WO2003104386A1 - Culture apparatus, artificial tissue and blood preparation - Google Patents

Abstract

An apparatus for culturing living cells, comprising culture tank (2) for accommodating a nutrient medium, circulation means (10) for effecting external circulation of the nutrient medium of the culture tank (2) and nutrient medium conditioning means (20) for conditioning the state of nutrient medium while the nutrient medium is being externally circulated. The nutrient medium of the culture tank (2) is externally circulated, and during the circulation outside the culture tank (2), the state of nutrient medium can be conditioned. Therefore, the state of nutrient medium in the culture tank (2) can be maintained at condition optimum for the cells accommodated in the culture tank (2) and thus the cells can be stably cultured continuously for a prolonged period of time.

Description

 Description Culture equipment, people: ι3ΐϋ and blood products

 The present invention relates to a culture device, an artificial tissue, and a clot preparation.

 Living organisms such as floating cells and bacteria are cultured while immersed in a culture solution containing nutrients and oxygen necessary for the cells. By the way, since oxygen contained in the culture solution is consumed with time, when cells are cultured for a long time, oxygen is supplied to the culture solution by the gas supply means, and the oxygen concentration in the culture solution is kept constant. The present invention relates to a culture apparatus provided with a gas supply means capable of maintaining a constant oxygen concentration in a culture solution.

 In recent years, regenerative medicine has been performed to transplant tissues and sputum to recover damaged or missing tissues and organs of the human body. S being used. Similarly, blood used for blood transfusions and components in the blood are covered by donated blood, which is chronically lacking. If such tissues, organs, blood, etc. can be cultured and manufactured artificially, we will be able to meet the demand, but stringers)! ^ To cultivate blood, etc. up to the number of cells that can be transplanted or transfused, a large number of cells must be continuously cultured for a long time, so artificially cultured tissues, ¾§, blood, etc. Not put into practical use. The present invention relates to an artificial yarn weaving artificially cultivated by the culture device and usable for regenerative medicine, and a blood product produced from blood vessel cells artificially cultured by the culture device. Background art

 As a culture apparatus provided with a gas supply means, there is a culture apparatus disclosed in Conventional Examples 1 and 2 (Patent Documents 1 and 2).

The culture unit of Conventional Example 1 includes a culture vessel for culturing cells and a circulation path for circulating the culture solution in the culture vessel to the outside. Gas exchange of the culture solution is performed in this circulation route. A gas exchange part is provided. In this gas exchange section, the Tef opening tube is immersed in the circulating culture solution, and the gas flowing in the Tef opening tube and the culture Gas exchanges are taking place between Sakuya.

 Therefore, by adjusting the gas flow rate, the amount of oxygen and nutrients supplied to the culture solution, and the amount of carbon dioxide and nitrogen compounds discharged from the culture solution can be adjusted. Therefore, since the oxygen concentration and the like in the culture solution can be kept constant, the cells can be cultured continuously for a long time.

 In the culture unit of Conventional Example 2, the culture tank is composed of a hollow cylindrical body and a hollow fiber accommodated in the cylindrical body, and cultures cells between the hollow fiber and the cylindrical body. The breeding medium is housed. The hollow fiber accommodated in the cylindrical body is communicated with a medium exchange device and a gas supply device provided outside the culture tank, and the hollow fiber is connected to the medium exchange device and the gas supply device. A circulating medium circulating between them is poured. The circulating medium is passed through a gas supply device and a medium exchanging device, so that the concentration and oxygen concentration of components necessary for culturing the cells contained in the circulating medium are kept higher than the growth medium.

 For this reason, when passing through the hollow fiber of the circulating medium force culture tank, components and oxygen necessary for culture are supplied from the circulating medium to the growth medium, and waste products and carbon dioxide discharged by the cells are transferred from the growth medium to the circulating medium. To be discharged. Therefore, by adjusting the flow rate of the circulating medium, the oxygen concentration, carbon dioxide concentration, etc. in the culture solution can be kept constant, so that cells can be cultured continuously for a long period of time.

Patent Literature 1

Japanese Unexamined Patent Publication No. 0 7-8 2 60

Patent Document 2

Japanese Laid-Open Patent Publication No. 0-9-9 8 7 6 9

However, the culture unit 1 can adjust the concentration of gaseous components in the culture solution, but the amino acids, hormones, and vitamins that serve as nutrients for the cells cannot be supplied from the gas to the culture solution. Once these nutrients are consumed, the cells can no longer grow. Since nitrogen compounds such as ammonia and urea discharged from the cells cannot be removed from the culture solution, the cells inhibit the growth when the concentration of these components exceeds a certain level. The culture unit of Conventional Example 1 is in a condition suitable for culturing a certain period of time, that is, concentration power of nitrogen compounds etc. in the culture medium. Cells can be cultured during this time, but cells cannot be cultured for longer periods. The period during which cells can be cultured is limited by the amount of the culture medium circulating through the culture unit, and the apparatus becomes large when culturing for a long time.

 In the culture unit of Conventional Example 2, the circulating medium is circulated, and the growth medium itself in which the cells are immersed is kept in the culture tank. Then, in the breeding culture medium, gas exchange is performed effectively in the part that is in contact with the hollow fiber, but the part that is isolated from the hollow fiber is hardly subjected to gas exchange and so on. The growth medium becomes uneven. Therefore, if the concentration difference between the growth medium near the hollow fiber and the circulation medium is reduced, the gas exchange efficiency will be low, so if the conditions near the hollow fiber are the same as the circulation medium, Even if the conditions are not suitable for culturing, gas exchange will not be performed. Then, in the culture unit of Conventional Example 2, even though the cells are proliferating in the vicinity of the hollow fiber during the long-term culture, the cells may be killed in other parts. If the number of dead cells increases, the hollow fibers may be clogged by these cells. If clogging occurs, the efficiency of gas exchange and the like will be further reduced. Even so, there is a possibility that the state of the culture solution becomes poor and cells cannot grow. Disclosure of the invention

 The culture apparatus of the first invention is an apparatus for culturing living cells, a culture tank for containing a culture solution, a circulation means for externally circulating the culture solution in the culture tank, and the culture solution And a culture solution adjusting means for adjusting the state of the culture solution during external circulation.

 According to the first invention, the state of the culture solution can be adjusted while the culture solution in the culture vessel is externally circulated and circulated outside the culture vessel. Therefore, since the state of the culture solution in the culture tank can be maintained under the optimum conditions for the cells in the culture tank, the cells can be cultured continuously for a long period of time in a stable state.

 The culture apparatus of a second invention is characterized in that, in the first invention, the culture medium adjusting means includes a gas concentration adjusting means for adjusting a gas concentration of the culture liquid.

According to the second aspect of the invention, the culture is performed by the gas concentration adjusting means while circulating outside the culture tank. Since the liquid can be saturated to a desired gas concentration, the gas concentration of the culture solution can be kept uniform and suitable for cell culture. Therefore, the cells can be cultured continuously for a long period of time in a stable state. In addition, since the gas concentration is saturated outside the culture tank, a gas concentration adjusting means having a desired ability can be employed regardless of the size of the culture tank. In addition, since no bubbles are generated in the culture tank, it is possible to prevent the cultured cells from being damaged by the bubbles and to reduce the stress applied to the cultured cells. Further, since the gas is saturated outside the culture tank, there is no concern that the gas concentration adjusting means will adversely affect cell culture even if the gas is directly supplied to the culture solution. Therefore, it is not necessary to use an air permeable tube when saturating the gas in the culture solution, so that the manufacturing cost of the apparatus can be kept low.

 A culture apparatus according to a third invention is characterized in that, in the first or second invention, the culture medium adjusting means includes a purification means for purifying the culture liquid.

 According to the third aspect of the invention, waste products discharged from the cells and substances harmful to the cells can be removed while circulating outside the culture tank. It is possible to maintain a state suitable for cell culture. Therefore, the cells can be cultured continuously for a long period of time in a stable state.

 The culture apparatus according to a fourth aspect of the present invention is the culture apparatus according to the first, second or third aspect, wherein the culture medium adjusting means includes a gas concentration adjusting means for adjusting a gas concentration of the culture liquid, and a purification apparatus for purifying the culture liquid. And the purifying means and the gas concentration adjusting means are arranged in series.

 According to the fourth invention, the gas concentration adjusting means and the purifying means are arranged in series, that is, the externally circulated culture solution is returned to the culture tank after passing through both the gas concentration adjusting means and the purifying means. In addition, the state of the culture solution in the culture tank can be reliably maintained in a state suitable for cell culture. Moreover, since it is compact and can be operated collectively, it can be manufactured as a disposable set. If the configuration of a disposable set is adopted, it is possible to prevent the problem of being susceptible to contamination at the connecting portion that occurs when the gas concentration adjusting means and the purifying means are arranged in series. Can do.

The culture apparatus according to a fifth invention is characterized in that, in the third or fourth invention, the purification means is a dialysis part for dialyzing the culture solution. According to the fifth invention, since the culture solution is purified by dialysis, even if cells are contained in the circulating culture solution, cell damage when passing through the dialysis part can be reduced. Since Shiga Koji is dialyzed outside the culture tank, it is sufficient to employ a dialysis section having the desired dialysis capacity regardless of the size of the culture tank. In addition, if the space between the hollow fibers is wide, it is possible to prevent the dialysis ability from being reduced due to clogging of cells to be cultured.

 The culture apparatus according to a sixth aspect of the present invention is the culture device according to the fifth aspect, wherein the dialysis part is composed of a hollow main body case and a hollow fiber disposed in the main body case, and the inner surface of the main body case and the hollow fiber A culture medium circulation passage through which the culture solution is passed is formed between the outer surface of the hollow fiber and a dialysis solution for dialyzing the culture solution is passed through the hollow fiber. .

 According to the sixth aspect of the invention, since the dialysate is allowed to flow inside the hollow fiber and the culture solution that is permeated between the main body case and the hollow fiber is allowed to flow, the flow resistance when the culture solution flows through the dialysis part is reduced. Can do. Therefore, the culture solution can be smoothly circulated externally. Even if the circulating medium contains cells, it can reduce the resistance received by the cells from the dialysis unit when passing through the dialysis unit. Cell damage can be reduced. Cells can also pass smoothly. The culture device according to a seventh aspect of the present invention is the culture apparatus according to the first, second, third or fourth aspect, wherein the culture medium adjusting means includes a nutrient supply section that supplies nutrients necessary for cell culture to the culture medium. Features.

 According to the seventh aspect of the invention, the nutrient replenishment unit can supply the culture solution with the nutrients consumed by the cells while circulating outside the culture tank. It can be cultured in a stable state.

 The culture device according to an eighth aspect of the present invention is the culture device according to the seventh aspect, wherein the nutrient replenishment section passes a culture medium passage section through which the culture medium is passed, and a nutrient medium passage through which a nutrient medium containing nutrients necessary for cell culture is passed. A path is provided, and the nutrient passage and the culture solution passage part are separated by a dialysis member.

According to the eighth aspect of the invention, since the culture solution passage part and the nutrient solution passage part are separated by the dialysis member, only the components whose concentration strength in the culture solution is reduced are contained in the culture solution. Can be supplied. Therefore, it is necessary to supply more nutrients than necessary. Therefore, it is possible to prevent the problem of cell damage that occurs.

 A culture apparatus according to a ninth aspect of the present invention is the first, second, third, fourth, or seventh aspect, further comprising an extraction means for extracting a culture solution from the culture tank, the extraction means having one end in the culture tank. An extraction path which is immersed in the liquid and disposed outside the culture vessel at the other end, a conveying means provided in the extraction path for conveying the culture solution, and a sterile gas is supplied into the extraction path. And a gas supply means, wherein the gas supply means supplies sterile gas between the other end and the transport means in the extraction path.

 According to the ninth aspect of the invention, if the culture solution is conveyed toward the other end by the conveying means, the culture solution in the culture tank can be extracted, so that the culture state of the cells in the culture tank can be confirmed. it can. Since sterilized gas is supplied between the conveying means and the other end of the extraction path, the sterilized gas is always supplied to the extraction path from the other end of the extraction path. It can prevent invasion of various bacteria. Then, it is possible to prevent the culture solution passing through the extraction route from being contaminated with various bacteria. Since the culture solution remaining in the extraction path is not contaminated with bacteria, the culture solution can be effectively used by returning the culture solution to the culture tank.

 The culture apparatus of the 10th invention is characterized in that, in the 1st, 2nd, 3rd, 4th, 7th or 9th invention, the culture tank is provided with a temperature control means for controlling the temperature of the culture solution. According to the tenth invention, since the temperature of the culture solution in the culture tank can be maintained at a temperature suitable for cell culture by the temperature control means, the cells can be continuously and stably for a long period of time. It can be cultured in a state.

 The culture apparatus of the 11th invention is characterized in that, in the 10th invention, the temperature control means includes a far-infrared radiation section that radiates far-infrared radiation.

 According to the first aspect of the invention, cells in the culture solution that are not limited to the culture solution can be heated by far infrared rays. Since the cells are directly heated, the influence of the temperature change of the culture solution on the temperature of the cells can be reduced. Since the cell temperature can be maintained at a temperature substantially equal to that of the far-infrared radiation portion, the cell temperature can be easily and reliably adjusted.

An incubator according to a first aspect of the present invention is the incubator according to the first aspect of the invention, wherein the far-infrared radiation portion has a peak of energy density of radiated far-infrared in a wavelength band of 7 to 12 m. It is characterized by being adjusted.

 According to the first and second inventions, cell proliferation and differentiation by far infrared rays can be promoted. Therefore, the cell culture efficiency can be improved.

 The culture apparatus of the 13th invention is characterized in that, in the 11th or 12th invention, the far infrared radiation portion is a planar heating element.

 According to the thirteenth invention, since the far-infrared radiation portion is a planar light emitting book, the apparatus can be configured more compactly.

 The culture device according to a fourteenth aspect of the present invention is the culture device according to the first aspect, wherein the planar heating element is provided between a pair of upper and lower base materials and the pair of upper and lower base materials, and emits far infrared rays when energized. And a power supply means for supplying electric power to the radiating portion, wherein the radiating portion is formed by printing a radiator on the inner surfaces of the pair of upper and lower substrates. It is characterized by being.

 According to the fourteenth aspect of the invention, if power is supplied to the radiating portion by the power supply means, it is possible to radiate far infrared rays from the radiating portion. In addition, since the radiating portion is formed by printing the radiating body on the inner surfaces of the pair of base materials above and below the radiating portion force, it is easy to manufacture the planar heating element and to manufacture it at low cost. Can do.

