JP2003325168A - Closed type cell culture system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tissue cell culture system for efficiently early proliferating a cell tissue in a damaged portion, namely in vivo, in regenerative medical engineering and for avoiding the outbreak of an infectious disease caused by bacteria in the damaged portion during a treatment. <P>SOLUTION: This closed type cell culture system is characterized by closing a tissue-lacked portion on the surface of the body or in the body to form the closed environment for preventing the invasion of bacteria and the like and circulating a solution suitable for the culture of cells in the closed tissue-lacked portion. A method for administering a medicine for promoting the cure of the tissue-lacked portion comprises utilizing the closed type cell culture system and dissolving the medicine in a perfusion liquid. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a system for inducing tissue regeneration at a damaged site by proliferating cell tissue at the damaged site in vivo.

[0002]

2. Description of the Related Art Conventionally, in the treatment of a body-damaged area, unnecessary substances such as foreign substances in the damaged area are removed and then disinfected with a disinfectant, etc. , We are trying to regenerate the skin by cell proliferation. However, self-renewal of the skin has various insurmountable and difficult problems such as the time required for healing and the occurrence of infections in the wound.

In order to solve such a problem, in recent years, as regenerative medical engineering, cells near the injured part are collected and proliferated in vitro to reconstruct a target tissue or organ outside the body. Then, a method of returning it to the defective portion, that is, a method of transplanting an autologous tissue by culture is performed. For example, a cell culture system including a container for cell growth medium and a wound dressing system (see, for example, Patent Document 1).
), A cultured skin substitute containing a human fibroblast and a production substance effective for wound treatment (see, for example, Patent Document 2), and the like. All of these methods are effective as methods for planar tissues, but it is difficult to culture multi-layer cells (for example, multi-layer cells of epidermal cells and dermal cells), and the cell growth rate is slow. There are various problems that must be solved.

Further, thick tissues have a different tissue structure from cells in vivo, and lack of blood vessels, which makes it difficult to supplement oxygen or nutrients, so that the survival rate of cells is not so high. I can't say that. in addition,
Tissues having a complicated three-dimensional structure, it is difficult for damaged areas, and the occurrence of infectious diseases during treatment cannot be avoided, and it is possible to solve the problems that have been encountered in the past. The current situation is that it is not.

[0005]

[Patent Document 1] Japanese Patent Publication No. 2001-507218

[Patent Document 2] Japanese Patent Laid-Open No. 2002-200161

[0006]

Therefore, the present invention is
In view of the above-mentioned present situation, in regeneration engineering, tissue cell culture in which early generation of injured cells, that is, efficient in vivo tissue in vivo, is avoided and bacterial infection in the injured cells is avoided during treatment. The challenge is to provide a system.

In order to solve such a problem, the present inventor has conducted diligent studies, and as a result, after removing unnecessary substances such as necrotic tissue at the wound of the defect damage portion, a bioabsorbable material that becomes a scaffold for cell proliferation. It is possible to efficiently regenerate the defect tissue on the spot by embedding cells in the defect space, closing the defect part from the outside, and circulating a solution suitable for cell culture to the closed part. Focusing on, the present invention has been completed.

[0008]

Therefore, according to the invention of claim 1 which is a basic aspect of the present invention, the invasion of bacteria and the like is prevented by sealing the body surface or the tissue defect portion in the body from the outside. The closed cell culture system is characterized in that the closed tissue is regenerated by circulating a solution suitable for cell culture into the closed tissue defect portion.

That is, the present invention provides a closed-type solution perfusion part by sealing the defective part itself from the outside to prevent infection from bacteria and to circulate a solution suitable for cell culture to that part. The feature is that sterilization and cell culture can be continuously performed by appropriately changing the components of the liquid.

Further, since cells generally divide and grow by adhering to a certain surface, a cell attachment scaffold is required for cell growth. In the present invention, such a scaffold is one in which a bioabsorbable material is embedded in the defect portion to promote cell growth. Therefore, the present invention according to claim 2 is the closed cell culture system according to the invention according to claim 1, wherein the bioabsorbable material is embedded in a closed environment to promote regeneration of defective tissue cells. .

