JP7064191B2 - Cell dispersal method and cell disperser - Google Patents

Description

The present invention relates to a cell dispersion method and a cell dispersion device. In particular, the present invention relates to a cell dispersion method and a cell disperser capable of efficiently dispersing a cell aggregate in which cells have aggregated, and is useful in the fields of drug discovery, pharmacy, medicine, biology and the like.

Since pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) can differentiate into cells of all tissues and organs in the living body, research and development have been actively carried out in recent years. There is. Since these pluripotent stem cells can be a new source of cells for cell therapy, expectations for clinical application are increasing. However, since pluripotent stem cells have the ability to differentiate into cells of all tissues, it is difficult to amplify and culture them in large quantities while maintaining an undifferentiated state, which is different from the conventional culture method. Where a method was required, Patent Documents 1 and 2 describe a cell device capable of efficiently culturing even human-derived ES / iPS cells that are extremely sensitive to shear stress. There is.

By the way, there are various methods for obtaining cells, including the methods of Patent Documents 1 and 2, but in many cases, the obtained cells are cell aggregates (tissue pieces) in which a plurality of cells are aggregated. ) Is formed. In order to utilize this in regenerative medicine, safety pharmacology tests, etc., it is necessary to disperse cell aggregates in individual cells. For example, Patent Documents 3 to 4 propose techniques for dispersing cells.

Japanese Patent No. 5958861 Japanese Patent No. 5959024 Japanese Unexamined Patent Publication No. 4-232834 Japanese Unexamined Patent Publication No. 2012-24061

Generally, in order to disperse cell aggregates in a single cell, the cells are dispersed by immersing the cell aggregates in an enzyme solution such as trypsin or collagenase and performing physical operations such as pipetting while keeping the cells warm at a constant temperature. Let me. Since the enzyme permeates from the outside of the cell aggregate, the cell gradually peels off from the outside, but it takes time to reach the inside of the cell aggregate or tissue, so physical manipulation of the cell It promotes dispersion.

However, in operations such as pipetting, cells that have been peeled off by enzymes may be destroyed by colliding with the vessel wall due to pipetting, and the recovery amount and recovery rate may decrease. In addition, experimental operations such as pipetting tend to vary from person to person, making it difficult to standardize the operations.

Further, Patent Documents 3 to 4 propose mechanical devices for cell dispersion, but the technique of Patent Document 3 has poor cell dispersion efficiency as pointed out in Patent Document 4, and Patent Document 4 The technique has a problem that the mechanism becomes complicated.

In view of such a situation, the present invention relates to a cell dispersion method and a cell dispersion device capable of efficiently dispersing a cell aggregate in which cells have aggregated to individual cells.

As a result of diligent studies by the present inventor on the above problems, it was found that by rotating the rotating body in a fixed tank, the aggregates of cells contained in the liquid in the tank are efficiently dispersed, and the present invention is completed. It came to.

The present invention includes, but is not limited to, the following aspects.
(1) A device that disperses cell aggregates into a single cell, and is a cylindrical tank that houses a treatment solution containing the cell aggregates, and is rotatably attached to the inside of the tank in a coaxial manner with the tank. The above-mentioned apparatus, which comprises a rotating member and, by rotating the rotating member, circulates a treatment liquid orbiting in the tank in a Taylor vortex state to disperse cell aggregates into a single cell.
(2) The cell dispersant according to (1), wherein the treatment liquid contains a cell aggregate degrading enzyme.
(3) The cell dispersant according to (1) or (2), further comprising a stirrer for rotating a cylindrical member.
(4) The cell dispersant according to any one of (1) to (3), wherein the rotating member has a cylindrical shape.
(5) A method of dispersing cell aggregates in a single cell, in which the treatment liquid is circulated along the inner surface of the tank in a cylindrical tank containing the treatment liquid containing the cell aggregates. The above method comprising dispersing the cell agglomerates into a single cell by means of.
(6) The method according to (5), which comprises rotating a cylindrical member coaxially and rotatably attached to the inside of the tank to bring the orbiting treatment liquid into a Taylor vortex state.
(7) The method according to (5) or (6), wherein the cell aggregate comprises pluripotent stem cells.

According to the present invention, a cell aggregate (tissue piece) in which cells have aggregated can be efficiently dispersed. Generally, a certain amount of shearing force is required to disperse cell aggregates and tissues to obtain single cells, but excessive stress on cells may reduce the survival rate of cells. In the present invention, since an appropriate shearing force is constantly applied to the cell aggregate contained in the liquid, it is considered that the cells are efficiently dispersed.

In the apparatus according to the present invention, the shearing force of the liquid generated when the rotating body in the fixed tank rotates is constant at any place in the tank. Therefore, it is possible to obtain an ideal liquid flow in which the strength of the local shearing force does not exist in the tank. Further, since the fixed tank and the rotating body do not have a structure in which cells collide with each other, it is possible to prevent the cell aggregate and the single cell after dispersion from being damaged by the collision.

