JP2003265164A - Cell culture device capable of loading mechanical stimulus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture system which reproduces a physiological mechanical stimulation pattern, has long-term sterility, and is versatile and can be applied to various cells. SOLUTION: A column-like device (1), a back pressure regulator (3), a valve (2) and (1)
3) A cell culture device comprising a pump (7), a pressure reservoir (6), a medium tank (4), a control computer (11), and a pressure sensor (10).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell culture device and a cell culture method capable of applying mechanical stimulation.

[0002]

2. Description of the Related Art In vivo tissues / cells are differentiated and proliferated by receiving static or dynamic mechanical stimulation as well as biochemical stimulation by various factors such as cytokines.
It exerts the biochemical and physiological functions of cells such as metabolism. The mechanical stimulus in the living body varies depending on the site, but the stimulus due to the hydrostatic pressure of several tens of atmospheric pressure and the pressure level itself is considerably lower than the hydrostatic pressure, but due to the stress pressure due to the flow of blood and body fluid. Stimulation is the main one.

Until now, several studies have been carried out on a cell culture system for the purpose of cell differentiation / proliferation using such mechanical stimulation.

As a culture system by stress stimulation, Le
ung et al. (Leung et al., Science 191 vol.
475-477, 1976) shows an example of culturing vascular smooth muscle by a method of stretching or vibrating a membrane to which cells are attached, Dunkelman et al. (Dunkelman N. et al.
S. Et al., Biotechnology and Bio
engineering 46, 299-305)
And Bujia et al. (Bujia J., Laryngo)
rhinoautologie Volume 73, 577-580
(1994, p. 1994) shows a culture example in which chondrocytes retained on a support are placed in a column, a culture medium is circulated by a pump, and stress stimulation is performed on the chondrocytes in the support by the flow of a liquid. This is a stimulus with a constant force in a constant direction, and the intensity of the stimulus and its stimulus pattern are far from those of the living body.

In addition, as a culture system by hydrostatic pressure stimulation, Wright et al. (Wright et al., Clinica
l Science 90, pp. 61-71, 1996.
Year) shows an example of stimulation load to chondrocytes by pressure load of gas cylinder type, Parkkinen et al. (Parkk).
inen et al., Archives of Biochem.
volume 300, pages 458-465, 1993.
(Year) shows an example of stimulating chondrocytes by a method of loading a column with a pressure of 5 MPa corresponding to the pressure in the joint with a hydraulic cylinder pump. In addition, Smith et al. (Sm
ith et al., Journal of Orthopaed
ic Research 14: 53-60, 19
1996) is a hydraulic cylinder type pressure load, which is 30 MPa centering on 10 MPa which is the physiological pressure of the joint.
Is applied to the chondrocytes to stimulate them.

These methods reproduce the in-vivo level pressure intensity itself, but the load pattern shows uneven pressure changes such as rapid pressure increase and decrease, which is also different from the original one. is there. Furthermore, this type of system shows only an example of stimulation for a short period of time, and has not been shown to be a culture system that ensures sufficient sterility to withstand long-term culture.

[0007]

As described above, it is hard to say that the existing cell culture system using mechanical stimulation reproduces a pattern close to the original mechanical stimulation load of the living body. From the aspect of cell culture, it is difficult to maintain sterility such that long-term culture for several weeks is possible, and the versatility that enables application to various cells is not sufficiently secured. I can say.

In view of the above situation, the present invention reproduces a physiological mechanical stimulation pattern, particularly a hydrostatic pressure in a joint, and
It is an object of the present invention to provide a culture system having a long-term sterility and versatility applicable to various cells.

