Composition and method for cryogenic grinding of tissue, and extracellular matrix prepared therefrom

Abstract

The present invention relates to a composition and a method for cryogenic grinding of tissue and, more specifically, to a composition and a method for cryogenic grinding of tissue, wherein the composition is applicable to both hard tissue and soft tissue and causes minimal damage to an extracellular matrix by comprising biologically derived tissue and a solid crystalline granular material which is used by being mixed with the biologically derived tissue during cryogenic grinding of the biologically derived tissue and is capable of decellularizing the tissue.

Description

Composition, method, and extracellular matrix prepared therefrom of tissue freeze-grinding

[0001] The present invention relates to a tissue freeze-grinding composition, a method, and an extracellular matrix produced therefrom.

[0002]

[0003] Regenerative medicine refers to cell therapy, gene therapy, tissue engineering therapy, etc., performed using human cells, etc., to regenerate, restore, or form human body structures or functions, or to treat or prevent diseases.

[0004] Regenerative medicine encompasses not only the structural replacement or restoration of human cells, tissues, and organs but also their functional replacement or restoration. It includes both technologies for producing these components in vitro to replace damaged body parts and technologies that promote the body's self-regeneration.

[0005] Among these regenerative medicines, 3D bioprinting is a type of 3D printing technology that fabricates tissues or organs by layering living cells or biomaterials into desired shapes or patterns. The bioink utilized in this process refers to an ink-type material composed of living cells, substances (extracellular matrix), and growth factors, which can be used to fabricate structures such as artificial organs and tissues through bioprinting. To maintain the physiological characteristics of the extracellular matrix (ECM) to be used as a biomaterial, manufacturing methods must be applied to maximize the three-dimensional structure of the ECM and the bioactive substances. Key processes in the manufacturing process are decellularization, a technique for removing cells from tissues to develop biomaterials free from antigenicity that can cause immune rejection, and sterilization.

[0006] However, in the production of extracellular matrix used as a biomaterial, a multi-step decellularization process is essential. Since the manufacturing process involves multiple steps, it is time-consuming, and there are problems such as protein denaturation and protein loss caused by the long production time.

[0007]

[0008] The purpose of the present invention is to provide a tissue freeze-grinding composition and a tissue freeze-grinding method with reduced manufacturing process time in order to solve the technical problem to be achieved by the present invention.

[0009] The purpose is to provide a tissue freeze-grinding composition and method that shortens the decomposition time of the extracellular matrix.

[0010] The purpose is to provide a tissue freeze-grinding composition and method that minimize protein denaturation and loss.

[0011] The purpose is to provide a tissue freeze-grinding composition and method applicable to various medical fields by forming an extracellular matrix protein raw material applicable to both hard and soft tissues.

[0012] The object of the present invention is to provide a pharmaceutical composition for tissue regeneration comprising an extracellular matrix obtained by using another tissue freeze-grinding composition or by a tissue freeze-grinding method.

[0013] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems can be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0014]

[0015] The present invention provides a tissue freeze-grinding composition comprising: a biological tissue; and a solid crystalline granular body that is mixed with the biological tissue during freeze-grinding of the biological tissue and is capable of decellularizing the tissue.

[0016] In the above composition, the biological tissue may be cut into pieces of a size of (0.6 cm to 3 cm) x (0.6 cm to 3 cm).

[0017] In the above composition, the solid crystalline granular body may be one or more selected from the group consisting of NaCl, Na2CO3, CaCl2, MgCl2, and sugar.

[0018] In the above composition, the biological tissue may be derived from one or more selected from the group consisting of bone, cartilage, mucous membrane, blood vessel, liver, lung, stomach, heart, small intestine, large intestine, duodenum, skin, salivary gland, tongue, esophagus, muscle, lymph node, fat, and cornea.

[0019] In the above composition, the biologically derived tissue and the solid crystalline granular body may be mixed in a weight ratio of 1:0.1 to 1:12.

[0020] In the above composition, the biological tissue is a hard tissue, and the biological tissue and the solid crystalline granular body may be mixed in a weight ratio of 1:0.2 to 1:5.

[0021] In the above composition, the biological tissue is soft tissue, and the biological tissue and the solid crystalline granular body may be mixed in a weight ratio of 1:1 to 1:5.

[0022] The present invention also provides a method for freeze-grinding a tissue, comprising the steps of: cutting a biological tissue into pieces of a predetermined size; mixing the cut biological tissue with a solid crystalline granular body; freeze-grinding the mixed composition; washing the freeze-grinded composition; and dissolving the washed composition in distilled water and freeze-drying it.

[0023] In the method of the present invention, the predetermined size may be (0.6 cm to 3 cm) Х (0.6 cm to 3 cm).

[0024] In the method of the present invention, the solid crystalline granular body may be one or more selected from the group consisting of NaCl, Na2CO3, CaCl2, MgCl2, and sugar.

[0025] In the method of the present invention, the biological tissue may be derived from one or more selected from the group consisting of bone, cartilage, mucous membrane, blood vessel, liver, lung, stomach, heart, small intestine, large intestine, duodenum, skin, salivary gland, tongue, esophagus, muscle, lymph node, fat, and cornea.

[0026] In the method of the present invention, the biologically derived tissue and the solid crystalline granular body may be mixed in a weight ratio of 1:0.1 to 1:12.

[0027] In the method of the present invention, the biological tissue is a hard tissue, and the biological tissue and the solid crystalline granular body may be mixed in a weight ratio of 1:0.2 to 1:5.

[0028] In the method of the present invention, the biological tissue is soft tissue, and the biological tissue and the solid crystalline granular body may be mixed in a weight ratio of 1:1 to 1:5.

[0029] The present invention also provides a pharmaceutical composition for tissue regeneration comprising an extracellular matrix prepared by the tissue freeze-grinding method of the present invention.

