JP2012147678A - Multiple culture apparatus for single sample - Google Patents

Abstract

[PROBLEMS] Development of closed-system culture devices for long-term automatic culture medium replacement, efficient culture methods, contamination, infectious disease countermeasures, and remote monitoring operations for medical device development.
A diaphragm pump function is attached to a culture vessel lid portion of a closed culture device, a medium inlet is attached to the center of the bottom of the culture vessel, a rectifying plate is attached in order to disperse a spring medium in all directions, and a discharge port is a culture vessel It is provided in the upper part, and it is a multiple culture method that rotates the porous scaffolding sheet for a long period of time.By removing floating wastes and subsidence wastes attached to the cultured cell sheet surface, and rotating and moving in the medium, Promote contact efficiency. A multi-culture device for a single specimen that can be monitored remotely by attaching a camera and pH measurement device to the cell culture state.
[Selection] Figure 2

Description

The present invention is a multi-culture apparatus for one specimen composed of a biocompatible material, which promotes cell proliferation and differentiation, and induces regeneration of living tissue, and remotely monitors the culture state, an endoscope camera, and a pH measurement apparatus. The present invention relates to a culture apparatus that performs culture in vitro and establishes a method for efficiently cultivating cell culture and cultures cells and tissues necessary for repairing a defective tissue of a human body.

There are two methods for inducing regeneration of living tissue. One is cell transplantation treatment in which cells are transplanted directly into the living body, and the other is to promote cell proliferation and differentiation by using a culture device composed of biocompatible materials, and to induce regeneration of living tissues in vitro. It is a method to do.

The present invention is a technique for performing cell / tissue culture in vitro using a culture apparatus, and the culture apparatus is focused on the development of a culture apparatus capable of ensuring the safety of a closed human cell culture apparatus or an automatic medium exchange method. .

Operations with general-purpose culture vessels such as ordinary culture dishes and culture flasks require specialized knowledge and skill of doctors, researchers, etc., which increases labor costs and management costs. Becomes expensive and extremely difficult to achieve in the medical business.

A culture solution is used for culturing cells and tissues, and in order to grow cells / tissues in a culture solution in a culture vessel containing cells / tissues, nutrients, oxygen, etc. are supplied from the culture solution to the cells / tissues, In order to promote the growth of cells and tissues by supplying nutrients and removing wastes, the exchange of culture medium is an extremely important matter for culturing cells and tissues, and automation is widely required.

As a device to apply mechanical pressure stimulus to in vitro culture of load-type cartilage (meniscus etc.), the research group of Harvard University in the United States and the research group of the University of Tokyo (Reference 3) have developed hydrostatic pressure culture devices. As an example of a culture system for hydrostatic pressure stimulation, an example of stimulation load on a chondrocyte through a culture medium by a pump is shown, and a load corresponding to the pressure in the joint is applied to the chondrocyte by applying a load to the culture medium. An example of the stimulation load is shown, and the medium is replaced by a plunger pump.

An example of a tilting culture device is shown for an experimental device for blood vessel culture. A gauze seeded with venous vascular endothelial cells and a gauze seeded with fibroblasts are stacked, and the gauze is placed on a flat plate and a medium reservoir at both ends. When gauze is hung in the upper medium tank and the tip is moistened with the medium, the gauze gradually gets wet from the end due to capillary action, and the medium flows into the lower medium tank. There is also an experimental apparatus in which the medium stored in the medium tank is returned to the upper medium tank by a pump and the medium is continuously supplied.

In order to prevent bacterial infection and contamination, the culture medium is replaced with a sealed container or a closed circuit circuit, and the medium replacement method using bellows (Patent Document 1) and (Patent Document 2) is used. In order to prevent the influence of the culture medium exchange method, and further the flow of the culture solution (Patent Document 3) using a tube pump (Patent Document 3) or a tube pump (Patent Document 4) (Patent Document 5) As the monitoring, there is a method of photographing the inside of the culture container with a camera and confirming the pH of the medium using a pH reagent (Patent Document 6).

There are also a method of laminating cell culture chambers having a culture fluid flow channel structure by perfusing culture fluid (Patent Document 7) and a cell culture cassette (Patent Document 8) sandwiching a cell culture carrier.

JP-A-4-356184 JP 2004-89138 A JP 2004-350557 A JP 2006-264647 A JP2007-20493 JP2007-110932A JP 2002-316136 A JP2007-167002

The present invention solves the problems of the prior art as described above, and as a regenerative medical culture apparatus shown in the NEDO roadmap, it is downsized (spread in research institutions), development of a low-cost apparatus, cassette type Practical use of culture containers, development of sealed containers, shortening of culture period, etc. are clearly shown as development guidelines for culture apparatuses. Furthermore, the Ministry of Health, Labor and Welfare states that it is desirable to dispose of specimens that are in contact with cultured cells and culture solutions as a culture apparatus for regenerative medicine.

In the present invention, there are no parts shared with cultured cells of other specimens in a multi-culture apparatus for one specimen, contamination with other cultured cells is prevented in advance, and all parts in contact with the cultured cells of the specimen can be discarded. It is.

The culture unit is a closed system that prevents bacteria from entering, the culture unit is sterilized in the clean bench, and the culture unit is also mounted in the clean and constant temperature case in the clean bench. Double-closed culture device equipped with a controller unit with a drive unit after double-closure with a closed culture unit and a closed thermostatic case and then removed from the clean bench.

Research and development of regenerative medicine is promoted by the Ministry of Home Affairs, Ministry of Education, Culture, Sports, Science and Technology, Ministry of Economy, Trade and Industry, and Ministry of Health, Labor and Welfare. The development of cell culture systems includes the development of closed and fully automatic cell culture devices, and moisture-proof packing. The development direction of the automatic monitoring device by the camera + remote operation technology and the development direction are clearly shown, and this guideline has been regarded as a priority issue to be solved.

The inventors have repeatedly discussed the subject of development of the present invention and have determined the following items to be described.
1. Automatic medium exchange (prevents infringement of cultured cells due to medium flow)
2. Efficient culture method (effective nutrient / oxygen supplementation method, shortening culture time)
3. Measures against infectious diseases (disposal of parts in contact with cultured cells and culture medium of the contributor)
4. Closed system / Fully automatic culture device 5. Remote operation of monitoring As a first step to solve these problems, we started automatic medium exchange.

The culture medium exchange method according to the present invention has developed a unique automatic culture medium exchange method from a completely different idea from the prior art. A diaphragm is attached to the lid of the culture vessel, a medium injection port is provided at the center of the bottom of the culture vessel, a rectifying plate is installed at a slightly spaced position, an upper discharge port is provided laterally below the diaphragm, and a linear actuator on the diaphragm And a joint that can be interlocked with the joint, and by expanding and contracting the linear actuator, the culture medium in the culture container is pressurized and depressurized to discharge and inject the medium, and the medium is automatically replaced.

