JP7588316B2 - Fiber mesh sheet, its manufacturing method, and cell culture chip using the fiber mesh sheet - Google Patents

Description

The present invention relates to a fiber mesh sheet, a manufacturing method thereof, and a cell culture chip using the fiber mesh sheet.

In recent years, there has been active development of organ-on-a-chip (OoC) chips for use in cell culture (see, for example, Patent Document 1). OoC is a cell culture chip that reproduces the tissue functions of a living body on a microscale by culturing cells in an artificial microspace that combines glass, resin, etc.

By administering drugs to cells cultured using such cell culture chips, it becomes possible to carry out tests that previously were conducted using animal tests using mice to evaluate the efficacy, toxicity, absorption, metabolism, excretion, etc. of drugs, using cells with functions closer to those found in the living body in an artificial chip outside the body.

JP 2019-180354 A

In order to provide cell sheets containing liver cells or intestinal cells with functions closer to those in vivo, for example, a conventional cell culture chip described in Patent Document 1 uses a micromesh sheet as a scaffold for culturing cells.

However, there are cases where two types of cells are cultured as separate upper and lower cell sheets, and different fluids are perfused separately from the top and bottom to evaluate drug permeability through the cell sheets. To do this, the mesh openings must be large enough to prevent the passage of a single cell, in order to culture the upper and lower cells separately, but this poses the problem that the test substance can easily become clogged in the mesh openings, inhibiting drug permeability.

In particular, this issue has become more pronounced in recent years as candidate compounds for new drugs have become increasingly polymeric.

The present invention aims to solve the above-mentioned problems of the conventional art by providing a fiber mesh sheet for use in a cell culture chip that can culture two types of cells separately above and below, even when the mesh openings are of an appropriate size to prevent clogging of the mesh openings with the test specimen.

In order to achieve the above object, the fiber mesh sheet of the present invention has a mesh structure in which two or more planar aligned fiber groups are laminated, in which a plurality of fibers made of a polymer material are aligned in one direction along the longitudinal direction of the fibers within a plane,
The longitudinal directions of the fibers of each of the adjacent two aligned fiber groups intersect at a crossing angle of 30° or more and 150° or less in a plan view seen from a direction perpendicular to the surface,
an upper portion of a cross section of the fibers of the aligned fiber group in the lowest layer on a side where there is an adjacent aligned fiber group is substantially circular, and a lower portion on a side where there is no adjacent aligned fiber group is substantially flat;
The cross sections of the fibers in the aligned fiber groups other than the bottom layer are substantially circular.

As described above, the fiber mesh sheet of the present invention makes it possible to separate, capture and recover substances the size of a single cell at the substantially flat lower portion of the aligned fibers in the lowest layer.
In addition, by using the fiber mesh sheet as a scaffold for culturing cells and constructing a cell culture chip, it becomes possible to culture two types of cells separately above and below even with mesh openings of an appropriate size that can prevent the test subject from clogging the mesh openings. This makes it possible to evaluate candidate compounds for new drugs, which are becoming increasingly high molecular weight in recent years.

1 is a schematic perspective view showing a configuration of a fiber mesh sheet according to a first embodiment. FIG. 1B is a schematic perspective view showing the structure and dimensions of the fibers constituting the first and second layers of the fiber mesh sheet of FIG. 1A. FIG. 4 is a flowchart of a method for manufacturing a fiber mesh sheet according to the first embodiment. 1 is an exploded perspective view showing a configuration of a cell culture chip according to a first embodiment. Table 1 shows the conditions for the comparative example and the examples, and the culture results when used in a cell culture chip.