As mentioned above, when disperse cell aggregates and tissue fragments into single cells in a test tube, shearing force was conventionally applied by taking in and out of liquid by pipetting, but this work varies from person to person and is standardized. It is difficult to describe in the procedure manual. In addition, the operator may unknowingly touch the tip of the pipette to the vessel wall or move it in and out at an unnecessarily high speed, so that the cells may be destroyed. On the other hand, according to the apparatus according to the present invention, there are few individual differences and cell-destroying actions, and a constant shearing force can be applied to efficiently obtain a homogeneous single-cell cell suspension. In particular, in the manufacturing process of products such as regenerative medicine, the yield of obtained cells can be improved, so that it is expected that the manufacturing efficiency will be improved and the cost will be significantly reduced.

When a rotating body installed in a cylindrical fixed tank is rotated, a laminar flow (couette flow) parallel to the rotation direction is generated in the liquid in the gap between the inner wall surface and the surface of the rotating body due to the frictional resistance of the surface. When the rotation speed is gradually increased, the liquid flow changes from the laminar flow to the vortex flow, and the parallel liquid flow becomes a complicated vortex flow (Taylor vortex flow). The details of why cells are efficiently dispersed by the present invention are not completely clear, and the present invention is not bound by this reasoning, but in the present invention, moderate physical stress is applied to cell aggregates. It is considered that the cell aggregates are efficiently dispersed by the above.

FIG. 1 shows the cell dispersant used in the experiment. FIG. 2 is a graph showing the recovered amount of iPS cells dispersed and treated in Experiment 1 (vertical axis: number of cells). FIG. 3 is a photograph of the culture solution treated in Experiment 1 observed under a microscope. FIG. 4 is a graph showing the experimental results of Experiment 2 (1). FIG. 5 is a graph showing the experimental results of Experiment 2 (1). FIG. 6 is a graph showing the experimental results of Experiment 2 (2). FIG. 7 is a graph showing the experimental results of Experiment 2 (3) (left: photo of myocardial troponin T cardiomyocytes and nuclei, right: number of cardiomyocytes). FIG. 8 is a graph showing the experimental results of Experiment 2 (4). FIG. 9 is a graph showing the experimental results of Experiment 2 (5).

The cell dispersant according to the present invention includes a fixed tank and a rotating member rotatably installed inside the fixed tank. In one aspect of the present invention, the rotating member is a cylindrical member having a cylindrical shape, and the gap between the inner wall surface of the fixed tank and the outer wall surface of the cylindrical member, the number of rotations of the cylindrical member, the physical characteristics of the liquid, and the like are liquid. It is an important factor that determines the flow.

In one embodiment of the present invention, the apparatus according to the present invention has a cylindrical fixed tank, a lid portion that can be attached to and detached from the fixed tank portion, a support column provided on the lid portion, and a cylinder rotatably attached to the support column. It is configured to have a shaped member (a rotating member that can rotate coaxially with the fixed tank).

Further, in the present invention, a stirrer for rotating the cylindrical member may be provided. As an example of the driving means of the stirrer, it is preferable to rotate the cylindrical member using a magnetic material (permanent magnet or the like).

The fixed tank is filled with a liquid containing a cell aggregate in which cells have aggregated. In the apparatus according to the present invention, the members (fixed tank, cylindrical member, lid, strut) to which the liquid containing the cell aggregates come into contact are inactive to the liquid component, have no cytotoxicity, and are sterilized (excluded). Also referred to as dyeing, sterilization or sterility), it is preferably formed of a material that is resistant to treatment. Examples of such a material include glass, synthetic resin, stainless steel and the like. Here, the capacity, shape, and the like of the fixed tank are appropriately determined according to the amount of liquid and the cylindrical member. Further, a convex portion may be provided in the center of the bottom surface of the fixed tank. This makes it possible to prevent the treatment liquid from stagnation at the center of the bottom surface of the fixed tank. The content (capacity) of the fixed tank is not particularly limited, but in a preferred embodiment, it may be, for example, 20 to 1000 ml, 30 to 800 ml, 40 to 600 ml, or 50 to 400 ml. The capacity of the fixed tank can also be determined based on the amount of liquid required in one treatment.

The column for rotatably installing the cylindrical member is fixed to the lid portion and is arranged along the central axis of the fixed tank. Then, the cylindrical member is rotatably attached to the support column. As a result, the cylindrical member can rotate coaxially with the fixed tank. However, as a modification, the support column can be provided upright in the center of the inner surface of the bottom of the fixed tank. At this time, it is preferable that the support column has a conical shape in which the portion fixed to the inner surface of the bottom of the culture tank is formed in a conical shape with an enlarged diameter in the downward direction. With such a shape, the stagnation of the liquid flow near the center of the bottom due to the rotation of the cylindrical member is reduced, and the cell aggregates can be prevented from settling at the bottom of the fixed tank. Further, the height of the strut is not particularly limited, and for example, the strut may be raised to support the cylindrical member relatively at the upper part, or the strut may be lowered to support the cylindrical member relatively close to the bottom. The structure may be supported at a position. The method of fixing the support column to the fixing tank is not particularly limited, but it can be fixed by a fixing tool such as a screw, or by using an adhesive (for example, a silicon rubber adhesive). It can be fixed by a method, and the fixed tank and the support can be integrated by integral molding.

