WO2022124847A1 - Composition for regenerating tissue, and method for producing same - Google Patents

Abstract

The present invention relates to a composition for regenerating a tissue, and a method for producing same, the composition comprising an undifferentiated fat tissue extract and cell-culture-derived extracellular matrix powder.

Description

Composition for tissue regeneration and manufacturing method thereof

This specification claims the benefit of the filing date of Korean Patent Application No. 10-2020-0173573 filed with the Korean Intellectual Property Office on December 11, 2020, the entire contents of which are incorporated herein by reference.

The present invention relates to a composition for tissue regeneration and a method for preparing the same.

Burns can be classified into scalding burns, contact burns, flame burns, flash burns, chemical burns, electrical burns, and radiographic burns according to the damage mechanism. Deep second- and third-degree burns, in which most of the protective epithelial layer and almost all dermal layers are destroyed, can rapidly pick up bacteria and cause infection.

Prevention of infection in burns is usually an important issue in the care of burn patients. In most burns, the infection can rapidly progress to life-threatening sepsis if left untreated. Therefore, prompt treatment of burn lesions is a problem, and U.S. Patent No. 3,761,590 describes a method for preparing a thick cream ointment containing silver sulfadiazine useful for conventional burn treatment.

However, these cream-based products are applied to the affected area using only sterilization technology. In addition, this type of drug administration operation is very time consuming, and the possibility of crosscontamination between patients due to multiple uses of the same container is conceivable. Cross-contamination normally discourages doctors from prescribing this product to patients, and after each treatment it is used only clinically rather than at home. Therefore, there is a risk that the patient may be exposed to infection during the new application of this cream, even if the burn has healed. Accordingly, there is a need in the art to develop inexpensive, less painful, easy-to-use, transparent, non-cross-contamination formulations for the treatment of burns.

That is, the current treatment material for burns has not been developed other than the dressing, and there is no treatment method other than a surgical method waiting for natural healing by covering the dressing while removing the scaly that is continuously formed. Therefore, in the treatment of burns, there is a need to develop a therapeutic agent for burns that is excellent in preventing infection and regenerating the skin.

An object of the present invention is to provide a composition for tissue regeneration capable of treating damaged or lost tissue, specifically, damaged or lost skin tissue, and a method for preparing the same.

One embodiment of the present invention, micronized (micronized) adipose tissue extract; And it provides a composition for tissue regeneration comprising a cell culture-derived extracellular matrix powder.

Another embodiment of the present invention, A) preparing an undifferentiated adipose tissue extract; B) preparing a cell culture-derived extracellular matrix powder; And C) mixing the undifferentiated adipose tissue extract and the cell culture medium-derived extracellular matrix powder; provides a method for producing a composition for tissue regeneration comprising a.

The composition for tissue regeneration according to the present invention has the advantage that effective regeneration of damaged or lost tissue can be made to help rapid recovery. In particular, the composition for tissue regeneration according to the present invention can effectively restore skin tissue damaged due to burns or the like. Furthermore, when the composition for tissue regeneration is applied as a spray, it is possible to prevent further infection of the affected area.

1 shows the results of treatment of the affected part of the pig to which the burn model according to the experimental example is applied.

In the present specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

In the present specification, when a member is said to be located “on” another member, this includes not only a case in which a member is in contact with another member but also a case in which another member is present between the two members.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention, micronized (micronized) adipose tissue extract; And it provides a composition for tissue regeneration comprising a cell culture-derived extracellular matrix powder.

The undifferentiated adipose tissue extract may serve to promote cell differentiation in the affected area. In addition, the cell culture-derived extracellular matrix powder can effectively perform the role of a substrate for cell differentiation in the affected area.

According to an exemplary embodiment of the present invention, the weight ratio of the undifferentiated adipose tissue extract and the cell culture medium-derived extracellular matrix powder may be 1:10 to 10:1. Within the weight ratio range, the composition for tissue regeneration effectively induces cell differentiation in the affected area, thereby effectively helping the regeneration of damaged or lost tissue.