As for the medium exchange method, a method of using a plunger pump, a centrifugal pump, a tube pump, a diaphragm pump, etc. as the culture container has been announced, as well as a method of repeatedly sucking and injecting the culture solution with a pipette nozzle from a manual spoid exchange. Depending on the amount of inflow and the shape of the culture vessel, the cultured cells may flow out, and the cells may be stimulated by the flow of the medium. However, the most important issue for changing the medium is not to apply unnecessary stress to the cells being cultured. That is.

In the medium exchange method of the present invention, the backflow prevention device on the medium injection side is closed when the medium is discharged, the backflow prevention device on the medium discharge side is closed when the medium is injected, and the flow of the medium is as in the case of injection from an external pump. There is no flow from the inlet to the outlet in the culture vessel. In the medium exchange method of the present invention, when the medium is discharged, the upper medium corresponding to the diaphragm insertion volume is discharged into the culture container, and the bottom medium does not flow out. At the time of injection, the rising volume of the diaphragm is injected, but at the time of injection, the backflow prevention device on the discharge side is closed and injected from the bottom of the culture vessel, but the medium does not flow out from the discharge port.

Furthermore, the medium that has flowed into the culture container collides with the rectifying plate and is dispersed in all directions, and the dispersed injected medium pushes up the medium in the culture container by the amount of injection. At the time of discharge, the culture medium in the culture container is pressurized, the infusion medium stays in place, the upper medium is discharged, and the infusion medium moves in layers from the bottom to the top, completely different from the conventional medium injection method. Different medium injection methods.

By changing the rotation drive (motor) to linear movement with a rotating cam and clamp, the shape of the direct acting actuator can be reduced, and the interlocking fixed position of the rotating cam and clamp can be changed, starting from the center of the rotating cam. The amplitude of the diaphragm can be varied at a fixed distance, and the amount of medium exchange in the culture vessel can be changed.

As a method of controlling the direct acting actuator, the ON time and OFF time of the direct acting actuator (motor) are adjusted by adjusting the ON time and OFF time by repeatedly turning the current ON and OFF in a pulsed manner. Adjustment is performed, the up / down time of the clamp is determined by the cam rotation time, the diaphragm amplitude is determined, and the cam driving time of the rotation is determined, thereby determining the medium exchange amount according to the driving time of the rotation.

By adjusting the diaphragm amplitude and clamp linear movement time, and setting the daily linear actuator drive time with a timer, the amount of medium exchange per day can be determined. By determining the time setting and the daily driving time, it is possible to determine the daily medium replacement amount. Further, if the number of driving days is determined, medium replacement can be performed automatically for a long period of time.

When static culture is performed on a culture dish or the like, nutrients, oxygen, etc. are supplied to the cells in one direction from the top of the cell sheet. In the case of static culture, fresh media and cultured cells such as waste products are used. However, it cannot be said that it has taken in nutrients, oxygen, etc. effectively.

In the multi-layer culture, cells are seeded on the front and back of the scaffold equipped with a porous sheet, and double culture on the front and back surfaces is performed, but suspended wastes adhere to the cultured cell surface on the back surface, Supply of nutrients, oxygen, etc. is hindered. This floating waste is currently processed by completely extracting the culture solution from the culture vessel, but it is extremely difficult to discharge the floating waste in the closed culture vessel.

The present invention reverses the porous sheet scaffold to the treatment of the floating waste, disengages the floating waste by reversing the back surface to the surface, and further rotates the scaffold fixing part very gently, It is possible to increase the contact efficiency, increase the supply of nutrients, oxygen, etc., increase the culture efficiency of cell culture, and shorten the culture period.

Since the culture apparatus is directly equipped with a diaphragm pump, the culture medium exchange structure of the automatic culture apparatus of the present invention is extremely simple, and the piping is only from the culture medium tank to the culture container and from the culture container to the discharge medium tank, It is a culture device with an extremely simple structure that does not require piping to the pump.

Simple culture device culture tank, culture vessel, drainage culture tank and piping, and accessories are all disposable, biocompatible material, flexible plastic, and durable synthetic resin bags. By using the draining medium tank, the culture apparatus of the present invention can completely seal a portion where the medium is perfused, and becomes a completely closed culture apparatus.

At the same time as the closed system culture apparatus described above, it is a multiple culture apparatus for one specimen, and all parts such as a culture vessel, a medium tank, a pipe for a discharge medium tank, a backflow prevention apparatus, etc. are a single sample apparatus, There is no risk of contamination between the cultured cells of the specimen and the cultured cells of other specimens.

For pH measurement of the medium discharged from the culture container, a pH measurement sensor is attached in the middle of the pipe after the medium passes through the discharge side check valve. A signal from a sensor that measures the state of the medium used for culture is sent to a measuring device, the signal is analyzed and recorded, and the culture state of the cultured cells is managed remotely.

Mounting the sensor of the pH measurement device in the middle of the piping of the backflow prevention device and the discharge medium tank from the culture device allows remote control of pH measurement without affecting the cells in the culture vessel. Is possible.

Make a hole in the side of the culture vessel and attach a camera fitting made of a biocompatible transparent material (acrylic, glass, etc.) that can be attached directly to the culture vessel. A visual camera is attached to a camera fitting made of a biocompatible transparent material (acrylic, glass, etc.), and information on the cell culture state in the culture vessel is imaged, analyzed, and recorded on a personal computer, and information on the culture status of the cells in the culture vessel is obtained. Remote control is performed with a personal computer for monitoring.

Attach a tube clamp in the middle of the reflux line of the culture medium, the injection side backflow prevention device of the injection pipe in the middle of the perfusion pipe and the culture vessel injection side pipe, and attach the tube clamp to the discharge side backflow prevention device of the culture container discharge side pipe, After culturing, both tube clamps are closed, the tube on the backflow prevention device is cut, and the joints of the two drive devices are removed. The culture vessel can be removed from the culture device in a closed state, and the cultured cells remain in the closed culture vessel. In the case of other medical facilities or remote places, the room can be moved and transported in a sterilized thermostatic case.

The culture apparatus according to the present invention can perform automatic medium exchange for a long time as a closed system culture apparatus with a small and simple configuration, and by rotating the porous scaffold sheet in a closed environment, two-layer culture on the front and back sides is also possible. Equipped with an endoscopic camera directly on the culture vessel, also equipped with a pH measurement device sensor, can be monitored remotely, and may promote cell / tissue differentiation and proliferation and effectively work on cell / tissue formation It is a culture device with a secret.