Examples of the bioabsorbable material serving as a scaffold for cell proliferation include polyglycolic acid fiber, lactic acid glycolic acid copolymer fiber or sponge, glycolic acid caprolactone copolymer fiber, polylactic acid fiber or sponge, Lactic acid caprolactone copolymer, polycaprolactone fiber, polydioxane fiber, collagen fiber or sponge, gelatin sponge, fibrin fiber sponge, polysaccharide fiber or sponge, tricalcium phosphate porous beads, calcium carbonate porous beads, hydroxyapatite, etc. Can be mentioned.

In the closed cell culture system of the present invention, the cell culture in a closed environment is perfused with a solution suitable for cell growth so that such cell growth is effectively performed. Therefore, the present invention according to claim 3 is the invention according to claim 1 or 2, wherein the solution suitable for cell culture is plasma solution serum separated from autologous blood, platelet concentrated serum, plasma liquid preparation, platelet concentrated serum. A closed cell culture system which is a plasma fractionation preparation, a blood blood protein fractionation component solution, a plasma expander, an osmotic isotonic infusion solution, and a cell culture medium.

The present invention according to claim 4 further comprises changing the circulating solution component for each treatment step.
It is a closed cell culture system that is programmed and has a cell proliferation environment suitable for analgesia of the defect, disinfection, and each tissue for regeneration. That is, in order to effectively perform cell culture in the defective portion, it is necessary to set the closed environment for cell growth to the most effective one. Therefore,
The present invention according to claim 4 is characterized in that it can meet such demands and can easily construct a cell proliferation environment suitable for analgesia and sterilization of a defective portion and each regeneration target tissue. .

The pH value, the carbon dioxide partial pressure, and the oxygen partial pressure of the cell culture environment change variously with the cell culture. Therefore, even if the composition of the solution suitable for cell culture is circulated, the closed environment must always be optimal for cell culture. To do so, the sensor is a physical factor in the culture medium in a closed environment, namely the pH value,
The carbon dioxide partial pressure and oxygen partial pressure are monitored, the gas exchanger provided at the inlet side of the culture environment is controlled by the signal, and the gas partial pressure of the closed environment is adjusted in real time according to the change of the culture solution. It is preferable to optimize the cell culture environment. Therefore, the present invention according to claim 5 incorporates a gas exchange device equipped with a monitoring device for measuring the pH value, the carbon dioxide gas partial pressure, and the oxygen partial pressure of the closed environment on the inlet side of the closed environment. It is a closed cell culture system that automatically adjusts the gas concentration based on the signal of to optimize the cell culture environment.

The present invention according to claim 6 is a closed cell culture system in which the pressure in a closed environment is controlled continuously or intermittently. That is, by changing the pressure in the closed environment variously by, for example, a pressure maintaining device, it is possible to secure a space necessary for the growth of nerve tissue and the like in the positive pressure state, and to promote the exudation of body fluid in the negative pressure state. It is possible to further promote cell growth on the surface of the damaged part. Further, by intermittently controlling the negative pressure, it is possible to perform the uniform and efficient replacement of the reflux liquid in the closed environment by temporarily reducing the volume in the closed environment.

Further, with regard to a wide range of severe burns, a serious loss of water in body fluid may occur at the damaged portion. Therefore, when culturing cells in a damaged area using the closed cell culture system of the present invention, an osmotic pressure-adjusted solution capable of appropriately maintaining the osmotic pressure of the perfusate on the surface of the damaged area due to burns is used. It is necessary to circulate as a perfusate. Therefore, the present invention according to claim 7 is a closed cell culture system having a circuit for circulating the osmotic pressure-adjusted perfusate.

Furthermore, when cell culture of a damaged part is performed using the closed cell culture system of the present invention, the damaged part is a relatively deep damage such as nerve cell tear, and such a nerve cell culture is performed. Alternatively, in the case of vascular cell culture, minimally invasive access is desirable. For such access, it is preferable to adopt the form of a puncture needle. Therefore, the present invention according to claim 8 is a closed cell culture system in which a cell culture space is formed deep inside a body cavity or in an organ by a combination of a puncture device and a balloon catheter.

That is, for the growth of nerve tissue cells or vascular tissue cells, it is necessary to secure a space for the growth of the cells. In order to create a space therefor, a puncture needle and a catheter with a balloon passing through its lumen are used to reach a target part in the body, and the balloon is expanded to create a void suitable for the growth of target cells. Further, it has a feature that the bioabsorbable material can be effectively left in the damaged part.