According to an exemplary embodiment of the present invention, the undifferentiated adipose tissue extract may include adipose tissue-derived extracellular matrix that passes through a filter pore of 100 μm to 300 μm. Specifically, the undifferentiated adipose tissue extract contains the extracellular matrix of adipose tissue having a size in the range of 100 μm to 300 μm, which contains a number of factors necessary for tissue regeneration in the damaged or lost affected area, Treatment can be effective.

According to an exemplary embodiment of the present invention, the undifferentiated adipose tissue extract may be derived from an allogeneic tissue or cell. Specifically, the undifferentiated adipose tissue extract may be derived from autologous adipose tissue. More specifically, the undifferentiated adipose tissue extract may be extracted using adipose tissue of a patient or animal to be treated.

According to an exemplary embodiment of the present invention, the cell culture-derived extracellular matrix may mean decellularized from a cultured tissue using allogeneic or autologous cells and/or tissues. That is, the cell culture medium is not a tissue extracted from a human body or an animal, but a cultured tissue that is cultured using cells or tissues in vitro and includes cells and extracellular matrix.

According to an exemplary embodiment of the present invention, the average particle diameter of the extracellular matrix powder derived from the cell culture may be 10 μm to 30 μm. Specifically, the cell culture-derived extracellular matrix powder may have an average particle diameter of 10 μm to 20 μm, or 10 μm to 15 μm. The cell culture-derived extracellular matrix powder is a solid particle, and has an average particle diameter in the above range, which may serve as a medium for cell differentiation in the affected area. When the average particle diameter of the cell culture-derived extracellular matrix powder is out of the above range, it is not possible to provide an environment in which cell differentiation in the affected area occurs effectively, and the recovery of the affected area may be delayed. The average particle diameter of the extracellular matrix powder derived from the cell culture may be calculated using a method of calculating the average particle diameter of particles collected in a unit area through a computer program.

According to an exemplary embodiment of the present invention, the cell culture-derived extracellular matrix powder may be derived from the same tissue or cell. Specifically, the cell culture-derived extracellular matrix powder may be derived from an autologous tissue. Specifically, the cell culture-derived extracellular matrix powder may be derived from allogeneic or autologous tissue and/or cells of the site requiring treatment, for example, when the composition for tissue regeneration is for skin regeneration, the cell culture-derived The extracellular matrix powder may be derived from skin cells and/or tissues. More specifically, the cell culture-derived extracellular matrix powder may be derived from the human body or the dermal cells of a patient to be treated.

Another embodiment of the present invention provides a method for preparing the composition for tissue regeneration.

Another embodiment of the present invention, A) preparing an undifferentiated adipose tissue extract; B) preparing a cell culture-derived extracellular matrix powder; And C) mixing the undifferentiated adipose tissue extract and the cell culture medium-derived extracellular matrix powder; provides a method for producing a composition for tissue regeneration comprising a.

The contents of the undifferentiated adipose tissue extract and the cell culture-derived extracellular matrix powder in the composition for tissue regeneration described above may be directly applied in the method for preparing the composition for tissue regeneration.

In one embodiment of the present invention, the undifferentiated adipose tissue extract may be prepared using the extracted adipose tissue. Specifically, the undifferentiated adipose tissue extract may be obtained by extracting adipose tissue using a general liposuction technique, and then micronizing it.

In one embodiment of the present invention, the adipose tissue may be autologous adipose tissue. Specifically, the adipose tissue may be the patient's own fat, and may be extracted through liposuction. In addition, the adipose tissue can be obtained by extracting the adipose tissue using liposuction, and then removing at least a portion of physiological saline and blood mixed with the adipose tissue. Specifically, the adipose tissue can be obtained by leaving the extract obtained using liposuction for a predetermined time to remove the blood and physiological saline layer separated from the fat layer. When physiological saline and blood are removed as described above, the following adipose tissue extract may be more effectively extracted, and the prepared adipose tissue extract may more effectively contain growth factors and active cells suitable for treatment.

