KR102171320B1 - A method for decellularizing of a tracheal mucosa tissue - Google Patents

Abstract

The present invention relates to a method for decellularizing tracheal mucosa tissue and an artificial organ mucosa sheet prepared therethrough. The method for decellularizing tracheal mucosa tissue of the present invention physically separates only tracheal mucosa tissue from which cartilage has been removed by freezing and thawing entire organ tissue, and treating a solution containing disodium carbonate and hydrogen peroxide in a short time. It is possible to minimize damage to the extracellular matrix while removing the cells of the organ mucosal tissue, so it can be usefully used in the reconstruction treatment for organ defects.

Description

A method for decellularizing of a tracheal mucosa tissue}

The present invention relates to a method for decellularizing tracheal mucosa tissue and an artificial organ mucosa sheet prepared therethrough.

An organ is a ring-shaped air passage that connects the mouth, nose, and lungs responsible for oxygen exchange from the outside, and consists of cartilage and mucous tissues. Among organ tissues, the mucous membrane is composed of pseudostratified ciliary columnar epithelium, has a submucosal structure in the lower part, and the respiratory mucosa secretes mucus to achieve a humidifying effect. It has a role to play.

Organ defects are divided into total defects due to congenital causes, partial defects due to tumors and trauma, depending on the cause of the onset, and treatment for airway defects varies depending on the cause of occurrence and the patient's age and condition, but generally the size of the damaged area, especially the length Depends on Among the types of organ defects, there are no treatments other than tracheal transplantation, so it is difficult to obtain a donor, and complications and mortality are high due to the occurrence of airway obstruction due to an immune response. In addition, partial organ defects are caused by trauma, tumors, tracheostomy, and the like, and up to now, short-stage anastomosis is known as the best treatment. However, these treatments shorten the entire organ length, which may cause rupture of the suture area during neck extension, requiring postoperative cervical fixation, and requires a detachment of the entire airway to relieve tension in the surgical site, which is highly likely to damage the occipital nerve. There is a problem.

In order to supplement the problems of existing treatments for organ defects, studies on reconstructing defects using decellularized biomaterials are being actively conducted. Decellularized biomaterials are tissues from which cells are removed through physical and chemical methods. There is no immune response, so there is no need to take immunosuppressants.Extracellular matrix (ECM) and growth factors are preserved to restore wound healing and tissue defects. Various studies are ongoing as a material to replace organ transplants. The most commercialized decellularized skin tissue as a decellularized biomaterial is a sheet type, and various products have been developed to facilitate storage in the operating room and easy to cut, so it can be used immediately for unexpected defects and is widely used in clinical practice. However, there is no commercially available decellularized mucosal tissue for reconstruction of mucosal defects, so decellularized dermal tissue is being used, but due to the different characteristics of the tissue, there is a limit to the treatment application.

As a decellularized biomaterial specific for organ reconstruction, a whole organ reconstruction method was attempted in which stem cells were cultivated in decellularized human organs and transplanted into patients. However, due to failure to regenerate the respiratory mucosa inside the organ, death due to airway obstruction and stenosis. Or taking an immunosuppressant has been reported.

Under this technical background, the present inventors have made diligent efforts to develop a biotransplant material that can be usefully used for treating organ defects, and as a result, developed a novel decellularization method using only organ mucosal tissues, and mucosal sheets manufactured through the method The present invention was completed by confirming that it contains an extracellular matrix and a degree of decellularization that is suitable for in vivo transplantation and useful for reconstruction treatment.

One object of the present invention is to provide a method for decellularizing organ mucosa tissue.

Another object of the present invention is to provide an artificial organ mucosa sheet prepared according to the decellularization method.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.

One aspect of the present invention for achieving the above object is a method for decellularizing tracheal mucosa tissue.

Specifically, the present invention

(a) treating the organ mucosal tissue with a solution containing a protease to separate the intercellular junctions in the tissue;

(b) removing cells in the tissue by treating the tissue of step (a) with a solution containing a surfactant; And

(c) It provides a method for decellularizing organ mucosa tissue comprising treating a solution containing disodium carbonate and hydrogen peroxide on the tissue from which the cells in step (b) have been removed.