 The culture device of the 15th invention is characterized in that, in the 14th invention, the radiator is a material having a positive temperature coefficient.

 According to the 15th invention, since the radiator is a material having a positive temperature coefficient, that is, has a self-temperature adjusting function, the state of the far infrared ray radiated to the cell can be kept constant. it can. Therefore, it is possible to prevent changes in the state of the far-infrared rays irradiated to the cells due to changes in the temperature of the far-infrared radiation part, thus preventing changes in the temperature of the cells and culturing the cells in a stable state. Can do.

 The culture apparatus according to a sixteenth aspect of the present invention is the first, second, third, fourth, seventh, ninth, or tenth invention, wherein the culture tank, the circulation means, and the culture medium adjustment means are connected in the state. It is formed in the size which can be accommodated in a clave apparatus.

According to the 16th invention, since it can be put into the autoclave apparatus without fouling the apparatus, it can be used as it is sterilized by the autoclave apparatus, and the culture solution and cells are contaminated with various bacteria. The probability of being done can be very low. The culture apparatus according to a seventeenth aspect of the present invention is the culture device according to the first, second, third, fourth, seventh, ninth, tenth or tenth invention, wherein the circulation means prevents the cells from flowing out of the culture tank. It has a prevention means.

 According to the seventeenth invention, when floating cells or bacteria are cultured, it is possible to prevent the floating cells from flowing out of the isobaric recharge tank. Cells can be reliably cultured, and cells can be cultured continuously for a long time. Moreover, it is possible to prevent dead cells from flowing out and being blocked in the circulation means. The culture apparatus according to an eighteenth aspect of the present invention is the culture apparatus according to the seventeenth aspect, characterized in that the outflow prevention means is an electromagnet.

 According to the eighteenth aspect of the present invention, it is not necessary to install inside the circulation path of the culture solution, that is, the electromagnet does not come into contact with the culture solution, so that it is possible to prevent the culture solution from being mixed. Shishika does not obstruct the flow of the culture solution, so that the culture solution can be smoothly circulated to the outside. Electromagnets do not prevent outflow by physical contact with cells to be cultured. In other words, since the cells are not in contact with the cells being cultured, even the very weak cells of the cell membrane can be prevented from flowing out of the culture tank and cultured without damaging them. 1st, 2nd, 3rd, 4th, 7th, 9th, 10th, 16th, and 17th inventions, the culture solution discharge part for discharging the culture solution in the culture tank to the outside, and the culture means for new culture from the outside A culture medium exchange means comprising a culture medium supply section for supplying the liquid is provided.

 According to the nineteenth aspect of the invention, a new culture solution can be supplied to the culture vessel by the culture solution supply unit while discharging the culture solution in use from the culture vessel by the culture solution discharge unit. For this reason, since the culture solution can be exchanged while the culture is continued, it is possible to culture even adherent cells and hematopoietic cells that require long-term culture. However, it is possible to automatically exchange the culture medium in use with a new culture medium by simply operating the culture medium discharge section and the culture medium supply section in synchronization. Therefore, no manual operation is required for exchanging the culture solution, and the risk of the culture solution and cells being cultured being mixed is reduced.

The culture apparatus according to a 20th invention is characterized in that, in the 19th invention, the culture medium discharge section includes an outflow prevention means for preventing the culture tank power from flowing out. According to the 20th invention, when the culture medium in the culture tank is discharged, it is possible to prevent floating cells and the like from flowing out of the culture tank, so that the culture medium can be replaced without stopping the cell culture. I can do it. Therefore; cells can be cultured continuously for a longer period of time. The culture device of the 21st invention is characterized in that, in the 20th invention, the outflow prevention means is an electromagnet.

 According to the second aspect of the invention, it is not necessary to install in the circulation path of the culture solution, that is, since the electromagnet does not contact the culture solution, it is possible to prevent the culture solution from being mixed. Yes. In addition, the electromagnet does not prevent the outflow by physical contact with the cells to be cultured. In other words, since it does not come into contact with the cells being cultured, it is possible to prevent the cells from flowing out of the culture tank even if the cell membrane is very weak.

 The culture apparatus according to a second invention is the culture apparatus according to the nineteenth, twenty-second or twenty-first invention, wherein the culture solution supply unit includes a sterilization unit for sterilizing the culture solution before supplying the culture solution to the circulation means. It is characterized by being.

 According to the 22nd invention, since the new culture solution is sterilized by the sterilization unit and then supplied to the culture tank, it is possible to prevent contamination by contamination when changing the culture solution. The liquid can be prevented from being mixed.

 The culture apparatus of the second invention is the first, second, third, fourth, seventh, ninth, tenth, tenth, tenth, seventeenth or nineteenth invention, wherein the culture vessel, the circulating means and the culture solution adjusting means A closed-loop circuit force S is formed by a path through which the culture medium flows, and the apparatus is provided with a caloric-pressure steam sterilization means for supplying the pressurized-pressure steam into the closed-loop circuit and sterilizing the inside of the closed-loop circuit. And

According to the second invention, if pressurized steam is supplied into the closed loop circuit by the force Q pressure steam sterilization means, the inside of the closed loop circuit can be sterilized without disassembling each means. Therefore, the entire apparatus can be sterilized easily and quickly. Further, since each means is not disassembled, that is, the manual operation in the sterilization operation of the culture apparatus can be eliminated, the risk of the culture tank and the like being mixed can be reduced. In addition, since the culture apparatus can be sterilized simply by operating the caloric steam sterilization means, the sterilization of the culture apparatus can be automated, and the operation of the culture apparatus can be completely automated. The culture apparatus of the 24th invention comprises the 1st, 2nd, 3rd, 4th, 7th, 9th, 10th, 10th, 17th, 19th Alternatively, the invention is characterized in that, in the invention, the circulation means includes a recovery means for recovering a desired component in the culture solution being cultured.

 According to the twenty-fourth invention, the protein that is an effective component in the culture solution and the component that produces cell force to be cultured can be recovered while the culture solution is circulated through the circulation path. The desired components can be recovered with high efficiency.

 The culture apparatus of the 25th invention is the cell supplying the cells to be cultured according to the 1st, 2nd, 3rd, 4th, 7th, 9th, 10th, 10th, 17th, 19th, 23rd, or 24th inventions A cell supply means for supplying a seeding solution containing cells to be cultured to the culture tank; and the seeding solution transferred by the cell supply means. It is characterized by comprising a discharge section that discharges inside.

 According to the 25th invention, the cells contained in the seeding solution can be supplied to the culture solution if the seeding solution is transported to the culture tank by the cell transporting means and discharged into the culture tank by the discharge unit. The culture apparatus according to the invention 26 is the culture device according to the 25th invention, wherein the cell transport means communicates between the storage section in which the seeding fluid force is stored and the discharge section, and the seeding solution. It is characterized by comprising: a pump for sending the gas from the storage part toward the discharge part; and a valve provided between the pump and the discharge part and connecting between the two.

 According to the twenty-sixth aspect of the invention, if the pump is operated with the valve closed, the pressure of the seeding solution in the seeding solution transfer path increases. If the valve is opened in this state, the seeding solution can be injected into the culture tank. Then, if a cell culture instrument or the like serving as a scaffold for culturing cells is provided in the culture tank, a seed solution, that is, cells can be seeded on the surface of the cell culture instrument. Therefore, even if it is an adherent cell that is not only floating cells, in other words, it is possible to inoculate without opening or closing the culture tank r, so that when cells are seeded, the cells and the culture medium are contaminated. Can be prevented.

 The culture apparatus of the second invention is the first, second, third, fourth, seventh, ninth, tenth, tenth, tenth, seventeenth, first, ninth, second, second, second or fifth invention, in the culture tank. It is provided with a culture device to which cells to be cultured are attached.

According to the twenty-seventh aspect of the invention, even cells that make up woven fli ^ can be cultured in a culture tank if they are attached to a culture instrument. This is not possible with conventional culture equipment It is possible to culture a large amount of the tissue and the adherent cells that make up the fl container.

 The culture apparatus according to a 28th invention is characterized in that, in the 27th invention, the culture instrument has a three-dimensional structure.

 According to the 28th invention, if cells are cultured on the surface of a culture instrument, a three-dimensional tissue can be constructed from various cells. Therefore, a large amount of three-dimensional tissues and organs necessary for regenerative medicine can be obtained. It becomes possible to supply. Further, since cells can be cultured along the shape of the culture instrument, a desired shape of tissue or organ can be formed by shaping the culture instrument into a desired shape.

 A culture apparatus according to a 29th aspect of the present invention is the culture apparatus according to the 28th aspect, wherein the culture instrument is formed by connecting a plurality of circular tubes.

 According to the 29th invention, since the culture instrument is a circular tube, the surface area of the culture instrument can be increased, so that a large amount of cells can be cultured. Moreover, since the culture fluid flows in the circular tube, a fresh culture fluid can be supplied to all cells. For this reason, nutrients and oxygen can be efficiently supplied to all cells to be cultured, so that the circulation efficiency of the culture solution can be increased and the culture conditions can be dramatically enhanced.

 A culture apparatus according to a 30th invention is the culture apparatus according to the 29th invention, wherein the culture instrument is disposed in the culture tank so that an axial direction thereof is parallel to an axial direction of the culture tank, A seeding device for seeding cells of cells to be cultured is provided on the surface of the culture device, and the seeding device supplies a liquid containing the cells to the tilting unit for tilting the culture tank. It comprises a cell supply unit and a moving unit for inserting and removing the cell supply unit with respect to the culture instrument provided in the culture vessel.

According to the 30th aspect of the invention, the cell supply unit is inserted into the culture instrument by the moving unit while the culture tank is tilted by the tilting unit, and includes cells of cells to be cultured from the cell supply unit into the culture instrument. If the liquid is dropped, the liquid can flow on the inner surface of the culture device, so that the cells can be seeded on the inner surface of the culture device. In addition, since the operation of the tilting unit, the moving unit, and the cell supply unit can be automated, cell culture using the culture apparatus can be automated. However, if it is automated, manual work can be eliminated, so that it is possible to prevent contamination of cultured cells and culture nights. A culture apparatus according to a 31st invention is characterized in that, in the 30th invention, the culture apparatus further comprises a rotating part for rotating the culture tank about its central axis.

 According to the third aspect of the invention, when the cell is seeded, if the culture tank is rotated in a tilted state, the liquid can flow over the entire inner surface of the culture device. Cells can be seeded.

 A culture apparatus according to a 32nd invention is the 30th or 31st invention, further comprising a cell recovery means for recovering cells of the cells cultured on the surface of the culture instrument, wherein the cell recovery means comprises the culture described above. It is characterized by comprising a stripping solution supply unit for supplying a liquid for separating the cells from the culture device to the device, and a recovery unit for collecting the liquid supplied from the stripping solution supply unit. According to the 32nd invention, if the stripping solution supply unit is inserted into the culture instrument, the stripping solution is supplied in that state, and the recovery unit collects the stripping solution together with the stripping solution. The power to collect r ' In addition, since the operation of the stripping solution supply unit and the recovery unit can be automated, the recovery of cells by the culture apparatus can be automated. However, automation can eliminate the need for manual work, thus preventing the cultured cells from being contaminated.

 A culture apparatus according to a 33rd invention is characterized in that, in the 27th invention, a material of the culture instrument is titanium or a titanium alloy.

 According to the 33rd invention, since the material of the culture device is titanium or titanium alloy having high biocompatibility, even if the cells to be cultured are seeded directly on the surface of the culture device, the cells are contaminated. Since it is not damaged, adherent cells such as fll ^ can be cultured for a long time. However, an extracellular matrix force is formed between the culture device and the cells, and a limestone layer is formed. For this reason, since the cell to culture can be reliably made to adhere to a culture instrument, a cell can be cultured in the stable state. In addition, even when used for a long period of time, the culture apparatus does not deteriorate or corrode, so that the culture apparatus can be used for a long period of time, and the cost of culturing cells can be reduced.

 The culture apparatus according to a 34th invention is characterized in that, in the 27th invention, the culture instrument has a titanium layer containing titanium on the surface thereof.

According to the 34th invention, since the layer containing titanium or titanium alloy having high biocompatibility is formed on the surface of the substrate of the culture device, the cells to be cultured are directly seeded on the surface of the culture device. The adherent cells of i can be cultured for a long period of time without being contaminated or damaged by the culture device. In addition, the culture device is completely covered with a layer containing titanium or a titanium alloy, and the layer of titanium or the like is completely covered with an extracellular matrix as the cell culture progresses. For this reason, since the substrate does not differ from the cells at all, the cells can be cultured in a stable state. In addition, the culture apparatus does not change or corrode even after long-term use. Therefore, the culture instrument can be used for a long time, and the cost of culturing cells can be reduced.

 A culture apparatus according to a 35th invention is characterized in that, in the 27th invention, a material of the culture instrument is a cell-derived polymer material.

 According to the 35th invention, since the material of the culture device is a cell-derived polymer material, even if the cells to be cultured are seeded directly on the surface of the culture device, the cells are contaminated by the culture device. Adhesive cells such as fli§ & that can be damaged can be cultured for a long time. In addition, extracellular matrix force is formed between the culture device and the cells, and a calcified layer is also formed. For this reason, the cells can be cultured in a stable state, although the cells to be cultured can be reliably attached to the culture apparatus. In addition, since the entire culture device is made of a cell-derived polymer material, and this cell-derived polymer material is absorbed by the living body, it can be transplanted as it is, so that the transplantation is performed reliably and easily. The culture device of the 36th invention can be characterized in that, in the 27th invention, the culture instrument has a layer of a cell-derived polymer material on the surface thereof.

According to the 36th invention, since the layer of the cell-derived polymer material is formed on the surface of the base material of the culture instrument, even if the cells to be cultured are seeded directly on the surface of the culture instrument, Since the cell force S is not contaminated or damaged by the instrument, adherent cells such as H swollen can be cultured for a long time. In addition, the culture device is completely covered with a layer of cell-derived polymer material, and the layer of cell-derived polymer material is completely covered with an extracellular matrix. For this reason, since the substrate does not come into contact with the cells at all, the cells can be cultured in a stable state. Furthermore, since the culture device does not change or corrode even if it is used for a long time, the culture device can be used for a long time, and the cost of culturing cells can be reduced. The culture device of the 37th invention is characterized in that, in the 35th or 36th invention, the cell-derived polymer material is collagen or a cell-absorbing polymer.