Although the above automatic cell culture device requires research on the effectiveness of regenerative medicine in the field of regenerative medicine, automatic operation such as closed-system culture device, long-term medium exchange, and rotation culture method is possible, and multiple culture for one specimen The device is being developed based on careful attention to contamination, infectious diseases, etc., and is assumed to be a culture device that is sufficient to support early realization of regenerative medicine.

The cost of constructing and maintaining the CPC room at the culture facility is very expensive, and the facility and maintenance costs are directly added to the medical costs, and the medical costs are soaring, causing problems in terms of price. Not only that, but it will also be a serious negative effect in the future. Since the culture apparatus of the present invention is a double-closed culture apparatus having a culture part and a closed thermostatic case, there is a high possibility that the culture can be performed outside the CPC room, and it can be a very useful culture apparatus.

The culture apparatus according to the present invention will be specifically described in Examples, but these Examples do not limit the present invention.
FIG. 1 is a configuration diagram of the entire reflux culture apparatus of the present embodiment, and shows the reflux (circulation) culture method according to claim 1. In order to recirculate the medium, the carbon dioxide gas in the carbon dioxide incubator 1 must be taken into the exhaust medium and reused by the method described in claim 2. In order to reproduce the blood flow of a living body ex vivo, the necessity of refluxing the medium must be supplied by continuous pulsatile flow for 60 minutes per minute, 24 hours, and 3 weeks of culture. For this purpose, an automatic reflux culture apparatus that circulates the culture medium is required, and a controller unit that remotely operates the culture unit and the drive unit installed in the carbon dioxide incubator 1.

A culture apparatus for perfusing a culture medium according to claim 3, wherein the culture part is a closed system, culture is performed in a sterilized constant temperature case, and medium change is automatically performed for a long time without opening and closing the constant temperature case. The diaphragm 23 is moved up and down by the linear actuator 13, the inside of the culture container is filled with the medium, the medium in the culture container is pressurized and depressurized, the amplitude, the driving speed and the driving time are set, and the exchange amount of the medium is determined. .

The perfusion-type closed culture apparatus described in claim 3 pumps out the medium from the medium tank 11 and is discharged to the discharge medium tank 12 after passing through the culture container 10. For this purpose, the inside of the medium tank 11 is decompressed, The discharge medium tank 12 is injected with the discharge medium, and the discharge medium tank is gradually pressurized and back pressure is applied, and the balance of the pressure is lost and adversely affects the flow of the medium. 5 is used to balance the pressure of the entire culture apparatus.

The features of the culture medium injection method in the culture container according to the structure in the culture container will be described with reference to the culture container sectional view of FIG. The culture vessel upper discharge port 32 is provided on the upper side of the culture vessel below the diaphragm, the medium injection port is provided at the center injection port 48 at the bottom of the culture vessel, and the rectifying plate 24 is installed immediately above, thereby rectifying the spring medium at the time of medium injection. The plate 24 is used to disperse the medium in all directions, and when discharged, the medium is pressurized and the injected medium is stopped at that state at the bottom. At the time of the next injection, the medium is similarly ejected from the culture container bottom center injection port 48, and the previously injected medium is pushed up.

The flow of the medium described above will be described with reference to experimental photographs in a prototype, FIGS. 18, 19, and 20. FIG. The experiment is performed with the linear motion motor rotating 5 times per minute, 1 rotation medium injection amount of 2.5 cc, and the scaffold rotating motor rotating once per minute. As a result of the experiment, the medium discharged from the medium injection port is dispersed in all directions by the rectifying plate 24 and stays at the bottom of the culture vessel. If the injection is continued, the medium becomes an injection method in which the medium rises from the lower layer to the upper layer. It was a gentle medium injection method.

In the experimental method, after filling the culture vessel 10 with clear water from the medium tank, the transparent water is colored and injected into the culture vessel 10. In FIG. 18, up to the current plate 24, in FIG. 19 from the scaffold to the lower surface, and in FIG. 20 from the scaffold to the top, the colored medium is continuously injected up to this point. The medium is injected in layers.

In FIG. 20, the experiment of confirming the influence of the stirring of the medium by the scaffold was performed by stopping the injection of the medium, driving the outer diameter of the rotating scaffold 20 mm, and rotating the scaffold for 10 minutes at 1 minute rotation. There is no change in the state of FIG. 20 and there is no disturbance of the medium, and the influence on the cultured cells due to the medium injection and scaffold rotation is assumed to be extremely weak.

The rotary actuator 14 adjusts the rotational speed of the rotary scaffold seeded with the cells, and performs settings such as rotation stop. The camera is fixed to the side of the culture vessel 10, and information from the endoscopic camera is imaged, analyzed, and recorded on the operation terminal (personal computer) 4 and information on the pH measurement sensor is stored in the pH measurement machine. Record and analyze. Multi-culture device for one specimen with monitor device.

Detailed description will be given with reference to FIG. 1 and FIG.
Using a geared motor with a rotational actuator (motor) in the range of 1 RPM to 70 RPM, the rotational power is changed to a linear motion by the cam 20 and the clamp 21, and the diaphragm 23 is added into the culture vessel 10 when the clamp is lowered. Press the pressure and insert to drain the medium in the culture vessel. When the clamp 21 is raised, the diaphragm 23 is raised, the inside of the culture vessel 10 is decompressed, and the medium is sucked into the culture vessel 10.

The amount of medium is determined by determining the width of the amplitude of the diaphragm 23 that transmits the linear motion of the clamp 21, the time of one stroke of the vertical motion of the clamp 21, and the driving time of the linear actuator 13 to determine the number of strokes of the vertical motion. Can be determined.

The determination of the automatic exchange of the medium amount is based on the amplitude control of the diaphragm 23 and the rotation control of the actuator (motor). The vibration width is determined by the position from the center of the rotational movement of the cam 20 at the fixed position of the cam 20 and the clamp 21. When fixed at a position 4 mm from the center, the amplitude is 8 mm.

The power supplied to the linear actuator 13 is supplied as an ON / OFF pulse current. When the ON time is shortened and the OFF time is lengthened, the movement of the linear actuator 13 is slowed down, and the time required for one stroke of the clamp 21 is slowed down. Also, if the ON time is lengthened and the OFF time is shortened, one stroke of the clamp is accelerated.

When the amplitude of the prototype shown in FIG. 14 is set to 8 mm and a 5 PPM actuator (motor) is passed through the linear actuator 13 to measure the amount of medium, 2.5 CC medium is discharged (replacement amount) in one stroke. It became 12.5CC in 1 minute. A cycle with an ON energization time of 0.5 seconds and an OFF disconnection time of 0.5 seconds is made and an experiment is performed with a 1 RPM actuator (motor). It took 5 strokes in 2 minutes, and the discharge amount per minute was 6.25CC.

