Cell Dispersion System

Abstract

To provide a technology for obtaining a cell with a size that can be used for the production of cell sheets from a cell aggregate by a simple process using only physical effects.SOLUTION: Provided is a cell dispersion system that crushes and disperses cell aggregates of pluripotent stem cells, somatic stem cells, progenitor cells, or somatic cells, the cell dispersion system having a first opening, a second opening, and a disperser having a narrow flow path of a predetermined length provided between the first opening and the second opening. The cell dispersion system moves a liquid containing cell aggregates from the first or second opening toward the second or first opening through the narrow flow path, and is capable of crushing and dispersing at least a portion of the cell aggregates into a size that can be used for the production of cell sheets using only physical effects.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a cell dispersion system. [Background technology]

[0002] In regenerative medicine, the use of various somatic cells, including pluripotent stem cells, somatic stem cells, and progenitor cells, has been explored. In recent years, the discovery of induced pluripotent stem cells (also known as iPS cells) has made it possible to clinically apply a variety of cells. Clinical applications include injection therapy of cell suspensions, as well as transplantation therapy of sheet-like tissues prepared from the desired cells to more efficiently transplant cells into the target tissue or organ. While these sheet-like tissues have traditionally been prepared by seeding cells onto biodegradable materials (scaffolds), technologies that enable the preparation of sheet-like tissues without the use of scaffolds have been developed (Non-Patent Documents 1 and 2), and these technologies are beginning to be applied clinically. These sheet-like tissues formed without the use of scaffolds are also called cell sheets. They are typically single-layer sheets of single cells formed by adhesion to each other directly or via an extracellular matrix to form a planar tissue. Clinical applications of these cell sheets are being explored for tissues such as the cornea, heart, esophagus, cartilage, periodontal tissue, middle ear, and lungs.

[0003] Thus, while regenerative medicine using cell sheets and the like is being widely considered for clinical application, it is necessary to efficiently obtain large amounts of cells suitable for the treatment target, especially when there are many patients to treat. Therefore, methods have been proposed in which autologous cells collected from the patient are cultured and expanded, or specific stem cells are cultured and expanded (Patent Documents 1 to 4).

[0004] However, it is known that when a three-dimensional culture method such as those described in Patent Documents 1 to 4 is employed, a large amount of cell aggregates consisting of a collection of a plurality of single cells can generally be formed. If the cell aggregates become too large, problems such as an insufficient supply of culture medium to the cells inside and the accumulation of waste products secreted by the cells may occur. Therefore, Patent Documents 1 to 4 describe dividing the cell aggregates into sizes that allow continuous culture using a mesh filter or the like. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6939643 [Patent Document 2] Patent No. 6292415 [Patent Document 3] Patent No. 6268342 [Patent Document 4] Patent No. 6277450

[0006] [Non-Patent Document 1] Shimizu T., et al., Curr. Pharm. Des., Vol. 15, 2807-2814(2009) [Non-patent document 2] Yamada N., et al., Makromol. Chem. Rapid Commun., Vol. 11, 571-576(1990) Summary of the Invention [Problem to be solved by the invention]

[0007] For example, when attempting to produce the aforementioned cell sheet using cell aggregates obtained by culture, given that cell sheets generally have a single-layer structure of single cells, the cell aggregates must be dispersed before use so that they contain a large number of single cells, preferably only single cells. Dispersion of such cell aggregates is generally carried out using protease.

[0008] However, proteolytic enzymes can destroy not only cell adhesion factors but also membrane proteins, potentially damaging cells. Furthermore, enzymatic reactions vary widely in their success and require complex processes, such as washing to remove enzymes, making them typically performed manually. This series of enzymatic reactions requires skilled operators, and the resulting cells may vary depending on the operator's level of proficiency. To provide regenerative medicine using cell sheets and other technologies to many patients, efficient processing of large quantities of cell aggregates is necessary. Therefore, there is a need for technologies that can produce various types of cells suitable for the production of cell sheets and other technologies using automated processes without using proteolytic enzymes. Furthermore, there is a need for technologies that can efficiently disperse cell aggregates during subculture, in order to obtain large quantities of cells by subculture.

[0009] Therefore, an object of the present invention is to provide a technique for obtaining cells of a size that can be used for producing cell sheets from cell aggregates through a simple process using only physical action. [Means for solving the problem]

[0010] The inventors have found through their research that when mesh filters such as those described in Patent Documents 2 to 4 are used and the mesh size is reduced, the cells themselves die to a certain extent due to destruction or other reasons. Therefore, various structures other than such filters were investigated. As a result, it was found that when a liquid containing cell aggregates is moved through a narrow channel of a predetermined length, it is possible to disrupt and disperse at least a portion of the cell aggregates to a size usable for cell sheet production simply by the physical action of passing through the narrow channel, without using proteolytic enzymes. The gist of the present invention is as follows.

[0011] (1) A cell dispersion system for crushing and dispersing cell aggregates of pluripotent stem cells, somatic stem cells, progenitor cells, or somatic cells, the cell dispersion system having a disperser with a first opening, a second opening, and a narrow flow path of a predetermined length provided between the first opening and the second opening, and moving a liquid containing cell aggregates from the first or second opening toward the second or first opening through the narrow flow path, and capable of crushing and dispersing at least a portion of the cell aggregates to a size that can also be used to produce cell sheets by physical action alone. (2) The cell dispersion system according to the preceding paragraph (1), wherein the dispersion device is connected to a first vessel and a second vessel having a syringe structure at the first and second openings, respectively, and the first vessel and the second vessel are in communication with each other via the narrow flow path, and the first vessel and the second vessel have spaces that can be filled with the liquid, which are determined by the gaskets of the first plunger and the second plunger, respectively, and by moving the first and second plungers complementarily, the liquid filled in the space of the first or second vessel is moved through the narrow flow path to the space of the second or first vessel. (3) A cell dispersion system according to the preceding paragraph (1) or (2), wherein the minimum width of the narrow channel is 0.20 to 2.0 times the particle diameter of the most frequent particle of 100 μm or more in the particle size distribution of the cell aggregates. (4) The cell dispersion system according to any one of (1) to (3) above, wherein the flow rate of the liquid passing through the narrow channel is 0.05 to 10 m / sec. (5) The cell dispersion system according to any one of (1) to (4), wherein the cross-sectional shape perpendicular to the longitudinal direction of the narrow channel is at least one selected from a single continuous circular ring, a single discrete circular ring, multiple discrete circular rings, a circle, and a square. (6) A cell dispersion system described in any one of the preceding paragraphs (1) to (5), wherein the minimum width of the narrow channel in the longitudinal direction is constant or gradually decreases from the first or second opening side toward the second or first opening side, and then gradually increases. (7) The cell dispersion system according to any one of (1) to (6) above, wherein at least a part of the wall surface constituting the narrow channel is provided with irregularities. (8) The cell dispersion system according to any one of (1) to (7) above, wherein the liquid is passed through the narrow channel twice or more times to gradually reduce the average particle size of the cell aggregates. (9) A cell dispersion system described in any one of the preceding paragraphs (2) to (8), in which a structure is provided near the first or second opening to direct the liquid flowing from the first or second vessel to the second or first vessel toward the peripheral wall of the narrow channel. [Effects of the Invention]

[0012] According to the present invention, a technique can be provided for obtaining cells of a size that can be used for producing a cell sheet from a cell aggregate through a simple process using only physical action. [Brief explanation of the drawings]