Step A) comprises: a1) a first filter having a pore diameter of 2 mm to 5 mm, a second filter having a pore diameter of 400 μm to 800 μm, and a pore diameter of 150 μm to 250 μm ( pore) sequentially using a third filter having a diameter, it may include pulverizing the adipose tissue in stages.

In an exemplary embodiment of the present invention, the first to third filters may be micronizers. In addition, the first to third filters are syringe filters, and may be crushed by passing the adipose tissue using a syringe. The syringe filter may be made of a stainless material, and it is possible to effectively pulverize adipose tissue by securing sufficient strength. In addition, the first to third filters may be a filter bag, which is a closed bag-shaped filter kit made of a flexible plastic material. The space may be in a partitioned form. In the case of using a filter bag, adipose tissue may be pulverized by injecting adipose tissue into the filter bag and passing it through the filter by applying external pressure (eg, pressure using a tool such as a silicone spatula).

In one embodiment of the present invention, step a1) may be to pulverize the adipose tissue by reciprocating the adipose tissue at least twice in each filter. Specifically, the first to third filters are syringe filters, and after mounting syringes at both ends, repeat the piston motion of the syringe to grind the extracted adipose tissue.

In one embodiment of the present invention, step a1) is at least in the first filter having a pore diameter of 2 mm to 5 mm, specifically 2 mm to 4 mm or 2 mm to 3 mm of the extracted adipose tissue. It may include a primary grinding step of passing it back and forth twice. The primary grinding step may be grinding the extracted adipose tissue by passing it through the first filter 2 to 7 times, or 2 to 3 times. In addition, the first grinding step may include removing the residue that did not pass through the first filter. The residue removed in the first grinding step may be fibers in adipose tissue. The fibers may interfere with the pulverization of adipose tissue using the second and third filters, and may also inhibit the activity of the tissue regeneration patch, so it is preferable to remove them.

In an exemplary embodiment of the present invention, in step a1), the adipose tissue pulverized through the first filter is passed through a second filter having a pore diameter of 400 μm to 800 μm reciprocally at least twice. It may include a grinding step. In the second grinding step, the adipose tissue pulverized through the first filter may be pulverized by passing it through the second filter 2 to 7 times, or 2 to 3 times. In addition, the second grinding step may include removing the residue that did not pass through the second filter.

In an exemplary embodiment of the present invention, in step a1), the adipose tissue pulverized through the second filter is passed through the third filter having a pore diameter of 150 μm to 250 μm reciprocally at least twice. It may include a grinding step. In the third grinding step, the adipose tissue pulverized through the second filter may be pulverized by passing it through the third filter 2 to 7 times, or 2 to 3 times. In addition, the third pulverization step may include removing residues that have not passed through the third filter.

In one embodiment of the present invention, in step a1), as described above, the adipose tissue is pulverized step by step using a filter having a small pore diameter, so that the time for fine pulverizing the adipose tissue can be greatly reduced, and the adipose tissue The cells in the extract can maintain high cellular activity. Specifically, when step a1) is used, there is an advantage in that a large amount of adipose tissue and active cells and active proteins effective for effective treatment can be secured in spite of the fine pulverization of the adipose tissue by a physical method. Furthermore, when step a1) is used, it is possible to collect the extracellular matrix and growth factors in an optimally active state, and also to have physical properties capable of performing (3D) bioprinting.

In one embodiment of the present invention, in step A), a2) the undifferentiated adipose tissue by the third filter is mixed with physiological saline and left to stand, an aqueous phase layer and an organic phase layer After phase separation, the step of removing the aqueous layer may be further included. In order to minimize impurities, step a2) may be repeated two or more times. Through this, it is possible to remove adipose tissue, blood, physiological saline, anesthetic, etc., which are excessively finely pulverized and have poor cell activity, which may interfere with the activity of the final tissue regeneration patch intended in the present invention. Through this, it is possible to obtain adipose tissue-derived extracellular matrix containing a large amount of factors that can effectively help cell differentiation.