Organ tissues are composed of cartilage and mucous tissues, and these are different in reactivity to decellularization agents depending on the density of the tissue, the presence of blood vessels, and the type of cells, so when decellularization is performed using the entire organ, the cells are removed. May be incomplete and there is a risk of damage to the extracellular matrix. Accordingly, in order to find a decellularization method suitable for organ mucosal tissue, experiments were performed by changing various variables such as treatment material, treatment sequence, concentration, temperature, time, etc., and the decellularization method of the present invention It is possible to minimize damage to the extracellular matrix while removing cells.

The method for decellularizing organ mucosa tissue according to the present invention includes the steps of (a) treating the organ mucosa tissue with a solution containing a protease to separate the intercellular junctions in the tissue. It is preferable that the organ mucosal tissue of step (a) is subjected to the step of freezing and then thawing the organ tissue before the solution treatment.

In the present invention, the organ mucosa tissue may be extracted from animals other than humans, and the animal may be a mammal or a bird. For example, mammals may be pigs, cattle, horses and sheep, and birds may be chickens, ducks, geese, and the like. In one embodiment of the present invention, organ tissue extracted from pigs was used. Extraction of the tracheal mucosa can be performed using various methods known in the art.

In the present invention, the organ mucosa tissue can be obtained by physically separating only the organ mucosa tissue from which cartilage has been removed by freezing and then thawing the extracted whole organ tissue. Since cartilage and mucous membranes in organ tissues have different reactivity to decellularization agents depending on the tissue density, the presence or absence of blood vessels, and types of cells, it is necessary to remove the cartilage in the organ tissues for efficient decellularization of the organ mucosa tissue. The present invention, which performs decellularization using only organ mucosal tissue, has the advantage of being able to reproduce the characteristics of organ mucosa tissue more similarly compared to the conventional decellularization method using whole organs.

The freezing of the organ tissue may be performed at -90 to -70°C, preferably -85 to -75°C, most preferably -80°C for 48 to 72 hours, and thawing is 15 to 35°C, preferably May be carried out at 20 to 30°C, most preferably at 25°C for 0.5 hours.

In one embodiment of the present invention, the organ tissue extracted from the pig was frozen at -80°C and thawed at 25°C, and then the cartilage tissue was physically removed to separate only the organ mucosa tissue.

In addition, the obtained organ mucosa tissue can be subdivided into a size sufficient to perform a decellularization process. A method known in the art may be used for the subdividing method.

The protease of step (a) may be dispase, trypsin, or any combination thereof, but is not limited thereto, and preferably trypsin. In the case of treatment with trypsin in step (a), a mixed solution with ethylenediaminetetraacetic acid (EDTA) may be used for effective separation of intercellular junctions in organ mucosal tissues.

The step (a) may be performed at 30 to 40° C., and when performed within the temperature range, the intercellular junction in the organ mucosa tissue may be efficiently separated. In performing step (a) according to the present invention, when performed at 30° C. or less, the activity of proteolytic enzymes decreases and may cause degeneration of mucous membrane tissue. Conversely, when performed at 40° C. or more, the activity of the enzyme There may be a problem of deterioration.

When the tissue is processed through the step (a), a step of washing the treated tissue may be additionally performed. A distilled water solution may be used for washing the tissue, and washing may be performed while stirring using a stirrer while the treated tissue is added to the distilled water. The temperature at which washing is performed may be performed at a stirring speed of about 150 rpm at 30 to 40° C. so as to correspond to step (a). This washing step can be carried out two or more times under the same conditions.

Step (a) of the present invention may further include the step of treating the organ mucosal tissue treated with the protease with ethanol.

The ethanol treatment step may be performed at 2 to 10° C., and hydrogen peroxide may be further treated.

In an embodiment of the present invention, step (a) may be performed in a state of treating a mixed solution of trypsin and EDTA and rotating at a rotation speed of 150 rpm for 4 hours at 36.5°C.

The method for decellularizing organ mucosa tissue according to the present invention includes the step of removing cells in the tissue by treating the tissue in step (a) with a solution containing a surfactant.

Surfactants are substances that have amphiphilic properties and play a role in removing cells by dissolving cell membranes and nuclear membranes of tissues. The surfactant may be a nonionic surfactant, preferably Triton X-100 (Triton X-100).

