Method for producing differentiated cells

Abstract

Provided is a method for producing differentiated cells, which makes it possible to separate differentiated cells and cells that are not differentiated satisfactorily from each other with a simple and inexpensive configuration without using a chemical substance. A method for producing differentiated cells according to the present invention is characterized by performing each of the following steps at least once: a differentiation induction step for inducing the differentiation of cells: and a size separation step for subjecting a particle suspension solution containing the cells obtained by the differentiation induction step to the separation into particles each having a size equal to or larger than a predetermined value and particles each having a size smaller than the predetermined value on the basis of a hydrodynamic effect.

Description

Method for producing differentiated cells

[0001] The present invention relates to a method for producing differentiated cells, and in particular to a step in the method for producing differentiated cells, of separating differentiated cells from cells that are not sufficiently differentiated.

[0002] In regenerative medicine, when differentiated cells obtained by a differentiation induction step are transplanted into a patient, it is necessary to separate the differentiated cells from the insufficiently differentiated cells, because if any insufficiently differentiated cells remain among the differentiated cells, this can cause tumor formation, etc. In other words, the production of differentiated cells includes a differentiation induction step and a separation step of separating the differentiated cells from the insufficiently differentiated cells.

[0003] Known methods for use in the separation step include the use of drugs to remove insufficiently differentiated cells. For example, Patent Document 1 listed below describes a drug for removing undifferentiated cells by selectively damaging cells expressing ABCB1 and ABCG2 transporters.

[0004] Furthermore, a flow cytometer (cell sorter) is known as an apparatus used in the separation step. For example, Patent Document 2 listed below describes a flow cytometer that irradiates cells with laser light and separates the cells by detecting scattered light that depends on the size of the cells.

[0005] International Publication No. WO 2018 / 043567 International Publication No. WO 2016 / 182034

[0006] Huang et al. , "Continuous Particle Separation Through Deterministic Lateral Displacement", Science Vol. 304, p p. 987-990, 2004

[0007] For example, as described in Patent Document 1, a method of removing insufficiently differentiated cells using a chemical agent can remove insufficiently differentiated cells from a sample after a differentiation induction step, but the chemical treatment with the chemical agent may affect differentiated cells. Furthermore, there is a problem in that insufficiently differentiated cells are removed by the chemical agent and cannot be reused. Therefore, there is a need for a technology that can remove insufficiently differentiated cells from a sample after a differentiation induction step and extract differentiated cells without using a chemical agent.

[0008] For example, the flow cytometer described in Patent Document 2 can separate differentiated cells from insufficiently differentiated cells without the use of chemicals. However, flow cytometers include a light irradiation means for irradiating light onto particles in a sample solution, a detection means for detecting scattered light or fluorescence emitted from the particles when irradiated with light, and a force generation means for applying a force to change the flow direction based on a signal from the detection means, resulting in a large, complex, and expensive device. Furthermore, while some flow cytometers can separate cells by detecting scattered light based on the size and shape of the cells, the use of scattered light results in a wide range of separation thresholds, which results in a problem of not necessarily high cell separation accuracy.

[0009] Furthermore, as mentioned above, flow cytometers are large, complex, and expensive, making it difficult to install multiple flow cytometers. For example, when producing differentiated cells from cells from multiple patients, it is difficult to assign a separate flow cytometer to each of the multiple samples after the differentiation induction process. Therefore, multiple samples are processed together using a single flow cytometer, which can lead to sample mix-ups and contamination. Furthermore, installation locations for large flow cytometers are limited, and the particle suspension obtained from the differentiation induction process must be transported to the flow cytometer installation location. This prevents the differentiation induction process and the separation process from being performed in the same location, potentially resulting in the separation of the work processes.

[0010] Furthermore, there is a possibility that the separation step cannot be performed at the appropriate timing because the processing time (machine time) of the device required for the separation step cannot be secured sufficiently. Furthermore, there are problems such as the practical difficulty of repeatedly performing the separation step on a single sample and the difficulty of incorporating a configuration for performing the separation step into the device that performs the differentiation induction step. Therefore, there is a demand for a technology that can individually separate multiple samples after the differentiation induction step using a simple and inexpensive device.

[0011] The present invention has been made in consideration of the various problems described above, and aims to provide a method for producing differentiated cells that is capable of separating differentiated cells from cells that are not sufficiently differentiated using a simple and inexpensive configuration without using chemicals.

[0012] As a result of intensive research in light of the above problems, the inventors noticed that some cells change in size before and after differentiation, and discovered that when separating differentiated cells from cells obtained by differentiation induction, by using a technology for size separation of particles based on hydrodynamic effects, it is possible to easily and efficiently separate and recover differentiated cells and cells that have not fully differentiated based on cell size.

[0013] The present invention was developed based on the above findings and provides a method for producing differentiated cells, which comprises performing each of the following steps at least once: a differentiation induction step for inducing cell differentiation; and a size separation step for separating particles having a size equal to or greater than a predetermined value from particles having a size less than the predetermined value based on hydrodynamic effects in a particle suspension containing cells obtained by the differentiation induction step.

[0014] As used herein, differentiated cells refer to cells that have undergone appropriate differentiation and progressed to a desired stage, while insufficiently differentiated cells refer to cells that have undergone differentiation but have not progressed sufficiently to a desired stage, and pre-differentiated cells that have not undergone differentiation.

[0015] According to the above process, cells whose size changes before and after differentiation can be separated into particles containing differentiated cells and particles containing cells that are not fully differentiated by size separation based on hydrodynamic effects.

[0016] A simple and inexpensive configuration using particle separation technology based on hydrodynamic effects makes it possible to accurately recover differentiated cells as particles of a predetermined size or larger, and cells that are not sufficiently differentiated as particles of a size smaller than the predetermined size.

[0017] Furthermore, particle separation devices that utilize the hydrodynamic effect are inexpensive and multiple particle separation devices can be easily prepared, so even if there are many samples containing differentiated cells, particle separation can be performed on each sample individually without concentrating them in one place, thereby reducing the risk of sample mix-ups or sample contamination.

[0018] Furthermore, by using a particle separation technique that utilizes the hydrodynamic effect, particle separation can be performed without chemically treating the sample with drugs, etc. This allows both differentiated and less differentiated cells to be recovered in a reusable state.

[0019] In the method for producing differentiated cells according to the present invention, in the size separation step, size separation may be performed using a DLD microchannel.

