JP6739739B2 - Particle sorting method and particle sorting apparatus for carrying out the method - Google Patents

Description

The present invention relates to a particle sorting apparatus and a particle sorting method, and more specifically, a particle sorting apparatus capable of sorting a large number of particles accurately, quickly, simply and simply in accordance with the particle size, the degree of aggregation and the density. And a method for separating particles.

Various techniques have been proposed for separating particles according to various factors such as density, agglomeration state, and particle size.
For example, in Patent Document 1, as a technique utilizing the difference in the sedimentation velocity of particles, a first coagulation tank into which raw water 11 and a coagulant are introduced and a downstream of the first coagulation tank are placed in communication with each other. A second flocculation tank having a larger internal volume than the first flocculation tank, a settling separation tank for separating flocs grown in the second coagulation tank, and a final settling for calming the treatment liquid separated in the settling separation tank An apparatus has been proposed in which at least four tanks are integrally formed.
Further, in Patent Document 2, as a technique utilizing centrifugal force, a base, a disk-shaped container that rotates on the base, and a suspension supply tank are provided, and the disk-shaped container surrounds the center of rotation. A plurality of fan-shaped centrifuge tanks arranged at equal intervals in the circumferential direction, and a particle supply tube, and the plurality of fan-shaped centrifuge tanks are each formed of a peripheral side wall and a bottom wall and are formed as recesses independent of each other. The particle supply cylinder is provided so as to discharge the suspension from the center of the disk-shaped container toward the fan-shaped centrifugal separation tank, and the suspension is supplied from the suspension supply tank. An apparatus has been proposed in which the particles contained therein are circumferentially partitioned on the inner surface of the outer wall of the centrifuge tank and are configured to be separated into a plurality of particle collection pockets.

JP, 2000-140510, A JP, 2006-239678, A

Enosawa, S.; et.al., Hepatocyte transplantation using the living donor reduced-graft in a baby with ornithine transcarbamylase deficiency: A novel source for hepatocytes. Liver Transplantation. 20,391-393, 2014 Sandi Sufiandi, Hiromichi Obara, Shin Enosawa, et.al., Improvement of infusion process in cell transplantation: Effect of shear stress on hepatocyte viability under horizontal and vertical syringe orientation., Cell Medicine, Vol. 7, pp. 59-66, 2015

However, the proposals in Patent Documents 1 and 2 have a problem that the sorting operation is still complicated and the sorting cannot be performed at a sufficiently required level.
For example, separation of cell particles as particles is also strongly demanded in the industry. The reason for this is that due to the development of regenerative medicine technology and the progress of immunological research, there is a high expectation for cell therapy in which bone marrow, pancreatic islets, and liver cells are transplanted into the body through blood vessels. In particular, regarding hepatocyte transplantation, the first clinical research report in Japan as a treatment for newborn infants with congenital liver disease has been made (Non-patent Document 1), and in the future as a development to ES cell/iPS cell utilization. Expectations are high. However, compared to the cancer cell-derived experimental cells used for research, the cells used for cell transplantation are extremely weak and the available amount of cells is limited.
Particularly in Japan, islets are used for transplantation medical treatment by separating cells from incompatible organs of pancreas transplantation and liver parenchymal cells from residual liver at the time of living liver transplantation for infants, and securing highly functional cells with high efficiency. Is a big issue. Therefore, it is necessary to separate the cell particles. Usually, the cell particles for cell transplantation are obtained by perfusing a tissue-degrading enzyme solution into the excised organ, dissolving it, and dispersing/extracting it. However, many cells lose their functions and are lost in this series of steps, and it is also very useful when the cell dispersion is transplanted together with the infusion solution from the portal vein, which is one of the inflowing blood vessels of the liver of the transplant destination, using the tubule. It is known that many cells are lost (Non-Patent Document 2). Against this background, it is important how to maintain the function of cells and deal with them without destroying or losing them, and how to separate cell particles that have lost function from functional cell particles. It is important to develop technology from such a viewpoint, and to overcome these problems, in particular, to gently and positively continuously operate the cell dispersion liquid to obtain functional cell particles and non-functional cell particles. It is required to establish the basic technology for efficient and efficient sorting.
From the above viewpoints, there has been a demand for the development of a particle separation method capable of separating a large number of particles accurately, quickly, simply and simply according to the particle size and the degree of aggregation.