[0009]

[Means for Solving the Problems] That is, the present invention provides a column-like device for holding cells and a culture carrier and growing the cells therein, a back pressure regulator connected to the outlet of the column-like device, and the bag. An electronically controllable valve installed on the outlet side of the column-like device to control the operation of the pressure regulator, an electronically controllable valve installed on the inlet side of the column-like device, on the inlet side of the column-like device A pump connected to the column-like device via the valve installed, a pressure reservoir installed between the valve and the pump connected to the inlet of the column-like device, an outlet of the column-like device The valve, the back pressure regulator, and the pressure riser connected to each other. A medium tank installed between the cell and the bar, which is electrically connected to the two valves, and operates the back pressure regulator by opening and closing the two valves to operate the cells in the column-like device. A cell culture device comprising a control computer for controlling the pressure pattern applied to the column, and a pressure sensor installed to measure the pressure in the column-like device and electrically connected to the control computer. is there.

In the cell culture device of the present invention, the pressure intensity applied to the cells is variable and cyclic pressure loading is possible. In this case, the variable range of the pressure intensity is 0 to 9 MPa (however, the preferable lower limit is 0.1 MPa).
It is possible to control the pressure load in the range of 10 Hz to 0.005 Hz (however, the preferred lower limit is 0.01 Hz and the preferred upper limit is 1 Hz) as the periodicity. Is.

The cell culture device of the present invention can be used to control the pressure load pattern applied to cells to the pressure load pattern occurring inside the living body. The pressure load pattern that occurs inside the living body usually refers to a sinusoidal pressure load pattern. In addition, in the cell culture device of the present invention, it is possible to cause the movement of the medium in the column-like device during both pressurization and non-pressurization.

The culture carrier in the column-like device is preferably made of a biocompatible material. The culture carrier in the column-like device is not particularly limited as long as it can well retain cells and the like, and specific examples thereof include a flat plate, a porous body, a nonwoven fabric, a mesh, a braid, and a gel. Examples of the biocompatible material include polyglycolic acid, polylactic acid, polyglycolic acid / polylactic acid copolymer, polyε-caprolactone, polylactic acid / polyε-caprolactone copolymer, alginic acid, cellulose, nitrocellulose, gelatin, Examples thereof include degradable materials such as collagen, non-degradable materials such as polyamide, polyester, polystyrene, polypropylene, polyacrylic acid, polyvinyl, polycarbonate and polyurethane.

The column-like device is preferably made of a transparent material. Specifically, tempered glass (borosilicate glass etc.) is mentioned.

The pump is preferably of the plunger type, hydraulic type or gas pressure type.

The cell culture procedure of the present invention may further include a back pressure regulator connected to the inlet of the column-like device so that the actuation can be operated by an electronically controllable valve located on the inlet side of the column-like device. preferable. This makes it possible to control the pressure load pattern more precisely.

When a back pressure regulator is also provided at the inlet of the column-like device, it is preferable that the two types of back pressure regulators be linked by a solenoid valve system.

The pump is preferably controlled by the control computer.

The cells retained on the culture carrier in the column-like device are preferably cells derived from mammals (eg, humans, dogs, cats, cattle, horses, pigs, etc., preferably humans), and cells derived from mammals. The bone marrow or tissue-derived stem cells or articular cartilage-derived cells are more preferable, and the human bone marrow- or tissue-derived stem cells or articular cartilage-derived cells are more preferable.

The basic principle of the cell culture device of the present invention is shown in FIG.
As shown in, the column-like device (1) is pressurized by the liquid supply from the pump (7) through the pressure reservoir (6), and the pressure corresponding to the capacity of the back pressure regulator (3), or the pump (7). ) The pressure corresponding to the pressure of the pressure reservoir (6) stored by the pressurization is generated. Control of the pressure load pattern is performed by turning on / off the valves (2) and (13) on the inlet side and the outlet side of the column-like device (1) or a back connected to this and the outlet of the column-like device (1). It is performed by combining the control by the pressure regulator (3). Therefore, the rising pattern of the pressure is defined by the flow rate of the medium pushed out from the pressure reservoir (6), and it is possible to arbitrarily create a rapid increase or a slow increase by changing the flow rate. The maintenance and lowering pattern of pressure is defined by the opening / closing of the valve (2) and the control pressure of the back pressure regulator (3) placed in the path for releasing the generated pressure. Gives a slower descent. With the mechanism as described above, it becomes possible to reproduce the rise and fall of the pressure in the living body by using the cell culture device of the present invention. Furthermore, the cell culture device of the present invention has a structure in which all parts in contact with the medium can be sterilized by an autoclave or ethylene oxide gas, and is made of a material that can withstand such a sterilization method. It is possible to culture cells throughout.
Also, general-purpose culture is possible by devising the column structure.