In one embodiment, the apparatus according to the present invention comprises a cylindrical member, wherein the cylindrical member is preferably made of a material that is inert and resistant to a liquid containing cell aggregates, for example. Examples include synthetic resin and stainless steel. The cylindrical member may be configured to include, for example, a cylindrical synthetic resin block having a hole (a recess that does not penetrate) into which a support column is inserted, which extends along the central axis of the cylinder. Then, the cylindrical member can be rotated with respect to the fixed tank by being rotatably attached to the support column. Further, the cylindrical member may be configured to include a magnet for applying a rotational force by a rotational mechanism. The device (fixed tank, lid, column, rotating member) according to the present invention can also be disposable.

As the material used in the apparatus of the present invention, a resin having light transmission (transparency) is preferable. The type of polymer is not particularly limited, but for example, polycarbonate, polystyrene, acrylic resin, polyvinyl chloride and the like are preferable, and polycarbonate and polystyrene are particularly preferable. Especially for the fixed tank, the processing state can be visually recognized by making the light transmissive.

The cells processed by the apparatus according to the present invention are not limited, and examples thereof include cells of animals, insects, plants and the like. Examples of the origin of animal cells include humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, cows, marmosets, African green monkeys, etc., but are not particularly limited. not. In particular, the apparatus according to the present invention can be applied to cells such as pluripotent stem cells (ES cells, iPS cells, etc.) and stem cells such as neural stem cells, which have been considered to have poor dispersion efficiency by conventional methods. The method for obtaining cells processed by the apparatus according to the present invention is not particularly limited, and can be applied to cells obtained by any method. In addition to cell aggregates derived from culture, tissue fragments derived from living organisms It is also possible to use the (section) for the treatment of dispersing in a single cell. In addition, by adapting the design and operating conditions of the device, it is possible to match the properties of cell aggregates (tissue pieces) such as cell aggregates caused by cells that are vulnerable to shear stress and, conversely, cell aggregates that are strongly aggregated. Processing becomes possible.

In the present invention, the enzyme may be added to the treatment liquid containing the cell aggregate. In the present invention, the cell aggregate is dispersed by a physical action, but the cell aggregate can be dispersed more efficiently by using an enzyme together. The enzyme used is not particularly limited, but in a preferred embodiment, trypsin, protease, collagenase and the like can be mentioned. Further, a chelating agent such as EDTA may be further added to the treatment liquid according to the present invention. The amount and concentration of the enzyme or chelating agent added may be appropriately adjusted, and if the cell aggregate is large and difficult to loosen, the amount of the enzyme added may be increased or the concentration may be increased.

Conditions such as processing time and processing temperature are not particularly limited and may be set as appropriate. The treatment time may be, for example, 5 seconds to 60 minutes, and in a preferred embodiment, 10 seconds to 50 minutes, 30 seconds to 40 minutes, and further 1 minute to 30 minutes. The treatment time can be appropriately set according to the cell type and the rotation speed. The treatment temperature is 0 to 70 ° C. in a preferred embodiment, and 5 to 50 ° C. or 10 to 40 ° C. is also preferable. The rotation speed of the device is based on the concentration and amount of the enzyme solution used when dispersing using a pipette, etc., and the amount of cells recovered when dispersed using a pipette when the treatment time is the same. Can be set to be equal to or greater than.

Further, in one embodiment, the present invention is a method for dispersing cells. In the cell dispersion method according to the present invention, a liquid containing a cell aggregate is placed in a fixed tank, and a cylindrical member in the fixed tank is rotated to generate a liquid flow between the fixed tank and the cylindrical member to cause cell aggregation. Includes dispersal of lumps. According to the present invention, cell aggregates can be uniformly dispersed easily and efficiently.

Liquid flow in the fixed tank In the device according to the present invention, when the rotating member (cylindrical member) installed in the fixed tank is rotated, the liquid in the gap between the wall surface of the fixed tank and the surface of the rotating body is formed by the frictional resistance of the surface. Generates a laminar flow (Quet flow) parallel to the direction of rotation. When the rotation speed is gradually increased, the liquid flow changes from the laminar flow to the vortex flow, and the parallel liquid flow becomes a complicated vortex flow (Taylor vortex flow).

The fixed tank (cylindrical radius: r0) having a cylindrical inner wall and the cylindrical member (cylindrical radius: ri) are coaxially and rotatably assembled with a predetermined gap (r0-ri). In a preferred embodiment, in the apparatus according to the present invention, the gap between the fixed tank and the cylindrical member is, for example, 0.1 to 100 mm, more preferably 1 to 50 mm, still more preferably 2 to 40 mm, still more preferably. It is 3 to 30 mm. Within such a range, the liquid can be efficiently flowed.

(Supply port, discharge port)
The apparatus according to the present invention may be provided with a supply port for supplying the fluid and a discharge port for discharging the fluid. A plurality of supply ports and discharge ports may be provided. Further, the supply port and the discharge port may be provided on the lid portion, or may be provided on the wall surface of the fixed tank or the like.

However, the fixed tank may be configured so that the wall surface is not provided with a supply port or a discharge port. By making the inner wall surface of the fixed tank a cylinder (a cylinder having no unevenness), it is possible to reduce the probability that the cell aggregates and dispersed cells will be damaged by collision during the treatment.

(Rotation mechanism)
The device according to the present invention may have a rotation mechanism for rotating a cylindrical member inside a fixed tank relative to a shaft. In a preferred embodiment, it is preferable that the rotation mechanism rotates the cylinder by using the magnetic force. In this case, it is preferable to provide a metal or a magnet on the cylindrical member.