The fiber mesh sheet according to the first aspect has a mesh structure in which two or more planar aligned fiber groups are laminated, in which a plurality of fibers made of a polymer material are aligned in one direction along the longitudinal direction of the fibers in a plane, and
The longitudinal directions of the fibers of each of the adjacent two aligned fiber groups intersect at a crossing angle of 30° or more and 150° or less in a plan view seen from a direction perpendicular to the surface,
an upper portion of a cross section of the fibers of the aligned fiber group in the lowest layer on a side where there is an adjacent aligned fiber group is substantially circular, and a lower portion on a side where there is no adjacent aligned fiber group is substantially flat;
The cross sections of the fibers in the aligned fiber groups other than the bottom layer are substantially circular.

In the fiber mesh sheet according to the second aspect, in the first aspect described above, the contact angle of the fibers of the substantially circular shape of the arranged fiber group other than the bottom layer with water may be 90° or more and 150° or less.

The fiber mesh sheet according to the third aspect may be the fiber mesh sheet according to the first or second aspect, in which the average diameter of the fibers in the arranged fiber group is 1 μm or more and 50 μm or less.

A method for producing a fiber mesh sheet according to a fourth aspect includes a step of laminating two or more planar aligned fiber groups, each of which has a plurality of fibers made of a polymeric material aligned in one direction along a plane, such that the longitudinal directions of the fibers in adjacent aligned fiber groups intersect at a crossing angle of 30° or more and 150° or less in a plan view seen from a direction perpendicular to the plane;
and a step of heat-treating the fibers at a temperature equal to or higher than the melting point of the polymeric material of the fibers and equal to or lower than a temperature (melting point + 100° C.) at which the fibers melt and break,
The heat treatment step causes the majority of the contact portions of the aligned fiber groups of the two adjacent layers to be entangled.

The method for producing a fiber mesh sheet according to the fifth aspect may be the fourth aspect described above, in which the lower part of the cross section of the fibers of the aligned fiber group of the lowest layer on the side where there is no adjacent aligned fiber group is made substantially flat by a process of heat treating the fibers at a temperature equal to or higher than the melting point of the polymeric material of the fibers and equal to or lower than the temperature at which the fibers melt and break (melting point + 100°C).

The cell culture chip according to the sixth aspect includes a fiber mesh sheet according to any one of the first to third aspects.

The fiber mesh sheet and its manufacturing method, and the cell culture chip according to the embodiment will be described below with reference to the attached drawings. Note that substantially the same components in the drawings are given the same reference numerals.

(Embodiment 1) <Fiber mesh sheet>
Fig. 1A is a schematic perspective view showing the configuration of a fiber mesh sheet 101 according to embodiment 1. Fig. 1B is a schematic perspective view showing the structure and dimensions of fibers constituting a first layer and a second layer of the fiber mesh sheet of Fig. 1A. For convenience, in the drawing, the plane of the aligned fiber groups 102 of the first layer and the aligned fiber groups 103 of the second layer is shown as an XY plane, and the stacking direction is shown as a Z direction.
The fiber mesh sheet 101 has a mesh structure in which two or more planar arranged fiber groups 102, 103 in which a plurality of fibers 1, 2 made of a polymer material are arranged in one direction in a plane (XY plane) are laminated. The longitudinal directions of the fibers 1, 2 of the adjacent two arranged fiber groups 102, 103 cross each other at a crossing angle θ of 30° to 150° in a planar view seen from a direction perpendicular to the plane (Z direction). The first layer arranged fiber group 102 is a fiber group made of a polymer material in which the longitudinal directions of the plurality of fibers 1 are aligned in the same direction in a thin line shape at equal intervals, and the cross-sectional shape of each fiber 1 is approximately circular at the top and approximately flat at the bottom. The upper part refers to the side where there is an adjacent arranged fiber group, that is, the positive direction in the Z direction, and the lower part refers to the side where there is no adjacent arranged fiber group, that is, the negative direction in the Z direction. The aligned fiber group 103 of the second layer is a fiber group made of a polymer material in which a plurality of fibers 2 are aligned in the same longitudinal direction at equal intervals in the form of thin lines, and the cross-sectional shape of the fibers 2 constituting the aligned fiber group 103 of the second layer is substantially circular. The majority of the lower portions of the fibers 2 of the aligned fiber group 103 of the second layer are entangled and joined to the upper portions of the fibers 1 of the aligned fiber group 102 of the first layer. Note that "entangled" means that the fibers 1 and 2 cross each other and are partially joined to each other at the points where they come into contact with each other.