The pump (7) is more preferably a plunger type pump, but the back pressure regulator (3) and the valve (2) may be used even if it is a conventional pressure load type by hydraulic pressure or gas pressure. And (1
By combining ON / OFF of 3), various load patterns such as a sinusoidal pressure load pattern can be reproduced.

One embodiment of the cell culture device of the present invention will be described in detail below. One embodiment of the cell culture device of the present invention is shown in FIG.
As shown in Fig. 1, the basic structure is a pump (7), back pressure regulators (3) and (14), column-like device (1), valves (2) and (13), pressure reservoir (6), medium tank (4). ), A control computer (11), preferably a pressure sensor (10) and a pH sensor. The column-like device (1) is pressurized by the liquid supply from the pump (7) through the pressure reservoir (6), and the pressure corresponding to the capabilities of the back pressure regulators (3) and (14), or the pump (7). )
The pressure corresponding to the pressure of the pressure reservoir (6) stored by the pressurization is generated. Control of the pressure load pattern is performed by turning on / off the valves (2) and (13) on the inlet side and the outlet side of the column-like device (1) or a back connected to this and the outlet of the column-like device (1). It is performed by combining the control by the pressure regulator (3) and the back pressure regulator (14) connected to the inlet of the column-like device (1). Therefore, the pressure rise pattern is defined by the flow rate of the medium extruded from the pressure reservoir (6),
By changing the flow rate, it is possible to arbitrarily create a sharp rise or a slow rise. The maintenance and lowering pattern of the pressure is regulated by the opening / closing by the control computer (11) of the valve (2) placed in the path for releasing the generated pressure and the control pressure of the back pressure regulator (3), and the control pressure is low. One can get a steep descent, and one can get a slow descent. With the mechanism as described above, it becomes possible to reproduce the rise and fall of the pressure in the living body by using the cell culture device of the present invention. The back pressure regulator (3) is used to increase and decrease the pressure.
It is also possible to perform opening / closing operations by controlling a computer (11) for controlling the valves before and after the column-like device (1) without using. However, as described in Examples, it is necessary to use the back pressure regulator (3) in order to reproduce a more physiological pressure load pattern.

The pressure sensor (10) and, if necessary, the pH sensor are installed at the entrance of the column-like device (1) or the like to perform real-time measurement. This measurement signal goes through a modulator and a control computer (11)
Is taken into.

The culture medium tank (4) is installed in a CO ₂ incubator and is brought into contact with the internal environment of the incubator through a filter (12) to control the temperature and pH of the culture medium.

The medium to be passed through the apparatus is not particularly limited as long as cells can be cultivated well.

Further, the cell culture apparatus of the present invention is operated by installing the entire apparatus, or at least the medium tank (4) and the column-like device (1) in a container capable of setting a temperature environment suitable for cells to be cultured. To do. Particularly, the medium tank (4) is installed and operated in a container such as a CO ₂ incubator that can provide an environment more suitable for cells.

Furthermore, the cell culture device of the present invention can be aseptically sampled during the culture, and can perform biochemical and physiological evaluations of cells and cell mixtures over time.