[0013] [Figure 1] 1A and 1B show an example of the configuration of a disperser and a vessel applicable to the cell dispersion system according to the embodiment, where FIG. 1A is a partial cross-sectional view in an exploded state, and FIG. 1B is a partial cross-sectional view in an assembled state. [Figure 2] FIG. 10 is a partially enlarged cross-sectional view showing the state in which the heads of the first vessel and second vessel are connected via a disperser. [Figure 3]1A and 1B show a first example of a narrow flow path forming member according to an embodiment, in which (a) is a partial cross-sectional view in an exploded state, and (b) is a partial cross-sectional view in an assembled state. [Figure 4] FIG. 10 is a perspective view showing a second example of a narrow flow path forming member according to the embodiment. [Figure 5] The upper side is a cross-sectional view taken along line II in FIG. 4, and the lower side is a cross-sectional view taken along line II-II in FIG. [Figure 6] FIG. 10 is a perspective cutaway cross-sectional view showing a third example of a narrow flow path forming member according to the embodiment. [Figure 7] The upper side is a cross-sectional view taken along line III-III in FIG. 6, and the lower side is a front view of the cross section in FIG. [Figure 8] This shows the process of passing a liquid containing cell aggregates from a first vessel through a narrow channel, where they are crushed and dispersed, and then stored in a second vessel. (a) is a cross-sectional view of the state in which the heads of the first vessel and second vessel are connected via a disperser, and (b) is a cross-sectional view of the state in which cell aggregates that have settled at the bottom of the first vessel are passed through a narrow channel, where they are crushed and dispersed, and then stored in a second vessel. [Figure 9] FIG. 1 is an overall perspective view of a cell aggregate dispersion device. [Figure 10] FIG. 10 is a perspective view showing a state in which a vessel is set in the cell aggregate dispersion device. [Figure 11] FIG. 3 is a cross-sectional view of a flow path forming member used in Reference Example 1. [Figure 12] (a) is an image showing the state of the cell aggregates before being crushed and dispersed in Example 1, and shows the particle size distribution; (b) is an image showing the state of the cell aggregates after being crushed and dispersed in Example 1, and shows the particle size distribution. [Figure 13] (a) is an image showing the state of the cell aggregates before being crushed and dispersed in Reference Example 1, and shows the particle size distribution; (b) is an image showing the state of the cell aggregates after being crushed and dispersed in Reference Example 1, and shows the particle size distribution. DETAILED DESCRIPTION OF THE INVENTION

[0014] A cell dispersion system according to an embodiment of the present invention crushes and disperses cell aggregates of pluripotent stem cells, somatic stem cells, progenitor cells, or somatic cells (hereinafter sometimes referred to as "cells"), and includes a disperser having a first opening, a second opening, and a narrow channel of a predetermined length disposed between the first and second openings. Here, the "predetermined length" means that the ratio of the effective length of the channel to the minimum width of the channel (aspect ratio) is sufficiently greater than 1. Then, by moving a liquid containing cell aggregates from the first or second opening toward the second or first opening through the narrow channel, it is possible to crush and disperse at least a portion of the cell aggregates to a size (hereinafter sometimes referred to as a "predetermined size") that can also be used to produce a cell sheet by physical action alone.

[0015] The pluripotent stem cells, somatic stem cells, progenitor cells, or somatic cells applicable to this cell dispersion system are not particularly limited. Examples include various pluripotent stem cells, various somatic stem cells, various progenitor cells, autologous somatic cells derived from a desired tissue, pre-proliferated undifferentiated pluripotent stem cells, pluripotent stem cells obtained by differentiating somatic stem cells or progenitor cells into somatic cells applicable to a diseased tissue, and somatic cells derived from somatic stem cells or progenitor cells. Pluripotent stem cells are capable of self-renewal and can differentiate into any cell type present in an adult body, such as embryonic stem cells (ES cells) and iPS cells. Somatic stem cells (also called adult stem cells or tissue stem cells) are capable of self-renewal and can typically differentiate into various cells within the tissue from which they originate. Progenitor cells are cells that emerge during the process from stem cells to terminally differentiated cells and are considered to have a more limited self-renewal capacity than somatic stem cells. These cells can be derived from mammals such as humans, dogs, and cats, but human-derived cells are preferred.

[0016] The liquid containing cell aggregates that can be applied to this cell dispersion system (hereinafter, sometimes referred to as "cell liquid") is not particularly limited, and a culture liquid containing cell aggregates after prior growth culture may be used as is, or a culture liquid obtained by prior growth culture and then replacing all or part of the culture liquid used for the growth culture with the same fresh culture liquid or a liquid other than the culture liquid containing a component that can maintain cell viability may be used. Examples of culture liquids containing cell aggregates after prior growth culture include cell aggregate (spheroid)-containing culture liquids obtained by culturing using the large-scale cell culture system described in Patent Document 3, etc.

[0017] The cell aggregates to be disrupted are masses of numerous cells, also called spheroids. The liquid used in the cell dispersion system contains numerous cell aggregates, and there is a size distribution. This distribution varies depending on the type of cell, but the average particle size of the cell aggregates is typically 100 μm or more, for example, 100 to 2000 μm. Note that disruption refers to the division, miniaturization, and reduction to single cells of cell aggregates without cell destruction, but does not mean that cell destruction is not involved at all.

[0018] The narrow channel may be narrow enough to crush and disperse at least a portion of the cell aggregates to a predetermined size. For example, it has a minimum width sufficient to crush at least a portion of a cell aggregate in a population containing a large number of cell aggregates to a predetermined size. Narrowing the channel allows shear forces to be applied to the cell aggregates passing through the channel. Furthermore, having a predetermined length for the narrow channel forms a narrow, continuous conduit, which is thought to enable efficient crushing and dispersion of the cell aggregates through the application of continuous shear forces. This minimum width can be determined appropriately depending on the type of cell, etc., but is preferably 0.20 to 2 times the particle size of the most frequent value of 100 μm or more in the particle size distribution of the cell aggregates. Cell aggregates 100 μm or more are targeted because they are the size of the cell aggregates to be crushed. The particle size distribution can be measured and calculated by image analysis using magnified images from a microscope or the like (for example, this can be performed using Cell3iMager, manufactured by SCREEN Holdings Co., Ltd.). The length of the narrow channel can be adjusted taking into account the cell type, the minimum width of the narrow channel, and the flow rate of the liquid containing the cell aggregates. However, the thickness of a stainless steel mesh filter, for example, does not provide sufficient disruption and dispersion to prevent cell destruction. When using such a mesh filter, mesh filters with openings of approximately 70 μm are typically used. However, some cell aggregates can pass through such openings. Those that cannot pass through are split and pass through, which can result in cell destruction. This tendency is exacerbated when the opening width is narrower than 70 μm. Increasing the opening width to prevent cell destruction results in cell aggregates being split into larger sizes. Among cell aggregates that can pass through without being split, those larger than the mesh opening width are forced to assume a long, thin shape after passing through the filter. After passing through the filter, shear forces are either absent or very small, maintaining their original shape and not being crushed to the desired size.On the other hand, in the case of the narrow channel described above, even if the minimum width of the channel cross section is approximately 70 μm, cell aggregates larger than the minimum width and able to pass through the narrow channel will deform as they enter the narrow channel. However, because the narrow channel is continuous for a certain length, deformation and shear forces act on the cell aggregates due to the walls of the continuous channel and the turbulent flow of the cell fluid, resulting in the cell aggregates being crushed and dispersed by the repeated shear and deformation forces. Furthermore, unlike forced separation using a mesh filter, the stresses caused by the channel walls and turbulence are not strong enough to destroy cells, which is thought to suppress cell destruction. If the dimensions of the narrow channel other than the minimum width are sufficiently larger than the particle size of the cell aggregates, cell aggregates will easily deform and pass through even if the minimum width of the channel is smaller than the particle size of the cell aggregates. The repeated shear and deformation forces applied as described above during passage through the channel allow for the crushing and dispersion of more cell aggregates.

[0019] Furthermore, the narrow channel may be narrow enough to crush and disperse at least a portion of the cell aggregates to a predetermined size. The channel structure is not particularly limited. However, from the viewpoint of efficiently crushing and dispersing the cell aggregates in a limited volume, the cross-sectional shape perpendicular to the length of the narrow channel is preferably at least one selected from a single continuous circular ring, a single discrete circular ring, multiple discrete circular rings, a circle, and a square. A single continuous circular ring, a single discrete circular ring, multiple discrete circular rings, or a combination of these with a square is more preferable. Furthermore, the cross-sectional shape may be the same and continuous along the length of the narrow channel, or it may vary. When the cross-sectional shape varies, it is preferable that the shape gradually decreases from the first or second opening side toward the second or first opening side, and then gradually increases.

