present invention provides a method for forming an artificial cell membrane chip, comprising: preparing a substrate having a plurality of wells for forming an artificial cell membrane, wherein a plurality of depressions are arranged in a row on a plate-shaped substrate, and grooves are formed between adjacent depressions in the row to connect the depressions; and inserting a strip-shaped sheet into the depressions and grooves, wherein in the insertion step, one sheet is inserted into two or more depressions and the grooves between those depressions, the sheet separates the depressions into two parts and has through holes that connect the two parts, and the well consists of the sheet and the depressions separated into two parts by the sheet.

[0012] There may be multiple rows of the aforementioned arrangement of depressions.

[0013] One of the sheets may be inserted into all of the recesses and grooves arranged in one of the rows.

[0014] The thickness of the sheet is preferably approximately the same as the distance between opposing groove walls of the groove.

[0015] The artificial cell membrane chip of the present invention is an artificial cell membrane chip comprising a plurality of wells for forming an artificial cell membrane, wherein the wells are provided with a first recess and a second recess separated from each other by a partition wall having through holes, the plurality of wells are arranged in a row on a plate-shaped substrate, the partition wall is formed by inserting a strip-shaped sheet into all of the plurality of wells arranged in a row, grooves are provided between adjacent wells, and the sheet is inserted into these grooves.

[0016] By inserting a single sheet into grooves that connect multiple depressions, multiple wells can be formed at once, allowing for the efficient and rapid formation of artificial cell membrane chips.

[0017] Figure 1 is a schematic diagram showing a substrate in which wells are individually fabricated. Figure 2 is a schematic diagram showing an artificial cell membrane chip in which wells are individually fabricated. Figure 3 is a schematic diagram showing a substrate according to an embodiment. Figure 4 is a schematic diagram showing an artificial cell membrane chip according to an embodiment. Figure 5 is a schematic diagram showing a substrate according to another embodiment.

[0018] The embodiments of the present invention will now be described in detail with reference to the drawings, but first, the individually fabricated forms will be described. The following description of preferred embodiments is essentially illustrative and is not intended to limit the present invention, its applications, or its uses.

[0019] (Individual Fabrication Method) Before describing the embodiment, first, the method of individually fabricating wells will be described based on Figures 1 and 2. The formation of the individually fabricated artificial cell membrane chip 60 begins with preparing a chip precursor substrate (a plate-shaped substrate 50) on which multiple adjacent and communicating first recesses 11 and second recesses 12 are arranged in a row. The first recesses 11 and second recesses 12 are formed by drilling roughly cylindrical holes in the substrate 50, and when observed from above the substrate 50, it appears as if an eight-shaped hole has been drilled in the upper surface of the substrate 50, with the first recesses 11 and second recesses 12 forming a single depression. A groove 13 is provided between the first recesses 11 and second recesses 12, and the first recesses 11 and second recesses 12 are in communication with each other by the groove 13. The individually fabricated chip precursor substrate is a state in which multiple sets of first recesses 11, second recesses 12 and grooves 13 are arranged in a row.

[0020] Next, sheets 20 are inserted into each of the multiple grooves 13. The sheets 20 have through holes in their central portions. The thickness of the sheets 20 is approximately the same as the distance between opposing groove walls of the grooves 13. When the sheets 20 are inserted into the grooves 13, wells 30 are formed, creating an artificial cell membrane chip 60. The wells 30 have a first recess 11 and a second recess 12 separated from each other by a partition wall (sheet 20) with a through hole.

[0021] (Embodiment 1) The method for forming an artificial cell membrane chip according to Embodiment 1 will be described with reference to Figures 3 and 4. The formation of the artificial cell membrane chip 61 according to Embodiment 1 begins with preparing a chip precursor substrate (a plate-shaped substrate substrate 51) on which a plurality of recesses, each consisting of adjacent and communicating first recesses 11 and second recesses 12, are arranged in a row. The substrate 51 is formed from, for example, plastic or glass. Similar to the individually fabricated form described above, the first recesses 11 and second recesses 12 are formed by drilling substantially cylindrical holes in the substrate 51. When observed from above the substrate 51, it appears as if an eight-shaped hole has been drilled in the upper surface of the substrate 51, and the communicating first recesses 11 and second recesses 12 form a single recess. A groove 15 is provided between the first recesses 11 and the second recesses 12, and the plurality of recesses are connected and communicate with each other by the groove 15. The groove 15 extends in a straight line. In this embodiment, all sets of multiple first recesses 11 and second recesses 12 arranged in a row on the chip precursor substrate are connected by a single groove 15.

[0022] Next, a sheet 21 is inserted into the groove 15. The sheet 21 is made of, for example, a thin plastic plate. The thickness of the sheet 21 is approximately the same as the distance between the opposing groove walls of the groove 15. As a result, even if a lipid bilayer-forming lipid solution is added to the first recess 11 and the second recess 12, the sheet 21 prevents the lipid bilayer-forming lipid solution from mixing into the adjacent first recess 11 and second recess 12. The sheet 21 also acts as a partition wall separating the first recess 11 and the second recess 12, but through holes are formed in the sheet 21 in the portion between each of the first recess 11 and the second recess 12.

