KR20180069779A - Method of preparing artificial tracheal scaffolds using animal tracheal tissues - Google Patents

Abstract

The present invention relates to a method for manufacturing an artificial organ support using an organ tissue of an animal. The artificial bio-organ support using the organ tissue of the animal manufactured by the method of the present invention can be safely used for tissue regeneration and healing rather than other materials made from existing artificial synthetic materials, has high biocompatibility, greatly reduces environmental pollution sources, and can be used as a raw material of bio-material better than an organ replacement material using existing artificial materials.

Description

[Method of preparing artificial tracheal scaffolds using animal tracheal tissues]

The present invention is an animal organ tissue (氣管組織, tracheal tissue) and an artificial organ having a multi-membrane structure using a cell injection technique, and a method for manufacturing the same.

Treatment for organ damage or organ stenosis due to congenital causes or tumors and trauma varies depending on the cause of occurrence and the age and condition of the patient, but generally depends on the size and length of the injury site. If only part of the tracheal wall is damaged, remove the part and leave it as it is if the size is small, or reconstruct it using various flaps.

In cases where both the inner diameter of the trachea is damaged, such as a tracheal stenosis, it is common to perform single-end anastomosis after resecting the lesion site. However, this method has a problem that is difficult to apply when the stricture area exceeds 5 cm. Therefore, when the length of the lesion is long, such as congenital organ stenosis in children, various methods such as tracheoplasty using various cartilage or allograft transplantation of a donor have been attempted. Allogeneic organ transplantation uses an organ of another person (brain death), and is relatively effective in organ plastic surgery, but uncertainty about the donor, the donor's medical history, immune rejection, the limitations of collection and the method of surgery are complicated, and several steps of surgery are required. It is necessary and a standard treatment has not been established due to disadvantages such as damage and infection that occur during the tissue collection process. In this way, in order to reduce the various side effects caused by the use of brain-dead tissues, manufacturing of artificial organs using various artificial materials has been attempted.

However, these artificial organs have poor mucosal regeneration, are susceptible to inflammation, and have many problems in clinical application due to the high possibility of formation of granulation tissues, separation of junctions, damage to surrounding tissues, and restenosis.

Important factors in the manufacture of artificial organs are the production of a cylindrical framework that acts as a cartilage of a normal organ, and the regeneration of the respiratory epithelial cells mucous membranes where mucosal ciliary movement occurs while granulation tissue and scar formation occurs as little as possible in the cylindrical skeleton. To induce. Artificial organs made of various materials that are currently being developed have not overcome these problems, and until now, only an experimental method for bronchus transplantation, an extreme treatment method, has been reported as a part of organ transplantation for organ treatment and reconstruction. . As a method for bronchial transplantation, bronchial transplantation through donation of brain dead is currently being performed, but bronchial transplantation of another person has been successful in the world to date due to immune rejection and organ infection due to excessive use of immunosuppressants. There is no. Another method for bronchial transplantation was to cultivate stem cells and nanofibers extracted from bone marrow to create an artificial respirator with a length of 7 cm and a diameter of 1.2 cm.However, it took several weeks for the stem cells to grow into new organs. Since it is required, it is limited to be applied in an emergency situation, and since the scaffold using nanofibers is also different from that of the normal bronchi, it is difficult to evaluate the long-term prognosis. In addition, in the case of transplantation of artificial organs using stem cells, since most of the scaffolds are produced by donating bronchi from brain dead, the supply of organs that can be transplanted is remarkably insufficient.

On the other hand, organ cartilage consists of only one cell called chondrocyte, and has low cell regeneration ability and repair rate because there are no blood vessels and lymphatic vessels in the tissue. In other words, damage and defects are not naturally healed or reconstructed. Therefore, in the case of organs containing more cartilage tissue than other organs, regeneration and treatment after surgery are not well performed.

The development of artificial organs using animals is advantageous in that customized bronchi can be secured without relying on organ donation or procedures such as cell culture to make organs.

