WO2020008805A1

WO2020008805A1 - Cell division device

Info

Publication number: WO2020008805A1
Application number: PCT/JP2019/022761
Authority: WO
Inventors: 淳史稲田; 伸彦加藤; 英俊高山
Original assignee: 富士フイルム株式会社
Priority date: 2018-07-05
Filing date: 2019-06-07
Publication date: 2020-01-09

Abstract

This cell division device includes: a flow path through which a cell suspension flows; a first mesh that has a first pore diameter and is disposed in the flow path; a second mesh that has a smaller pore diameter than the first pore diameter and is disposed in the flow path at a location downstream of the first mesh in the flow direction of the cell suspension; a first flow path section extending from the upstream side of the first mesh in the flow direction to the first mesh; and a second flow path section extending from the upstream side of the second mesh to the second mesh, the second flow path section being a flow path section between the first mesh and the second mesh. The linear velocity of the cell suspension when the cell suspension flowing through the second flow path section reaches the second mesh is equal to or less than the linear velocity of the cell suspension when the cell suspension flowing through the first flow path section reaches the first mesh.

Description

Cell division device

技術 The disclosed technology relates to a cell division device.

The following technology is known as a technology relating to a cell dividing device that divides a cell aggregate (cell mass) in which a plurality of cells aggregate into smaller cell aggregates.

For example, Japanese Patent Application Laid-Open No. 2016-136956 (Patent Document 1) describes an apparatus for reducing the size of a macroscopic aggregate of cells to a smaller cell aggregate having a size equal to or smaller than a predetermined value. The apparatus has a first reduced size mesh located near the inlet in the cleaning chamber and a second reduced size mesh located near the outlet in the cleaning chamber. The opening of the first reduced mesh is larger than the opening of the second reduced mesh.

Furthermore, WO 2014/136581 (Patent Document 2) describes a culture system having a passage filter portion having a mesh capable of dividing a cell mass of pluripotent stem cells.

Japanese Patent Application Laid-Open No. 2017-22187 (Patent Document 3) discloses a first dividing mechanism for dividing a cell mass composed of stem cells established by an initialization culture device into a plurality of cell masses, and a method for expanding and culturing cells by an expansion culture device. There is described a stem cell manufacturing system including a second dividing mechanism for dividing a cell mass composed of stem cells into a plurality of cell masses. At least one of the first and second dividing mechanisms includes a divider having a through hole therein, and the through hole has a large hole diameter portion and a small hole diameter portion alternately.

In culturing stem cells such as iPS cells (induced pluripotent stem cells), if the size of cell aggregates (spheres) generated by culturing the cells becomes excessive, the cell aggregates adhere and fuse to each other, and the cells start to differentiate. Or the cells in the central part of the cell aggregate are necrotic. Therefore, in order to prevent the size of the cell aggregate from becoming excessively large, the cell aggregate is divided (disintegrated) into a plurality of smaller-sized cell aggregates at an appropriate time during the cell culture period. Division processing is being performed.

As a method of dividing the cell aggregate, a method of mechanically dividing the cell aggregate by passing a cell suspension containing the cell aggregate through a mesh having a plurality of openings (mesh) has been proposed. . However, according to this method, there is a problem that cells accumulate on the mesh surface as the throughput increases. This problem becomes more pronounced when mass culture is performed. That is, when a large-scale culture is performed, the amount of cells deposited on the mesh surface increases with an increase in the cumulative processing amount, the substantial pore diameter of the mesh changes every moment, and the area of a region effectively functioning as a mesh. Changes every moment. As a result, the size variation of the cell aggregate after the division processing may be large. In addition, there is a possibility that the ratio of cell aggregates whose size after the splitting process deviates from the target size becomes large. In addition, cells deposited on the mesh surface may be extruded and deformed by the subsequent cell suspension, which may cause a problem such that cell quality is reduced and cell proliferation is reduced.

技術 The disclosed technology has been made in view of the above points, and has as its object to suppress the accumulation of cell aggregates on the mesh surface.

The cell dividing device according to the disclosed technology includes a channel through which a cell suspension flows, a first mesh having a first pore diameter disposed in the channel, and a first mesh in the channel. A second mesh having a pore diameter smaller than the first pore diameter, the second mesh being arranged downstream in the flow direction of the cell suspension, and reaching the first mesh from the upstream side in the flow direction with respect to the first mesh. A first channel section, a channel section between the first mesh and the second mesh, and a second channel extending from the upstream side in the flow direction to the second mesh with respect to the second mesh. And a flow path section. The linear velocity of the cell suspension flowing through the second channel section when the cell suspension reaches the second mesh is determined by the linear velocity of the cell suspension flowing through the first channel section reaching the first mesh. When the linear velocity of the cell suspension is the same or less.

This makes it possible to suppress the accumulation of cell aggregates on the mesh surface.

において In the cell division device according to the disclosed technology, the area of the second mesh may be equal to or larger than the area of the first mesh.

Accordingly, the linear velocity of the cell suspension flowing through the second flow path section when the cell suspension reaches the second mesh is reduced by the cell suspension flowing through the first flow path section. It can be the same or less than the linear velocity of the cell suspension when it reaches the mesh.

In the cell division device according to the disclosed technology, the pressure difference between the upstream side and the downstream side of the second mesh is equal to or smaller than the pressure difference between the upstream side and the downstream side of the first mesh. You may.

In the cell division device according to the disclosed technology, the length of the first flow path section along the flow direction is the length of the flow path in the direction intersecting the flow direction at the portion where the first mesh is arranged. It is preferably at least 0.3 times. This allows the cell suspension flowing through the first flow path section to stabilize its flow before it reaches the first mesh, stably dividing the cell aggregate by the first mesh. It can be carried out.