The step (b) may be performed at 20 to 30° C., and when performed within the temperature range, cells in the organ mucosa tissue may be efficiently removed.

According to an embodiment of the present invention, step (b) may be performed in a state in which a solution containing Triton X-100 is treated and rotated at a rotation speed of 150 rpm for about 40 hours at 25°C.

The method for decellularizing organ mucosa tissue according to the present invention includes the step of (c) treating the tissue from which the cells of step (b) are removed with a solution containing disodium carbonate and hydrogen peroxide.

In the decellularization method of the present invention, the treatment of the solution containing disodium carbonate and hydrogen peroxide may be performed for the purpose of decellularization and disinfection of organ mucosa tissue. Preferably, the solution containing disodium carbonate and hydrogen peroxide may be a mixture of disodium carbonate and hydrogen peroxide in a volume ratio of 1:1 to 2, preferably in a volume ratio of 2:3, more preferably disodium Carbonate and hydrogen peroxide may be included in a volume ratio of 2:3, and citric acid and sodium carbonate may be further included, but the present invention is not limited thereto.

In addition, the solution containing disodium carbonate and hydrogen peroxide is not limited thereto, but may be commercially available peracid-based disinfectants or sterilizing agents, and in one embodiment of the present invention, 2:3 Decellularization was performed by treatment with Perasafe™ (Rely+On Perasafe; DuPont, Wilmington, DE, USA) containing disodium carbonate/hydrogen peroxide, citric acid and sodium carbonate in a volume ratio.

The solution containing disodium carbonate and hydrogen peroxide is excellent in decellularization of organ mucosa compared to conventional formulations used for decellularization of organ tissues, and the degree of preservation of extracellular matrix and growth factors in organ mucosa tissue Has excellent characteristics. In addition, there is an effect that can simultaneously achieve decellularization and disinfection effects at low cost.

The step (c) may be performed at 20 to 30° C., and when performed within the temperature range, cells in the organ mucosa tissue may be efficiently removed.

According to an embodiment of the present invention, step (c) may be performed in a state of rotating at a rotation speed of 150 rpm for 1 hour at 25° C. by processing Perasafe™.

In the decellularization method of the present invention, a tissue washing step may be performed before and after each step, and the tissue washing may be performed by treating distilled water and a solution containing an antibiotic therein.

In another aspect, the present invention provides an artificial organ mucosa sheet prepared by the decellularization method.

The artificial organ mucosa sheet according to the present invention exhibits a degree of decellularization suitable for transplantation in a living body and minimizes damage to the extracellular matrix, so that it can be easily used for reconstruction for the treatment of organ defects.

In the present invention, the tracheal mucosa sheet has characteristics similar to that of the tracheal mucosa, and is used as a term meaning a biological scaffold or a support used for the reconstruction treatment of an organ defect. The sheet may further include a cell layer provided for transplantation as a substitute for the organ mucosa. The term "organ mucosa sheet" may be used interchangeably with "artificial organ mucosa sheet".

The tracheal mucosa sheet according to the present invention can be used as an implant material (replacement of tracheal mucosa) for patients whose organs are damaged or missing. At the time of transplantation, it is preferable to use a surgical suture to fix the graft to surrounding tissues to promote survival. Provides a material for transplantation (tracheal mucosa-like sheet) applicable to patients with organ defects requiring organ mucosa transplantation. The biological tissue sheet of the present invention can be used for regeneration (reconstruction) of organ mucous membranes and the like, and, for example, the biological tissue sheet of the present invention can be directly implanted into a defective part of an organ.

The thickness of the mucous membrane sheet may be 350 to 550 μm.

The decellularization method for preparing the artificial organ mucosa sheet of the present invention is as described above.

The method for decellularizing tracheal mucosa tissue of the present invention physically separates only tracheal mucosa tissue from which cartilage has been removed by freezing and thawing entire organ tissue, and treating a solution containing disodium carbonate and hydrogen peroxide in a short time. It is possible to minimize damage to the extracellular matrix while removing the cells of the organ mucosal tissue, so it can be usefully used in the reconstruction treatment for organ defects.

