Cell/tissue culture apparatus

The invention provides a cell/tissue culture apparatus for applying a physical stimulation necessary for the proliferation and growth of a cell or tissue to a material to be cultivated serving as a cell or tissue to be cultivated, thereby realizing the culture as desired and protecting the material to be cultivated from contamination of various bacteria and so forth. The cell/tissue culture apparatus comprises a cylindrically formed material to be cultivated (matrix 18), a chamber (culture chamber 8) for accommodating therein the material to be cultivated, a circulation path (first circulation path 32) connected to the material to be cultivated through which a culture fluid is circulated in an inner side of the material to be cultivated, a pump (tube pump 36) for controlling a velocity of the culture fluid, control means (controller 60) and so forth, wherein the culture fluid (30) having different flows is supplied to the inner side and an outer side of the material to be cultivated, thereby applying an optional physical stimulation to the material to be cultivated serving as the cell or tissue to be cultivated, thereby realizing the culture as desired and protecting the material to be cultivated from contamination of various bacteria and so forth.

TECHNICAL FIELD

The invention relates to an apparatus for cultivating a cell or tissue (hereinafter referred to as cell/tissue culture apparatus) for use in the culture of a cell or tissue, and so forth to which a tissue engineering is applied, more particularly relates to a cell/tissue culture apparatus for efficiently realizing a metabolism function of a cell or tissue when performing an in vitro culture of the cell or tissue of a living body such as human body and so forth, and applying a physical stimulation necessary for prolongation, differentiation, and acceleration of a cell to a material to be cultivated.

BACKGROUND ART

There has been conventionally employed a method of performing an in vitro culture of a cell or tissue of a living body such as human body, wherein a temperature, a humidity, a carbon dioxide concentration, an oxygen concentration in an incubator (culture housing) are maintained at proper conditions, and the cell is cultivated in the incubator. The cell or tissue is placed in a culture fluid in a suspending state, and it is fixed to an interior or surface of a gel in which the culture fluid ingredient is contained, thereby proliferating and growing the cell or tissue, or the cell or tissue is transplanted in a material, that is exemplified as a matrix or scaffold, a carrier or a mold and so forth (hereinafter referred to as “matrix”), thereby proliferating and growing the cell or tissue in the matrix.

Meanwhile, it is important to apply a physical stimulation to a cell or tissue to be cultivated in addition to an environment condition such as a temperature, a humidity, a carbon dioxide concentration, an oxygen concentration for proliferating and growing the cell or tissue. Such a physical stimulation is an indispensable constituent for facilitating differentiation and growth of the cell or tissue and for growing the cell or tissue to be rendered closer to that in the living body. For a technology for applying a physical stimulation to the cell or tissue for proliferating and growing the cell or tissue, there are, for example, JP 2001-504697A entitled “Application of shear flow stress to chondrocytes”, U.S. Pat. No. 6,121,042 entitled “Apparatus and method for simulating in vivo conditions while seeding and culturing three-dimensional tissue constructs”, and so forth.

Although it is necessary to add a dynamic condition such as a physical stimulation to a static condition, a so-called culture environment such as a temperature, a humidity, a carbon dioxide concentration, an oxygen concentration for proliferating and growing the cell or tissue, there is a possibility that the control of the dynamic condition together with the static condition renders a control mode complex, and a factor caused by the invasion of various bacteria, and so forth increases. It is an important challenge to protect a material to be cultivated from contamination of various bacteria.

Accordingly, it is a first object of the invention to provide a cell/tissue culture apparatus capable of realizing a desired culture by applying a physical stimulation, which is necessary for proliferation and growth of the cell or tissue, to a material to be cultivated serving as a cell or tissue to be cultivated.

It is a second object of the invention to provide a cell/tissue culture apparatus for protecting the material to be cultivated from contamination of various bacteria and so forth.

DISCLOSURE OF THE INVENTION

To achieve the first object of the invention, a cell/tissue culture apparatus of the invention is characterized in comprising a cylindrically formed material to be cultivated (matrix18), a chamber (culture chamber8) for accommodating therein the material to be cultivated, and a circulation path (first circulation path32) connected to the material to be cultivated through which a culture fluid is circulated in an inner side of the material to be cultivated.

