JPH0534945B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method and apparatus for adhering adhesion-dependent cells to carrier particles.

[Conventional technology]

Generally, when culturing cells, if the cells to be cultured are suspension proliferating cells, that is, cells that can proliferate while suspended in a liquid medium, the cells that are to be cultured must be placed in the liquid medium that is their nutrient source. The cells are brought into contact with the medium by being suspended, but if the cells are adhesion-dependent cells, that is, cells that require adhesion to a substrate for growth and proliferation in a liquid medium, the cells are allowed to adhere to the surface of a suitable substrate. It is necessary to bring the cells into contact with the liquid culture medium after the preparation. Recently, small-diameter carrier particles (hereinafter simply referred to as "carrier particles") have been used as substrates for adhesion-dependent cells because they can easily obtain a large adhesion area. There is.

[Problem that the invention seeks to solve]

However, in order to culture target adhesion-dependent cells (hereinafter simply referred to as "cells") with high efficiency, it is necessary to prepare a liquid medium containing the cells to be cultured when adhering them to carrier particles. If the carrier particles can be dispersed with a high degree of dispersion in the culture medium, the effective area of the surface of the carrier particles that can be used for adhesion of the cells will be increased, which is extremely advantageous in increasing culture efficiency.

Conventionally, the common method for adhering cells to be cultured to carrier particles is to stir a mixture of cells to be cultured and carrier particles in a liquid medium using a rotary blade; however, this method is a method in which dispersion is performed by applying shear force to a mixed system consisting of a liquid medium, cells, and carrier particles. is difficult to adhere to carrier particles. Therefore, in order to make the cells adhere to the carrier particles, it is necessary to repeat the standing step and the stirring step, and as a result, the number of cells adhering to the carrier particles tends to be uneven, and eventually the carrier particles The effective usable area of the surface is small, and the adhesion efficiency has not been sufficiently increased.

Although various measures have been researched to solve these problems, at present, no method or device for adhering cells to carrier particles that essentially solves these problems has yet been developed. be.

[Means for solving problems]

The present invention was completed as a result of intensive research based on the above circumstances, and its purpose is to enable cells to adhere to carrier particles with high efficiency in a liquid medium. An object of the present invention is to provide a bonding method and device.

The method of the present invention is characterized by a liquid medium,
A container filled with a mixed system consisting of cells and carrier particles so as to be substantially full is rotated so as to rotate around an axis held at an angle in the range of 0 to 55 degrees with respect to the horizontal. The point is that the cells are constantly rotated together with the container, thereby adhering the cells to the carrier particles within the mixing system.

The device of the present invention is also characterized by a container having a rotating shaft supported at an angle in the range of 0 to 55 degrees with respect to the horizontal, and a drive mechanism that rotates the container around the axis. The driving mechanism has a function of constantly rotating a mixing system consisting of a liquid medium, cells, and carrier particles filled in the container so as to substantially fill the container together with the container. It is in.

The present invention will be explained in detail below.

In the present invention, as shown in FIGS. 1 and 2, for example, in a cylindrical sealed container 1,
A liquid medium 3 containing cells and carrier particles 2,
The container 1 is filled so that the internal space is substantially filled, and the axis X of the container 1 is set at 0 to 55 degrees with respect to the horizontal plane (H).
The container 1 is rotated around this axis X at a constant rotational speed while being held at an angle (α) preferably in the range of 0 to 45°, particularly preferably 0 to 5°. At this time, if the angle (α) is too large, the dispersibility of the cells and carrier particles in the liquid medium will decrease, that is, the cells and carrier particles 2 will be concentrated in a part of the liquid medium 3, and the efficiency will be reduced. It becomes difficult to perform proper adhesion.

By rotating the container 1 in this way,
After an initial period immediately after the start of rotation, container 1
Due to the viscosity of the liquid medium 3, the mixed system of the liquid medium 3 and the cells and carrier particles 2 in the container 1 comes to rotate steadily around the axis X integrally with the container 1 without mechanical flow. . Therefore, the cells and carrier particles 2 change their position with respect to the outside of the container 1 without changing their relative position with respect to the liquid medium 3 in the container 1, and therefore the cells and carrier particles 2 in the container 1 are sequentially Gravity acts from different directions, which causes the cells and carrier particles 2 to be dispersed in the liquid medium 3 with a high degree of dispersion. Such a state can be realized by appropriately selecting the specific gravity and size of the cells and carrier particles 2, the specific gravity and viscosity of the liquid medium 3, the rotation speed of the container 1, etc.