 According to the thirty-seventh aspect of the invention, collagen and cell-absorbing polymers have a very high cell affinity with cells, so that the culture environment of adherent cells can be further improved. Further, since the affinity between the culture apparatus and the cells is further increased, a large number of cells can be cultured with low cost and efficiency.

 A culture apparatus according to a thirty-eighth aspect of the present invention is the culture apparatus according to the twenty-seventh aspect, wherein a material of the culture instrument is a calcium compound.

 According to the thirty-eighth aspect of the invention, since the material of the culture device is a calcium compound, even if the cells to be cultured are seeded directly on the surface of the culture device, the cells to be cultured directly on the surface of the culture device are Even after seeding, the cells can be contaminated or damaged by the culture device. Therefore, it is possible to culture adherent cells such as 1 thigh for a long time. In addition, since the entire culture device is made of a strong ruthenium compound and this calcium compound is absorbed by the living body, it can be transplanted as it is, so that the transplantation can be performed reliably and easily. A culture apparatus according to a thirty-ninth aspect of the present invention is the culture apparatus according to the twenty-seventh aspect, wherein the culture device has a calcium compound layer on a surface thereof.

 According to the thirty-ninth invention, since the calcium compound layer is formed on the surface of the base material of the culture device, even if the cells to be cultivated are directly seeded on the surface of the culture device, As a result, the cells are not contaminated or damaged, and adherent cells such as fl fibers can be cultured for a long time. In addition, the culture device is completely covered with a layer of calcium compound, and this layer of calcium compound is completely covered with an extracellular matrix. For this reason, since the substrate does not come into contact with the cells at all, the cells can be cultured in a stable state. Furthermore, even if it is used for a long time, it does not deteriorate or corrode the culture equipment. Therefore, the culture instrument can be used for a long time, and the cost of culturing cells can be reduced.

 The culture apparatus according to a 40th aspect of the present invention is the culture apparatus according to the 27th aspect, wherein a material of the culture instrument is a synthetic biodegradable polymer.

According to the 40th invention, since the material of the culture instrument is a synthetic biodegradable polymer, Even if cells to be cultured are seeded directly on the surface of the device, the cell force S is not contaminated or damaged by the culture device, so that adherent cells such as can be cultured for a long time. However, since the entire culture device is formed of a synthetic biopolymer, and this synthetic biodegradable polymer is absorbed by the living body, it can be transplanted as it is, so that transplantation is performed reliably and easily. be able to.

 The culture apparatus according to a 41st invention is characterized in that, in the 27th invention, the culture instrument has a layer of a synthetic biodegradable polymer on the surface thereof.

 According to the 41st invention, since the layer force of the synthetic biodegradable polymer is formed on the surface of the base material of the culture device, the culture device can be used even if the cells to be cultured are seeded directly on the surface of the culture device. Thus, the cells are not contaminated or damaged, and adherent cells such as H§§ can be cultured for a long period of time. In addition, the culture device is completely covered with a layer of synthetic biodegradable polymer, and the layer of synthetic biodegradable polymer is completely covered with an extracellular matrix. For this reason, the cells can be cultured in a stable state because they do not come into contact with the cells that are spread on the substrate. In addition, the culture equipment will not be altered or corroded even after long-term use. Therefore, the culture instrument can be used for a long time, and the cost of culturing cells can be reduced.

 The culture apparatus of the 42nd invention is characterized in that, in the 40th or 41st invention, the synthetic biopolymer is poly-L-lactic acid or polydaricholic acid.

 According to the forty-second invention, poly-L-lactic acid-polyglycolic acid has a very high cell affinity with cells, so that the culture environment for adherent cells can be further improved. In addition, since the affinity between the culture apparatus and the cells is further increased, it is possible to cultivate a large number of cells efficiently and at low cost.

 The culture apparatus of the 43rd invention is characterized in that, in the 27th invention, the culture device has a layer of a tissue induction material having a tissue induction ability on the surface thereof.

According to the fourth aspect of the invention, since the tissue-inducing material layer is provided on the surface of the culture instrument, the cell affinity between the culture instrument and the cells is high. Cells to be cultured are seeded directly on the surface of the culture instrument. However, since cells are not contaminated or damaged by the cultivating device, adherent cells such as rn ^ can be cultured for a long time. In addition to improving the culture environment for adherent cells, it can be used directly in the living body for regenerative medicine. Can be reduced. In addition, not only the growth of cells is promoted by the effect of tissue-inducing materials, but also differentiation of undifferentiated mesenchymal cells into various tissues can be induced. The culture apparatus of the 44th invention is characterized in that, in the 43rd invention, the job induction material is an extracellular matrix.

 According to the 44th invention, since the material such as the yarn weaving fluid is an extracellular matrix and the cell affinity between the culture device and the cells is very high, the culture environment of adherent cells can be further improved. And since the affinity between the culture device and the cells is further increased, it becomes a powerful ability to culture a large number of cells efficiently and at low cost. In addition, the differentiation of undifferentiated mesenchymal cells into various tissues can be achieved not only by promoting the cell proliferation ability due to the influence of the extracellular matrix. Therefore, it is possible to easily supply cells necessary for regenerative medicine in addition to the ability to culture while maintaining the differentiation state of the cells.

 A culture apparatus according to a 45th aspect of the present invention is the culture apparatus according to the 27th aspect, wherein the material of the culture instrument is a silicon-containing material.

 According to the 45th invention, since silicon has a limestone function and has a very high cell affinity with cells, cells can be cultured even if cells to be cultured are seeded directly on the surface of a culture device. Adherent cells such as Hi ^ can be cultured for a long period of time without being contaminated or damaged. In addition, an extracellular matrix is formed between the culture apparatus and the cells, and a limestone layer strength s is formed. For this reason, since the cell to culture can be reliably made to adhere to a culture instrument, a cell can be cultured in the stable state. In addition, since the culture apparatus does not deteriorate or corrode even when used for a long time, the culture apparatus can be used for a long time, and the cost of culturing cells can be reduced. Furthermore, since the silicon-containing material contains silicon, it emits far-infrared rays when the temperature rises, so that an effect of promoting cell differentiation and proliferation can be obtained.

 The culture apparatus of a 46th invention is characterized in that, in the 27th invention, the culture instrument has a layer of a silicon-containing material on the surface thereof.

According to the 46th invention, since silicon has a calcification ability and has a very high cell affinity with cells, even if cells to be cultured are seeded directly on the surface of the culture device, Adherent cells such as fli ^ can be cultured for a long time without contaminating or damaging the cells. In addition, the culture device is completely covered with a layer of silicon-containing material, and this This layer of silicon-containing material is completely covered by the extracellular matrix over the course of cell culture. For this reason, Sii does not do anything with the cells, so it is possible to cultivate the cells in a stable state τ '. In addition, the culture apparatus will not be altered or corroded even after long-term use. Therefore, the culture instrument can be used for a long time, and the cost of culturing cells can be reduced. Furthermore, since the silicon-containing material contains silicon, it emits far-infrared rays when the temperature rises, so that an effect of promoting cell differentiation and proliferation can be obtained.

 The culture apparatus of a 47th invention is characterized in that, in the 45th or 46th invention, the silicon-containing material is zeoli.

 According to the 47th invention, since zeolite has a very high cell affinity with cells, even if the cells to be cultured are seeded directly on the surface of the culture device, the cells may be contaminated by the culture device.

Adhesive cells such as flt ^ that can be damaged can be cultured for a long time. Zeolite contains silicon, so it emits far-infrared rays when the temperature rises, so that cell differentiation and proliferation can be obtained.

 The culture apparatus according to a 48th invention is characterized in that, in the 27th invention, the material of the culture instrument is an artificial ceramic.

 According to the 48th invention, since the artificial ceramic has a very high cell affinity with the cells, even if the cells to be cultured are seeded directly on the surface of the culture apparatus, the cell is contaminated or damaged. Adherent cells such as ¾§ can be cultured for a long time. However, an extracellular matrix is formed between the culture device and the cells, and a calcified layer is further formed. For this reason, since the cells to be cultured can be reliably attached to the culture apparatus, the cells can be cultivated in a stable state. In addition, even when used for a long time, the culture equipment strength S does not deteriorate or corrode, so that the culture equipment can be used for a long time, and the cost of culturing cells can be reduced. Furthermore, artificial ceramics emit far-infrared rays when the temperature rises, so that the effect of promoting cell proliferation can be obtained.

 A culture apparatus according to a 49th aspect of the present invention is the culture apparatus according to the 27th aspect, wherein the culture instrument has an artificial ceramic layer on a surface thereof.

According to the 49th invention, since artificial ceramics have a very high affinity for cells, even if cells to be cultured are seeded directly on the surface of the culture device, the cell force S is contaminated by the culture device. Adherent cells such as those that can not be damaged can be cultured for a long time. The In addition, the culture device is completely covered with an artificial ceramic layer, and this artificial ceramic layer is completely covered with an extracellular matrix as the cells are cultured. For this reason, since the cells do not come into contact with the base material, it is possible to cultivate the cells in a stable state. In addition, the culture apparatus will not be altered or corroded even after long-term use. Therefore, the culture instrument can be used for a long time, and the cost of culturing cells can be reduced. Furthermore, artificial ceramics emit far-infrared rays when the temperature rises, so that the effect of promoting cell proliferation can be obtained.

 The culture apparatus of the 50th invention is the 2nd, 3rd, 3rd, 3rd, 4th, 3rd, 5th, 3rd, 6th, 3rd, 8th, 9th, 40th, 4th, 1st, 4th, 4th, 4th, 4th, 6th, 4th, 8th or 4th 9 The invention is characterized in that the culture instrument is a porous body.

 According to the 50th invention, since the culture device is porous, the cells attached to the surface of the culture device are also attached to the cell attached to the culture device only with the cells on the surface. Fresh culture fluid can be supplied. For this reason, nutrients and oxygen can be efficiently supplied to all the cells to be cultured, so that the circulation efficiency of the culture solution is improved and the culture conditions are dramatically enhanced.

 The culture apparatus of the 5th invention is the 2nd, 3rd, 3rd, 3rd, 4th, 3rd, 5th, 3rd, 6th, 3rd, 8th, 9th, 40th, 4th, 1st, 4th, 4th, 4th, 4th, 6th, 4th, 8th or 4th 9 The invention is characterized in that the culture device has a network structure. -According to the fifth aspect of the invention, since the culture device has a network structure, the cells attached to the surface of the culture device are attached to the culture device only with the cells on the surface. It is possible to supply a fresh culture solution to a part of cells. For this reason, nutrients and oxygen can be efficiently supplied to all of the cells to be cultured, so that the circulation efficiency of the culture solution is improved and the culture conditions are dramatically enhanced.

 The culture apparatus of the fifth invention is the culture apparatus according to the first, second, third, fourth, seventh, ninth, tenth, tenth, tenth, seventeenth, nineteenth, seventeenth, nineteenth, second, third, second, second, fifth, or second-second inventions. It is characterized by culturing by immersing floating cells in the liquid.

According to the 52nd invention, since floating cells can be cultured in a large amount, various active ingredients produced in the process of culturing floating cells can be recovered. Therefore, pharmaceuticals, antibodies, vaccines and the like can be produced from the collected components. The culture apparatus of the 53rd invention is characterized in that, in the 52nd invention, the floating cells include hematopoietic stem cells.

 According to the 53rd invention, if hematopoietic stem cells are contained, they are erythroblasts, myeloblasts, megakaryoblasts, monoblasts, erythrocytes that are differentiated into lymphoblasts, nucleated leukocytes It can produce neutrophils, eosinophils, basophils, platelets, monocytes and lymphocytes. Therefore, a mixed solution that can be used as a transfusion preparation can be produced.

 The culture device of the 54th invention is characterized in that, in the 52nd or 53rd invention, the floating cells include blasts of blood cells.

 According to the 54th invention, if erythroblasts, myeloblasts, megakaryoblasts, monoblasts, lymphoblasts, etc. are included, erythrocytes that are direct blood cells, neutrophils that are nucleated leukocytes Can produce eosinophils, basophils, platelets, monocytes and lymphocytes. Therefore, a mixed solution that can be used as a transfusion preparation can be produced.

 The culture apparatus according to a 55th invention is characterized in that, in the 27th, 52nd, 53rd, or 54th invention, the cells attached to the culture device and the floating cells floating in the culture medium are mixed and cultured. To do.

 According to the 55th invention, so-called mixed culture can be performed in which floating cells are cultured in a culture solution while culturing adherent cells with a culture device provided in a culture tank. For this reason, since the factor that activates and promotes differentiation of the foliage vesicles is produced and secreted from the adherent cells, it is possible to promote the culture of the floating cells. In addition, since floating cells produce bioactive factors, the activity and differentiation of adherent cells can be promoted by the action of the bioactive factors. Therefore, since the culture of each cell can be promoted as compared with the case where only floating cells or adherent cells are cultured, each cell can be efficiently cultured. The culture device of the 56th invention is characterized in that, in the 55th invention, the cell attached to the culture instrument is a bone marrow stromal cell, and the cell floating in the culture solution contains a hematopoietic stem cell. .

According to the 56th invention, if hematopoietic stem cells are mixed and cultured in a culture solution while culturing bone marrow stromal stem cells with a culture device, the factor that activates hematopoietic stem cells from bone marrow stromal stem cells or promotes differentiation. Is produced and secreted, which promotes the production of erythrocytes, nucleated leukocytes, neutrophils, eosinophils, basophils, platelets, monocytes and lymphocytes from hematopoietic stem cells. Also, Biologically active factors produced by hematopoietic stem cells promote the separation of bone marrow stromal stem cells into osteoblasts, chondroblasts, fibroblasts, fat cells and other cells. Therefore, it is faster and more efficient than culturing hematopoietic stem cells and bone marrow stromal stem cells alone. Differentiation can be induced.

 The culture apparatus of the 57th invention is the culture apparatus according to the 55th or 56th invention, wherein the cells attached to the culture instrument are bone marrow stromal cells, and the floating cells include blasts of blood cells. It is characterized by.

 According to the 57th invention, blood blast cells such as erythroblasts, myeloblasts, megakaryoblasts, monoblasts, and lymphoblasts are cultured in the culture medium while culturing bone marrow stromal stem cells with a culture device. When bone marrow tissue containing selenium is mixed and cultured, bone marrow stromal stem cells activate erythroblasts and other factors, and factors that promote differentiation are produced and secreted. Production of white blood cells such as neutrophils, eosinophils, basophils, platelets, monocytes and lymphocytes is promoted. In addition, the physiologically active factors produced by erythroblasts promote the differentiation of stromal stem cells into osteoblasts, chondroblasts, fibroblasts, fat cells and other cells. For this reason, it is possible to produce erythrocytes, which are blood cells that are faster and more efficient and more active than culturing bone marrow tissue and bone marrow stromal stem cells alone, and efficiently induce differentiation of osteoblasts and the like. can do.