With the cell/tissue culture apparatus having such a configuration, the culture fluid is supplied to the interior of the cylindrical material to be cultivated accommodated in the chamber through the circulation path. Owing to the flow of the culture fluid, a necessary nutrition is supplied to the material to be cultivated through the interior thereof, and also a sheer stress corresponding to the flow of the culture fluid is applied to the material to be cultivated through the interior thereof as a physical stimulation, thereby facilitating the growth of cell or tissue of the material to be cultivated. The physical stimulation mimics a physical stimulation applied to a specific part of a human body, thereby forming a cell or tissue of, e.g., a blood vessel.

A cell/tissue culture apparatus of the invention is characterized in comprising a cylindrically formed material to be cultivated (matrix18), a chamber (culture chamber8) for accommodating therein the material to be cultivated, supply means (third circulation path54) for supplying a culture fluid to the chamber or circulating the same in the chamber, and a circulation path (first circulation path32) connected to the material to be cultivated accommodated in the chamber through which the culture fluid is circulated in an inner side of the material to be cultivated.

With the cell/tissue culture apparatus having such a configuration, the material to be cultivated is accommodated in the chamber and the culture fluid is supplied or circulated from the supply means. In this case, the supply means supplies the culture fluid successively to the chamber or it circulates the culture fluid between the chamber and a storage tank of the culture fluid. Further, the culture fluid flows to the inner side of the material to be cultivated through the circulation path, and it is supplied to the outer side of the material to be cultivated from the supply means. The sheer stress serving as a physical stimulation is applied to the inner and outer surfaces of the material to be cultivated through the flow of the culture fluid together with the supply of a necessary nutrition. That is, although the sheer stress is applied to the inner and outer surface layers of the material to be cultivated corresponding to the flow of the culture fluid, since the material to be cultivated is cylindrical, the flow of the culture fluid is restricted to one direction, at the inner surface layer of the material to be cultivated, and the sheer stress corresponding to such a flow is applied to the inner side of the material to be cultivated, thereby facilitating the growth of the cell or tissue. That is, according to the physical stimulation through the flow of the culture fluid, it can mimic a physical stimulation applied to a specific part of the human body, thereby forming the cell or tissue, e.g., of a blood vessel.

A cell/tissue culture apparatus of the invention is characterized in comprising a cylindrically formed material to be cultivated (matrix18), a chamber (culture chamber8) for accommodating therein the material to be cultivated, a first circulation path (first circulation path32) connected to the material to be cultivated through which a culture fluid30is circulated in an inner side of the material to be cultivated, and a second circulation path (bypasses40,42) through which the culture fluid30is circulated in an outer side of the material to be cultivated.

With the cell/tissue culture apparatus having such a configuration, since the first and second circulation paths are arranged such that the first circulation path supplies the culture fluid to the inner side of the material to be cultivated and the second circulation path supplies the culture fluid to the outer side of the material to be cultivated, they may be configured independently from each other or may branch off from a common circulation path. In this case, the culture fluid flowing from the first circulation path is restricted in flow to a certain direction by the cylindrical material to be cultivated while the culture fluid flowing from the second circulation path to the interior of the chamber is not fixed in direction. The sheer stress corresponding to the difference in flow is applied to both the inner and outer surface layers of the material to be cultivated owing to such a flow of culture fluid which is configured different in two-flow system, thereby facilitating the growth of cell or tissue of the material to be cultivated. The physical stimulation caused by the culture fluid having different fluid flows can mimic a physical stimulation to be applied to a specific part of the human body.

Since the second circulation path side is opened to the interior of the chamber according to the first and second circulation paths which branch off from a common circulation path, if a fresh culture fluid is supplied from the supply means to the interior of the chamber, the fresh culture fluid is taken into the first circulation path side through the second circulation path, so that nutrition and so forth necessary for culture is supplied to the material to be cultivated together with the physical stimulation, thereby facilitating metabolism of the material to be cultivated. In the case where the first and second circulation paths are independently configured, even if a fresh culture fluid is supplied to the first or second circulation path, or it is replaced with another culture fluid, the same operation can be effected.