Therefore, an object of the present invention is to provide a particle sorting method and a sorting apparatus capable of sorting a large number of particles accurately, quickly, simply and simply according to their density, particle size and degree of aggregation. is there.

As a result of intensive studies to solve the above problems, the present inventors have found that a liquid-liquid formed between a particle-dispersed fluid such as a cell culture solution containing a large number of cell particles and a fluid having a high density for cells/solutions. It is thought that the interface problem can be solved by utilizing the interface, and as a result of various studies on the method of utilizing the liquid-liquid interface, the two-phase fluid induced by the liquid-liquid two-phase fluid having the liquid-liquid interface is induced in the curved pipe. It was found that the above object can be achieved without destroying the cell particles by positively using the secondary flow, and the present invention has been completed.
That is, the present invention provides each of the following inventions.
1. A particle sorting apparatus for sorting a large number of particles,
A large number of particles described above, the first flow liquid, and a second flow liquid insoluble in the first flow liquid and having different densities can be added, and have a predetermined ratio of these particles for transfer. A transfer preparation unit that holds the liquid for transfer,
A flow passage for circulating the transfer liquid, the flow passage being a curved body having a predetermined curved shape, and a separation unit connected to the flow passage, and the separated particles are collected for each separation section. A particle sorting apparatus having a collecting unit.
2. A particle separation method for separating a large number of particles,
A transfer preparation step for adjusting a transfer liquid having a large number of particles, a first flow liquid, and a second flow liquid insoluble in the first flow liquid and having different densities at a predetermined ratio. ,
A separation step of circulating the transfer liquid in a flow path which is a tubular body having a curved shape having a predetermined curved shape for separating the particles, and a separation step of separating particles. A method for separating particles, comprising a collecting step of collecting particles for each separation section.
3. The particles are cell particles, and the sorting step is a step of sorting the cell particles into living cells and dead cells, and the cells are sorted according to a difference in density and/or a difference in particle size. The method for separating particles according to 2, which is a step.

The sorting apparatus of the present invention is capable of sorting a large number of particles accurately, quickly, simply and simply according to the particle size and the degree of aggregation.
Further, according to the fractionation method of the present invention, a large number of particles can be fractionated accurately, quickly, simply and simply according to the particle size and the degree of aggregation.

FIG. 1 is a schematic diagram showing a main part of the particle sorting apparatus of the present invention. FIG. 2 is an enlarged view of the II portion of FIG. FIG. 3 is a diagram (internal cross-sectional view) schematically showing a separation step, (a) shows a state before separation, (b) shows an initial separation, (c) shows a middle separation period, (D) shows the end of separation. FIG. 4 is a graph showing the measurement results of particle distribution in the particle sorting apparatus, and (a) is a graph showing the result of particle distribution at each position when the particle diameter dp=70 μm and the radius of curvature R=98 mm. (B) is a graph showing the results at De=50 using a main part having a radius of curvature R=98 mm with a particle size dp=31 and a particle size of 70 μm.

1: Particle sorting device, 10: Transfer preparation part, 20: Separation part, 50 Transfer liquid

Hereinafter, the present invention will be described in more detail based on the embodiments.
<Overall structure>
The particle sorting device of the present embodiment is, as shown in FIG. 1, a particle sorting device that sorts a large number of particles, and the large number of particles described above, the first liquid flow, and the first liquid flow. A second liquid solution that is insoluble and can have different densities can be added, and a transfer preparation unit that holds the second liquid solution in a predetermined ratio to transfer it, and a flow passage through which the second liquid solution flows In addition, the flow passage includes a sorting unit that is a curved body having a predetermined curved shape, and a collecting unit that is connected to the flow passage and that collects the sorted particles for each sorting section.
Further description will be given below. The particles, the first liquid flow, the second liquid flow, and the transfer liquid will be described later.