From the experimental results, the exchange amount in the energization time of 0.5 seconds is 6.25 ÷ 60, which is about 0.104 CC, and the energization time of the pulse circuit is increased to 0.5 seconds and the OFF stop time is increased to 20 seconds. When it is performed in a cycle, it becomes about 0.42 CC in 1 minute, and further, the daily medium replacement amount can be determined by setting the daily energization time with a timer. When the energization time of one day is set to 10 minutes at 1 minute 0.42 CC, the medium exchange amount is 4.2 CC, and the medium exchange can be continued during the culture period. Regarding the medium injection, a very small amount of medium is injected, and further, a non-invasive and extremely gentle medium can be exchanged by the rectifying plate 24 in four directions.

A porous scaffold sheet is placed on the scaffold fixing part 26 shown in FIG. 5 and fixed to the scaffold fixing part 26 with a scaffold fixing 0-ring 28. After seeding the cells, the lid part is fixed to the culture vessel. Further, a rotary scaffold fixture 25 equipped with a scaffold support 27 is mounted on the side surface of the culture vessel 10, and the scaffold support 27 and the shaft of the rotary actuator 14 are connected by a joint 19 and rotated.

The rotary scaffolding fixture 25 is rotated in the culture vessel 10 filled with the culture medium by the controller 3 for the rotary actuator, the front and back surfaces of the cell culture are switched by rotating the front and back surfaces of the cell culture, and the floating waste material attached to the back surface culture surface. To leave.

By rotating the rotary scaffold fixture 25, the inside of the culture medium in the culture vessel 10 is rotated and brought into contact with the fresh culture medium on the cell culture surface to supply nutrients and oxygen. Moreover, the small culture container 10 can be effectively utilized by stacking the scaffold fixing portions 26 in many stages.

The rotating mesh columnar culture device 29 described in claim 16 will be described with reference to FIG. It was configured in a cylindrical shape with a mesh of a biocompatible material that can pass through the medium. A culture in which cells are seeded on atelocollagen or a three-dimensional culture in which cells are seeded on a three-dimensional porous scaffold is inserted into the mesh cylinder, and rotational culture is performed. By changing the position of the contact surface between the adherent cells and the mesh by rotation, the adherent cells are not adhered, and nutrients, oxygen, etc. are supplied from the medium from all directions of the three-dimensional body.

The pulse waveform of the scaffold rotation speed can be adjusted, the rotation speed can be varied from 70 rotations per minute to one rotation per hour, the experiment of FIG. 20 is performed at 1 RPM, and the medium is hardly stirred, and the medium by rotation It seems that there is no adverse effect on cultured cells due to pressure from

An endoscopic camera is mounted on the endoscopic camera mounting tool 18 of the culture container 10 of the one-sample culture apparatus shown in FIGS. 1 and 2, and the inside of the culture container is photographed. At the time of shooting, the rotating scaffold fixture can be rotated so that the culture sheet can be adjusted in front of or obliquely to the camera, the image from the light source attached to the endoscopic camera, the amount of transmission from the light source attached to the opposite side, and the cells Analyze the density and compare it with materials such as past microscope images.

Furthermore, the light source attached to the culture device can be attached not only to one point but also to many parts. The wavelength of the light source is also studied, the captured image is analyzed, and the camera operation terminal can be operated remotely. Cell image processing and image data are taken in, and the situation analysis of the culture, the progress analysis of the culture, the flow rate of the medium, and the scaffold rotation are determined.

The pH sensor is inserted into the pH sensor insertion container 17 in the middle of the piping from the backflow prevention device 15 on the culture container upper discharge port 32 side to the discharge medium tank 12, and the pH change amount of the used medium is measured. Information is automatically sent to the machine 5 and the result of measurement by the pH measuring machine is analyzed to determine the amount of medium to be injected.

The cell seeding jig 33 described in claim 20 will be described with reference to FIGS. 7 and 8 as a cell seeding method on a rotary scaffold. FIG. 8 is a cross-sectional view of a culture vessel and a cross-sectional view of a cell seeding jig. The cell seeding jig 33 is inserted into the culture container in a state where the scaffold fixing part 26 is dipped in the culture medium. The cell seeding jig 33 is inserted and brought into contact with the scaffold fixing part, the cell turbid medium 34 is injected into the cell seeding jig 33, and the injected cell turbid medium 34 is cell seeded. The cells do not flow out from the jig 33 into the culture vessel 10, and the cells settle and adhere to the scaffold sheet.

21 is a perfusion culture apparatus according to claim 3, in which the culture part of FIG. 9 is assembled by sterilization in a clean bench, the medium is injected into the medium tank 11, and the cell seeding is performed on the scaffold sheet 44 of the scaffold fixing part 26 After seeding the cells with the jig 33, the closed culture unit is assembled in the state shown in FIG.

As shown in FIG. 10, the closed culture unit shown in FIG. 9 is fixed to the sealed constant temperature case 36 by interlocking the cam 20 and the clamp 21 with the joint 19, and the scaffold support 27 is fixed to the joint 19. By doing so, the culture part and the sealed thermostatic case 36 are double sealed.

The double-sealed culture part is taken out from the clean bench, and fixed with a controller joint 38 with a controller part 37 with a drive unit which is taken out from the sealed thermostatic case 36 as shown in FIG.

The backflow prevention device 15 described in claim 22 will be described with reference to FIGS. 12 and 13. FIG. 13 is a cross-sectional view of the backflow prevention device, and FIG. 13 is a cross-sectional view of the backflow prevention device. An auxiliary discharge port 41 connected to the backflow prevention discharge port 40 is provided, and the spherical valve 42 has a different specific gravity. It is made of resin, ceramic, or metal. It is also possible to change the pressure applied to the culture vessel by changing the inlet diameter. The internal structure on the inlet side is a mortar-shaped portion 43.

The fact that the auxiliary discharge port 41 is provided on the side of the backflow prevention discharge port 40 is that when a resin spherical valve 42 having a light specific gravity is used, the medium valve is lifted by the momentum of the medium injection, and the spherical valve 42 opens the backflow prevention discharge port 40. As a solution to this problem, an auxiliary discharge port 41 is provided, the backflow prevention inlet 39 is a mortar-shaped portion 43, and the spherical valve 42 is always a backflow prevention inlet. It falls into 39 and closes the backflow prevention inlet 39.