In addition, in the case of parenchymal organs such as liver, pancreas, kidney, etc., by culturing the target cells in each organ by providing a culture space in the same manner, in vitro organs can be obtained in organs with reduced function or in a defective state. Culturing cells by culturing technology Cells having an excellent functional structure can be grown by the above.

The present invention also provides a method capable of effectively administering an effective drug when culturing cells in a damaged area. That is, in the closed cell culture system provided by the present invention, a cell growth factor or a vascular growth factor is added to the perfusate in order to promote conversion to a medium component optimal for the growth of each cell or to promote healing. The defect can be perfused. Therefore, the present invention according to claim 9 is also a method for administering a drug, which utilizes the closed cell culture system according to claim 1 to dissolve a therapeutic agent in perfusate and accelerate treatment of a defect site. is there. In order to avoid the occurrence of improper drug compounding problems caused by the mixing of drugs when changing the therapeutic drug, in this case, cells in a closed environment are subjected to intermittent negative pressure cycles. The perfusate circulating in the culture space can be positively replaced to uniformly and efficiently exchange the drug solution.

Drugs that can be administered in such cases include antiseptics, local anesthetics, anti-inflammatory analgesics, antibiotics, peripheral vasodilators, various cell growth factor preparations, nerve growth factor preparations,
Examples include vascular growth factor preparations (various cell growth inhibitor preparations at the time of removing cancer cells), immunosuppressants, and the like.

In addition to these drugs, various cell adhesive molecules such as fibronectin and hydronectin may be contained in the perfusate. A gene can also be introduced, and examples of such a gene include naked DNA, adenovirus vector gene, retrovirus vector gene, liposome embedding gene, hydrogel embedding gene and the like.

Claim 1 as another aspect of the present invention
The invention described in 0 is a treatment from injured cell culture by collecting autologous cells in advance, culturing and proliferating the cells in vitro, suspending the cells in a perfusate, and returning the cells to a closed culture system through the perfusion system. Simultaneously with the above, it is also a method of transferring and proliferating cells which promotes healing of a damaged part by carrying out culture proliferation and engraftment in combination with tissues cultured in vitro.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The closed cell culture system provided by the present invention will be described below in detail with reference to the drawings.

FIG. 1 is a schematic exploded schematic view for explaining the closed cell culture system provided by the present invention. That is, the closed cell culture system (1) provided by the present invention
Is basically a solution suitable for cell culture to the closed tissue defect portion by providing a closed environment in which the invasion of bacteria and the like is protected by sealing the tissue defect portion (2) inside the body or the body from the outside. The closed cell culture system is characterized in that the defective tissue is regenerated by circulating.

More specifically, the closed cell culture system (1) provided by the present invention (in the figure, the closed state is released and shown in a schematic development view) is a body surface or a tissue in the body. A closed environment in which the invasion of bacteria and the like is prevented by means (3) (an adhesive sheet such as a surgical film in the figure) for sealing the defect (2) from the outside, that is, the tissue defect (2) on the body surface is surgically A closed environment is created by covering and sealing with a film (3), and a solution suitable for cell culture is introduced into the closed tissue defect part (2) from a perfusion solution introduction tube (10) (direction of arrow A in the figure). To perfuse the perfusate discharge tube (11) (in the direction of arrow B in the figure).

The perfusion solution perfused into the system (1) from the perfusion solution introduction tube (10) is immersed in, for example, the bioabsorbable material for epidermis (6) through the disc 2 (5), and the perfusion solution is immersed. The liquid is further immersed in the buried bioabsorbable material (7) which is buried in the body tissue defect portion (2) thereunder, and the cells in the body tissue defect portion (2) are provided with an environment suitable for cell growth on the spot. This results in efficient cell growth.

On the other hand, the perfusate in which the buried bioabsorbable material (7) was dipped and the body tissue defect portion (2) was filled, passed through the air space between the disks 1 (4) and 2 (5). , Is discharged from the perfusion liquid discharge tube (11) provided on the disk 1 (4), and the perfusion is completed.