<Transfer preparation section>
The transfer preparation unit puts the particles, the first flowing liquid, and the second flowing liquid into a container configured by one chamber or a pipe line to form a liquid-liquid interface as a transfer liquid. It is a part that forms a liquid-liquid two-phase fluid.
In the present embodiment, as shown in FIG. 1, the first flowing liquid supply pipe and the second flowing liquid supply pipe arranged at a predetermined angle with respect to the first flowing liquid supply pipe are both the same. It is composed of a transfer pipe, which is connected at one place and which transfers the above-mentioned first liquid stream and the above-mentioned second liquid stream together as a liquid for transfer to a separation section.
The first flowing liquid supply pipe, the second flowing liquid supply pipe, and the transfer pipe are connected at a predetermined angle as shown in FIG. 2, but the first flowing liquid supply pipe and the transfer pipe are connected. And the angle θ2 between the second flowing liquid supply pipe and the transfer pipe are the same in the present embodiment. In this embodiment, θ1=θ2=θ3=120°. Further, in the present embodiment, each tube is arranged on one plane so that it can be seen as one tube when viewed in the direction of FIG. 1, but it is not limited to this and three-dimensional angles are provided. May be. Further, the angles may not be the same as in the present embodiment, but may be set to different angles.
The pipe diameters of the first flowing liquid supply pipe, the second flowing liquid supply pipe, and the transfer pipe are not particularly limited, but in the present embodiment, they are all the same. It is possible to form a thick transfer pipe by matching the Dean number that defines the magnitude of the secondary flow velocity, and it is also possible to use any pipe diameter by combining the flow rates. For simplicity, all pipe diameters are the same. In this embodiment, each has an inner diameter of 2 mm.
Further, although not particularly shown here, the first liquid flow supply pipe and the second liquid flow supply pipe are respectively containers for storing the first liquid flow and a container for storing the second liquid flow, and a pump, etc. Is connected to the transfer force adding means.
The length of the transfer pipe is preferably set such that the two liquid-liquid phases are stable after the first liquid flow and the second liquid flow are combined, and should be set according to the transfer speed. It is preferable to set the length so as to secure an approach section according to the Reynolds number. In addition, in this embodiment, it is set to 50 mm.

<Separation part>
As shown in FIG. 1, the separation unit 20 has a main portion 21 which is a semicircularly curved flow passage, and a straight line extending from an end of the main portion 21 and connected to a collecting portion (not shown). And a linear end portion 22 that extends.
In the present embodiment, both the main portion 21 and the end portion 22 are made of tubular members, and the inner diameters of these tubes are the same as the inner diameter of the transfer tube 13. The main portion 21 has a radius of curvature of 98 mm and is a semicircle (θ=180°). The length of the end portion 22 is the same as that of the transfer pipe 13 in the transfer preparation unit 10 for the same reason.
Although the main portion 21 has a semicircular shape in the present embodiment, the shape is not limited to this, and various shapes can be used. For example, it has an S shape, a spiral shape, or the like. In short, the main part is not particularly limited as long as it has a curved shape capable of producing a secondary flow described later.

<Collection Department>
Although a collector is not particularly shown, a known liquid-liquid recovery device can be adopted without particular limitation. For example, a device using a micro rectangular tube or a difference in density between the first and second liquid streams separated into two is used to transfer the liquid for transfer from the end portion into two phases. It can be configured by a pool for collecting and storing in a separated state, and a suction device for continuously sucking and collecting the second flowing liquid having a low density in the pool. In addition to this, instead of sucking, a flow path for recovery is provided separately in the liquid-liquid interface region and the peripheral region of the pipe wall using the flow of the transfer liquid, and the transfer liquid is provided in each flow channel. By allowing the liquids of the respective phases to flow in as they are, it is possible to separately collect the particle groups existing at the liquid-liquid interface and the particle groups existing in the peripheral region of the tube wall.

<Particle separation method>
The particle sorting method of the present embodiment is a particle sorting method for sorting a large number of particles, and can be performed using the above-described particle sorting apparatus.
Then, a transfer liquid having a large number of particles, the first flow liquid, and the second flow liquid which is unnecessary for the first flow liquid and has different densities at a predetermined ratio is prepared. Transfer preparation process,
A flow passage for circulating the transfer liquid, which is tubular and has the curved flow passage having the predetermined curved shape (the shape of the main portion which is the above-described curved body), and is used for circulating the transfer liquid. It can be carried out by performing a separation step of separating the particles and a collection step of collecting the separated particles for each separation section.