[0030]

[0031] According to one embodiment of the present invention, a method for freezing and grinding tissue with reduced manufacturing process time can be provided.

[0032] According to one embodiment of the present invention, a method for freezing and grinding tissues can be provided that shortens the decomposition time of the extracellular matrix.

[0033] According to one embodiment of the present invention, a tissue freeze-grinding composition and method that minimize protein denaturation and loss can be provided.

[0034] According to one embodiment of the present invention, an extracellular matrix protein raw material applicable to both hard tissues and soft tissues is formed, thereby providing a tissue freeze-grinding composition and method applicable to various medical fields.

[0035] An extracellular matrix prepared using a tissue freeze-grinding composition according to one embodiment of the present invention, or by a tissue freeze-grinding method, can be utilized for tissue regeneration.

[0036]

[0037] FIG. 1 is a diagram showing the manufacturing process of a tissue freeze-grinding composition according to one embodiment of the present invention.

[0038] Figure 2 is a diagram showing the amount of DNA remaining in the pulverized product according to the freeze-pulverization time.

[0039] Figure 3 is a graph showing the DNA content according to the size of the tissue.

[0040] Figure 4 is a graph showing the DNA content according to the type of crystal.

[0041] Figure 5 is a graph showing the DNA content according to the mixing ratio of tissue and crystals (additives) in cartilage tissue.

[0042] Figure 6 is a graph showing the DNA content according to the mixing ratio of tissue and crystals (additives) in mucosal tissue.

[0043] Figures 7 and 8 are graphs comparing the DNA, collagen, and elastin content of the final products after an optimized decellularization process in cartilage tissue and mucosal tissue, respectively.

[0044] Figure 9 is a graph showing the residual DNA content according to the type of tissue that underwent decellularization.

[0045] Figure 10 is a scanning electron microscope image of the product immediately after the freeze-grinding step, depending on the presence or absence of crystalline granules.

[0046] Figure 11 is a scanning electron microscope image of the extracellular matrix obtained by freeze-drying the product obtained by freeze-grinding mucosal tissue through decellularization by osmotic action, and the extracellular matrix (DFdECM) obtained by freeze-grinding mucosal tissue together with crystalline granules through a washing step.

[0047] Figures 12a and 12b are figures evaluating the ability to regulate chondrocyte differentiation by the final product after the decellularization process of stem cells.

[0048] Figures 13 and 14 are drawings evaluating the ability to regulate chondrocyte differentiation by the final product of bone tissue formation (in vivo).

[0049] Figures 15a and 15b are the results of histoimmunochemical staining to evaluate the ability of the final product after the decellularization process of stem cells to regulate chondrocyte differentiation, and Figure 15c is the result of protein quantification analysis.

[0050] Figure 16a is the result of a histochemical analysis performed to evaluate (in vivo) the expression pattern of inflammatory response-related factors after transplantation of stem cells and the final product after decellularization, and Figure 16b is the image quantitative analysis.

[0051] FIG. 17 is a flowchart of a tissue freeze-grinding method according to one embodiment of the present invention.

[0052]

[0053] Hereinafter, the tissue freeze-grinding composition and tissue freeze-grinding method according to the present invention will be described in detail with reference to the attached drawings.

[0054] It should be noted that when assigning reference numerals to the components of each drawing, the same components are assigned the same reference numeral whenever possible, even if they are shown in different drawings. Furthermore, in describing the embodiments of the present invention, if it is determined that a detailed description of related known components or functions would hinder understanding of the embodiments of the present invention, such detailed description is omitted.

[0055] In describing the components of the embodiments of the present invention, terms such as first, second, A, B, (a), (b), etc., may be used. These terms are intended merely to distinguish the components from other components, and the essence, order, or sequence of the components is not limited by such terms. Furthermore, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0056] A tissue freeze-grinding composition according to one embodiment of the present invention may include a biological tissue and a solid crystalline granular body capable of decellularizing the tissue, which is mixed with the biological tissue when freeze-grinding the biological tissue.

[0057] Here, the solid crystalline granular body can be mixed with the tissue during freeze-grinding and may include an ionic solid capable of decellularizing the tissue, and may include an ionic lattice structure material capable of decellularizing the tissue. Additionally, the crystalline granular body may include a solid alkali metal salt or an alkaline earth metal salt.

[0058] In addition, the solid crystalline granular body can be prepared as a water-soluble compound having a crystalline structure. The above tissue is decellularized, and the crystalline granular body of a water-soluble compound having a crystalline structure is, for example, LiF, CaF2, CuCl, MgO, RbCl, NaI, KF, CsI, LiCl, RbBr, NH4Br, NaClO4, NaClO3, KClO4, Na2S, NaCN, KCN, NH4SCN, SrCl2, BaCl2, LiBr, NH4I, MgCl2, SrNO32, BaNO32, Na2SO3, K2SO3, NaNO2, KNO2, NH4NO3, LiNO3, RbNO3, CsNO3, KH2PO4, NH42SO4, Na2SiF6, NH4Cl, AgCl, CsCl, NaBr, KBr, NaF, KI, Na2SO4, NaNO3, NaHSO3, NaMnO4, BaCl2, SrCl2, PbCl2, CuCl2, KNO3, S9, KCl, CaO, PbS, NiO, ZnS, SrO, CdS, BaO, CH3COONa, Na2SO4, Na2S2O3, NH4NO3, KNO3, Na2CO3, K2CO3, Na3PO4, Ca(NO3)2, MgSO4, ZnSO4, Sr(NO3)2, BaCl2, FeCl3, NiSO4, Pb(NO3)2, LiCl, Na2SeO4, Na3AsO4, K2SO4, AlCl3·6H2O, NaOH, NaHCO3, NaI, NaCl, NaClO4, Na2Cr2O7, KMnO4, Na2B4O7·10H2O, NaNO3, KSCN, NH4H2PO4, Na2O2, Ca(OH)2, Na2SnO3, KHC4H4O6, Ba(NO3)2, C12H25NaO4S, C6H7NaO6, Al(NO3)3·9H2O, FeSO4·7H2O, Fe(NO3)3·9H2O, CdCl2·2.It may be at least one of 5H2O, Na2MoO4·2H2O, Na2WO4·2H2O, SrCl2·6H2O, KClO3, NH4C2H3O2, NaClO, Na2SiO3, ZnCl2, C6H11NaO7, Ca(C2H3O2)2, and NaBF4. For example, it may be one or more selected from the group consisting of NaCl, KCl, Na2CO3, CaCl2, MgCl2, and sugar. For example, it may be one or more selected from the group consisting of NaCl, Na2CO3, CaCl2, MgCl2, and sugar. Preferably, it may be one or more selected from the group consisting of NaCl, Na2CO3, and CaCl2.