 The blood product of the 58th invention comprises the culture apparatus of the 1st, 2nd, 3rd, 4th, 7th, 9th, 10th, 10th, 17th, 19th, 23rd, 24th, 25th or 27th inventions. When culturing hematopoietic stem cells, it contains a blood component produced in the culture solution.

 According to the 58th invention, it is possible to produce a large amount of blood components from a small amount of hematopoietic stem cells, but it is also possible to easily produce a blood product containing a desired component in large amounts. However, since hematopoietic stem cells can be ingested from people who need blood transfusions and blood products, and the hematopoietic stem cells can be cultivated to produce blood for blood transfusions and blood products, it is possible to produce safe blood for blood transfusions, etc. Can be built.

 5th 9th Artificial Artificial & Weaving is 1st, 2nd, 3rd, 4th, 7th, 9th, 10th, 1st, 6th, 1th, 1th, 9th, 2nd, 3rd, 4th, 25th, or 27th invention The apparatus is characterized in that it is formed by culturing cells seeded in the culture instrument.

According to the 59th invention, a desired string can be produced from a small amount of cells. Necessary for live medical treatment)]] can be produced in large quantities at a low price. However, fllgg can be produced by ingesting cells of the desired tissue from a person in need of transplantation and culturing the cells. It is also possible to prevent the occurrence of s.

 The artificial tissue of the 60th invention is characterized in that, in the 59th invention, the cell is an undifferentiated cell.

 According to the 60th invention, if the culture conditions are adjusted, it is possible to produce d ^ and the like required for regenerative medicine in a large amount at low cost, in addition to being able to differentiate into desired cells. it can. However, if cells taken from a person who needs transplantation are used, compatibility with the living body can be increased, and rejection can also be prevented. Brief Description of Drawings

FIG. 1 is a schematic block diagram of the culture apparatus 1 of the present embodiment.

FIG. 2 is a schematic explanatory diagram of the culture tank 2 installed in the culture apparatus 1 of the present embodiment.

FIG. 3 is a detailed explanatory view of the culture medium adjusting means 20.

FIG. 4 is a schematic explanatory diagram of the gas concentration adjusting means 22 of another embodiment.

FIG. 5 is a schematic block diagram of the culture apparatus 1 provided with the extraction means 80.

FIG. 6 is a schematic block diagram of the culture apparatus 1 provided with the cell supply means 90.

FIG. 7 is a schematic explanatory diagram of the planar heating element 71.

FIG. 8 is a schematic front longitudinal sectional view of the cell seeding means 60.

FIG. 9 is a schematic side longitudinal sectional view of the cell seeding means 60.

FIG. 10 is a schematic explanatory diagram showing a state where the cell seeding means 60 is rotated.

FIG. 11 is a schematic front longitudinal sectional view of a cell seeding means 60 according to another embodiment.

FIG. 12 is a schematic side longitudinal sectional view of cell seeding means 60 according to another embodiment.

FIG. 13 is a schematic explanatory diagram of a state where the cell seeding means 60 according to another embodiment is rotated. Fig. 14 is a graph showing (A) the weight ratio of each component of black body powder, and (B) a graph showing the results of FT-IR analysis of black body powder.

Fig. 15 is a graph showing (A) the weight ratio of each component of black body powder, and (B) a graph showing the results of FT-IR analysis of black body powder. Fig. 16 is a graph showing (A) the weight ratio of each component of natural ceramics, and (B) a graph showing the results of FT-IR analysis of natural ceramics.

Figure 17 is a graph showing the results of FT-IR analysis of zeolite.

Fig. 18 is a graph showing (A) the weight ratio of each component of artificial special ceramics, and (B) a graph showing the results of FT-IR analysis of artificial special ceramics.

FIG. 19 is a diagram comparing (A) the turbidity of the culture solution when culturing E. coli using the culture device and flask of the present invention, and (B) the culture device and dish of the present invention. FIG. 3 is a diagram comparing the total amount of yeast in the culture medium when yeast is used and cultured.

FIG. 20 is a diagram comparing (A) the number of cells in the culture medium and (B) comparing the amount of antibody produced when cultivating hyperpridoma cells using the culture apparatus and dish of the present invention. FIG.

Figure 21 is a diagram comparing (A) the number of platelets per total culture volume when hematopoietic stem cells were cultured using the culture apparatus and dish of the present invention, and (B) per hematopoietic stem cell number. It is the figure which compared the number of platelets. BEST MODE FOR CARRYING OUT THE INVENTION

 Next, an embodiment of the present invention will be described with reference to the drawings.

 The culture apparatus of the present invention is an apparatus for culturing cells, and is provided in a culture tank for containing a culture solution, a circulation means for circulating the culture solution externally, and a path for circulating the culture solution externally. It is provided with a culture medium adjusting means, and is characterized in that the culture medium is externally circulated and the state of the culture liquid is adjusted by the culture medium adjusting means during the external circulation.

FIG. 1 is a schematic explanatory diagram of the culture apparatus 1 of the present embodiment. In the same figure, the code | symbol 2 has shown the culture tank of the culture apparatus 1 of this embodiment. The culture tank 2 is a container made of, for example, glass stainless steel, and is composed of a main body 2a for containing a culture solution and a lid 2b that can seal the inside of the main body 2a. When the lid 2b is attached to the main body 2a, the inside of the culture tank 2 can be hermetically sealed from the outside. Note that the material of the culture tank 2 is not limited to the above, and does not corrode even after long-term use. The material is not particularly limited as long as it does not alter the culture solution.

 As shown in FIGS. 1 and 2, the lid 2 b of the culture tank 2 is air-tightly attached to one end of the piping 1 1 of the circulating means 10 passing through the lid 2 b. The other end of 1 is connected to the suction port of pump l ip. On the other hand, one end of the pipe 1 2 is attached to the discharge port of the pump l ip, and the other end of the pipe 1 2 is airtightly attached to the lid 2b. Then, one end of the pipe 11 and the other end of the self-pipe 12 are both immersed in the culture solution in the culture tank 2. Therefore, if the pump lip is operated, the culture solution in the culture tank 2 can be circulated outside the culture tank 2 through the pipes 11 and 12.

 Further, as shown in FIG. 1, the culture medium adjusting means 20 for adjusting the state of the culture liquid flowing in the pipe 12 is interposed in the pipe 12. This culture medium adjusting means 20 includes a dialysis unit 21 for removing wastes and the like contained in the culture liquid, a gas concentration adjusting means 22 for adjusting the gas concentration of the culture liquid, and a culture medium for cell culture. It is equipped with a nutrient supply unit 2 3 that supplies the necessary nutrients.

 Therefore, the culture solution in the culture tank 2 can be externally circulated by operating the pump l ip to supply the culture solution to the culture solution adjusting means 20. Then, if the culture solution is externally circulated while culturing the cells in the culture tank 2, the waste product discharged by the cells when the culture solution passes through the culture solution adjusting means 20 is clarified by the dialysis unit 21. The concentration of oxygen, nitrogen, carbon dioxide, etc. in the culture medium is adjusted by the gas concentration adjusting means 22 and the nutrients consumed by the cells are supplied to the culture medium by the nutrient supply unit 23 Then, by circulating the culture solution externally, the culture solution can be adjusted to a state before starting cell culture, in other words, in a state suitable for cell culture, and returned to the culture tank 2.

 Therefore, according to the culture device 1 of the present embodiment, the force of maintaining the state of the culture solution in the culture tank 2 at the optimum condition for cell culture τ ′ can be achieved. Can be cultured in the state. And since it can culture continuously for a long period of time, it is possible to culture a large amount of cells.

Then, as shown in Fig. 1, if one end of the pipe 1 1 is placed in the upper part of the culture tank 2 and the other end of the pipe 1 2 is placed in the lower part of the culture tank 2, the culture medium can be circulated externally. A flow from the bottom to the top can be generated in the culture tank 2. In addition, a rotor 4 with metal braces attached to both ends in the axial direction is arranged at the bottom of the main body 2a of the culture tank 2, and the culture tank If the motor 5 with the magnet 5 b attached to the main shaft is installed directly under the rotor 4 outside of 2, the magnet 5 b rotates when the motor 5 is driven, and the magnet 5 b Since the rotor 4 in the culture tank 2 can be rotated by the magnetic force, a flow swirling in the horizontal plane can be generated in the culture solution in the culture tank 2. Then, if the rotor 4 is rotated while the culture medium is externally circulated, the culture medium that is externally circulated and returned to the culture tank 2 is retained in the culture tank 2 by the action of the two flows. Since the mixing with the existing culture solution can be promoted, the state of the culture solution in the culture tank 2 can be made to be uniform. Moreover, since it is possible to eliminate the culture solution that is retained in the culture tank 2 and is not externally circulated, the state of the culture solution, that is, the culture conditions of the cells can be maintained well. Since the flow generated by external circulation of the culture solution is used for mixing the culture solution, the mixing of the culture solution can be promoted even if the rotational speed of the rotor 4 is lowered. Then, the flow rate of the culture solution in the culture tank 2 can be reduced compared to the case where convection is generated and mixed in the culture tank 2 only by the rotation of the rotor 4, so that even cells that are very sensitive to external pressure can be used. The culture can be performed while keeping the state of the culture solution in the culture tank 2 uniform.

 The rotor 4 need not be provided, but if the rotor 4 is provided, the culture medium can be homogenized quickly and efficiently.

 In addition, as shown in FIGS. 1 and 2, when culturing cells that flow with the culture solution, such as floating cells and bacteria, as an outflow prevention means at one end of the culture tank 2 in the pipe 11. An electromagnet 6 is preferably provided. Usually, suspended cells and bacteria are charged and repelled by the electromagnet 6, so that the suspended cells and the like can be prevented from passing through the position where the electromagnet is provided.

 Then, even when floating cells, bacteria, etc. are cultured while circulating the culture solution externally, it is possible to retain the floating cells etc. in the culture tank 2, and the dead cells will flow out and be piped. Blockage within 1 1, 1 2 can also be prevented.

However, since the electromagnet 6 may be attached to the outer peripheral surface of the pipe 11, the flow of the culture solution is not hindered. And the electromagnet 6 does not prevent the outflow by physical contact like the Phil evening. In other words, since the cells are not in contact with the cells being cultured, cells such as very weak cell membranes such as erythrocytes, macrophages, and plasma plates can be prevented from flowing out of the culture tank 2 and can be cultured without being damaged. In addition, if the suspension cell is strong against external pressure, a device that prevents the outflow of suspended cells by physical contact, such as a film or membrane, may be used as an outflow prevention means. .

 Next, the culture medium adjusting means 20 will be described in detail.

 As shown in FIGS. 1 and 3, the dialysis section 21 of the culture medium adjusting means 20 is interposed in the pipe 12. The dialysis unit 21 is composed of a hollow main body case 21a and a hollow fiber 21b disposed in the main body case 21a.

 The inside of the main body case 21a is in communication with the pipe 12 and is configured such that the culture fluid flows through the culture fluid circulation passage 21h between the inner surface thereof and the hollow fiber 21b.

 The hollow fiber 2 lb is made of a material having a semi-permeable function such as polypropylene, cellulose diastate, or silicone rubber, and has a bicarbonate buffer solution, a phosphate buffer solution, etc. Dialysate is passed.

 Therefore, when the culture solution passes through the culture passage 2 lh, the culture solution is dialyzed through the hollow fiber 21b, and wastes such as ammonia and nitrogen compounds contained in the culture solution move to the dialysate. From this, the culture solution can be purified. Therefore, deterioration of the culture solution can be prevented, and the cell can always be kept in a state suitable for cell culture, so that the cell can be cultured continuously for a long time in a powerful and stable state.

 The potato dialysis unit 2 1 force is provided outside the recharge tank 2, that is, because the culture solution is dialyzed outside the culture tank 2, the size of the dialysis unit 2 1 and the dialysis unit 2 1 The material to be used can be freely selected regardless of the size of the culture tank 2 and the nature of the culture solution. Therefore, since the dialysis unit 21 having a desired dialysis ability can be employed, the culture medium can be surely dialyzed to a desired condition for purification. If the dialysis part 21 has a sufficiently high permeation capacity compared to the size of the culture tank 2, the dialysis capacity can be increased in accordance with the increase in the number of cells. It is also possible to continuously culture these cells.

In addition, since the dialysate is passed through the hollow fiber 21b and the culture fluid is passed through the culture fluid circulation passage 2 lh, the flow resistance when the culture fluid flows through the dialysis section 21 can be reduced, and the culture fluid can be reduced. Can be circulated smoothly outside. Since the flow resistance when flowing through the dialysis unit 21 is small, even if cells are contained in the circulating culture medium, it passes through the dialysis unit 21. When you can reduce the resistance that the cell receives, when passing through the dialysis part 21 can be less damaged cells.

 Furthermore, if the dialysis section 21 1 force S has a wide space between hollow fibers, it is possible to prevent cells to be cultured from being clogged with 2 lb of hollow fibers and lowering the dialysis performance.

 In addition, the culture solution may be passed through the hollow fiber 21b and the dialysate may be passed through the culture fluid circulation passage 2 lh. With the configuration as in the present application, the flow of the culture solution is larger than when passing through the hollow fiber 21b. Since the stakes are reduced, dialysis efficiency can be increased, and when cells are contained in the circulating culture medium, cell damage can be reduced. If the inner diameter of the hollow fiber 21b is increased, it is possible to prevent cells to be cultured from clogging with 2 lb of the hollow fiber and reducing the dialysis ability s.

 Furthermore, instead of the hollow fiber 21b, a configuration in which the path through which the culture solution is passed and the path through which the dialysate is passed may be separated in the main body case 21a by a dialysis membrane or the like.

 Furthermore, the method of purifying the culture solution is not limited to the method of digestion by dialysis as described above, and an ammonia adsorption column, affinity column, molecular sieve, or the like may be used.

As shown in FIG. 1 and FIG. 3, in the pipe 12, a gas concentration adjusting means 22 is interposed downstream of the dialysis unit 21. The gas concentration adjusting means 20 includes a hollow case 22a, and the inside of the case 22a communicates with the pipe 12. The culture medium supplied from the upper part of the case 22a is discharged from the lower part of the case 22a. Then, one end of a pipe 26 whose other end is connected to the gas supply means G is disposed at the upper part of the case 22a. The gas adjusting means G supplies a saturated gas adjusted so that a desired gas concentration, for example, C 0 ₂ is 5% and air is 95% into the case 22a.