In the cell division device according to the disclosed technology, the length of the second flow path section along the flow direction is the length of the flow path in the direction intersecting the flow direction at the portion where the second mesh is arranged. It is preferably at least 0.3 times. This allows the cell suspension flowing through the second channel section to stabilize its flow before it reaches the second mesh, stably dividing the cell aggregate by the second mesh. It can be carried out.

In the cell division device according to the disclosed technology, the area of the cross section that intersects the flow direction of the first flow path section extends over the entire first flow path section, and the portion where the first mesh of the flow path is arranged May be the same as the area of the cross section intersecting the flow direction.

Thereby, the effect of stabilizing the flow of the cell suspension flowing through the first channel section can be promoted.

The cell division device according to the disclosed technology includes a first division module including a first channel section and a first mesh, a second division section including a second channel section and a second mesh, and a first division module. May include a second divided module configured as a separate body, and a pipe connecting the first divided module and the second divided module.

Thereby, the first divided module and the second divided module can be individually exchanged. Further, it is possible to flexibly cope with a case where the pore size of at least one of the first mesh and the second mesh is adjusted according to the size of the cell aggregate contained in the cell suspension to be treated. It is. Further, it is possible to flexibly cope with a case where another divided module is provided between the first divided module and the second divided module.

において In the cell division device according to the disclosed technology, the opening ratio of the first mesh is preferably 60% or more and 80% or less, and the opening ratio of the second mesh is preferably 55% or more and 77% or less.

This makes it possible to perform an appropriate division process, and suppress the accumulation of cell aggregates on the mesh surface.

In the cell division device according to the disclosed technology, the first mesh and the second mesh are each configured to include a fibrous member, and are at least 4.5 times the wire diameter of the fibrous member constituting itself. It is preferable to have a plurality of openings having a large hole diameter.

This makes it possible to appropriately divide the cell aggregate in each of the first mesh and the second mesh.

According to the disclosed technique, it is possible to suppress the accumulation of cell aggregates on the mesh surface.

It is sectional drawing which shows an example of a structure of the cell division | segmentation apparatus which concerns on embodiment of the technique of indication. FIG. 4 is a plan view of a first mesh and a second mesh according to an embodiment of the disclosed technology. It is an enlarged view of the part enclosed with the broken line in FIG. 2A. It is sectional drawing which shows the mode of the division | segmentation process using the cell division | segmentation apparatus which concerns on embodiment of the technique of indication. It is sectional drawing which shows the mode of the division | segmentation process using the cell division | segmentation apparatus which concerns on a comparative example. It is sectional drawing which shows an example of a structure of the cell division | segmentation apparatus which concerns on embodiment of the technique of indication. It is sectional drawing which shows an example of a structure of the cell division | segmentation apparatus which concerns on embodiment of the technique of indication. It is sectional drawing which shows the mode of the division | segmentation process using the cell division | segmentation apparatus which concerns on embodiment of the technique of indication. It is sectional drawing which shows an example of a structure of the cell division | segmentation apparatus which concerns on embodiment of the technique of indication. It is sectional drawing which shows an example of a structure of the cell division | segmentation apparatus which concerns on embodiment of the technique of indication.

Hereinafter, embodiments of the disclosed technology will be described with reference to the drawings. In the drawings, substantially the same or equivalent components or portions are denoted by the same reference numerals.

[First Embodiment]
FIG. 1 is a cross-sectional view illustrating an example of a configuration of a cell division device 1 according to the first embodiment of the disclosed technology.

The cell dividing device 1 has the container 10 that forms the cell suspension flow path 30. At one end of the container 10, an inlet 11 for introducing a cell suspension containing cell aggregates into the inside of the container 10 is provided, and at the other end of the container 10, a divided cell suspension An outlet 12 for allowing the liquid to flow out of the container 10 is provided. The cross section of each position of the flow path 30 that intersects the flow direction F1 of the cell suspension is, for example, circular.

The cell division device 1 has a first mesh 21 and a second mesh 22 disposed between the inflow port 11 and the outflow port 12 in the middle of the channel 30. 2A is a plan view of the first mesh 21 and the second mesh 22, and FIG. 2B is an enlarged view of a portion P1 surrounded by a broken line in FIG. 2A. As shown in FIG. 2B, each of the first mesh 21 and the second mesh 22 has a plurality of openings (mesh) 201 formed by, for example, plain weaving a plurality of fibrous members 200. The weaving method of the fibrous member 200 is not limited to plain weaving. The material of the fibrous member 200 is not particularly limited, but is preferably made of a material having high corrosion resistance. For example, nylon or stainless steel can be suitably used. In the present embodiment, the outer shapes of the first mesh 21 and the second mesh 22 are circular in conformity with the cross-sectional shape of the flow channel 30 that intersects the flow direction F1 of the cell suspension. The mesh used in the cell division device according to the embodiment of the disclosed technology does not include a nonwoven fabric.

The first mesh 21 and the second mesh 22 are installed in the container 10 such that the main surface having the plurality of openings 201 extends in a direction intersecting with the flow direction F1 of the cell suspension. . When the cell suspension passes through the first mesh 21 and the second mesh 22, the cell aggregates contained in the cell suspension are mechanically divided. That is, the cell aggregate is divided by a two-stage division process using two meshes.

The second mesh 22 is disposed downstream of the first mesh 21 in the flow direction F1 of the cell suspension. That is, the first mesh 21 is provided on the inflow port 11 side of the channel 30, and the second mesh 22 is provided on the outflow port 12 side of the channel 30. Therefore, the cell suspension flowing in the flow path 30 along the flow direction F1 passes through the first mesh 21 and then passes through the second mesh 22.

径 The diameter of the opening 201 of the first mesh 21 (hereinafter, referred to as the pore diameter L) is, for example, smaller than the average diameter of the cell aggregate before the division processing. The pore size L of the second mesh 22 is smaller than the pore size L of the first mesh 21, and is determined according to the target size of the cell aggregate after the division. In each of the first mesh 21 and the second mesh 22, the hole diameter L is preferably at least 4.5 times the wire diameter d of the fibrous member 200 constituting the mesh. This makes it possible to appropriately divide the cell aggregate in each of the first mesh 21 and the second mesh 22.