[0059] The tissue freeze-grinding composition and tissue freeze-grinding method according to one embodiment of the present invention shorten the decomposition time of the extracellular matrix, thereby reducing the manufacturing process time, and minimize protein denaturation and loss through detergent-free washing and shortened manufacturing time. In addition, an extracellular matrix protein raw material applicable to both hard and soft tissues is formed, making it applicable to various medical fields.

[0060] The biological tissue used in the present invention may be, for example, a biological tissue derived from a vertebrate, but is not limited thereto. Preferably, it may be a biological tissue derived from any one of mammals, fish, and birds, as it has a low rejection reaction. Most preferably, it may be a biological tissue derived from a pig or a human.

[0061] As a site of a biologically derived tissue, a site having an extracellular matrix structure may be used. Examples of such sites include, but are not limited to, the liver, kidney, ureter, bladder, urethra, tongue, tonsil, esophagus, stomach, small intestine, large intestine, anus, pancreas, heart, blood vessel, spleen, lung, brain, bone, spinal cord, cartilage, testis, uterus, fallopian tube, ovary, placenta, cornea, skeletal muscle, tendon, kidney, skin, fat, salivary gland, trachea, and cornea. Preferably, the site of the biologically derived tissue may be any one of the esophagus, trachea, heart, liver, lung, and brain.

[0062] The living tissue can be transplanted into a recipient and regenerated. The living tissue may be provided as either hard tissue or soft tissue. Hard tissue is a tissue with a hardness, such as bone or cartilage, and soft tissue is a tissue with a softness, such as mucosa, blood vessels, liver, lungs, stomach, heart, small intestine, large intestine, duodenum, skin, salivary glands, tongue, esophagus, muscle, lymph nodes, fat, and cornea, but is not limited thereto.

[0063] In addition, the biological tissue may be prepared by chopping it into a predetermined size. Here, the predetermined size may refer to a size in which the biological tissue is chopping to facilitate decellularization. Preferably, the predetermined size may be 1 cm x 0.5 cm.

[0064] Figure 2 is a diagram showing the amount of DNA remaining in the pulverized product according to the freeze-pulverization time.

[0065] To optimize the decellularization of tracheal cartilage and esophageal mucosal tissues obtained from pigs, the degree of intracellular nucleus removal according to freeze-grinding time was visually confirmed through DAPI staining. As shown in Figure 2, when cells within the tissue retain their intact shape, nuclei are observed as distinct circles; however, after the nuclei are destroyed, although DAPI may stain the nuclear remnants, the distinct circular shape of the nuclei disappears and is no longer observed. It was observed that the number of blue-stained nuclei gradually decreased as grinding time increased, and after 30 minutes, while some blue color appeared, the distinct shape of the nuclei was not observed. From this, the grinding time at which cells within the tissue are sufficiently destroyed during the freeze-grinding process may be, for example, 60 minutes.

[0066] Average DNA content (ng / mg of dried tissue) Margin of error native 17 2.47±10.51 small 69.22±6.48 large 71.63±1.64 X-large 46.98±3.76

[0067]

[0068] Table 1 and Figure 3 above are a table and a graph showing DNA content according to tissue size.

[0069] Here, small refers to tissues cut into pieces of 0.1 cm to 0.2 cm, large refers to tissues cut into pieces of 0.5 cm x 0.5 cm, and X-large refers to tissues cut into pieces of 1 cm x 0.5 cm. As can be seen from Table 1 and the graph in Figure 3 above, it can be confirmed that the DNA content of X-large was the lowest.

[0070] Preferably, the size of the biologically derived tissue that is easy to decellularize can be prepared as (0.6 cm to 3 cm) × (0.6 cm to 3 cm). A more preferred tissue size may be 1 cm × 0.5 cm, corresponding to X-large.

[0071] To optimize the types of additives during freeze-grinding, various types of water-soluble crystalline granules were mixed with biological tissues, freeze-grinded, and decellularized, and the residual DNA amount was quantified.

[0072] Table 2 and Figure 4 below are a table and a graph showing the average DNA content of tissues according to the type of additive.

[0073] Average DNA content (ng / mg of dried tissue) Margin of error Cartilage native 3 24.33±17.56 NaCl 7 2.09±6.99 KCl 8 18.70±3.28 Na2CO 3 54.33±8.42 CaCl 2 68.37±2.18 MgCl 2 151.40±12.53 Sugar 10 2.34±7.62

[0074]

[0075] As shown in Table 2 and Figure 4, DNA of 50 ng or less per 1 mg of dried tissue was detected in NaCl, Na2CO3, and CaCl2. It can be confirmed that the DNA content of tissues with added NaCl, Na2CO3, and CaCl2 is low, and that they are effective for decellularization. Additives mixed during freeze-grinding can play a role in tearing cell membranes during the physical grinding process, and accordingly, it may be advantageous for particles with an angular structure.