[0020] A cell dispersion system according to an embodiment will be described below with reference to the drawings.

[0021] Fig. 1 shows an example of the configuration of a disperser and vessel applicable to a cell dispersion system according to an embodiment. In the figure, reference numeral 1a denotes a first syringe, 1b denotes a second syringe, 2 denotes a first vessel, 3 denotes a second vessel, and 4 denotes a disperser. Fig. 1(a) is a cross-sectional view showing a disperser 4 in a disassembled state, and the first syringe 1a and the second syringe 1b in a state not connected to the disperser 4, and Fig. 2(b) is a cross-sectional view showing the disperser 4, the first syringe 1a, and the second syringe 1b in an assembled state.

[0022] As shown in FIG. 1 , the first syringe 1a has a cylindrical outer tube 5 and a front flange 6 and a back flange 7 integrally formed at both ends of the outer tube 5. The head 8, which serves as a discharge port and is located at the center of the front flange 6, has a standard luer lock structure. The plunger 9 has a shaft 10 with a gasket 11 at the tip thereof that elastically fits slidably within the outer tube 5, and a plunger button 12 at the base end. In this example, the elastic fit between the gasket 11 and the outer tube 5 is achieved by two O-rings 11B attached to the outer periphery of the gasket 11, but this is not limiting. The second syringe 1b also has the same structure as the first syringe 1a. That is, the first vessel 2 and the second vessel 3 have the same structure. However, the two syringes may have different structures, for example, different volumes.

[0023] In this example, the tip 11A of the gasket 11 is shaped as a conical convex portion, and the tip-side inner surface of the outer tubular portion 5, i.e., the inner surface 6A on the side where the front flange 6 is provided, is shaped as a conical concave portion that receives the tip 11A, so that the liquid inside the outer tubular portion 5 can be dispensed without leaving any residue. However, the tip surface of the tip 11A and the inner surface 6A need only have corresponding shapes so that the liquid can be dispensed without leaving any residue, and for example, both may be flat. In addition, the head 8 integrally provided on the outer surface of the front flange 6 has a luer lock structure.

[0024] The capacity and inner diameter of the first vessel 2 and the second vessel 3 can be determined appropriately taking into consideration the amount of cell fluid to be treated, etc. For example, a capacity of 5 to 100 mL and an inner diameter of 5 to 50 mm can be used. The materials constituting the first syringe 1a and the second syringe 1b are not particularly limited, and inorganic materials such as metal, glass, and ceramic, and organic materials such as synthetic resin can be appropriately selected and used depending on the function of each component. When sterilization is performed using an autoclave, it is necessary to use heat-resistant materials that can withstand the sterilization conditions. Furthermore, when sterilization is performed using electromagnetic waves or gas, it is necessary to use materials that can withstand the respective processing conditions.

[0025] As shown in Figures 1 to 3, the disperser 4 has a narrow flow path 41 that can crush cell aggregates by passing cell fluid through the vessels 2 and 3, and can connect the heads 8, 8 of the vessels 2 and 3 to each other. The disperser 4 has a narrow flow path forming member 40, a first member 13, and a second member 14. The first member 13 has a flow path 15 at its center and a hollow housing portion 16 at one axial end. A female thread portion 17 is formed on the inner periphery of the housing portion 16, and a connection portion 18 with the head 8 at the other axial end. The second member 14 has a flow path 19 at its center and a male thread portion 20 at one axial end that screws into the female thread portion 17 of the first member 13 and a connection portion 21 with the head 8 at the other axial end. A recess 22 that opens at the tip end is formed in the male thread portion 20 of the second member 14, the narrow flow path forming member 40 is placed in the recess 22, and in a state where the male thread portion 20 of the second member 14 is screwed onto the female thread portion 17 of the first member 13, a packing 43 provided on the narrow flow path forming member 40 is compressed to seal between the inner bottom surface of the housing portion 16 of the first member 13 and the inner bottom surface of the recess 22 of the second member 14. In addition to this, the gap between the first member 13 and the second member 14 may be sealed by (1) tapering the tip side outer peripheral surface 20A of the male thread portion 20 and the bottom side inner peripheral surface 16B of the housing portion 16, and / or the base side outer peripheral surface 20B of the male thread portion 20 and the tip side inner peripheral surface 16A of the housing portion 16, or (2) providing and compressing a packing having a center hole that communicates with the flow paths 15, 19 on both axial end sides or one end side of the narrow flow path forming member 40. Furthermore, when the axial length of the narrow flow passage forming member 40 is to be shortened, a spacer having a central hole communicating with the flow passages 15 and 19 may be provided at a position adjacent to the narrow flow passage forming member 40 .

[0026] As shown in Fig. 3, the narrow flow path forming member 40 has an inner member 42, a packing 43, and an outer member 44. The inner member 42 has a cylindrical portion 45 with a cylindrical structure, and a flange 46 and a recess 47 with a circular cross section at one axial end (base end) of the cylindrical portion 45. The side wall of the recess 47 has a plurality of small holes 48 that communicate between the inside and outside of the recess 47 (in the example shown in Figs. 2 and 3, eight small holes with a circular cross section are provided). A spiral convex portion 49a is provided on the outer peripheral surface 49 in a range from the other axial end (tip) to a portion close to the small holes 48. The bottom surface 47a of the recess 47 is a circular flat surface. The packing 43 has a central hole 50 through which the cylindrical portion 45 of the inner member 42 can be inserted. The outer member 44 has a cylindrical structure, has a hollow portion formed therein extending from one axial end to the other, and has a spiral groove portion 51a formed on an inner circumferential surface 51 forming the hollow portion. Then, a packing 43 is disposed between the inner member 42 and the outer member 44, the columnar portion 45 of the inner member 42 is inserted into the hollow portion formed by the center hole 50 of the packing 43 and the inner circumferential surface 51 of the outer member 44, and the annular surface of the flange 46 on the columnar portion 45 side and the annular base end surface 44a of the outer member 44 are brought into contact with each other via the packing 43. By compressing the packing 43 as described above, a narrow flow passage 41 having a single continuous annular cross section surrounded by the outer circumferential surface 49 of the inner member 42 and the inner circumferential surface 51 of the outer member 44 is formed. The narrow flow path 41 opens at a portion surrounded by the small holes 48 of the inner member 42, the outer peripheral edge of the tip surface 45a of the inner member 42, and the inner peripheral surface 51 of the outer member 44, forming an opening. In other words, these openings correspond to the first opening and the second opening of the disperser 4. Furthermore, as shown in FIG. 2, when the cell fluid is flowed into the disperser 4 from the syringe 1a, the cell fluid strikes the bottom surface 47a of the inner member 42, changes its flow direction from the axial direction to a direction perpendicular to the axial direction, and flows from the small holes 48 toward the inner peripheral surface 51 of the outer member 44, which is the outer peripheral wall of the narrow flow path 41. In other words, the bottom surface 47a forms a structure that directs the cell fluid toward the peripheral wall of the narrow flow path 44.

[0027] The axial length of the narrow flow passage 41 can be determined by taking into account various conditions, as described above, and can be, for example, 3 to 20 mm. The minimum width of the narrow flow passage 41 is calculated by the outer diameter of the cylindrical portion 45 of the inner member 42 and the inner diameter of the inner circumferential surface 51 of the outer member 55 at a position concentric with the outer diameter. This minimum width can be determined by taking into account various conditions, as described above. In the example shown in Figures 2 and 3, the maximum outer diameter of the cylindrical portion 45 of the inner member 42 and the minimum inner diameter of the inner circumferential surface 51 of the outer member 55 are constant. Furthermore, the helical ridge portion 49a and the helical groove portion 51a are provided so that their protrusions and recesses correspond to each other, forming a continuous, curved flow passage in the axial cross section with approximately the same width in the axial direction (see Figure 3(b)). Such a flow passage can be formed, for example, by forming the helical ridge portion 49a and the helical groove portion 51a into a male and female thread structure with the same pitch, and making the nominal diameter of the male thread structure smaller than that of the female thread structure. In addition to the illustrated structure, the spiral ridge portion 49a and the spiral groove portion 51a may be configured as male and female thread structures with different pitches, with the outer diameter (thread diameter) of the male thread structure being smaller than the inner diameter (thread diameter) of the female thread structure, so that the two do not thread together but are assembled by dropping the male thread structure into the female thread structure. The size of the small holes 48 is not particularly limited and can be determined appropriately taking into account the number of small holes 48, the minimum width of the narrow flow path 41, the average particle size of the cell aggregates, the permeability of the cell fluid, and other factors. For example, by making the cross-sectional area surrounded by the spiral ridge portion 49a and the spiral groove portion 51a and the combined cross-sectional area of ​​all the small holes 48 approximately equal, the change in flow rate through the small holes 48 can be minimized.

