JP7291509B2 - Device and method for diluting cell suspension - Google Patents

Description

The present invention relates to devices and methods for diluting cell suspensions.

In recent years, attempts have been made to transplant various cells in order to repair damaged tissues and the like. For example, use of fetal myocardial cells, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. for the repair of myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction. has been attempted (Non-Patent Document 1).

As part of such attempts, cell structures formed using scaffolds and sheet-like cell cultures in which cells are formed into sheets have been developed (see, for example, Patent Documents 1 to 3).

Along with the establishment of new treatment methods based on regenerative medicine, freezing and preserving autologous cells to form three-dimensional structures such as artificial tissues and sheet-like cell cultures, and direct cell transplantation, In recent years, there have been increasing opportunities to thaw such cryopreserved cells to collect autologous cells and use them for treatment.

Forming a sheet-like cell culture by this method requires more cells than usual, so in order to recover many viable cells, physico-chemical processes are applied to the cells during freezing and/or thawing. It is devised so as not to damage it as much as possible.

Under these circumstances, the present inventors proposed a cryopreserved cell thawing system that includes an operating unit that injects a diluent and an arithmetic control unit that controls the diluent injection speed of the operating unit (Patent Document 4). . By controlling the diluent injection rate in such a system, it is possible to reduce the number of cells that die due to rapid changes in osmotic pressure, thereby increasing the number of viable cells that can be recovered.

Japanese Patent Publication No. 2007-528755 Japanese Unexamined Patent Application Publication No. 2010-81829 JP 2010-226991 A JP 2015-181433 A

Haraguchi et al., Stem Cells Transl Med. 2012;1(2):136-41

The system as described above has a complicated structure for realizing the operation section and the control section, is expensive to manufacture, and is complicated to operate. In addition, the distribution of the diluent in the cell suspension may vary during the dilution process.

The present invention has been made in consideration of such points, and an object thereof is to provide a means which has a simple structure and can efficiently dilute a cell suspension by a simple operation. .

That is, the present invention relates to the following.
[1] A device for diluting a cell suspension, comprising a first channel, a second channel, and a communication channel that communicates the first channel and the second channel, and the The above device, wherein the diluent is configured to be added through the communicating channel to the cell suspension flowing in the other channel.
[2] The device according to [1], wherein the one or more communication paths are arranged along the direction of the flow path.
[3] The device according to [1] or [2], which is configured such that the addition rate gradually increases in the direction of the flow path.
[4] The device according to any one of [1] to [3], wherein the cross-sectional area of the communicating path gradually increases in the direction of the flow path.
[5] The device according to any one of [1] to [4], which is configured to have a downward slope from one flow path to the other flow path.
[6] The device according to [5], wherein the gradient gradually increases in the direction of the flow path.

[7] The device according to any one of [1] to [6], wherein the injection rate of the diluent is configured so that the maximum osmotic pressure load during dilution is 40 to 250 mOsm/sec.
[8] The device according to any one of [1] to [7], wherein the first channel and the second channel are spirally arranged.
[9] The device according to any one of [1] to [8], wherein the first channel and the second channel are adjacent to each other, and the communication channel is provided in the wall separating the two channels [10] ] The device further includes a container portion forming a sealed space, the cell suspension is diluted on the lower side of the container portion, and the diluted cell suspension is accumulated on the upper side of the container portion. , [1] to [9].
[11] A method for diluting a cell suspension, wherein the diluent flowing through one channel is added to the cell suspension flowing through the other channel through a communication channel.

According to the present invention, since it has a simple structure, the manufacturing cost can be kept low, and the cell suspension can be efficiently diluted with a simple operation, so it depends on the skill of the operator. work level can be uniformed.

In addition, according to the present invention, cells can be recovered while maintaining high viability even after freezing and thawing. can be secured.

Furthermore, according to the present invention, the diluent can be added while the cell suspension is being moved, and variations in the distribution of the diluent in the cell suspension can be suppressed. improves. A large amount of cell suspension can be efficiently diluted at once by continuously flowing the cell suspension and the diluent through the channel.

FIG. 1 is a conceptual diagram of a device according to a first embodiment of the invention. FIG. 2 is a conceptual diagram of a device according to a second embodiment of the invention.