Grillo HC, Donahue DM, Matisen DJ. Postintubation tracheal stenosis, treatment and results. J Thorac Cardiovasc Surg. 1995; 109:486-493 Grillo HC. Slide tracheoplasty for long segmental congenital tracheal stenosis. Ann Thorac Surg. 1994;58:613-621 Kim JG, Suh SW, Shin JY, Kim JH, Choi YS, Kim HJ. Replacement of a tracheal defect with a tissue-engineered prosthesis; Early results from animal experiments. J Thorac Cardiovasc Surg. 2004;128:124-129

Accordingly, the present inventors have made efforts to solve the problems of the conventional synthetic raw materials such as polyethylene glycol and polyurethane, as a result of which the problems of the existing synthetic raw materials such as continuous foreign substance reaction, low biocompatibility and difficulty in supply can be greatly reduced. The present invention was completed by developing technology for animal-derived biomedical materials.

It is an object of the present invention to provide a method of manufacturing an artificial organ support using an animal organ tissue.

Another object of the present invention is to provide an artificial organ support using the organ tissue of an animal prepared by the above method.

In order to achieve the above object, the present invention

(a) recovering the organ tissue of a mammal and then decellularizing it to prepare a support for the organ tissue; And

(b) It provides a method of manufacturing an artificial organ scaffold using the organ tissue of an animal comprising the step of transplanting tissue cells to the decellularized organ tissue scaffold of step (a) to recellularize.

In one embodiment of the present invention, the mammal is made of pig, horse, cow, dog, rabbit, cat, goat, sheep, goat, deer, bear, tiger, lion, monkey, gorilla, chimpanzee, wolf, fox, and mouse. It is characterized in that at least one selected from the group.

In one embodiment of the present invention, it is characterized in that it further comprises the step of removing cells remaining in the organ tissue after recovering the organ tissue of the mammal in step (a).

In one embodiment of the present invention, sterilizing the decellularized organ tissue support of step (a) by exposure to gamma irradiation, electron beam irradiation, or ethylene oxide It characterized in that it further comprises.

In one embodiment of the present invention, the tissue cells of step (b) are mesenchymal stem cells, adult stem cells, bone-marrow stem cells, and embryonic stem cells. It is characterized in that it is a stem cell selected from the group consisting of cells (Embryonic Stem Cells).

In addition, the present invention provides an artificial organ support using the organ tissue of an animal prepared according to the above method.

The artificial organ scaffold using the organ tissue of an animal manufactured by the method of the present invention can be safely used for tissue regeneration and healing than materials manufactured from existing artificial synthetic materials, has high biocompatibility, and can significantly reduce environmental pollutants. In addition, it is useful because it can contribute to promoting public health care business and improving public welfare by providing high-quality biomaterials compared to institutional replacements using existing artificial materials.

1 is a graph showing the results of measuring IL-2 and IFN-gamma through ELISA for a blood sample of a beagle dog after transplanting a pig organ into an organ of a beagle dog.
2A is a photograph of an organ of a normal pig (three-way hybrid, wild type) after transplanting the organ of a beagle dog and observing the organ of a beagle dog with an endoscope.
FIG. 2B is a photograph of an organ of a transgenic pig (GT-KO) with suppressed immune rejection reactions transplanted into an organ of a beagle dog, and then the organ of the beagle dog was observed with an endoscope.
Figure 3 is a photograph of tissue observation by cutting the tissue after transplanting the organ tissue of a pig into an organ of a beagle dog.
4 is a diagram showing the results of genetic analysis by collecting tissue from a transplant site in order to confirm the organ transplantation by the insertion of foreign xenogenes in the organ transplantation of a pig organ tissue in a beagle dog.

Hereinafter, the present invention will be described in detail.

The present inventors made artificial organs using organs extracted from pigs as a raw material, and applied fibrin and hyaluronic acid synthetic gel to the surface of the organs until the normal respiratory mucosal epithelium grows and enters. And the formation of granulation tissue was to be suppressed, and the present invention was completed by developing a multi-membrane artificial organ made of a porous membrane by using a cell injection method for decellularization and recellularization of heterogeneous bronchi.

Accordingly, an object of the present invention is to provide an artificial organ having a multi-membrane structure and a method of manufacturing the same through the method of decellularizing porcine organ tissue and applying a fibrin/hyaluronic acid synthetic gel and injecting cells.