1 is a classification of five decellularization protocols designed in Examples of the present invention and a control (Native, original tissue) and experimental group (freeze-thaw only FT group; wFTP, mFTD and mFTP group subjected to a decellularization process) Is shown.
Figure 2 shows the organ mucosa sheet of the pig produced by the decellularization method of the present invention.
Figure 3 shows the dsDNA content of the decellularized organ mucosa sheet prepared according to the present invention.
4 shows the result of confirming the degree of nuclear inclusion in the sheet using the H&E staining result of the mFTP group among the decellularized organ mucosa sheets prepared according to the present invention.
5 shows the result of confirming the degree of nuclear inclusion in the sheet using the H&E staining result of the decellularized organ mucosa sheet prepared according to the present invention.
Figure 6 shows the proteoglycan and collagen content of the mucosal sheet of the decellularized organ prepared according to the present invention, immunohistochemical analysis results. In Safranin O staining, blue means collagen, and in other staining results, dark brown means positive (Scale bar=100 μm).
7 shows the staining results for the leukocyte common antigen CD45 of the mucosa of the decellularized organ prepared according to the present invention.
8 shows the results of analyzing the collagen content in the mucosa of the decellularized organ prepared according to the present invention.
9 shows the results of SEM analysis of the lumen and cross sections of mucous tissues in order to observe the morphological characteristics of the decellularized organ mucosa prepared according to the present invention (Scale bar = 1 μm).
10 and 11 show the results of analyzing the cytotoxicity and biocompatibility of the original tissue and the decellularized organ mucosa prepared according to the present invention.

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. .

The Wilcoxon signed-rank test was used to confirm the difference in dsDNA weight, collagen content, cell viability, and proliferation results between the experimental groups, and p<0.05 was considered statistically significant. SPSS software (v19; IBM Crop., Armonk, NY, USA) was used for statistical analysis, and all data are expressed as mean ± standard deviation.

Example 1. Preparation of organ mucosa tissue

In order to prepare the tracheal mucosa tissue subject to decellularization of the present invention, a trachea of 15 to 20 cm from under the larynx to the carina is filled with ice at a meat processing company (Forkland, Cheonan). It was purchased and used while stored in an ice box.

The purchased pig organ tissue was immersed in a solution containing 25 μg/mL of 1% Antibiotic-Antimycotic (Gibco, Grand Island, NY, USA) and stirred at 150 rpm overnight to remove plasma, debris and microorganisms.

The tracheal mucosa was then divided into the following five groups according to the decellularization protocol. The organ mucosa tissue obtained by physically removing cartilage tissue after organ washing was defined as the Native group. All organs of the group except for the Native group were frozen at -80℃ and then thawed in a warm bath at 25℃ for 30 minutes. It was rough. The decellularization protocol used in the present invention is shown in FIG. 1.

1. Trachea mucosa separated from cartilage after trachea cleaning: Native

2. Tracheal mucosa separated from cartilage after freezing (Thawing): FT

3. Decellularization with Perasafe-based surfactant-enzyme-containing solution after freeze-thaw and before tracheal mucosa separation: wFTP

4. Decellularization with DNase-based surfactant-enzyme-containing solution after freeze-thaw, separation of organ mucosa: mFTD

5. After freeze-thaw, separation of tracheal mucosa, and decellularization with Perasafe-based surfactant-enzyme-containing solution: mFTP

Example 2. Decellularization of organ mucosa tissue

2-1. Decellularization Protocol Processing Sequence-FTP Group ( wFTP , mFTP )

In the decellularization step, wFTP and mFTP groups using a Perasafe-based surfactant-enzyme-containing solution were subjected to decellularization in the same manner except for the order of the decellularization protocol (applying the decellularization protocol before separation of the organ mucosa from the organ: wFTP After separating the tracheal mucosa from the trachea, applying a decellularization protocol: mFTP). The organ mucosa tissue of the pig obtained in Example 1 was immersed in distilled water and washed at 4° C. for 30 minutes, and this was repeated twice. The washed tissue was treated with a mixed solution containing 0.25% trypsin and 0.05% EDTA (Gibco) for 4 hours at 36.5° C., and washed twice with distilled water for 30 minutes and 1 hour each. Next, the tissue was treated with 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) overnight and 24 hours at 25° C., and twice each with distilled water for 30 minutes and 1 hour. Washed repeatedly. Next, the tissue treated with Triton-X 100 was treated with a Perasafe-based surfactant-enzyme-containing solution, specifically, a solution containing disodium carbonate and hydrogen peroxide, citric acid, and sodium carbonate in a volume ratio of 2:3 (Perasafe™) for 1 hour. After treatment, it was washed twice with distilled water for 30 minutes and 1 hour. The washed tissue was stored on PBS containing 1% Antibiotic-Antimycotic solution as an antibiotic at 4°C. All the above processes were carried out under stirring at a speed of 150 rpm at 25°C (VWR International, Pittsburgh, PA, USA).