[0027]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

(Embodiment 1) The individual units used in Embodiments 2 to 4 will be described below. The pump (7) uses a plunger type and the pressure strength is 2500 ps.
A product manufactured by Chemco Co., Ltd., which can apply a pressure load up to i (about 16 MPa) was used. In this product, parts of the plunger part that come into contact with the medium can be disassembled and sterilized individually by autoclaving or ethylene oxide gas. The back pressure regulators (3) and (14) used were manufactured by Upchurch. As the back pressure regulator (3) for pressure generation, a control pressure of 1000, 750 or 500 psi was used according to the pressure to be generated. 5, 20, 40, 7 for back pressure regulator (14) for pressure relief
5, 100 or 250 psi was used depending on the desired pressure drop pattern.

The column-like device (1) is manufactured by Waters Co., which has a cylindrical double structure in which the inner column part is made of borosilicate glass and the outer shield part is made of polycarbonate, and is resistant to pressure and heat. Was used.

The line connecting the column-like device (1), back pressure regulator (3), pump (7) and culture medium tank (4), tubing pump (5), pressure reservoir (6), sterile water tank (8) is A PEEK material made by Upchurch, which is resistant to heat, was used, and a connection device made by Upchurch was used for connection to each unit.

The pressure sensor (10) manufactured by Generex Co., Ltd. was used and installed at the inlet of the culture column. A modulator manufactured by Bio-Rad Co. was used to import the measurement signal into a computer.

The medium tank was installed in a CO ₂ incubator, and the temperature and pH of the medium were controlled by contacting with the internal environment of the incubator through a filter (12) having a pore size of 0.22 μm. The medium was Ham's F-12 medium (Gibco) containing 10% fetal bovine serum.
BRL) was used to aseptically fill the system.

(Examples 2 and 3 and Comparative Examples 1 and 2) Using the cell culture apparatus of Example 1, pressure was applied to the inside of the culture column. It was possible to generate various pressure load patterns depending on the time of pressure load, the magnitude of pressure, and the presence or absence of a back pressure regulator. Typical ones are shown in A to D of FIG.

When a hydrostatic pressure of 5 MPa was generated at intervals of 1 second, the pressure dropped sharply unless the back pressure regulator (3) was used, and the pressure generation pattern was sharp (Fig. 2). In A, Comparative Example 1), a pressure pattern close to a sine wave was observed when the back pressure regulator (3) was used (Fig. 2-B, Example 2). Further, when hydrostatic pressure of 5 MPa was generated at intervals of 15 seconds, the pressure dropped sharply when the back pressure regulator (3) was not used (Fig. 2-
C, Comparative Example 2). When the back pressure regulator (3) was used under the same pressure load condition, a pressure pattern as shown in FIG. 2-D was obtained (Example 3). This was very similar to the physiological pressure loading pattern in the joint (Fig. 3).

FIG. 2-A: Without using the back pressure regulator (3), after applying a hydrostatic pressure of 5 MPa for 1 second, 0
In the case of holding at MPa for 1 second (Comparative Example 1). FIG. 2-B: A case where a hydrostatic pressure of 5 MPa was applied for 1 second and then held at 0 MPa for 1 second using a back pressure regulator (3) (Example 2). Fig.2-C: Without using the back pressure regulator (3), after applying a hydrostatic pressure of 5 MPa for 15 seconds, it was 1 at 0 MPa.
When held for 5 seconds (Comparative Example 2). Fig. 2-D: Using a back pressure regulator (3), a hydrostatic pressure of 5 MPa was applied for 15 seconds, and then 1 at 0 MPa.
When held for 5 seconds (Example 3).

Example 4 Chondrocytes collected from a 4-week-old bovine forelimb joint loading part were placed in a PLGA sponge disk having a diameter of 6.5 mm and a thickness of 3 mm at 2 × 10 ⁷ cells / c.
The cells were inoculated so as to have m ³ and cultured under pressure using the cell culture device of Example 1. A pattern was repeated, in which a load of 5 MPa was applied for 5 minutes, and then the pressure was maintained at atmospheric pressure (0 MPa) for 55 minutes. After culturing for 2 weeks under these conditions, the effect of cyclic hydrostatic pressure on the formation of cartilage tissue was evaluated biochemically and histologically.