In addition, the present invention provides an artificial organ support using animal organ tissue, which is a material for organ transplantation with high biocompatibility.

According to an aspect of the present invention, the present invention provides a method of manufacturing an artificial organ support using an animal organ tissue comprising the following steps.

(a) recovering the organ tissue of a mammal and then decellularizing it to prepare a support for the organ tissue; And (b) transplanting the tissue cells to the decellularized organ tissue support of step (a) and recellularization.

Hereinafter, the present invention will be described in detail according to each step of the present invention.

(a) After recovering the organ tissue of a mammal, decellularization is performed to prepare a support for the organ tissue.

Organ tissues usable in the present invention are selected from the group consisting of pigs, horses, cows, dogs, rabbits, cats, sheep, goats, goats, deer, bears, tigers, lions, monkeys, gorillas, chimpanzees, wolves, foxes, and mice. Organ tissue of mammals, but is not limited thereto.

In the present invention, the method of recovering the organ tissues of pigs is collected by a sterile surgical method, and the age and size of the pigs at the time of recovery are not particularly limited.

According to a preferred embodiment of the present invention, the present invention further comprises the step of removing the cells remaining in the recovered pig organ tissue after recovering the pig organ tissue. The remaining cells are, for example, organic substances such as epithelial tissue, connective tissue, muscle tissue, nerve tissue, cartilage tissue, etc. that can remain in the organ tissue of the pig. The removal of the foreign matter may be largely a mechanical method such as a method using detergents, a chemical method, a protein decomposition method, and a combination thereof, but is not limited thereto.

In order to maintain the physiological properties of the extracellular matrix to be used as a biomaterial, a manufacturing method must be applied to retain as much as possible the three-dimensional structure of the extracellular matrix and physiologically active substances. Important steps in the manufacturing process are technologies that remove cells from pig tissues (decellularization) and sterilization techniques to develop biomaterials from which antigenicity that can cause immune rejection reactions has been removed. Methods for removing cells and antigenic substances from tissues include peracetic acid, hypertonic solution or stock solution treatment, digestive enzymes such as trypsin or chollagenase, detergents such as sidium deoxycholate solution or Triton X-100, and protein digestion methods. It is a combination method, but is not limited thereto.

Unlike conventional methods, pig organ-derived biomaterials are made from extracellular matrix derived therefrom by culturing chondrocytes separated into cartilage tissue at high density. Unlike conventional materials that only use extracellular matrix, the extracellular matrix secreted by cells is collected, then decellularized and sterilized, so that decellularization efficiency can be increased and damage to the extracellular matrix can be minimized. Maintain the physiological activity of the vagina as much as possible.

Sterilization and disinfection methods for removing tissue toxins, viruses, and bacterial DNA from the prepared pig's decellularized organ tissue scaffold include gamma irradiation, electron beam irradiation, and ethylene oxide. ) Including methods by exposure, etc.

Tensile test, residual stress test, bending stiffness test of organ cartilage tissue, solubility test of tracheal cartilage tissue, or scanning electron Includes scanning electron microscope (SEM) inspection.

Step (b): recellularization by transplanting tissue cells to the decellularized organ tissue support of step (a)

Cells for re-cells of the decellularized organ tissue scaffold of pigs prepared in the present invention include all mammalian mesenchymal stem cells, adult stem cells, and bone marrow stem cells, including humans. (Bone-Marrow Stem Cells) and embryonic stem cells (Embryonic Stem Cells), including all tissue cells, including stem cells derived from living organisms.

To prepare the target tissue for recellularization on the prepared pig's decellularized organ tissue support, the recipient's organ mucosa was cut into small sections, washed with phosphate buffered saline (PBS), and 0.2% Protease (Sigma). -Aldrich, St Louise, MO, USA) and. Treated with 0.2% Collagenase P (Boehringermannheim, Indianapolis, IN).