[0020] According to the above process, size separation can be performed by a particle separation technique utilizing hydrodynamic effects, particularly by a technique using a DLD microchannel.

[0021] In the method for producing differentiated cells according to the present invention, the size separation step may be performed using a particle separation device, wherein the particle separation device includes a pressure generating unit for discharging gas, a particle suspension storage unit having an inlet and an outlet and into which the particle suspension is injected, a buffer storage unit having an inlet and an outlet and into which a buffer solution is injected, a branch pipe including a first conduit connected to the outlet of the pressure generating unit, a second conduit branching from the first conduit and connected to the inlet of the particle suspension storage unit, and a third conduit branching from the first conduit and connected to the inlet of the buffer solution storage unit, and a particle suspension inlet connected to the outlet of the particle suspension storage unit. a DLD micro-channel chip including: a buffer solution inlet connected to the outlet of the buffer solution storage section; a DLD flow path section having a DLD micro-channel structure in which a plurality of fine pillars are arranged, along which the particle suspension introduced via the particle suspension inlet and the buffer solution introduced via the buffer solution inlet flow in parallel while coming into contact with each other; an outlet for discharging the buffer solution containing particles having a size equal to or larger than a predetermined value that have been transferred from the particle suspension to the buffer solution in the DLD flow path section; and an outlet for discharging the particle suspension after the particles having a size equal to or larger than the predetermined value have been transferred to the buffer solution in the DLD flow path section.

[0022] According to the above process, size separation can be performed using a particle separator having a DLD microchannel chip, which is available at a reasonable price and has excellent hygienic properties.

[0023] The method for producing differentiated cells according to the present invention may include a differentiation progression step in which differentiation of cells separated as particles of a size less than the predetermined value in the size separation step is advanced, and a second size separation step in which particles of a size equal to or greater than the predetermined value and particles of a size less than the predetermined value are separated from a particle suspension containing cells obtained by the differentiation progression step based on hydrodynamic effects.

[0024] In this specification, the differentiation progression process refers to at least one of a process of inducing differentiation of cells that have not been induced to differentiate (pre-differentiation cells) and a process of promoting differentiation of cells that have not progressed sufficiently in differentiation.

[0025] According to the above process, insufficiently differentiated cells separated as particles of a size less than a predetermined value can be reused, and after the insufficiently differentiated cells are differentiated, size separation based on hydrodynamic effects can be performed to separate them into particles containing differentiated cells and particles containing insufficiently differentiated cells.

[0026] The method for producing differentiated cells according to the present invention may include a differentiation progression step in which the differentiation of cells separated as particles having a size equal to or greater than the predetermined value in the size separation step is advanced, and a second size separation step in which particles having a size equal to or greater than a second predetermined value, which is larger than the predetermined value, and particles having a size less than the second predetermined value are separated from a particle suspension containing cells obtained by the differentiation progression step based on hydrodynamic effects.

[0027] According to the above process, differentiated cells separated as particles of a size equal to or larger than a predetermined value can be further differentiated, and then size separation based on hydrodynamic effects can be performed using a second predetermined value larger than the predetermined value as a separation threshold, thereby separating the cells into particles containing differentiated cells and particles containing cells that are not sufficiently differentiated.

[0028] In the method for producing differentiated cells according to the present invention, the predetermined value may be 5 μm or more and 100 μm or less.

[0029] According to the above process, when performing size separation based on the hydrodynamic effect, it is possible to set a separation threshold that allows appropriate separation of differentiated cells from cells that are not sufficiently differentiated.

[0030] FIG. 4 is a flow diagram illustrating a method for producing differentiated cells in an embodiment of the present invention. FIG. 5 is a diagram illustrating an example of the configuration of a particle separation device that can be used in an embodiment of the present invention. FIG. 6 is a diagram illustrating the configuration of a DLD micro-channel chip incorporated in a particle separation device that can be used in an embodiment of the present invention. FIG. 7 is a schematic diagram illustrating the principle of particle separation in the DLD micro-channel chip shown in FIG. 3. FIG. 8 is a photograph of a liquid containing particles having a size equal to or greater than the separation threshold in Example 1 of the present invention. FIG. 9 is a photograph of a liquid containing particles having a size less than the separation threshold in Example 1 of the present invention. FIG. 10 is a graph showing the results obtained in Example 1 of the present invention. FIG. 11 is a photograph of a liquid containing particles having a size equal to or greater than the separation threshold in Example 2 of the present invention. FIG. 12 is a photograph of a liquid containing particles having a size less than the separation threshold in Example 2 of the present invention. FIG. 13 is a graph showing the results obtained in Example 2 of the present invention. FIG. 14 is a photograph of cells four days after differentiation induction in Example 3 of the present invention. FIG. 15 is a photograph of cells recovered as particles having a size less than the separation threshold two days after culturing in Example 3 of the present invention.

[0031] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0032] In this embodiment, a method for producing differentiated cells is provided, which involves performing a differentiation induction process to induce cell differentiation and a size separation process to separate particles having a size equal to or greater than a predetermined value from particles having a size less than the predetermined value based on hydrodynamic effects in a particle suspension containing cells obtained by the differentiation induction process, each at least once.

[0033] In the differentiation induction step of this embodiment, differentiation of cells is induced under differentiation induction conditions. The cells used in this embodiment are not particularly limited as long as they change in size (particle size) as they differentiate, and may be stem cells that differentiate into various cells such as muscle cells, adipocytes, and nerve cells, or pluripotent stem cells such as ES cells and iPS cells.

[0034] The differentiation induction method in the differentiation induction step is not particularly limited, and a differentiation induction method suitable for the type of cells to be differentiated may be carried out. For example, differentiation induction may be carried out by adding a stimulating factor that initiates and promotes cell differentiation or by contacting the cells with specific cells.

[0035] In the size separation step of this embodiment, the cells obtained in the differentiation induction step are separated into differentiated cells and insufficiently differentiated cells. Differentiated cells refer to cells that have undergone appropriate differentiation and progressed to the desired stage. On the other hand, insufficiently differentiated cells refer to cells that have undergone differentiation but have not progressed sufficiently to the desired stage, and pre-differentiated cells that have not been induced to differentiate.

[0036] In this embodiment, cells whose size changes before and after differentiation can be used as the separation target. For example, cells whose size after differentiation is larger than their size before differentiation can be used as the separation target of the present invention. In this case, when comparing pre-differentiated cells, cells in the middle of differentiation (cells that have not fully differentiated), and differentiated cells (cells after differentiation), the cells in the middle of differentiation are larger in size than pre-differentiated cells, and differentiated cells are larger in size than cells in the middle of differentiation. However, cells whose size after differentiation is smaller than their size before differentiation can also be used as the separation target of the present invention.