According to an exemplary embodiment of the present invention, in step A), a3) after filtering the adipose tissue undifferentiated by the third filter through a fourth filter having a pore diameter of 25 μm to 125 μm, the filtrate The method may further include removing and collecting the residues collected in the fourth filter. Specifically, after filtering the adipose tissue undifferentiated by the third filter through the fourth filter, the filtered material may be discarded to obtain a material collected in the filter. Specifically, in step a3), the undifferentiated adipose tissue is mixed with physiological saline through step a1), filtered using the fourth filter, separated and removed from the filtered material, and then collected in the fourth filter The material used may be used as an undifferentiated adipose tissue extract. The material to be filtered and removed may include cells, adipose tissue, blood, physiological saline, anesthesia, and the like, which are too finely pulverized to have poor cell activity. Through this, it is possible to obtain a higher purity adipose tissue-derived extracellular matrix.

According to an exemplary embodiment of the present invention, step B) includes: b1) culturing the cell layer on a cell culture substrate having a predetermined pattern and rigidity, and then separating the cell culture substrate and the cell layer to obtain a cell culture; b2) decellularizing the cell culture to obtain a decellularized cell culture; and b3) freeze-drying the decellularized cell culture and pulverizing it to obtain a cell culture-derived extracellular matrix powder.

According to an exemplary embodiment of the present invention, the cell culture substrate, a substrate having a melting point at a temperature of any one of 0 ℃ to 30 ℃, hydrophobic to hydrophilic at a temperature of any one of 0 ℃ to 30 ℃ It may be a substrate that is formed, or a substrate that is degraded by an enzyme.

According to an exemplary embodiment of the present invention, step b1) may be performed in an atmosphere of 35 °C to 40 °C. This is when the cell culture substrate is a substrate having a melting point at a temperature of any one of 0 °C to 30 °C, or a substrate that is converted from hydrophobicity to hydrophilicity at a temperature of any one of 0 °C to 30 °C, the temperature range In an atmosphere of (ie, 35° C. to 40° C.), the cell culture substrate maintains a solid form or hydrophobicity, and the cultured cells are actively activated, thereby promoting the formation of a cell layer.

According to an exemplary embodiment of the present invention, the cell culture substrate may be a hydrogel substrate comprising a hydrogel as a main component.

The "hydrogel" is a material that can contain a large amount of water, and is a material that can easily transmit and move substances necessary for cell survival, such as oxygen, water, water-soluble nutrients, polypeptides such as enzymes and cytokines, and waste products, or It can mean the form. The hydrogel may be a hydrogel particle formed by solidifying an aqueous solution containing colloidal particles. The hydrogel is, for example, polyacrylamide (poly(acrylamide)), polyacrylic acid (poly(acrylic acid)), polyhydroxyethyl methacrylate (poly(hydroxyethyl methacrylate)), polyvinyl alcohol (poly(vinyl alcohol)), poly(lactic acid), water-soluble, hydrophilic, or water-absorbing synthetic polymers such as poly(glycolic acid); And it may be a particle consisting of a hydrogel chemically cross-linked with polysaccharides, proteins, and nucleic acids. Examples of the polysaccharide include, but are not limited to, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, starch, glycogen, agar, pectin, and cellulose. In addition, collagen and its hydrolyzates gelatin, proteoglycan, fibronectin, vitronectin, laminin, entactin, tenascin, thrombospondin , von Willebrand factor (von Willebrand factor), osteopontin (osteopontin), fibrinogen (fibrinogen) and the like may be mentioned, but is not limited thereto.

According to an exemplary embodiment of the present invention, the cell culture substrate may include at least one of polyphosphazene-based hydrogel, pluronic-based hydrogel, and poly(N-isopropylacrylamide). Specifically, the cell culture substrate may be a polyphosphazene-based hydrogel substrate, a pluronic-based hydrogel substrate, or a poly(N-isopropylacrylamide) substrate.