(Surface roughness)
In the apparatus according to the present invention, the surface roughness Ra of the inner wall surface of the fixed tank and the surface of the cylindrical member is preferably 1/10 or less of the gap (r0-ri), more preferably 5 nm to 1 mm, and more preferably 10 nm to. 100 μm is more preferable. Within such a range, the rotational energy of the cylindrical member can be efficiently applied to the liquid, and damage to cell aggregates and dispersed cells due to surface roughness can be reduced.

(Liquid flow)
In the present invention, a liquid containing a cell aggregate is placed in a concentric cylindrical space formed by a cylindrical fixed tank and a cylindrical member, and the cylindrical member is rotated at an angular velocity ω. Generally, when the rotation speed is low, a laminar Couette flow is generated, and when the rotation speed is further increased, a laminar Taylor vortex flow is generated.

Here, a specific relational expression when a Taylor vortex is generated is shown. The Taylor number is generally defined as Ta in the following equation.
Ta = {riω (r0-ri) / ν} {(r0-ri) / ri} ^0.5 = Re {(r0-ri) / ri} ^0.5
[In the formula, ω: angular velocity, Re: Reynolds number, ri: radius of cylindrical member, r0: radius of fixed tank, ν: kinematic viscosity of fluid]
In the present invention, the liquid flow in the fixed tank is preferably laminar-controlled rather than turbulent-controlled. Here, the Reynolds number (Re) is represented by r _i ω d / ν, and if the numerical value is small, it tends to be laminar-dominated.
Re = riωd / ν
[Ri: radius of cylinder, ω: rotational angular velocity, d: mean value of gap width (ro-ri), ν: kinematic viscosity coefficient]
Further, the critical Taylor number Tac in which the Taylor vortex is generated is shown as follows. This critical Taylor number Tac is determined by η = ri / r0. In the present invention, considering the flow efficiency of the liquid by the cylindrical member, the η is preferably 0.5 or more, and in this case, the Tac is a value in the vicinity of 50 (about 40 to 70). It can be approximated to 50. Here, ωc is the angular velocity at which the Taylor vortex is generated, Rec is the critical Reynolds number, ri is the radius of the cylinder 2, r0 is the radius of the cylinder 1, and ν is the kinematic viscosity of the fluid.
Tac = {riωc (r0-ri) / ν} {(r0-ri) / ri} 0.5 = Rec {(r0-ri) / ri} 0.5.

From each of the above equations, the relationship between the Taylor number Ta, the critical Taylor number Tac, the Reynolds number Re, the critical Reynolds number Rec, the angular velocity ω, and the angular velocity ωc where the Taylor eddy is generated is as follows.
Ta / Tac = Re / Rec = ω / ωc
Taylor vortex flow is likely to occur in the range of 1 ≦ Ta / Tac = Re / Rec = ω / ωc <25. If Ta / Tac is less than 1, Taylor vortex flow does not occur, and if it is 25 or more, turbulence is likely to occur.

In the present invention, it is preferable to disperse the cell aggregates in the liquid containing the cell aggregates under the condition that turbulence does not occur, and under the condition that the Taylor vortex is generated in the liquid containing the cell aggregates. , It is preferable to carry out a dispersion treatment of cell aggregates. By generating a Taylor vortex in the liquid, a large shearing force can be applied to the cell aggregates contained in the liquid, and the probability of damage to the cell aggregates (dispersed cells) during the treatment is reduced. Therefore, it is possible to increase the processing efficiency (recovery efficiency and recovery amount) of cell aggregates. Specifically, by generating a Taylor vortex, the liquid moves not only in the orbital direction, but also in the height direction and the radial direction of the cylinder (orbiting while drawing a spiral orbit). This complex moving liquid exerts a large shearing force on the cell aggregates to efficiently disperse the cell aggregates. On the other hand, in the Taylor vortex flow, the liquid moves in a complicated manner, but unlike the turbulent flow, it moves with a certain regularity. Therefore, it is possible to reduce the probability that the cell aggregates (dispersed cells) contained in the liquid collide with each other, and also prevent the generation of a large (damaging cells) shearing force locally. be able to. From the above, by utilizing the Taylor vortex flow, it is possible to increase the dispersion efficiency of cell aggregates and reduce the possibility of cell damage. Furthermore, by utilizing a fixed tank having a cylindrical inner surface and a cylindrical shape having a cylindrical outer surface, the present invention is mechanically colliding with cell aggregates, especially in the direction of liquid movement. The number of elements is reduced, and the cell aggregate can be dispersed so as not to damage the cell aggregate.

In the present invention, the shape and operating conditions of the device can be set so that the Taylor vortex flow is generated in the liquid containing the cell aggregate. Since the shear force required for dispersion, the optimum liquid component (type and amount of additive component), and processing time differ depending on the cell type and aggregation status, the optimum device shape, rotation speed, and processing time are different. It will change. By using the above equation, for example, the setting condition of the rotation speed can be narrowed down from the information on the kinematic viscosity of the liquid and the information on the shape of the device, so that the condition setting of the dispersion processing becomes easy. Alternatively, it is possible to design the device based on the information on the required processing amount in one processing and the required shearing force, and it is also possible to design in response to the scale-up request under the same conditions. In particular, by using a fixed tank having a cylindrical inner surface or a cylindrical shape having a cylindrical outer surface, it becomes easy to apply the above theoretical formula.