[0037] In the size separation process, the particle suspension containing the cells obtained in the differentiation induction process is separated into particles with a size equal to or larger than a predetermined value and particles with a size smaller than a predetermined value. For example, a particle suspension (cell dispersion) is prepared by dispersing the particles containing the cells obtained in the differentiation induction process in a liquid such as a buffer solution. Note that in this embodiment, it is not necessary to attach antibody labels or fluorescent labels to the cells contained in the particle suspension.

[0038] The particle suspension contains cells, cell clumps, and cell-containing clumps obtained by the differentiation induction process as particles, and particles of a size equal to or greater than a predetermined value are mixed with particles of a size less than a predetermined value. This particle suspension is subjected to a process of separating particles of a size equal to or greater than a predetermined value as differentiated cells and particles of a size less than a predetermined value as cells that have not yet differentiated. Among differentiating cells, some cells change size before and after differentiation, while others change size due to aggregation or fusion of multiple cells. The size separation process in this embodiment separates particles based on size, regardless of the differentiation morphology.

[0039] In the size separation process, particles contained in the particle suspension are separated into particles having a size equal to or larger than a predetermined value and particles having a size smaller than the predetermined value based on a hydrodynamic effect. The hydrodynamic effect is an action such as resistance associated with the flow of a liquid. In this embodiment, a microchannel utilizing hydrodynamic laminar flow separation such as DLD (Deterministic Lateral Displacement), Dean flow, or pinched flow is used as the particle separation based on the hydrodynamic effect. By flowing the particle suspension through the microchannel, particles can be separated based on their size.

[0040] In this embodiment, utilizing the fact that cell size changes due to differentiation, for a particle suspension containing cells obtained by a differentiation induction step, particles of a size equal to or greater than a predetermined value can be separated as differentiated cells from the cells contained in the particle suspension based on the hydrodynamic effect, and particles of a size less than the predetermined value can be separated as insufficiently differentiated cells. For example, if insufficiently differentiated cells are removed by chemical treatment using a drug or the like, it is difficult to reuse the insufficiently differentiated cells, but if size separation based on the hydrodynamic effect is performed, the insufficiently differentiated cells can be recovered in a reusable state.

[0041] Furthermore, particle separation technology based on the hydrodynamic effect can easily separate large and small particles using a specific size boundary. In this case, simply by passing a particle suspension through a microchannel having a separation threshold, a liquid containing particles with a size equal to or greater than a predetermined value can be easily separated from a liquid containing particles with a size less than a predetermined value. Particle separation based on the hydrodynamic effect enables highly accurate separation of particles with a size equal to or greater than a predetermined separation threshold from particles with a size less than the separation threshold. Microchannels can be used, for example, in cartridge form, which has the advantages of being affordable and disposable, making them hygienic.

[0042] The predetermined value used as a criterion for separating differentiated cells from insufficiently differentiated cells can be set appropriately depending on the size of the cells before and after differentiation. Although the size of cells before and after differentiation varies depending on the type of cell, the predetermined value is preferably, for example, a value in the range of 5 μm to 100 μm, more preferably a value in the range of 5 μm to 30 μm, and even more preferably a value in the range of 10 μm to 20 μm.

[0043] Cells that are smaller than a predetermined size and separated as insufficiently differentiated cells in the size separation step may be differentiated again and then separated based on size to obtain differentiated cells. For example, a differentiation promotion step may be performed to promote the differentiation of insufficiently differentiated cells that have been separated as particles smaller than a predetermined size. The differentiation promotion step includes both a step of inducing differentiation of cells that have not been induced to differentiate (pre-differentiated cells) and a step of promoting differentiation of insufficiently differentiated cells.

[0044] In the differentiation progression step, insufficiently differentiated cells separated as particles with a size smaller than the predetermined value may be subjected to differentiation induction again under differentiation induction conditions. That is, the above-mentioned differentiation induction step may be performed again on insufficiently differentiated cells separated as particles with a size smaller than the predetermined value.

[0045] Furthermore, since insufficiently differentiated cells separated as particles smaller than a predetermined value have been treated in the differentiation induction step and differentiation has already begun, differentiation of the cells may proceed simply by placing them under conditions that promote differentiation. In such cases, in the differentiation progression step, differentiation of insufficiently differentiated cells separated as particles smaller than a predetermined value may be promoted under conditions that promote differentiation.

[0046] By performing a size separation step again on the cells obtained by the differentiation progression step, it is possible to separate differentiated cells from insufficiently differentiated cells. The size separation step performed here is a second size separation step (second size separation step), and it is possible to differentiate the insufficiently differentiated cells separated in the first size separation step (first size separation step) described above, and recover the differentiated cells.

[0047] The differentiated cells separated as particles having a size equal to or larger than a predetermined value in the size separation step can be used in various fields such as drug discovery, research, and regenerative medicine, but further separation based on size may also be carried out.

[0048] Differentiated cells separated as particles with a size equal to or larger than a predetermined value may change in size as differentiation progresses. For example, some differentiated cells increase in size as differentiation progresses, and cell size may correlate with the degree of differentiation.

[0049] For example, a differentiation process may be performed on cells separated as differentiated cells that are equal to or larger than a predetermined size, in which differentiation of the cells is promoted under conditions that promote differentiation that has already begun, and the cells obtained by the differentiation process may then be subjected to a size separation process again. The differentiation process performed in this case may be a process of inducing differentiation of cells that have not been induced to differentiate (pre-differentiation cells) (i.e., a second differentiation induction process), or a process of promoting differentiation of cells that have not yet progressed sufficiently. Note that the second differentiation induction process may involve differentiation induction of cells under different differentiation induction conditions than those used in the previous differentiation induction process.

[0050] The size separation step performed again here is a second size separation step (second size separation step), and uses a predetermined value (second predetermined value) greater than the predetermined value (first predetermined value) of the first size separation step (first size separation step) as a criterion to separate the cells obtained in the differentiation progression step into cells with sizes equal to or greater than the second predetermined value and cells with sizes less than the second predetermined value. This allows for the separation of differentiated cells corresponding to multiple stages of differentiation. Specifically, differentiated cells separated at a differentiation stage corresponding to the first predetermined value can be further separated based on a differentiation stage corresponding to the second predetermined value. Furthermore, after the differentiation progression step is further performed, a third size separation step may be performed based on a third predetermined value greater than the second predetermined value. There is no upper limit to the number of predetermined values ​​that can be used as criteria, and any number of predetermined values ​​can be set depending on the purpose.