 Note that the gas concentration of the saturated gas is not limited to the above concentration, and the oxygen concentration may be increased, and the gas concentration may be optimized for the state of the cells to be cultured. For example, the oxygen concentration may be 30% or nitrogen may be 65%.

If the saturated gas is sent from the gas supply means G into the case 22a of the gas concentration adjusting means 20, the saturated gas can be brought into contact with the culture solution in the case 22a, so that the gas concentration in the culture solution is the saturated gas. The gas concentration can be adjusted to be equivalent to the above. Therefore, the culture solution Saturate to the desired gas concentration and keep the gas concentration in the culture medium uniform and suitable for cell culture. Can do.

 In addition, the gas concentration adjusting means 2 2 is provided outside the culture tank 2, that is, since the gas concentration of the culture solution can be adjusted outside the culture tank 2, the gas concentration adjusting means 2 2 The size can be freely selected regardless of the size of the culture tank 2. Therefore, since the gas concentration adjusting means 22 having a desired gas exchange capability can be adopted, it is possible to reliably adjust the culture solution to the desired gas concentration. If the gas concentration adjustment means 2 2 is sufficiently high compared to the size of the recharge tank 2, the gas exchange capacity is increased in accordance with the increase in the number of cells. Therefore, it is possible to culture a large number of cells continuously.

 In addition, when the circulating culture solution does not contain cellular force, an aeration part 22b having a large number of pore forces s formed at one end of the pipe 26 is provided, and this aeration part 22 is cultured in the case 22a. A configuration may be adopted in which the gas is discharged into the culture solution from a large number of pores of the aeration section 22b by being immersed in the solution (see FIG. 4 (A)). In this case, if the saturated gas is sent from the gas supply means G to the aeration unit 22b, the culture solution in the case 22a can be aerated, so the contact area force between the culture solution and the saturated gas increases, so oxygen is added to the culture solution. It is possible to increase the efficiency of dissolving the gas and so on, and to increase the gas comfort factor. Since the culture solution does not contain cells, bubbles generated when aerated are not damaged or stressed.

 In addition, if the sterilizing filter 27 for sterilizing the saturated gas is provided in the pipe 26 between the case 22a and the gas supply means G, the culture fluid can be contaminated even if the saturated gas contains germs. Can be prevented.

 Furthermore, as shown in FIG. 4 (B), an air permeable tube 22d (for example, a tube made of Teflon (registered trademark of DuPont)) is provided in the case 22a, and the culture solution is passed through the tube. If the configuration is such that saturated gas is supplied into the case 22a, it is possible to prevent direct contact between the culture solution and the saturated gas, so that the culture solution is more reliably prevented from being contaminated. be able to

And, in the pipe 1 2, the dialysis part 2 1 and the gas concentration adjusting means 2 2 are arranged in series, In other words, the culture medium circulating outside is passed through both the dialysis unit 21 and the gas concentration adjusting means 2 2 and then returned to the culture tank 2, so that the state of the culture medium in the culture tank 2 is suitable for cell culture. It can be reliably kept in a state. Since it is compact and can be manufactured collectively, it can be manufactured as a disposable set. In particular, if a configuration of a disposable set is adopted, it is possible to prevent the problem of contamination from the connection part that occurs when the dialysis part 21 and the gas concentration adjusting means 22 are arranged in series.

 As shown in FIG. 1 and FIG. 3, a nutrient solution replenishing section 23 is interposed on the downstream side of the gas concentration adjusting means 2 2 in the pipe 12. This nutrient supply unit 23 includes a culture portion 23a through which a culture solution flows, and a nutrient channel 23b through which nutrient solutions including nutrients such as site-in, hormones, and vitamins necessary for cell culture, The two passages are separated from each other by, for example, a dialysis member formed of a material having a semi-permeable function such as polypropylene, cellulose diastate, or silicone rubber.

 For this reason, when the culture solution passes through the culture solution passage part 23a, the nutrient consumed by the cells and reduced in the concentration force s moves from the nutrient to the culture solution via the dialysis member 23c. Nutrients necessary for cell culture can be supplied. Therefore, it is possible to prevent the nutrients in the culture medium from being depleted and to always maintain a state suitable for cell culture, so that cells can be cultured continuously for a long period of time in a stable state.

 In addition, the nutrient supply part 2 3 is provided outside the culture tank 2, that is, the nutrient solution is supplied to the culture solution outside the culture tank 2. The ability can be freely selected regardless of the size of the culture tank 2 or the like. Therefore, since the nutrient supply unit 23 having the desired ability can be employed, the concentration of the nutrient contained in the culture solution can be reliably supplied to the desired concentration. If the nutrient supply unit 2 3 has a sufficiently high capacity compared to the size of the culture tank 2, the amount of nutrients to be supplied to the culture solution in accordance with the increase in the number of cells. Since it can be adjusted, it is possible to continuously culture large numbers of cells.

In addition, since the culture solution passage portion 23a and the nutrient solution passage portion 23b are separated by the dialysis member 23c, only the components having a reduced concentration in the culture solution among the nutrients contained in the nutrient solution are supplied to the culture solution. Can do. Therefore, it is possible to prevent cell damage caused by supplying the nutrient nutrient solution more than necessary. The configuration of the nutrient supply unit 23 may be substantially the same as the configuration of the dialysis unit 21, and the nutrient may be poured into the hollow fiber.

 In addition, if the culture medium adjusting means 20 is provided with a recovery means 28 such as a dialyzer, an affinity column, a molecular sieve or the like for recovering the desired components in the culture liquid, the culture medium is circulated externally. In the meantime, it is possible to recover the protein that is the active ingredient in the culture medium and the ingredient produced by the cultured cells, so that the desired ingredient such as the active ingredient can be recovered with high efficiency and reliability. Without stopping the culture, it is possible to collect only the desired components. In addition, if cultured for a long period of time, even if it is purified by the dialysis unit 21 or supplemented with nutrients by the nutrient replenishment unit 23, the culture solution will deteriorate, but the culture has the following structure. If a culture medium exchange means having a liquid supply section 30 and a culture medium discharge section 40 is provided, the culture medium can be replaced with a fresh culture medium while culturing cells. As shown in FIG. 1, one end of the pipe 33 of the culture solution supply unit 30 is attached to the pipe 12 via the communication valve 31. The other end of the pipe 33 communicates with a culture medium storage unit (not shown). This pipe 3 1 is provided with a heater 3 4, a pump 3 5, a vacuum pump, which warms the culture solution supplied from the culture solution storage unit to about 35 ° C. between the culture storage unit and the communication valve 3 1. Bacteria filter 3 2 is installed and installed in that order from the culture medium reservoir. On the other hand, the culture medium drain 2 40 is connected to the culture tank 2 lid 2 b in an airtight manner. One end thereof is immersed in the culture solution in the culture tank 2. A pump 4 2 is interposed at the other end of the pipe 41, and a sterilizing filter is interposed between the pump 42 and the culture tank 2.

For this reason, the culture medium supply means 30 and the pipe 12 are communicated by the communication valve 3 1, and the fresh culture medium is supplied from the culture medium reservoir to the heater 3 4 and the sterilization filter 3 2 by the pump 3 5. If the culture medium is supplied to the pipe 12 and the culture medium discharger 40 is used to discharge the culture medium in use from the culture tank 2, the culture medium can be replaced without interrupting the culture in the culture tank 2. It can be carried out. Therefore, since the culture solution can be exchanged while culturing continues, it is possible to cultivate even adherent cells and hematopoietic cells, which are necessary for long-term cultivation. In addition, the culture medium can be automatically exchanged for a new culture medium only by operating the culture medium discharge section 40 and the culture medium supply section 30 in synchronization. Therefore, it is difficult to replace ± recharge solution In short, the risk of contamination of the culture medium and cells in culture can be reduced.

 Since each pipe 3 3 and 4 1 is provided with a sterilizing filter 3 2 and 4 3, it is more reliable that the culture medium is contaminated with germs during the replacement. Can be prevented.

 When culturing floating cells, if the electromagnet 6 is provided at one end of the pipe 41, it floats with the culture solution when the culture solution in the culture tank 2 is discharged. Since it can be prevented from flowing out of the tank 2, the culture solution can be exchanged without suspending the culture even for floating cells.

 Note that the amount of the culture solution in the culture tank 2 can be adjusted by operating only the culture solution supply unit 30 or only the culture solution discharge unit 40.

 When cells are cultured for a long period of time, it is necessary to periodically extract the culture medium and examine the state of the cells and the culture liquid. However, if an extraction means 50 having the following configuration is provided, Extraction is possible without contamination and without waste of the culture medium.

 As shown in FIG. 5, one end of the extraction path 81 is airtightly attached to the lid 2 b of the culture tank 2, and one end thereof is immersed in the culture solution in the culture tank 2. The other end of the extraction path 81 is disposed outside the culture tank 2, and the other end is disposed in a sterilized extract receiving container 82 such as a cylinder.

 This extraction path 81 is provided with a pump 83 force, and a sterilizing filter 54 is connected between the pump 83 and the other end of the extraction path 81 from a gas supply unit (not shown). The sterilization gas is supplied. This sterile gas is always supplied to the extraction path 81. It is intervened.

 For this reason, if the pump 83 is activated, the culture medium in the culture tank 2 can be discharged into the extraction liquid receiving container 82 through the extraction path 81. The culture state of the vesicle can be confirmed.

In addition, the sterilized gas force S is always supplied to the extraction path 8 1, and the sterilized gas is supplied from the other end of the extraction path 8 1 into the extract receiving container 8 2, and always receives the extract liquid. Container 8 2 is filled. Then, since outside air cannot enter the extraction path 81 and the extract receiving container 8 2, it is possible to prevent bacteria from entering the extraction path 81 and the extract receiving container 8 2. I can do it. Therefore, when the extracted culture solution passes through the extraction path 81 and enters the effluent receiving vessel 82, it can be prevented from being contaminated with various germs.

 Furthermore, if the extraction path 8 1 or the extract receiving container 8 2 is actually kept in a sterile condition, the culture fluid discharged into the extraction path 8 1 or the extract receiving container 8 2 may be contaminated with various bacteria. Absent. Then, when more than necessary culture fluid is extracted, the culture fluid remaining in the extraction path 81 and the extract receiving vessel 8 2 can be returned to the culture vessel 2 to effectively use the culture fluid. I can help. When the culture solution is returned, gas in the extraction path 81 and the extract receiving container 8 2 may also enter the culture tank 2, but there is no inside of the extraction path 81 and the extract receiving container 8 2. Since it is filled with fungus gas, it does not enter with the gas. In particular, when recovering the culture solution while continuously culturing, there is no concern that the recovered culture solution is contaminated, and thus the culture solution can be used safely.

 In addition, as shown in FIG. 6, it is preferable to provide a cell supply means 90 that can supply cells to be cultured in the culture medium in the culture tank 2, because the cell culture can be completely automated. The

 In FIG. 1, reference numeral 91 denotes a storage unit in which a seeding solution containing cells to be cultured is accommodated. One end of the cell transfer path 92 is attached to the storage unit 91, and one end thereof is immersed in the seeding solution in the storage unit 91. The other end of the cell transfer path 92 is airtightly attached to the lid 2 b of the culture tank 2, and one end thereof is disposed in the culture tank 2. A discharge section 93 such as a known nozzle is provided at the other end of the cell transfer path 92. The cell transport path 92 is provided with 94 pumps for transporting the seeding solution in the storage section 91 toward the discharge section 93.

 For this reason, if the pump 94 is operated, the seeding solution can be conveyed to the culture tank 2, and the cells contained in the seeding liquid are transferred to the culture medium 2 by being discharged from the discharge part 93 into the culture tank 2. Can be supplied. Accordingly, since the supply of cells can be automated, cell culture can be completely automated.

Then, if the cell transfer path 92 between the pump 94 and the discharge part 93 is provided with a valve 95 such as a pinch valve that cuts off the communication between the two, the valve 95 is closed. If the pump 94 is operated in this state, the pressure of the seeding liquid in the seeding liquid transfer path 2 can be increased. Then, if the valve 95 is opened while the seeding fluid pressure is high, the seeding fluid Can be injected into the culture tank 2 vigorously. Then, if a cell culture instrument or the like serving as a scaffold for culturing cells is provided in the culture tank 2, a seed solution, that is, cells can be seeded on the surface of the cell culture instrument. Therefore, not only floating cells but also adherent cells can be seeded automatically, in other words, without opening and closing the culture tank 2, so that the cells and culture medium are contaminated when seeding the cells. Can be prevented.

 Furthermore, the culture apparatus 1 of the present embodiment can be used inside a facility that can maintain a constant environment around the culture apparatus 1 such as an incubator or a warmer. If the temperature control means 70 is provided as follows, the culture apparatus 1 can be used at a desired location.

 As shown in FIG. 1, a planar heating element 7 1 of temperature control means 70 is provided on the outer surface of the culture tank 2. The planar heating element 71 is connected to a control unit 78 that adjusts the power supplied to the planar heating element 71. This control unit 78 adjusts the temperature of the sheet heating element 71 by adjusting the power supplied to the sheet heating element 71 in accordance with the temperature of the culture solution in the culture tank 2.

 As shown in FIG. 7, the planar heating element 71 includes a radiating portion 73 that generates heat when energized and emits far-infrared rays toward the inside of the culture tank 2. In other words, the planar heating element 71 can heat the culture solution not only with the heat generated by the radiating portion 73 but also with the far infrared rays emitted when it generates heat.

 However, far infrared rays radiated from the planar heating element 71 propagate in the culture medium in the culture tank 2, and a part of the energy of the far infrared rays is directly supplied to the cells. . For this reason, the cells to be cultured can be heated by far infrared rays that are transferred only by heat transfer from the culture medium, and as long as they are irradiated to the far infrared force culture, even if the cells around the cells to be cultured are cultured. Even if the temperature of the liquid changes, the temperature change of the cell itself can be suppressed.