The use of a material having a different specific gravity for the spherical valve 42 of the backflow prevention device 15 changes the pressure applied to the culture medium. In the case of the spherical valve 42 having a large specific gravity, the pressure applied to the culture medium from the diaphragm 23 increases. By changing the discharge diameter, the pressure applied to the culture medium in the culture vessel can be changed even with the spherical valve 42 having the same weight. Selection of these pressures is necessary for culturing arterial blood vessels, load-type cartilage and the like. The important point of this culture apparatus is that the medium exchange amount can be accurately performed by preventing the accuracy of the backflow prevention device 15 and the pressure difference between the medium tank 11 and the discharge medium tank 12.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited.
FIG. 1 is an overall configuration diagram of a reflux (circulation) culture apparatus according to the present invention, which is a basic configuration of a reflux system culture apparatus from a culture vessel 11 to a culture vessel 10 and from the culture vessel 10 to the culture vessel 11. For exchanging the medium, the driving time and the number of times of driving are set for the diaphragm 23 attached to the culture vessel 10 by the controller 2 for the linear actuator, and the diaphragm 23 is moved up and down by driving the linear actuator 13. When descending, the descending insertion volume integral medium of the diaphragm 23 is discharged into the culture vessel 10, and when rising, the diaphragm 23 is pulled up and the medium of the increased volume of the culture vessel 10 is aspirated.

In the culture method, the scaffold sheet 44 is fixed to the scaffold fixing part 26, the cells are seeded on the scaffold sheet 44, and the joint is fixed to the rotary actuator 14 by the joint 19 to perform rotary multi-layer culture. The flow of the culture medium is sucked from the culture medium tank 11 through the backflow prevention device 15 to the culture container 10 and is discharged from the culture container 10 to the culture medium tank 11 through the backflow prevention apparatus 15 when discharged.

In order to reuse the medium after cell culture, it is necessary to take carbon dioxide, oxygen, etc. in the carbon dioxide incubator 1 into the medium, and the medium used with the gas in the carbon dioxide incubator 1 by the method described in claim 2 is used. The contact surface is increased, carbon dioxide is taken into the working medium, and the medium is circulated.

FIG. 2 is an overall configuration diagram of the perfusion culture apparatus according to the present invention, and culture is possible in the closed thermostatic case 36 described in claim 3, and the culture vessel 10 is transferred from the culture vessel 11 to the culture vessel 10, and the discharge culture vessel from the culture vessel 10. 12, the culture medium is perfused, and the culture section becomes a closed culture apparatus.

In both the circulation type and perfusion type shown in FIGS. 1 and 2, the medium amount is determined by setting the vertical movement distance of the linear actuator 13 from the linear actuator controller 2 and determining the amplitude of the diaphragm 23, and setting the diaphragm 23 amplitude. Set the driving time of the day and determine the number of days of culture.

The scaffold sheet 44 is fixed to the scaffold fixing portion 26 of the rotary scaffold fixture 25 of FIG. 5 with the scaffold fixing 0 ring 28 of a biocompatible material. After inserting and fixing in the culture vessel 10 from the rotary scaffold insertion port, and seeding the cells, the scaffold support 27, the cylinder of the rotary actuator 14 and the joint 19 are fixed together. The rotation speed and the number of rotations per day are set by the controller 3 for the rotation actuator, the number of culture days is determined, and the rotation actuator 14 is commanded to perform automatic culture.

During the culture, the endoscope camera is fixed to the endoscope camera mounting tool 18. Information from the endoscope camera is sent to a camera operation terminal (personal computer) 4, and processing such as photographing, analysis, analysis, and recording is performed by remote control to observe the culture state of cells / tissues in the culture vessel 10.

Using a light source attached to the culture container 10 on the opposite side of the endoscope camera, etc., the light transmittance and reflectance of the culture sheet, and the light source cycle are changed, and the rotating scaffold fixture 25 is further rotated. Analyze the video from the direction.

A pH sensor is inserted into the pH sensor insertion container 17, the pH of the cultured medium is measured, information is sent to the pH measuring machine 5, and the culture state is analyzed as a result of the pH measurement.

FIG. 3 is an explanatory drawing in which the linear actuator 13 (rotation) is changed to a linear motion by the cam 20 and the clamp 21. The amplitude of the diaphragm 23 can be changed at a clamp fixing position 22 where the cam 20 and the clamp 21 are fixed.

FIG. 4 is a cross-sectional view of the culture vessel 10 and explains the characteristics of the culture vessel. The vertical movement of the clamp 21 is passed through the clamp stabilizer 46 and transmitted to the diaphragm 23. The clamp stabilizer 46 adjusts the clamp stabilizer 46 with the vertical stabilizer height adjustment screw 47 in accordance with the vertical amplitude of the clamp 21.

When the diaphragm 23 is raised, the culture medium is injected from the center inlet 48 on the bottom surface of the culture vessel, dispersed in all directions by the rectifying plate 24, and the culture medium rises in layers at the upper outlet 32 of the culture vessel. The scaffold support 27 having the scaffold sheet 44 fixed to the scaffold fixing part 26 is fixed to the rotary actuator 14 by the joint 19, and the scaffold sheet 44 fixed to the scaffold fixing part 26 is rotated.

FIG. 5 is an explanatory view of the scaffold support 27 that fixes the scaffold sheet 44 to the scaffold fixing portion 26 with the scaffold fixing tool 25 and the scaffold fixing 0-ring 28.

In FIG. 6, atelocollagen or a three-dimensional porous scaffold in which chondrocytes are seeded is inserted into the mesh column part 30 with the mesh rotation column culture tool 29, and then rotated and contacted with the medium from all directions.

7 and 8 are cross-sectional views of the culture vessel 10 of the present invention and an explanatory view of a cell seeding jig 33 for fixing the scaffold sheet 44 to the rotary scaffold fixture 25 and seeding cells.

The medium is inserted up to the medium injection line, the cell seeding jig 33 is inserted into the culture container 10, and the inside of the culture container and the outer diameter of the cell seeding jig 33 and the airtight 0-ring 35 attached are sealed. The cell turbid medium 34 is injected into the seeding jig 33, and the injected cell turbid medium 34 does not flow out of the cell seeding jig 33, and the cells sink and adhere to the scaffold sheet 44.

FIG. 9 is a culture part of a perfusion culture apparatus, and the culture part is assembled as a completely closed culture part.

FIG. 10 is a diagram in which the closed culture unit shown in FIG. 9 is inserted into the sealed constant temperature case 36 and fixed by the joint 19.

FIG. 11 is a completed view of a multi-culture apparatus for one sample in which a closed thermostatic case 36 in which the closed culture unit of FIG. 9 is inserted into a closed thermostatic case 36 and a controller unit 37 with a drive device are fixed by a controller joint 38.