[0051] In the above-described method, the differentiation progression step is performed on cells separated as differentiated cells with a size equal to or greater than a predetermined value, and the cells obtained by the differentiation progression step are then subjected to a size separation step. However, the size separation step may be performed without the differentiation progression step. In this case, a second size separation step based on a second predetermined value can be performed immediately after the first size separation step based on a first predetermined value, thereby separating the cells obtained by the differentiation induction step into cells with a size less than the first predetermined value, cells with a size equal to or greater than the first predetermined value but less than the second predetermined value, and cells with a size equal to or greater than the second predetermined value. An additional size separation step may be performed based on a third predetermined value greater than the second predetermined value. There is no upper limit to the number of predetermined values ​​that can be used as references, and any number of predetermined values ​​can be set depending on the purpose.

[0052] 1 is a flow diagram illustrating the method for producing differentiated cells according to this embodiment. As shown in FIG. 1, this embodiment involves a differentiation induction step (step S11) for inducing cell differentiation, followed by a size separation step (step S13) for separating the particle suspension containing cells obtained by the differentiation induction step into particles with a size equal to or greater than a predetermined value and particles with a size less than the predetermined value based on hydrodynamic effects. This separates the particle suspension into a liquid containing particles with a size equal to or greater than a predetermined value and a liquid containing particles with a size less than the predetermined value.

[0053] Furthermore, if necessary, a size separation step (second size separation step) may be performed again (step S15). In this case, the second size separation step may be performed on a liquid containing particles with a size equal to or greater than a predetermined value, or on a liquid containing particles with a size less than the predetermined value. Furthermore, a differentiation promotion step may be performed before performing the second size separation step. The differentiation promotion step performed in this case may be a step of inducing differentiation of cells that have not been induced to differentiate (pre-differentiation cells) (i.e., a second differentiation induction step), or a step of promoting differentiation of cells whose differentiation has not progressed sufficiently. Furthermore, if necessary, further size separation steps and differentiation promotion steps may be performed.

[0054] Thus, in the method for producing differentiated cells according to the present embodiment, cells obtained by differentiation induction are size-separated based on the hydrodynamic effect, allowing not only differentiated cells but also insufficiently differentiated cells to be recovered in a reusable state. This allows further differentiation progression steps (differentiation induction steps or steps for promoting differentiation that has already begun) and size separation steps to be carried out on the separated differentiated cells and insufficiently differentiated cells, respectively. By smoothly repeating the differentiation progression step and size separation step, the production rate (recovery rate) of differentiated cells can be significantly improved.

[0055] Furthermore, the method for producing differentiated cells according to this embodiment can use a microchannel capable of separating particles based on hydrodynamic effects. The microchannel can be implemented, for example, on a disposable chip measuring several centimeters square. Microchannel chips are small and inexpensive. Therefore, even when there are multiple samples from which differentiated cells must be separated, multiple microchannel chips can be prepared. By simply flowing particle suspensions prepared from each sample individually through the microchannels, differentiated cells can be easily separated from insufficiently differentiated cells. Thus, when separating differentiated cells, there is no need to aggregate each sample; each sample can be individually flowed through the microchannel for separation, preventing sample mix-ups and sample contamination.

[0056] In this embodiment, particle separation is performed based on the hydrodynamic effect, but particle separation may be performed in combination with other particle separation techniques. For example, particle separation may be performed with a low degree of accuracy using a flow cytometer (cell sorter), and then particle separation may be performed with a high degree of accuracy based on the hydrodynamic effect in the size separation step of this embodiment.

[0057] Next, a particle separation device that can be used in the size separation process of this embodiment will be described. As described above, in the size separation process of this embodiment, particle separation techniques that utilize hydrodynamic effects, such as techniques that utilize microchannels, such as DLD, Dean flow, and pinched flow, can be used, and DLD microchannels can be particularly preferably used.

[0058] The configuration of a particle separation device that can be used in the size separation step in this embodiment will be described with reference to Fig. 2. Fig. 2 is a diagram showing an example of the configuration of the particle separation device in this embodiment.

[0059] 2 is generally configured to include a pressure generating unit 20 that discharges compressed air to a particle suspension storage unit 30 and a buffer solution storage unit 40, a particle suspension storage unit 30, a buffer solution storage unit 40, a DLD microchannel chip (separation unit) 50, a particle suspension recovery unit 60, and a buffer solution recovery unit 70. In the following description, the pressure generating unit 20 side is defined as the upstream side, and the particle suspension recovery unit 60 side and the buffer solution recovery unit 70 side are defined as the downstream side, based on the flow directions of the compressed air, particle suspension, and buffer solution.

[0060] Pressure generating unit 20 is configured to discharge compressed air. The compressed air discharged from pressure generating unit 20 is sent to particle suspension storage unit 30 and buffer solution storage unit 40 through compressed air flow pipe 21, branch pipe 22, tube 23, and tube 24.

[0061] The pressure generating unit 20 is not particularly limited as long as it has the function of discharging compressed air, and may be, for example, a reciprocating air compressor equipped with a motor and a cylinder, or may be a rotary, centrifugal, axial flow, etc. The pressure generating unit 20 may be configured to maintain the pressure of the compressed air within a predetermined pressure range by feedback control, and may be configured to stop compressing the air when the operating time exceeds a predetermined time or based on the measurement results of the flow rate of the particle suspension or buffer solution.

[0062] The branch pipe 22 branches the compressed air flow pipe (first pipe line) 21 into two branches. A tube (second pipe line) 23 is connected to one of the branches, and a tube (third pipe line) 24 is connected to the other of the branches.

[0063] An adapter 23a is attached to the downstream end of the tube 23. The adapter 23a is detachably attached to the inlet of the barrel 31 of the particle suspension container 30.

[0064] An adapter 24a is attached to the downstream end of the tube 24. The adapter 24a is detachably attached to the inlet of the barrel 41 of the buffer solution storage section 40.

[0065] The particle suspension storage unit 30 includes a cylindrical barrel 31 having an inlet and an outlet. The barrel 31 stores a particle suspension containing cells obtained by the differentiation induction step.