The opening ratio of the first mesh 21 is preferably 60% or more and 80% or less, and the opening ratio of the second mesh 21 is preferably 55% or more and 77% or less. The aperture ratio A of the first and second meshes can be represented by the following equation (1). However, in equation (1), B is the total area of the openings of the mesh, and C is the area of the entire mesh. The aperture ratios of the first and second meshes may be calculated by deriving a hole diameter L and a wire diameter d from a microscopic image of the mesh, and inputting the derived hole diameters L and the wire diameter d into computer software. Good. By setting the range of the aperture ratio of the first mesh 21 and the second mesh 22 to the above range, appropriate division processing can be performed, and accumulation of cell aggregates on the mesh surface can be suppressed.
A = B / C (1)

The cell dividing device 1 has a first flow path section 31 extending from the upstream side in the flow direction F1 of the cell suspension to the first mesh 21 with respect to the first mesh 21. In addition, the cell dividing device 1 is a flow path section between the first mesh 21 and the second mesh 22, and from the upstream side in the flow direction F1 of the cell suspension with respect to the second mesh 22. It has a second flow path section 32 leading to the second mesh 22.

The linear velocity V2 [cm / s] of the cell suspension when the cell suspension flowing through the second flow path section 32 reaches (contacts) the second mesh 22 is equal to the first flow path section 31. It is preferable that the linear velocity V1 of the cell suspension when the flowing cell suspension reaches the first mesh 21 is equal to or smaller than V1. The linear velocities V1 and V2 can be expressed by the following equations (2) and (3), respectively.
V1 [cm / s] = Q1 [mL / s] / S1 [cm ² ] (2)
V2 [cm / s] = Q2 [mL / s] / S2 [cm ² ] (3)

However, in the expression (2), Q1 [mL / s] is a flow rate of the cell suspension reaching the first mesh 21 per unit time. In the equation (2), S1 is an area of a cross section of the flow channel 30 where the first mesh 21 is arranged, which crosses the flow direction F1 of the cell suspension. In the cell division device 1 according to the present embodiment, the area S1 is the same as the effective area of the first mesh 21. In the equation (3), Q2 [mL / s] is a flow rate of the cell suspension reaching the second mesh 22 per unit time. In the equation (3), S2 is an area of a cross section of the flow channel 30 where the second mesh 22 is arranged, which crosses the flow direction F1 of the cell suspension. In the cell division device 1 according to the present embodiment, the area S2 is the same as the effective area of the second mesh 22. In addition, the effective area of the first mesh 21 and the second mesh 22 is an area of a region of the mesh that can come into contact with the cell suspension. In the channel 30, the flow rate of the cell suspension passing through an arbitrary cross section per unit time is constant, so that Q1 and Q2 are equal.

The linear velocities V1 and V2 can be controlled by, for example, the areas S1 and S2. For example, by making the area S1 and the area S2 the same, the linear velocity V1 and the linear velocity V2 can be made the same. According to the cell division device 1 according to the present embodiment, since the area S1 and the area S2 are the same, the linear velocity V1 and the linear velocity V2 become equal. On the other hand, by making the area S2 larger than the area S1, the linear velocity V2 can be made smaller than the linear velocity V1 (see FIG. 8).

The pressure difference (transmembrane pressure) ΔP2 between the upstream side and the downstream side of the second mesh 22 is equal to the pressure difference (transmembrane pressure) ΔP1 between the upstream side and the downstream side of the first mesh 21. Preferably, it is the same or smaller. By making the linear velocity V1 and the linear velocity V2 the same, the pressure difference (transmembrane pressure) ΔP2 and the pressure difference (transmembrane pressure) ΔP1 can be made the same. Further, by making the linear velocity V2 smaller than the linear velocity V1, the pressure difference (transmembrane pressure difference) ΔP2 can be made smaller than the pressure difference (transmembrane pressure difference) ΔP1.

{Circle around (1)} The first channel section 31 is an approach section until the cell suspension flowing from the inlet 11 contacts the first mesh 21. By securing an appropriate length as the length of the first flow path section 31 along the flow direction F1, the cell suspension flowing through the first flow path section 31 is converted into the first mesh 21. By the time, the flow can be stabilized, and the cell aggregate can be stably divided by the first mesh 21. The length A1 of the first flow path section 31 along the flow direction F1 is the length W1 (first length) of the flow path 30 in the direction intersecting the flow direction F1 at the portion where the first mesh 21 is arranged. It is preferably at least 0.3 times (equivalent to the diameter of the mesh 21).

In addition, in the present embodiment, the first flow path section 31 is defined by the cylindrical portion of the container 10. Therefore, the area of the cross section of the first flow path section 31 that intersects with the flow direction F1 of the cell suspension extends over the entire area of the first flow path section 31 and the first mesh 21 of the flow path 30. Is the same as the cross-sectional area S1 (corresponding to the effective area of the first mesh 21) of the cross-section that intersects with the flow direction F1 of the cell suspension at the position where is disposed. In this way, by making the area of the cross section of the first flow path section 31 that intersects with the flow direction F1 of the cell suspension constant, the flow of the cell suspension flowing through the first flow path section 31 is reduced. The effect of stabilization can be promoted.

Similarly, the second flow path section 32 is a run-up section until the cell suspension that has passed through the first mesh 21 contacts the second mesh 22. By securing an appropriate length as the length of the second flow path section 32 along the flow direction F1, the cell suspension flowing through the second flow path section 32 is transferred to the second mesh 22. By this time, the flow can be stabilized, and the cell aggregates can be stably divided by the second mesh 22. The length A2 of the second flow path section 32 along the flow direction F1 is the length W2 of the flow path 30 in the direction intersecting the flow direction F1 at the portion where the second mesh 22 is arranged (the second length W2). It is preferably at least 0.3 times (equivalent to the diameter of the mesh 22).