[0028] In addition to the embodiment shown in the figure, the narrow flow path forming member 40 shown in FIG. 3 can employ, for example, the following modifications. The shape of the small holes 48 in the inner member 42 is not limited to a circular cross section, but may be a square cross section or other shapes. The outer peripheral surface 49 and inner peripheral surface 51 of the cylindrical portion 45 that forms the narrow flow path 41 are formed with a spiral ridge portion 49a and a spiral groove portion 51a, respectively, and each peripheral surface 49, 51 is provided with irregularities, but these may not be necessary. Furthermore, the shape is not limited to a spiral shape, and closed annular irregularities may be provided at equal or arbitrary intervals in the axial direction. Furthermore, the width of the narrow flow path 41 is generally constant in the axial direction, but the outer diameter of the cylindrical portion 45 and the inner diameter of the inner peripheral surface 51 may be varied so that they gradually decrease from the side of the small holes 48 and then gradually increase. In this case, an axially symmetrical structure is preferable. Furthermore, the inner member 42 may be arranged symmetrically in the axial direction, that is, the flange 46, recess 47, and small hole 48 of the inner member 42 may be provided on both sides in the axial direction.

[0029] 3(b), the case where cell fluid is delivered from the recess 47 side of the inner member 42 will be described as follows. The cell fluid delivered from the recess 47 side of the inner member 42 strikes the bottom surface 47a of the recess 47 of the inner member 42, changes its flow direction, and flows into the narrow channel 41, which has a single continuous ring-shaped cross section perpendicular to the axial direction, through each of the small holes 48, which become first openings. The cell fluid then strikes the inner circumferential surface 51 of the outer member 44, changes its flow direction, passes through the narrow channel 41 along the axial direction, and flows out from an opening (second opening) surrounded by the outer periphery of the tip surface 45a of the inner member 42 and the inner circumferential surface 51 of the outer member 44. As the cell aggregates pass through the narrow channel 41, which has a circular cross-section perpendicular to the axial direction and a predetermined axial length, they are subjected to shear stress when they collide with the inner circumferential surface 51 and the outer circumferential surface 49, and to shear forces due to shear flows generated by the fluid flow. This is thought to prevent cell destruction and break them down to a predetermined size. Furthermore, the presence of a structure that changes the flow direction of the cell fluid on the outside and inside of the small holes 48, which serve as the first opening, is thought to more effectively apply shear forces and promote efficient cell aggregate crushing. Cell aggregates larger than the size of the small holes 48 are thought to be broken down into smaller cell aggregates or deformed depending on their size before passing through the small holes 48. In this example, the provision of irregularities on the peripheral surfaces 49 and 51 that make up the narrow channel 41 is thought to further efficiently crush the cell aggregates.

[0030] On the other hand, when cell fluid is delivered from the second opening, the flow is in the opposite direction. In this case, some of the cell fluid flows directly into the second opening, while others impact the tip surface 45a of the inner member 42, change direction, and then change direction again before flowing into the second opening. The cell fluid that flows into the narrow channel 41 from the second opening passes through the narrow channel 41 along the axial direction, changes direction near the inside of the small hole 48, and flows out of the first opening, the small hole 48. In this way, even when flowing from the opposite direction, by flowing in near the outside of the second opening, passing through the narrow channel 41, and flowing out of the first opening, shear force is efficiently applied to the cell aggregates, and the cell aggregates are efficiently crushed while suppressing cell destruction, resulting in a predetermined size.

[0031] In addition to the narrow flow passage forming member 40 having the structure shown in FIGS. 2 and 3, for example, narrow flow passage forming members 40a and 40b having the structures shown in FIGS. 4 to 7 can be used.

[0032] The narrow flow path forming member 40a shown in Figures 4 and 5 has a cylindrical shape and a structure in which a plurality of narrow hollow portions are formed that extend parallel to the axial direction from an end face 92 at one end in the axial direction to an end face 93 at the other end. These hollow portions form narrow flow paths, and in the figures, they correspond to the reference numerals 90a to 90c and 91a to 91c. Each narrow flow path opens at both end faces 92 and 93, forming an opening at each of these ends. One of these openings is referred to as a first opening, and the other as a second opening. The narrow channels 90a-90c (outer narrow channels) and the narrow channels 91a-91c (inner narrow channels) have discontinuous circular cross sections (also referred to as axially orthogonal cross sections) perpendicular to the axial direction (corresponding to the length of the narrow channels), i.e., discrete circular cross sections. The outer narrow channels 90a... and the inner narrow channels 91a... are arranged concentrically around the central axis of the cylindrical structure, forming double circular rings. In the example shown in Figures 4 and 5, each circular ring is divided into three equal parts, but this is not limited thereto. The number may be two, four, or more, and these divisions may be equal or unequal. Furthermore, there is no particular limitation on the arrangement of the outer narrow channels 90a... and the inner narrow channels 91a... in the radial direction of the axially orthogonal cross section. The width of the outer narrow channels 90a can be calculated from the diameter of the axial cross section of the outer peripheral surfaces 94 and inner peripheral surfaces 95 that form the outer narrow channels 90a. Similarly, the width of the inner narrow channels 91a can be calculated from the diameter of the axial cross section of the outer peripheral surfaces 96 and inner peripheral surfaces 97 that form the inner narrow channels 91a. In the example shown in Figures 4 and 5, the diameter of the axial cross section of each peripheral surface is the same along the axial direction, so these widths are the same along the axial direction. The minimum width can be calculated from the diameter of any axial cross section, and is the same for each narrow channel. The minimum width of each narrow channel may be the same or different as long as it can ultimately crush and disperse cell aggregates to a predetermined size, but is preferably the same. The axial length of the outer narrow channels 90a and inner narrow channels 91a can be determined taking into account various conditions as described above, and can be, for example, 3 to 20 mm.

[0033] In the narrow flow path forming member 40a shown in Figures 4 and 5, in addition to the embodiment shown in the figures, for example, the following modifications can be adopted. In the example shown in the figures, each peripheral surface is smooth, but any of them may be provided with irregularities. Furthermore, the width of the outer narrow flow paths 90a,... and the inner narrow flow paths 91a,... is the same at any position in the axial direction, but the diameter of the peripheral surfaces 94,... in a cross section perpendicular to the axial direction may be changed so that it gradually becomes smaller from the end face 92 side toward the end face 93 side and then gradually becomes larger. In this case, the position where the width becomes smallest is preferably equidistant from both end faces 92, 93. Alternatively, one axial end of the outer narrow flow passages 90a,... may be continuous with the first opening and the second axial end may be closed, while the second axial end of the inner narrow flow passages 91a,... may be continuous with the second opening and the first axial end may be closed, so that the outer narrow flow passages 90a,... and the inner narrow flow passages 91a,... are connected to each other in the axial middle or in the vicinity of the one axial end of the inner narrow flow passages 91a,...