[0066] The barrel 31 is supported with its longitudinal direction set approximately vertically so that the inlet is located at the top and the outlet is located at the bottom. The particle suspension is injected through the inlet of the barrel 31 with the adapter 23 a removed, and after injection, the adapter 23 a is airtightly attached to the inlet of the barrel 31.

[0067] The buffer solution storage section 40 includes a cylindrical barrel 41 having an inlet and an outlet. The barrel 41 stores the buffer solution.

[0068] Barrel 41 is supported with its longitudinal direction set approximately vertically so that the inlet is located at the top and the outlet is located at the bottom, similar to barrel 31. With adapter 24a removed from the inlet of barrel 41, the buffer solution is injected, for example, in an amount equal to the particle suspension. After injection, adapter 24a is airtightly attached to the inlet of barrel 41.

[0069] When adapter 23a of tube 23 is airtightly attached to barrel 31 and adapter 24a of tube 24 is airtightly attached to barrel 41, the internal space communicating from compressed air flow pipe 21 to barrel 31 and barrel 41 via branch pipe 22 becomes equal-pressure and airtight. When compressed air is discharged from pressure generating unit 20 in this state, the air pressure in the airtight internal space increases uniformly. As a result, air pressure acts on the particle suspension contained in barrel 31 of particle suspension container 30 via tube 23, and the particle suspension contained in barrel 31 of particle suspension container 30 is discharged from the outlet. Similarly, air pressure acts on the buffer solution contained in barrel 41 of buffer solution container 40 via tube 24, and the buffer solution contained in barrel 41 of buffer solution container 40 is discharged from the outlet.

[0070] The DLD microchannel chip 50 is supported by a leak-proof chip holder or the like, and as shown in Figure 3, is roughly configured to include a particle suspension inlet 51, a buffer solution inlet 52, a particle suspension outlet 53, a buffer solution outlet 54, and a DLD channel section 55 having a microchannel.

[0071] The particle suspension inlet 51, the buffer solution inlet 52, the particle suspension outlet 53, and the buffer solution outlet 54 are each connected to the DLD flow path section 55. The particle suspension inlet 51 is an inlet for allowing the particle suspension to flow into the DLD flow path section 55. The buffer solution inlet 52 is an inlet for allowing the buffer solution to flow into the DLD flow path section 55. The particle suspension outlet 53 is an outlet for allowing the particle suspension that has flowed and moved within the DLD flow path section 55 to flow out of the DLD flow path section 55. The buffer solution outlet 54 is an outlet for allowing the buffer solution that has flowed and moved within the DLD flow path section 55 to flow out of the DLD flow path section 55.

[0072] The particle suspension inlet 51 is connected to the outlet of the barrel 31 of the particle suspension storage unit 30 via a tube 81. The buffer solution inlet 52 is connected to the outlet of the barrel 41 of the buffer solution storage unit 40 via a tube 82. The DLD channel unit 55 has a DLD microchannel structure in which a plurality of fine pillars are arranged. It is desirable that the DLD channel unit 55, the channel from the outlet of the barrel 31 of the particle suspension storage unit 30 to the tube 81, the particle suspension inlet 51, and the DLD channel unit 55, and the channel from the outlet of the barrel 41 of the buffer solution storage unit 40 to the tube 82, the buffer solution inlet 52, and the DLD channel unit 55 be filled with the buffer solution in advance.

[0073] When the air pressure increased by the discharge of compressed air by the pressure generating unit 20 acts, the particle suspension in the barrel 31 of the particle suspension storage unit 30 is introduced from the outlet through the tube 81 and the particle suspension inlet 51 into the DLD flow path unit 55, and the buffer solution in the barrel 41 of the buffer solution storage unit 40 is introduced from the outlet through the tube 82 and the buffer solution inlet 52 into the DLD flow path unit 55.

[0074] DLD flow path section 55 has a plurality of fine pillars (micropillars) arranged based on the principle of the DLD method as described in, for example, Non-Patent Document 1. The particle suspension introduced through particle suspension inlet 51 and the buffer solution introduced through buffer solution inlet 52 flow in parallel as laminar flows while in contact with each other within DLD flow path section 55, and move in the directions of particle suspension outlet 53 and buffer solution outlet 54.

[0075] According to the DLD method, when a particle dispersion is flowed through a group of pillars P arranged according to a predetermined rule, as shown in Figure 4, small particles move along the direction of the flow, while large particles move obliquely relative to the direction of the flow because their movement along the direction of the flow is hindered by the presence of the pillars P. By appropriately setting a threshold (separation threshold) determined by the spacing G of the pillars P and the shift amount d, it is possible to separate particles with a size below the separation threshold from particles with a size equal to or greater than the separation threshold.

[0076] In the DLD flow path section 55, particles contained in the particle suspension and having a size equal to or larger than the separation threshold advance obliquely with respect to the flow direction of the particle suspension and move into the buffer solution, which flows in parallel with the particle suspension as a laminar flow while contacting the particle suspension. As a result, the buffer solution containing particles that have moved from the particle suspension to the buffer solution is discharged from the buffer solution outlet 54. Meanwhile, in the DLD flow path section 55, particles contained in the particle suspension and having a size smaller than the separation threshold advance along the flow direction of the particle suspension and are discharged from the particle suspension outlet 53 together with the particle suspension.

[0077] In this way, particles contained in the particle suspension introduced into the DLD microchannel are separated by the action of a hydrodynamic effect as they pass through the DLD channel section 55. Specifically, particles with a size smaller than a predetermined value (separation threshold) pass through the DLD channel section 55 and are discharged from the particle suspension outlet 53, and particles with a size equal to or larger than the predetermined value (separation threshold) pass through the DLD channel section 55 and are discharged from the buffer solution outlet 54.

[0078] In the DLD flow path section 55, the particle suspension and the buffer solution flow in parallel as laminar flows while contacting each other and slightly mix with each other. Therefore, the particle suspension discharged from the particle suspension outlet 53 may contain a portion of the buffer solution, and the buffer solution discharged from the buffer solution outlet 54 may also contain a portion of the particle suspension.