[0076] When freeze-grinding, the decellularization effect can be optimized according to the mixing ratio of tissue and crystalline granules. The tissue can be prepared as either hard tissue or soft tissue, and depending on the type of tissue, the crystalline granules mixed with the tissue can be mixed at a predetermined ratio. In addition, the in vivo safety of the extracellular matrix, that is, the standard for the absence of immune rejection, may be a residual DNA content of 50 ng / mg or less.

[0077] Table 3 and Figure 5 below are a table and graph showing the quantitative analysis of decellularization efficiency according to the amount of crystalline granules relative to the weight of hard tissue.

[0078] Tissue : Additives (w / w) Average DNA Content (ng / mg of dried tissue) Margin of Error Native cartilage 4 15.95±16.3 31:0.28 9.98±8.9 51:0.5 4 1.21±5.6 01:13 7.19±5.7 11:24 5.13±3.9 21:5 20.02±3.88

[0079]

[0080] In a composition and method according to one embodiment of the present invention, the biological tissue is a hard tissue, and the biological tissue that is a hard tissue and the crystalline granular body may be mixed in a weight ratio of, for example, 1:0.1 to 1:12, 1:0.1 to 1:11, 1:0.1 to 1:10, 1:0.1 to 1:9, 1:0.1 to 1:8, 1:0.1 to 1:7, 1:0.1 to 1:6, 1:0.1 to 1:5, or 1:0.2 to 1:5. Preferably, the biological tissue that is a hard tissue and the crystalline granular body may be mixed in a weight ratio of 1:0.5 to 1:5.

[0081] Table 4 and Figure 6 below are a table and graph showing the quantitative analysis of decellularization efficiency according to the amount of crystalline granules relative to the weight of soft tissue.

[0082] Tissue : Additives (w / w) Average DNA content (ng / mg of dried tissue) Margin of error Mucosal native 98 4.1±8 1.71:12 89.8±9.11:23 2.3±4.81:5 10 2.4±0.7

[0083]

[0084] In a composition and method according to one embodiment of the present invention, the biological tissue is a hard tissue, and the biological tissue and crystalline granular body may be mixed in a weight ratio of, for example, 1:0.1 to 1:12, 1:0.1 to 1:11, 1:0.5 to 1:10, 1:1 to 1:10, 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1:7, 1:1 to 1:6, or 1:1 to 1:5. Preferably, the biological tissue and crystalline granular body may be mixed in a weight ratio of 1:1 to 1:5.

[0085] As shown in Table 4 above, the average DNA content was measured to be lowest at a weight ratio of crystalline granules to soft tissue of 1:2. Therefore, most preferably, the soft tissue, which is a biologically derived tissue, and the crystalline granules may be mixed in a weight ratio of 1:2.

[0086] Hard tissues can influence physical fragmentation between hard tissues during the hard tissue grinding process. Therefore, in hard tissues, increasing the mixing ratio of crystalline granules relative to the tissue can proportionally increase decellularization efficiency. On the other hand, soft tissues have soft physical properties, so even when ground while frozen, they may not break into smaller tissues due to friction between soft tissue particles. It is the crystalline granules in contact with the fragments that tear the cell membranes contained within the soft tissue, and decellularization efficiency can be enhanced only if the fragmentation force is effectively transmitted to the crystalline granules. Accordingly, if the amount of crystalline granules surrounding the tissue is too large, it becomes difficult to transmit the fragmentation force, which may actually lead to a decrease in grinding efficiency.

[0087]

[0088] <Example 1>

[0089] Preparation Example

[0090] Airway cartilage tissue and esophageal mucosal tissue were each cut into pieces approximately 5mm x 5mm in size using a knife. The cut tissue pieces were prepared with NaCl in a weight ratio of 1:1 to 1:5. They were placed in a dedicated container for freeze-grinding and freeze-grinded for one hour.

[0091] The freeze-ground product is placed in a 50 mL conical tube, added to 30 mL of distilled water, and stirred at 900 rpm for 2 hours using a 2D orbital shaker. After 2 hours, centrifuge at 3739 g-force for 10 min. Carefully remove the supernatant, add 30 mL of clean distilled water, and stir for 5 minutes. Repeat the process of centrifuging and removing the supernatant three more times. After removing the supernatant, dissolve the aggregate in 10 mL of clean distilled water, freeze at -80 ℃, freeze-dry, and store. The extracellular matrix produced through this process is named DFdECM.

[0092]

[0093] control group

[0094] As a control, 5g of tissue pieces cut into 5mm x 5mm sizes were placed in a 50mL conical tube, treated with 30mL of 5% SDS solution, and stirred for 48 hours. After stirring, the supernatant was removed, and the samples were washed twice with 30mL of PBS. The extracellular matrix produced through this process was named DTdECM.

[0095] In addition, to determine whether crystalline granules added during the freeze-grinding process of the extracellular matrix affect tissue fragmentation, the first product, which is a freeze-grinding product of mucosal tissue obtained after freeze-grinding mucosal tissue alone; the second product, which is a freeze-grinding product of mucosal tissue containing crystals, which is an intermediate product obtained after a freeze-grinding step including NaCl; the third product, which is a DFdECM derived from mucosal tissue obtained after a washing step of the second product; and the fourth product, which is an extracellular matrix decellularized by osmotic action obtained after a washing step of freeze-grinding the first product without NaCl using a NaCl solution (0.57M) in which 1g of NaCl is dissolved in 30mL of water, were each freeze-dried and their morphologies were confirmed using a scanning electron microscope.

