JPH04158782A - Cell culture device - Google Patents

Abstract

PURPOSE:To carry out supply of nutrient by a flexible tube member by consti tute the title device so that the second rotating member may rotate around the axis in the horizontal direction by transmitting force when the first rotating member revolves around the axis in the vertical direction. CONSTITUTION:A revolving frame 200 and revolving frame member 21 0 are rotated by a driving device 105 and a rotating member 220 kept in a state where one end of a flexible cylindrical body 300 is fixed to a fixing support 103 and the other end is fixed to the rotating member 220 are revolved around the vertical axis Lv. The flexible cylindrical body 300 and tube member 250 inserted in the interior rotates the culture vessel 230 without causing twist. The culture vessel has two chambers partitioned by a semi-permeable membrane and feeding and discharging tubes 2501 and 2502 for packing a cell and culture medium into either one of these chambers and packing the culture medium while pouring into the other are connected to the culture vessel. The culture vessel and tubes connected thereto are integrally and exchangeably provided in the objective device.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application 1] The present invention relates to a cell culture device, particularly a cell culture device capable of infinite sedimentation.

[Prior Art] Conventionally, such a cell culture apparatus has been disclosed, for example, in Japanese Patent Application Laid-Open No.
The one described in Japanese Patent No. 92278 is known.

In this method, a culture container filled with a mixed system of liquid medium and cells is rotated around a substantially horizontal axis to obtain an infinite sedimentation state.

When supplying nutrients, etc. to the culture container, the tip of the tube fixed to the fixed part is positioned inside the culture container, and a mechanical seal is placed between the fixed side containing this tube and the rotating side of the culture container. to obtain a sealed condition.

[Problems to be Solved by the Invention] However, although cell culture apparatuses using such conventional rotary culture vessels have excellent cell culture efficiency, as described above, the sealing between the stationary side and the rotating side is difficult. Since a mechanical seal had to be used to obtain the desired condition, there were the following problems.

(1) Abrasion of the rubber lip of the mechanical seal is unavoidable, and this contaminates the liquid culture medium. Alternatively, depending on the material of the lip part, it may dissolve into the liquid medium and adversely affect cell culture.

(2) It is difficult to sterilize the mechanical seal.

(3) It is difficult to use disposable cell culture containers that do not require cleaning, sterilization, etc. each time.

The object of the present invention is to solve such conventional problems.

To provide a cell culture device that can replenish nutrients and perform cell culture reliably while maintaining a sealed state without using mechanical seals, etc., and in which all wetted parts can be made into disposable parts. It's about doing.

1 Means for Solving the Problems 1 In order to achieve the above object, the present invention provides a fixing column that is provided on a fixing member with its axis oriented in a substantially vertical direction and has a tube passage formed therein, and a fixing column that has a tube passage formed therein. a first rotating member rotatably supported around an axis; a tube passage is formed by the first rotating member being rotatably supported around a substantially horizontal axis, and a culture container can be attached to the first rotating member; The second rotary member is inserted into the tube passage of the fixed strut and the tube passage of the second rotary member, and is maintained in a bent state forming an approximately 90'' angle between the fixed strut and the second rotary member. a flexible tube member that is connected to the culture container, and a drive means that rotationally drives the first rotating member; - The first step is to fill the cells and culture medium.
The chamber and the second chamber are connected to tubes for supplying and discharging the culture medium, and the culture medium and the tubes connected to it can be replaced as a unit. Features.

[Function 1] According to the present invention, when the first rotating member is revolved around the axis in the substantially vertical direction by the driving means, the second rotating member is rotated in the substantially horizontal direction due to the transmission force of the flexible tube member. is rotated around its axis.

At this time, the flexible tube member rotates around the horizontal axis while revolving around the vertical axis, and is not twisted. As a result, it is possible to connect one end of the visible tube member to a fixing member and the other end to a culture container, and it is possible to maintain a sealed state without using a mechanical seal or the like.

[Examples] Examples of the present invention will be described below with reference to the accompanying drawings.