[0079] The DLD micro-channel chip 50 has a layered structure in which, for example, a particle suspension inlet 51, a buffer solution inlet 52, a particle suspension outlet 53, a buffer solution outlet 54, and a DLD channel section 55 are formed in a channel-processed chip, and a flat chip having holes for allowing liquid to flow at positions corresponding to the particle suspension inlet 51, the buffer solution inlet 52, the particle suspension outlet 53, and the buffer solution outlet 54 is bonded onto the channel-processed chip. That is, the DLD micro-channel chip 50 is configured so that the particle suspension or the buffer solution can flow through the particle suspension inlet 51, the buffer solution inlet 52, the particle suspension outlet 53, and the buffer solution outlet 54, respectively, via holes formed on one side of the DLD micro-channel chip 50. The DLD micro-channel chip 50 is known, available at an affordable price, and is also hygienic because it is disposable. Furthermore, the DLD micro-channel chip 50 may be selected depending on the size of the cells before and after differentiation, and it is particularly preferable to select a DLD micro-channel chip 50 having a separation threshold that coincides with a predetermined value that serves as a criterion for separating differentiated cells from cells that are not sufficiently differentiated. For example, a DLD micro-channel chip 50 having a separation threshold of 10 μm or 20 μm can be used.

[0080] Particle suspension recovery unit 60 is connected to particle suspension outlet 53 via tube 83. The particle suspension that passes through DLD flow path 55 and is discharged from particle suspension outlet 53 is recovered in particle suspension recovery unit 60 through outlet 83d of tube 83. The particle suspension recovered in particle suspension recovery unit 60 is a particle suspension from which particles with a size equal to or larger than a predetermined value (separation threshold) have been removed, and which also contains particles with a size smaller than the predetermined value (separation threshold).

[0081] The buffer solution outlet 54 is connected to the buffer solution recovery unit 70 via a tube 84. The buffer solution discharged from the buffer solution outlet 54 through the DLD flow path unit 55 is recovered in the buffer solution recovery unit 70 through an outlet 84d of the tube 84. The buffer solution recovered in the buffer solution recovery unit 70 is a buffer solution into which particles having a size equal to or larger than a predetermined value (separation threshold) have migrated, and contains particles having a size equal to or larger than the predetermined value (separation threshold).

[0082] The DLD micro-channel chip 50 may be formed into a cartridge, for example, a cartridge that integrates the barrel 31, the barrel 41, the tube 81, the tube 82, the DLD micro-channel chip 50, the tube 83, and the tube 84. By forming a configuration including the DLD micro-channel chip 50 into a cartridge in this way, the DLD micro-channel can be easily handled and can also be made disposable.

[0083] 2 shows only one DLD micro-channel chip 50, but it is also possible to connect two or more DLD micro-channel chips 50 in series. For example, by arranging the DLD micro-channel chips 50 so that the separation threshold (second predetermined value) of the downstream DLD micro-channel chip 50 is larger than the separation threshold (first predetermined value) of the upstream DLD micro-channel chip 50, particles can be separated into those with a size less than the first predetermined value, those with a size equal to or greater than the first predetermined value but less than the second predetermined value, and those with a size equal to or greater than the second predetermined value.

[0084] Examples of the present invention will be described below. In Examples 1 to 3 below, size separation of pre-differentiated and post-differentiated cells was performed using a DLD microchannel chip based on the size change before and after differentiation. For size separation, the particle separation device 10 shown in FIG. 2 was used.

[0085] Example 1: Recovery of myotube cells differentiated from mouse myoblasts In Example 1, mouse myoblasts (C2C12 cells) were used. C2C12 cells can be induced to differentiate into myotube cells. C2C12 cells fuse with each other through differentiation induction, and change into multinucleated myotube cells (muscle fibers) with a tubular structure. Compared to the size of C2C12 cells before differentiation, the size of myotube cells after differentiation is larger. Here, we examined whether it is possible to accurately separate and recover differentiated cells by utilizing the fact that the size of C2C12 cells increases with cell maturation due to differentiation induction.

[0086] In Example 1, C2C12 cells were seeded and cultured until confluent, followed by a differentiation induction step under differentiation induction conditions. Four days or more after the start of differentiation induction, cells containing differentiated myotubes were detached from the culture dish using trypsin, and the detached cells were dispersed in a liquid to prepare a particle suspension. The particle suspension was then subjected to a size separation step using a particle separation device 10 equipped with a DLD microchannel chip 50 having a separation threshold Dc of 20 μm.

[0087] The particle separation device 10 is configured to discharge a discharge liquid containing particles of a size equal to or larger than the separation threshold Dc (here, 20 μm) from an outlet (outlet 84d of the particle separation device 10 shown in Figure 2), and to discharge a discharge liquid containing particles of a size smaller than the separation threshold Dc from an outlet (outlet 83d of the particle separation device 10 shown in Figure 2).

[0088] The C2C12 cells used in Example 1 are cells with a size less than the separation threshold Dc (= 20 μm), while the differentiated cells (myotube cells) obtained from the C2C12 cells by the differentiation induction step are cells with a size equal to or greater than the separation threshold Dc (= 20 μm). That is, cells before differentiation are smaller in size than the separation threshold Dc, while differentiated cells change to be larger in size than the separation threshold Dc. Therefore, ideally, cells before differentiation should be able to be separated by size as particles with a size less than the separation threshold Dc, and differentiated cells should be able to be separated by size as particles with a size equal to or greater than the separation threshold Dc.

[0089] FIG. 5 is a photograph of the liquid collected through outlet 84d (a liquid containing particles with a size equal to or greater than the separation threshold Dc), and FIG. 6 is a photograph of the liquid collected through outlet 83d (a liquid containing particles with a size less than the separation threshold Dc). In FIGS. 5 and 6, the cell shapes are shown in bright field, and the cell nuclei are shown as merged images of SYBR® Green-stained fluorescent images. The scale bars in the lower right corners of FIGS. 5 and 6 indicate a length of 0.1 mm. In the particle separation device 10 used in Example 1, outlet 84d for particles with a size equal to or greater than the separation threshold Dc is located on the left side of the device, and outlet 83d for particles with a size less than the separation threshold Dc is located on the right side of the device. The "left side" in FIG. 5 and the "right side" in FIG. 6 represent the positions of the outlets from which the liquid is discharged.

[0090] The liquid collected through outlet 84d (see FIG. 5) contains particles with a size equal to or larger than the separation threshold Dc. The liquid collected through outlet 83d (see FIG. 6) does not contain particles with a size equal to or larger than the separation threshold Dc. In other words, differentiated cells should ideally be able to be collected through outlet 84d.

[0091] The number of nuclei in the cells contained in the liquid recovered through outlet 84d (see Figure 5) and the number of nuclei in the cells contained in the liquid recovered through outlet 83d (see Figure 6) were actually investigated, and the results are shown in Figure 7.