In this fiber mesh sheet, the longitudinal directions of the fibers 1 and 2 of the two adjacent layers of aligned fiber groups 102 and 103 cross at a crossing angle θ of 30° to 150° in plan view from the direction perpendicular to the surface (Z direction). This makes it possible to suppress both the passage of cells and clogging of the test object.

The contact angle of the approximately circular shape of fiber 2 of the aligned fiber group 103 in the second layer (not the bottom layer) with water may be 90° or more and 150° or less. This further promises to obtain a cell sheet with functions closer to those found in vivo by wetting the lateral direction of the approximately circular shape of fiber 2 and contacting the cell type through the openings in the mesh.

Furthermore, the average diameter of the fibers 1, 2 in the aligned fiber groups 102, 103 in the first and second layers may be, for example, 1 μm or more and 50 μm or less.
The average diameter is the average value of the diameters of the fibers 1 and 2. The diameters of the fibers 1 and 2 are the diameters of the cross sections perpendicular to the length direction of the fibers. When such cross sections are not circular, the maximum diameter may be regarded as the diameter. Also, the width in the direction perpendicular to the length direction of the fibers when viewed from the normal direction of one of the main faces of the aligned fiber groups 102 and 103 in the first and second layers may be regarded as the diameter of the fibers. The average fiber diameter is, for example, the average value of the diameters of any 10 fibers included in the aligned fiber groups 102 and 103 in the first and second layers, determined by image processing measurement.
In addition, a third or more layers of aligned fiber groups may be provided above the second layer of aligned fiber groups 103, that is, in the positive direction of the Z direction.

<Method of manufacturing fiber mesh sheet>
2 is a flow chart of a method for manufacturing the fiber mesh sheet 101 according to the first embodiment. (1) S01 is a step of preparing a film. The surface of the film is preferably one that has a suitable releasability due to fluorine treatment or the like. This is because, when fibers are spun onto the film in each of steps S02 and S04 described below, an adhesive function for the fibers is required, and when the fiber mesh sheet is later incorporated into a cell culture chip, a function for peeling the fiber mesh sheet from the film is required. (2) S02 is a step of spinning the first layer of aligned fibers 102. A solution of a polymer material used as the fiber mesh sheet 101, which is melted by heating or swollen with an organic solvent, is applied in the same direction in thin lines at equal intervals on the film prepared in step S01.
Here, the polymer material supplied in a molten or liquid state is naturally cooled or naturally dried to form fibers in a solid state only.
In the first embodiment, polystyrene with low cytotoxicity is used as the polymer material, and a solution of pellet-shaped polystyrene swollen to 30% by weight in DMF (N,N-dimethylformamide), an organic solvent, is used to coat fibers with a diameter of 5 μm in the same direction at intervals of 30 μm. The average diameter of the fibers may be 1 μm or more and 50 μm or less.