In the figure, 100 is a roughly rectangular machine base, and machine base 1
00 includes a substantially rectangular support frame 101 as a fixing member.
is fixedly supported by a column 102.

A fixed column 103 having an axis LV in the vertical direction is press-fitted into an opening of the support frame 101 .

The fixed support 103 is formed hollow in this embodiment,
This hollow portion constitutes a tube passage through which a tube member, which will be described later, is inserted.

A revolving frame 200 having a U-shaped cross section is rotatably supported on the outer periphery of the fixed column 103 via a bearing 104 .

The revolution mount 200 has a hub 201 for supporting the bearing 104
a rectangular bottom portion 202 provided with a rectangular bottom portion 202;
It consists of side parts 203, 203 extending vertically from both ends of the bottom part 202, and a gear 204 is fixed to the lower side of the bottom part 202. The gear 204 is a drive tooth of a drive device 105 that is fixed to the support pedestal 101 and consists of a motor and a speed reducer! It meshes with +06.

Further, at the upper portions of both side surfaces 203 and 203, a substantially square revolving frame 210 is supported by shafts 211, 211 around the horizontal axis Ls+ such that the angle can be freely adjusted. It has opposing long side frames 212.212 and dagger frames 213.213, and the long side frames 212.212 are supported by shafts 211, 211 on the side parts 203.203 of the above-mentioned revolution mount 200. The long side frames 212 and 212 each have an arc-shaped elongated hole 212A with the axis LHI as the center of the arc,
21.2A is provided and bolts 214, 214 provided on the side portions 203.203 are arranged to pass therethrough. After adjusting the angle θ of the revolving frame 210 with respect to the horizontal direction, the revolving frame 210 is held at that angle by tightening the bolts 214 and 214.

Therefore, the above-mentioned revolution mount 200 and revolution frame 21O
This constitutes the first rotating member.

Furthermore, a balance weight 215 is attached to one side of the dagger frame 213 of the revolving frame body 210.

Further, a through hole 213A is formed on the other side, and a hub 213B is provided outside around this through hole 213A.

A rotating member 220 as a second rotating member is rotatably supported on the hub 213B via a bearing 216 around a horizontal axis IIL perpendicular to the horizontal axis L□. has been done. This rotating member 220
is provided with a disc-shaped container attachment part 221, and
The rotating member 220 is formed entirely hollow. In this embodiment, this hollow portion constitutes a tube passage through which the tube member is inserted, as will be described later.

Further, in this embodiment, the container mounting portion 221 can accommodate a flat cylindrical resin culture container 230 centered on the above-mentioned horizontal axis, ox.

That is, the flange 22 is provided on the outer circumference of the container attachment portion 221.
1A is formed and the protrusion provided on the outer periphery of the culture container 230 is held between the frame member 222, and the bolt 22 is
3 is tightened to the flange 221A.

As shown in FIG. 3, the culture container 230 according to this embodiment is a semi-transparent III! It consists of two chambers separated by 231, one of which is the first chamber 2, which is filled with a culture medium in which cells are dispersed.
32, and the other chamber consists of a second chamber 233 connected to supply and discharge tubes 2501 and 250□ for filling with the culture solution while flowing.

Oxygen-containing gas (air, a mixed gas of air and carbon dioxide, etc.) is supplied to one or both of the two chambers, usually the first chamber 232. #1. The element-containing gas flows through a tube 234 made of an air permeable material such as silicone rubber provided in the room, passes through the silicone rubber wall, and dissolves into the culture solution (for this purpose, the culture vessel 230 is , tubes 2503 and 2504 for supplying oxygen-containing gas are connected.

The semipermeable membrane 231 used in the present invention includes regenerated cellulose,
Examples include semipermeable membranes made of cellulose such as cellulose acetate, as well as semipermeable membranes made of polyester, polyacrylonitrile, and the like. The pore size of the semipermeable membrane is such that cells cannot pass therethrough and is usually 10 μm or less. Note that 30% or more, preferably 50% or more of the surface forming the boundary between the first chamber 232 and the second chamber 233 is a semipermeable membrane.