[0092] In Figure 7, the horizontal axis represents classes divided by the number of nuclei in the cells, and the vertical axis represents the proportion of the number of nuclei contained in each class (number of nuclei in the cells × number of cells × 100 ÷ total number of nuclei). The reason for focusing on the number of nuclei is that it corresponds to the number of cells before fusion. The liquid recovered through outlet 84d (see Figure 5) is represented by open bars, and the liquid recovered through outlet 83d (see Figure 6) is represented by filled bars.

[0093] The number of nuclei in each of 85 cells contained in the liquid recovered through outlet 84d (see Figure 5) was counted. As a result, as shown in Figure 7, the proportion of nuclei in cells with only one nucleus (unfused cells) was 27%, and the proportion of nuclei in cells with two or more nuclei (fused cells) was 73%.

[0094] On the other hand, the number of nuclei in each of 86 cells contained in the liquid recovered through outlet 83d (see Figure 6) was counted. As a result, as shown in Figure 7, the proportion of nuclei in cells with only one nucleus (unfused cells) was 90%, and the proportion of nuclei in cells with two or more nuclei (fused cells) was 10%.

[0095] As described above, when particles with a size equal to or larger than the separation threshold Dc and particles with a size smaller than the separation threshold Dc are separated from the particle suspension obtained by the differentiation induction process based on the hydrodynamic effect, it was found that the suspension can be appropriately separated into a liquid in which the proportion of nuclei in fused cells (differentiated cells) with two or more nuclei is only 10% (only 10% of the pre-fusion cells have fused) and a liquid in which the proportion of nuclei in fused cells (differentiated cells) with two or more nuclei is 73% (73% of the pre-fusion cells have fused).

[0096] Both the cells contained in the liquid recovered through outlet 84d (see FIG. 5) and the cells contained in the liquid recovered through outlet 83d (see FIG. 6) can be recovered without chemical treatment using chemicals. This allows, for example, the insufficiently differentiated cells contained in the liquid recovered through outlet 83d (see FIG. 6) to be reused and re-cultured for differentiation induction or differentiation promotion treatment. In this way, differentiated cells can be extracted from the particle suspension and re-recovered insufficiently differentiated cells. By reusing the re-recovered insufficiently differentiated cells, the number of recovered differentiated cells can be increased.

[0097] (Example 2: Recovery of adipocytes differentiated from mouse embryonic fibroblasts) In Example 2, mouse embryonic fibroblasts were used. Mouse embryonic fibroblasts can be induced to differentiate into adipocytes. Mouse embryonic fibroblasts accumulate lipid droplets during differentiation induction to become adipocytes. Compared to the size of mouse embryonic fibroblasts, which are cells before differentiation, the size of adipocytes, which are cells after differentiation, is larger. Here, by utilizing the fact that size increases upon differentiation induction, as in mouse embryonic fibroblasts, it was verified whether it was possible to accurately separate and recover differentiated cells.

[0098] In Example 2, mouse embryonic fibroblasts were seeded and cultured until confluent, after which a differentiation induction step was performed by adding isobutylmethylxanthine, insulin, dexamethasone, and bexarotene. One week after the start of differentiation induction, cells containing adipocytes that had accumulated lipid droplets through differentiation were detached from the culture dish, and the detached cells were dispersed in a liquid to prepare a particle suspension. The particle suspension was then subjected to a size separation step using a particle separation device 10 equipped with a DLD microchannel chip 50 having a separation threshold Dc of 20 μm.

[0099] The particle separation device 10 is configured to discharge a discharge liquid containing particles of a size equal to or larger than the separation threshold Dc (here, 20 μm) from an outlet (outlet 84d of the particle separation device 10 shown in Figure 2), and to discharge a discharge liquid containing particles of a size smaller than the separation threshold Dc from an outlet (outlet 83d of the particle separation device 10 shown in Figure 2).

[0100] The mouse embryonic fibroblasts used in Example 2 are cells with a size less than the separation threshold Dc (= 20 μm), while the differentiated cells (adipocytes) obtained from the mouse embryonic fibroblasts by the differentiation induction step are cells with a size equal to or greater than the separation threshold Dc (= 20 μm). That is, cells before differentiation are smaller in size than the separation threshold Dc, while differentiated cells change to be larger in size than the separation threshold Dc. Therefore, ideally, cells before differentiation should be able to be separated by size as particles with a size less than the separation threshold Dc, and differentiated cells should be able to be separated by size as particles with a size equal to or greater than the separation threshold Dc.

[0101] FIG. 8 is a photograph of the liquid recovered through outlet 84d (liquid containing particles of a size equal to or greater than the separation threshold Dc), and FIG. 9 is a photograph of the liquid recovered through outlet 83d (liquid containing particles of a size less than the separation threshold Dc). The scale bars at the bottom right of FIGS. 8 and 9 indicate a length of 0.1 mm. In the particle separation device 10 used in Example 2, outlet 84d for particles of a size equal to or greater than the separation threshold Dc is located on the left side of the device, and outlet 83d for particles of a size less than the separation threshold Dc is located on the right side of the device. The "left side" in FIG. 8 and the "right side" in FIG. 9 respectively represent the positions of the outlets from which the liquid is discharged.

[0102] The liquid collected through outlet 84d (see FIG. 8) contains particles with a size equal to or larger than the separation threshold Dc. The liquid collected through outlet 83d (see FIG. 9) does not contain particles with a size equal to or larger than the separation threshold Dc. In other words, differentiated cells that contain many lipid droplets and have increased in size should ideally be able to be collected through outlet 84d.

[0103] The results of investigating the diameter of cells and the number of lipid droplets contained in the liquid recovered through outlet 84d (see Figure 8), and the diameter of cells and the number of lipid droplets contained in the liquid recovered through outlet 83d (see Figure 9) are shown in Figure 10.

[0104] In Figure 10, the horizontal axis represents the measured cell diameter, and the vertical axis represents the number of lipid droplets in the cells. The liquid collected through outlet 84d (see Figure 8) is plotted as open dots, and the liquid collected through outlet 83d (see Figure 9) is plotted as filled dots.

[0105] Of the cells contained in the liquid collected through outlet 84d (see FIG. 8), the diameter of each of 11 cells was measured and the number of lipid droplets within the cells was counted. As a result, as shown in FIG. 10, the liquid collected through outlet 84d (see FIG. 8) contained cells with a large size equal to or greater than the separation threshold Dc (= 20 μm) and a large number of lipid droplets. As shown by the open star marks in FIG. 10, the average cell diameter of the 11 cells was 32 μm, and the average number of lipid droplets was 15.