[0034] 4 and 5, the case of using the narrow channel-forming member 40a to transport cell fluid from an end face 92 at one axial end to an end face 93 at the other axial end will be described as follows. Some of the cell fluid transported from the end face 92 side flows directly into the openings (first openings) of the end face 92 of the outer narrow channels 90a,... and the inner narrow channels 91a,..., while some of the cell fluid strikes the end face 92, changes direction, and flows into the first opening. The cell fluid that flows from the first openings into the outer narrow channels 90a,... and the inner narrow channels 91a,... passes along the axial direction through the outer narrow channels 90a,... and the inner narrow channels 91a,..., and flows out from the openings (first openings) of the respective end faces 93. As the cell aggregates pass through the outer narrow channels 90a,... and the inner narrow channels 91a,..., each having a double discrete annular cross-sectional shape perpendicular to the axial direction and a predetermined length in the axial direction, they are subjected to shear stress when they collide with the outer peripheral surface 94,... and the inner peripheral surface 95,..., and the outer peripheral surface 96,... and the inner peripheral surface 97,..., and are subjected to shear forces due to shear flows generated by the liquid flow. This is thought to prevent cell destruction and break them down to a predetermined size. Furthermore, having a structure that can change the flow direction of the cell solution near the first opening is thought to more effectively apply shear forces and promote efficient cell aggregate disruption. Cell aggregates larger than the size of the first opening are thought to be broken down into smaller cell aggregates or deformed depending on their size before passing through the first opening. Since this embodiment has the same shape and structure in the axial direction, the same applies when pumping cell solution from end face 93 to end face 92.

[0035] The narrow flow path forming member 40b shown in Figures 6 and 7 is composed of a cylindrical inner portion 40c and a cylindrical outer portion 40d that is provided to support the inner portion 40c at its outer periphery and surround the entire outside of the inner portion 40c. The member 40b has end faces 99, 100 at both axial ends. The end faces 99, 100 are formed on the outer portion 40d. Openings 101, 102 are formed at the centers of the end faces 99, 100. One of the two openings 101, 102 is referred to as a first opening, and the other as a second opening. A hollow portion surrounded by the inner portion 40c and the outer portion 40d is formed inside the member 40b, and this hollow portion forms a narrow flow path 105. The narrow flow path 105 has a plurality of narrow hollow sections 98a, 98b, 98c, 98d arranged in an annular shape extending parallel to the axial direction from one axial end of the member 40b to the other, and disk-shaped narrow hollow sections 103, 104 that are continuous with both axial ends of these hollow sections 98a, ... and extend in a direction perpendicular to the axial direction. The narrow flow path 105 is open and communicates with openings 101, 102 on both end faces 99, 100.

[0036] The narrow flow passage 105, consisting of the hollow portions 98a, 98b, 98c, and 98d, has a discontinuous circular cross section perpendicular to the axial direction (corresponding to the length of the narrow flow passage), i.e., a discrete circular cross section. The hollow portions 98a are arranged on the same circumference centered on the central axis of the cylindrical inner portion 40c. In the example shown in Figures 6 and 7, the circular cross section has an arc shape divided equally into four parts, but this is not limited to this. The number of parts may be two, three, five, or more, and these divisions may be equal or uneven. The width of the hollow portions 98a can be calculated from the diameter of the axial cross section of the inner circumferential surface 106 of the outer portion 40d and the outer circumferential surface 107 of the inner portion 40c, which form the hollow portions 98a. In the examples shown in Figures 6 and 7, the diameter of each peripheral surface's cross section perpendicular to the axial direction is the same along the axial direction, so the width is the same along the axial direction. The minimum width can be calculated from the diameter of any cross section perpendicular to the axial direction, and is the same for each hollow section 98a,... The minimum width for each hollow section 98a,... may be the same or different as long as it can ultimately crush and disperse the cell aggregates to a predetermined size, but it is preferable that they are the same. The axial length of the narrow channel 105 in the portion composed of hollow sections 98a,... whose cross section perpendicular to the axial direction is a discrete annular shape can be determined taking into account various conditions as described above, and can be, for example, 3 to 20 mm.

[0037] The narrow flow passage 105 in the portion formed by the hollow portions 103, 104 has a circular cross section perpendicular to the axial direction and a rectangular cross section parallel to the axial direction, resulting in an overall disk shape. The width of the narrow flow passage 105 in the portion formed by the hollow portions 103, 104 can be calculated from the shortest distance between both axial end faces 108, 109 of the inner portion 40c and the opposing inner bottom faces 110, 111 in the axial direction of the outer portion 40d. In the example shown in the figure, the end faces 108, 109 and the bottom faces 110, 111 are parallel, the shortest distance between them is the same, and the entire hollow portions 103, 104 have the same width. The widths of the hollow portions 98a,... and the hollow portions 103, 104 may be the same or different, but are preferably the same. In the narrow flow path 105 formed by the hollow portions 103 and 104, the size in the direction perpendicular to the axial direction can be determined by the diameter of both inner bottom surfaces 110 and 111, and this size can be determined taking into account various conditions as described above, and can be, for example, 3 to 20 mm.

[0038] Here, the cell fluid flowing through the hollow portions 103 and 104 flows, for example, from the opening 101 and radially outward from the center of the opening 101, so the length direction of the narrow flow path is perpendicular to the axial direction of the member 40b. Therefore, the cross-sectional shape perpendicular to the length direction of the hollow portions 103 and 104 is rectangular. In this way, the cross-sectional shape of the narrow flow path 105 perpendicular to its length direction is a cross-sectional shape that combines a single discrete annular shape and a rectangular shape.

[0039] Furthermore, when the cell fluid Y is introduced into the disperser 4 from the syringe 1a as shown in FIG. 8 (described later), the cell fluid Y flows into the narrow channel 105 from the opening 101 of the outer portion 40d, strikes the end face 108 of the inner portion 40c, changes its flow direction from the axial direction to a direction perpendicular to the axial direction, and flows toward the inner circumferential surface 106 of the outer portion 40d, which is the outer peripheral wall side of the narrow channel 105, whose cross section perpendicular to the axial direction has a discrete annular shape. That is, the axial end face 108 of the inner portion 40c constitutes a structure that directs the cell fluid Y toward the peripheral wall side of the narrow channel 105 in the axial direction. Note that when the cell fluid Y is introduced into the disperser 4 from the syringe 1b, the axial end face 109 of the inner portion 40c constitutes a structure that directs the cell fluid Y toward the peripheral wall side of the narrow channel 105 in the axial direction.

[0040] In the narrow flow path forming member 40b shown in Figures 6 and 7, in addition to the embodiment shown in the figures, for example, the following modifications can be adopted. In the example shown in the figures, the wall surfaces forming each hollow portion 98a,... are smooth, but any of them may be provided with irregularities. Furthermore, in the example shown in the figures, the width of the narrow flow path 105 is the same at any position in the axial direction and at any position perpendicular to the axial direction. However, (1) in the discrete annular hollow portions 98a,..., the diameter in a cross section perpendicular to the axial direction of the inner circumferential surface 106,... and the outer circumferential surface 107 may be changed so that the diameter gradually decreases from the end face 108 side toward the end face 109 side and then gradually increases. Alternatively, (2) the width of each hollow portion may be changed so that the diameter gradually decreases from the opening 101 side toward the opening 102 side and then gradually increases. In this case, the position where the width becomes smallest is preferably equidistant from both end faces 108, 109. Although both end faces 108 and 109 are flat, they may be formed into a conical or truncated conical shape, and bottom faces 110 and 111 may be inclined to correspond to this.

[0041] The following describes the case where a cell fluid is sent from an opening 101 on one end face 99 in the axial direction toward an opening 102 on the other end face 100 using the narrow channel-forming member 40b shown in Figures 6 and 7. Some of the cell fluid sent from the end face 99 side flows directly into the opening 101 (first opening), while others strike the end face 99, change flow direction, and flow into the first opening. The cell fluid that flows from the opening 101 into the narrow channel 105 in the portion formed by the hollow portion 103 strikes the end face 108, changes flow direction, and flows toward the inner circumferential surface 106. Next, the cell fluid strikes the inner circumferential surface 106, changes direction, and finally flows into the narrow channel 105 in the portion formed by the hollow portions 98a, ..., and flows toward the other end face 100 along the axial direction. The cell aggregates then strike bottom surface 111, change direction, and flow into narrow channel 105, which is the portion formed by hollows 103 and 104. They then exit from the periphery of bottom surface 109 and finally through opening 102, which is formed at the center of bottom surface 109, i.e., end surface 100. As they pass through narrow channel 105, which has a cross-sectional shape perpendicular to the axial direction that is a double discrete annular shape and a portion formed by hollows 98a,..., each having a predetermined length in the axial direction, and a portion formed by disk-shaped hollows 103 and 104, the cell aggregates are crushed to a predetermined size while suppressing cell destruction by being subjected to shear stress when colliding with the wall surface and shear forces due to shear flows generated by the liquid flow. Furthermore, by providing a structure that can change the flow direction of the cell liquid near opening 101 or in narrow channel 105, the shear forces are more effectively applied, promoting efficient crushing of cell aggregates. It is believed that cell aggregates larger than the size of opening 101 may be divided into smaller cell aggregates or deformed depending on the size of the aggregates before passing through opening 101. Note that, since this embodiment has the same shape and structure in the axial direction, the same can be considered when the cell fluid is sent from end face 100 to end face 99.