The tube 250 has one end connected to a supply source (not shown),
The other end is connected to the culture container 230 as described above, and is made of a flexible member such as silicone rubber. A plurality of flexible tubes 250 are bundled together in the hollow part of the fixed column 103 and the rotating member 22
It is inserted into the hollow part of 0 and supported.

Although it is not necessary if the flexible tube 250 is fixedly supported in the hollow part of the fixed column 103 and the rotating member 220 and has sufficient strength, In order to reliably transmit the signal to the rotating member 220, in this embodiment, both ends of the flexible cylindrical body 3000 bent at 90 degrees are fitted to the ends of the fixed column 103 and the rotating member 220. Flexible cylinder 30
0 is preferably flexible against bending and strong against torsion. As long as it has such properties, it is possible to use a thick rubber tube, a coil spring, or the like, and the shape thereof may also be bellows-like.

In this embodiment having the above configuration, the culture container 230 filled with a mixed system of a predetermined liquid medium and cultured cells so as to be substantially filled with the tube member 250 is integrally connected to the tube member 250. In this state, the tube member 250 is as described above (
First, the supply source side end of the tube member 250 is inserted from the right side to the left side of the rotating member 220, and then from the upper side to the lower side of the fixed column 1f13, and then the culture vessel 230
is housed and fixed in the container attachment part 221. The distal end of the tube member 250 is unshielded and connected to a supply source (not shown). The culture vessel 230 is then rotated about a horizontal axis by the drive device 105 while feeding nutrients compatible with the cells to be cultured from a supply source through the tube member 250.

That is, when the drive device 105 operates, the drive gear 106
and the revolving mount 200 via the driven gear 204 that meshes with it.
, and in turn, the revolution frame member 210 is rotated.

Now, in the state shown in the second figure, the revolving frame member 210 is rotated clockwise by 1
If it rotates, one end of the flexible cylinder 30 (l is fixed to the fixed support 103 and is immovable, and the other end is fixed to the rotating member 220, so the rotating member 220 and, by extension, the culture container 230) are vertical. It revolves once around the axis Lv, rotates around the vertical axis, and rotates once clockwise around the horizontal axis LH1 when viewed from the side.

As a result, the culture container 230 can be rotated without twisting the flexible cylinder 300 and the tube member 250 inserted therein. Note that when the flexible cylinder 300 is not used, the tube member 250 can function as a torque transmission member.

The rotation speed of this culture container 230 is 10 to 10, which is suitable for cell culture.
In order to rotate at about 20 rpm, the revolving frame member 210 may be rotated at about <10 to 20 rpm.

According to this embodiment, the culture container 230 to which the tube member 250 is connected can be rotated around the horizontal axis 182 in a completely sealed state to obtain an infinite subsidence state.

In order to more reliably obtain this infinite sedimentation state, in this embodiment, the culture container 230 is oriented along the horizontal axis. It is tilted at a predetermined angle θ with respect to the center, so that the influence of centrifugal force can be canceled.

Here, assuming that the radius of rotation r, the angular velocity ω, and the acceleration of gravity of the Lv of the culture container 230 are g, the inclination angle θ that balances the centrifugal force is expressed by the following equation.

θ=jan-'(1027g) Then, once the optimum value of the inclination angle θ is determined, the angle of the revolution frame member 210 may be set according to the optimum value using the elongated hole 212A.

The culture container 230 and the tube members 250 integrally connected thereto are stored and used in a sterilized state. In addition, it is preferable to use a disposable set to prevent contamination between cell lofts and to save time and effort for cleaning.

In the above-described embodiment, the fixed support 103 and the rotating member 220 are formed hollow to form a tube passage to facilitate the insertion of the tube members. A passageway may be formed in the member.

Kama 11 Egami The following cells were cultured using the culture device of the present invention.