2-2. Decellularization Protocol Treatment Solution- mFTD group

The mFTD group was decellularized in the same order as the mFTP group of Example 2-1, except that a DNase-based surfactant-enzyme-containing solution was used in the decellularization step (after separation of the organ mucosa from the organ. Decellularization protocol applied). The organ mucosa tissue of the pig obtained in Example 1 was immersed in distilled water and washed at 4° C. for 30 minutes, and this was repeated twice. The washed tissue was treated with a mixed solution containing 0.25% trypsin and 0.05% EDTA (Gibco) for 4 hours at 36.5° C., and washed twice with distilled water for 30 minutes and 1 hour each. Next, the tissue was treated with 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) overnight and 24 hours at 25° C., and twice each with distilled water for 30 minutes and 1 hour. Washed repeatedly. The tissue treated with Triton X-100 was treated with a DNase-based surfactant-enzyme-containing solution, specifically DNase I (100 U/mL, Sigma-Aldrich) at 37° C. for 6 hours, and distilled water for 30 minutes and 1 hour Washing was repeated twice each. The washed tissue was stored on PBS containing 1% Antibiotic-Antimycotic solution as an antibiotic at 4°C. All the above processes were carried out under stirring at a speed of 150 rpm at 25°C (VWR International, Pittsburgh, PA, USA).

Example 3. Decellularized Agency Mucosal sheet Characteristics check

The characteristics of the organ mucosa sheet subjected to decellularization of Example 2 were confirmed.

First, as a result of measuring the size and thickness of the prepared mucous membrane sheet, the length was 9 to 10 cm, the width was 2 to 3 cm, and the thickness was confirmed to be 375 to 518.75 ㎛. 2 shows a mucosal sheet (mFTP) to which a decellularization protocol was applied after separating the organ mucosa from the organs of the prepared mucosal sheets.

3-1. DNA quantification

The DNA of the tracheal mucosa sheet prepared according to the present invention was isolated using the DNeasy Blood and Tissue Kit (Qiagen), and the total in the mucosa sheet according to the manufacturer's instructions using the Quan-iT Picogreen dsDNA Assay kit (Thermo Fisher Scientific). DNA content was analyzed. Specifically, the decellularized organ mucosa tissue of Example 2 and the non-decellularized organ mucosa native tissue were digested overnight at 65° C. with 100 μl of proteinase K, and the provided buffer and DNA was isolated using mini-columns. Then, the isolated DNA was quantified by measuring fluorescence emission at 520 nm using a Quan-iT Picogreen dsDNA assay kit (Thermo Fisher Scientific) and a microplate reader (Synergy H1; Biotek, Vermont, USA). The results are shown in FIG. 3.

As shown in Figure 3, the total amount of DNA in the original tissue of the organ mucosa that was not decellularized was 3373.8 ng/mL on average, whereas (Native: 3996.8 ± 1810.2 ng/mg, FT: 2524.8 ± 568.1 ng/mg, p< 0.01), the average of the total amount of DNA in the mucosal sheet decellularized according to the method of the present invention is 14.153 ng/mL, and the range is 1.329 to 31.704 ng/mL, and the DNA in the organ mucosa tissue according to the method of decellularization of the present invention Was remarkably removed, which corresponds to 50 ng/mL or less (Biomaterials, vol. 32, no. 12, pp. 3233-3243, 2011), which is a criterion for decellularization suitability of the scaffold for explantation. In particular, compared to the DNase-based surfactant-enzyme-containing solution (mFTD), the Perasafe-based surfactant-enzyme-containing solution has remarkably high decellularization efficiency (wFTP, mFTP), and further, after organ freeze-thaw, the decellularization solution In the mFTP group from which the tracheal mucosa was isolated before treatment, it was confirmed that dsDNA in the tracheal mucosa was most significantly removed.