In one aspect, the present invention is a device for diluting a cell suspension, comprising a first flow path, a second flow path, and a communication path that connects the first flow path and the second flow path, The device is configured such that the diluent flowing through one channel is added to the cell suspension flowing through the other channel through the communicating channel.
In one aspect of the present invention, there is provided a method for diluting a cell suspension, wherein the diluent flowing through one channel is added to the cell suspension flowing through the other channel through a communication channel. Regarding. In one aspect of the invention, the cell suspension is obtained by thawing cryopreserved cells.

In the present invention, "cryopreserved cells" usually mean cryopreserved cells themselves, but may also mean one cryopreserved unit of cryopreserved cells. In this case, a cryopreservation unit means a group of cells, eg a tube, that are cryopreserved together as a group. Therefore, in this case, when the "cryopreserved cells" are thawed, a frozen "cell suspension" is obtained.

In the present invention, the "osmotic pressure load at the time of dilution" means the rate of change per unit time of osmotic pressure due to the addition of the diluent. The osmotic pressure load varies depending on factors such as the rate of addition of the diluent (addition amount per unit time) and the difference in osmotic pressure between the diluent and the liquid to be diluted (eg, thawed cell suspension). "Maximum osmotic pressure load" means the maximum numerical value among the osmotic pressure loads that change due to the addition of the diluent from the start of dilution to the end of dilution.

The present invention is characterized by diluting a cell suspension obtained by thawing cryopreserved cells so that the change in osmotic pressure during dilution is sufficiently slowed. When thawing cryopreserved cells to collect viable cells, culture medium is added to the thawed cell suspension for the purpose of reducing the effects of cytotoxic components in the thawed cell suspension. dilute with The present inventors have found that rapid dilution in the dilution process reduces viability of viable cells due to damage to cells due to abrupt changes in the osmotic pressure of the suspension.

If the change in osmotic pressure during dilution is slow enough, damage to cells is reduced and the recovery of viable cells is increased. Any method known in the art may be used to slow down the change in osmotic pressure, such as slowing down the rate of adding the diluent or using a diluent with a small difference in osmotic pressure between the cell suspension and the diluent. For example, it is preferable not to use a diluent having cytotoxicity, and not to cause damage other than osmotic pressure.

Diluents that can be used in the present invention are not particularly limited, but those that do not physically and chemically damage cells are preferred. Examples of diluents include, but are not limited to, liquid media such as DMEM medium, Hank's balanced salt solution, buffer solutions such as PBS, isotonic solutions such as physiological saline, and distilled water. . Further, other components such as albumin may be added to these.

In this specification, the unit Osm is used as a unit of osmotic pressure, and 1 Osm means an osmotic pressure equivalent to the osmotic pressure of an ideal solution of 1 mol/L. The osmotic pressure load at the time of dilution is expressed as a change in osmotic pressure per second under room temperature conditions (unit: Osm/sec). may be used, including, but not limited to, diluent addition rate, volume or weight increase rate, and the like.

The osmotic pressure load may be obtained by measuring changes in osmotic pressure in real time, or by obtaining osmotic pressure at two points in time and calculating the average change per unit time between them. When the diluent is added at a constant rate in a certain time span, as long as the same diluent is added, the osmotic pressure load at the start of the diluent addition is generally It is considered that the pressure load reaches the maximum value and the osmotic pressure load decreases as the amount of the diluent added increases. Therefore, in one embodiment, when the same diluent is added at a constant rate, the osmotic pressure at the start of adding the diluent and the osmotic pressure after a unit time (for example, 1 second in the above example of the present invention) and the difference in osmotic pressure at both time points is taken as the maximum osmotic load at that dilution.

According to the present invention, the diluent flowing in one channel is added to the cell suspension flowing in the other channel through the communicating channel, so that the cell suspension flows from the beginning to the end of the channel. The diluent can be added while moving towards (running). Therefore, when the present invention is used to add a diluent, variations in the distribution of the diluent in the cell suspension are less likely to occur. Then, the osmotic pressure load on the starting end side of the channel of the cell suspension becomes the maximum value of the osmotic pressure load, and as the channel progresses toward the terminal side, the amount of the diluent added increases, It is thought that the osmotic pressure load gradually decreases.