Further, when the culture solution in the culture tank 2 rises to a predetermined temperature, the energy absorbed in the culture solution is reduced among the energy released from the planar heating element 71 in the form of far infrared rays. Then, most of the energy released as far infrared rays from the planar heating element 71 is supplied to the cells to be cultured and used for heating the cells themselves. Since the energy of far infrared rays depends on the temperature of the planar heating element 71, it is heated by far infrared rays. The cell temperature can be increased to approximately the same temperature as the planar heating element 71. Further, the peak of the energy density of far-infrared rays emitted from the surface of the planar emission book 71 is adjusted to be formed in a wavelength band of 7 to 12 m. this? The wavelength of ~ 12 m is a wavelength band called growth light, which is effective for the growth of plants, etc., but animals, especially rabbit cells, such as hepatocytes, skin cells, osteoblasts, immune cells In the culture of stem cells that can proliferate and differentiate into multiple advanced cells, such as cells that have the ability to proliferate, hematopoietic stem cells, stromal stem cells, etc. In this way, cell proliferation and separation are promoted compared to the case of not irradiating far infrared rays. This cell growth and differentiation is promoted by activating genes, growth factors, and differentiation-inducing factors that affect growth and differentiation, and conversely, by the action of genes and inhibitory factors that suppress growth and differentiation. It is thought that far infrared rays function directly as growth factors and differentiation-inducing factors. Cells that were undifferentiated and capable of differentiating into cells of various tissues, such as bone marrow, terminal blood,) 1 cord blood, tissue-derived stem cells, embryonic stem cells, etc. were irradiated with far infrared rays When cultured in a state, it is possible to promote the proliferation of very sensitive and undifferentiated cells themselves, as well as to promote differentiation from undifferentiated cells to desired cells. From undifferentiated cells, desired sorted cells can be easily cultured in large quantities. In addition, if cells that have already differentiated into various tissues but can proliferate, such as hepatocytes and osteoblasts, are cultured in the state of being irradiated with far-infrared rays, proliferation can be activated by acting for a long period of time.

That is, if the temperature control means 70 as described above is provided in the culture tank 2 of the culture apparatus 1 of the present embodiment, the cell proliferation and differentiation promoting efficiency can be improved as compared with the case where the temperature control means ⁷⁰ is not provided. It can be improved.

 If the pipes 11 and 12 and the culture medium adjusting means 20 are covered with a heat insulating material, it is possible to prevent the temperature of the culture medium from changing during external circulation.

 Furthermore, if the temperature control means 70 is also provided in the dialysis part 21, the gas concentration adjusting means 22, and the nutrient supply part 23, the temperature change of the culture solution can be prevented more reliably.

In addition, the radiator 75 used for the planar heating element 71 is based on a material that emits far-infrared rays, such as bonbon black, carbon graph eye, ceramic powder, alumina, zircon, and polyethylene glycol. Positive temperature of semiconductors used as materials PTC materials and radiation materials can be used without using special control mechanisms or sensors if materials containing materials with a low self-temperature control function (PT C: Pos it iveTemperature Coef iiciennt) function are used. The temperature can be reliably maintained in the vicinity of the predetermined temperature. In this case, the far-infrared wavelength and energy density distribution emitted from the radiator 75 can be maintained in a predetermined state. Therefore, the state of the far infrared ray due to the temperature change of the planar sample 71 can be changed, that is, the state of the far infrared ray emitted to the cell can be kept constant, so that the temperature change of the cell can be prevented. Can cultivate cells in a stable state.

 The far-infrared radiation portion need not be the planar heating element 71 as described above, and is not particularly limited as long as it can irradiate cells with far-infrared rays having a predetermined wavelength.

 Before starting the culture and after the completion of the culture, it is necessary to sterilize the flow path of the culture solution and other parts to prevent germs from growing in the culture apparatus 1, but the volume of the culture tank is 10 If it is smaller than L, the culture apparatus 1 can be accommodated in the autoclave apparatus in a state in which the ± recharge tank 2, the circulation means, and the culture solution adjustment means 20 are all connected. Since it can be sterilized by putting it in the autoclave apparatus without disassembling, the sterilization operation of the culture apparatus 1 becomes easy.

 Moreover, since it is not necessary to disassemble the connected device, if the sample is sterilized by an autoclave device before starting the culture operation and used as it is, the probability that the culture solution and cells will be contaminated by various bacteria is very high. Can be lowered.

 If the culture tank 2 is 20 L or more, do not show the piping 11 1 and pressurized steam supply means that can generate pressurized steam. If pressurized steam is supplied into the closed loop circuit by the pressurized steam sterilization means, each means can be sterilized without disassembling each means. Then, even if the culture apparatus 1 is large, the entire apparatus can be easily and quickly sterilized, so that the risk of the culture tank 2 and the like being mixed can be reduced. Further, since the culture apparatus can be sterilized only by operating the caloric steam sterilization means, the sterilization of the culture apparatus 1 can be automated, and the operation of the culture apparatus 1 can be completely automated.

Further, as shown in FIG. 8 and FIG. 9, in the culture apparatus 1 of the present embodiment, a culture tank 2 equipped with a culture instrument 50 that can attach cells and serve as a scaffold for proliferation is used. In this way, the cells are cultured in a state where they are suspended in a culture solution for a short period of time, such as bacteria and floating rice fields. It is suitable for culturing adherent cells that require a period of several weeks to one month for culturing, such as osteoblasts and dermal fibroblasts that can only be made of cells, and that require a scaffold for growth.

 In FIG. 8 and FIG. 9, reference numeral 50 indicates a culture instrument provided in the culture tank 2. This culture instrument 50 is formed by connecting a plurality of circular tubes made of pure titanium or titanium alloy to each other, and is held in the culture tank 2 by a support member 51.

 For this reason, even if the cells to be cultured are attached to the surface of the culture apparatus 50 and placed in the culture tank 2 and immersed in the culture solution, even the adherent cells constituting the tissue or organ can be obtained. Can be cultured. In addition, the cultivating apparatus 1 of the present embodiment is also capable of culturing continuously for a long period of time. The adherent cells can be cultured in large quantities until the desired size is reached.

 In addition, since the culture device 50 is formed by connecting circular tubes and the surface area of the culture device 50, that is, the portion where cells can be used as a scaffold can be increased, a large amount of cells can be cultured. I can do it. Shiga can also flow the culture medium through the circular tube, so that fresh culture medium can be supplied to all cells. For this reason, nutrients and oxygen can be efficiently supplied to all of the cells to be cultured, so that the circulation efficiency of the culture solution can be improved and the culture conditions can be dramatically increased.

The culture device 50 is not limited to the above structure, and various shapes can be used. For example, if a culture device 50 with a honeycomb structure or a porous structure having a through-hole penetrating in the vertical direction is used, the culture liquid in the vertical direction flows smoothly, so that all cells of the cultured cell Nutrients and oxygen can be supplied efficiently. In addition, if a culture device 50 having a desired three-dimensional structure, for example, various shapes, is used, cells grow along the shape of the culture device 50, and a three-dimensional tissue having a desired shape is obtained. This makes it possible to supply a large amount of 3D organization and) required for regenerative medicine. Furthermore, a membrane-like member may be used as the shape of the culture instrument 50, and in that case, a membrane-like fiber can be formed. In particular, if a membrane-like member having a network structure, a so-called mesh, is used, a fresh culture solution can be supplied to the cells through the culture device 50. Fine parts Fresh culture fluid can also be supplied to the cells that are attached to the culture apparatus 50 that is composed only of cells.

 In addition, pure titanium and titanium alloys are used as the material for the circular tube of the culture instrument 50. Pure titanium and titanium alloys are cytophilic, so even if the cells to be cultured are seeded directly on the surface of the culture device 50, the cells will not be contaminated or damaged. The ability to culture adherent cells such as n§§ for a long time. Moreover, an extracellular matrix is formed between the culture device 50 and the cells, and a calcified layer force is formed. For this reason, since the cells to be cultured can be reliably attached to the culture apparatus 50, the cells can be cultured in a stable state for a long period of time. Pure titanium and titanium alloys are extremely chemically stable and do not change or corrode even after long-term use. This prevents cells from being damaged by corrosion or alteration of culture equipment 50. be able to. Since the same culture instrument 50 can be used any number of times, it is possible to reduce the cost of culturing cells.

 Note that the culture apparatus 50 may not be entirely formed of titanium or a titanium alloy, but may be formed by forming a layer containing titanium or a titanium alloy on the surface of the base material. Also in this case, since the cell is contaminated or damaged by the culture instrument 50, it is possible to culture adherent cells such as fli§§ for a long period. Further, the culture device 50 is completely covered with a layer containing titanium or a titanium alloy, and the layer of titanium or the like is completely covered with an extracellular matrix. For this reason, since the substrate does not come into contact with the cells at all, even if it is used for a long time, the culture device 50 does not deteriorate or corrode, so the culture device 50 can be used for a long time, and the cells are cultured. You can reduce the cost of cost. Furthermore, the material of the culture apparatus 50 is composed of cell-derived polymeric materials such as collagen, cell-absorbing polymer, and chitin, calcium phosphate compounds such as hydroxypatite, and synthetic biodegradation such as poly-L-lactic acid and polyglycolic acid. Any material may be used as long as it has a high cell affinity, such as a functional polymer.

Even if the base material of the culture apparatus 50 is a silicon compound, glass, magnetic material, ceramics, or other material having no cell affinity, cell-derived polymer material or calcium phosphate-based material is formed on the surface thereof. The formation of a layer of a tissue-inducing material having a stringer-inducing ability such as a compound, a synthetic biodegradable polymer, or an extracellular matrix can increase the cell affinity of the culture device 50. You can.

 If the material of the instrument itself is a cell-derived polymer material or has a cell-derived polymer material layer on the surface of the instrument, the culture instrument 50 The adherence cells such as fli§§ can be cultured for a long period of time because the cell affinity between the cells and the cells is further increased, and the cells are not contaminated or damaged by the culture apparatus 50. In addition, an extracellular matrix is formed between the culture device 50 and the cells, and a calcified layer is further formed. For this reason, since the cells to be cultured can be reliably attached to the culture apparatus 50, the cells can be cultured in a stable state.

 In addition, if the culture apparatus 50 is made of an S cell-derived polymer material, the cell-derived polymer material is absorbed by the living body. Since 0 can be used as it is, that is, the culture device 50 can be transplanted as it is, it is possible to perform transplantation reliably and easily.

 In particular, if the cell-derived polymer material is collagen, the cell affinity with the cells is very high, so that the culture environment for adherent cells can be further improved. Further, since the affinity between the culture apparatus 50 and the cells is further increased, a large amount of cells can be efficiently cultured at a low cost.

 In addition, when the material of the device itself is a calcium phosphate compound or a device having a calcium phosphate compound layer on the surface of the device, the calcium phosphate compound is compatible with cells. Because of the sexual growth, an extracellular matrix can be formed directly on the surface of the culture device 50. Therefore, since the cells to be cultured are not contaminated or damaged by the culture device 50, adherent cells such as organs can be cultured for a long time. In addition, if the entire culture device 50 is formed of a calcium phosphate compound, the calcium phosphate compound is absorbed by the living body, so that the culture device 50 can be transplanted as it is. It can be done easily.

When the culture instrument 50 is a synthetic biodegradable polymer of the material itself or having a synthetic biodegradable polymer layer on the instrument surface, the synthetic biodegradable polymer is Since the cell affinity is high, an extracellular matrix can be directly formed on the surface of the culture device 50. Therefore, culture using culture apparatus 50 Since cells are not contaminated or damaged, adherent cells such as can be cultured for a long time. In addition, if the entire culture device 50 is formed of a calcium phosphate compound, the synthetic biodegradable polymer is absorbed by the living body, so that the culture device 50 can be used as it is in the living body for regenerative medicine. In other words, since it can be transplanted as it is with the culture apparatus 50, the transplantation can be performed reliably and easily.

 In particular, if the synthetic biodegradable polymer is poly (L) acid or polyglycolic acid, the cell affinity between the culture device 50 and the cells is very high, so that the culture environment for adherent cells should be further improved. Can do. And since the affinity between the culture apparatus 50 and the cells is further increased, it becomes a powerful ability to culture a large amount of cells efficiently and at a low cost.

 Further, as a culture instrument 50, a silicon-containing material, for example, glass gellite, a black body powder having a composition as shown in FIGS. 14 to 16 or natural ceramics is used. Also good. In this case, since it has the silicon power S calcification ability contained in blackbody powder, natural ceramics, etc., a limestone cocoon layer is formed on the surface of the culture device 50 by a calcium phosphate compound or the like. Then, the affinity force S between the culture device 50 and the cells is increased, and therefore, the cells cultured by the culture device 50 are not contaminated or damaged. Therefore, (11 ^ etc. adherent cells can be cultured for a long time

Since the force τ 'can be ensured that the cells to be cultured adhere to the culture apparatus so, the force culturing the cells in a stable state for a long period of time can be achieved.

 Moreover, as shown in Fig. 14 to Fig. 17, blackbody powder, natural ceramics, zeolite, etc. have a wavelength in the range of about 833 to 1429 cm-1, that is, the far-infrared region in the vicinity of about 7 to 12. It has the characteristic of absorbing specifically. In other words, black body powder, natural ceramics, zeolite, and the like have a property of strongly emitting far-infrared rays of about 7 to 12 m, so that the culture device 50 emits far-infrared rays when the temperature rises. Then, since the far-infrared power is supplied from the culture instrument 50 to the cells attached to the surface of the culture instrument 50, an effect of promoting cell differentiation and proliferation can be obtained.

Then, the surface of the culture apparatus 50 is coated with a mixture of glass, natural ceramics, zeolite or other powders such as those described above, or black body powder and an adhesive such as silica gel. The same effect can be obtained by forming a layer containing body powder, natural ceramics, etc., that is, a layer of a material containing silicon. Further, as the culture device 50, an artificial ceramic, for example, an artificial ceramic having a composition as shown in FIG. 18 may be used. Also in this case, the force of forming a limestone cocoon layer by the calcium phosphate compound or the like on the surface of the culture device 50 increases the affinity between the culture device 50 and the cells. Therefore, adherent cells such as Bigg that are not contaminated or damaged by the culture apparatus 50 can be cultured for a long period of time. Since the cells to be cultured can be reliably attached to the culture apparatus 50, the cells can be cultured in a stable state for a long period of time.

 Moreover, as shown in FIG. 18, the artificial ceramic has a characteristic of specifically absorbing the wavelength in the region of about 833 to 1429 cm-1, that is, about 7 to: the far infrared region of L 2 m. Yes. In other words, since artificial ceramics has a property of strongly emitting far-infrared rays in the vicinity of about 7 to 12, the culture instrument 50 emits far-infrared rays when the temperature power increases. Then, since the far infrared rays are supplied from the culture instrument 50 to the cells attached to the surface of the culture instrument 50, an effect of promoting the differentiation and proliferation of the cells can be obtained.

 The same effect can be obtained even if the artificial ceramic layer is formed by coating the surface of the culture apparatus 50 with a mixture of the above artificial ceramic powder and an adhesive such as silica gel. it can.