3-2. Histological analysis

The degree of decellularization of the organ mucosa sheet prepared according to the present invention was confirmed using H&E staining. Specifically, the mucosa sheet prepared in Example 2 was fixed with a 10% formaldehyde solution (Sigma-Aldrich), inserted into paraffin, and cut into 4 μm-thick sections. Then, after deparaffining and dehydration of the section slide, stained with H&E (hematoxylin and eosin) and Masson's trichrome, the stained section was observed at 400 magnification with an optical microscope (Carl Zeiss, Gottingen, Germany) to confirm the degree of nuclear staining. The results are shown in FIGS. 4 and 5.

4 and 5, it was confirmed that the nuclei were not stained in the mucosal tissue decellularization group wFTP, mFTD or mFTP.

In addition, safranin-O staining was performed to measure the content of proteoglycan and collagen in the tissue. Further, for immunohistochemical analysis, each section after deparaffining and dehydration of the section slide was washed with PBS containing 0.05% Triton X-100 (PBST; Sigma-Aldrich), and 37° C. and a pepsin solution (GBI Labs , Bothell, WA, USA) incubated for 20 minutes and washed three times with PBST. Each section was first tested for collagen type I, collagen type IV, cytokeratin V, fibronectin, laminin, epidermal growth factor and CD45 (leukocyte common antigen) (Origene, Rockville, MD, USA). Incubated overnight at 4° C. with the antibody. After washing with PBST, each section was treated with a biotinylated secondary antibody (GBI Labs) for 1 hour and streptavidin-HRP (GBI Labs) for 1 hour, and then visualized using a DAB solution (GBI Labs). After staining, each section was mounted on a gelatin-coated slide to obtain an image using a bright field microscope (Optinity KCS-31S; Korea Lab Tech, Seoul, Korea) and OptiView 3.7 software (Korea Lab Tech). The results are shown in FIG. 6.

As shown in FIG. 6, it was confirmed through the safranin-O staining result that collagen fibers remained even after the organ mucosa tissue was decellularized according to the decellularization method according to the present invention. In addition, through the immunohistochemical staining results, it was confirmed that extracellular matrix (ECM) such as collagen type I, collagen type IV, cytokeratin V, fibronectin, and laminin were all present in the decellularized cell membrane. Vascular endothelial growth factor (VEGF) was also confirmed to be positive at the decellularized site.

In addition, the staining results for the leukocyte common antigen CD45 of the mucosa of the decellularized organ prepared according to the present invention are shown in FIG. 7. As shown in FIG. 7, it was confirmed that the mFTP group was CD45 negative, and showed very low antigenicity compared to the wFTP group treated with the FT group and perasafe before organ mucosa separation.

3-3. Collagen content analysis

The extracellular matrix content in the organ mucosa sheet prepared according to the present invention was measured. Specifically, the contents of the decellularized organ mucosa sheet of Example 2 and the collagen of the original tissue that were not decellularized were prepared according to the manufacturer's instructions using the Sircol Soluble Collagen Assay Kit (Biocolor, Cat. No S1000). Measured. First, 1 mg of a lyophilized sample was hydrolyzed with 1 ml of a pepsin solution (0.1 mg/mL pepsin in 0.5 M acetic acid) for 1 week at 4°C. After neutralization, collagen was separated using a separation and concentration reagent, and 100 μL of a sample was added to 10 mL dye, stirred for 30 minutes, and centrifuged at 19,000 g for 10 minutes. Dye was released from the pellet using an alkaline reagent, and absorbance at 555 nm was measured using a microplate reader (Synergy H1; Biotek, Vermont, USA). Absolute values were obtained using a standard graph containing collagen type I provided with the kit in the range of 5 μg to 100 μg per 0.3 mL. The results are shown in FIG. 8.

As shown in FIG. 8, it was confirmed that the collagen content in the organ mucosa sheet prepared according to the decellularization method according to the present invention did not show a significant difference compared to the control group in which decellularization was not performed (p>0.05). Through the above results, it was confirmed that the decellularization method according to the present invention effectively preserves the extracellular matrix that plays a supporting role in the growth and development of cells.