[0096] Quantification of DNA and extracellular matrix proteins was performed as follows. To determine the amount of DNA residue in the decellularized extracellular matrix, 10 mg of freeze-dried extracellular matrix was placed in an Eppendorf tube. 1 mL of RIPA aqueous solution was added and stirred at 4°C for 2 hours. After stirring, 50 μL of the solution was taken and analyzed using the Quant-iT™ PicoGreen™ dsDNA Reagent and Kit (Invitrogen™). Fluorescent quantification was performed using the Quant-iT™ PicoGreen™ dsDNA Reagent and Kit (Invitrogen™) to determine the DNA residues in native tissue, DFdECM, and DTdECM. Collagen and elastin were quantified colorimetrically using the Collagen Assay kit (abcam) and F2000 Fastin Elastin, respectively.

[0097] DNA analysis was performed as follows. Approximately 1 mg of dried DFdECM, after the decellularization process was completed, was dispersed in 50 μL of PBS, and the nuclei were stained by treating with DAPI solution. After staining for 10 minutes, approximately 5 μL of the DFdECM dispersion was dropped onto a slide glass, and the remaining nuclei were observed under a fluorescence microscope.

[0098] The lysis of the extracellular matrix was performed as follows. A solution of 1 mg of pepsin dissolved in 0.1 N HCl was prepared. 10 mg of freeze-dried extracellular matrix was added to 1 mL of the pepsin solution and thawed at 4°C for 24 hours. After confirming that the extracellular matrix was thawed, it was frozen and stored at -80°C.

[0099] The preparation of a culture medium containing extracellular matrix was performed as follows. A stem cell culture medium, a culture medium for the differentiation of stem cells into chondrocytes (hereinafter referred to as the chondrocyte differentiation culture medium), and a culture medium mixed with lysed extracellular matrix (hereinafter referred to as the extracellular matrix / stem cell culture medium or the extracellular matrix / chondrocyte culture medium) were prepared, respectively. When mixing with lysed extracellular matrix, the culture medium (stem cell culture medium or chondrocyte differentiation culture medium) was mixed with 10 mg / mL of lysed extracellular matrix at a volume ratio of 1:9, and then the pH was adjusted to 7.4 using 10N NaOH.

[0100] Stem cell aggregates were prepared as follows: 2 x 10⁶ 51 mL of a stem cell dispersion of cells / mL was placed in a 15 mL conical tube and centrifuged at 300 g for 5 minutes. The supernatant was removed, and the cell aggregates were dispersed into either the extracellular matrix / stem cell culture medium or the extracellular matrix / chondrocyte culture medium, respectively. The mixture was then centrifuged at 600 g for 5 minutes to form cell aggregates. The aggregates contained in the culture medium were transferred directly to a 37°C, 5% CO2 incubator and cultured for 7, 14, and 21 days. During the culture period, the culture medium was replaced every two days, and a medium excluding the extracellular matrix was used for replacement.

[0101] Immunohistochemical staining was performed as follows. Stem cell aggregates were harvested after 7, 14, and 21 days from the culture date, washed with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde. The fixed cell aggregates were prepared into OCT blocks, sliced ​​to a thickness of 4 µm, and frozen sections were prepared. The sections were reacted with a primary antibody related to chondrocyte differentiation at 4°C for 12 hours, washed with phosphate-buffered saline, and treated with a fluorescence-labeled secondary antibody at room temperature for 1 hour. The nuclei were stained with DAPI and observed under a fluorescence microscope. Here, the type of primary antibody may be at least one of Collagen 2 alpha 1 (a1560, abclonal; 1:200), Collagen X (ab49945, abcam; 1:1000), Aggrecan (AHP0022, invitrogen; 1:200) and Sox 9 (ab185966, abcam; 1:500).

[0102] Quantitative analysis of proteins related to chondrocyte differentiation was performed as follows. After 7, 14, and 21 days from the start of stem cell aggregate culture, the aggregates were homogenized in RIPA buffer using a homogenizer. The supernatant was taken and collagen 2, aggrecan, and sox9 were quantified colorimetrically, respectively. Here, the colorimetric quantification kits may be the Human Collagen Type 2 Alpha 1 ELISA Kit, Human Aggrecan core protein ELISA Kit, Human Transcription factor SOX-9 ELISA Kit, Human Collagen alpha-1 (X) chain ELISA Kit, or Abclonal.

[0103] Subcutaneous transplantation in small animals was performed as follows. To determine whether stem cell aggregates prepared in a culture medium containing extracellular matrix differentiate into chondrocytes in vivo, cell aggregates were prepared using the same method, and three aggregates were encapsulated within Matrigel (Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free). The Matrigel containing the aggregates was exposed to the culture medium for 24 hours under different conditions, after which it was washed with PBS. An incision of approximately 1 cm was made in the dorsal skin of mice (Bala / c nu, 8 weeks), Matrigel was inserted, and the skin was sutured.

[0104] Histopathological analysis was performed as follows. After 2 and 4 weeks had elapsed, respectively, following the transplantation of Matrigel containing aggregates, mice were anesthetized and the transplanted Matrigel was retrieved. The retrieved Matrigel was fixed in a 4% formaldehyde solution for at least 24 hours, and OCT blocks were prepared. After preparing 4 μm thick frozen sections, each was stained with H&E, and to confirm whether the transplanted stem cell aggregates had differentiated into chondrocytes, they were stained with Alcian blue and safranin O and observed under a light microscope. In addition, immunohistochemical staining was performed on the expression factors Collagen 2 alpha 1 (a1560, abclonal; 1:200), Collagen X (ab49945, abcam; 1:1000), Aggrecan (AHP0022, invitrogen; 1:200), and Sox 9 (ab185966, abcam; 1:500). After reacting with the primary antibody at 4°C for 12 hours, the samples were washed with phosphate-buffered saline (PBS), treated with a fluorescently labeled secondary antibody at room temperature for 1 hour, and the nuclei were stained with DAPI and observed under a fluorescence microscope.