In addition, in the present embodiment, the second flow path section 32 is defined by the cylindrical portion of the container 10. Accordingly, the area of the cross section of the second flow path section 32 that intersects with the flow direction F1 of the cell suspension extends over the entire area of the second flow path section 32 so that the second mesh 22 of the flow path 30 is formed. Is the same as the area S2 (corresponding to the effective area of the second mesh 22) of the cross section intersecting with the flow direction F1 of the cell suspension at the position where is disposed. As described above, by keeping the area of the cross section of the second flow path section 32 that intersects with the flow direction F1 of the cell suspension constant, the flow of the cell suspension flowing through the second flow path section 32 is reduced. The effect of stabilization can be promoted.

FIG. 3 is a cross-sectional view showing a state of a dividing process using the cell dividing device 1. The cell suspension 101 containing the cell aggregates 100 that has flowed into the container 10 from the inlet 11 reaches the first mesh 21 via the first channel section 31. The cell aggregate 100 is divided by passing through the first mesh 21, and the average diameter of the cell aggregate 100 is reduced. After that, the cell suspension 101 reaches the second mesh 22 via the second channel section 32. The cell aggregate 100 is divided by passing through the second mesh 22, and the average diameter of the cell aggregate 100 is further reduced. The cell suspension 101 that has passed through the second mesh 22 is discharged to the outside of the container 10 via the outlet 12.

According to the cell division device 1 according to the present embodiment, the cell aggregate 100 is divided by a two-stage division process using two meshes. The hole diameter L of the second mesh 22 is smaller than the hole diameter L of the first mesh 21. Therefore, the average diameter of the cell aggregate 100 before the division treatment is X1, the average diameter of the cell aggregate 100 after passing through the first mesh 21 is X2, and the cell aggregate 100 after passing through the second mesh 22. X1> X2> X3 when the average diameter of X3 is X3.

Here, FIG. 4 is a cross-sectional view showing a state of a division process using the cell division device 1X according to the comparative example. The cell division device 1X according to the comparative example has a single mesh 22X. The pore size of the mesh 22X is the same as the pore size of the second mesh 22 provided in the cell division device 1 according to the embodiment of the disclosed technology.

According to the cell division device 1X according to the comparative example, the divergence between the average diameter of the cell aggregate to be subjected to the division processing and the pore size of the mesh is larger than that of the cell division device 1 according to the embodiment of the disclosed technology. As a result, as shown in FIG. 4, the cell aggregate 100 is more likely to be deposited on the surface of the mesh 22X. Therefore, when a large number of cells are processed using the cell division device 1X according to the comparative example, the area of the region effectively functioning as a mesh and the pore size of the mesh 22X change every moment as the cumulative processing amount increases. As a result, there is a possibility that the size of the cell aggregate 100 after the division processing varies greatly. In addition, the ratio of the cell aggregates 100 whose size after the division process deviates from the target size increases. Further, as shown in FIG. 4, a part of the cells deposited on the surface of the mesh 22X is extruded and deformed by the subsequent cell suspension 101, and as a result, the quality of the cells is reduced, and the proliferation of the cells is reduced. May decrease.

On the other hand, according to the cell dividing device 1 according to the embodiment of the disclosed technology, as described above, the cell aggregate 100 is divided stepwise so that the average diameter gradually decreases, Of the cell aggregate 100 can be suppressed. Therefore, even when a large number of cells are processed using the cell division device 1, the area and the hole diameter L of the effective area of the first mesh 21 and the second mesh 22 change with an increase in the cumulative processing amount. Can be suppressed. As a result, as compared with the cell division device 1X according to the comparative example, the variation in the size of the cell aggregate 100 after the division processing can be reduced. In addition, according to the cell dividing device 1 according to the embodiment of the disclosed technology, since the accumulation of the cell aggregates 100 on the mesh surface can be suppressed, a part of the cells deposited on the mesh surface can be removed from the subsequent cell suspension. It can be prevented from being deformed by being pushed out by the suspension 101. Therefore, it is possible to suppress a decrease in cell quality and a decrease in cell proliferation.

Further, according to the cell division device 1 according to the embodiment of the disclosed technology, the cell suspension 101 flowing through the second flow path section 32 reaches the second mesh 22 when the line of the cell suspension 101 is drawn. The velocity V2 is the same as the linear velocity V1 of the cell suspension 101 when the cell suspension 101 flowing through the first flow path section 31 reaches the first mesh 21. As a result, damage to the cells can be reduced as compared with the case where the linear velocity V2 is higher than the linear velocity V1. The plurality of cells constituting the cell aggregate 100 are considered to be damaged to some extent by passing through the first mesh 21. Therefore, the cells that pass through the second mesh 22 are further suppressed from being damaged, so that the cell survival rate can be increased.

According to the cell division device 1 according to the embodiment of the disclosed technology, the length A1 of the first channel section 31 along the flow direction F1 is the portion of the channel 30 where the first mesh 21 is arranged. Is 0.3 times or more the length W1 (corresponding to the diameter of the first mesh 21) in the direction intersecting the flow direction F1. By ensuring an appropriate length as the length of the first flow path section 31 along the flow direction F1, the cell suspension 101 flowing through the first flow path section 31 is converted into a first mesh. By the time the flow reaches the cell 21, the flow can be stabilized, and the cell aggregate 100 can be stably divided by the first mesh 21.