First, the particles and the flowing liquid used in the method of the present invention will be described.
(Particles, sorting target)
In the present invention, particles to be used for sorting include cell particles (more specifically, liver cells, pancreatic islets, stem cells, differentiated cells, etc.), various polymer particles (more specifically, styrene particles, microchannel method). Polymer particles, etc.), microorganisms (algae, Euglena etc.), polymer-encapsulated particles, metal particles and the like.
The particle size of these particles is not particularly limited, and particles having various particle sizes can be separated.
Further, the separation according to the present invention can be carried out for each aggregate unit of particles existing in the flow liquid, with the size and density of the aggregate unit. That is, it can be said that the size and density of each set of particles are targets of classification. Here, the aggregation unit corresponds to one of the particles if there is only one particle, and the cluster if there are a plurality of particles that aggregate to form one cluster.
(First flow)
The first liquid flow can be set arbitrarily, but it is preferable that the first liquid has a high affinity with the above particles. For example, when cell particles are used as the particles, a culture solution (containing water as a main component and a cell culture component) can be used, and in the case of polymer particles, a good solvent for the raw material polymer of the polymer particles. A liquid can be used.
(Second flow)
The second flowing liquid is insoluble in the first flowing liquid and has a higher density than the first flowing liquid and particles, but the density is not particularly limited, and the second flowing liquid is It suffices that a difference be provided between the first flowing liquid and the second flowing liquid to the extent that a liquid-liquid interface is formed and a two-phase fluid is formed.
Further, the density of the second liquid flow is preferably higher than the density of the particles used. As a specific example of the second liquid flow, for example, when cell particles are used as particles, a fluorine-inert liquid having a density higher than that of cells (a commercially available product such as a product name “Florinato” manufactured by 3M) can be used. Can be mentioned.

(Transfer liquid)
The transfer liquid is a liquid flow in which two phases are laminated by the above-mentioned first liquid flow and second liquid flow, and has a configuration as shown in FIG. .. That is, as shown in FIG. 3, the transfer liquid 50 is composed of the first flowing liquid 51 and the second flowing liquid 53 located in the lower layer of the first flowing liquid 51. It is a flowing liquid in the formed state.

Hereinafter, each step will be described.
(Transfer preparation process)
First, the first flowing liquid is supplied to the transfer pipe 13 by the first flowing liquid supply pipe 11, and the second flowing liquid is supplied to the transfer pipe 13 by the second flowing liquid supply pipe 12, The first liquid and the second liquid are caused to collide at the entrance of the two liquids and the two liquids are combined to separate into two phases as shown in FIG. 3A to form a transfer liquid having a liquid-liquid interface. .. At this time, the supply rate of the first flow liquid and the supply speed of the second flow liquid are such that the first and second flow liquids are not mixed with each other, and in the case of particle damage and cell particles, The rate is preferably such that cell death does not occur.
For example, because the balance between the secondary flow (spiral flow in each phase) and the buoyancy and drag of particles is important, the size of the secondary flow is estimated by the Dean number, and the buoyancy (gravity) is calculated from the particle density. The velocity can be determined so that the balance between them can be optimized by measuring the drag force based on the size and the viscosity.