[0105] According to the above example, the DNA content of DFdECM, the final product obtained by a decellularization method optimized for airway cartilage and esophageal mucosal tissues, and the major extracellular matrix components (collagen, elastin) were compared with DTdECM, obtained by decellularizing with the surfactant SDS. As shown in Figures 7 and 8, a smaller amount of DNA residue was detected in DFdECM relative to 1 mg of dry tissue weight compared to DTdECM, while the content of collagen and elastin, the major components of the extracellular matrix, was found to be higher. The same analysis was performed on the esophageal mucosa, and similar to the cartilage tissue, DFdECM showed a low DNA residue and excellent collagen and elastin content.

[0106] Figure 9 is a graph showing the residual DNA content according to the type of tissue that underwent decellularization.

[0107] The above DFdECM production conditions, which were performed on cartilage tissue, were applied identically to skin, brain, liver, bone, lung, and muscle (stomach) tissues to perform decellularization, and the amount of DNA reduced by tissue type was measured under conditions where the tissue:additive (w / w) ratio was 1:2. As a result of decellularization, the residual DNA content in skin and brain tissues decreased to 50 ng / mg or less, and in liver and bone tissues, it decreased to a level of about 100 ng / mg.

[0108] From this, it was confirmed that the mixing of crystalline granules can contribute to effective cell removal by inducing the physical breakdown of cell structures regardless of tissue type.

[0109] Figures 10 and 11 are the results of scanning electron microscope examination of intermediate products of the decellularization process of mucosal tissue to determine whether crystalline granules added during the freeze-grinding process of the extracellular matrix affect tissue disruption during the decellularization process.

[0110] According to Fig. 10, a network of tissues was observed in the first product obtained after freeze-grinding mucosal tissue alone, and in the second product obtained after freeze-grinding including crystals, a structure in which the tissue was torn and attached around the crystalline particles was observed. Through this, it can be seen that the crystals cause physical damage to the tissue during the freeze-grinding process, thereby enhancing the decellularization effect.

[0111] In addition, to compare the decellularization effect caused by osmotic pressure as the NaCl used in the freeze-grinding process dissolves in distilled water during the washing step, the final product, the fourth product, obtained by freeze-grinding mucosal tissue and then performing a washing step using a solution in which the same amount of NaCl was dissolved in 30 mL of distilled water, was freeze-dried and compared with the third product.

[0112] According to Figure 11, when mucosal tissue is crushed alone and then washed with an NaCl solution, a porous structure is observed on the surface of the tissue. However, in the extracellular matrix obtained by washing freeze-crushed mucosal tissue containing crystals with distilled water, a shape appearing to be torn or broken was mainly observed. From this, it can be seen that the process of freezing-crushing with crystals mixed with the tissue enhances the effect of physically crushing the tissue, and that during this process, torn cell membranes and internal nuclear membranes flow out during the washing process, leading to decellularization.

[0113] Cell aggregates were prepared by mixing DFdECM and DTdECM obtained from tracheal cartilage tissue with adipose tissue-derived stem cells, respectively, and the effect of the extracellular matrix obtained from each process on stem cell differentiation was investigated. A control group (Media) was prepared by preparing aggregates using only stem cells and culturing them in a chondrocyte differentiation medium. Figures 12a and 12b illustrate the evaluation of the ability to regulate chondrocyte differentiation by the final product following the decellularization process of stem cells. Lacunar structures were confirmed in the H&E staining results of the three types of stem cell pellets. Here, lacuna refers to a general term for small openings, empty cavities, or hollows or depressions located in the middle or between other body tissues. Compared to the Media group, the morphology and size of the lacuna were observed to be more distinct and larger in the DTdECM and DFdECM groups. To determine whether lacuna structures were formed as stem cells differentiated into chondrocytes within the tissue, chondrocyte differentiation markers SOX9, Aggrecan, and Collagen 2 were observed using immunofluorescence staining.

[0114] Cell aggregates obtained by mixing DFdECM and DTdECM, respectively, with adipose tissue-derived stem cells were mixed into Matrigel and implanted into the dorsal subcutaneous tissue of mice. After 4 weeks, the Matrigel was recovered and H&E staining was performed. Figures 13 and 14 show the evaluation (in vivo) of the ability to regulate chondrocyte differentiation by the final product of bone tissue formation.

[0115] As shown in Figures 13 and 14, unlike the in vitro results, almost no lacuna structures were observed in the group transplanted with only stem cell pellets (Only). In the group mixed with DTdECM, cross-sections of the cell pellets similar to the in vitro results were observed until week 2, but from week 4, the chondrogenic differentiation pattern disappeared and angiogenesis became dominant. On the other hand, in the DFdECM group, cross-sections similar to DTdECM were observed at week 2, but by week 4, clearly formed lacuna structures were observed throughout the area. Clear lacuna structures were also confirmed after 4 weeks in the group in which stem cell pellets mixed with DFdECM were prepared in stem cell growth medium (MSC media).

[0116] Safranin O staining was performed to confirm the distribution of cartilage tissue within the cell pellet. Figures 15a, 15b, and 15c show the results of histochemical staining and protein quantification analysis performed to evaluate the ability of the final product after the decellularization process of stem cells to regulate chondrocyte differentiation. According to Figures 15a and 15b, the lacuna-like form observed in the H&E staining was most clearly observed in the group treated with red DFdECM. Almost no red color was expressed in cell aggregates composed solely of stem cells or in cell aggregates mixed with DTdECM.