Further, according to the cell division device 1 according to the embodiment of the disclosed technology, the length A2 of the second flow path section 32 along the flow direction F1 is the same as the second mesh 22 of the flow path 30. The length W2 (corresponding to the diameter of the second mesh 22) in the direction intersecting with the flow direction F1 at the portion where the flow has occurred is 0.3 times or more. By securing an appropriate length as the length of the second flow path section 32 along the flow direction F1, the cell suspension 101 flowing through the second flow path section 32 is converted into a second mesh. By the time the flow reaches the cell 22, the flow can be stabilized, and the cell aggregate 100 can be stably divided by the second mesh 22.

Further, according to the cell division device 1 according to the embodiment of the disclosed technology, the first channel section 31 and the second channel section 32 each have a cross section that intersects the flow direction F1 of the cell suspension. The area is fixed. Thereby, the effect of stabilizing the flow of the cell suspension 101 flowing through the first channel section 31 and the second channel section 32 is promoted.

[Second embodiment]
FIG. 5 is a cross-sectional view illustrating an example of a configuration of a cell division device 1A according to the second embodiment of the disclosed technology. The cell division device 1A further includes a third mesh 23. The third mesh 23 is disposed between the first mesh 21 and the second mesh 22. The hole diameter L of the third mesh 23 is smaller than the hole diameter L of the first mesh 21 and larger than the hole diameter L of the second mesh 22.

{Circle around (1)} The cell division device 1A has a first flow path section 31 extending from the upstream side in the flow direction F1 of the cell suspension to the first mesh 21 with respect to the first mesh 21. In addition, the cell dividing device 1A is a flow path section between the first mesh 21 and the third mesh 23, and is located on the upstream side in the flow direction F1 of the cell suspension with respect to the third mesh 23. It has a second flow path section 32 reaching the third mesh 23. Further, the cell dividing device 1A is a flow path section between the third mesh 23 and the second mesh 22, which is located on the upstream side in the flow direction F1 of the cell suspension with respect to the second mesh 22. It has a third flow path section 33 leading to the second mesh 22.

The linear velocity V2 [cm / s] of the cell suspension when the cell suspension flowing through the second flow path section 32 reaches (contacts) the third mesh 23 is less than the first flow path section 31. It is preferable that the linear velocity V1 of the cell suspension when the flowing cell suspension reaches the first mesh 21 is equal to or smaller than V1. The linear velocity V3 [cm / s] of the cell suspension when the cell suspension flowing through the third flow path section 33 reaches (contacts) the second mesh 22 is determined by the second flow path section. It is preferable that the linear velocity V2 of the cell suspension when it reaches the third mesh 23 is equal to or smaller than the linear velocity V2.

According to the cell division device 1A according to the second embodiment of the disclosed technology, the cell aggregate has three meshes arranged such that the pore diameter decreases stepwise along the flow direction F1 of the cell suspension. Are divided by three-stage division processing. Thereby, the effect of suppressing the accumulation of cell aggregation on the mesh surface can be promoted.

The number of mesh steps may be four or more. In this case, the pore diameter of the mesh arranged on the downstream side in the flow direction F1 of the cell suspension is preferably smaller than the pore diameter of the mesh arranged on the upstream side. The linear velocity of the cell suspension when the cell suspension reaches the mesh arranged on the downstream side is determined by the linear velocity of the cell suspension when the cell suspension reaches the mesh arranged on the upstream side. Preferably, it is equal to or less than the linear velocity. In order to realize this, for example, the cross-sectional area of the flow channel 30 at the portion where the mesh on the downstream side is arranged is equal to or greater than the cross-sectional area of the flow channel 30 at the portion where the mesh on the upstream side is arranged. May be larger.

[Third Embodiment]
FIG. 6 is a cross-sectional view illustrating an example of a configuration of a cell division device 1B according to the third embodiment of the disclosed technology. The cell dividing device 1B includes a first dividing module 41 and a second dividing module 42. The first division module 41 and the second division module 42 are configured separately from each other, and are connected to each other via a pipe 50. The first division module 41 and the second division module 42 can be separated from each other.

The first division module 41 has a first container 10A that forms the flow path 30 of the cell suspension. At one end of the first container 10A, a first inlet 11A for introducing a cell suspension containing cell aggregates into the inside of the first container 10A is provided. At the end, there is provided a first outlet 12A for allowing the cell suspension to flow out of the first container 10A. The first division module 41 has the first mesh 21 disposed between the first inlet 11A and the first outlet 12A in the flow channel 30.

２The second division module 42 has the second container 10B that forms the cell suspension flow path 30. At one end of the second container 10B, a second inlet 11B for introducing a cell suspension containing cell aggregates into the inside of the second container 10B is provided. A second outlet 12B for discharging the cell suspension to the outside of the second container 10B is provided at the end. The second division module 42 has the second mesh 22 disposed between the second inlet 11B and the second outlet 12B in the flow channel 30.

孔 The pore diameter L of the first mesh 21 is, for example, smaller than the average diameter of the cell aggregate before the division processing. The pore size L of the second mesh 22 is smaller than the pore size L of the first mesh 21, and is determined according to the target size of the cell aggregate after the division.

１The first outlet 12A of the first split module 41 and the second inlet 11B of the second split module 42 are connected by a pipe 50. The pipe 50 is preferably made of, for example, a flexible member.

The first division module 41 has the first flow path section 31 extending from the upstream side in the flow direction F1 of the cell suspension to the first mesh 21 with respect to the first mesh 21. The second division module 42 is a flow path section between the first mesh 21 and the second mesh 22 and is located on the second mesh 22 from the upstream side in the flow direction F1 of the cell suspension. It has a second flow path section 32 leading to the second mesh 22.

The linear velocity V2 [cm / s] of the cell suspension when the cell suspension flowing through the second flow path section 32 reaches (contacts) the second mesh 22 is equal to the first flow path section 31. It is preferable that the linear velocity V1 of the cell suspension when the flowing cell suspension reaches the first mesh 21 is equal to or smaller than V1. The linear velocities V1 and V2 can be represented by the above equations (2) and (3), respectively. In the present embodiment, the area S1 in the expression (2) is the same as the effective area of the first mesh 21. The area S2 in the equation (3) is the same as the effective area of the second mesh 22.