Incidentally, the disk 1 provided in the system
The two-layer disc of (4) and disc 2 (5) may have a mechanism for holding the perfusate introduction tube (10) and the perfusate discharge tube (11), respectively, and the material thereof is a non-permeable plastic. It can be molded integrally with the tube.

As described above, the system (1) according to the present invention circulates the perfusate into the closed environment at the body tissue defect portion, whereby the living body becomes a scaffold to which cells adhere to the body tissue defect portion. Cellular growth is efficiently promoted by the absorbable material, and when the cell growth is completed, the bioabsorbable material that serves as a scaffold is decomposed and absorbed, and the tissue of the body defect site is regenerated in a sterile state. Of.

FIG. 2 shows a schematic cross section obtained by accessing the closed tissue culture system (20) of the present invention from the body surface. That is, in the case of injury such as trauma, burns, pressure ulcers, body tissues including the epidermis (21) and the dermis (22) are lost, and the injured part is exposed on the body surface. In this case, the wound surface is filled with the two-layer disc wound surface adapter (24) of the closed-type tissue culture system (20) of the present invention having the coating film (23), and the perfusate is perfused. Even in this case, a bioresorbable material is embedded as a suitable scaffold biomaterial (25) for promoting cell growth in a tissue requiring cell growth to form a cell growth part (26). It's good to leave.

In this case, the perfusate is the perfusate inlet (2) provided at the center of the closed tissue culture system (20).
8) It is perfused from 8), flows to the damaged part, is attached to the scaffold biomaterial (25) which is a bioabsorbable material, and diffuses to the peripheral part. After that, from the single edge (27) of the peripheral portion to the two-layer disc (2
It passes through the gap of 4) and is discharged as drainage from the perfusate outlet (29).

Therefore, since the injured surface is in a sealed state, infection from bacteria or the like can be prevented, and the perfusate and the scaffolding biomaterial can effectively proliferate cells at the damaged part.

The closed-type tissue culture system of the present invention for accessing the body surface is adapted to the size of each injured part,
It can be designed to any desired size. Therefore, in the case of a wide range of burns and the like, the closed tissue culture system of the present invention having a two-layer disc is combined to match the area and shape of the burn, and at the same time, the perfusate is perfused into each system. The same effect as when used alone can be obtained.

On the other hand, in the case of performing cell proliferation in a relatively deeply damaged area in the body such as nerve cell tear or vascular tissue tear, it is desirable to access with minimally invasiveness.
A closed cell culture system in the form of a needle is used.
Specifically, create a space for proliferation of nerve tissue cells and vascular tissue cells with a combination of a puncture needle and a catheter with a balloon that passes through the lumen, and place the scaffold biomaterial at the site expanded with the balloon. The perfusion fluid can be circulated by the perfusion catheter. The puncture needle used in this case can be flexibly bent, and the catheter can be placed along the damaged portion for the purpose of cell proliferation.

The concept of cell proliferation by the system of the present invention for cell proliferation in a relatively deeply damaged part in the body, for example, nerve cell disruption or vascular tissue disruption is shown in FIGS. That is, using a puncture needle (30) as shown in FIG. 3, the puncture needle is pierced from the skin surface to introduce a tip to a target portion for forming a void for cell proliferation, and a balloon catheter is introduced from the tip hole of the puncture needle. While retracting the tip of the puncture needle, the catheter balloon part (31) is exposed and the balloon part (32) is inflated.

Then, after expanding the balloon to create a void (intra-tissue space) for cell proliferation, the balloon is contracted and only the balloon catheter is removed, as shown in FIG. After that, a bioabsorbable material (33) serving as a scaffold for cell proliferation is inserted from the lumen of the puncture needle (30). The bioabsorbable material may be left indwelling through a puncture needle, which is a catheter that is contained in the lumen in advance.

[0038] A bioabsorbable material that serves as a scaffold for cell proliferation is left in the injured part, the catheter for embedding the bioabsorbable material is removed, and then a perfusion solution suitable for cell proliferation is perfused as shown in FIG. , Double lumen catheter for perfusion (3
4) is inserted, then only the perfusion catheter (34) is left, the puncture needle is pulled out from the body, and the perfusion fluid inlet (3
5) Connect the outlet (36) to the perfusion system and start the perfusion. This state is schematically shown in FIG.