For biochemical evaluation, the amount of proteoglycan produced by chondrocytes was determined by chondroitin-4-sulfate ELIS.
It was evaluated by quantifying according to A. The cartilage tissue after culturing was extracted with a buffer solution for extraction (4M guanidine hydrochloride, 0.05%
M sodium acetate, 0.01M disodium ethylenediaminetetraacetic acid, 0.1M aminohexanoic acid, 0.005
M benzamidine hydrochloride, pH 5.8) at 4 ° C, 48
After gently shaking for solubilization for a period of time, proteoglycan was precipitated with an ethanol solution containing 1.3% potassium acetate. The precipitate was adsorbed on a 96-well plate for ELISA and left overnight at 4 ° C. ELISA cleaning solution (0.0
After washing each well with a phosphate buffer containing 5% Tween 20, 200 μl of a phosphate buffer containing 0.1% bovine serum albumin was added to each well and left for 2 hours. E
After washing with a LISA washing solution, a biotin-labeled anti-chondroitin-4-sulfate monoclonal antibody (Seikagaku Corporation) diluted 500-fold was added in an amount of 100 μl per well and left for 2 hours. Horseradish peroxidase-streptavidin conjugate (GIBCO / BRL Laborat) diluted 1000 times after washing with ELISA washing solution
ory) was added in an amount of 100 μl per well and left for 30 minutes. After washing with an ELISA washing solution, a chromogenic substrate (Genzy
me / Techne) was added in an amount of 100 μl per well and left at room temperature for 30 minutes, and then 2N sulfuric acid was added at 50 per well.
The reaction was stopped by adding μl. After stopping the reaction, the absorbance at 450 nm was measured by an auto plate reader. When the amount of chondroitin-4-sulfate produced by the cells was measured by the above method, the amount produced by the chondrocytes cultured under cyclic pressure loading was cultivated for 2 weeks under atmospheric pressure without pressure loading. It was about 1.5 times higher than that produced by chondrocytes (Fig. 4).

For histological evaluation, the cartilage tissue thus prepared was subjected to HE
(Hematoxylin-eosin) staining and evaluation were performed by counting the number of viable cells. Table 1 shows the number of viable cells per 1 mm ² of frozen sections of a cartilage tissue cultured under cyclic hydrostatic pressure and a cartilage tissue cultured under atmospheric pressure. As can be seen from Table 1, the number of viable cells loaded with hydrostatic pressure was about 1.6 times the number of viable cells not loaded. In the cartilage tissue loaded with hydrostatic pressure, the cells were more uniformly dispersed in the support.

[0039]

[Table 1]

[0040]

INDUSTRIAL APPLICABILITY The present invention is a system applicable to culturing cells of all parts of a living tissue, and among them, it is particularly applicable to culturing chondrocytes. The chondrocytes in the living body are constantly subjected to a high pressure load, and undergo cyclic pressure changes due to walking and the like. The present invention has the ability to faithfully reproduce this pressure load, and it is strongly suggested that it works effectively for the proliferation, function expression and maintenance of chondrocytes, and in turn, the promotion of cartilage tissue formation.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a cell culture device which is a preferred embodiment of the present invention.

FIG. 2 is a diagram showing a pressure load pattern generated by the cell culture device according to one embodiment of the present invention or a cell culture device for comparison.

FIG. 3 is a diagram showing a physiological pressure load pattern in a living body and a pressure load pattern by the cell culture device according to one embodiment of the present invention.

FIG. 4 is a graph showing the amount of chondroitin-4-sulfate produced by cultured cells in the cell culture device of one embodiment of the present invention and a cell culture device for comparison and control.