The linear velocities V1 and V2 can be controlled by, for example, the areas S1 and S2. For example, by making the area S1 and the area S2 the same, the linear velocity V1 and the linear velocity V2 can be made the same. According to the cell dividing device 1B according to the present embodiment, since the area S1 and the area S2 are the same, the linear velocity V1 and the linear velocity V2 are equal. On the other hand, by making the area S2 larger than the area S1, the linear velocity V2 can be made smaller than the linear velocity V1 (see FIG. 8).

{Circle around (1)} The first channel section 31 is a run section until the cell suspension flowing from the inlet 11A comes into contact with the first mesh 21. By securing an appropriate length as the length of the first flow path section 31 along the flow direction F1, the cell suspension flowing through the first flow path section 31 is converted into the first mesh 21. By the time, the flow can be stabilized. Thereby, division of the cell aggregate by the first mesh 21 can be performed stably. The length A1 of the first flow path section 31 along the flow direction F1 is the length W1 (first length) of the flow path 30 in the direction intersecting the flow direction F1 at the portion where the first mesh 21 is arranged. It is preferably at least 0.3 times (equivalent to the diameter of the mesh 21).

Similarly, the second flow path section 32 is a run-up section until the cell suspension that has passed through the first mesh 21 contacts the second mesh 22. By securing an appropriate length as the length of the second flow path section 32 along the flow direction F1, the cell suspension flowing through the second flow path section 32 is transferred to the second mesh 22. By the time it reaches, its flow can be stabilized. Thereby, division of the cell aggregate by the second mesh 22 can be performed stably. The length A2 of the second flow path section 32 along the flow direction F1 is the length W2 of the flow path 30 in the direction intersecting the flow direction F1 at the portion where the second mesh 22 is arranged (the second length W2). It is preferably at least 0.3 times (equivalent to the diameter of the mesh 22).

FIG. 7 is a cross-sectional view showing a state of a dividing process using the cell dividing device 1B. The cell suspension 101 including the cell aggregates 100 flowing into the first division module 41 from the first inlet 11A reaches the first mesh 21 via the first channel section 31. . The cell aggregate 100 is divided by passing through the first mesh 21, and the average diameter of the cell aggregate 100 is reduced. The cell suspension 101 that has passed through the first mesh 21 flows into the second divided module 42 via the first outlet 12A, the pipe 50, and the second inlet 11B. The cell suspension 101 flowing into the second division module 42 reaches the second mesh 22 via the second flow path section 32. The cell aggregate 100 is divided by passing through the second mesh 22, and the average diameter of the cell aggregate 100 is further reduced. The cell suspension 101 that has passed through the second mesh 22 is discharged to the outside of the second division module 42 via the second outlet 12B.

As described above, according to the cell division device 1B according to the present embodiment, the cell aggregate 100 is divided by a two-stage division process using two meshes. The hole diameter L of the second mesh 22 is smaller than the hole diameter L of the first mesh 21. Therefore, the average diameter of the cell aggregate 100 before the division treatment is X1, the average diameter of the cell aggregate 100 after passing through the first mesh 21 is X2, and the cell aggregate 100 after passing through the second mesh 22. X1> X2> X3 when the average diameter of X3 is X3.

According to the cell division device 1B according to the present embodiment, it is possible to suppress the accumulation of cell aggregates on the mesh surface, as in the cell division device 1 according to the first embodiment.

The first division module 41 and the second division module 42 are configured separately from each other and can be separated from each other. Therefore, the first divided module 41 and the second divided module 42 can be individually exchanged. Also, it is possible to flexibly adjust the pore diameter L of at least one of the first mesh 21 and the second mesh 22 according to the size of the cell aggregate 100 included in the cell suspension 101 to be treated. It is possible to respond. Moreover, it is possible to flexibly cope with a case where another divided module is provided between the first divided module 41 and the second divided module 42. Further, by forming the pipe 50 connecting the first divided module 41 and the second divided module 42 with a flexible material, for example, a cell culture that automatically performs a series of processes required for cell culture When the cell dividing device 1B according to the present embodiment is incorporated in a device (not shown), the degree of freedom in the arrangement of the cell dividing device 1B can be increased.

[Fourth embodiment]
FIG. 8 is a cross-sectional view illustrating an example of a configuration of a cell division device 1C according to a fourth embodiment of the disclosed technology. Similar to the cell division device 1B according to the third embodiment, the cell division device 1C includes a first division module 41 having a first mesh 21 and a first flow path section 31, and a second flow path section 32. And a second division module 42 having the second mesh 22.

Here, let S1 be the area of the cross section of the flow channel 30 where the first mesh 21 is arranged, which crosses the flow direction F1 of the cell suspension. The area of the cross section of the flow channel 30 where the second mesh 22 is arranged, which crosses the flow direction F1 of the cell suspension, is defined as S2. In the cell division device 1 </ b> C according to the present embodiment, the area S <b> 1 is the same as the effective area of the first mesh 21, and the area S <b> 2 is the same as the effective area of the second mesh 22. In the cell dividing device 1C according to the present embodiment, the area S2 (effective area of the second mesh 22) is larger than the area S1 (effective area of the first mesh 21). Thereby, the linear velocity V2 [cm / s] of the cell suspension when the cell suspension flowing through the second flow path section 32 reaches (contacts) the second mesh 22 is reduced by the first flow path. The linear velocity V1 of the cell suspension when the cell suspension flowing in the section 31 reaches the first mesh 21 becomes smaller.