The cell/tissue culture apparatus of the invention is characterized in that the second circulation path is bypasses (40,42) which are formed by allowing the second the second circulation path to branch off from the first circulation path. That is, with the cell/tissue culture apparatus having such a configuration, the culture fluid is forced to flow to the inner side of the material to be cultivated in the chamber through the first circulation path while it is supplied to the outer side of the material to be cultivated through the bypasses. As a result, the sheer stress serving as a physical stimulation through the flow of the culture fluid is applied to the inner and outer surfaces of the material to be cultivated together with necessary nutrition. In this case, the culture fluid flowing from the circulation path is restricted to a certain direction in its flow owing to the cylindrical material to be cultivated while the culture fluid flowing from the bypasses to the interior of the chamber is not fixed in direction. The sheer stress corresponding to the difference in fluid flow is applied to the inner and outer surface layers of the material to be cultivated owing to such a flow of culture fluid which is configured different in two-flow system, thereby facilitating the growth of cell or tissue.

Meanwhile, a small amount of cell is attached to a matrix serving as a material to be cultivated at the early stage of the culture, and hence the attachment of a material (i.e., a so-called an extracellular matrix) produced by the cell is small. The matrix serving as the material to be cultivated is formed of, e.g., spongy or cloth-shaped, wherein a culture fluid flows freely from the inner side of a wall surface of the matrix serving as the cylindrical material to be cultivated to the outer side thereof or from the outer side thereof to the inner side thereof in such a spongy state. However, since gaps in the matrix are filled with extracellular matrix after the middle stage of the culture where the cell is increased to form a tissue, the flow of the culture fluid between the wall surfaces of the cylindrical material to be cultivated is restricted. At this time, since it is difficult to allow a fresh culture fluid to flow to the inner surface side of the material to be cultivated through the first circulation path alone, a fresh culture fluid has to be supplied from another route.

Accordingly, if the bypasses which branch off from the first circulation path are used, a fresh culture fluid supplied to the interior of the chamber is taken in the bypasses and it can be circulated in the material to be cultivated through the first circulation path so that the fresh culture fluid can be supplied to the material to be cultivated. Further, the problem of the pressure difference between the inner and outer sides of the material to be cultivated can be resolved by the provision of the bypasses, and hence the inner and outer sides of the material to be cultivated can be maintained substantially at the same pressure, thereby obviating the inconvenience caused by the pressure difference.

The cell/tissue culture apparatus of the invention is characterized in that a distribution of the amount of flow of the culture fluid is differentiated between the first circulation path and the bypasses, thereby applying different shear stresses are applied to the outer side and the inner side of the material to be cultivated. That is, the culture fluid flowing from the first circulation path is restricted to a certain direction in flow owing to the cylindrical material to be cultivated while the culture fluid flowing from the bypasses to the interior of the chamber is not fixed in direction, and further, the distribution of the amount of flow of the culture fluid is differentiated, thereby applying the sheer stress corresponding to a velocity of flow and flow rate of the culture fluid to the inner and outer surface layers of the material to be cultivated. For example, it is possible to apply a large sheer stress to the inner side of the material to be cultivated and a very small sheer stress can be applied to the outer side thereof.

The cell/tissue culture apparatus of the invention is characterized in further comprising a pump (tube pump36) for supplying the culture fluid under pressure which flows to the material to be cultivated under pressure, and the supply state under pressure is variable. That is, the culture fluid needs to be supplied under pressure, and the supply state under pressure can be varied, thereby applying a sheer stress as desired to the material to be cultivated.

The cell/tissue culture apparatus of the invention is characterized in further comprising control means (controller60) for controlling a pressurized supply force of the pump with an optional pattern. That is, the culture fluid can be supplied under pressure to the circulation path in response to the revolution of the pump, and the supply state under pressure can be controlled in response to the rotary pattern, thereby applying a sheer stress serving as a physical stimulation as desired to the material to be cultivated. Further, the physical stimulation is allowed to be varied.

The cell/tissue culture apparatus of the invention is characterized in further comprising, to achieve the second object, a culture unit in which the chamber is formed is rendered in a hermetically sealed state, and the culture unit is detachable. That is, the material to be cultivated can be moved per unit of culture unit, and the hermetical sealing is made easily so that the material to be cultivated can be protected from contamination of various bacteria and so forth.