(Separation process)
The separation step is performed by causing the transfer liquid to flow through the flow passage formed by the tubular main portion 21 in the separation unit 20 described above.
The flow direction at this time is the direction of the arrow in FIG. 1, and the speed thereof is arbitrarily set depending on the inner diameter of the main part, the viscosities of the first and second flowing liquids, and further the particle size and density of the particles. It is something.
Further, when the particles are cell particles, the separation step includes an aggregate (dead cell particles) formed by aggregating only one cell particle (normal cell particle) and a plurality of particles. Classify into and. It is known that normal cell particles exist independently one by one, but dead cell particles aggregate to form clusters. In the sorting step of the sorting method of the present invention, formation of these clusters is known. Can be performed efficiently, and normal cell particles and dead cell particles can be positioned in a separated state in a state where they can be separated and collected easily.
Explaining the mechanism that can be separated in this way, centrifugal force is induced in the fluid and particles in the curved pipe with particles, and in the pipe, a pair of vertically symmetrical vortices ( Dean vortex) is formed.
Generally, in a curved pipe, the Dean number is defined by the following equation as a dimensionless number representing the instability of the flow due to the centrifugal force, and the Dean vortex strength depends on the Dean number.
Particle suspensions are polydisperse systems in which particles or agglomerates of particles of various sizes are mixed. These particle groups are dispersed and transferred from the transfer pipe 13 to the main portion 21, and a transfer liquid 50 that is a two-phase fluid having a liquid-liquid interface 53 is formed as shown in FIG. ..
Then, the transfer liquid 50 transferred to the main part proceeds in the direction of the arrow in FIG. 1 in the distribution pipe, which is the main part, and the arrows shown in FIGS. 3(b) and 3(c) in each of the two phases. Direction Dean vortices are generated and each particle is affected not only by the main flow direction (arrow direction in FIG. 1) but also by the Dean vortices shown in FIGS. 3B and 3C. Moreover, since the main portion 21 is curved, the liquid-liquid temporary surface 53 tilts as shown in (b) and wavy phenomenon occurs as shown in (c), and the two-phase liquid flow level (water level) A position different from that in the case of (a) in which the stretching force due to the bending is not applied also occurs in the position of the flowing liquid corresponding to (a). Due to these synergistic effects, particles having a small density and particle size (normal cells in FIG. 3) ride on the flow of the Dean vortex and spirally move in the flow path of the main part 21, while the density and particle size are Large particles (dead cells in FIG. 3) travel along the flow path while remaining on the outer wall side without riding on the flow of the Dean vortex. For this reason, as shown in (d) at the end, particles having a small density or particle size (normal cells in FIG. 3) are located at the liquid-liquid interface, and the density or particles are formed on the wall side of the tube constituting the end. Particles with large diameter (dead cells in FIG. 3) will be located.

(Collection process)
In the collecting step, the particles or particle groups (clusters) separated as shown in FIG. 3(d) in the above-described separating step are collected according to a conventional method.
Examples of the recovery method include a method in which the outer wall side and the liquid-liquid interface in each phase are separately sucked and recovered in different containers.

<Effect, application>
The fractionation device and the fractionation method of the present invention hardly break the particles, and can separate and collect the particles in the size and the agglomerated state.
Thus, for example, when applied to cell particles, it becomes possible to perform live/dead separation of cells, which is important for cell transplantation and cell medicine, continuously and with low invasiveness without using a centrifuge or the like. Furthermore, it becomes possible to select and extract a cell cluster of an appropriate size that is important for cell transplantation.
Furthermore, by using it as a culture space, it is possible to control the supply of oxygen and the environment of various liquid factors, and to supply a culture environment that can reduce physical irritation such as bottom contact.

The present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the spirit of the present invention.
For example, in the above-described embodiment, the tube having a circular cross section is used as the shape of each tube, but a rectangular tube or an elliptic tube in which a liquid-liquid two-layer interface is formed, or a tube having an optimized shape may be used. Good.
Further, if a liquid-liquid interface can be formed and a secondary flow can be formed, an open type flow path having a U-shaped cross section or a U shape is not necessary even if the flow path is a closed type flow path such as a pipe. Thus, the flow passage can be formed.

Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

[Example 1]
The device shown in FIGS. 1 and 2 was produced. The inner diameter of each tube was r = 2 mm, and three types of radius of curvature R = 47 mm, 98 mm, and 142 mm were prepared and used.
Purified water was used as the first flow, a fluorine-inert liquid (trade name "Fluorinert FC-40" manufactured by 3M Co.) was used as the second flow, and fluorescent particles were used as the particles. The particles used have the central particle size dp=70 μm (density 1500 kg/m ³ ) and dp=31 μm (density 1050 kg/m ³ ) in order to be similar to the condition that particle clusters and individual particles are mixed. And were mixed in equal parts by weight and used.
First, both particles were dispersed in purified water to prepare a 0.01 wt% suspension of particles, 20 ml of which was filled in a syringe, and a fluorine-inert liquid was also filled in the same amount in a similar syringe. The liquid was fed to each of the supply pipes by a syringe pump while being connected to the first and second flow liquid supply pipes.
The inspection area has a curved flow path inlet with θ=0°, θ=0° ((a) in FIG. 1), 90° ((b) in FIG. 1), 180° ((c) in FIG. 1) Checked in 3 places. In addition, the experiment was conducted under the conditions of De=20, 35, 50, 65, 80 where the liquid-liquid interface can be stably formed. For observation, a zoom lens and a digital camera (SONY ILCE-QX1) were used to take a moving image of the upper surface of the channel to measure the particle distribution in the channel, and the particle distribution was measured by image processing from consecutive images.
Fig. 4 (a) shows the result graph of the particle distribution at each position when the particle diameter dp = 70 µm and the radius of curvature R = 98 mm.
Immediately after the flow into the main portion 21 (θ=0°), the distribution has a bilateral symmetry, and a large number of particles are shown corresponding to the local flow velocity in the center where the flow velocity is fast.
On the other hand, in the center of the main part 21 (θ=90°), the distribution is asymmetrical, and many particles are gathered near the outer peripheral wall surface of the curved pipe, and the particles are unevenly distributed outside the flow path, and 0.8≦a About 50% of the particles are concentrated in the range of /r≦1. This is considered to be a strong indication of the Dean flow effect.
Further, at the position of θ=180° on the downstream side, the maximum value is shown at (a/r=0.8) at a position slightly away from the wall surface, and these results show that particles can be separated.
Further, FIG. 4B shows the results at De=50 using particles having a particle diameter dp=31 and particles having a particle diameter of 70 μm and a main part having a radius of curvature R=98 mm. As is clear from the results shown in FIG. 4(b), at dp=70 μm (particles with a large particle size), the particles are 0.6≦a/r≦1, that is, unevenly distributed near the inner wall of the tube, At dp=31 μm (particles with a small particle size), some concentration was shown on the inside, but no uneven distribution of particles was found at specific locations. From this result, it was found that the fractionation device and the fractionation method of the present invention are useful for fractionating particles.

Claims (3)

A particle sorting method for sorting a large number of cell particles, comprising:
A large number of the above-mentioned cell particles, a culture solution as the first flow, and a fluorine-inert liquid as the second fluid , which is insoluble in the first flow and has a higher density than the cell particles. And a transfer preparation step for adjusting a transfer liquid having a predetermined ratio of
A flow passage through which the transfer liquid is passed, which is tubular, and is passed through the flow passage that is a curved body having a predetermined curved shape to kill the cell particles with living cells. A method for separating particles into cells, which comprises a step of separating cells into cells by a difference in density and/or a difference in size of cell particles , and a collecting step of collecting the separated particles for each of the divided sections. .. The feed rate of the first liquid stream and the feed rate of the second liquid stream in the transfer preparation step are such that the size of the secondary flow is estimated by the Dean number, and the buoyancy is determined from the particle density and the drag force is determined from the size and the viscosity. The particle fractionation method according to claim 2, wherein the particle fractionation method is determined by measuring A particle fractionation device for carrying out the particle fractionation method according to claim 1 , comprising:
A transfer preparation unit that can be charged with the above-mentioned large number of cell particles, the first flow solution, and the second flow solution, and that holds them for transferring a transfer solution having these in a predetermined ratio. A first flowing liquid supply pipe, a second flowing liquid supply pipe arranged at a predetermined angle with respect to the first flowing liquid supply pipe, a first flowing liquid supply pipe and a second flowing liquid supply A transfer pipe which is connected at a connecting point of the pipes, both of which have a predetermined angle, and which transfers the above-mentioned first flowing liquid and the above-mentioned second flowing liquid together as a transferring liquid to the separation section. A transfer preparation unit for performing the transfer preparation step,
In order to perform the sorting step, the flow passage for circulating the transfer liquid is formed, and the flow passage has a main portion that is a curved flow passage and an end portion that linearly extends from an end of the main portion. And a collecting unit that is connected to the flow passage and that collects the separated particles for each separating section for performing the collecting step.