する As described above, by making the linear velocity V2 lower than the linear velocity V1, damage to cells can be reduced as compared with the case where the linear velocity V2 becomes higher than the linear velocity V1. The plurality of cells constituting the cell aggregate are considered to be damaged to some extent by passing through the first mesh 21. Therefore, the cells that pass through the second mesh 22 are further suppressed from being damaged, so that the cell survival rate can be increased.

According to the cell dividing device 1C according to the present embodiment, similarly to the cell dividing device 1 according to the first embodiment, it is possible to suppress the accumulation of cell aggregates on the mesh surface.

[Fifth Embodiment]
FIG. 9 is a cross-sectional view illustrating an example of a configuration of a cell division device 1D according to a fifth embodiment of the disclosed technology. The cell division device 1D further includes a third division module 43. The third division module 43 is disposed between the first division module 41 and the second division module 42.

The first division module 41, the second division module 42, and the third division module 43 are configured separately from each other, and are connected to each other via

pipes

50A and 50B. The first division module 41, the second division module 42, and the third division module 43 can be separated from each other.

３The third division module 43 has a third container 10C that forms the flow path 30 of the cell suspension. At one end of the third container 10C, a third inflow port 11C for introducing a cell suspension containing cell aggregates into the inside of the third container 10C is provided. At the end, a third outlet 12C for allowing the cell suspension to flow out of the third container 10C is provided.

The first outlet 12A of the first split module 41 and the third inlet 11C of the third split module 43 are connected by a pipe 50A. The third outlet 12C of the third split module 43 and the second inlet 11B of the second split module 42 are connected by a pipe 50B. It is preferable that each of the

pipes

50A and 50B is formed of a flexible member.

The third division module 43 has the third mesh 23 disposed between the third inflow port 11C and the third outflow port 12C in the flow path 30. That is, the third mesh 23 is disposed between the first mesh 21 and the second mesh 22. The hole diameter L of the third mesh 23 is smaller than the hole diameter L of the first mesh 21 and larger than the hole diameter L of the second mesh 22.

The first division module 41 has the first flow path section 31 extending from the upstream side in the flow direction F1 of the cell suspension to the first mesh 21 with respect to the first mesh 21. The third division module 43 is a flow path section between the first mesh 21 and the third mesh 23, and is located on the third mesh 23 from the upstream side in the flow direction F1 of the cell suspension. It has a second flow path section 32 reaching the third mesh 23. The second division module 42 is a flow path section between the third mesh 23 and the second mesh 22, and from the upstream side in the flow direction F <b> 1 of the cell suspension with respect to the second mesh 22. It has a third flow path section 33 leading to the second mesh 22.

Here, let S1 be the area of the cross section of the flow channel 30 where the first mesh 21 is arranged, which crosses the flow direction F1 of the cell suspension. The area of the cross section of the flow channel 30 where the second mesh 22 is arranged, which crosses the flow direction F1 of the cell suspension, is defined as S2. The area of the cross section of the flow channel 30 where the third mesh 23 is arranged and which crosses the flow direction F1 of the cell suspension is S3.

In the cell division device 1D according to the present embodiment, the area S1 is the same as the effective area of the first mesh 21, the area S2 is the same as the effective area of the second mesh 22, and the area S3 is 3 is the same as the effective area of the mesh 23. In the cell division device 1D according to the present embodiment, the area S3 (the effective area of the third mesh 23) is larger than the area S1 (the effective area of the first mesh 21). The area S2 (effective area of the second mesh 22) is larger than the area S3 (effective area of the third mesh 23). Thereby, the linear velocity V3 [cm / s] of the cell suspension when the cell suspension flowing through the second flow path section 32 reaches (contacts) the third mesh 23 is reduced by the first flow path. The linear velocity V1 of the cell suspension when the cell suspension flowing in the section 31 reaches the first mesh 21 becomes smaller. The linear velocity V2 [cm / s] of the cell suspension when the cell suspension flowing through the third flow path section 33 reaches (contacts) the second mesh 22 is determined by the second flow path section. The linear velocity V3 of the cell suspension when it reaches the third mesh 23 when flowing through the cell 32 becomes smaller.

According to the cell division device 1D according to the fifth embodiment of the disclosed technology, the cell aggregate has three meshes arranged such that the pore diameter decreases stepwise along the flow direction F1 of the cell suspension. Are divided by three-stage division processing. Thereby, the effect of suppressing the accumulation of the cell aggregate 100 on the mesh surface can be promoted. Further, by setting the linear velocity V3 to be lower than the linear velocity V1 and setting the linear velocity V2 to be lower than the linear velocity V3, damage to cells can be reduced.

[Example]
The cell culture was performed under a plurality of conditions in which the number of mesh stages, the pore size and the opening ratio, and the linear velocity of the cell suspension when reaching each mesh were varied. The evaluation results are shown in Table 1 below. The evaluation was performed from the viewpoint of the recovery rate and the quality of the cells after culture. The quality of the cells is reflected in the shape and size of the cell aggregates. Since the variation in the size of the cell aggregate immediately after the division greatly affects the variation in the size after the culture, the quality was evaluated based on the variation in the size of the cell aggregate immediately after the division.
The cell recovery rate was a value obtained by dividing the cell count measurement result after the division treatment by the cell count measurement result before the division treatment.
The variation in cell aggregate size is calculated by sampling a predetermined amount of the cell suspension after the division process, measuring the diameter by approximating the sphere diameter of all the cell aggregates included in the image analysis, and calculating the variation coefficient by statistical processing. (Standard deviation ÷ mean) was applied.
The size of cell aggregates can be determined by capturing all cell aggregates contained in a given amount of cell suspension into an image using Cell Imager (SCREEN), determining the viability, and determining the cell diameter distribution of only viable cells. And the arithmetic mean was calculated.
The aperture ratio of the mesh was calculated from the derived hole diameter L and wire diameter d by deriving the hole diameter L and wire diameter d of the mesh from a microscope image of the mesh.