The cell/tissue culture apparatus of the invention is characterized in further comprising photographing means (CCD camera86) for photographing the material to be cultivated in the chamber. That is, the photographing of the material to be cultivated in the chamber and the obtaining of image data supplies important data for the proliferation and growth of the material to be cultivated.

The cell/tissue culture apparatus of the invention is characterized in further comprising the culture unit which is transparent as a part or as a whole and photographing means, wherein the material to be cultivated is accommodated and cultivated in the chamber formed in the culture unit, and the material to be cultivated can be photographed by the photographing means from an outside of the chamber. That is, the material to be cultivated can be photographed from the outside of the chamber without disturbing a culture environment in the chamber, thereby obtaining its image data.

Other objects, features, advantages and so forth of the invention are more clarified from the description of the mode for carrying out the invention and the embodiment as illustrated in the drawings.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention and the mode for carrying out the invention are now described in detail with reference to the embodiment as illustrated in the attached drawings.

FIGS. 1to6show the cell/tissue culture apparatus according to an embodiment of the invention, whereinFIG. 1shows a culture unit and so forth on a culture circuit,FIG. 2shows an internal construction of the culture unit,FIG. 3is a sectional view of the culture unit taken along the line III—III shown inFIG. 2,FIG. 4is a sectional view of the culture unit taken along the line IV—IV shown inFIG. 2,FIG. 5shows the culture circuit and a controller, andFIG. 6shows the culture circuit and the controller.

A culture unit2for cultivating a material to be cultivated serving as a cell or tissue of a living body such as a human and so forth is provided in the cell/tissue culture apparatus, and the culture unit2has a high grade heat resistant property and is made up of a synthetic resin material or a metal material from which a material adversely affecting on a living body does not liquate out, for example, fluorine resin, PEEK, high grade heat resistance polypropylene, silicone, stainless steel and so forth.

The culture unit2comprises a container4and a cover6which is detachably attached to the container4, and a culture chamber8serving as a closed culture space is formed in the interior of the culture unit2. According to the embodiment, the cover6is detachably fixed to the container4by a plurality of fixing bolts10, and O rings12are interposed between the container4and the cover6, so that a sufficient airtightness is maintained in the culture chamber8.

The culture chamber8comprises a circular portion14at an inner side thereof and a rectangular portion16at an opening side thereof, and a matrix18serving as a cylindrical material to be cultivated is accommodated in the rectangular portion16side. That is, the matrix18is cylindrical and it is molded as a mold of a blood vessel to be molded as means for proliferating the blood vessel, and the cell of the blood vessel of a human is sowed in the matrix, according to this embodiment.

A pair of culture ports20,22serving as supporting means of the matrix18are provided, and they penetrate the rectangular portion16of the container4from an outer surface side thereof and fixed to the rectangular portion16of the container4. The culture ports20,22project in the culture chamber8by a predetermined length and they oppose each other, wherein an opening end of a through hole19is engaged with the projecting portion thereof so that the matrix18is held at the rectangular portion16side of the culture chamber8. In this embodiment, although the end of the matrix18is brought into contact with an inner wall of the culture chamber8, it can be held in a non-contact state. The culture chamber8maybe set to have the shape and size corresponding to the size of the matrix18to be cultivated.

According to the embodiment, the culture ports20,22have flanges26formed on the midway of a pipe24, and the pipe24penetrates a transparent hole28formed in the rectangular portion16side of the culture chamber8and fixed to the transparent hole28, and integrated with the culture unit2. The flanges26serve as positioning means for restricting the projecting length of the culture ports20,22relative to the interior of the rectangular portion16. According to the embodiment, although the culture ports20,22are formed separately from the culture unit2, they may be integrated with a member constituting the culture unit2.

A first circulation path32through which a culture fluid30is circulated in the through hole19of the matrix18is formed in the culture ports20,22, and the first circulation path32is made up of a circulation tube34. A tube pump36serving as supply means under pressure of the culture fluid30is attached to the first circulation path32for facilitating proliferation and growth of the cell or tissue on the matrix18, and a rotating force is applied to the tube pump36by a motor38serving as driving means, whereby fluid flow which is the same as the blood flow of a human body is formed by controlling the rotation of the driving means.