Cultured cells: rV-79J derived from Chinese hamster lung fibroblasts Liquid medium: rEagle-MEMJ containing fetal bovine serum with IO capacity to dissolve oxygen and carbon dioxide (viscosity: O
, 01poise, specific gravity: 1.01) Carrier particles: r
cytodex m J (Pharmacia)
(Particle size: 180 μm, specific gravity: 1.03) First chamber 232
In a flat cylindrical culture container with an internal volume of 400 mj2, 1. Fill the liquid medium with OOOmg (approximately 4 x 10') of carrier particles, and add 2.7
After seeding O' cultured cells, in an environment at a temperature of 37°C,
The culture vessel was rotated at a rotation speed of 15 r, p, m for 72 hours.

During this period, 2i rEagl without fetal bovine serum
The e-MEMJ medium was circulated in the second chamber 233 and replaced with fresh medium every 24 hours.

Oxygen was supplied through a mixture of 5% carbon dioxide and 95% air by volume through a silicon tube (1 x 1.5 mm, 4 m).

As a result, cells on all carrier particles had grown to 6×10' cells.

Cultured cells of Osa ■Yu: Anti-tissue plasminogen activator (anti-TPA) monoclonal antibody-producing hybridoma Liquid medium: RPMI1640 particles containing dissolved oxygen and carbon dioxide gas and 10% by volume tallow serum (stir bar): "Biosilon" (Polystyrene, particle size 160-300
μm.

(manufactured by Nippon Intermed Co., Ltd.) in the same flat cylindrical culture container as in Experimental Example 1, with a dry weight of 0.
Fill the liquid medium with 6g of particles and add 2x10°
After seeding the cultured cells so that the number of cells/■β was obtained and sealing the cells to prevent air from entering, the culture container was rotated at a rotation speed of 15 r, p, and m for 15 days at a temperature of 37°C. I did it.

During this period, RPM11 at 2°C without fetal bovine serum.
640 medium was circulated and replaced with fresh medium every 2 days.

Oxygen is silicon tube < I X 1.5+++m,
4m) D) A mixture of 5% by volume carbon dioxide gas and 95% by volume air was supplied through the mixture.

As a result, the hybridomas proliferated smoothly, reaching lXl0' cells/llepsilon on the 7th day, and maintained this density until the 15th day. In addition, hybridomas are subjected to ELI after 7 days of culture.
The amount of antibody measured by SA method was 30-40 μg/m
℃ produced monoclonal antibodies.

Higashi 1 Hidan Cultured Cells: Human Peripheral Blood Lymphocytes (Reference 1) Liquid Medium Dioxygen and carbon dioxide dissolved in RPM11640 containing lo volume % human AB type serum, recombinant interleukin-
2 (rlL-2) was added. (Reference 2) Particles (stir bar)
= “Bio Glass J (Glass.

Particle size 150-210 μ decoy, Solohill Engineering Co., Ltd.) 1.000 mg in the same flat cylindrical culture container as in Experimental Example 1
The particles were added to fill a liquid medium, and 4 x 10' cultured cells were seeded in this, followed by static culture at a temperature of 37°C. After 3 days, the incubator was rotated at 15r for 24 days.
Rotated at p, m.

During this time, 2i RPMr1640 medium not containing human AB type serum and rlL-2 was circulated and replaced with fresh medium every 2 days.

For oxygen, use a silicon tube (I x 1.5a+m, 4m
) was supplied through a mixture of 5% by volume carbon dioxide and 95% by volume air.

As a result, the lymphocytes proliferated smoothly and reached 3X on the 12th day.
It reached 10' cells/lIβ and maintained this density until the 24th day. After culturing, collect the cells and incubate with 1.2 x 10I
O lymphocytes (LAN cells) were obtained. In addition, as a result of measuring the cytotoxic activity of this cell by the llCr release method (Reference 3), it was 80.0% (E/TIO: 1) against (lG56 (human lung squamous cell carcinoma cells), 562 (human leukemia) It had a high activity of 60.0% (E/T20:1) against cells).