(3) S03 is a process of rotating the film on which the first layer of aligned fibers 102 has been spun in S02 by 90° in-plane. (4) S04 is a process of spinning the second layer of aligned fibers 103 on the film rotated 90° in-plane in S03. A solution of a polymer material used for the fiber mesh sheet 101, which has been melted by heating or swollen with an organic solvent, is applied in the form of thin lines at equal intervals in the same direction on the film prepared in S03.
In the first embodiment, polystyrene with low cytotoxicity was used as the polymer material as in the step S02, and a solution in which pellet-shaped polystyrene was swollen to 30% by weight in DMF (N,N-dimethylformamide), an organic solvent, was used to apply fibers with a diameter equivalent to 5 μm in the same direction at intervals equivalent to 30 μm. (5) S05 is a step of heating the fiber mesh sheet on the film created up to the step S04. Specifically, by heating for a certain period of time at a temperature equal to or higher than the melting point of the polymer material (polystyrene in the first embodiment) and equal to or lower than the temperature at which the fibers melt and break (melting point + 100°C), the majority of the contact points at the upper part of the aligned fiber group 102 of the first layer and the lower part of the aligned fiber group 103 of the second layer are entangled, and the lower part of the aligned fiber group 102 of the first layer is made substantially flat. The entanglement of the contact points between the upper part of the aligned fiber group 102 of the first layer and the lower part of the aligned fiber group 103 of the second layer may be, for example, a joining by melting intersecting fibers.
In this manner, a fiber mesh sheet is obtained.

<Cell culture chip>
Further, a cell culture chip 300 using the fiber mesh sheet 101 according to the first embodiment will be described below.
3 is an exploded perspective view showing a configuration of the cell culture chip 300 according to the embodiment 1. For convenience, in the drawing, the in-plane of the first substrate 11 etc. is shown as an XY plane, and the direction perpendicular thereto is shown as a Z direction.
The cell culture chip 300 according to the first embodiment includes a main body and a fiber mesh sheet 101 produced by a fiber mesh sheet manufacturing method. The main body has a main surface parallel to the XY plane, and has a layered structure in which a first substrate 11, a first partition layer 12, a second partition layer 14, and a second substrate 15 are layered in this order along a predetermined direction (the Z-axis direction in the figure). The fiber mesh sheet 101 is sandwiched between the first partition layer 12 and the second partition layer 14 of the main body.

The components that make up this cell culture chip 300 are described below.

<First Substrate>
The first substrate 11 is a plate-like member formed using a material such as glass. The material of the first substrate 11 is not limited to glass, and any material such as resin or ceramics may be used. In addition, the first substrate 11 is formed from a material that does not have cytotoxicity because it comes into contact with the cells when the cells are cultured.
The first substrate 11 is in the form of a plate having a rectangular main surface in the embodiment 1. The first substrate 11 is provided with a hole 31 penetrating the first substrate 11 along a predetermined direction so as to communicate with the first partition layer 12 to be laminated thereon.

<First partition layer>
In a case where a part of the first partition layer 12 is exposed without overlapping with the first substrate 11, for example, the first partition layer 12 may be configured to directly communicate with the first partition layer 12 without passing through the hole 31 of the first substrate 11. The first partition layer 12 is a plate-like member formed of a silicone resin.
The first partition layer 12 has a first through hole that penetrates the first partition layer 12 in the thickness direction (Z-axis direction) at least partially in the first substrate 11. The first through hole corresponds to the first flow path 33, which will be described in detail later.
Both ends of the first through hole correspond to two of the holes 31 formed in the first substrate 11. Furthermore, the first partition layer 12 is provided with holes 32 that penetrate the first partition layer 12 in the thickness direction so as to correspond to the remaining two holes 31 other than the two that correspond to the first through holes and to communicate with the second partition layer 14 to be laminated thereon.

<Fiber mesh sheet>
The fiber mesh sheet 101 has a first main surface 101a on the first partition layer 12 side and a second main surface 101b on the second partition layer side. The first main surface 101a has a substantially circular cross-sectional shape of the fibers of the aligned fiber group of the second layer in FIG. 1A, and the second main surface 101b has a flat cross-sectional shape of the fibers of the aligned fiber group of the first layer in FIG. 1A. In addition, a predetermined opening is formed through the first main surface 101a and the second main surface 101b, which are facing back to back. Note that "facing back to back" means that the first main surface 101a and the second main surface 101b are facing back to back.
Here, the predetermined opening is set to be smaller than the cell diameter of the cells cultured using the cell culture chip 300. Therefore, the fiber mesh sheet 101 has a semi-permeable function of preventing cells larger than the predetermined opening from passing from the first main surface 101a to the second main surface 101b, or from the second main surface 101b to the first main surface 101a, and allowing solution components (e.g., test substances and medium components) smaller than the predetermined opening to pass through.