Further, according to the embodiment, bypasses40,42serving as the second circulation path through which the culture fluid30is circulated outside the matrix18, i.e. at the outer wall side thereof branch off from the first circulation path32, and they are connected to bypass ports44,46which are opened to the container4of the culture unit2. The bypass ports44,46project from the outer surface of the container4of the culture unit2, and through holes48are formed in the interior of the bypass ports44,46. Accordingly, the culture fluid30of the first circulation path32flows toward the matrix18as shown in arrows A, B, and passes through the bypasses40,42and the bypass ports44,46as shown in arrows C, D, then it flows inside the culture chamber8. Although each tubular diameter of the culture ports20,22and the bypass ports44,46can be set at an optional value, the tubular diameter of the bypass ports44,46side is set to be smaller than that of the culture ports20,22according to the embodiment, so that a flow rate of the culture fluid30flowing through the bypass ports44,46is restricteted.

Meanwhile, since the bypasses40,42are opened to the culture chamber8, the culture fluid30supplied to the culture chamber8can be taken into the bypasses40,42so that the culture fluid30is circulated through the first circulation path32, thereby circulating the culture fluid30in the inner side of the matrix18. That is, assuming that a fresh culture fluid30is supplied to the culture chamber8, the culture fluid30can be circulated through the first circulation path32and the matrix18through the bypasses40,42.

Further, ports50,52for circulation are formed at the circular portion14side of the culture chamber8in a direction perpendicular to the matrix18inside the culture chamber8, and a third circulation path54serving as supply means through which the culture fluid30is supplied is formed in the ports50,52for circulation. The ports50,52for circulation project from the outer surface of the container4of the culture unit2, and have through holes56therein, and the third circulation path54is constituted by circulation tube58mounted on the ports50,52for circulation, whereby the fresh culture fluid30is supplied from the supply means (e.g., a culture fluid bag94) of the culture fluid. Depicted by arrows E, F show the culture fluid30flowing to the culture chamber8through the third circulation path54and the flowing direction thereof. That is, although the culture fluid30at the first circulation path32side is supplied to the inner side of the matrix18, since the culture fluid30at the third circulation path54side is supplied to the outer side of the matrix18though the culture chamber8, and to the first circulation path32and the inner side of the matrix18through the bypasses40,42, the fresh culture fluid30is constantly circulated in the inner side of the matrix18.

A controller60serving as means for controlling the rotation of the motor38, monitoring the proliferation and growth of the cell or tissue and so forth is provided in the culture unit2. The controller60has a control part62for controlling the rotation of the motor38in the manner that a driving current is forced to flow to the motor38and so forth, e.g., as shown inFIG. 5, whereby a program control for setting a rotary pattern and so forth is executed by the control part62. The motor38has a rotary sensor64for detecting the rotation thereof, and a temperature sensor66for detecting a temperature of the motor, wherein each signal for detecting the rotation and the temperature of the motor is supplied to the control part62as a control input. The control part62has a CPU serving as processing means, a ROM, a RAM, and so forth serving as a memory, and a program such as rotation conditions and so forth is set from an externally connected input unit68. The control part62has a display part70, an operation switch74for giving an operation instruction, operation display means, for example, a display lump76, a warning buzzer78serving as warning means, an external storage device80serving as means for storing various data, a database82, and so forth.

Further, according to the embodiment, a transparent wall portion84thorough which the interior of the culture chamber8can be seen is provided on a part or the whole of the culture unit2at the cover6side, and a CCD camera86serving as photographing means for photographing the matrix18inside the culture chamber8is installed in the vicinity of the transparent wall portion84, wherein image obtained by the CCD camera86is supplied to an image processing unit88where it is processed, and also a processed image is displayed on an image display unit90and is also supplied to a decision unit92as decision information. The state of the proliferation and growth of the cell or tissue is decided by the decision unit92in response to the change of color, the shape and so forth of the matrix18based on the processed image, and the result of decision is supplied to the control part62. If the state of the matrix18in the culture chamber8is grasped as image information, and it is observed through this image information, the state of proliferation and growth of the cell or tissue on the matrix18and each stage of the growth of the cell or tissue can be visually grasped with accuracy, and an appropriate countermeasure can be taken corresponding to the state of the proliferation and growth of the cell or tissue.