[Explanation of symbols]

1: Column-like device 2: Valve 3: Back pressure regulator 4: Medium tank 5: Tubing pump 6: Pressure reservoir 7: Pump 8: Sterile water tank 9: Filter 10: Pressure sensor 11: control computer 12: Filter 13: Valve 14: Back pressure regulator

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Tetsuya Tateishi             670-3 Shimohirooka, Tsukuba City, Ibaraki Prefecture (72) Inventor Takashi Ushida             1-817-204 Takezono, Tsukuba City, Ibaraki Prefecture (72) Inventor Hideo Niwa             2568-3 Uozumicho, Akashi City, Hyogo Prefecture (72) Inventor Ken Fukuchi             3-123 Asagiri-cho, Akashi-shi, Hyogo 304 Saison Asagiri (72) Inventor Masao Sato             6-31-17 Shioya, Tarumi-ku, Kobe-shi, Hyogo (72) Inventor Kenji Yamashita             332-3 Kamugawakubocho, Takamatsu City, Kagawa Prefecture F term (reference) 4B029 AA02 AA21 BB11 CC02 CC10                       DA04 DF07                 4B065 AA90X BA23 BA24 BC05                       BC07 BC18 BC42 CA46

Claims (18)

[Claims] 1. A column-like device for holding cells and a culture carrier and growing cells therein, a back pressure regulator connected to an outlet of the column-like device, and a back pressure regulator for operating the back pressure regulator. The electronically controllable valve installed on the outlet side of the column-like device, the electronically controllable valve installed on the inlet side of the column-like device, the valve via the valve installed on the inlet side of the column-like device A pump connected to a column-like device, a pressure reservoir installed between the pump and the valve connected to the inlet of the column-like device, the valve connected to the outlet of the column-like device, and the back pressure. A medium tank installed between the regulator and the pressure reservoir, Note that it is electrically connected to the two valves, and controls the operation of the back pressure regulator by opening and closing the two valves to control the pressure pattern applied to the cells in the column-like device. A cell culture device comprising a computer and a pressure sensor installed to measure the pressure in the column-like device and electrically connected to the control computer. 2. The cell culture device according to claim 1, wherein the pressure intensity is variable and cyclic pressure loading is possible. 3. The cell culture device according to claim 2, wherein the variable range of the pressure intensity is 0 to 9 MPa, and the pressure load can be controlled in the range of 10 Hz to 0.005 Hz as periodicity. 4. The cell culture device according to claim 1, which is for controlling the pressure pattern to a pressure pattern occurring inside a living body. 5. The medium can be moved in the column-like device both when pressurized and when not pressurized.
The cell culture device according to any one of 1. 6. The culture carrier in the column-like device is made of a biocompatible material.
The cell culture device according to the item. 7. The cell culture device according to claim 1, wherein the column-like device is made of a transparent material. 8. The cell culture device according to claim 1, wherein the pump is of a plunger type. 9. The pump according to claim 1, which is of a hydraulic type.
8. The cell culture device according to any one of 8 above. 10. The cell culture device according to claim 1, wherein the pump is a gas pressure type. 11. A back pressure regulator connected to the inlet of a column-like device so that its operation can be operated by an electronically controllable valve installed on the inlet side of the column-like device. The cell culture device according to item 1. 12. The cell culture device according to claim 11, wherein two types of back pressure regulators are linked by a solenoid valve system. 13. The cell culture device according to claim 1, wherein the pump is controlled by a control computer. 14. The cell culture device according to claim 1, wherein the cells retained in the culture carrier in the column-like device are cells of mammalian origin. 15. The cell culture device according to claim 1, wherein the cells retained in the culture carrier in the column-like device are human-derived cells. 16. The cell culture device according to claim 1, wherein the cells retained in the culture carrier in the column-like device are human-derived bone marrow or tissue-derived stem cells. 17. The cell retained in the culture carrier in the column-like device is a human articular cartilage-derived cell.
The cell culture device according to any one of 13 above. 18. A cell culture method comprising culturing cells with the cell culture device according to claim 1.