[0023] In this way, when the sheet 21 is inserted into the groove 15, multiple wells 31 are formed at once, creating an artificial cell membrane chip 61. The wells 31 have a structure in which a first recess 11 and a second recess 12 are separated from each other by a partition wall (sheet 21) with through holes.

[0024] In this embodiment, multiple wells are created at once by separating multiple depressions into two parts with a single sheet, allowing for the formation of artificial cell membrane chips quickly and efficiently. In the process of inserting the sheet into the grooves, insertion failures can occur, resulting in no wells being formed. Because the sheet is longer in this embodiment than when each well is formed individually, the failure rate of sheet insertion is higher in this embodiment. As an example of the specific sheet configuration, the sheet is made of polyimide, has a thickness of 5 μm, a width of 3 mm in the insertion direction, a through hole with a diameter of 0.2 mm, and a length of approximately 100 mm. In the case of individual creation, for example, the length would be 6 mm. However, if a larger number of wells are formed at once than the ratio of the failure rate in this embodiment to that of individual creation, the number of well formation failures will be lower than in the case of individual creation, resulting in a higher success rate of well formation than individual creation. In addition, in the case of individual creation, the area near the through hole is grasped with tweezers or the like when inserting into the groove, which can cause the through hole to deform or collapse, but this is less likely to occur in this embodiment.

[0025] (Embodiment 2) The method for forming an artificial cell membrane chip according to Embodiment 2 differs from Embodiment 1 in that there are multiple rows of rows of multiple depressions, but the other configurations, structures, and processes are the same as in Embodiment 1. Therefore, the differences between Embodiment 2 and Embodiment 1 will be explained below.

[0026] In Embodiment 2, as shown in Figure 5, a chip precursor substrate is created by arranging multiple rows of recesses, each consisting of an adjacent and communicating first recess 11 and second recess 12, on a substrate 52, and multiple rows of recesses are created. In each of the rows of recesses, grooves 15 are formed between adjacent recesses within the row, and each groove 15 extends in a straight line.

[0027] In this embodiment, a chip precursor substrate is prepared, and sheets 21 similar to those in Embodiment 1 are inserted into each groove 15 to form an artificial cell membrane chip.

[0028] Embodiment 2 also achieves the same effects as Embodiment 1.

[0029] (Other Embodiments) The embodiments described above are illustrative examples of the present invention, and the present invention is not limited to these examples. These examples may be combined with or partially replaced with well-known technologies, conventional technologies, or prior art. Modified inventions that can be easily conceived by a person skilled in the art are also included in the present invention. For example, the groove 15 may be formed by physically or chemically carving it from one side of the substrate 51 (substrate 52), or it may be formed by joining two members, each having a groove wall that forms the groove 15. In addition, the two members may be joined with the sheet 21 sandwiched in between, and the state obtained by such a process, in which the sheet 21 is inserted into the groove 15, is also included in the present invention.

[0030] 11 First recess 12 Second recess 13 Groove 15 Groove 20 Sheet (separator) 21 Sheet (separator) 30 Well 31 Well 50 Substrate (plate-shaped base material) 51 Substrate (plate-shaped base material) 52 Substrate (plate-shaped base material) 60 Artificial cell membrane chip 61 Artificial cell membrane chip

Claims

1. A method for forming an artificial cell membrane chip having a plurality of wells for forming an artificial cell membrane, comprising the steps of: preparing a substrate having a plurality of depressions arranged in a row on a plate-shaped base material, with grooves formed between adjacent depressions in the row to connect the depressions; and inserting a strip-shaped sheet into the depressions and grooves, wherein in the insertion step, one sheet is inserted into two or more depressions and the grooves between those depressions, the sheet separates the depressions into two parts and has through holes that connect the two parts, and the wells consist of the sheet and the depressions separated into two parts by the sheet, a method for forming an artificial cell membrane chip.

2. A method for forming an artificial cell membrane chip according to claim 1, wherein there are multiple rows of the aforementioned arranged depressions.

3. A method for forming an artificial cell membrane chip according to claim 2, wherein one sheet is inserted into all of the recesses and grooves arranged in one row.

4. The method for forming an artificial cell membrane chip according to claim 1, wherein the thickness of the sheet is substantially the same as the distance between opposing groove walls of the groove.

5. An artificial cell membrane chip comprising a plurality of wells for forming an artificial cell membrane, wherein the wells are provided with a first recess and a second recess separated from each other by a partition wall having through holes, the plurality of wells are arranged in a row on a plate-shaped substrate, the partition wall is formed by inserting a strip-shaped sheet into all of the plurality of wells arranged in a row, and grooves are provided between adjacent wells, into which the sheet is inserted, the artificial cell membrane chip.

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