[0117] Immunohistochemical staining and ELISA protein quantification were performed on specific markers expressed during differentiation into chondrocytes. As a result, a large number of SOX9-expressing cells were observed in the group mixed with DFdECM. There was no difference in the expression pattern of Aggrecan between groups, while Collagen 2 showed high expression levels in stem cell aggregates and stem cell aggregates mixed with DFdECM. On the other hand, observation of Collagen 10 expression revealed a specifically high expression pattern only in stem cell aggregates mixed with DTdECM. Collagen 10 is a marker specifically expressed in hypertrophic chondrocytes, which are cells observed during the process of bone tissue development. This result may suggest that when stem cells are treated with DTdECM, they initially differentiate into chondrocytes, but some cells have the potential to differentiate into osteocytes. According to the above results, DFdECM may be a more advantageous material for differentiating stem cells into cartilage tissue because the preservation rate of original tissue-derived components is superior to the preservation rate of tissue-derived components due to the conventional decellularization process by the detergent-free decellularization process performed during production.

[0118] To confirm the in vivo immune response to DFdECM, CD68 / iNOS and CD68 / CD163 were stained as M1 / M2 specific markers for macrophages, respectively. Figures 16a and 16b show the results of histochemical and image quantification analyses performed to evaluate (in vivo) the expression patterns of inflammation-related factors after transplantation of stem cells and the final products following decellularization. According to Figure 16b, M1 macrophages co-expressing CD68 and iNOS, which induce inflammation, were observed most frequently in the group treated with stem cell aggregates mixed with DFdECM. Conversely, M2 macrophages co-expressing CD68 and CD163, which suppress inflammation and are involved in tissue regeneration, were found to be highest in the group treated with DFdECM.

[0119] From the above results, it was confirmed that the tissue freeze-grinding composition and method according to one embodiment of the present invention can efficiently remove cells from tissue, have a low rate of damage or loss of extracellular matrix, exhibit excellent effects in inducing and regulating stem cell differentiation, and produce an extracellular matrix with excellent biocompatibility.

[0120] FIG. 17 is a flowchart of a tissue freeze-grinding method according to one embodiment of the present invention.

[0121] The tissue freeze-grinding method according to FIG. 17 may include the step of cutting the tissue, which can be transplanted and regenerated, into a predetermined size (cutting step) (S100), the step of mixing the tissue cut into a predetermined size with a crystalline granular body in a predetermined content ratio (mixing step) (S200), the step of freeze-grinding the mixed composition (freeze-grinding step) (S300), the step of washing the freeze-grinded composition (washing step) (S400), and the step of dissolving the washed composition in distilled water and freeze-drying it (drying step) (S500).

[0122] The description regarding the tissue freeze-grinding method is the same as that in the tissue freeze-grinding composition, unless contradictory.

[0123] A method for freezing and grinding a tissue according to one embodiment of the present invention may include a cutting step of cutting the tissue into a predetermined size (S100). The cutting step may cut the tissue into a predetermined size, and more specifically, the tissue may be a tissue capable of being transplanted and regenerated, and may include at least one of hard tissue and soft tissue. The tissue may be prepared by cutting it into a predetermined size, wherein the predetermined size may refer to a size in which the tissue is chopping to facilitate decellularization, for example, the predetermined size may be (0.6 cm to 3 cm) × (0.6 cm to 3 cm). Preferably, the predetermined size may be 1 cm × 0.5 cm.

[0124] The chopped biological tissue can be mixed with solid crystalline granules (S200). The solid crystalline granules are mixed with the biological tissue to decellularize the tissue during freeze-grinding. The biological tissue and the solid crystalline granules can be mixed in a weight ratio of 1:0.1 to 1:12. Most preferably, the biological tissue and the solid crystalline granules can be mixed in a weight ratio of 1:2. Of course, the content ratio of the crystalline granules can be composed differently depending on the type of tissue.

[0125] In a method for freeze-grinding a tissue according to one embodiment of the present invention, the solid crystalline granular body may be one or more selected from the group consisting of NaCl, Na2CO3, CaCl2, MgCl2, and sugar. Preferably, the solid crystalline granular body may be one or more selected from the group consisting of NaCl, Na2CO3, and CaCl2.

[0126] In a method for freezing and grinding tissue according to one embodiment of the present invention, the biological tissue is a hard tissue, and the biological tissue and the solid crystalline granular body may be mixed in a weight ratio of 1:0.2 to 1:5.

[0127] In a method for freezing and grinding a tissue according to one embodiment of the present invention, the biological tissue and the solid crystalline granular body may be mixed in a weight ratio of 1:0.1 to 1:12.

[0128] A composition mixed with a biological tissue and a solid crystalline granule can be freeze-ground (S300). The composition mixed with the crystalline granule can be placed in a dedicated container for freeze-grounding and freeze-ground for a predetermined time. To freeze-ground, it can be frozen at -196°C, and the predetermined time may be 10 minutes, but is not limited thereto.

[0129] The freeze-ground composition may be washed (S400). The washing step may include a stirring step in which the freeze-ground composition is stirred with distilled water and a separation step in which the stirred composition is centrifuged to remove the supernatant, and the washing may be repeated a predetermined number of times according to the choice of a person skilled in the art. Preferably, the predetermined number of times may be three, but is not limited thereto.

[0130] The stirring step may involve placing the freeze-ground composition into distilled water and stirring for a predetermined time. The freeze-ground composition may be placed into distilled water and stirred at a predetermined rpm for a predetermined time. Preferably, 1 g of the composition may be washed in 30 mL of distilled water at 900 rpm for 2 hours. A separation step may be performed by centrifuging the composition stirred in the stirring step for a predetermined time to remove the supernatant. The separation step may involve centrifuging for a predetermined time to remove the supernatant.

[0131] After removing the supernatant from the composition, it can be dissolved in clean distilled water and freeze-dried at a predetermined temperature (S500). Preferably, 1 g of the composition can be dissolved in 10 mL of distilled water, frozen at minus 80 °C for at least 24 hours, and then freeze-dried.

[0132] The present invention provides an extracellular matrix prepared using the tissue freeze-grinding composition of the present invention, or prepared by a tissue freeze-grinding method.