As described above, by using the closed cell culture system of the present invention, the body surface or the tissue defect part in the body can be sealed from the outside to form a closed environment in which invasion of bacteria and the like is prevented. It is possible to circulate a solution suitable for cell culture to the closed tissue defect portion to grow cells in the defect portion and regenerate tissue.

In the closed cell culture system provided by the present invention, the solution components to be circulated are changed step by step to provide analgesia of the defective portion, disinfection, and the construction of a cell growth environment suitable for each regeneration target tissue. It is also possible. For example, as such means, as shown in FIG. 7 as a schematic conceptual diagram, the type of the perfusate is switched from a plurality of containers (40) (two in the figure) containing the cell culture perfusate. Through a 3-way stopcock (41)
After controlling the flow rate by the flow rate controller (42), the patient (4
6) Closed cell culture system (closed incubator)
After perfusing the inside of (43), the culture solution after perfusion can be discarded (45) through the pump (44) into the drainage container.

In this case, the discharge amount of the pump can be controlled by using, for example, a pressure sensor or the like, and thereby the pressure of the target damaged portion for cell proliferation can be kept constant.

In the case of using the plurality of perfusates, not only perfusing a plurality of components suitable for cell growth but also a bactericidal agent for disinfecting a contaminated wound site or the growth of bacteria is suppressed. Perfusion of antibiotics to induce perfusion at an early stage, and further various cell growth factors, nerve growth factors, and blood vessel growth factors (various cell growth suppression factors at the time of cancer cell removal) for the purpose of promoting better healing
Can be perfused with a perfusate containing

For more effective treatment, a perfusion solution containing a local anesthetic, an anti-inflammatory analgesic, an antibiotic agent, a peripheral vasodilator, an immunosuppressant, etc. can be perfused.

Further, in the cell culture environment, the pH value, the carbon dioxide gas partial pressure, and the oxygen partial pressure inside the culture environment change variously as the cells are cultured. Therefore, it is preferable that the closed environment, which is the culture environment, is always the optimum environment for cell culture. For that purpose, a sensor (47) for measuring and monitoring physical factors in the culture medium in a closed environment, that is, pH value, carbon dioxide partial pressure, and oxygen partial pressure is provided by a sensor, and a signal from the monitor is used to enter the inlet of the culture environment. It is possible to optimize the cell culture environment by controlling the gas exchanger (48) provided on the side and adjusting the gas partial pressure of the closed environment in real time according to the change of the culture solution.

The closed cell culture system provided by the present invention can also perfuse a perfusate suitable for cell culture using a constant pressure continuous aspirator, an infusion bag and an infusion tube circuit that are commonly used in hospitals and the like. it can. The schematic concept is shown in FIG. Note that, in FIG. 8, reference numerals in the drawing have the same meanings as in FIG. 7.

Also in FIG. 8, the basic concept is as shown in FIG.
Similar to the system shown in FIG. 7, the part for discarding the culture solution after perfusion into the drainage container through the pump provided in FIG. 7 was connected to a constant pressure continuous aspirator (51) commonly used in hospitals and the like. Only the parts are different.

As described above, according to the present invention, after removing unnecessary substances such as necrotic tissue at the wound of the defect damaged portion, a bioabsorbable material that serves as a scaffold for cell proliferation is embedded in the defect space, The cells can be closed from the outside and a solution suitable for cell culture is circulated to the closed portion, so that defective cells can be efficiently proliferated in situ to regenerate the tissue.

[0048]

EXAMPLES Specific examples of cell proliferation of the present invention will be described below with reference to Examples. Example 1 Treatment of Damaged Site after Surgery for Grafted Skin The hemorrhage from the wound surface after skin removal surgery was immediately stopped by using fibrin spray, and the peripheral area was disinfected. Then
After filling the recessed portion with collagen beads, an open-cell collagen sheet having a hole in the center was formed into a larger shape along the wound surface, and the wound surface was covered. The two-layer adapter was also formed into a shape similar to that of the collagen sheet and then laminated on it, and a surgical film having openings only at the entrance and exit of the adapter was covered to form a closed space between the collagen beads and the sheet.
If there is pain in the skin-cutting area, a plasma preparation containing xylocaine as a local anesthetic and a mixed antibiotic of three kinds is circulated in the closed space, and after 3 hours, the local anesthetic and the antibiotic solution are stopped, and a base is added. Fibroblast growth factor (bFG
Perfusion was performed by switching to F).