Further, according to the cell/tissue culture apparatus of the invention, it is configured, e.g., as shown inFIG. 6, such that it is accommodated in a culture housing93forming an optimum culture environment. The third circulation path54has the culture fluid bag94for storing the culture fluid30therein, a gas absorption tube96for allowing the culture fluid circulated through the third circulation path54to absorb gas, a fluid supply valve98for circulating the culture fluid30, and a pressure regulating valve100. A piston104which is advanced or retracted by a piston driving unit102is provided in the fluid supply valve98, wherein when the piston104is advanced, a valve106is opened and a valve108is shut while when the piston104is retracted, the valve106is shut and the valve108is opened, thereby effecting delivery of a predetermined amount of culture fluid30like the heart. A valve112which is advanced or retracted by a valving unit110is provided in the pressure regulating valve100, wherein when the valve112is advanced, the pressure regulating valve100is shut and when the valve112is retracted, the pressure regulating valve100is opened, thereby effecting the pressure regulation of the culture fluid30in the third circulation path54.

The culture housing93is provided with a heater114serving as heating means, a fan116serving as blowing means, a humidifier118serving as means for setting a humidity as desired, and a temperature sensor120, and to which N2is supplied from an N2supply unit122, O2is supplied from an O2supply unit124, and CO2is supplied from a CO2supply unit126, thereby forming an optimum culture environment adapted for the proliferation and growth of the cell or tissue.

A culture processing using the cell/tissue culture apparatus is next described with reference to flowcharts shown in FIG.7and FIG.8. InFIGS. 7 and 8, depicted by circled a, b and c show connecting symbols of the divided flowcharts extending overFIGS. 7 and 8.

In step S1, the controller60is set at an operation state, and an operation program is inputted through the input unit68, thereby setting conditions such as rotary periods T1, T2, T3of the motor38and continuation time T12, T22, T32and so forth. After setting such conditions, when the operation switch74turns ON in step S2, the program goes to step S3where an operation indication is displayed on the display part70and the display lump76is lit up.

In step S4, the motor38rotates so that variable driving of the motor38is effected at period T1, at the speed ranging from the maximum speed R×K1to the minimum speed R×K1×C1, then the program goes to step S5, where during the rotation of the motor, it is decided whether anomaly of the rotation of the motor occurs or not based on detection outputs from the rotary sensor64and the temperature sensor66, and when there dose not occur anomaly, the program goes to step S6. In step S6, image data is fetched from the image processing unit88, and it is stored in the memory in the control part62, and the external storage device80, then it is registered in the database82.

In step S7, it is decided whether the continuation time T12elapses or not, and the processings of the steps S4to S6are continuously executed until the continuation time T12elapses.

Upon elapse of the continuation time T12, the program goes to step S8where the variable driving of the motor38is effected at period T2, at the speed ranging from the maximum speed R×K2to the minimum speed R×K2×C2, then the program goes to step S9, where during the rotation of the motor, it is decided whether anomaly of the rotation of the motor occurs or not based on detection outputs from the rotary sensor64and the temperature sensor66in the same manner as step S5, and when there dose not occur anomaly, the program goes to step S10. In step S10, image data is fetched from the image processing unit88, and it is stored in the memory in the control part62, and the external storage device80, then it is registered in the database82in the same manner as step S6.

In step S11, it is decided whether the continuation time T22elapses or not, and the processings of the steps S8to S10are continuously executed until continuation time T22elapses.

Upon elapse of the continuation time T22, the program goes to step S12where the variable driving of the motor38is effected at period T3, at the speed ranging from the maximum speed R to the minimum speed R×C3, then the program goes to step S13, where during the rotation of the motor, it is decided whether anomaly of the rotation of the motor occurs or not based on detection outputs from the rotary sensor64and the temperature sensor66in the same manner as step S9, and when there dose not occur anomaly, the program goes to step S14. In step S14, image data is fetched from the image processing unit88, and it is stored in the memory in the control part62, and the external storage device80, then it is registered in the database82in the same manner as step S10.

In step S15, it is decided whether the continuation time T32elapses or not, and the processings of the steps S12to S14are continuously executed until the continuation time T32elapses.

Upon elapse of the continuation time T32, the program goes to step S16where a completion display is effected, then the program goes to step S17where it is decided whether the operation switch74turns OFF or not, and the processings of the steps S12to S16are executed until the operation switch74turns OFF.