However, when the present invention is applied to a cell type that is weak against shearing force, it is also possible to carry out a cell dispersion treatment under the condition that Taylor eddy current does not occur.
In the present invention, the amount of the liquid or the shape of the device can be adjusted so that the liquid does not entrap air (so that the liquid does not entrap air bubbles) during the treatment. Specifically, when the liquid and the cylindrical member are provided in the fixed tank (before operation), the liquid processing amount may be adjusted so that the liquid level does not exceed the upper surface of the cylindrical member. Alternatively, when the cylindrical member is rotated to disperse the cell aggregates, the liquid processing amount and the rotation speed may be adjusted so that the liquid level does not exceed the upper surface of the cylindrical member. As a result, the probability that air is caught in the liquid can be reduced, and the probability of damaging the cell agglomerates can be reduced. Alternatively, it is also possible to inject the liquid so that the fixed tank (the gap between the fixed tank and the cylindrical member) is filled with the liquid. For example, the lid is provided with a member (valve, filter, etc.) that allows air to pass through but does not allow liquid to pass through, and a liquid injection port. be able to. According to this, it is possible to prevent the liquid from entraining air during the treatment. In this case, it is also possible to disperse the cells while the fixed tank is laid down sideways.

In the present invention, the fixed tank may be configured to have various sensors for measuring the state of the liquid. However, in this case, a sensor using a member that does not form a large unevenness (such as a pH sensor / DO sensor using a fluorescent patch or a temperature sensor that can measure from outside the tank) can be applied. As a result, it is possible to measure the state of the liquid during the dispersion treatment while controlling the liquid flow, so that it is possible to accurately determine the treatment conditions and control the quality of the cells. When using a fluorescent patch, it is preferable to set the position where the fluorescent patch is provided so that the influence on the liquid flow is small. It can be provided near the bottom surface).

In the present invention, the treatment time of the liquid containing the cell aggregate is not particularly limited, but it can be adjusted depending on the cell type and the like, and is preferably 100 seconds or more, for example.
The details of why cells are efficiently dispersed by the present invention are not completely clear and the present invention is not bound by this reasoning, but in the present invention, moderate physical stress is applied to the cells. It is considered that the cells are efficiently dispersed.

Hereinafter, the present invention will be described in more detail while showing specific experimental examples, but the present invention is not limited to the following specific experimental examples. Further, unless otherwise specified in the present specification, the concentration and the like are based on weight, and the numerical range is described as including the end points thereof.

Experiment 1: Cell dispersal of induced pluripotent stem cells (1) Cell aggregates Induced pluripotent stem cells (iPS cells, Ff-i14: Kyoto University) grown on a 6-well plate using a scaffolding material (iMatrix, Nippi). Was maintained and cultured in a medium (StemFit AK03N, Ajinomoto).

In a single-use 100 mL bioreactor for iPS cell culture (hereinafter referred to as 100 mL reactor, Able), 100 mL of StemFit AK03N and Y27632 (ROCK inhibitor manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to a final concentration of 10 μM were placed at 37 ° C., pH 7.2, The dissolved oxygen concentration (DO) was adjusted to 40% (atmospheric saturation) in advance. Fresh iPS cells were harvested from 6-well plates and seeded in a pre-arranged 100 mL reactor at a density of 2.0 × ¹⁰⁷ / mL. After sowing, the 100 mL reactor was maintained at 37 ° C., pH 7.2, DO = 40% (atmospheric saturation), and cultured at 40 rpm with stirring. A part of the culture solution was collected on the first day of culture, and it was confirmed that cell aggregates were growing. On the second day of culturing, the entire amount of the culture broth containing the cell aggregate was collected in a centrifuge tube and centrifuged at 190 G for 1 minute to remove the supernatant. Resuspend cell aggregates in the same amount of StemFit AK03N fresh and kept at 37 ° C, reinject into a 100 mL bioreactor, and incubate at 37 ° C, pH 7.2, DO = 40% (atmospheric saturation), 40 rpm. Continued. The same operation was repeated on the 3rd day of the culture, and after continuing the culture for 4 days, cell aggregates of iPS cells were obtained.

(2) Cell Dispersion Device Figure 1 shows a photograph of the appearance of the device used in the experiment. As shown in FIG. 1, a cylindrical member is provided concentrically inside a tubular housing (made of transparent resin, material: polycarbonate, inner diameter: about 38 mm), and the cylindrical member (made of translucent resin, made of translucent resin). (Material: Polypropylene) is rotatably attached to the inside of the cylindrical housing and can be sealed by the upper lid member (capacity: about 20 ml). As for the cylindrical rotating member (cylinder) provided inside the cell processing apparatus, three types having outer diameters (outer diameters) of 22 mm, 25 mm, and 28 mm were prepared and used in the following experiments.

Here, a magnet is embedded in a cylindrical member rotatably attached to the inside of the device, and the cylindrical member inside the device can be rotated by placing the device on a magnetic stirrer.