Examples of cells that can be used in this method include, but are not limited to, adherent cells (adherent cells). Adherent cells include, for example, adherent somatic cells (e.g., cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, etc.) and stem cells (e.g., myoblasts, tissue stem cells such as cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) and so on. Somatic cells may be differentiated from stem cells, particularly iPS cells. Non-limiting examples of cells that can form sheet-like cell cultures include myoblasts (e.g., skeletal myoblasts), mesenchymal stem cells (e.g., bone marrow, adipose tissue, peripheral blood, skin, hair roots, etc.). , muscle tissue, endometrium, placenta, those derived from umbilical cord blood, etc.), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (e.g., oral mucosal epithelium) cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (e.g., vascular endothelial cells, etc.), hepatocytes (e.g., hepatocytes, etc.), pancreatic cells (e.g., pancreatic islet cells, etc.), renal cells, adrenal glands cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells and the like. In the present specification, those that form a monolayer cell culture, such as myoblasts or cardiomyocytes, are preferred, and skeletal myoblasts or iPS cell-derived cardiomyocytes are particularly preferred.

As mentioned above, the cell suspension obtained by thawing may contain cytotoxic components (such as DMSO), and dilution can reduce the effect of the cytotoxic components. The present invention is characterized by sufficiently slowing the change in osmotic pressure during dilution, ie, sufficiently reducing the osmotic pressure load, thereby increasing the viability of thawed cells.

In the present invention, the threshold for "sufficiently slow change in osmotic pressure" can vary depending on the cells used, melting conditions, temperature, and the like. For example, when using a normal cryopreservation solution (e.g., DMEM medium containing about 10% DMSO) and a normal diluent (e.g., commercially available DMEM medium), in certain embodiments, the maximum osmotic load is about 250 mOsm/sec or less. and is preferably about 220 mOsm/sec. It is more preferably about 100 mOsm/sec or less, and even more preferably about 50 mOsm/sec or less.

Also, if the dilution takes too long, the cells may be damaged due to reasons other than osmotic load, for example, due to the influence of cytotoxic components in the cell suspension. Diluted is preferred. In the method of the present invention, the lower limit of the maximum osmotic load may not be set, but a normal cryopreservation solution (e.g., DMEM medium containing about 10% DMSO) and a normal diluent (e.g., When using a commercially available DMEM medium or the like), it is preferably about 2 mOsm/sec or more, more preferably about 20 mOsm/sec or more, and even more preferably about 40 mOsm/sec or more.

Therefore, in a preferred embodiment of the method of the present invention, the maximum osmotic load is between 2 mOsm/s and 250 mOsm/s, more preferably between 2 mOsm/s and 220 mOsm/s, even more preferably between 20 mOsm/s and 100 mOsm/s. and most preferably 40 mOsm/sec to 50 mOsm/sec from the viewpoint of the amount of viable cells that can be recovered.

As mentioned above, in the methods of the invention, the diluent initially added to the thawed cell suspension is most likely to affect the maximum osmotic load. Therefore, in one aspect of the present invention, the maximum osmotic pressure load is the maximum osmotic pressure load from the start of dilution to 3-fold dilution, and in another aspect, 2-fold dilution is the maximum osmotic load during

In a preferred embodiment of the present invention, dilution is performed while measuring the osmotic pressure and adjusting the maximum osmotic load. The osmotic pressure measurement may be performed continuously at all times, or may be performed at specific time intervals such as every second, every 10 seconds, every 30 seconds, or every minute. Any method known in the art can be used to measure the osmotic pressure, and the osmotic pressure can be measured using, for example, an osmometer, although the method is not limited to this.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail based on preferred embodiments with reference to the drawings. Note that the size of each member in the drawings is appropriately emphasized for explanation, and does not represent the actual ratio or size.

<First Embodiment>
First, a device according to a first embodiment of the present invention will be described. FIG. 1 is a conceptual diagram of a device according to a first embodiment of the invention. FIG. 2 is a conceptual diagram showing a modification of the device according to the first embodiment of the invention.