 Further, when the culture instrument 50 has a layer of a tissue-inducing material on the surface of the instrument, since the cell affinity between the tissue-inducing material such as the extracellular matrix and the cell is high, the culture environment of the adherent cell is reduced. In addition to improvement, it can be used directly in living bodies for regenerative medicine, and the cost of culturing cells can be reduced. In addition, not only the proliferation of cells is promoted by the effects of tissue-inducing materials, but also differentiation of undifferentiated mesenchymal cells into various tissues can be induced. Therefore, not only can culture be performed while maintaining the cell separation state, but also cells necessary for regenerative medicine can be easily supplied.

Then, if desired adherent cells are seeded and cultured in the culture device 50 of the culture apparatus 1 of the present embodiment, the state of the culture solution that does not cause bubbles or fast convection in the culture tank 2 is obtained. Since the state can be maintained in a state suitable for culturing cells with almost uniform strength, and the culture medium can be changed while culturing cells, various adherent cells can be used for a very long time (weeks to months). But can also be cultured. Then, a small amount of adherent cells can be used for regenerative medicine. Since it is possible to produce a tissue with a size that can be used, it is possible to manufacture a large amount of man-made stringers and artificial strings required for regenerative medicine. Shiga can also take cells of the desired string from a person who needs to be transplanted and culture the cells)]! ^ To increase the compatibility with the living body. Can also prevent rejection.

 In addition, if undifferentiated cells such as stromal stem cells are used as the cells to be cultured, they can be differentiated into desired cells by adjusting the culture conditions. Thread II and others can be produced from readily available cells. An artificial tissue that can be applied to any stringer can be produced by growing undifferentiated cells while remaining undifferentiated.

 It is preferable to install the culture tank 2 in the following cell seeding means 60 because the seeding of the cells in the culture device 50 can be automated.

 The cells can be seeded on the culture device 50 by the above-described cell supply means 90. However, as the culture device 50, a plurality of circular tubes are arranged so that their axial directions coincide with each other and have a cross-section. When using the tubes formed so that they are arranged in the shape of circular tubes visually, the following cell seeding means 60 can be used to more reliably seed the cells in the culture device 50. Power S can be.

 As shown in FIG. 8 and FIG. 9, a culture instrument 50 is mounted in the culture tank 2 so that the central axis thereof coincides with the central axis of the culture tank 2.

 In the figure, reference numeral 61 denotes a base of the cell seeding means 60. On the upper surface of the base 61, there is provided an evening table 62 that can rotate on the upper surface of the base 61. The turntable 62 is a stand that supports the culture tank 2. For this reason, if the stand S supporting the culture tank 2 is placed on the turn tray 62, the culture tank 2 can be rotated about its axis.

 As shown in FIGS. 8 and 9, the base 61 is provided with a support frame 63, and the support frame 63 has a culture device provided in the vertical direction, that is, in the culture tank 2. A horizontal axis 64 4 extending in the radial direction of the culture tank 2 is provided so as to be movable in the axial direction of 50. The support frame 63 and the horizontal axis β4 are the moving parts referred to in the claims.

The horizontal axis 64 is attached with 65 cell supply units. This cell supply The part 65 is a hollow member, and is provided with an injection part 65a force s to which a liquid containing cells to be cultured is supplied. On the other hand, a plurality of cylindrical seeding parts 65b extending in the axial direction of the culture device 50 are provided at the lower part of the cell supply part 65.

 The plurality of seeding parts 65b are all provided side by side in the same plane as the central axis of the culture device 50. The distance from the central axis of the culture tank 2 to each seeding part 65b is the central axis of the culture tank 2 and the multiple circular pipes located in the same plane among the multiple circular tubes of the culture device 50. It is arranged to be the same length as the distance to.

 In addition, the base 61 is provided with a tilting shaft 66 at a position eccentric to the central axis of the culture tank 2 so as to be able to protrude from the lower surface of the base 61.

 Next, the operation of seeding cells with the cell seeding means 60 will be described.

 FIG. 10 is a schematic explanatory view showing a state where the cell seeding means 60 is tilted. First, all the culture solution in the culture tank 2 is discharged, and then the lid 2 a of the culture tank 2 is removed.

 Next, when the horizontal axis 64 is lowered along the support frame 63, the seeding part 65b of the cell supply part 65 is inserted into each circular tube of the culture device 50. In this state, when the tilting shaft 6 6 protrudes from the lower surface of the base 6 1, the base 6 1 of the portion where the tilting shaft 6 6 is provided rises, and the culture tank 2 tilts together with the base 6 1 (FIG. 10). )

 When the liquid containing the cell force to be cultured is poured from the injection part 65a of the cell supply part 65, the liquid flows into the culture instrument 50 from the seeding part 65b through the cell supply part 65. Since two culture tanks are present, the liquid flows through the inner surface of the culture device 50, so that cells can be seeded on the inner surface of the culture device 50.

Next, when the horizontal axis 64 is raised along the support frame 63, the cell supply unit 65 is detached from the culture instrument 50. After that, the turntable 62 is rotated so that the row of circular tubes is located in the same plane as the seeding part 65b. Then, when the horizontal axis 64 is lowered again along the support frame 63, it is inserted into each circular tube of the seeding part 65b force S culture instrument 50 of the cell supply part 65. Then, when the liquid containing the cell force to be cultured is poured from the injection part 65a of the cell supply part 65, it flows into the liquid force resuscitation device 50 from the seeding part 65b and reaches the inner surface of the culture tool 50. Flowing By repeating the above work, cells can be seeded on the inner surface of all the tubes, If the turntable 62 is rotated while liquid is flowing on the surface, the cells can be seeded on the entire inner surface of the circular tube of the culture device 50.

 Also, instead of the liquid containing the cells of the cells to be cultured, a stripping solution (trypsin EDTA) for stripping from the culture device 50 is injected into the injection unit 65b of the cell supply unit 65, and the culture device 5 from the seeding unit 65b. The cells can be detached from the culture device 50 by being suspended in the cell.

 If a recovery unit 67 for recovering the stripping solution is provided at the bottom of the culture tank 2 or the like, the cells detached from the culture device 50 can be recovered together with the stripping solution.

 That is, the cell seeding means 60 can be used as a cell recovery means, and in this case, the cell supply section 65 becomes the stripping solution supply section referred to in the claims.

 The cell seeding means 60 has a structure as shown in FIG. 11 to FIG. 13 and is close to and spaced from the lower end of the culture device 50 at the bottom of the culture vessel 2, and the lower end of the culture device 50 is If a bottom lid 68 8 that can be hermetically sealed or opened is provided, the exfoliation solution can be stored inside the culture device 50, so that it is possible to ensure cell detachment. In addition, the consumption of the stripping solution can be reduced.

 Furthermore, the culture tank 2 may be installed above the collector 62 of the cell seeding means 60 not only during seeding and collection but also when cells are being cultured. However, it may be installed on the cell seeding means 60 only when the cells are seeded and collected. As described above, when the culture apparatus 1 of the present embodiment is used, there is no generation of bubbles or fast convection in the culture tank 2, and the state of the culture solution is almost uniform and suitable for cell culture. In addition, since the culture medium can be exchanged while culturing the cells, various floating cells can be cultured for a very long period (several weeks to several months). As a result, it is possible to produce a large number of floating cells themselves, and various active ingredients produced in the process of floating cell growth can be collected. From these collected components, antibodies, vaccines, pharmaceuticals, etc. Can be manufactured.

In particular, when the floating cells contain hematopoietic stem cells, the hematopoietic stem cells not only proliferate but also differentiate into various blood cells, and almost all cells contained in the blood, such as erythroblasts, are contained in the culture medium. Sphere, myeloblast, megakaryoblast, monoblast, lymphoblast differentiated erythrocyte erythrocyte, nucleated leukocyte neutrophil, eosinophil, basophil, platelet, monocyte Lymphocytes are produced. And if you continue to culture for a long time, The blood component power of S is produced.

 Then, by treating the ± nutrient solution, it is possible to extract a desired component, so that a blood product containing a desired component can be easily produced. In addition, since the culture apparatus 1 of the present embodiment can extract, exchange, and add a culture solution while continuing cell culture, the cells can be continuously collected while collecting blood cells and the like contained in the culture solution. Can also be cultured.

 In addition, hematopoietic stem cells can be ingested from people who need blood transfusions and blood products, and the stem cells can be cultured to produce blood and blood products for transfusion. Blood for transfusion and the like can be produced.

 In addition, if the floating cells include erythroblasts, myeloblasts, megakaryocytes, monoblasts, lymphoblasts, etc., erythrocytes that are blood cells, neutrophils that are nucleated leukocytes, eosinophils Furthermore, since it can produce halophilic salts, blood platelets, monocytes and lymphocytes, it can produce a mixed solution that can be used as a transfusion preparation.

 It is also possible to carry out a loose mixed culture in which suspended cells are cultured in a culture solution while culturing adherent cells with a culture device 50 provided in the two culture vessels. In this case, since the factor that activates the floating cell or promotes differentiation is produced and secreted from the adherent cell, the culture of the buoyant magnetic field can be promoted. In addition, since floating cells produce bioactive factors, the activity and differentiation of adherent cells can be promoted by the action of the bioactive factors. Therefore, since the culture of each cell can be promoted compared to the case where only floating cells or adherent cells are cultured, each cell can be cultured efficiently.

For example, if hematopoietic stem cells are mixed and cultured in a culture solution while culturing bone marrow stromal stem cells using a culture device 50, factors that activate hematopoietic stem cells or promote differentiation from bone marrow stromal stem cells are produced and secreted. Therefore, hematopoietic stem cells are promoted to produce erythrocytes, nucleated leukocytes, neutrophils, eosinophils, basophils, platelets, monocytes and lymphocytes. In addition, the bioactive factor produced by hematopoietic stem cells promotes the separation of bone marrow stromal stem cells into osteoblasts, chondroblasts, fibroblasts, adipocytes and other cells. For this reason, it is possible to produce erythrocytes, etc., which are faster and more efficient and more active than culturing hematopoietic stem cells and bone marrow stromal stem cells alone, and efficiently differentiate osteoblasts etc. Can be guided. In addition, while culturing bone marrow stromal stem cells with culture apparatus 50, bone marrow containing blood blasts such as erythroblasts, myeloblasts, megakaryoblasts, monoblasts, and lymphoblasts in the culture medium. When tissue is mixed and cultured, bone marrow stromal stem cells activate erythroblasts, etc., and factors that promote differentiation are produced and secreted. From the myeloma, erythrocytes, nucleated leukocytes, neutrophils, Production of eosinophils, basophils, platelets, monocytes and lymphocytes is promoted. In addition, differentiation of stromal stem cells into osteoblasts, chondroblasts, fibroblasts, adipocytes and other cells is promoted by the action of physiologically active factors produced by erythroblasts and the like. For this reason, it is possible to produce erythrocytes, which are blood f¾ cells that are faster and more efficient and more active than culturing bone marrow tissue and bone marrow stromal stem cells alone, and efficiently differentiate osteoblasts and the like efficiently. Can be guided.

 Since the culture apparatus 1 of the present embodiment has the above-described configuration, it is possible to prevent the deterioration of the culture solution, which is a problem when mixed culture is performed.

 【Example】

 Below, the Example which culture | cultivates a cell with the culture apparatus of this invention is described.

 In the following examples, when the volume of the culture tank is 10 L or less, the above-described means and devices (for example, a circulation means, a gas concentration adjusting means, etc.) are connected in an autoclave. Using a cultivated culture apparatus, sterilization was carried out at 121 degrees for 20 minutes.

 When housed in the autoclave, the electrical system portion of the pH meter or dissolved oxygen meter, which is the top sound of the culture apparatus, is removed because it is non-heat-resistant and moisture-resistant. In addition, each culture medium and gas injecting or discharging tube in the culture medium exchanging apparatus or sampling apparatus is accommodated in the autoclave while being covered with aluminum foil to prevent contamination.

 In addition, the culture medium circulation device, the gas saturation device, the temperature control device, the leakage prevention device, the culture solution exchange device, and the cell recovery device in the following examples are respectively the circulation means 10 and the culture solution adjustment means 20 described above. Corresponds to temperature control means 70, electromagnet 6, culture solution supply means, cell recovery means.

 (Example 1)

An example of producing cytokines by culturing transformed E. coli using the culture apparatus of the present invention will be described. In this example, the culture tank has a volume of 1 L. Are using.

 Further, as a comparative example, a comparison was made with the case where cytokines were produced by culturing E. coli transformed in a flask.

 The cytokine production was compared by comparing the turbidity of the cultures. The turbidity was compared using a Beckman absorptiometer (DU series 500, Beckman, Fuller ton, CA).

(1) First, after sterilizing the ± recharge apparatus in an autoclave, the culture solution (dried bouillon “Nissi” 05511, Usui Pharmaceutical Co., Ltd., Tokyo) is supplied to the culture apparatus, and then transformed into a specific one by transformation. E. coli that express cytokines was injected into the culture vessel. (Concentration OD ₅₅₀ = 0. 032)

(2) In this state, the _cells were cultured continuously for 3 hours and grown to OD _55Q = _0.535 .

 (3) As shown in Fig. 19 (A), the turbidity of the culture solution used in the culture apparatus of the present invention is about 3 times higher than that in the culture flask in 2 hours after the start of the culture. It became more than 4 times 2 hours after the start of culture. That is, in the culture apparatus of the present invention, it can be confirmed that Escherichia coli is cultured in a large amount and more efficiently than the flask culture even at the same time.

(4) After growing to OD ₅₅₀ = 0.535, induce protein expression at IPTG final concentration of 0.5 mM, induce the expression for 4 hours, and then collect bacteria in the collection device. Cain can be purified.

 (5) As described above, according to the culture apparatus of the present invention, E. coli can be cultured in large quantities and efficiently, and cytokines can be efficiently produced in large quantities. And can be clinically applied as a recombinant drug for various cytokines.

 (Example 2)

 An example of culturing a gene-introduced yeast and producing a cytokine using the culture apparatus of the present invention will be described. In this example, a culture tank having a volume of 1 L is used.

In addition, as a comparative example, it was compared with the case where yeast transformed in a dish was cultured to produce a cypress force-in. The amount of yeast was compared by comparing the turbidity of the cultures. The turbidity was compared using a Beckman absorptiometer (DU series 500, Beckman, Fullerton, CA).