[0106] On the other hand, among the cells contained in the liquid collected through the outlet 83d (see FIG. 9), the diameter of each of 12 cells was measured and the number of lipid droplets within the cells was counted. As a result, as shown in FIG. 10, the liquid collected through the outlet 83d (see FIG. 9) contained cells with a small size below the separation threshold Dc (= 20 μm) and a small number of lipid droplets. As shown by the black star mark in FIG. 10, the average cell diameter of the 12 cells was 13 μm, and the average number of lipid droplets was 5.

[0107] As described above, when the particle suspension obtained by the differentiation induction process was separated into particles of a size equal to or larger than the separation threshold Dc and particles of a size smaller than the separation threshold Dc based on hydrodynamic effects, the number of lipid droplets contained in cells of a size equal to or larger than the separation threshold Dc was significantly greater than the number of lipid droplets contained in cells of a size smaller than the separation threshold Dc (p<0.05), and it was found that the liquid could be appropriately separated into a liquid containing cells with a small number of lipid droplets and insufficient differentiation and a liquid containing cells with a large number of lipid droplets and advanced differentiation (differentiated cells).

[0108] Both the cells contained in the liquid recovered through outlet 84d (see FIG. 8) and the cells contained in the liquid recovered through outlet 83d (see FIG. 9) can be recovered without chemical treatment using chemicals. This allows, for example, the insufficiently differentiated cells contained in the liquid recovered through outlet 83d (see FIG. 9) to be reused and re-cultured for differentiation induction or differentiation promotion treatment. In this way, differentiated cells can be extracted from the particle suspension and re-recovered insufficiently differentiated cells. By reusing the re-recovered insufficiently differentiated cells, the number of recovered differentiated cells can be increased.

[0109] (Example 3: Re-culturing after size separation step) In Example 3, it was confirmed that separated cells could be re-cultured using mouse myoblasts (C2C12 cells). Figure 11 is a photograph of cells four days after differentiation induction, and Figure 12 is a photograph of the liquid recovered through outlet 83d (liquid containing particles with a size below the separation threshold Dc) two days after cell culture. The scale bars in the lower right corners of Figures 11 and 12 indicate a length of 0.1 mm.

[0110] As in Example 1, cells containing differentiated myotubes (see FIG. 11 ) were detached from the culture dish using trypsin four days or more after the start of differentiation induction. The detached cells were dispersed in a liquid to prepare a particle suspension. The particle suspension was then subjected to a size separation process using a particle separation device 10 equipped with a DLD microchannel chip 50 having a separation threshold Dc of 20 μm. Cells that were not sufficiently differentiated and recovered as particles with sizes below the separation threshold Dc were primarily recovered. These cells could be reused and cultured again (see FIG. 12 ).

[0111] The above-described embodiments have been described to facilitate understanding of the present invention, and are not intended to limit the present invention. Therefore, each element disclosed in the above-described embodiments is intended to include all design modifications and equivalents that fall within the technical scope of the present invention.

[0112] DESCRIPTION OF SYMBOLS 10 Particle separation device 20 Pressure generation unit 21 Compressed air flow pipe (first pipe) 22 Branch pipe 23 Tube (second pipe) 24 Tube (third pipe) 23a, 24a Adapter 30 Particle suspension storage unit 31, 41 Barrel 40 Buffer solution storage unit 50 DLD microchannel chip (separation unit) 51 Particle suspension inlet 52 Buffer solution inlet 53 Particle suspension outlet 54 Buffer solution outlet 55 DLD channel unit 60 Particle suspension recovery unit 70 Buffer solution recovery unit 81, 82, 83, 84 Tubes 83d, 84d Outlet

Claims

1. A method for producing differentiated cells, comprising: a differentiation induction step of inducing cell differentiation; and a size separation step of separating particles having a size equal to or greater than a predetermined value and particles having a size less than the predetermined value from a particle suspension containing the cells obtained by the differentiation induction step based on a hydraulic effect, wherein each of the steps is performed one or more times.

2. The method for producing differentiated cells according to claim 1, wherein in the size separation step, size separation is performed by a DLD microchannel.

3. In the size separation step, size separation is performed using a particle separation device, the particle separation device comprising: a pressure generating unit for discharging gas; a particle suspension storage unit having an inlet and an outlet into which the particle suspension is injected; a buffer solution storage unit having an inlet and an outlet into which a buffer solution is injected; a branch pipe including a first pipe connected to the outlet of the pressure generating unit, a second pipe which is one of the bifurcations from the first pipe and is connected to the inlet of the particle suspension storage unit, and a third pipe which is the other of the bifurcations from the first pipe and is connected to the inlet of the buffer solution storage unit; a DLD flow path unit having a DLD microchannel structure in which a plurality of fine pillars are arranged such that a particle suspension introduced through the particle suspension inlet and a buffer solution introduced through the buffer solution inlet flow in parallel while contacting each other, an outlet for discharging the buffer solution containing particles having a size equal to or greater than the predetermined value that have moved from the particle suspension to the buffer solution in the DLD flow path unit, and an outlet for discharging the particle suspension after the particles having a size equal to or greater than the predetermined value have moved to the buffer solution in the DLD flow path unit; and a DLD microchannel chip. The method for producing differentiated cells according to claim 2, characterized by having the above components.

4. A differentiation progression step of promoting the differentiation of the cells separated as particles having a size less than the predetermined value in the size separation step; and a second size separation step of separating particles having a size equal to or greater than the predetermined value and particles having a size less than the predetermined value from a particle suspension containing the cells obtained by the differentiation progression step based on a hydraulic effect. The method for producing differentiated cells according to any one of claims 1 to 3, characterized by performing the above steps.

5. A differentiation progression step of promoting the differentiation of cells separated as particles having a size equal to or greater than the predetermined value in the size separation step; and a second size separation step of separating, from the particle suspension containing the cells obtained by the differentiation progression step, particles having a size equal to or greater than a second predetermined value greater than the predetermined value and particles having a size less than the second predetermined value based on a hydraulic effect. The method for producing differentiated cells according to any one of claims 1 to 3, characterized by performing the steps.

6. The method for producing differentiated cells according to any one of claims 1 to 3, characterized in that the predetermined value is 5 μm or more and 100 μm or less.

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