Separately, the tissue of the dermal layer was collected, and the fibroblasts finely suspension-cultured in vitro with fetal bovine serum were replaced with the serum of the patient and perfused. Cell transplantation was carried out by injecting the cell turbid solution into a closed type incubator through a three-way stopcock under a perfusate bag. Then, when the cell proliferation of dermal cells was completed, bFGF was exchanged for epidermal growth factor (EGF) and the epithelial cells were cultured until they became uniform with the surrounding skin surface.

Example 2 A puncture needle which can be flexibly bent from the skin on the surface of the nerve reconstructed nerve of the femoral rupture nerve of the lower limbs,
Puncture the nerve so that it reaches the nerves at both ends. A balloon having a length that can reach both nerve stumps is selected, and a balloon catheter is inserted through a puncture needle to inflate the balloon to create a space between nerve stumps.

A parallel collagen fiber bundle, which serves as a scaffold for nerve growth, is inserted into the void with a catheter housed in the lumen. Further, this catheter is replaced with a thin catheter for perfusion, the puncture needle is removed, and only the catheter for perfusion is left indwelling.

Nerve growth factor ( ^10-8 mol) is added to the serum obtained from the patient's blood, perfusion is started, and the circulation circuit is switched to when the internal air is released.

A slight positive pressure is maintained so that the space between nerve stumps does not collapse during perfusion. When the cells have grown sufficiently, the perfusion catheter is removed and completed.

Example 3 Treatment of Severe Burn Injury After the skin and dead tissue were excised for severe burn of the abdomen, the burn wound surface was covered with a collagen fiber sheet,
Further, a two-layer perfusion adapter is attached on it. The wound surface is first disinfected with an isotonic plasma bulking formulation containing povidone iodine solution.

A human plasma preparation having an osmotic pressure equal to that of the wound surface is perfused in order to suppress the loss of body fluid from the wound surface of the patient. In addition, add antibiotic injections to prevent infection,
In addition, if the patient complains of pain, xylocaine, a local anesthetic, is injected into the perfusate to provide analgesia.

Xylocaine after the patient's condition has stabilized,
Administration of antibiotics was discontinued and fibroblast growth factor (bFG
F), cell growth factors such as platelet-derived cell growth factor (PDGF) and epidermal growth factor are added to the perfusate. When the epidermis on the wound surface has sufficiently grown, the closed cell culture device is removed, and the wound surface is exposed to the outside air, and the rest of the natural healing is awaited.

Example 4: A rabbit liver is punctured with a catheter introducer equipped with a puncture needle while observing an ultrasonic tomographic image. The puncture needle was removed while leaving the introducer sheath, and a balloon catheter for removing a Fogerty thrombus was inserted instead, and the balloon was inflated until the diameter became about 2 cm to secure a cell culture space in the liver. After shrinking the balloon, the Fogerty catheter was removed, and a Teflon (registered trademark) tube containing a cotton-like collagen fiber pellet was inserted into the culture space to fill the collagen fiber pellet. Remove the Teflon (registered trademark) tube for inserting collagen and use 2 for perfusion.
The catheter was placed so that the perfusion port of the heavy lumen was located in the culture space, the catheter introducer sheath was also removed, leaving only the double lumen catheter, and fixed to the skin by suturing, and at the same time fixed with a film for external fixation of the catheter. .

While the serum prepared from the blood of the rabbit was perfused into the cell culture space, hepatocytes were cultured in the rabbit liver for 30 days.

After the treatment for 30 days, the rabbits were sacrificed, and the autopsy of the treated liver revealed that hepatocytes were proliferated and cultured in the collagen fibers.

[0060]

As described above, according to the closed cell culture system of the present invention, the defective portion is closed from the outside to prevent infection from bacteria, and a solution suitable for cell culture is circulated to the closed portion. This has the advantage that the defective cells can be efficiently proliferated on the spot to regenerate the tissue.

Further, by changing the perfusate variously, the cell growth factor or the therapeutic drug can be effectively administered to the target site, and there is an advantage that more effective tissue regeneration can be secured. ing.

[Brief description of drawings]

FIG. 1 is a schematic schematic exploded view for explaining a closed cell culture system of the present invention.