The fiber mesh sheet 101 also functions as a scaffold for the cells being cultured in the cell culture chip 300. Therefore, the fiber mesh sheet 101 may be made of a material that is less toxic to the cells being cultured and that can be adhered to the cells.

The fiber mesh sheet 101 is positioned at locations corresponding to the first through hole and the second through hole described later, and is sandwiched between the first partition layer 12 and the second partition layer 14 outside the first through hole and the second through hole when viewed in a planar view from the stacking direction (Z direction).
In this manner, where the first through hole and the second through hole overlap, the first through hole and the second through hole are each defined by the fiber mesh sheet 101. In this manner, a first flow path 33 is formed having a first main flow path 36 defined by the main surface of the first substrate 11, the first through hole, and the first main surface 101a. In other words, the first main flow path 36 is formed between the first substrate 11 and the fiber mesh sheet 101 by the first through hole.

The first main flow passage 36 is a part of the first flow passage 33 formed by the first through hole. The first main flow passage 36 is in contact with the first flow passage 33 thus defined, in particular the first main flow passage 36, and extends within and along the first main flow passage 36.
The first flow path 33 has a first inlet 34 at one end corresponding to the hole 31 and a first outlet 38 at the other end, each of which communicates with the outside of the cell culture chip 300 via the hole 31 .
The first flow path 33 has a first inlet flow path 35 connecting from the first inlet 34 to the first main flow path 36, and a first outlet flow path 37 connecting from the first outlet 38 to the first main flow path 36. The first inlet flow path 35 and the first outlet flow path 37 are defined by the second partition wall layer 14 instead of the fiber mesh sheet 101 with respect to the first main flow path 36.

<Second partition layer>
The second partition layer 14 is a plate-like member formed of a silicone resin. The second through hole corresponds to the second flow path 41, as will be described in detail later. Both ends of the second through hole correspond to the hole 31 formed in the first substrate 11 and the hole 32 formed in the first partition layer 12.

<Second Substrate>
The second substrate 15 is a plate-like member formed using a material such as glass. The material of the second substrate 15 is not limited to glass, and any material such as resin, ceramics, etc. may be used. In the first embodiment, the second substrate 15 is a plate-like member having a rectangular main surface.

In this manner, the second flow path 41 is formed, which has a second main flow path 44 defined by the main surface of the second substrate 15, the second through hole, and the second main surface 101b.
In other words, the second main flow path 44 is formed by the second through hole between the second substrate 15 and the fiber mesh sheet 101. The second main flow path 44 is a part of the second flow path 41 formed by the second through hole.

The fiber mesh sheet 101 is arranged between the first flow path 33 and the second flow path 41 such that the first main flow path 36 of the first flow path 33 is located on the first main surface 101a and the second main flow path 44 of the second flow path 41 is located on the second main surface 101b.
Therefore, via the fiber mesh sheet 101, the first main flow path 36 and the second main flow path 44 can exchange components that are smaller than a predetermined pore size, such as test substances and culture medium components that flow through each flow path.

In addition, the second flow path 41 has a second inlet 42 at one end corresponding to holes 31 and 32, and a second outlet 46 at the other end, which are connected to the outside of the cell culture chip 300 via holes 31 and 32, respectively.
The second flow path 41 has a second inlet flow path 43 that connects from the second inlet 42 to the second main flow path 44, and a second outlet flow path 45 that connects from the second outlet 46 to the second main flow path 44. The second inlet flow path 43 and the second outlet flow path 45 are defined by the first partition wall layer 12 instead of the fiber mesh sheet 101 with respect to the second main flow path 44.