[0133] The present invention provides a pharmaceutical composition for wound healing, wound treatment, tissue regeneration, epidermal regeneration, dermal regeneration, neovascularization, or skin appendage regeneration comprising an extracellular matrix prepared using the tissue freeze-grinding composition of the present invention or prepared by a tissue freeze-grinding method.

[0134] The present invention provides a pharmaceutical composition for tissue regeneration comprising an extracellular matrix obtained using a tissue freeze-grinding composition according to one embodiment of the present invention.

[0135] The present invention provides a pharmaceutical composition for tissue regeneration comprising an extracellular matrix prepared by a tissue freeze-grinding method according to one embodiment of the present invention.

[0136] The extracellular matrix (ECM) refers to an aggregate of biomolecules filling the intracellular or extracellular space of tissues, and includes collagen, elastin, etc.

[0137] In the present specification, the extracellular matrix may be a decellularized extracellular matrix. An extracellular matrix prepared using the tissue freeze-grinding composition of the present invention or by a tissue freeze-grinding method may be a decellularized extracellular matrix.

[0138] A tissue freeze-grinding composition according to one embodiment of the present invention shortens the decomposition time of the extracellular matrix, thereby reducing the manufacturing process time, and minimizes protein denaturation and loss through detergent-free washing and shortened manufacturing time. In addition, an extracellular matrix protein raw material applicable to both hard and soft tissues is formed, making it applicable to various medical fields.

[0139] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0140] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

[0141]

[0142] [National R&D projects that supported this invention]

[0143] [Project ID] 2710024174

[0144] [Assignment No.] 00070684

[0145] [Ministry Name] Ministry of Science and ICT

[0146] [Project Management (Specialized) Agency Name] Pan-Governmental Regenerative Medicine Technology Development Agency

[0147] [Project Name] Inter-Ministerial Regenerative Medicine Source Technology Development Project (R&D)

[0148] [Project Title] Development of Scaffolds for Regeneration of Intractable Tissue-Type Respiratory Systems via Stem Cell Spheroids and Decellularized Hybrid Ink Bioprinting

[0149] [Name of Project Performing Organization] Seoul National University Industry-Academic Cooperation Foundation

[0150] [Research Period] April 1, 2022 ~ December 31, 2026

Claims

1. A biologically derived tissue; and A tissue freeze-grinding composition comprising a solid crystalline granular body capable of decellularizing the tissue, which is mixed with the biological tissue during freeze-grinding of the biological tissue.

2. A tissue freeze-grinding composition according to claim 1, wherein the biological tissue is cut into pieces of a size of (0.6 cm to 3 cm) × (0.6 cm to 3 cm).

3. In Claim 1, The above solid-state crystalline granular body is one or more selected from the group consisting of NaCl, Na2CO3, CaCl2, MgCl2, and sugar, in a tissue freeze-grinding composition.

4. In Claim 1, A tissue freeze-grinding composition in which the above-mentioned biological tissue is derived from one or more selected from the group consisting of bone, cartilage, mucous membrane, blood vessel, liver, lung, stomach, heart, small intestine, large intestine, duodenum, skin, salivary gland, tongue, esophagus, muscle, lymph node, fat, and cornea.

5. In Claim 1, A tissue freeze-grinding composition in which the above-mentioned biological tissue and the above-mentioned solid-state crystalline granular body are mixed in a weight ratio of 1:0.1 to 1:

12.

6. In Claim 1, A tissue freeze-grinding composition in which the above-mentioned biological tissue is a hard tissue, and the above-mentioned biological tissue and the above-mentioned solid crystalline granular body are mixed in a weight ratio of 1:0.2 to 1:

5.

7. In Claim 1, A tissue freeze-grinding composition in which the above-mentioned biological tissue is soft tissue, and the above-mentioned biological tissue and the above-mentioned solid crystalline granular body are mixed in a weight ratio of 1:1 to 1:

5.

8. A step of cutting the biologically derived tissue into predetermined sizes; A step of mixing the chopped biological tissue and the solid crystalline granular body; Step of freeze-grinding the mixed composition; A step of washing the freeze-ground composition; and A method for freeze-grinding tissue, comprising the step of dissolving the washed composition in distilled water and freeze-drying it.

9. In Claim 8, A method for freezing and grinding tissue, wherein the above-mentioned predetermined size is (0.6 cm to 3 cm) x (0.6 cm to 3 cm).

10. In Claim 8, A method for freezing and grinding a tissue, wherein the solid crystalline granular body is one or more selected from the group consisting of NaCl, Na2CO3, CaCl2, MgCl2, and sugar.

11. In Claim 8, A method for freezing and grinding tissue, wherein the above-mentioned biological tissue is derived from one or more selected from the group consisting of bone, cartilage, mucous membrane, blood vessel, liver, lung, stomach, heart, small intestine, large intestine, duodenum, skin, salivary gland, tongue, esophagus, muscle, lymph node, fat, and cornea.

12. In claim 8, A method for freeze-grinding tissues in which the above-mentioned biological tissue and the above-mentioned solid crystalline granular body are mixed in a weight ratio of 1:0.1 to 1:

12.

13. In Claim 8, A method for freeze-grinding tissue, wherein the above-mentioned biological tissue is a hard tissue, and the above-mentioned biological tissue and the above-mentioned solid crystalline granular body are mixed in a weight ratio of 1:0.2 to 1:

5.

14. In Claim 8, A method for freeze-grinding tissue, wherein the above-mentioned biological tissue is soft tissue, and the above-mentioned biological tissue and the above-mentioned solid crystalline granular body are mixed in a weight ratio of 1:1 to 1:

5.

15. A pharmaceutical composition for tissue regeneration comprising an extracellular matrix prepared by the method of Claim 8.

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