(1) First, after sterilizing the culture apparatus in an autoclave, a culture solution (YPDBR OTH SIGMA-ALDRICH) is supplied to the culture apparatus, and then yeast that expresses a specific sycaine by transformation. (Concentration OD _5M = 2.130) was injected into the culture vessel. (Concentration OD ₅₅₀ = 0.078) (2) In this state, the cells were cultured continuously for 2 days and grown to OD ₅₅₀ = 1.784.

 (3) After 24 hours from the start of culture, the turbidity of the culture solution used in the culture device of the present invention was about 22 times that of the culture solution used for dish culture. As shown in Fig. 19 (B), when the total amount of yeast multiplied by the turbidity and the culture volume is compared, the total amount of yeast in the culture solution used in the culture apparatus of the present invention is 178.383. The total amount of yeast in the culture medium used for cultivation was about 95.5 times. That is, in the culture apparatus of the present invention, it can be confirmed that yeast is cultured in a large amount and efficiently compared to the dish culture.

(4) After growing to OD ₅₅₀ = 1.784, the bacteria can be recovered with a recovery device, so that the cytokine can be purified.

 (5) As described above, according to the culture apparatus of the present invention, yeast can be cultured in large quantities and efficiently, and cytokines can be produced in large quantities and efficiently. It can be formulated and clinically applied as a recombinant drug for various cytokines.

 (Example 3)

 An example will be described in which a hyperpridoma cell is cultured using the culture apparatus of the present invention to produce an antibody that can be used as a pharmaceutical product. In this example, a culture tank having a volume of 100 L is used.

 (1) First, a cell carrier combined with a pure titanium tube was inserted into a culture tank, and the culture tank, circulation apparatus, and gas saturation apparatus of the culture apparatus were sterilized with an autoclave.

(2) Then, 50,000 hybridoma cells producing antibodies that can be used as pharmaceuticals were suspended in 50,000 Zml, and 100 μΐ each was injected into a cell carrier tube using a cell seeding device. (3) As shown in Fig. 20 (A), it can be confirmed that the number of cells increases over time when the hyperidoma cells are continuously cultured. Then, as shown in FIG. 20 (B), it can be confirmed that the amount of antibody production increases dramatically as the number of hyperidoma cells increases.

 (4) One week after culturing, the antibody can be obtained by removing the culture solution from the outlet and connecting it to an ultrafiltration device for filtration.

 (5) As described above, according to the culture apparatus of the present invention, a large amount of hybridoma cells can be cultured efficiently, and an antibody that can be used as a pharmaceutical can be efficiently produced in large quantities. The ability to formulate an antibody that can be used as a drug, and clinical application as various antibody drugs.

 (Example 4)

 An example in which hematopoietic stem cells are cultured to produce platelets using the culture apparatus of the present invention will be described. In this example, a culture tank having a volume of 1 L is used.

 In addition, as a comparative example, a comparison was made with the case where hematopoietic stem cells were cultured in a dish to produce platelets.

 The production of platelets was compared with the number of platelets relative to the total amount of culture solution.

(1) Sterilize the culture apparatus in an autoclave, remove the rabbit femur and blood, suck the bone marrow from the femur using a syringe, suspend it in MEM medium, mix with collagen gel, and use titanium as a cell carrier. It was built in the center of a culture tank sterilized on a mesh.

 (2) The culture solution is automatically poured into the culture tank until it is full, cultured while circulating with a circulation device, and after 1 week, the culture solution containing the cells is collected, and a density gradient using Ficoll is collected. The platelet fraction was obtained by centrifugation.

 (3) As shown in FIG. 21, it can be confirmed that the culture apparatus of the present invention can generate a much larger amount of platelets as compared with the case of culturing in a dish.

(4) According to the culture device of the present invention, a large amount of platelets can be efficiently produced from a small amount of hematopoietic stem cells. Therefore, by formulating the platelets prepared according to this example, surgery and It can allow for other and autologous platelet transplantation as a treatment for thrombocytopenia. Industrial applicability

 According to the culture apparatus of the present invention, not only bacteria and floating cells, but also adherent cells that form tissues and organs can be cultured efficiently for a long period of time. The ability to produce large quantities of such products efficiently. In particular, since a large amount of blood cells can be cultured from a small amount of hematopoietic stem cells, a blood product for transfusion can also be produced. In addition, since a large amount of cells can be cultured, an antibody that can be used as a pharmaceutical product that produces cells can be efficiently produced in large quantities.

Claims

The scope of the claims

1 An apparatus for culturing cells of a living body, comprising a culture tank for containing a culture solution, a circulation means for externally circulating the culture solution in the culture tank, and a period during which the culture solution is externally circulated And a culture medium adjusting means for adjusting the state of the culture medium.

 2. The culture apparatus according to claim 1, wherein the culture medium adjusting means includes gas concentration adjusting means for adjusting a gas concentration of the culture liquid.

 3. The culture apparatus according to claim 1 or 2, wherein the culture liquid adjusting means includes a purification means for purifying the culture liquid.

 4 The culture medium adjusting means comprises gas concentration adjusting means for adjusting the gas concentration of the culture liquid and purification means for purifying the culture liquid, and the purification means and the gas concentration adjusting means are arranged in series. 4. The culture apparatus according to claim 1, 2 or 3, wherein 5. The culture apparatus according to claim 3 or 4, wherein the purification means is a dialysis unit that dialyzes the culture solution.

 6 The dialysis part includes a hollow main body case and a hollow fiber disposed in the main body case, and the culture solution passes between the inner surface of the main body case and the outer surface of the hollow fiber. 6. The culture apparatus according to claim 5, wherein a culture fluid circulation passage is formed, and a dialysate for dialyzing the culture fluid is passed through the hollow fiber.

7. The culture apparatus according to claim 1, 2, 3 or 4, wherein the culture solution adjusting means includes a nutrient supply unit that supplies nutrients necessary for cell culture to the culture solution. 8 The nutrient supply unit includes a culture solution passage unit through which the culture solution is passed, and a nutrient channel through which a nutrient containing nutrients necessary for cell culture is passed. The culture apparatus according to claim 7, wherein the fluid passage part is separated by a dialysis member.

9 provided with an extraction means for extracting a culture solution from the culture tank, the extraction means having an extraction path in which one end is immersed in the culture solution in the culture tank and the other end is disposed outside the culture tank; A transport means for transporting the culture solution provided in the extraction path; and a gas supply means for supplying a sterile gas in the extraction path. The gas supply means is provided in the extraction path. The culture apparatus according to claim 1, 2, 3, 4, or 7, wherein a sterile gas is supplied between the other end and the conveying means.

 10. The culture apparatus according to claim 1, 2, 3, 4, 7 or 9, wherein the culture tank is provided with a temperature control means for controlling the temperature of the culture solution.

11. The culture apparatus according to claim 10, wherein the temperature control means includes a far-infrared radiation unit that emits far-infrared radiation.

 12. The incubator according to claim 11, wherein the far-infrared radiation portion is adjusted to have a peak of energy density of emitted far-infrared energy in a wavelength band of 7 to L2m.

 13. The culture apparatus according to claim 11 or 12, wherein the far-infrared radiation portion is a planar heating element.

 14 Said surface! The dog heating element includes a pair of upper and lower ¾W, a radiating unit that radiates far-infrared rays when energized, and a power supply unit that supplies power to the radiating unit. 14. The culture apparatus according to claim 13, wherein the radiating portion is formed by printing the inner surface of the pair of upper and lower substrates and a radiator.

15. The radiator according to claim 14, wherein the radiator is a material having a positive temperature coefficient.

16. In a state where the culture tank, the circulation means, and the culture solution adjusting means are connected, the culture tank, the culture tank, and the culture medium adjustment means are formed in a size that can be accommodated in an autoclave device. The culture apparatus according to 7, 9, or 10.

 17. The circulation means comprises an outflow prevention means for preventing cells from flowing out of the culture tank, 1, 2, 3, 4, 7, 9, 10 or 16 Description

18. The culture apparatus according to claim 17, wherein the outflow prevention means is an electromagnet.

19 It is provided with a culture medium exchange means comprising a culture medium discharge section for discharging the culture medium in the culture tank to the outside and a culture liquid supply section for supplying a new culture liquid from the outside to the circulation means. The culture apparatus according to claim 1, 2, 3, 4, 7, 9, 10, 10, 16 or 17 20. The culture apparatus according to claim 19, wherein the culture discharge unit includes an outflow prevention means for preventing cells from flowing out of the culture kiln.

 21. The culture apparatus according to claim 20, wherein the outflow prevention means is an electromagnet. 22 The sterilization unit sterilizes the culture solution before the culture solution supply unit supplies the culture solution to the circulation unit. The culture apparatus according to claim 19, 20, or 21. 23 In the culture tank, the circulation means, and the culture medium adjusting means, a closed loop circuit is formed by a path through which the culture medium flows, and pressurized steam is supplied into the closed loop circuit so that the inside of the closed loop circuit is The culture apparatus according to claim 1, 2, 3, 4, 7, 9, 10, 10, 16, 17 or 19 comprising an autoclave for sterilization. 24. The circulation means comprises a recovery means for recovering a desired component in the culture solution being cultured. 1, 2, 3, 4, 7, 9, 1 The culture apparatus according to 0, 16, 17, 19 or 23.

 A cell supply means for supplying the cells to be cultured is provided, the cell supply means transporting a seeding solution containing the cells to be cultured to the culture tank, and transported by the cell supply means. And a discharge section for discharging the seeded solution into the culture tank. 1, 2, 3, 4, 7, 9, 1 0, 1 6, 1 7, 1 9, 2. The culture apparatus according to 3 or 24.

 26 The cell transport means includes a seeding liquid transport path for communicating between the storage unit in which the seeding solution is stored and the discharge unit, and a bonnet for sending the seeding solution from the storage unit toward the discharge unit. 26. The culture apparatus according to claim 25, further comprising: a pump and a valve provided between the pump and the discharge portion to cut off communication between the two.

 27. A culture instrument to which cells to be cultured are attached is provided in the culture tank. 1, 2, 3, 4, 7, 9, 10, 10, 16, 17, 7, 19 , 2 3, 2 4 or 2 5.

 28. The culture apparatus according to claim 27, wherein the culture instrument has a three-dimensional structure.

29. The culture apparatus is formed by connecting a plurality of circular tubes. 30 The culture apparatus is disposed in the culture tank so that the axial direction thereof is parallel to the axial direction of the culture tank, and the cells of the cells to be cultured are seeded on the surface of the culture apparatus. A seeding means, the seeding means provided in the culture tank, a tilting part for tilting the culture tank, a cell supply part for supplying a liquid containing cells to the culture instrument, and the culture tank 30. The culture apparatus according to claim 29, further comprising a moving unit for inserting and removing the cell supply unit with respect to the culture instrument.

 31. The culture apparatus according to claim 30, further comprising a rotating unit that rotates the culture tank around a central axis thereof.

 32. A cell recovery means for recovering the cells of the cells cultured on the surface of the culture instrument, wherein the cell recovery means supplies the culture instrument with a liquid for separating the cells from the culture instrument. 32. The culture apparatus according to claim 30, wherein the culture apparatus comprises: a section; and a collection section that collects the liquid supplied from the stripping solution supply section.

33. The culture apparatus according to claim 27, wherein a material of the culture instrument is titanium or a titanium alloy.

 34. The culture apparatus according to claim 27, wherein the culture instrument has a titanium layer containing titanium on a surface thereof.

 35. The culture apparatus according to claim 27, wherein the material of the culture instrument is a cell-derived polymer material.

 36. The culture apparatus according to claim 27, wherein the culture instrument has a layer of a cell-derived polymer material on a surface thereof.

 37. The culture apparatus according to claim 35 or 36, wherein the cell-derived polymer material is collagen or a cell-absorbing polymer.

 38 The material of the culture device is a calcium compound,

39 The culture apparatus according to claim 27, wherein the culture instrument has a calcium compound layer on a surface thereof.

 40. The culture apparatus according to claim 27, wherein a material of the culture instrument is a synthetic biodegradable polymer.

41 The culture device has a synthetic biodegradable polymer layer on a surface thereof. The culture device according to claim 27.

 42. The culture apparatus according to claim 40 or 41, wherein the synthetic biodegradable polymer is poly L-lactic acid or polyglycolic acid.

 43. The culture apparatus according to claim 27, wherein the culture instrument has a layer of paper weaving induction material having a tissue induction ability on a surface thereof.

 44. The woven fabric-inducing material is an extracellular matrix.

45 The material of the culture device is a silicon-containing material.

46. The culture apparatus according to claim 27, wherein the culture instrument has a layer of a silicon-containing material on a surface thereof.

 47. The silicon-containing material is zeolite, 45 or 46

48. The material according to claim 27, wherein the material of the culture instrument is artificial ceramics.

49. The culture apparatus according to claim 27, wherein the culture instrument has an artificial ceramic layer on a surface thereof.

 50. The culture according to claim 27, 33, 34, 3 5, 36, 38, 39, 40, 41, 43, 45, 46, 48 or 49, wherein the culture device is a porous body.

51. The culture according to claim 27, 33, 34, 35, 36, 38, 39, 40, 41, 43, 45, 46, 48 or 49, wherein the culture instrument has a network structure.

52 The suspension according to claim 1, 2, 3, 4, 7, 9, 10, 16, 17, 19, 23, 24, 25, or 27, wherein suspended cells are immersed in the culture medium. Culture

53. The culture apparatus according to claim 52, wherein the floating cells include hematopoietic stem cells.

54. The floating cell according to claim 52 or 52, comprising a blast of a blood cell. 5 The culture apparatus according to 3.

 55. The culture apparatus according to claim 27, 52, 53, or 54, wherein the cells attached to the culture device and the floating cells floating in the culture medium are mixed and cultured.

 56. The culture apparatus according to claim 55, wherein the cells attached to the culture device are bone marrow stromal cells, and the cells floating in the culture solution contain hematopoietic stem cells.

 57. The culture apparatus according to claim 55 or 56, wherein the cells attached to the culture device are bone marrow stromal cells, and the floating cells include blasts of blood cells.

58 When hematopoietic stem cells are cultured by the culture device according to claim 1, 2, 3, 4, 7, 9, 10, 1 6, 1 7, 1 9, 2 3, 2 4, 2 5 or 2 7 A blood product comprising a blood component produced in the culture solution.

 59 In the culture apparatus according to claim 1, 2, 3, 4, 7, 9, 10, 1 6, 1 7, 1 9, 2 3, 2 4, 2 5 or 2 7, seeded in the culture instrument Artificial shrine characterized by culturing cells.

 60. The artificial tissue according to claim 59, wherein the cell is an undifferentiated cell.