12 and 13 show the backflow prevention device 15 of the present invention, and the spherical valve 42 is made of a biocompatible material such as resin, ceramic, metal, and the like, and the spherical valve 42 having a different specific gravity is selected and used depending on the culture. In order to reduce the resistance to the circulation of the culture medium, if the resin spherical valve 42 having a low specific gravity is used, the resin spherical valve is pushed up by the culture medium due to the low specific gravity, and the backflow prevention discharge port 40 of the backflow prevention device 15 is opened. In some cases, the medium flow was stopped. For this purpose, an auxiliary discharge port 41 in contact with the backflow prevention discharge port 40 is provided.

The pressure in the culture vessel 10 can be changed by using a spherical valve in which the density of the spherical valve 42 in the backflow prevention device 15 is changed or by changing the diameter of the backflow prevention inlet 39. For example, if the diameter of the backflow prevention injection port 39 of the discharge side backflow prevention device is made smaller than the backflow prevention injection port of the injection side backflow prevention device 15, the pressure in the culture vessel 10 becomes higher than the pressure at the time of discharge. .

The important points of the closed culture apparatus of the present invention are the pressure balance between the medium tank 11 and the discharge medium tank 12 and the precision of the backflow prevention apparatus 15. The internal diameter of the circulation pipe is about 4 mm, and the backflow prevention device 15 is extremely small compared to the conventional industrial use. The dedicated backflow prevention device 15 is necessary, and the valve also uses the spherical valve 42. If the pressure is received from the backflow prevention discharge port 40, the mortar-shaped portion 43 falls into the backflow prevention injection port 39 to reliably close the backflow prevention injection port 39.

FIG. 18, FIG. 19, and FIG. 20 are photographs of the medium injection state in the experimental apparatus of the present invention, in which the medium is layered and pushed upward. It is the most important medium injection method of the present invention.

FIG. 21 shows a method of moving a cultured tissue to a target place after culturing. The tube clamp 16 is closed, the culture vessel 10 is sealed, the tube opposite to the culture vessel 10 and the clamp is cut, and the entire culture vessel is sealed. Move to the desired location in the state. When the target location was a remote location, the sealed culture vessel was placed in a constant temperature case and allowed to move in a sealed state.

Regenerative medicine is a dream medicine, and the potential global market size related to TE (Tissue Engineering) is estimated to be 48 trillion in biological tissue engineering sponsored by the US NSF in 1988. Therefore, workshops were held and many TE venture companies were established.

In Japan, a TE venture company was created around 2.000 based on the results of researchers as a dream medicine, and a TE venture company that received a loan from the Ministry of Health, Labor and Welfare also appeared. Currently.

The basic principle of cell culture is said to be the three basic elements of cells, materials, and stimulating factors. For the time being to solve ethical problems, etc., separation and differentiation induction of somatic stem cells and somatic cells apart from embryonic stem cells (ES) cells The technology is the starting point for regenerative medicine, and is a source of cultured cells, development of serum-free medium, development of materials for three-dimensional scaffolds, biochemical factors and physical stimulating factors for cell differentiation and proliferation.

These cells, materials, and stimulating factors are in place, and researchers who are familiar with cell culture in the laboratory have succeeded in culturing cells and tissues, and the results are not immediately utilized for commercialization. In order to realize regenerative medicine at an early stage, cell-based culture medical devices, that is, industrial use is possible only by enabling aseptic and unattended cell / tissue culture in a safe environment. Thus, as a first step in the development of a cultured medical device, a culture solution is used as a culture medium and a nutrient or oxygen transmission medium in cell / tissue culture. The culture period requires several weeks, and the multi-culture apparatus for one specimen of the present invention has been developed as an important issue on how to provide the cells with nutrients and oxygen necessary for cell / tissue culture.

When the culture apparatus for regenerative medicine is one sample, one apparatus, and the culture period is 3 to 4 weeks, only about 20 cell / tissue cultures can be regenerated per year with one culture apparatus. However, if the culture apparatus is poor and expensive, the depreciation cost of the culture apparatus is added to the medical expenses as it is, and an inexpensive culture apparatus is strongly desired for realizing regenerative medicine.

The single-sample culture device with a monitor device according to the present invention is a small and light-weight culture device, and also has a monitoring device that can be operated remotely, and can cultivate multiple and multi-layer culture sheets, and grow as a medical device through future monitoring. Thus, the culture device can be sufficiently expected to be approved as a regenerative medical culture device.

The economic scale of regenerative medicine is said to be 48 trillion, of which the economic scale of culture devices, culture media, scaffolds, etc. is also said to be 20 to 30 trillion. Although necessary, the multi-culture apparatus for one specimen of the present invention has sufficient potential as a regenerative medical apparatus.

  Overall structure of reflux culture equipment   Perfusion culture device concept   Connection diagram of culture vessel, linear actuator and rotary actuator   Cross section of culture vessel   Rotating scaffold fixture   Mesh rotating column culture device   Cell seeding jig side view and culture vessel cross section   Cell seeding jig cross section and culture vessel cross section   Closed culture section   Fixed culture unit in a sealed thermostat case   Double closed culture device set configuration diagram   Backflow prevention device side view   Cross section of backflow prevention device   Prototype incubator front view   Prototype incubator top view   Controller photo   Rotating scaffold two-stage jig photo   Culture container medium injection experiment, rotating scaffold effect experiment photo   Culture container medium injection experiment, rotating scaffold effect experiment photo   Culture container medium injection experiment, rotating scaffold effect experiment photo   Moving culture container photo

1: Carbon dioxide incubator 2: Controller for linear actuator 3: Controller for rotary actuator 4: Camera terminal controller (PC)
5: pH measuring device 10: Culture container 11: Medium tank 12: Medium tank for discharge 13: Direct acting actuator 14: Rotating actuator 15: Backflow prevention device 16: Tube clamp 17: pH sensor insertion container 18: Endoscopic camera mounting tool 19: Joint 20: Cam 21: Clamp 22: Clamp fixing position 23: Diaphragm 24: Rectifying plate 25: Rotating scaffold fixture 26: Scaffold fixing part 27: Scaffold support 28: 0 ring for fixing scaffold 29: Rotating cylinder made of mesh Cultivation tool 30: Mesh cylindrical part 31: Medium insertion port 32: Culture container upper discharge port 33: Cell seeding jig 34: Cell turbid medium 35: Airtight 0 ring 36: Sealed constant temperature case 37: Controller with drive unit 38: Controller joint 39: Backflow prevention inlet 40: Backflow prevention outlet 4 : Auxiliary outlet 42: spherical valve 43: bowl-shaped portion 44: scaffold sheet 45: vent 46: Clamp stable fixture 47: vertical stabilizer instrument height adjustment screw 48: the culture vessel bottom central inlet

Claims (22)