Reference 1 Preparation of human peripheral blood lymphocytes Healthy peripheral blood (300+1j2) was diluted with the same amount of PBS- and 50m0 was added to lymphocyte separation medium (Lym
phocyte 5eparatfon Medium
, manufactured by Organo Techno Co., Ltd. 1 (hereinafter referred to as rLsMJ)
Layer on 30 mβ and centrifuge at 1,350 rpm 11.20°C
, 30 minutes), the lymphocyte layer was collected and washed with Hank's buffer containing lactalbumin (hereinafter referred to as "Hank's buffer"), and then 20 mn of 0.75 wt% ammonium chloride/Tris-HCl buffer (pH 7, Add 6) and heat to 37℃
and heated for 5 minutes. Next, the cells were centrifuged to remove hemolyzed red blood cells and washed with Hank's solution to obtain 4 x 10' lymphocytes.

Reference 2 Preparation of liquid culture medium The following was dissolved in 900 mj2 of water, the pH was adjusted to 7.0 with IN sodium hydroxide, and the medium was then filtered through a 0.22 μl filter.

Powder RPMI1640 10.4g Sodium bicarbonate 2.0g Penicillin G 1.25x lo'U streptomycin 125mg HEPE5
5. OgAB type serum
100mρrIL-2 (recombinant)
5 X 10@U anti-CD3 monoclonal antibody 10μ
gReference 3 ``Measurement of injury rate by Cr release method QG56 (human lung squamous cell carcinoma cells) and ni5 were used as target cells.
62 (human leukemia cells) and labeled these target cells with @l and r. Next, the obtained LAN cells and
5×10″ each of target cells labeled with 1′Cr were placed in QG
56 is cell number ratio (LAN cells:QG56) 5:1,1
0:1, 20:1, K562 was mixed at a cell number ratio (LAN cells: K562) of 2.5 to 1, 5:1, 10+1, placed in a 96-well microplate, and incubated in a carbon dioxide incubator for 37 hours. Cultured at ℃. After 4 hours, the radioactivity released from each well was measured using a gamma counter, and the injury rate was determined according to the following formula.

Here, the maximum release amount is the amount of 1Cr released when labeled cells are lysed by adding O,IN hydrochloric acid, and the spontaneous release amount is the amount of 8 l Cr released when only the medium is added instead of LAN cells.
The test release amount is 1 when LAN cells are added.
l Cr release amount.

[Advantageous Effects of the Invention 1] As is clear from the above description, according to the device of the present invention, cells can be uniformly dispersed in a liquid medium and constantly contacted with a fresh medium through a semipermeable membrane, In addition, the culture medium can be supplied and discharged from a continuous tube without twisting without using a mechanical seal, while maintaining a sealed state, and cells can be cultured efficiently over a long period of time.

In addition, the culture container and the multiple tubes connected to it can be sterilized and made into a disposable set, which eliminates the risk of contamination between lots of culture and contributes to the improvement of medical technology. be.

[Brief explanation of drawings]

FIG. 1 is a side sectional view showing an embodiment of the present invention, FIG. 2 is a plan view thereof, and FIG. 3 is a sectional view showing an embodiment of the culture container of the present invention. 100... Machine base, 101... Support pedestal (fixed member), 103... Fixed column, 200... Revolution pedestal (first rotating member), 210...
Revolution frame member (first rotating member), 220... rotating member (
221... Container attachment part, 230... Culture container, 250... Tube member, 300... Flexible cylindrical body.

Claims (1)

[Claims] 1) provided on the fixed member with its axis oriented in a substantially vertical direction;
a fixed column in which a tube passage is formed; a first rotating member rotatably supported by the fixed column about its axis; and a first rotating member rotatably supported by the first rotating member about its substantially horizontal axis. , a second rotating member having a tube passage formed therein so that a culture container can be attached thereto; a second rotating member that is inserted into the tube passage of the fixed column and the tube passage of the second rotating member; a flexible tube member connected to the culture container while being maintained in a bent state with a temperature of approximately 90° C. between the tube member and a drive means for rotationally driving the first rotating member; , consists of two chambers separated by a semipermeable membrane, one of which is the first chamber filled with cells and culture medium, and the other connected with supply and discharge tubes to be filled with the culture medium while flowing. A cell culture device comprising a second chamber, wherein the culture container and the tube connected thereto are replaceable as one unit.