(3) Dispersion of cell aggregates 25 mL of the culture solution containing the cell aggregates on the 4th day of culture was evenly dispensed into a centrifuge tube. The centrifuge tube was centrifuged at 190 G for 3 minutes to remove the supernatant, suspended in an equal volume of phosphate buffered saline (PBS, Nacalai Tesque), and centrifuged again at 190 G for 3 minutes. After centrifugation, the supernatant was removed again, and an enzyme solution (protease, TrypLE Select, Gibco) was added. As the enzyme solution, 12.5 mL of the enzyme solution (containing 10 μM Y27632, hereinafter referred to as 0.5 × TrypLE Select) diluted 2-fold with PBS containing 0.5 mM EDTA was added.

When the cell disperser is not used (also referred to as the manual method or manual method), the centrifuge tube is kept warm at 37 ° C for 5 minutes, and then the treatment solution is vigorously taken in and out using a pipetter (hereinafter referred to as pipetting). Cell aggregates were dispersed. The pipette was a pipette aid (10 mL) manufactured by Drummond Scientific Company. After incubating at 37 ° C. for 5 minutes again, the dispersion of cell aggregates by pipetting was repeated until the aggregates became invisible visually. In order to meet the conditions, use a 10 mL pipette to maximize the suction / discharge speed setting of the automatic pipette, place the tip of the pipette on the bottom of the centrifuge tube, repeat the inflow and outflow of the liquid 5 times, and then use the enzyme 5 Treatment was performed for 1 minute to obtain a uniform suspension of single cells (when this is repeated twice, the liquid is taken in and out 10 times, and the enzyme treatment is performed for 10 minutes).

When using a cell disperser (hereinafter also referred to as the device method), a magnetic stirrer (MD-200, MD-200,) in which cell aggregates suspended in 0.5 × TrypLE Select are placed in the cell disperser and placed in a humidified carbon dioxide incubator. The cells were placed on a magnetic stirrer (Yamato Kagaku), and the cylindrical member was rotated at 1200 rpm at room temperature to bring the treatment liquid into a Taylor vortex state, and the enzyme treatment was performed for about 5 minutes.

The flow velocity and shear stress in the shear direction under the conditions of 1000 rpm to 1500 rpm were calculated as follows from the results of particle image velocimetry (PIV) performed in advance. In the particle image flow velocity measurement, a liquid with a specific density of 1.101 g / mL and a viscosity of 3.72 cP was used.
(Flow velocity)
・ Cylinder outer diameter 22 mm: 0.392 to 0.491 m / s
・ Cylinder outer diameter 25 mm: 0.331 to 0.623 m / s
・ Cylinder outer diameter 28 mm: 0.750 to 0.751 m / s
(Shear stress)
・ Cylinder outer diameter 22mm: 87.6-121.5Pa
・ Cylinder outer diameter 25mm: 88.4-162.6Pa
・ Cylinder outer diameter 28mm: 301.5-302.2Pa
Immediately after the treatment, the cell suspension after the dispersion treatment was added with an equal amount of StemFit AK03N containing Y27632 having a final concentration of 10 μM, filtered through a cell strainer (Corning, mesh size: 40 μm), and filtered at 190 G for 3 minutes. Centrifuged. After centrifugation, the supernatant was removed and suspended in StemFit AK03N containing Y27632 at a final concentration of 10 μM.

(4) Analysis after dispersion treatment A part of the treatment solution obtained in (3) above is appropriately diluted with PBS and stained with trypan blue, and the number of living cells is visually observed on a hemocytometer under a microscope. Counted. The results are shown in Fig. 2. Although the recovery efficiency tended to decrease slightly as the outer diameter of the cylinder became smaller, the recovery efficiency was about the same as that of the manual method.

As shown in FIG. 3, the cell aggregates before the dispersion treatment were spheres having a diameter of about 100 to 300 μm, but it was confirmed that the cell aggregates were reduced by the dispersion treatment. That is, when the cell dispersant according to the present invention was used, there was a tendency that cell aggregates tended to remain as the outer diameter of the cylinder became smaller.

(5) Regeneration and growth of dispersed treated single cells The proliferative ability of the single cells (iPS cells) obtained in (3) above was confirmed. Specifically, when the cell suspension obtained in (3) above was re-seeded in a 100 mL bioreactor under the same conditions as in (1) above, the same degree of growth was shown after culturing for 4 days. That is, it was confirmed that the iPS cells dispersed up to a single cell by the cell dispersant device of the present invention maintain their original proliferative ability.

Experiment 2: Cell dispersion of myocardial cells (1) Dispersion of cell aggregates of myocardial cells Generally, human iPS cells can be differentiated into various cells by inducing differentiation, and these cells are used for regenerative medicine and safety pharmacology. It is expected to be used for tests, etc. Since differentiation induction by three-dimensional floating stirring suspension culture is accompanied by cell agglomeration formation as in undifferentiated amplification, efficient dispersion of cell agglomerates after differentiation induction is effective recovery and yield of target cells. It is indispensable for securing.

Therefore, in this experiment, we induced differentiation of human iPS cells into cardiomyocytes by adding growth factors and low-molecular-weight compounds in a time-specific manner in a three-dimensional floating culture environment, and obtained cell aggregates of cardiomyocytes. , A dispersion experiment was performed in the same manner as in Experiment 1. Specifically, a cell aggregate of cardiomyocytes induced to differentiate from iPS cells (Ff-i14, Kyoto University) was subjected to a dispersal experiment into single cells using the disperser described in Experiment 1. Experiments were carried out using cell aggregates (cell masses of cardiomyocytes) 14 days after induction of differentiation.