That is, in a plan view seen from the stacking direction (Z direction), the first inlet flow path 35 and the second inlet flow path 43 do not overlap, and the first outlet flow path 37 and the second outlet flow path 45 do not overlap. As a result, in the first inlet flow path 35 and the first outlet flow path 37, a part of the first flow path 33 is formed by the main surface of the second partition layer 14 on which the second through hole is not formed.
In the second inlet flow passage 43 and the second outlet flow passage 45, a part of the second flow passage 41 is formed by the main surface of the first partition layer 12 where the first through holes are not formed.

Comparison of Examples and Comparative Examples
A comparative example and an example of the first embodiment will be described below with reference to Table 1 in FIG.
In both the comparative example and the example, a fiber mesh sheet was prepared by the fiber mesh sheet manufacturing method described in this embodiment 1, and the first layer was mounted on the cell culture chip described in this embodiment 1 so as to face the second main flow path, and the second layer was mounted facing the first main flow path.
However, in the comparative example, although entanglement between the first and second layers is formed by controlling the heating temperature/time in S05 of the fiber mesh sheet manufacturing method described in this embodiment, both the first and second layers have an approximately circular cross-sectional shape.
In addition, for the cells to be seeded into the cell culture chip, low invasive cells and high invasive cells are used as cell type X to be seeded on the first main flow path side, and low invasive cells and high invasive cells are used as cell type Y to be seeded on the second main flow path side, and the culture is evaluated by changing the order in which the cells are seeded on the first main flow path side and the second main flow path side.

In this embodiment, for both cell type X and cell type Y, cells with a size equivalent to 20 μm are used, and a 1 μM polymer compound solution (equivalent to a molecular weight of 750,000) is used as the test substance.

The fiber mesh sheet produced by the fiber mesh sheet manufacturing method described in this embodiment has openings with a diameter of 5 μm, spaced at intervals of 30 μm, and measuring 25 μm square, which is larger than the size of a single cell (20 μm), and is less likely to become clogged with the 1 μM polymer compound solution (equivalent to a molecular weight of 750,000) used as the test substance.

On the other hand, the culture evaluation results show that cell type X and cell type Y are not mixed and are each formed into a sheet, with A to E indicating that A: cell type X and cell type Y are not mixed and are each formed into a sheet at 100% B: cell type X and cell type Y are not mixed and are each formed into a sheet at less than 100% and 80% or more C: cell type X and cell type Y are not mixed and are each formed into a sheet at less than 80% and 60% or more D: cell type X and cell type Y are not mixed and are each formed into a sheet at less than 60% and 40% or more E: cell type X and cell type Y are not mixed and are each formed into a sheet at less than 40%.

In Comparative Examples 1 and 2 in Table 1, when cell type X seeded on the first main channel side and cell type Y seeded on the second main channel side are both low invasive cells, the culture result is C, regardless of the order in which the cells are seeded.

This is presumably because the opening size of the fiber mesh sheet was larger than the size of a single cell to be seeded, allowing some cells to slip through from the main channel side where they were seeded to the opposing main channel side.

In contrast, in Comparative Examples 3 and 4, when highly invasive cells were used as cell type Y to be seeded on the second main flow path side, the culture results deteriorated to D when the cells were seeded in the order of first main flow path side → second main flow path side, and the culture results deteriorated further to E when the cells were seeded in the order of second main flow path side → first main flow path side.
This is presumably because in Comparative Example 3, most of the low invasive cells seeded on the first main flow path were formed into a sheet first, but most of the highly invasive cells seeded next on the second main flow path slipped through to the first main flow path and were mixed in.

In addition, in Comparative Example 4, it is presumed that almost all of the highly invasive cells seeded on the second main channel side slipped through the fiber mesh sheet to the first main channel side, which caused the cell type X seeded next on the first main channel side to not form a sheet.