As shown in FIGS. 1A-1F, the device includes a first channel 1 , a second channel 2 , and a communication channel 3 that communicates the first channel 1 and the second channel 2 . In the figure, the upper side shows a top view showing the arrangement of the first channel 1, the second channel 2, and the communication path 3, and the lower side shows a cross-sectional view taken along line X-X' in the top view. When using the device, for example, the cell suspension is injected from the beginning (injection port 4) of the first channel 1, the diluent is injected from the beginning (injection port 5) of the second channel 2, and the diluted solution is continuously added. By adding to the cell suspension through the passage 3, the cell suspension can be diluted, and the diluted cell suspension can be collected at the terminal side of the channel. Cells in the logarithmic growth phase are preferably used from the viewpoint of the amount of viable cells recovered.

Since the cell suspension is diluted by the diluent that has passed through the communication path 3 while proceeding through the first channel 1, the distribution of the diluent in the cell suspension is less likely to vary. That is, when a diluent is dropped (added) to a cell suspension contained in a centrifuge tube or the like, the distribution of the diluent tends to vary between the upper and lower sides of the cell suspension, but in the present invention, Since the diluent is added while moving the cell suspension, the distribution of the diluent is less likely to vary.

In addition, in the present invention, since the cell suspension and the diluent can be flowed through the channel without interruption (continuously), a large amount of cell suspension can be efficiently diluted at once. . That is, when a diluent is added dropwise to a cell suspension contained in a centrifuge tube or the like, the cell suspension is diluted only by the volume of the centrifuge tube. Also, when diluting a large amount, efficiency is poor, such as using a plurality of centrifuge tubes and adding the diluent to each. Also, when using a large-capacity centrifuge tube, the distribution of the diluent tends to vary between the upper and lower sides of the cell suspension.

As shown in FIG. 1A, the communication path 3 is a flow path for bypassing the first flow path 1 and the second flow path 2, and is called a communication path to distinguish it from other flow paths. At least one communication path 3 may be arranged between the first flow path 1 and the second flow path 2 . As described above, the maximum osmotic pressure load is related to the diluent addition speed (addition amount per unit time), and the addition speed is related to the cross-sectional area of the communication passage 3 . Therefore, it is preferable to set the cross-sectional area of the communication path 3 so that the osmotic pressure changes sufficiently slowly.

The cell suspension is diluted by flowing (adding) the diluent flowing through one channel (second channel 2) into the cell suspension flowing through the other channel (first channel 1). can be configured to occur. If a reverse flow occurs, the cell suspension that has passed through the communication channel 3 will be caught in the flow of the diluent, causing a rapid change in osmotic pressure. In addition, by allowing dilution to occur only in the other channel, cells remaining after the dilution process is completed can be recovered simply by flowing a fluid (e.g., gas, diluent, etc.) into the other channel. can be done. Therefore, a cell non-adhesive treatment may be applied to the inner surface of the channel or communicating path.

As a means for causing the inflow of the liquid, the injection pressure of the diluent into the second channel 2 is made higher than the injection pressure of the cell suspension into the first channel 1 to generate a pressure difference. be done. It can also be realized by replacing the pressure difference with a flow velocity difference. In this case, the first flow path 1, the second flow path 2, and/or the communication path 3 can be configured as a cylindrical closed space, thereby facilitating propagation of fluid pressure within the space.

Moreover, as another means, there is a configuration in which the end side of the second flow path 2 is closed. In this case, even if the injection pressure (or flow velocity) to the two flow paths is the same, the internal pressure on the side of the second flow path 2 increases, so the pressure difference between the side of the first flow path 1 whose end side is open occurs. Furthermore, as another means, there is a configuration in which there is a downward slope from one flow path to the other flow path. This can be achieved, for example, by providing a height difference between the second flow path 2 and the first flow path 1, or by providing a downward slope in the communication path 3 from the second flow path 2 to the first flow path 1. can.

As shown in FIG. 1B, a plurality of communication paths 3 can be arranged discretely along the flow path direction (direction in which liquid flows). In this case, the cell suspension is diluted with the diluent that has passed through the plurality of communication paths 3 while proceeding through the first flow path 1 from the starting end toward the terminal end. That is, the cell suspension traveling in the first flow path 1 is diluted with the diluent that has passed through the first communication path 3, and this diluted cell suspension is transferred to the following second communication path 3 It is further diluted with the diluent that has passed through the Therefore, the amount of diluent per unit volume of the cell suspension can be gradually increased, and the osmotic pressure load can be gradually decreased. The distance between the communication paths 3 can be freely set.