According to an exemplary embodiment of the present invention, the polyphosphazene-based hydrogel includes a temperature-sensitive polyphosphazene-based compound, and the melting point may be adjusted to about 4°C to about 10°C. For example, a temperature-sensitive and cross-linkable phosphazene-based hydrogel disclosed in Korean Publication No. 10-2017-0061530, a phosphazene-based hydrogel having an ionic group capable of controlling the decomposition rate disclosed in Korean Publication No. 10-2014-0016521 Polymer, the biodegradable temperature-sensitive polyphosphazene-based hydrogel disclosed in Korean Publication No. 10-2007-0076386 may be included in the polyphosphazene-based hydrogel of the present invention.

According to an exemplary embodiment of the present invention, the pluronic-based hydrogel can be adjusted to about 0 ℃ to 30 ℃ the temperature of the melting point by using a pluronic polymer. The pluronic polymer is polyethylene oxide (PEO) - polypropylene oxide (PPO) - any polymer having a structure (PEO-PPO-PEO) of polyethylene oxide (PEO) can be used. For example, F38, F68, F77, F77, F98, F108, F127 derivatives starting with F, L31, L42, L43, L44, L62, L72, L101 derivatives starting with L, etc., P75 starting with P, P103, P104 derivatives, etc. (all trade names) may be included. More specifically, F68 having a molecular weight of about 8,700 Daltons and F127 having a molecular weight of about 12,600 Daltons approved by the US Food and Drug Administration (FDA) among the fluoric polymers may be used.

However, the cell culture substrate is not limited to polyphosphazene-based hydrogels and pluronic-based hydrogels, and if it is a hydrogel having a melting point at a temperature of 30° C. or less, the first substrate and/or additional first It can be applied as a base material.

Also, according to an exemplary embodiment of the present invention, the cell culture substrate may be a poly(N-isopropylacrylamide) substrate. In addition, the cell culture substrate may be surface-treated with poly(N-isopropylacrylamide). Specifically, the cell culture substrate may be a surface coated with poly (N-isopropyl acrylamide) creat-bonded or surface-coated. Since the poly(N-isopropylacrylamide) is converted from hydrophobicity to hydrophilicity in an atmosphere of 30 °C or less, specifically about 20 °C to about 30 °C, the cell culture substrate is poly(N-isopropylacrylamide) In the case of a substrate or a substrate surface-treated therewith, the cell culture substrate can be easily removed using physiological saline at about 20°C to about 30°C. In addition, through the poly(N-isopropylacrylamide), a cell layer can be better formed on the cell culture substrate, and when the cell culture substrate is removed, the poly(N-isopropylacrylamide) is also formed in the cell layer. can be easily separated. However, the present invention is not limited thereto, and any material having a property of converting from hydrophobicity to hydrophilicity at a temperature of 30° C. or less can be applied similarly to the poly(N-isopropylacrylamide).

According to an exemplary embodiment of the present invention, the cell culture substrate may be a hydrogel substrate including an enzyme-susceptible peptide. For example, the cell culture substrate may be a hydrogel substrate comprising an enzyme-sensitive peptide and poly(ethylene glycol) (PEG) gel. In addition, the cell culture substrate may be a hydrogel substrate to which an amino acid sequence that can be degraded by matrix metallopro teinases (MMPs), elastase and/or plasmin is attached. Specifically, the cell culture substrate is an amino acid sequence attached PEG-succinimidyl propionate hydrogel (PEG-succinimidyl propionate hydrogel) substrate, for example, a synthetic tetrapeptide Ala-Pro-Gly-Leu (4armPEG10k-LGPA) It may be based on a PEG-succinimidyl propionate hydrogel to which a functionalized PEG-amine is attached. Alternatively, the cell culture substrate is an amine-reactive PEG-monoacrylate and a collagenase sensitive peptide (Gly-Gly-Leu'Gly-Pro-Ala-Gly-Gly-Lys), or an integrin binding domain peptide (integrin-binding domain peptide) (Tyr-Ile-Shy-Ser-Arg) may be attached to a PEG-hydrogel base.