[0042] 3 to 7, the materials constituting the narrow flow path forming member 40, ..., first member 13, and second member 14 are not particularly limited, and inorganic materials such as metals and ceramics, and organic materials such as synthetic resins can be appropriately selected and used depending on the function of each member, but it is preferable to use a material that is coated for the parts that come into contact with the cell liquid, which can suppress the adhesion of cell aggregates and their fragments. Furthermore, it is necessary to use materials that are adaptable to each condition depending on the sterilization treatment conditions.

[0043] Next, an overview of the cell dispersion system will be described with reference to Figure 8. First, a cell solution containing a predetermined number of cells is poured into the first vessel 2. As the cell solution, a culture solution containing cell aggregates obtained by culturing using a large-scale cell culture system such as that described in Patent Document 3 may be used as is. The number of cells contained in the cell solution filled into the first vessel 2 can be determined appropriately so as to include the number of cells required for transplant surgery, etc. Furthermore, it is preferable to use cell aggregates of cardiomyocytes with a cTnT positivity rate of 80% or more.

[0044] As shown in FIG. 8(a), the head 8 of the first vessel 2 containing the cellular fluid Y is connected to one of the connectors 18 of the disperser 4, and the other connector 21 of the disperser 4 is connected to the head 8 of the second vessel 3. The first vessel 2 is placed with the head 8 facing downward and left stationary. When left stationary in this manner, cell aggregates X settle at the bottom of the head 8 side of the first vessel 2. In this state, the plunger 9 of the first vessel 2 is pushed in and the plunger 9 of the second vessel 3 is simultaneously pulled back in the withdrawal direction, moving both plungers 9, 9 in a complementary manner. This generates a pressure difference between the upstream and downstream sides, and moves the cellular fluid Y in the first vessel 2 to the second vessel 3 (FIG. 8(b)). At this time, for example, when the narrow channel-forming member 40 shown in FIGS. 3 to 7 is used, the cellular fluid Y pushed out of the first vessel 2 passes from the head 8 through the conduit 15 of the first member 13 and reaches the narrow channel-forming member 40. As described above, the cell fluid Y flows in through the first opening, passes through the narrow channels 41, and flows out through the second opening. The cell aggregates X are crushed and dispersed to a predetermined size, resulting in a dispersion Z in which the cell aggregates X are crushed and dispersed (FIG. 8(b)). The supply rate (ml / sec) of the cell fluid Y can be determined appropriately depending on the type of cell, the structure of the narrow channels, and the like. In the case of iPS cell-derived cardiomyocytes, it can be set to, for example, 0.1 to 5 ml / sec. In addition, in the case of iPS cell-derived cardiomyocytes, the flow rate (linear velocity) of the cell fluid Y passing through the narrow channels can be set to, for example, 0.05 to 10 m / sec, preferably 0.1 to 1 m / sec. This flow rate can be calculated, for example, from the supply rate (ml / sec) and the cross-sectional area through which the cell fluid passes at the same axial position from the first opening. If the cross-sectional area varies at a predetermined position, the largest supply rate among the cross-sectional areas is adopted.

[0045] If passing the cell fluid Y through the narrow channel once is not sufficient to disrupt and disperse the cell aggregates X to the desired size, the cell fluid Y may be passed through the narrow channel two or more times. In this case, from the state shown in FIG. 8(b), the plunger 9 of the second vessel 3 is pushed in while the plunger 9 of the first vessel 2 is simultaneously retracted in the withdrawing direction, creating a pressure difference between the upstream and downstream sides. This moves the cell fluid Y (dispersion fluid Z) that has been disrupted and dispersed once in the second vessel 3 to the first vessel 2, returning to the state shown in FIG. 8(a). At this time, for example, if the narrow channel-forming member 40 shown in FIGS. 3 to 7 is used, shear force is again applied to the insufficiently disrupted cell aggregates in the dispersion fluid Z as the dispersion fluid Z passes through the narrow channel 41, promoting disruption. The above procedure can be repeated until the cell aggregates X are disrupted and dispersed to the desired size, gradually reducing the average particle size of the cell aggregates X. However, if the number of repetitions is too many, the number of cells that are destroyed or that are not destroyed but die tends to increase, so it is preferable to determine the number of repetitions appropriately depending on the cell tumor used, etc. For example, in the case of cardiomyocytes derived from iPS cells, the number of repetitions is preferably 5 or less.

[0046] The disrupted cell aggregates obtained through the above-described series of steps have a size that is suitable for use in the production of cell sheets, for example, and are also applicable to the production of three-dimensional cell tissues, in addition to the production of cell sheets.

[0047] The above-mentioned series of steps can be performed automatically, for example, by a cell aggregate dispersion device A shown in Figures 9 and 10. This device A is an application of the inter-vessel cell fluid transfer device described in Patent Documents 3 and 4.

[0048] The cell aggregate dispersion device A shown in Figures 9 and 10 is an example of an automated device that can connect the heads 8, 8 of two vessels with a connector 4, hold the front flange 6 of the outer tube 5 so that it cannot move axially, grasp the plunger button 12 to drive the plunger 9 in the axial direction, and move a predetermined amount of cell liquid from one vessel to the other at a constant speed, thereby crushing and dispersing the cell aggregates.

[0049] Cell aggregate dispersion device A has a common base 29 for a first mechanism 27 and a second mechanism 28 that face each other in the same vertical direction, a vessel attached to each, and drives a plunger 9 forward and backward with the outer cylinder 5 fixed. Here, "forward" means moving in the direction of discharging cell fluid from the head 8 of the vessel, and "backward" means moving in the direction of sucking cell fluid from the outside, meaning moving in the opposite direction to forward.

[0050] As shown in Figures 9 and 10, in the cell aggregate dispersion device A, the first mechanism 27 includes a fixed part 30 that fixes the outer cylindrical part 5 of the first vessel 2 and a movable part 31 that drives the plunger 9 forward and backward, and a first drive mechanism that drives the movable part 31 is provided on the backside of the movable part 31. The second mechanism 28 also includes a fixed part 33 that fixes the outer cylindrical part 5 of the second vessel 3 and a movable part 34 that drives the plunger 9 forward and backward, and a second drive mechanism that drives the movable part 31 is provided on the backside of the movable part 31. The movable part 31 and the first drive mechanism of the first mechanism 27 have the same structure as the movable part 34 and the second drive mechanism of the second mechanism 28, but are upside down. The movable part 31 of the first mechanism 27 and the movable part 34 of the second mechanism 28 are driven forward and backward in synchronization so that the flow rates through the dispersion device 4 are consistent. Even when the cross-sectional areas of the respective vessels are different, the movable part 31 and the second drive mechanism are driven forward and backward in synchronization so that the flow rates are consistent depending on the cross-sectional areas. The first and second drive mechanism sections can be configured in the same manner as those described in Patent Documents 3 and 4, for example.

[0051] In the portion of base portion 29 covered by protective covers 36, a vertical support plate is fixed in an upright position to the center of the upper surface of horizontal base plate 37, and two reinforcing plates are erected along both side edges on the back side of this support plate and fixed to base plate 37 and the support plate, resulting in a highly rigid U-shaped structure in plan view. A first drive mechanism is disposed at the upper front surface of this support plate, and a second drive mechanism is disposed at the lower front surface, and a fixing portion 30 of first mechanism 27 and a fixing portion 33 of second mechanism 28 are disposed between the two drive mechanism portions at the center of the front surface.