During such a rotating operation period, focusing on the cells and carrier particles 2 in the mixed system, one cell or carrier particle is relatively It is assumed that the cells and the carrier particles 2 are uniformly dispersed in the liquid medium 3 due to the circular movement shown by the arrow A.
This dispersion occurs extremely quickly depending on the conditions, and the cells and carrier particles 2 also become dispersed in the axis can be dispersed into

In the present invention, there is no need for a special method to fill a container with a liquid medium, cells, and carrier particles; for example, a premixed system of liquid medium, cells, and carrier particles can be filled. Alternatively, the cells may be added to the liquid medium and then the carrier particles are added, or the carrier particles may be added to the liquid medium and then the cells are added.

In the application of the present invention, the cells used are not limited in any way as long as they have adhesive properties to carrier particles, such as human uterine cancer cells.
HeLa, Chinese hamster lung fibroblasts V-79, human fetal lung cells MRC-5, chimpanzee liver fibroblasts, human foreskin cells, chicken fetal fibroblasts, primary monkey kidney cells, mouse metastatic fibroblasts, subbrain Examples include pituitary tumor cells and lymphoid cells.

In the application of the present invention, the liquid medium used is not particularly limited, and any known medium can be used as is, for example, 1 to 30% (V/V)
Minimal essential medium containing calf serum or fetal bovine serum
medium: general-purpose cell culture basic medium), 1-30%
Examples include Dulbecco's modified Eagle's medium containing (V/V) calf serum or fetal bovine serum, HB 101 (manufactured by Hana Biologics), HB 102 (manufactured by Hana Biologics), and the like. The specific gravity of these liquid media is generally 1.00 to 1.05.
Note that it is usually necessary to dissolve oxygen and carbon dioxide gas in these liquid media. Further, the temperature of the liquid medium during adhesion of cells to carrier particles is usually 30 to 40°C, preferably 37°C.

In the application of the present invention, the carrier particles used are not particularly limited as long as they are suitable for cell adhesion and proliferation, such as synthetic polymer materials such as polystyrene, proteins,
Examples include particles whose surfaces are made of natural polymeric materials such as polysaccharides. Advantageously, such carrier particles are magnetic,
When such carrier particles are used, the carrier particles can be easily and quickly collected, moved, processed, and otherwise handled using a magnet.
Examples of magnetic carrier particles include those formed by binding magnetic powder with the above-mentioned polymeric material, and those formed by covering a magnetic core with the above-mentioned polymeric material. Specific examples of the magnetic material for forming the magnetic core include iron, cobalt, nickel, alloys thereof, low carbon steel, silicon steel, γ-iron oxide, ferrite, and magnetite. The specific gravity of the carrier particles varies depending on the viscosity and specific gravity of the liquid medium, but is generally within the range of 1.0 to 1.5. In addition, the particle size of the carrier particles is 40~
The diameter is preferably about 500 μm, and the shape is preferably spherical, but it may also be granular, cylindrical, or irregularly shaped.

The amount of cells seeded per ml of liquid medium is generally 1 x 10 ⁴ to 1 x 10 ⁶ cells, and the number of carrier particles used is usually 1 x 10 4 to 1 x 10 ⁴ per ml of liquid medium.
The number of cells seeded ^per carrier particle is generally about 1 to 10.

In the present invention, it is desirable that the container is filled with a mixed system of liquid culture medium, cells, and carrier particles so that there are no spaces,
This makes it possible to easily rotate the mixing system together with the container, and even if there is a space, if it is small, the disturbance in the liquid medium due to the existence of the space will prevent the mixing system inside the container from being disturbed. Since it is limited to a small area of the upper layer portion, the effect of the present invention is not actually invalidated. However, as the proportion of space within the container increases, it tends to become difficult to rotate the mixing system together with the container without substantially disturbing it, making it impossible to achieve the object of the present invention. Therefore, in the present invention, a substantially full state is defined as a case where the filling ratio of the mixed system in the container is 80% by volume or more, preferably 90% by volume or more, and particularly preferably 98% by volume or more.

In the present invention, the rotational speed of the container is selected in consideration of the size and specific gravity of the cells or carrier particles, the viscosity of the liquid medium, the shape and size of the container, etc., and cannot be unconditionally defined. Usually 5-50r.pm, preferably 10-30r.p.
Rotate the container so that the rotation speed is approximately m.m.

In addition, the shape of the container is, as in the above example,
A cylindrical shape is preferable, but there is no problem with a prismatic or spherical shape, and the rotation method is not particularly limited. In other words, the container can be rotated substantially in a vertical plane so that the top and bottom of the container are reversed. For example, as shown in FIG.
The container 12 may be held at the tip of an arm 11 that rotates around the cylindrical container 20 for circular movement in a vertical plane.Alternatively, as shown in FIG. It may also be rotated to rotate around Y.