The criteria for the recovery are as follows.
A: The recovery rate is 75% or more B: The recovery rate is 60% or more and less than 75% C: The recovery rate is less than 60% The evaluation criteria for the quality are as follows.
A: Coefficient of variation of diameter distribution of cell aggregate is less than 0.32 B: Coefficient of variation of diameter distribution of cell aggregate is 0.32 or more and less than 0.35 C: Coefficient of variation of diameter distribution of cell aggregate is 0. 35 or more

では In Examples 1 to 3, the number of mesh steps was two, and in Example 4, the number of mesh steps was three. In each of Examples 1 to 4, each mesh was arranged so that the pore diameter of the mesh gradually decreased from the upstream side to the downstream side in the flow direction of the cell suspension. In Example 1, the linear velocity when the cell suspension reached the mesh on the upstream side was the same as the linear velocity when the cell suspension reached the mesh on the downstream side. In Examples 2 to 4, the linear velocity when the cell suspension reached the downstream mesh was set lower than the linear velocity when the cell suspension reached the upstream mesh.

In Comparative Example 1, the number of mesh steps was one. In Comparative Example 2, the number of mesh steps was set to two, and the linear velocity when the cell suspension reached the downstream mesh was higher than the linear velocity when the cell suspension reached the upstream mesh. did.

From the upstream side to the downstream side in the flow direction of the cell suspension, the cell dividing device is configured so that the pore size of the mesh gradually decreases, and when the cell suspension reaches the downstream side mesh. In Examples 1 to 4 in which the linear velocity was the same as or lower than the linear velocity when the cell suspension reached the mesh on the upstream side, the A judgment was made for at least one of the recovery rate and the quality. No C-determination was obtained. On the other hand, in Comparative Example 1, both the recovery rate and the quality were determined to be C, and in Comparative Example 2, both the recovery rate and the quality were determined to be B.

The disclosure of Japanese Patent Application No. 2018-128454 is incorporated herein by reference in its entirety.

All documents, patent applications, and technical standards described herein are to the same extent as if each individual document, patent application, and technical standard were specifically and individually stated to be incorporated by reference. Incorporated herein by reference.

1, 1A, 1B, 1C, 1D Cell dividing device 10 Container 11 Inflow port 11A First inflow port 11B Second inflow port 12 Outflow port 12A First outflow port 12B

Second outflow port

50, 50A, 50B Piping 21 First mesh 22 Second mesh 22X Mesh 23 Third mesh 30 Flow path 31 First flow path section 32 Second flow path section 33 Third flow path section 41 First split module 42 Second Division module 43 Third division module 100 Cell aggregate 101 Cell suspension 200 Fibrous member 201 Opening F1 Flow direction L Hole diameter d Wire diameter

Claims

A channel through which the cell suspension flows,
A first mesh having a first hole diameter arranged in the flow path;
A second mesh having a pore diameter smaller than the first pore diameter, the second mesh being arranged on the downstream side in the flow direction of the cell suspension with respect to the first mesh in the flow channel;
A first flow path section extending from the upstream side in the flow direction to the first mesh with respect to the first mesh,
A second flow path section between the first mesh and the second mesh, the second flow path section extending from the upstream side in the flow direction to the second mesh with respect to the second mesh; When,
Including
When the linear velocity of the cell suspension flowing through the second flow path section reaches the second mesh, the cell suspension flowing through the first flow path section has a linear velocity. A cell division device that is equal to or less than the linear velocity of the cell suspension when reaching the first mesh.
The cell division device according to claim 1, wherein the area of the second mesh is equal to or larger than the area of the first mesh.
The pressure difference between the upstream side and the downstream side of the second mesh is the same as or smaller than the pressure difference between the upstream side and the downstream side of the first mesh. Cell division device.
The length of the first flow path section along the flow direction is at least 0.3 times the length of the flow path in a direction intersecting the flow direction at a portion where the first mesh is arranged. The cell division device according to any one of claims 1 to 3.
The length of the second flow path section along the flow direction is at least 0.3 times the length of the flow path in a direction intersecting the flow direction at a portion where the second mesh is arranged. The cell division device according to any one of claims 1 to 3.
The area of the cross section of the first flow path section that intersects with the flow direction is over the entire area of the first flow path section, and the flow direction at the portion of the flow path where the first mesh is arranged. The cell division device according to any one of claims 1 to 5, wherein the cell division device has the same area as an intersecting cross section.
A first division module including the first flow path section and the first mesh;
A second division module including the second flow path section and the second mesh and configured as a separate body from the first division module;
Piping connecting the first split module and the second split module;
The cell division device according to any one of claims 1 to 6, comprising:
An aperture ratio of the first mesh is 60% or more and 80% or less;
The cell division device according to any one of claims 1 to 7, wherein an aperture ratio of the second mesh is 55% or more and 77% or less.
The first mesh and the second mesh are each configured to include a fibrous member, and have a plurality of openings having a hole diameter 4.5 times or more the wire diameter of the fibrous member constituting the first mesh and the second mesh. The cell division device according to any one of claims 1 to 8, comprising a unit.
A third mesh having a pore size smaller than the first pore size and larger than the second mesh size, disposed between the first mesh and the second mesh in the flow path; ,
A third flow path section between the first mesh and the third mesh, the third flow path section extending from the upstream side in the flow direction to the third mesh with respect to the third mesh; ,
Further comprising
When the linear velocity of the cell suspension flowing through the third flow path section reaches the third mesh, the cell suspension flows through the first flow path section. The same or less than the linear velocity of the cell suspension when reaching the first mesh,
When the linear velocity of the cell suspension flowing through the second flow path section reaches the second mesh, the cell suspension flowing through the third flow path section is The cell dividing device according to any one of claims 1 to 9, wherein the linear velocity of the cell suspension when reaching the third mesh is equal to or smaller than the linear velocity.