  A device for culturing cells and tissues in an environment where the inside of the carbon dioxide incubator is controlled with sterilization, temperature, humidity, oxygen, carbon dioxide, nitrogen partial pressure, etc., from the medium tank to the culture container injection side, from the culture container discharge side A circulation pipe is provided in the culture medium tank, and a backflow prevention device is provided in the middle of the culture medium injection side pipe and the culture medium discharge side pipe. A stretchable synthetic rubber diaphragm made of a biocompatible material is fixed to the culture container lid, and the synthetic rubber A joint made in a state that can be interlocked with the cylinder of the direct acting actuator to the diaphragm made of plastic, and expanding and contracting the cylinder of the direct acting actuator, pressurizes and depressurizes the culture medium in the culture vessel via the synthetic rubber diaphragm Then, the medium in the culture container is replaced. During decompression of the culture container internal medium, the volume is expanded, the medium is injected, the cylinder of the linear actuator is extended, a synthetic rubber diaphragm is inserted inside the culture container, the volume of the culture container internal medium is reduced, and the medium is reduced. The culture medium in the culture vessel is automatically replaced. The medium inlet is discharged from the center of the bottom of the culture container, and the medium outlet is discharged from the upper side of the culture container. A baffle plate is provided at a position where a gap is formed above the center culture medium inlet at the bottom of the culture vessel, and the injection medium is dispersed in all directions. In addition, an insertion window is provided on the side of the culture container so that the rotating scaffold fixture can be inserted from the side of the culture vessel, a sheet-like scaffold is fixed to the scaffold fixture, and the rotary scaffold fixture that is attached to the scaffold support is attached to the scaffold fixture. Fix to the culture vessel. Cultured cells are seeded on a sheet-like scaffold fixed to a culture vessel. A part of the scaffold support of the rotary scaffolding fixture is configured so as to come out of the culture vessel, and is secured to the cylinder of the rotary actuator so that it can be interlocked with a joint. The culture vessel is equipped with a diaphragm pump, and the culture medium is circulated from the culture vessel to the culture vessel and from the culture vessel to the culture vessel to cultivate the cells and tissues. After culturing, all the parts that come into contact with the cultured cells and the culture medium can be discarded. Multi-culture apparatus for one specimen.   The piping tube is made of a gas permeable membrane from the culture vessel to the carbon dioxide gas, oxygen and the like through the discharge medium. Further, the discharge medium is returned from the culture container to the medium tank and the discharge medium is exchanged with the gas in the carbon dioxide incubator. The multiple culture apparatus for one specimen according to claim 1, wherein the medium is flowed on a shower-like or convex surface and returned to the culture tank in order to widen the contact surface and obtain carbon dioxide and the like.   In a sterilized sealed thermostatic case with adjustable temperature, piping from the culture vessel composed of a deformable container of biocompatible material to the culture vessel, and piping from the culture vessel to a draining culture chamber composed of a deformable container A culture vessel for culturing cells and tissues is equipped with a backflow prevention device on the medium injection side from the medium tank and on the medium discharge side from the culture vessel, and a stretchable synthetic rubber diaphragm made of a biocompatible material on the culture container lid The diaphragm made of synthetic rubber is fixed in a state where it can be linked with the cylinder of the linear actuator, and the cylinder in the culture vessel is pressurized through the diaphragm made of synthetic rubber by extending and contracting the cylinder of the actuator. The medium is exchanged by reducing the pressure and changing the volume of the medium in the culture vessel. A culture medium is injected into the culture container from the center of the bottom of the culture container, and a rectifying plate is provided at a position spaced from the center injection hole at the bottom of the culture container, and the injection medium is dispersedly injected in all directions. The medium at the time of discharge is discharged from the upper side of the culture vessel, and the medium is automatically changed. In addition, an insertion window is provided on the side of the culture container so that the rotating scaffold fixture can be inserted from the side of the culture container, and a sheet-like scaffold is fixed to the scaffold fixing part, and a scaffold support is attached to the scaffold fixing part and fixed to the culture container. The cultured cells are seeded on the sheet-like scaffold. A part of the scaffold support of the rotary scaffolding fixture is configured so as to come out of the culture vessel, and can be fixedly interlocked with the cylinder and joint of the rotary actuator. The culture medium is perfused from the culture medium tank to the culture container, and the culture medium is perfused from the culture container to the discharge medium tank to automatically change the culture medium in the culture container, thereby culturing cells and tissues. Multi-culture apparatus for one specimen that can be disposed of after the culture is complete, all parts that have come into contact with the cultured cells and culture medium.   A closed-system culture in which a ventilation tube is provided with a biocompatible material tube at the lid of the culture medium tank and the culture medium tank made of biocompatible hard glass, etc., so that no pressure difference occurs between the culture medium tank and the culture medium tank for discharge. A multi-culture apparatus for one specimen according to claim 3, wherein the apparatus is an apparatus.   A culture tank made of synthetic rubber (including a gas permeable membrane) and a discharge medium tank as a biocompatible material are configured as a closed culture apparatus, and the container is deformed and reduced according to the discharge amount when the medium is discharged due to decompression. 4. The single culture multiple culture apparatus according to claim 3, wherein the discharge medium tank is deformed and expanded when the discharge medium is injected into the discharge medium tank and pressurized, and the container itself is a closed system culture apparatus that supports decompression and pressurization.   Attach a tube clamp between the backflow prevention device and the injection port to the tube piped from the culture medium tank to the culture vessel inlet, and attach a tube clamp in the middle of the piping tube from the culture vessel outlet to the backflow prevention device.・ After completing the tissue culture, close the tube clamp and cut the tube. It is possible to remove the joint from the actuator and move the cultured cells / tissues from the culture facility to the target place in the sterilized thermostat case without moving the cultured cells / tissues out of the culture vessel. The multiple culture device for one specimen according to claim 1 or 3.   In order to weaken the influence of the medium inflow from the medium inlet at the center bottom of the culture vessel, a rectifying plate is provided at the inlet. A space is provided between the culture vessel inner bottom surface / culture vessel inner side surface and the rectifying plate, and after the medium is injected, the flow is dispersed in all directions by the rectifying plate, so that the physical influence due to the medium inflow to the cell / tissue culture can be reduced. The single culture multiple culture apparatus according to claim 1 or 3, which is weakened.   The single culture multiple culture according to any one of claims 1 and 3, wherein a geared motor used for a linear motion actuator (motor) and a rotation actuator (motor) has a rotation speed of IRPM to 70 RPM. apparatus.   