FIG. 2 is a schematic cross-sectional view in which the closed cell culture system of the present invention is accessed on the body surface.

FIG. 3 is a diagram explaining the concept of cell proliferation by the system of the present invention for cell proliferation in nerve cell tear, which is a deeply damaged area in the body.

FIG. 4 is a diagram illustrating the concept of cell proliferation by the system of the present invention for cell proliferation in nerve cell tear, which is a deeply damaged area in the body.

FIG. 5 is an explanatory view of the concept of cell proliferation by the system of the present invention due to cell proliferation in nerve cell tear, which is a deeply damaged part in the body.

FIG. 6 is an explanatory view of the concept of cell proliferation by the system of the present invention due to cell proliferation in nerve cell tear, which is a deeply damaged area in the body.

FIG. 7 is a schematic explanatory diagram of a case where a plurality of perfusates are circulated by the closed cell culture system of the present invention.

FIG. 8 is a schematic explanatory view of the closed cell culture system of the present invention in which a perfusate is perfused using a constant pressure continuous inhaler, an infusion bag, and an infusion tube circuit.

[Explanation of symbols]

1 Closed cell culture system 2 Body tissue defect 3 Surgical film 7 Buried bioabsorbable material 20 Closed cell culture system 23 Covering film 24 dual layer disc 25 Scaffolding biomaterial 26 Cell Proliferation Department 30 puncture needle 32 balloon 33 Bioabsorbable material 34 double lumen catheter 40 perfusate bag 41 3-way stopcock 42 Flow rate adjustment 43 Closed Cell Culture Substrate 44 pumps 45 disposal 46 patients 47 pH gas monitor 48 gas exchanger 51 Constant Pressure Continuous Aspirator

Continued front page    F-term (reference) 4B065 AA90X AA93X BA30 BB01                       BB25 BB40 BC02 BC06 BC07                       BC14 BC15 BC50 CA43 CA44                 4C081 AC08 BA12 CE02                 4C087 BB34 BB35 BB38 MA17 MA66                       NA05 NA10 ZA01 ZA89

Claims (10)

[Claims] 1. A closed environment in which the invasion of bacteria and the like is protected by sealing a tissue defect portion on the body surface or in the body from the outside, and a solution suitable for cell culture is circulated to the closed tissue defect portion. A closed cell culture system characterized by performing regeneration of defective tissue. 2. The closed cell culture system according to claim 1, wherein a bioabsorbable material is embedded in a closed environment to promote regeneration of defective tissue cells. 3. A solution suitable for cell culture is serum separated from autologous blood, blood plasma preparation, platelet concentrated serum, plasma fraction preparation, blood protein fraction component solution, plasma expander, osmotic isotonic infusion solution. The closed cell culture system according to claim 1, which is a cell culture medium. 4. The method according to claim 1, wherein the circulating solution component is changed for each treatment stage to provide analgesia of the defect, disinfection, and a cell growth environment suitable for each tissue to be regenerated and programmed. The closed cell culture system according to any one of claims. 5. A connected gas exchange device equipped with a monitor device for measuring pH value, carbon dioxide gas partial pressure, and oxygen partial pressure of the closed environment is installed on the inlet side of the circuit connected to the closed environment. The closed cell culture system according to claim 1, wherein the gas concentration is automatically adjusted based on the signal to optimize the cell culture environment. 6. The closed cell culture system according to claim 1, wherein the pressure in the closed environment is controlled continuously or intermittently. 7. The closed type tissue culture system according to claim 1, further comprising a circuit for perfusion of an osmotic pressure adjusted perfusion solution. 8. The closed cell culture system according to claim 1 or 2, which has a form in which a cell culture space is formed deep inside a body cavity or in an organ by a combination of a puncture device and a balloon catheter. 9. A method for administering a drug, which utilizes the closed cell culture system according to claim 1 to dissolve a therapeutic drug in a perfusate to promote treatment of a defect site. 10. Self-collecting cells in advance, culturing and proliferating the cells in vitro, suspending them in a perfusate, returning to a closed cell culture system through a perfusion system, and simultaneously treating the damaged cell culture. A method for transferring and proliferating cells which promotes healing of a damaged part, which comprises performing cell culture proliferation and engraftment in a complex manner by adding cells cultured in vitro.