When the operation switch74turns OFF, the program goes to step S18where stoppage of the rotation of the motor, and the cancellation of the operation display and completion display are effected, thereby completing the culture program.

In the case where the anomaly of the rotation of the motor is turned out in step S5, step S9, and step S13, the program goes to step S19where the rotation of the motor stops, then the program goes to step S20where a notice of generation of anomaly is displayed on the display part70as a warning display, and the warning buzzer78is sounded.

A, B, C and D inFIG. 9show the operation of the culture program, wherein A shows ON, OFF of the operation display, B shows ON, OFF of the completion display, C shows image decision outputs of V1, V2, V3, D shows a speed of the motor38, wherein inFIG. 9D, depicted by I indicates a first stage showing the processing in step S4, II indicates a second stage showing the processing in step S8, and III indicates a final stage showing the processing in step S12.

Meanwhile, in this embodiment, the coefficients K1, K2, and C1, C2, C3used in the processings in step S4, step S8and step S12are set values stored in the memory of the control part62but they may be changed by inputting them optionally from the input unit68. Further, according to the embodiment, although it is explained that the circulating direction of the culture fluid30is set to one direction, but the direction may be changed to a reverse direction or reciprocal direction.

In the practical culture, the matrix18in which the cell or tissue is transplanted is accommodated in the culture chamber8after removing the cover6, and it is installed in the culture housing93. After a temperature, a humidity, a carbon dioxide concentration, an oxygen concentration and so forth in the culture housing93are set to optimum values or conditions, the culture fluid30having a flow rate optimum for the cell or tissue is supplied to the matrix18, thereby executing the culture program.

When the culture fluid30is supplied to the matrix18through the circulation paths32,54, the flow of the culture fluid30acts on the inner and outer surfaces of the matrix18, so that the cell or tissue on the matrix18proliferates and grows while it is subjected to the application of a shear stress in the circumferential direction of the culture chamber8.

Described next is the shear stress to be applied to the matrix18with reference to FIG.10. Assuming that a viscosity coefficient of the culture fluid30is μ, velocity of flow of the culture fluid30is u, variation of the velocity of flow is du, a difference between the different flow velocities is dz, a shear stress τ acting on the surface layer of the matrix18serving as a blood vessel is expressed as follows.
τ=μ·du/dz

In this case, the velocity of flow u on the outer wall surface of the matrix18is substantially zero, and a shear stress τOUTacting on this part is very small, but a large shear stress τINacts on the surface layer of the inner side of the matrix18. Such a shear stress τ becomes a physical stimulation to contribute to the proliferation and grows of the cell or tissue.

In this case, the shear stress τINacting on the inner side of the matrix18, i.e., on the inner surface layer of the through hole19and the shear stress τOUTacting on the inner surface layer of the outer side of the matrix18are set in the manner that since the matrix18is cylindrical at the inner side of the matrix18, a flowing direction is restricted, and the velocity of flow of the culture fluid30is high and the shear stress τINbecomes large while since the velocity of flow of the culture fluid30at the outer side of the matrix18is slow and has no specific direction, the shear stress τOUTis small. As a result, a large shear stress is applied to the inner side of the matrix18while a small shear stress is applied to the outer side of the matrix18. That is, since the cell or tissue which grows in the cylindrical matrix18is subjected to an application of a large shear stress at the inner side of the matrix18owing to the flow of fluid while it is hardly subjected to an application of a shear stress at the outer side of the matrix18owing to the flow of fluid, it grows as a blood vessel inner cuticle tissue at the inner side of the matrix18while as a blood vessel outer cuticle tissue at the outer side of the matrix18. Since the blood vessel tissue of a human is differentiated in inner and outer side thereof, the tissue on the matrix18obtained by such culture forms a tissue close to the blood vessel of a living body.

If a flow rate of the culture fluid30which is allowed to flow by the tube pump36is controlled by a program by which the flow rate is changed to an optimum flow rate as the culture time elapses, a flow of fluid same as that of a living body is obtained. In this case, it is efficient that the flow rate and the velocity of flow of the culture fluid30flowing toward the inner side of the matrix18is controlled to become an optimum value in response to the stage of growth of the cell to the tissue.