According to an exemplary embodiment of the present invention, the cell culture substrate may be a hydrogel substrate containing carboxylmethyl cellulose (CMC) or alginate (Al).

Specifically, according to an exemplary embodiment of the present invention, the carboxylmethyl cellulose or alginate may be conjugated with tyramine. That is, the cell culture substrate may be a hydrogel substrate comprising carboxylmethyl cellulose (CMC-ty) conjugated with tyramine. In addition, the cell culture substrate may be a hydrogel substrate comprising alginate (Al-ty) conjugated with tyramine.

The cell culture substrate may be a hydrogel substrate containing at least 0.5% CMC-ty or Al-ty. Specifically, the cell culture substrate may be a hydrogel substrate containing 0.5% to 4% CMC-ty or Al-ty. The % may be wt% or vol%.

According to an exemplary embodiment of the present invention, the enzyme may be a carboxylmethyl cellulose degrading enzyme or an alginate degrading enzyme. Specifically, when the cell culture substrate is a carboxylmethyl cellulose substrate, the enzyme may be a carboxylmethyl cellulose degrading enzyme. In addition, when the cell culture substrate is an alginate substrate, the enzyme may be an alginate degrading enzyme.

According to an exemplary embodiment of the present invention, the cell culture substrate is a substrate having a melting point at a temperature of any one of 0 ℃ to 30 ℃ or from 0 ℃ to 30 ℃ hydrophobic to hydrophilic at any one point. and b1) may be to separate the cell layer by providing a temperature below the melting point of the cell culture substrate or a temperature converted to hydrophilicity. Specifically, step b1) may be to contact the cell culture substrate with a solution having a temperature below the melting point of the cell culture substrate or a temperature at which the cell culture substrate is converted to hydrophilicity. In this case, the solution having a temperature below the melting point of the cell culture substrate may be physiological saline having a temperature of about 4 °C to about 30 °C. Alternatively, step b1) may be to lower the ambient temperature to a temperature below the melting point of the cell culture substrate or to a temperature at which the cell culture substrate is converted to hydrophilicity. In this case, the ambient temperature (air temperature) may be about 4 ℃ to about 30 ℃.

According to an exemplary embodiment of the present invention, the cell culture substrate is a substrate that is decomposed by an enzyme, and in step b1), the cell culture substrate is contacted with a solution containing the enzyme to separate the cell layer may be doing Specifically, step b1) may be to contact the cell culture substrate with a solution containing an enzyme that decomposes the cell culture substrate.

According to an exemplary embodiment of the present invention, decellularization in step b2) can be applied without limitation to decellularization techniques known in the art. In the case of decellularization of a cell culture as in the present invention, since decellularization is easier than in the case of decellularizing the extracted human or animal tissue, the components contained in the cell layer and/or the extracellular matrix of the tissue are preserved as much as possible and decellularized. By doing so, there is an advantage that the active ingredient can be left as much as possible.

According to an exemplary embodiment of the present invention, in step b3), the decellularized cell culture may be freeze-dried to be solidified and then pulverized. The freeze-drying may be performed using a freeze-drying method generally known in the art. Further, in step b3), the freeze-dried decellularized cell culture is subjected to mechanical grinding such as a ball mill method in a liquid nitrogen atmosphere, and has an average particle diameter of 10 μm to 30 μm, 10 μm to 20 μm, or 10 μm to 15 μm. A cell culture-derived extracellular matrix powder can be obtained.

According to an exemplary embodiment of the present invention, the cell culture substrate may have a predetermined pattern or rigidity suitable for implementing the characteristics of the cell layer. For example, the cell culture substrate may have a pattern and/or stiffness similar to that of dermal tissue.

According to an exemplary embodiment of the present invention, the composition for tissue regeneration may be a composition for skin treatment. Specifically, the composition for tissue regeneration may be used for the treatment of dermal tissue, and in particular, it may be effective in regenerating dermal tissue in the affected area of a burn patient.

Another embodiment of the present invention provides a spray composition for treating burns comprising the composition for tissue regeneration. The composition of the spray composition for treatment of burns may be adjusted to be suitable for a general spray method or an aerosol spray method.