Furthermore, in the present invention, adhesion of cells to carrier particles can also be performed while exchanging the liquid medium.

FIG. 6 shows an example of the apparatus according to the present invention, and in the example shown in this figure, the container is constituted by a bottom plate 30 and a bottomed cylindrical container body 31. That is, the container main body 31 is pressed against the bottom plate 30 via an O-ring 33 by a presser mechanism 32 fixedly provided on the bottom plate 30, and the internal space thereof is used as a bonding operation area. The bottom plate 30 is a rotating sleeve 35
The rotary sleeve 35 is rotatably held by a mantle member 38 held by a stand 37 via a dry bearing 36, and the axis Z of the rotary sleeve 35 is 0 with respect to the horizontal plane (H). ~
The angle (α) is maintained at an angle of 55°, preferably between 0 and 45°, particularly preferably between 0 and 5°. Bottom plate 30
has an opening in its center, and this opening is blocked by a filter member, such as a mesh 40, which allows the liquid medium to pass through but not cells and carrier particles.
An inner conduit 41 extending inside the mesh 40 is fixed to the mesh 40. This inner conduit 41 is connected to the container body 31.
It has an opening 43 at one end located inside the rotary sleeve 35 , and the other end extends outwardly through the rotary sleeve 35 such that the end communicates with a supply pipe 51 from the outside at the connecting seal portion 50 . is rotatably connected to. An outer conduit 4 is provided on the inner periphery of the rotating sleeve 35.
2 is fixedly provided so as to have a double pipe structure surrounding the inner conduit 41, one end opening 44 of the outer conduit 42 is connected to communicate with the opening of the bottom plate 30, and the other end is connected to the outside at the connecting seal part 50. It is rotatably connected to the discharge pipe 52 so as to communicate therewith. 60 is a driven gear fixed to the rotating sleeve 35, and this driven gear 60 meshes with a driving gear 61 driven by a motor (not shown).

In the device configured as above, the drive gear 6
1 is driven, the rotating sleeve 35 rotates, thereby rotating the container constituted by the bottom plate 30 and the container body 31 around its axis Z, and at the same time, the inner conduit 41 and the outer conduit 42 are rotated. also rotates together.

However, in this lever device, when the container is filled with a mixed system of liquid culture medium, cells, and carrier particles so as to substantially fill the container, the container is held at zero horizontally.
Since the adhesion operation is performed while rotating around the axis Z, which is supported at an angle in the range of ~55°, cells and carriers can be highly dispersed in the container without applying any shearing force. The particles can be dispersed, and therefore, the frequency of contact between cells and carrier particles can be sufficiently increased while dispersing cells and carrier particles, and as a result, cells can be efficiently adhered to carrier particles.

Furthermore, in this device, since the liquid medium can be continuously exchanged, culturing subsequent to adhesion can be performed efficiently.

〔Example〕

Examples of the present invention will be described below.

Example 1 Cells: "V-79" cells derived from Chinese hamster lung fibroblasts Liquid medium: 10% dissolved oxygen and carbon dioxide
(V/V) "MEM" medium containing fetal bovine serum (viscosity: 0.01poise, specific gravity: 1.01) Carrier particles: dextran particles "Cytodex3"
(manufactured by Pharmacia) (Particle size: 180 μm, specific gravity: 1.03) 750 mg dry weight in a cylindrical container with a capacity of 300 ml.
(approximately 2.5 × 10 ⁶ cells) of the above carrier particles were added and the liquid medium was filled, 8 × 10 ⁶ cells were seeded therein, and the mixture was sealed to prevent air from entering.
The temperature is 37℃ with the rotation axis of the container held horizontally.
The container was rotated at a rotational speed of 15 rpm for 4 hours in an environment of 150 rpm to allow the cells to adhere to the carrier particles.

As a result, the proportion of carrier particles to which cells did not adhere was approximately 5%, and the number of adhered cells in each carrier particle to which cells adhered was 2 to 4 (3.2 on average).
and the number of cells on all carrier particles was 7.5×10 ⁶ .

Example 2 A cell adhesion operation was carried out using the same cells, liquid medium, and carrier particles as in Example 1 using an apparatus having a container having an internal volume of 1 as shown in FIG. That is, using 3 g (approximately 1.0 x 10 ⁷ cells) of carrier particles in dry weight for 1 liquid medium, 3 x 10 ⁷ cells were seeded, and these liquid medium Filled with a mixed system of cells and carrier particles, the container was rotated at a rotational speed of 12 rpm for 4 hours at a temperature of 37°C with the rotation axis of the container set horizontally, until the cell carrier particles The gluing operation was performed.