20 mL of cell aggregates (100 mL) induced to differentiate into cardiomyocytes in a 100 mL vessel were used under each condition. The cell aggregates to be used were transferred to a conical tube (50 mL volume, Corning) and centrifuged at 190 G for 1 minute at room temperature. The supernatant was removed by suction, resuspended in 20 mL of PBS (Nacalai Tesque), and then centrifuged again at room temperature at 190 G for 1 minute. The supernatant was removed by suction, and 12.5 ml of 0.05% TE (0.25% trypsin / EDTA diluted 5-fold with PBS (-)) was added and resuspended.
(Cell dispersion by manual method: Manual) A 50 mL conical tube containing the above enzyme and cell aggregates was placed in a constant temperature bath at 37 ° C. and allowed to stand for 10 minutes (stirred with a pipette every 2 minutes). After 10 minutes, the cells were removed from a constant temperature bath, and pipetting was repeated in a clean bench using a 10 mL pipette (falcon) until the cell aggregates became invisible to the naked eye.
(Cell dispersion by device method: Device) The above enzyme (0.05% TE 12.5 mL) and cell aggregates are placed in a cell disperser (outer diameter of rotating member: 28 mm) and placed in a 37 ° C CO2 incubator at 1000 rpm, 1500 rpm, 2000 rpm. The mixture was stirred for 10 minutes at the same rotation speed. Under either condition, the processing liquid circulating in the apparatus is in a Taylor vortex state.

To the above-mentioned treatment liquid after cell dispersion (manual method and device method), 12.5 mL of 10% FBS DMEM (sigma) was added, and the mixture was centrifuged at 190 G at room temperature for 3 minutes. After removing the supernatant by suction, the cells were resuspended in 15 mL of 10% FBS DMEM, passed through a 40 μm strainer, and the cell number was counted for the cell suspension.

The results are shown in FIGS. 4 to 5, and it was confirmed that the present invention can disperse the cell aggregates of cardiomyocytes. In particular, by treating human iPS cell-derived cell aggregates with proteolytic enzyme at 1500 rpm using a cell disperser with a cylinder outer diameter of 28 mm, it became possible to recover the same number of cells as the manual method.
Data in Fig. 4 (bar graph) ・ 1000 rpm: 2.6 × ¹⁰⁷ cells
・ 1500rpm: 3.3 × ¹⁰⁷ cells
・ 2000rpm: 2.9 × 10 ⁶ cells
-Manual work: 2.5 x ¹⁰⁷ cells
Data in Fig. 5 (bar graph) ・ Manual: 3.4 × 10 ⁷ cells ± 1.3 × 10 ⁷ cells
・ Device: 3.7 × 10 ⁷ cells ± 1.3 × 10 ⁷ cells

(2)
From the above (1), it was clarified that the yield equivalent to that of manual work can be obtained by dispersing the cell aggregates using the cell disperser. In general, the cell aggregate of cardiomyocytes after induction of differentiation is not composed only of cardiomyocytes (the purity of cardiomyocytes is not 100%), so that the ratio of target cells in the recovered cells is evaluated. It is essential. Therefore, the proportion of cardiomyocytes contained in the cells dispersed by the device method and the manual method was evaluated by a flow cytometer.

Specifically, the recovered cells (1.0 × ¹⁰⁷ cells) were fixed with 4% paraformaldehyde and stained with an anti-myocardial troponin mouse monoclonal antibody (ThermoFisher Scientific, MS295-P1 clone 13-11). After that, the cells were stained with Cy3-labeled anti-mouse antibody, and myocardial purity was measured by FACS. A mouse IgG1 antibody (DAKO, X0931) was used as an isotype control, and MofloXDP (Beckman Coulter) was used as a FACS.

The results are shown in FIG. 6. The cells dispersed in (1) contained about 90% of myocardial troponin T-positive cardiomyocytes, and a difference was observed between the two methods. I didn't. That is, it was clarified that the cardiomyocytes, which are the target cells, can be recovered by the dispersion using the cell disperser as well as the manual dispersion.
Data in Fig. 6 (bar graph) ・ Manual: 88.7 ± 3.0%
・ Device: 93.0 ± 1.6%

(3)
It was confirmed whether the cardiomyocytes recovered by the cell disperser could be cultured in the same manner as the cardiomyocytes recovered manually. That is, since the dispersion of cell aggregates involves physical stress on the cells, there is a concern that excessive stress may cause damage to the cells, and therefore evaluation after culturing the cells after recovery is indispensable. Therefore, cardiomyocytes collected manually with a cell disperser were cultured for the same period, and the number of cardiomyocytes after culturing was evaluated.

The cells collected under each condition were seeded in a 24-well plate (Falcon) (1.0 × 105 cells / well, medium: ¹⁰ % FBS DMEM). 2-3 days after seeding, the cells were fixed with 4% paraformaldehyde, stained with anti-myocardial troponin T rabbit polyclonal antibody (Abcam), and then stained with FITC-labeled anti-rabbit antibody. Nuclei were stained using Hoechst 33258 (Invitrogen).