3-4. Electron microscope analysis

In order to observe the morphological characteristics of the mucosa of the mFTP group, the lumen and cross sections of the mucosal tissue were analyzed by SEM. The sample was fixed in 2.5% glutaraldehyde at room temperature for one day, washed with distilled water, and then dehydrated using 100% ethanol. The sample was coated with a platinum sputter-coater (208HR; Cressington Scientific Instruments, Watford, UK) and then mounted on an aluminum stub. Images were obtained using a field-radiation scanning electron microscope (FE-SEM; JSM-6700F; JEOL, Tokyo, Japan). The results are shown in FIG. 9.

As shown in FIG. 9, it was confirmed that the lumen of the mucosal tissue of the mFTP group showed a smooth surface, whereas the cross section showed a fibrous, porous structure, which was advantageous for cell adhesion.

3-5. Cytotoxicity, cell suitability analysis

To confirm the cytotoxicity of intact and decellularized tissues, tonsil-derived mesenchymal stem cells (TMSCs) and tonsil-derived mesenchymal stem cells (TMSCs) were used using Transwell inserts (Corning, Corning, NY, USA) with a pore size of 3 μm. The membrane was co-cultured. Specifically, TMSC was placed in the lower compartment and tracheal mucosal tissue was placed in the upper compartment, and cultured in Dulbecco's modified Eagle medium (DMEM), and DMEM medium was used as a negative control. The degree of TMSC proliferation on the 1st, 2nd, and 3rd days was measured with an EZ-Cytox cell viability assay kit (Daeil Lab Service, Seoul, Korea), and the cytotoxicity of each organ mucosal tissue was calculated using the following equation.

[Equation 1]

RGR (relative growth rate, relative cell growth) = (average O.D of each group) / (average O.D of negative control) × 100%

Cytocompatibility was evaluated by the method of Butler CR, et al (Biomaterials 2017; 124:95-105). Specifically, the original tissue and the decellularized tissue were cut into 5mm discs using a biopsy punch (Ellis Instrument, Madison, NJ, USA). Prior to seeding the cells, mucosal tissues were cultured in 96-well plates with the lumen side facing up and incubated overnight in 200 μL of DMEM. Thereafter, TMSCs were seeded at 1×10 ⁴ cells/well and left overnight. After moving the mucosal tissue to a new plate while maintaining the lumen side up, cell viability was measured on the 1st, 2nd, and 3rd days using an EZ-Cytox cell viability assay kit. The results are shown in FIGS. 10 and 11.

As shown in FIG. 10, the cytotoxicity of each group confirmed through RGR was not significantly different between the original tissue and the decellularized tissue on the 1st, 2nd, and 3rd days. On the other hand, as shown in Figure 11, as a result of confirming the cell viability of TMSC measured on the 1st, 2nd, and 3rd days, the decellularized mucosal tissue showed a higher TMSC proliferation rate than the original tissue (p<0.01), of which perasafe It was confirmed that the mFTP group treated after separation of the tracheal mucosa showed a remarkably high TMSC proliferation rate.

Example 4. In vivo Biocompatibility analysis

The in vivo biocompatibility of the organ mucosa sheet prepared by the tax saturation process according to the invention was analyzed. All experiments were conducted under the approval of the Animal Management Committee of Ewha Medical Research Institute. 18 C57BL/6 male mice (Orient Bio, Seoul, Korea) weighing 20-25 g were divided into 3 groups transplanted with SHAM (negative control), native mucosa, and decellularized organ mucosa tissue. Decellularized organ mucosal tissues include organ mucosa separated from cartilage after freezing-thawing (FT group), and organ decellularized with Perasafe-based surfactant-enzyme-containing solution after freezing-thawing, organ mucosa separation. Mucosa (mFTP group) was used.

All animals were placed in a light-dark cycle (12 hours) for at least 7 days before the experiment, and food and water were fed freely. All three groups made a 1cm vertical incision at 0.5cm away from the dorsal midline, and in the other two groups except for the SHAM group, the subcutaneous layer was separated on both sides to make a pocket, and then a dehydrated circular mucosal tissue with a diameter of 8mm was inserted. After 3 days, 2 weeks, and 5 weeks, the mice were sacrificed and the mucosa was recovered and histological analysis was performed. In the care and use of laboratory animals, the National Institute of Health (NIH)'s Care and Use of Laboratory Animals and the Ewha Institute of Medicine's animal testing guidelines were followed.