[0052] The fixed portion 30 of the first mechanism 27 and the fixed portion 33 of the second mechanism 28 are directly or indirectly attached to a common fixed stage 54 fixed parallel to and spaced apart from each other on the surface side of the support plate. In this embodiment, the fixed portion 33 is directly attached to the lower part of the fixed stage 54, and the fixed portion 30 is attached to a movable stage 55 that is provided above the fixed stage 54 and is movable in the vertical direction. In order to accommodate errors in the dimensions of the disperser 4 and the connection depth of the disperser 4 relative to the vessel head 8, the fixed portion 33 and the fixed portion 30 may be connected via a spacing adjuster, as necessary. For example, the spacing adjuster described in Patent Documents 3 and 4 can be used.

[0053] The movable part 31 of the first mechanism 27 is provided on the stage 38, and is configured so that the plunger holding part 57, which holds the plunger button 12 of the plunger 9, can be fixed by finely adjusting its position in the vertical direction relative to the stage 38. Specifically, an adjustment plate 58 is configured to be joined to the surface of the stage 38 so that it can be moved and guided in the vertical direction, and a clamping screw 62 is threaded into the stage 44 through a vertically extending elongated hole opened in the center of the adjustment plate 58. The clamping screw 62 can be easily tightened and loosened manually by holding a handle 63 provided on the clamping screw 62. Here, the plunger holding part 57 is configured with an engagement recess 64 that engages with the plunger button 12 from the front side, as shown in FIG. 10.

[0054] Similarly, the movable part 34 of the second mechanism 28 is provided on the stage 51, and is configured so that a plunger holding part 65 that holds the plunger button 12 of the plunger 9 can be fixed by finely adjusting its position in the vertical direction relative to the stage 51. Specifically, an adjustment plate 66 is configured to be joined to the surface of the stage 51 so as to be movable in the vertical direction, and a clamping screw 70 is threaded into the stage 51 through a vertically extending elongated hole 69 that opens in the center of the adjustment plate 66. The clamping screw 70 can be easily tightened and loosened manually by holding a handle 71 provided on the clamping screw 70. Here, as shown in FIG. 10, the plunger holding part 65 is configured with an engagement recess 72 that engages with the plunger button 12 from the front side.

[0055] The fixed portion 30 of the first mechanism 27 is provided with an outer cylinder portion holding portion 73 that holds the outer cylinder portion 5 of the first vessel 2. Specifically, as shown in FIG. 10 , the outer cylinder portion holding portion 73 is composed of a U-shaped recess 74 that receives the outer cylinder portion 5, a flange fitting groove 75 into which the front flange 6 is fitted, and a holding member 76 that presses and holds the front flange 6 from the outside. The holding member 76 is configured so that one end is horizontally rotatable and the other end can be elastically engaged and disengaged with a hook 77. Here, it is also preferable to provide two outer cylinder portion holding portions 73, one at the top and one at the bottom, so that the back flange 7 can also be held at the same time.

[0056] Similarly, the fixed portion 33 of the second mechanism 28 is provided with an outer cylinder portion holding portion 78 that holds the outer cylinder portion 5 of the second vessel 3. Specifically, as shown in FIG. 10 , the outer cylinder portion holding portion 78 is composed of a U-shaped recess 79 that receives the outer cylinder portion 5, a flange fitting groove 80 into which the front flange 6 is fitted, and a holding member 81 that presses and holds the front flange 6 from the outside. The holding member 81 is configured to hold one end so that it can rotate horizontally, and to allow the other end to be elastically engaged and disengaged with a hook 82. Here, it is also preferable to provide two outer cylinder portion holding portions 78, one at the top and one at the bottom, so that the back flange 7 can also be held at the same time.

[0057] Furthermore, although not shown, a limit mechanism is provided to limit the range of movement of each movable part for safety and to determine the home position. For example, the limit mechanisms described in Patent Documents 3 and 4 can be used for this limit mechanism. It is also possible to use a simple container as the lower second vessel 3, and pass the cell aggregates from the first vessel 2 through a narrow channel in the disperser 4 to crush them to a predetermined size, and then collect the dispersed cell liquid directly in the desired container. In other words, cell aggregate crushing and dispersion operations are possible even with an apparatus equipped only with the first mechanism 27 of the cell aggregate dispersion apparatus A.

[0058] To attach the first vessel 2 and the second vessel 3 to the cell aggregate dispersion device A configured as described above, first, as shown in Figure 10, connect the heads 8, 8 of the first vessel 2 and the second vessel 3 with the disperser 4, release the holding members 76, 81, and loosen the fastening screws 62, 70. With this in mind, fit the front flange 6 of the first vessel 2 into the flange fitting groove 75 and engage the plunger button 12 in the engagement recess 64, fit the front flange 6 of the second vessel 3 into the flange fitting groove 80, and engage the plunger button 12 in the engagement recess 72, and then close the holding members 76, 81 to hold the front flange 6. Then, tighten the fastening screws 62, 70 to set them in place.

[0059] When cell aggregates are crushed and dispersed using the cell aggregate dispersion device A, the first syringe 1a, disperser 4, and second syringe 1b, connected as shown in FIG. 8(a), are set in the cell aggregate dispersion device A as described above and allowed to stand. This state is maintained for a while, allowing the cell aggregates to settle inside the first vessel 2. Then, the movable part 31 of the first mechanism 27 is advanced to push in the plunger 9 of the first vessel 2, while the movable part 34 is retracted to move the plunger 9 of the second vessel 3 in the withdrawal direction. The two plungers 9, 9 are then moved complementarily to crush and disperse the cell aggregates contained in the cell solution, and the dispersion liquid in which the crushed cell aggregates have been dispersed is collected in the second vessel 3. If the crushed material has reached the specified size, the crushing and dispersion process is terminated. If the crushed material has not reached the specified size, the crushed material is subjected to the crushing and dispersion process again.

[0060] When the dispersion liquid (cell liquid) contained in the second vessel 3 is to be subjected to another crushing and dispersion process, the movable part 31 of the first mechanism 27 is subsequently moved backward to move the plunger 9 of the first vessel 2 in the pull-out direction, and at the same time, the movable part 34 is advanced to move the plunger 9 of the second vessel 3 in the push-in direction, crushing and dispersing the cell aggregates contained in the dispersion liquid (cell liquid) to a predetermined size, and the dispersion liquid in which the crushed material of the predetermined size has been dispersed is contained in the first vessel 2. By repeating the above crushing and dispersion process as necessary, the original cell aggregates can be crushed and dispersed to a predetermined size. [Example]

[0061] Hereinafter, the embodiments of the present invention will be described in detail based on examples.

[0062] Example 1 First, a 10 ml volume of the first vessel 2 was filled with 1 x 10 viable cells. 7 A cell solution containing cell aggregates of 253G1 iPS cells and 10 ml of medium (StemFit AK02N, supplemented with 10 mM Y-27632) was placed in the cell aggregate dispersion device. Using a disperser 4 with a narrow channel-forming member 40 as shown in FIG. 3, a first syringe 1a, the disperser 4, and a second syringe 1b with a 10 ml capacity second vessel were connected as shown in FIG. 1, and the cell aggregate dispersion device was attached to the cell aggregate dispersion device A described above and allowed to stand. Next, the movable parts 31 and 34 of the cell aggregate dispersion device A were moved as described above to move the plungers 9 and 9 complementarily so that the flow rate through the narrow channel 41 was 0.16 m / sec. This moved the cell solution through the narrow channel 41, disrupting the cell aggregates. The dispersed cell solution (dispersion solution) was then placed in the second vessel 3. The resulting dispersion and the cell suspension before treatment were imaged using a Cell3iMager (manufactured by SCREEN Holdings Co., Ltd.) to measure particle size distribution. The particle size distribution was measured using the particles that had passed through a 40 μm strainer. The results for the dispersion before and after the crushing and dispersion treatment are shown in Figure 12(a) and Figure 12(b), respectively. Furthermore, live cells were confirmed by measurement using trypan blue staining.