Images of stained cells were obtained in each well using ImageXpress (Molecular Device) (49 fields of view were imaged with a 20x lens), and in each well using MetaXpress and Acuity Softwear (both Molecular Device). The number of myocardial troponin T-positive cells was counted.

The results are shown in FIG. 7, and no difference was observed in the number of cardiomyocytes between the device method and the manual method. In other words, it is suggested that the new cell aggregate dispersion method using the cell disperser was recovered with less influence on the target cells, cardiomyocytes, and as a result, the cardiomyocytes were maintained and cultured in good condition in the same manner as the manual recovery method. Was done.
Data in Fig. 7 (bar graph) -Manual: 13690 ± 2475 cells
・ Device: 13356 ± 2734 cells

(4)
In general, a small amount of undifferentiated iPS cells remain in the cell aggregate after induction of differentiation into cardiomyocytes. Therefore, the residual ratio of undifferentiated iPS cells was evaluated by a flow cytometer for the single cells recovered manually with a cell disperser.

The recovered cells (1.0 × ¹⁰⁷ cells) were fixed with 4% paraformaldehyde, stained with FITC-labeled anti-Tra-1 60 mouse monoclonal antibody (BD, 560380), and remained. Undifferentiated iPS cells were measured by FACS. A FITC-labeled mouse IgMκ antibody (555583) was used as an isotype control. MofloXDP (Beckman Coulter) was used as the FACS analysis device.

The results are shown in FIG. 8, and Tra-1 60-positive undifferentiated iPS cells were not detected in the cells recovered by any method. When the positive ratio in the control (Isotype control) was set to 1% or less, the number of Tra-1 60 positive cells was less than 0.2%, which was below the detection limit. That is, it was clarified that the dispersion of cell aggregates after induction of myocardial differentiation by the cell disperser enables cell recovery without affecting the residual ratio of undifferentiated iPS cells.
Data in Figure 8 (bar graph)-Manual: Tra-1 60, 0.13 ± 0.03; isotype, 1.00 ± 0.01
-Device: Tra-1 60, 0.18 ± 0.10; isotype, 0.99 ± 0.01

(5)
As described above, the evaluation by the flow cytometer did not detect Tra-1 60-positive undifferentiated iPS cells in the cells after the cell disperser and manual induction of myocardial differentiation and the dispersal of cell aggregates, but they were more sensitive. The survival of iPS cells was evaluated by the expression of Lin28 mRNA, which is an index.

The collected cells were seeded in a 6-well plate (Corning) (4 × 10 ⁶ cells per well, medium: 10% FBS DMEM). Cells were collected 1 day and 4 days after the start of culture using RNA Protect Cell Regent (Qiagen), and total RNA was collected using Rneasy mini kit (Qiagen). For the first sample (Day 0 sample), total RNA was recovered from the cells immediately after recovery in the same manner as above.

Quantitative RT-PCR was performed using TaqMan ™ Fast Advenced Master Mix (Applied Biosystems).
The results are shown in FIG. 9, and the expression of Lin28 was observed in the cells immediately after recovery in both methods, and the expression level was not different between the two methods.

On the other hand, when the recovered cells were cultured and the expression level of Lin28 on the 4th day was compared, the expression level of Lin28 was significantly higher in the cells recovered by the cell disperser than in the cells recovered by hand. It was a low price. This suggests that the proliferation of residual iPS cells in the cells recovered using the cell dispersant was suppressed. It is widely known that human pluripotent stem cells are prone to cell death in a single state. With manual dispersal, the dispersal level tends to vary, so the remaining undifferentiated iPS remains in the cell aggregate to avoid cell death and re-proliferate after culturing, whereas recovery with a cell disperser device It is considered that this result is influenced by the difficulty in survival and reproliferation of residual iPS cells in a single cell state because they can be dispersed more uniformly.

Data in Fig. 9 (bar graph) (Day 0) Manual: 0.032 ± 0.023; Device: 0.013 ± 0.010
(Day 1) Manual: 0.024 ± 0.017; Device: 0.015 ± 0.007
(Day 4) Manual: 0.076 ± 0.036; Device: 0.028 ± 0.007

Claims (7)

A cell disperser that disperses cell aggregates into a single cell.
A cylindrical tank that stores the treatment liquid containing cell aggregates,
A rotating member that is rotatably attached to the inside of the tank coaxially with the tank,
The cell dispersant device for dispersing cell aggregates in a single cell by rotating the rotating member to orbit the treatment liquid orbiting in the tank in a Taylor vortex state. The cell dispersant according to claim 1, wherein the treatment liquid contains an enzyme for dispersing cell aggregates. The cell dispersant according to claim 1 or 2, wherein the treatment liquid contains a chelating agent. The cell dispersant according to any one of claims 1 to 3 , further comprising a stirrer for rotating a rotating member. The cell dispersant according to any one of claims 1 to 4 , wherein the rotating member has a cylindrical shape. A method of dispersing cell aggregates into a single cell,
Dispersing the cell aggregates into a single cell by circulating the treatment solution along the inner surface of the tank in a cylindrical tank containing the treatment liquid containing the cell aggregates .
The rotating member coaxially and rotatably attached to the inside of the tank rotates to bring the orbiting treatment liquid into a Taylor vortex flow state.
The above method, including. The method of claim 6 , wherein the cell aggregate comprises pluripotent stem cells.

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