Similarly, in Comparative Examples 5 and 6, when highly invasive cells are used as cell type X seeded on the first main channel side and less invasive cells are used as cell type Y seeded on the second main channel side, Comparative Example 5, in which the more invasive cells are seeded first, is classified as E, and Comparative Example 6, in which the more invasive cells are seeded second, is classified as D, just like Comparative Examples 3 and 4 above.

On the other hand, in Comparative Examples 7 and 8, when highly invasive cells were used for cell type X seeded on the first main channel side and cell type Y seeded on the second main channel side, the culture result was E, regardless of the order in which the cells were seeded. Similarly, it is presumed that the culture seeded first slipped through the fiber mesh sheet to the main channel side where the next cells were seeded, minimizing the area in which the next cell type seeded was formed into a sheet.

On the other hand, the culture results improved in all of the Examples corresponding to the Comparative Examples, and this tendency was particularly noticeable in the Examples in which highly invasive cell types were seeded first from the second main channel side (Examples 4, 6, and 8).
In other words, by using the fiber mesh sheet 101 in the embodiment and seeding the highly invasive cell type Y first on the second main flow path side, the cell type Y is cultured in sheet form on the second main flow path side without slipping through the fiber mesh sheet 101, and then the cell type X to be seeded on the first main flow path side is cultured in sheet form regardless of whether the cell type X has a high or low invasiveness, so that it is presumably possible to stack cells of cell types X and Y on both the first main flow path side and the second main flow path side in sheet form.

This means that highly invasive cell types exhibit the effect of easily passing through the approximately circular side, but having difficulty passing through the approximately flat side.
In other words, it is presumed that highly invasive cell types are more likely to get stuck in the gaps in the mesh by wetting laterally rather than on the approximately circular surface, making them more likely to slip through the openings, whereas the approximately flat side is more likely to wet in the surface direction than the approximately circular side, thereby preventing the cell types from getting stuck in the gaps in the mesh.

In addition, when highly invasive cell type X is seeded on the first main flow path side, it is expected that a cell sheet with functions closer to those in the body can be obtained by wetting the lateral direction of the approximately circular shape and coming into contact with cell type Y previously seeded on the second main flow path side through the openings in the mesh.
In order to obtain such an effect, in the embodiment, the most desirable contact angle with water for the substantially circular shape is set to 90°, but the same effect can be expected if the contact angle is between 90° and 150°.

In the embodiment, cell type X and cell type Y with a cell size equivalent to 30 μm are used, and 1 μM (equivalent to a molecular weight of 750,000) is used as the test substance. The spacing of the fiber mesh sheet 101 is set to 30 μm so as to prevent clogging of the test substance, but this can be set appropriately.
Specifically, the lower limit must be greater than the size at which the analyte used becomes clogged depending on its molecular weight and concentration, while the upper limit should be set to a distance that prevents the passage of the seeded cells.
In addition, in the embodiment, polystyrene fibers having a diameter of 5 μm are used, but this can be set appropriately.

Specifically, the thickness is 1 μm to 50 μm, which is expected to function as a scaffold for the cells to be cultured. The material is not limited to polystyrene, and may be a polylactic acid or silicone-based material with low cytotoxicity, but since flexibility is required to function as a scaffold for cells, a polymer material is preferable.
In addition, the in-plane rotation angle in step S03 of the fiber mesh sheet manufacturing method described in this embodiment is not limited to 90°, and as long as it is between 30° and 150°, it is possible to suppress both the passage of cells and clogging of the test object.

If it is necessary to increase the thickness of the fiber mesh sheet depending on the cell culture chip used, the film rotation and spinning can be repeated in the same manner after step S04 of the fiber mesh sheet manufacturing method described in this embodiment. In this case, it is desirable to standardize the rotation angle and fiber spacing to prevent clogging of the test object.