The medium exchange amount is determined by changing the flow rate of the medium according to the amplitude of the diaphragm interlocking with the cylinder of the linear actuator. The actuator (motor) switches to linear motion with a cam and clamp. In order to vary the linear motion amplitude, it is possible to change the position of the interlocking fixed point between the rotary cam and the clamp, and the amplitude is variable at a fixed point position away from the fixed point near the center of the rotary cam. 4. The multi-culture apparatus for one specimen according to claim 1, wherein the flow rate of the medium is variable at the fixed position of the rotary cam and the clamp, and the amplitude of the diaphragm is variable.   In addition to the change in the flow rate described in claims 8 and 9, the medium flow rate within a predetermined time is determined at the rotational speed of the rotary actuator (motor). In order to vary the rotation speed, the current flowing through the rotary actuator (motor) is adjusted in a pulsed manner by ON / OFF time adjustment. If the ON time is shortened and the OFF time is lengthened, the rotation of the actuator (motor) within a certain period of time will slow down, the flow of the medium will be small, the ON time will be long, and if the OFF time is shortened, the rotation actuator ( The rotation time of the motor) becomes faster, and the medium flow rate within a certain time increases. The multiple culture apparatus for one sample according to claim 1 or 3, wherein the culture medium flow rate is varied by the rotation speed of a rotary actuator (motor).   The driving time of the linear actuator is set by a timer in the method for varying the medium flow rate described in claim 8, claim 9, or claim 10. The daily medium exchange amount and the culture period can be determined by setting the timer according to the setting method described in claims 8, 9 and 10 and the driving time of the direct acting actuator of the day. The multiple culture apparatus for one specimen according to claim 3.   In the control of the rotational speed for rotating the motor one time according to the eighth aspect, the current is supplied in a pulse manner as in the method of the tenth aspect. By shortening the ON time and lengthening the OFF time, the rotation speed of the rotating scaffolding fixture will be slow, and by shortening the ON time and shortening the OFF time, the rotation speed of the scaffold will be faster and the scaffold rotation speed. The multi-culture apparatus for one specimen according to claim 1 or 3, wherein said variable is variable.   When the rotation time of one rotation and the number of rotations per day described in claim 12 are determined, the driving time of the day is determined, and by setting the driving time of the day as a timer, The multiple culture apparatus for one specimen according to claim 1 or 3, wherein the culture period can be determined.   The rotary scaffolding fixture is composed of a scaffolding support that is directly connected to the sheet-like scaffolding fixing part and the rotary actuator (motor). The sheet-like scaffolding fixing part is circular, and is engraved 2 to 4 mm smaller than the outer shape. Drill a small through hole of about 5 mm, place the scaffold on the engraved part, and fix the scaffold with a soft material resin (silicone 0 ring, etc.) made of biocompatible material. Silicone with a 0 ring slightly larger than the engraved hole outline The multi-culture apparatus for one specimen according to claim 1 or 3, wherein the culture scaffold is fixed using elasticity of resin or the like, and the rotary scaffold is cultured.   By fixing the sheet-like scaffold fixing part seeded with cells in a state of being spaced apart from the sheet-like scaffold fixing part seeded with cells on the rotary scaffolding fixture according to claim 14, two or three stages A multi-culture apparatus for one sample that can perform stacked multi-stage multi-culture in the same culture vessel. The rotating scaffolding fixture is made of a biocompatible material and the mesh mesh that can pass through the medium is made into a cylindrical shape. The rotating mesh is made up of a rotating support that can be detached on both sides of the cylindrical mesh, and the cells are seeded on a three-dimensional porous scaffold or atelocollagen. By inserting the object into the cylindrical mesh and rotating the rotating scaffold fixture, the position of the contact portion between the cultured cells and the cylindrical mesh is changed, and this is for one specimen that prevents adhesion between the adherent cells and the mesh. Multiple culture equipment.   Open a window for endoscopic observation on the side of the culture vessel, attach the endoscope camera to the biocompatible transparent member in a removable state, and perform image, analysis, analysis, and recording inside the culture vessel by computer operation, The multi-culture apparatus for one specimen according to claim 1 or 3, which is equipped with a monitor device capable of remotely observing an image of cell culture in a culture container.   The culture vessel is attached to the side surface of the culture container according to claim 17 opposite to the side surface where the endoscopic camera is mounted, or in a state where a plurality of light sources can be detached as required. Adjust the angle of the rotating scaffold on which the cells were seeded, and also observe the culture state of the cultured cells and tissues from the opposite side and side of the endoscopic camera at various angles to observe the culture state. The multiple culture apparatus for one specimen with a monitor device according to claim 1 or 3, wherein monitoring is performed using a light source on the opposite side to the light source on the camera side.   A sensor of a pH measuring device is mounted between the backflow prevention device mounted on the discharge side pipe from the culture container of the multi-culture device for one specimen described above and the discharge medium tank, and the discharged medium after culture is measured. The multiple culture apparatus for one specimen with a monitor device according to claim 1 or 3, wherein confirmation and recording are performed by remote operation, and the culture state of the cultured cells is confirmed by remote operation.   The cell seeding jig is a cylindrical shape that is slightly smaller than the inner diameter of the culture vessel. The inner diameter is the same as the inner diameter of the rotating scaffolding fixture, and the same through hole is made to maintain the airtightness of the inner diameter of the culture vessel and the outer diameter of the cell seeding jig. For this purpose, the groove is cut so that a silicon ring or the like can be attached to any place of the outer diameter, the airtight 0 ring is attached, the rotating scaffold fixture in the culture vessel is leveled, and the medium covers the scaffold sheet. After injecting the medium, insert a cell seeding jig equipped with an airtight 0-ring so that it is in contact with the rotating scaffold fixing jig, inject the cell turbid medium into the inner diameter of the cell seeding jig, let the cells settle, and scaffold sheet Cell seeding jig that adheres cells to cells.   The closed-system perfusion culture apparatus described in claim 3 is divided into three parts: a culture part, a sealed thermostatic case, and a controller with a drive device. The sterilized culture part parts are assembled in a clean bench and are placed in a culture tank in the clean bench. After inoculating the medium and seeding the cells on the rotating scaffolding sheet of the culture vessel, the closed culture unit is assembled, the closed culture unit is attached to the drive joint in the sealed thermostatic case, and the closed closed culture unit and the closed thermostatic case are double closed The single-culture multiple culture apparatus according to claim 3, wherein the double-closed culture apparatus is constructed as a double closed system culture apparatus having a structure and taking out from the clean bench and connecting a controller with a driving device to a controller joint that is exposed outside the sealed thermostat case. .   The backflow prevention device uses a spherical valve made of biocompatible materials (synthetic resin, ceramic, metal) having different specific gravity, and determines the injection / discharge direction. An auxiliary discharge port is provided at the discharge port, and the injection port side is formed in a mortar shape, and the spherical valve falls into the injection port to prevent backflow. The pressure in the culture vessel can be varied by changing the inlet diameter of the backflow prevention device. The single-sample multiplex culture apparatus according to claim 1 or 3, wherein the spherical valve is a backflow prevention device having a sphere larger than the inlet diameter.