The recovered mucosal tissue was fixed in 4% paraformaldehyde solution (Biosesang, Seong-nam, Korea), and then inserted into paraffin and cut into 4 μm thick sections. Then after de-paraffin and the dehydrating of the segment slide H & E (hematoxylin and eosin) to the dye and the original tissue, and to a histological score that was implanted mucosal tissue and graded as shown in Table 1 in after 3 days, 2 weeks, 5 weeks vivo Biocompatibility was analyzed.

Score 3 2 One 0 Day 3 Cellular infiltration (40x) >150 cells 75-150 cells 1-75 cells 0 cells Degradation No scaffold present Some scaffold present Mostly present No degradation Encapsulation No encapsulation Minimal encapsulation Moderate encapsulation Dense encapsulation Inflammatory cell aggregation (40x) 0 cells 1-75 cells 75-150 cells >150 cells Day 14 Cellular infiltration (40x) >150 cells 75-150 cells 1-75 cells 0 cells Collective tissue organization Highly organized
connective tissue
present Moderately organized
connective tissue
present Unorganized connective tissue throughout disrupted original scaffold Original scaffold intact Degradation No scaffold present Some scaffold present Mostly present No degradation Encapsulation No encapsulation Minimal encapsulation Moderate encapsulation Dense encapsulation Multinuclear Giant Cell (40x) 0 cells 1 cell 2-5 cells >5 cells Blood vessel distribution
(vascularity, 40x) >10 vessels 6-10 vessels 2-5 vessels 0-1 vessel Day 35 Collective tissue organization Highly organized
connective tissue
present Moderately organized
connective tissue
present Unorganized connective tissue throughout disrupted original scaffold Original scaffold intact Degradation No scaffold present Some scaffold present Mostly present No degradation Encapsulation No encapsulation Minimal encapsulation Moderate encapsulation Dense encapsulation Blood vessel distribution
(vascularity, 40x) 0 cells 1 cell 2-5 cells >5 cells

As a result, it was confirmed that the mFTP group treated with perasafe after tracheal mucosal separation of the decellularized group showed 14.5 points and 21.5 points in the frozen and thawed FT group, indicating that the mFTP group showed remarkably excellent biocompatibility. In particular, it was confirmed that acute inflammatory cell aggregation appeared more in the original tissue transplant group on the third day.

From the above results, in the method of decellularizing the organ mucosa, after washing the organ tissue, the cartilage tissue is physically removed to obtain the organ mucosa, and the mucous tissue is frozen at -80°C and thawed at 25°C. , After separation of organ mucosa, it was confirmed that the biocompatibility of the decellularized mFTP group was most excellent by treating a Perasafe-based solution containing disodium carbonate and hydrogen peroxide.

The above results show that as the decellularization method of the present invention optimizes the treatment material and the treatment sequence, it is possible to minimize damage to the extracellular matrix while removing cells of the organ mucosa tissue within a short time, and the artificial organ mucosa prepared therefrom This suggests that the sheet can be usefully used in reconstruction treatment for organ defects.

As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (11)

(a) after trachea washing, freezing and thawing;
(b) obtaining organ tissue by physically removing cartilage from the organ of step (a);
(c) treating the tissue of step (b) with a solution containing a protease to separate the intercellular junctions in the tissue; And
(d) treating the tissue of step (c) with a surfactant solution containing disodium carbonate, hydrogen peroxide, citric acid, and sodium carbonate to decellularize the tissue. The method of claim 1, wherein the trachea in step (a) is extracted from pigs. delete delete The method of claim 1, wherein the proteolytic enzyme in step (c) is at least one selected from the group consisting of dispase and trypsin. The method of claim 1, wherein the step (c) is performed at 30 to 40°C. The method of claim 1, further comprising: decellularizing by treating Triton X-100 before treatment with the surfactant solution in step (d). The method of claim 1, wherein disodium carbonate and hydrogen peroxide in the surfactant solution of step (d) are mixed in a volume ratio of 1:1 to 2, decellularization of organ mucosa tissue. delete The method of claim 1, wherein the step (d) is performed at 20 to 30°C. An artificial organ mucosa sheet prepared by the decellularization method of any one of claims 1 to 2, 5 to 8, and 10.

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