As described above, a method of slowing down the rate of adding the diluent can be used as a means of slowing down the change in osmotic pressure. takes time. However, as the dilution progresses, the ratio of the diluent to the cell suspension increases, so it can be said that even if a larger amount of the diluent is added, the cell suspension is unlikely to be rapidly diluted. Therefore, in the present invention, the time required for the dilution process can be greatly shortened by configuring the addition speed of the diluent to increase gradually in the direction of the flow path as the dilution progresses. can.

As shown in FIG. 1C, a plurality of communicating paths 3 are arranged discretely along the direction of the flow path, and the cross-sectional area of the communicating path 3 gradually increases in the direction of the flow path. can be done. As a result, the addition speed of the diluent can be made to gradually increase in the direction of the flow path. Alternatively, as shown in FIG. 1D, the distance between the plurality of communication paths 3 may be configured to gradually decrease in the direction of the flow path. That is, as shown in FIGS. 1C and 1D, the cross-sectional area of the communication path 3 (cross-sectional area per unit length in the flow direction) can be configured to gradually increase in the flow direction. .

As shown in FIG. 1E, the communication path 3 can also be arranged between the first flow path 1 and the second flow path 2 across the flow direction. Further, as shown in FIG. 1E, the cross-sectional area per unit length of the communication path 3 can be configured to gradually increase in the direction of the flow path. 1C to 1E are examples of means for gradually increasing the rate of addition of the diluent. As another example, the gradient from the second flow path 2 to the first flow path 1 is It can also be configured to gradually increase in the direction. This can be realized, for example, by gradually increasing the height difference between the flow paths in the direction of the flow path, or by gradually increasing the gradient of the communication path 3 in the direction of the flow path.

FIG. 1F shows a device with a first channel 1 and a second channel 2 arranged in a spiral. The cell suspension and diluent are injected from inlets 4 and 5 provided in the center of the device, respectively, and are diluted through a communication channel 3 (not shown) while progressing spirally through the channel. be. The second channel 2 can also be arranged inside the first channel 1 so that centrifugal force is applied to the diluent to add it to the outer suspension. As shown, the channels can also increase in the direction of the channel. The first channel 1 and the second channel 2 may be arranged in any way as long as the cell suspension and the diluent run side by side.

The device may include at least the first channel 1, the second channel 2, and the communication channel 3 that communicates the first channel 1 and the second channel 2, and the diluent flowing through one channel is Any modification is possible as long as it is configured to be added to the cell suspension flowing in the other flow path through the communicating path. The embodiments described above with reference to FIGS. 1A-1F in which one or more communicating passages are arranged along the direction of the flow path have been described in detail for the sake of easy understanding of the present invention, and are not necessarily It is not limited to what has been described. The first channel 1, the second channel 2, and the communication channel 3 may be designed in any way as long as they are constructed so that the change in osmotic pressure during dilution is sufficiently slow.

As described above, according to the present invention, since it has a simple structure, the manufacturing cost can be kept low, and the cell suspension can be efficiently diluted with a simple operation. work level can be uniformed.

In addition, according to the present invention, cells can be recovered while maintaining high viability even after freezing and thawing. can be secured.

Furthermore, according to the present invention, the diluent can be added while the cell suspension is being moved, and variations in the distribution of the diluent in the cell suspension can be suppressed. improves. A large amount of cell suspension can be efficiently diluted at once by continuously flowing the cell suspension and the diluent through the channel.

<Second embodiment>
Next, a device according to a second embodiment of the invention will be described. FIG. 2 is a conceptual diagram of a device according to a second embodiment of the invention. Differences from the first embodiment will be described in detail below, and descriptions of the same items will be omitted.

As shown in FIG. 2, the device according to the second embodiment of the present invention includes a first channel 1, a second channel 2, a communication channel 3, an inlet 4, an inlet 5, a container portion 6, and an outlet 7. include. The container part 6 has a cylindrical container shape, and a closed space is formed inside. The container part 6 is partitioned into an upper stage and a lower stage by a partition plate 61, and a spiral first flow path 1 and a spiral second flow path 2 are arranged on the lower stage side as shown in FIG. 1F. . The injection port 4 and the injection port 5 are provided at the center of the lower surface of the container portion 6 . The communication passages 3 can, for example, be arranged as described above with reference to FIGS. 1A-1F.