[0063] The shape, structure, and dimensions of the narrow-flow-path forming member 40 used in this example are as follows: The cylindrical portion 45 has a male-threaded structure with M3.5 threads and a pitch of 0.35 mm toward the tip of the small holes 48. The small holes 48 have a circular cross section of 0.7 mm in diameter, and eight small holes 48 are formed at equal intervals around the circumference, with their centers positioned 1.35 mm from the top surface of the flange 46. The distance from the center of the small holes 48 to the tip 45a is 3.5 mm. The outer member 44 has a female-threaded structure with M4 threads and a pitch of 0.5 mm along the entire axial direction of the inner circumferential surface 51, and its axial length is 3.5 mm. The male-threaded and female-threaded portions can be combined by dropping in rather than by screwing. The thickness of the packing 43 is 1 mm. As a result, the width of the narrow flow path is 0.2 to 0.5 mm (minimum width is 0.2 mm), and the axial length (the distance from a position of small hole 48 close to flange 46 to tip 45a of cylindrical portion 45) is 2.5 mm. As shown in FIG. 12(a), the most frequent particle diameter of 100 μm or more in the particle size distribution of the cell aggregates before dispersion treatment is 420 μm, and the minimum width of the narrow channel, 0.2 mm, is 0.48 times that particle diameter.

[0064] (Reference example 1) Cell aggregates were disrupted in the same manner as in Example 1, except that a flow path forming member 200 shown in FIG. 11 was used instead of the narrow flow path forming member 40 shown in FIG. 3, and the cell solution was delivered at a flow rate of 0.5 ml / sec. Images were taken before and after treatment, and the particle size distribution was measured, in the same manner as in Example 1. The results are shown in FIG. 13. The results for the cell solution before treatment are shown in FIG. 13(a), and the results for the treated solution after the same treatment are shown in FIG. 13(b).

[0065] 11 has a structure in which a 1 mm thick disk (A) 201 having a 1 mm diameter through-hole 203 in its center and a 1 mm thick disk (B) 202 having a 4 mm diameter through-hole 204 in its center are stacked in the order A, B, A, B, A, A, and A, forming a continuous large step in the flow path. The flow velocity when passing through the 1 mm diameter through-hole is 0.16 m / sec. As shown in FIG. 13(a), the most frequent particle diameter of 100 μm or more in the particle size distribution of the cell aggregates before dispersion treatment is 420 μm, and the minimum width of the flow channel, 1 mm, is 2.4 times that particle diameter.

[0066] As shown in Figure 12, by passing the cell aggregates through a narrow channel, the shadows of the cell aggregates become lighter before and after the dispersion treatment, and the number of cell aggregates 40 μm or smaller increases dramatically before and after the dispersion treatment, indicating that the cell aggregates are crushed and dispersed to a size suitable for cell sheet production. In contrast, in Reference Example 1, as shown in Figure 11, multiple large steps were provided in the channel to increase the shear force load on the cell aggregates. However, as shown in Figure 13, no significant change was observed in the images of the cell aggregates before and after the treatment, and although the number of cell aggregates 40 μm or smaller increased to some extent, it was found that a sufficient crushing effect was not achieved compared to Example 1. This is thought to be because the channel width was only 1 mm, which was relatively larger than the cell aggregates and not narrow. [Explanation of symbols]

[0067] A cell aggregate dispersion device, X cell aggregate, Y cell solution, Z dispersion solution, 1a first syringe, 1b second syringe, 2 first vessel, 3 second vessel, 4 dispersion device, 5 outer cylinder, 6 front flange, 7 back flange, 8 head, 9 plunger, 10 shaft, 11 gasket, 11A tip, 11B O-ring, 12 plunger button, 13 first member, 14 second member, 15 flow path, 16 housing, 16A tip side inner surface, 16B bottom side inner surface, 17 female thread portion, 18 connection portion, 19 flow path, 20 male thread portion, 21 connection portion, 22 recess, 27 first mechanism, 28 second mechanism, 29 base portion, 30 fixed portion, 31 movable portion, 33 fixed portion, 34 movable portion, 36 protective covers, 37 Base plate, 38 stage, 40 narrow flow path forming member, 40a narrow flow path forming member, 40b narrow flow path forming member, 40c inner portion, 40d outer portion, 41 narrow flow path, 42 inner member, 43 packing, 44 outer member, 44a base end surface, 44b tip surface, 45 cylindrical portion, 45a tip surface, 46 flange, 47 recess, 47a bottom surface, 48 small hole, 49 outer peripheral surface, 49a spiral ridge, 50 center hole, 51 inner peripheral surface, 51a spiral groove, 54 fixed stage, 55 movable stage, 57 plunger holding portion, 58 adjustment plate, 62 clamping screw, 63 handle, 64 engagement recess, 65 plunger holding portion, 66 adjustment plate, 70 clamping screw, 71 handle, 72 engagement recess, 73 Outer tube holding portion, 74 U-shaped recess, 75 flange fitting groove, 76 holding member, 77 hook, 78 outer tube holding portion, 79 U-shaped recess, 80 flange fitting groove, 81 holding member, 82 hook, 83 grounding pad, 90a outer narrow channel, 90b outer narrow channel, 90c outer narrow channel, 91a inner narrow channel, 91b inner narrow channel, 91c inner narrow channel, 92 end surface, 93 end surface, 94 outer circumferential surface, 95 inner circumferential surface, 96 outer circumferential surface, 97 inner circumferential surface, 98a hollow portion, 98b hollow portion, 98c hollow portion, 98d hollow portion, 99 end surface, 100 end surface, 101 opening, 102 opening, 103 hollow portion, 104 hollow portion, 105 Narrow flow path, 106 inner peripheral surface, 107 outer peripheral surface, 108 end surface, 109 end surface, 110 inner bottom surface, 111 inner bottom surface, 200 flow path forming member, 201 disk (A), 202Disk (B), 203 through hole, 204 through hole.

Claims

1. A cell dispersion system for disrupting and dispersing cell aggregates of pluripotent stem cells, somatic stem cells, progenitor cells, or somatic cells, comprising: the cell dispersion system includes a dispersion device having a first opening, a second opening, and a narrow channel of a predetermined length provided between the first opening and the second opening; a cross-sectional shape perpendicular to the length direction of the narrow flow path is at least one selected from a single continuous circular ring, a single discrete circular ring, and multiple discrete circular rings, The minimum width of the cross section of the narrow flow channel in the cross-sectional shape is set to be in a range of 0.20 times or more of the particle diameter of the most frequent value of 100 μm or more in the particle size distribution of the cell aggregates, and smaller than the particle diameter; A cell dispersion system that moves a liquid containing cell aggregates from the first or second opening toward the second or first opening through the narrow flow path, and is capable of crushing and dispersing at least a portion of the cell aggregates to a size that can also be used to create cell sheets using only physical action.

2. the disperser is connected to a first vessel and a second vessel having a syringe structure at the first and second openings, respectively, and the first vessel and the second vessel are in communication with each other via the narrow channel; the first vessel and the second vessel have spaces that can be filled with the liquid and are defined by gaskets of the first plunger and the second plunger, respectively; 2. The cell dispersion system according to claim 1, wherein the liquid filling the space of the first or second vessel is moved through the narrow channel to the space of the second or first vessel by moving the first and second plungers in a complementary manner.

3. 3. The cell dispersion system according to claim 1, wherein the flow rate of the liquid passing through the narrow channel is 0.05 to 10 m / sec, and the supply rate is 0.1 to 5 ml / sec.

4. The cell dispersion system according to any one of claims 1 to 3, wherein the minimum width of the narrow channel in the longitudinal direction is constant or gradually decreases from the first or second opening side toward the second or first opening side and then gradually increases.

5. 5. The cell dispersion system according to claim 1, wherein at least a part of the wall surface constituting the narrow channel is provided with irregularities.

6. The cell dispersion system according to any one of claims 1 to 5, wherein the liquid is passed through the narrow channel two or more times to gradually reduce the average particle size of the cell aggregates.

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