Hereinafter, examples will be given to describe the present invention in detail. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention is not to be construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present invention to those of ordinary skill in the art.

[Example]

Obtaining Undifferentiated Adipose Tissue Extract

Using liposuction, 70 ml to 150 ml of inhaled material is extracted from the abdomen of the pig to which the burn model has been applied, and after leaving it for about 5 minutes, the saline and blood mixed with the adipose tissue are removed to remove the sediment, and the adipose tissue was obtained.

Then, the syringe filled with fat and a new syringe are mounted on the two-way inlet of the connector with a stainless syringe filter (Adnizer, SKT-AN-2400, BSL) with a pore diameter of 2.4 mm, and the adipose tissue removes the filter by piston movement. After passing through three times to be pulverized, fibers that did not pass through the filter were removed. Then, the adipose tissue from which the fibers were removed was pulverized sequentially using a syringe filter having a pore diameter of about 500 μm and a syringe filter having a pore diameter of about 200 μm, and the residue that did not pass through the filter was removed. . The pulverized adipose tissue was mixed with physiological saline and left to stand to remove the aqueous layer including cells and blood twice, thereby obtaining an undifferentiated adipose tissue extract.

Obtaining dermal cell-derived extracellular matrix

To prepare a hydrogel sheet, a gelatin mold was prepared and the bottom of the mold formed a micro pattern. In an atmosphere of about 25 °C, an alginate solution containing alginate in an MES buffer of pH 6-7 in an amount of about 2 wt% was placed in the gelatin mold. Then, the nitrocellulose membrane was covered to flatten the upper surface of the prepared hydrogel sheet, and a plastic mesh and a glass plate for fixing it were laminated. Then, an aqueous alginate crosslinking solution was added dropwise to hydrogel the alginate solution in the mold. Thereafter, an alginate hydrogel sheet was prepared by dissolving the gelatin mold using a washing buffer (Saline, PBS, ddH ₂ O) at about 37°C.

Then, in an atmosphere of about 37 ° C., a cell layer was formed by culturing the dermal cells of the pig to which the image model was applied on the patterned surface of the prepared Pluronic hydrogel sheet. Then, the hydrogel sheet was removed to obtain a cell culture derived from dermal cells.

The obtained cell culture was subjected to decellularization treatment using a decellularization solution, and then freeze-dried using a freeze-dried preservation solution. Then, the freeze-dried decellularized cell culture was ground in a ball mill cooled with liquid nitrogen to obtain an extracellular matrix derived from a dermal cell culture having an average particle diameter of about 15 μm.

Preparation of a composition for tissue regeneration

A composition for tissue regeneration was prepared by mixing 10 ml of the adipose tissue extract obtained as described above and 10 ml of an extracellular matrix derived from a dermal cell culture at a concentration of 25 mg/ml to be homogenized using a syringe syringe.

[Experimental example]

After injecting the composition for tissue regeneration obtained according to the example into a spray device together with argon, and spraying it on the affected area of a pig to which the burn model of 2nd to 3rd degree was applied, the regeneration effect of the affected area was observed.

As a comparative example, the regenerative effect of the affected area was observed by dressing only the affected area of the pig to which the 2nd to 3rd degree burn model was applied without separate treatment.

1 shows the results of treatment of the affected part of the pig to which the burn model according to the experimental example is applied. Specifically, the white-looking wrinkles in the photo on Day 28 show that the epithelialization of the wound has been completed due to the proliferation and migration of the epithelial cells. Moreover, unlike the healing process in which the general wound healing shrinkage occurs and leaves a scar by closing in a diamond shape as in the comparative example, in the case of the Example, it is regenerated without shrinkage (it closes when the square shape is maintained), and the scar is minimized. It was confirmed that it was played with That is, when comparing the results after 35 days after the treatment of the affected area, in the case of the comparative example in which only the dressing was performed without any additional treatment, the scar was clearly visible, whereas the wound treated with the composition for tissue regeneration according to the example was recovered with minimal scarring. could confirm that