As a result, the proportion of carrier particles to which cells did not adhere was approximately 4.8%, and the number of adherent cells in each carrier particle to which cells adhered was 2 to 5 (average 2.8).
and the number of cells on all carrier particles was 2.76×10 ⁷ .

Comparative Example 1 Using a spinner bin with an internal volume of 2, Example 1
The cell adhesion procedure was performed using the same cells, liquid medium, and carrier particles as in . That is, 3 g of carrier particles by dry weight was added to 1 part of the liquid medium in a spinner bottle, and then 3 × 10 ⁷ cells were seeded, and the rotation speed of the rotor was set to 30 r.pm in an environment at a temperature of 37°C. It was rotated for a period of time.

As a result, the percentage of carrier particles to which cells did not adhere was approximately 48.6%, and the number of adherent cells in each carrier particle to which cells adhered was 0 to 7 (average 1.4).
and the number of cells on all carrier particles was 1.52×10 ⁷ .

Example 3 A cell adhesion operation was carried out using the same cells, liquid medium, and carrier particles as in Example 1 using an apparatus having a container having an internal volume of 1 as shown in FIG. That is, for each liquid medium, 3 g (approximately 1.0 x 10 ⁷ cells) of carrier particles are used, and 3 x 10 ⁷ cells are seeded, and these liquid mediums are grown in such a way that no air enters the container. , filled with a mixed system of cells and carrier particles, and kept at a temperature of 37°C with the axis of rotation of the container set at an angle of 45° with respect to the horizontal plane.
The container was rotated at a rotational speed of 15 rpm for 4 hours in an environment of 150 rpm to allow the cells to adhere to the carrier particles.

As a result, the proportion of carrier particles to which cells did not adhere was approximately 5%, and the number of adhered cells in each carrier particle to which cells adhered was 2 to 4 (average 2.5).
and the number of cells on all carrier particles was 2.4×10 ⁷ .

〔Effect of the invention〕

As explained in detail above, according to the present invention,
The container is filled with a mixed system of liquid medium, cells, and carrier particles so as to be substantially full, and the container is rotated about an axis held at an angle of 0 to 55 degrees with respect to the horizontal. Since the rotation is performed, cells and carrier particles can be dispersed with a high degree of dispersion in the container without applying substantially shearing force. Therefore, while dispersing cells and carrier particles, the frequency of contact between cells and carrier particles can be reduced. can be made large enough. As a result, cells can be efficiently adhered to carrier particles, and as a result, it is possible to provide an adhesion method and device that can achieve highly efficient adhesion of cells to carrier particles.
Since cells can be adhered to carrier particles with high efficiency in this way, the effective area of carrier particles can be increased, and cells can be efficiently attached during the cell culture process that normally follows the cell adhesion operation. It becomes possible to culture.

[Brief explanation of drawings]

FIGS. 1 and 2 are an explanatory perspective view and a sectional view of an example of the present invention, FIG. 3 is an explanatory view of the movement of carrier particles in a liquid medium, and FIG.
5 and 5 are respectively an explanatory cross-sectional view and a perspective view showing another example of the present invention, and FIG. 6 is an explanatory cross-sectional view showing an example of the apparatus according to the present invention. 1, 12, 20... Container, 2... Adhesion-dependent cells and carrier particles, 3... Liquid medium, 30... Bottom plate, 31... Container body, 32... Presser mechanism, 33
...O-ring, 35...Rotating sleeve, 36...
...Dry bearing, 37...Stand, 38...
...Sheath member, 40...Mesh, 41...Inner conduit, 42...Outer conduit, 50...Connection seal portion, 5
1... Supply pipe, 52... Discharge pipe, 60... Driven gear, 61... Drive gear.

Claims (1)

[Scope of Claims] 1. Around an axis in which a container filled with a mixed system consisting of a liquid culture medium, adhesion-dependent cells, and carrier particles so as to be substantially full is held at an angle in the range of 0 to 55 degrees with respect to a horizontal plane. carrier particles for adhesion-dependent cells, characterized in that the mixing system is rotated so as to rotate on its own axis, and the mixing system is constantly rotated together with the container, thereby causing the adhesion-dependent cells to adhere to the carrier particles within the mixing system. How to adhere to. 2 A container having a rotation axis supported at an angle in the range of 0 to 55 degrees with respect to a horizontal plane, and a drive mechanism for rotating the container so as to rotate around the rotation axis, the drive mechanism has the function of constantly rotating a mixed system consisting of a liquid medium, adhesion-dependent cells, and carrier particles, which is filled in the container so as to substantially fill the container, together with the container. A device for adhering sex cells to carrier particles.