The isolated cells were added to Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY, USA) medium containing 10% FBS (Fetal bovine serum, Life Technologies, Grand Island, NY, USA) and then put in trypan blue ( Life Technologies, Grand Island, NY, USA) and counted with a hemocytometer. After that, put 5×10 ⁶ cells/25 cm ² in the flask together with the medium, incubate in a 37°C incubator (95% air, 5% CO ₂ ), change the medium every 2-3 days, and change the medium for about 4-5 days. Passage to.

Bronchial endothelial cell transplantation (seeding) is carried out by the manual seeding (static seeding and gravity seeding) technique to separate and culture the recipient's bronchial mucosal epithelial cells aseptically prepared porcine bronchial graft material (seeding).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Cutting and decellularization of porcine organ tissue

Organ tissues of general pigs (Farm Story Hanneng Co., Ltd., Chungju, Chungbuk, Samwon hybrid, wild type) and transformed pigs (GT-KO, National Institute of Livestock Science) with suppressed immune rejection reactions were treated with sterile surgical instruments. Cleanly remove the connective tissues and muscles attached to the organ tissues of the body. Thereafter, the connective tissue and the organ tissue from which the muscle has been removed are washed twice in sterile distilled water. Soak the prepared tissue in 4% sidium deoxycholate solution for about 4 hours to remove the cells remaining in the organ tissue. Afterwards, washing is performed several times aseptically using sterile distilled water. As a treatment for removing cell nuclei and DNA ^{, the prepared tissue is immersed in 1M NaCl +} DNAse I solution for about an hour. Organ tissues with only cartilage left in the culture medium are stored in a refrigerator or frozen at -80℃. Gamma irradiation was performed to remove tissue toxins, viruses, and bacterial DNA from the prepared pig's decellularized organ tissue scaffold.

Example 2: Recellularization of pig organ tissue scaffolds

In this study, tracheal mucosa cells of a 10 kg beagle dog (Orient Bio Co., Ltd., Seongnam, Gyeonggi Province, Beagle), which are recipient animals, were used. The tracheal mucosa was cut into 2 x 2 cm in size, washed with phosphate buffered saline (PBS), 0.2% Protease (Sigma-Aldrich, St Louise, MO, USA) and 0.2% Collagenase P (Boehringermannheim, Indianapolis). , IN, USA), the cells isolated after treatment were Dulbecco's modified Eagle medium (DMEM) containing 10% FBS (Fetal bovine serum, Life Technologies, Grand Island, NY, USA) (Life Technologies, Grand Island, NY, USA), and then stained with trypan blue (Life Technologies, Grand Island, NY, USA), and counted with a hemocytometer. 5×10 ⁶ cells/25 cm ² were added to the flask with the medium and cultured in a 37°C incubator (95% air, 5% CO ₂ ).

Conventional MTT using the principle that yellow dye (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) turns dark blue by succinate dehydrogenase of living cell mitochondria to measure cell viability. assay was used. The monolayer cultured chondrocytes were removed using 0.05% trypsin, and the viability of the cells was checked with trypan blue ^{, adjusted to 2×10 4} cells per well, and cultured in a 96 well plate for one day. MTT (Methylthiazol tetrazolium 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) dye (Sigma-Aldrich, St Louise, MO, USA) was added at a concentration of 10%, and an incubator (37℃, 5 % CO ₂ ) After 4 hours incubation, the cells were dissolved in a buffer solution and absorbance was measured at a wavelength of 540 nm. Afterwards, pigs (Samwon hybrid, wild type) and transgenic pigs with suppressed immune rejection reactions (GT-KO) prepared aseptically prepared bronchial mucosal epithelial cells of beagle dogs separated and cultured by manual seeding (static seeding and gravity seeding) technique. Cells were transplanted to the bronchial graft material. The separated and cultured cell suspension was applied on the lumen and outer surfaces of the graft material using a pipette, and then the graft material was placed in a media Petri dish and incubated for several minutes after incubation to allow cell adhesion.