Claims (15)

1. micronized adipose tissue extract; and a cell culture-derived extracellular matrix powder, a composition for tissue regeneration.
2. The method according to claim 1,

   The weight ratio of the undifferentiated adipose tissue extract and the cell culture medium-derived extracellular matrix powder is 1:10 to 10:1, the composition for tissue regeneration.
3. The method according to claim 1,

   The undifferentiated adipose tissue extract is a composition for tissue regeneration, characterized in that it contains adipose tissue-derived extracellular matrix that passes through a filter pore of 100 μm to 300 μm.
4. The method according to claim 1,

   The undifferentiated adipose tissue extract is a composition for tissue regeneration, characterized in that it is derived from autologous adipose tissue.
5. The method according to claim 1,

   The cell culture-derived extracellular matrix powder has an average particle diameter of 10 μm to 30 μm, a composition for tissue regeneration.
6. The method according to claim 1,

   The cell culture-derived extracellular matrix powder is a composition for tissue regeneration, characterized in that it is derived from the same type of tissue or cell.
7. A) preparing an undifferentiated adipose tissue extract;

   B) preparing a cell culture-derived extracellular matrix powder; and

   C) mixing the undifferentiated adipose tissue extract and the cell culture medium-derived extracellular matrix powder; comprising, a method for producing a composition for tissue regeneration.
8. 8. The method of claim 7,

   Step A) comprises: a1) a first filter having a pore diameter of 2 mm to 5 mm, a second filter having a pore diameter of 400 μm to 800 μm, and a pore diameter of 150 μm to 250 μm ( pore) sequentially using a third filter having a diameter, step-by-step pulverizing adipose tissue;
9. 9. The method of claim 8,

   In step A), a2) the adipose tissue undifferentiated by the third filter is mixed with physiological saline and left to stand, phase-separated into an aqueous phase layer and an organic phase layer, and then the aqueous layer is separated The method of manufacturing a composition for tissue regeneration, characterized in that it further comprises the step of removing;
10. 9. The method of claim 8,

    In step A), a3) the adipose tissue undifferentiated by the third filter is filtered through a fourth filter having a pore diameter of 25 μm to 125 μm, and then the filtrate is removed and collected in the fourth filter The method of manufacturing a composition for tissue regeneration, characterized in that it further comprises; collecting the residue.
11. 8. The method of claim 7,

    B) step is,

    b1) culturing a cell layer on a cell culture substrate having a predetermined pattern and rigidity, then separating the cell culture substrate and the cell layer to obtain a cell culture;

    b2) decellularizing the cell culture to obtain a decellularized cell culture; and

    b3) after freeze-drying the decellularized cell culture, pulverizing it to obtain a cell culture-derived extracellular matrix powder;
12. 12. The method of claim 11,

    The cell culture substrate is a substrate having a melting point at a temperature of any one of 0 °C to 30 °C, a substrate that is converted from hydrophobicity to hydrophilicity at a temperature of any one of 0 °C to 30 °C, or decomposed by an enzyme A method for producing a composition for tissue regeneration, characterized in that it is a substrate.
13. 12. The method of claim 11,

    The cell culture substrate is a substrate having a melting point at any one point of 0 °C to 30 °C or a substrate that is converted from hydrophobicity to hydrophilicity at a temperature of any one point of 0 °C to 30 °C,

    Step b1) provides a temperature below the melting point of the cell culture substrate or a temperature converted to hydrophilicity to separate the cell layer, a method for producing a composition for tissue regeneration.
14. 12. The method of claim 11,

    The cell culture substrate is a substrate that is degraded by an enzyme,

    In step b1), the cell culture substrate is brought into contact with a solution containing an enzyme to separate the cell layer, the method for producing a composition for tissue regeneration.
15. 12. The method of claim 11,

    Step b3) is a method for producing a composition for tissue regeneration, characterized in that the freeze-dried decellularized cell culture is mechanically pulverized in a liquid nitrogen atmosphere.

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