Upon completion of the execution of the culture program, the culture unit2operates such that a circulation tube58of the circulation path54connected to the ports50,52is shut, for example, as shown in FIG.11. According to the embodiment, a pinch cock128, a cock valve130, which overstride the culture unit2and serve as shutting means, are attached to the circulation tube58, and if the shutting operation is effected, the culture unit2can be separated from the circulation path54and the culture chamber8can be kept in a hermetically sealed state, so that the culture fluid30is not leaked out from the culture chamber8. In this case, the circulation path32is separated from the tube pump36and the circulation tube34is detached from the tube pump36and it is better to be moved together with the culture unit2.

Further, if the culture unit2is sterilized by a sterilizing method using an autoclave, and so forth, UV sterilization, gummer rays sterilization, and so forth, the interior of the culture unit2can be maintained in an aseptic condition for a long period of time. According to the embodiment, although the pinch cock128and the cock valve130are employed as means for shutting the circulation tube58, other closing means may be used.

In this case, the circulation tube34at the first circulation path32side may be shut by other shutting means as a pinch cock and a cock valve and so forth in the same manner as the circulation tube58.

Although the bypasses40,42are configured to be formed by branching off from the first circulation path32in the embodiment, they may be formed separately independently from the first circulation path32as the second circulation path or they may be formed by providing separate openings at side wall portions of the culture ports20,22.

In the case where the bypasses40,42are configured as the independent second circulation path, if supply means for supplying a fresh culture fluid is installed at the first circulation path32or bypasses40,42sides, the fresh culture fluid together with a physical stimulation such as a necessary shear stress and so forth can be supplied to the inner side of the matrix18through the first circulation path32.

Further, the tube pump36has been used as supply means under pressure according to the embodiment, for example, a plunger type pump may be used.

As mentioned in detail above, the following effects can be obtained by the invention.

a It is possible to apply a physical stimulation such as a shear stress caused by the velocity of flow of the culture fluid, and so forth to the material to be cultivated in the chamber in a non-contact state. As a result, it is possible to apply the shear stress, which imitates a physical stimulation on the living body, can be applied to the material to be cultivated, thereby contributing to the facilitation of the culture.

b The manner of flow of the culture fluid is differentiated between the inner side and an outer side of the cylindrical material to be cultivated, and the shear stress can be independently applied to the inner side and outer side of the cylindrical material to be cultivated.

c The culture fluid is supplied to the inner side of the cylindrical material to be cultivated through the first circulation path and it is supplied to the outer side of the cylindrical material to be cultivated through the second circulation path so that the shear stress can be independently applied to the inner side and the outer side of the cylindrical material to be cultivated, and a fresh culture fluid can be supplied from the first circulation path to the material to be cultivated through the second circulation path or bypasses.

d A shear stress corresponding to each stage of the growth of the cell or tissue can be applied to the material to be cultivated through the flow of the culture fluid.

e The culture unit for accommodating therein the material to be cultivated can be moved while it is independently separated from or detachably attached to the culture circuit, thereby protecting the material to be cultivated from contamination of the bacteria and so forth.

f A physical stimulation as desired can be applied to the material to be cultivated, so that a physical stimulation corresponding to the part of a living body can be realized and the facilitation of the culture can be enhanced.

g Each stage of the growth of the cell or tissue can be accurately grasped by image.

Although the configurations, operations and effects of the cell/tissue culture apparatus serving as the mode for carrying out the invention are described with reference to the embodiment as shown in the attached drawings, the invention is not limited to such a mode for carrying out the invention and the embodiment, but it includes all the configurations, which can be predicted or conjectured by a person skilled in the art, such as various configurations, modifications and so forth which can be conjectured by the object, the mode for carrying out the invention, and the embodiment of the invention.

INDUSTRIAL APPLICABILITY

As mentioned above, the cell/tissue culture apparatus of the invention is useful for the culture technology of the cell or tissue to which a tissue engineering is applied, more particularly it is adapted for performing an in vitro culture of the cell or tissue of a living body such as human body, and is also adapted for efficiently realizing a metabolism function of a cell or tissue and applying a physical stimulation necessary for prolongation, differentiation, and acceleration of the cell to the material to be cultivated.