Example 3: Animal experiments using artificial organs

Transplant materials made using organ tissues of pigs (three-way hybrids, wild type) and transgenic pigs (GT-KO) with suppressed immune rejection reactions were tested in animals to evaluate the in vivo immune rejection reaction, stability and efficacy. The organ tissues of pigs from which the internal mucosa tissue was removed, leaving only cartilage, was prepared by separating and culturing the mucosal cells of the beagle dog, which is the target animal for transplantation, followed by cell transplantation, and stored in an incubator until transplantation. For the recipient animal, 3 10kg beagle dogs were prepared and splenectomy was performed 7 days before the transplant.

The operation was performed as follows. General anesthesia was performed by injecting 0.7 ml of tiletamin/zolazepam and 0.2 ml of xylazine per 1 kg of the body weight of the experimental animals. After completely removing the hairs of the anterior neck with a hair removal agent, it was corrected by lying on the operating table. After disinfecting the surgical site with 70% isoprophyl alcohol and betadine, a disinfectant cloth was applied. When the anesthesia was deep or breathing was not smooth, a tube was intubated into the trachea orally to maintain breathing with an AMBU bag. After the beagle dog's neck is extended in a supine position to ensure sufficient visibility in the foreground, 2% xylocaine is injected locally, and then the skin under the thyroid cartilage is removed by vertical incision in a size of 4 cm, and the subcutaneous tissue and the dural fascia are removed. The strap muscle was exposed. While paying attention to the damage to the internal jugular vein and carotid artery, the supernatant was exfoliated to expose the trachea, and a 2cm long tracheal tissue was excised in the longitudinal direction from the position of the 3-5th trachea to induce a defect. A pre-prepared bronchus was placed on the defect of the organ of the beagle dog, and a simple continuous suture was performed on the peritracheal tissue using 5-0 Maxon. Fibrin-glue was applied over the suture to prevent air leakage. After sufficiently disinfecting the wound area and suturing the enlarged muscle, the skin incision was sutured using 3-0 Maxon. The wound area was disinfected every day for one week after surgery, and antibiotics and analgesic anti-inflammatory drugs were administered.

Example 4: Evaluation of immune rejection and inflammatory response through blood test

Blood was collected from the jugular vein of beagle dogs before and after transplantation of artificial organ tissues on the 7th and 28th days, and hematology and blood chemistry tests were performed. The amount of inflammatory cytokines in blood was measured by ELISA, immune cell infiltration into the organ tissue transplant site was measured by H & E staining, and the type of immune cells infiltrated through immunostaining was analyzed. The numerical values of all test items obtained in the experiment were statistically compared and analyzed using the one-way ANOVA method.

Example 5: Evaluation of intratracheal inflammatory response of beagle dogs through tracheal endoscopy

On the 28th, 60th, and 90th days after surgery on a beagle dog, 100mg (2ml) and 24mg (1ml) of ketamine chloride (Ketamine chloride, Yuhan, Seoul, Korea) and xylazine chloride (Beagle Korea, Seoul, Korea) were administered. After mixing and intramuscular injection into the thigh of the left leg to induce anesthesia, an endotracheal tube (No. 7) was intubated. After that, while maintaining anesthesia with an inhalation anesthetic, a bronchoscopy was performed by inserting an endoscope through an endotracheal tube.

As a result, on the 28th day (POD28, Fig. 2A) after the artificial organ transplantation surgery, the organ tissues (matrix) of the transplanted pig (three-way hybrid, wild type) and the transformed pig (GT-KO) with suppressed immune rejection reaction It could be observed in the lumen of the trachea of the beagle dog, but granulomas and crust formation were not observed severely. In the examination at 60 days after surgery (POD 60, FIGS. 2A and 2B), organ grafts of pigs (three-way hybrids, wild type) and transgenic pigs (GT-KO) with suppressed immune rejection reactions were compared to the anterior organ matrix of beagle dogs. It was observed that mucous membrane regeneration was in progress on the surface of, and in the examination on the 90th day (POD 90, FIGS. 2A and B), it was confirmed that mucosal regeneration was completed and the graft site was not well distinguished. It was confirmed that the tracheal mucosa of the beagle dog was completely restored on the 90th day (POD 90) after the operation (FIGS. 2A and 2B).