The cell suspension and diluent are placed in a syringe or the like and injected into injection ports 4 and 5 from the bottom side of container section 6 via a tube or the like. At this time, the syringe may be manipulated to create a pressure difference in the injection pressure. The cell suspension and diluent injected from the injection ports 4 and 5 are diluted through the communication passage 3 while spiraling through the channel. The diluted cell suspension that has flowed out from the terminal end of the channel passes through a plurality of holes 62 provided along the circumferential direction on the outside of the partition plate 61 while flowing on the outside of the container part 6. It flows into the space on the upper side of the container part 6 .

That is, the device according to the present embodiment is configured such that dilution occurs on the lower side of the container section 6 and the diluted cell suspension accumulates on the upper side of the container section 6 . The diluted cell suspension advances spirally and convection also occurs on the upper side of the container part 6, so that the washing effect of the cells is enhanced. The diluted cell suspension is discharged from the discharge port 7 provided on the side surface of the container part 6 . At this time, the container part 6 may be turned upside down to discharge the diluted cell suspension from the discharge port 7 . By arranging the discharge port 7 on the upper side of the container part 6, contamination can be prevented when the container part 6 is installed on a workbench.

If the device can be hygienically handled, the configuration of the upper side and the lower side can be exchanged, and the diluted cell suspension can be accumulated in the lower side of the container part 6 and discharged from the openable discharge port 7. good. The plurality of holes 62 may be openable and closed to prevent mixing of pre-diluted and diluted liquids. The cross-section of the container part 6 may be square, and in that case, the channels are arranged linearly and a folded part is formed in the middle, thereby efficiently arranging a plurality of linear channels on the bottom surface of the container part 6. be able to. When the cross section of the container part 6 is circular, the bottom surface of the container part 6 can be efficiently used by arranging the channel in a spiral shape, and sharp curves are less likely to be formed, so cells are less likely to remain.

As described above, according to the present invention, since it has a simple structure, the manufacturing cost can be kept low, and the cell suspension can be efficiently diluted with a simple operation. work level can be uniformed.

In addition, according to the present invention, cells can be recovered while maintaining high viability even after freezing and thawing. can be secured.

Furthermore, according to the present invention, the diluent can be added while the cell suspension is being moved, and variations in the distribution of the diluent in the cell suspension can be suppressed. improves. A large amount of cell suspension can be efficiently diluted at once by continuously flowing the cell suspension and the diluent through the channel.

In the present invention, each component can be replaced with any one capable of exhibiting a similar function, or any component can be added.

1 First channel 2 Second channel 3 Communication channel 4 Inlet (first channel)
5 inlet (second flow path)
6 container part 61 partition plate 62 hole 7 outlet

Claims (8)

A device for diluting a cell suspension, comprising:
including a first flow path, a second flow path, and a communication path that connects the first flow path and the second flow path,
The diluent flowing in one channel is added to the cell suspension flowing in the other channel through the communicating channel, and the adding speed is increased by gradually increasing the cross-sectional area of the communicating channel in the direction of the channel. , is configured to gradually increase in the direction of the flow path,
Said device. 2. The device according to claim 1, wherein the one or more communicating paths are arranged along the flow direction. 3. The device of claim 1 or 2 , configured to provide a downward slope from one flow path to the other flow path. 4. The device of claim 3 , wherein the gradient is configured to gradually increase in the direction of the flow path. A device according to any one of claims 1 to 4 , wherein the first channel and the second channel are arranged in a spiral. The device according to any one of claims 1 to 5 , wherein the first channel and the second channel are adjacent to each other, and the communicating channel is provided in the wall separating the two channels. The device further includes a container that forms a closed space, the cell suspension is diluted on the lower side of the container, and the diluted cell suspension is accumulated on the upper side of the container. Device according to any one of claims 1-6 . A method for diluting a cell suspension, wherein the device according to any one of claims 1 to 7 is used to pass a diluent flowing through one channel through a communicating channel to flow through the other channel. The above method, wherein the cell suspension is added, and the addition rate is gradually increased in the direction of the flow path.

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