<Capturing and recovering at the single cell level>
The effects shown in the cell culture chip of the fiber mesh sheet in the embodiment are also effective in capturing and recovering cells.
Specifically, for example, when a fiber mesh sheet is used as a separation membrane for capturing and recovering single-cell-sized substances (e.g., red blood cells with a diameter equivalent to 8 μm) from a sample (e.g., blood), in a comparative example constituted only by fibers having a substantially circular cross-sectional shape, the single-cell-sized substances are more likely to get stuck in the gaps in the mesh due to wetting in the lateral direction rather than the surface of the substantially circular shape, making it possible to capture the single-cell-sized substances but difficult to recover them.
On the other hand, in the embodiment having fibers with an approximately flat cross-sectional shape, the fibers are more likely to wet in the surface direction than the fibers with an approximately circular cross-sectional shape, which suppresses the phenomenon of the fibers getting stuck in the gaps in the mesh, making it possible to simultaneously capture and recover substances the size of a single cell.

Note that this disclosure includes appropriate combinations of any of the various embodiments and/or examples described above, and can achieve the effects of each embodiment and/or example.

The fiber mesh sheet according to the present disclosure can be used to contribute to new developments in pharmaceutical development, for example, by constructing a test system using cells cultured using a cell culture chip.

1, 2 Fiber 101 Fiber mesh sheet 102 First layer of aligned fibers 103 Second layer of aligned fibers S01 Film preparation S02 First layer spinning S03 90° rotation S04 Second layer spinning 300 Cell culture chip 11 First substrate 12 First partition layer 14 Second partition layer 15 Second substrate 31 Hole 32 Hole 33 First flow path 34 First inlet 35 First inlet flow path 36 First main flow path 37 First outlet flow path 38 First outlet 41 Second flow path 42 Second inlet 43 Second inlet flow path 44 Second main flow path 45 Second outlet flow path 46 Second outlet

Claims (4)

A mesh structure in which two or more layers of planar aligned fiber groups are laminated, in which a plurality of fibers made of a polymer material are aligned in one direction along the longitudinal direction of the fibers within a plane,
The longitudinal directions of the fibers of each of the adjacent two aligned fiber groups intersect at a crossing angle of 30° or more and 150° or less in a plan view seen from a direction perpendicular to the surface,
an upper portion of a cross section of the fibers of the aligned fiber group in the lowest layer on a side where there is an adjacent aligned fiber group is substantially circular, and a lower portion on a side where there is no adjacent aligned fiber group is substantially flat;
The cross section of the fibers of the arranged fiber group other than the bottom layer is substantially circular,
The average diameter of the fibers in the aligned fiber group is 1 μm or more and 50 μm or less.
Fiber mesh sheet. The fiber mesh sheet according to claim 1, wherein the contact angle of the substantially circular fibers of the aligned fiber groups other than the bottom layer with water is 90° or more and 150° or less. a step of laminating two or more planar aligned fiber groups, each of which has a plurality of fibers made of a polymeric material aligned in one direction along a plane, such that the longitudinal directions of the fibers in adjacent aligned fiber groups intersect at a crossing angle of 30° or more and 150° or less in a planar view seen from a direction perpendicular to the plane;
a step of heat-treating the fibers at a temperature equal to or higher than the melting point of the polymeric material of the fibers and equal to or lower than a temperature (melting point + 100° C.) at which the fibers melt and break;
Including,
The heat treatment step entangles the majority of the contact portions of the aligned fiber groups of the two adjacent layers ,
A method for manufacturing a fiber mesh sheet, comprising the steps of: heating the fibers at a temperature equal to or higher than the melting point of the polymer material of the fibers and lower than the temperature at which the fibers melt and break (melting point + 100°C) to make the lower part of the cross section of the fibers of the lowest layer of aligned fiber groups on the side where there are no adjacent aligned fiber groups approximately flat . A cell culture chip comprising the fiber mesh sheet according to claim 1 or 2 .

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