Example 6: tissue examination

After transplanting the organ tissue of the pig into the organ of a beagle dog, on the 28th, 60th, and 90th day, after sacrifice according to the regulations of the animal experiment, the tissue was excised from the lower part of the cricoid cartilage to the lower part of the matrix graft part, including enough normal tracheal rings. . Thereafter, the tissue was stained with H&E (hematoxylin-eosin) to check the condition of the graft junction, the degree of regeneration of the mucous membrane, and the inflammatory response, and the microscopic morphological changes and the presence of mucosal regeneration were observed by imaging with an electron microscope (Fig. 3). .

Example 7: DNA test of pig-derived organs

In order to confirm that the organ of the beagle dog was derived from a pig, the tissue from the transplant site was collected and genetic analysis was performed.

Specifically, a solution (100 mM NaCl, 10 mM Tris·Cl, 25 mM EDTA, 0.5 % SDS, 0.1 mg/ml proteinase K), pulverized, and incubated overnight at 37°C. The same amount of phenol chloroform (Phenol chloroform, Sigma, USA) was added to the grinding solution. Isopropyl alcohol in the same amount as the aqueous layer was added, followed by centrifugation (2000 g) for 5 minutes. The pellet was washed in 70% ethanol and then centrifuged (200g) for 5 minutes. DNA was dissolved with distilled water, and genetic testing was performed using the primers shown in Table 1 below, and 16s ribosomal RNA of pigs and cytochrome b DNA of beagle dogs were tested.

primer order Dog (cytochrome b) forward: 5′-CCT TAC TAG GAG TAT GCT TG-3′ (SEQ ID NO: 1)
reverse: 5'-TGG GTG ACT GAT GAA AAA G-3' (SEQ ID NO: 2) Pig (16S)
forward: 5′- CAA CCT TGA CTA GAG AGT AAA ACC-3′ (SEQ ID NO: 3)
reverse: 5'- GGT ATT GGG CTA GGA GTT TGT TAT TAG T-3' (SEQ ID NO: 4)

As a result, it was confirmed that all beagle dogs survived during the experiment, and that the surgical site healed well without organ stricture or necrosis. In endoscopy, there was a white xenograft on the surface of the airway epithelial cells 28 days after transplantation, but it was confirmed that the mucous membrane was covered over the transplantation 60 days after the transplantation, and 90 days after the transplantation, the airway epithelium was confirmed to be regenerated normally. . From the results of the PCR test, it was possible to confirm the organ components of the pig in the organ of the beagle dog transplanted with the organ tissue of the pig (Fig. 4).

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Method of preparing artificial tracheal scaffolds using animal <130> PN1806-193 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cytochrome b forward primer <400> 1 ccttactagg agtatgcttg 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> cytochrome b reverse primer <400> 2 tgggtgactg atgaaaaag 19 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 16S forward primer <400> 3 caaccttgac tagagagtaa aacc 24 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 16S reverse primer <400> 4 ggtattgggc taggagtttg ttattagt 28

Claims (2)

Recovering the trachea tissue of the pig, removing the cells remaining in the trachea tissue, decellularizing the tissue to irradiate the supporter of the trachea tissue with the gamma ray only;
Applying a fibrin and hyaluronic acid synthesis gel to the surface of the support of the organ tissue irradiated with the gamma rays; And
Implanting tissue cells into an organ tissue support having only cartilage, to which fibrin and hyaluronic acid synthesis gel are applied, to recellularize the tissue,
The method of claim 1,
The organ mucosal tissues of the dogs were cut into pieces and then washed with phosphate buffered saline (PBS), treated with 0.2% Protease and 0.2% Collagenase P, and the treated dog's mucosal tissues were treated with 10% FBS (Fetal bovine The cells were cultured in an incubator at 37 ° C, 95% air, 5% CO 2 at 37 ° C, subcultured for 4-5 days, and cultured for 2-3 days in a Dulbecco's modified Eagle medium (DMEM) , And the cultured dog tracheal mucosal cells are seeded on the tracheal tissue support with the fibrin and hyaluronic acid synthesis gel applied thereon to the cartilage support by passive seeding technique. A method for manufacturing an engine support.
An artificial organ support prepared by the method of claim 1.

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