Preparation method of human-derived acellular dermal matrix

Abstract

The present invention relates to a method for preparing an acellular dermal matrix derived from a human body, in which the acellular dermal matrix is washed for the second time with an alcohol-based degreasing solution, thereby not only having excellent safety against cytotoxicity, but also being able to effectively remove residual fat.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing human-derived acellular dermal matrix. BACKGROUND

[0002] Acellular dermal matrix (ADM) is widely used in soft tissue reconstruction surgery by removing cells in the dermis after removing the epidermis from the skin tissue of a donated cadaver to eliminate an immune rejection reaction, and is also widely used as an allograft for burn treatment. The dermal tissue is composed of 80 to 90% of collagen, elastin, and glycosaminoglycan.

[0003] On the other hand, LifeCell Corporation developed a process of treating sliced skin tissue with 1M NaCl, incubating at a temperature of 37℃ for 18 to 32 hours in the case of human skin and for 35 to 55 hours in the case of pig skin to remove epidermal tissue, and then treating with a decellularization solution containing 0.5% SDS HBSS and 1mM EDTA for 1 hour at a condition of 40±5 RPM to perform decellularization, and then treating with a cryopreservation solution. However, since the epidermis is removed using NaCl, a process of only separating the epidermis is separately required, and there is a problem in that not only the process time is lengthened, but also the epidermis and the dermis are not well separated.

[0004] In order to solve the above problems, LNC Bio Corporation replaced NaCl with neutral proteolytic enzymes such as neutral protease, thermolysin, and trypsin, and Decell Technologies Inc. replaced NaCl with a hypotonic solution to shorten the process time, but if the use concentration and the treatment time are not adjusted, there is a problem in that tissue damage can occur and fat remains in the dermis.

[0005] Therefore, there is an urgent need to develop a method that can remove residual fat without damaging the tissue. SUMMARY

[0006] TECHNICAL PROBLEM

[0007] The present application was proposed to solve the problems as described above, and an object thereof is to provide a method for preparing human-derived acellular dermal matrix, which can effectively remove fat remaining in the dermis.

[0008] TECHNICAL SOLUTION

[0009] To achieve the object, a method for preparing human-derived acellular dermal matrix according to an embodiment of the present application includes the following steps: step (i), preparing human-derived skin tissue which has not been subjected to a separate de-epidermization and de-fat process; step (ii), subjecting the skin tissue to an impregnation treatment in a hypotonic solution containing a surfactant; step (iii), subjecting the skin tissue subjected to the impregnation treatment in the step (ii) to a first washing with an isotonic solution; and step (iv), subjecting the skin tissue subjected to the first washing in the step (iii) to a second washing with a de-lipid solution.

[0010] Effects of the Invention

[0011] According to an embodiment of the present application, the method for preparing human-derived acellular dermal matrix subjects the second washing to a de-lipid solution of an alcohol, thereby not only having excellent safety against cytotoxicity but also effectively removing residual fat.

[0012] Also, the third washing is performed with purified water, thereby reducing the amount of IPA residue to a low toxicity, and thus safety can be improved.

[0013] Also, it is possible to minimize damage to the tissue, reduce the amount of detergent residue in the tissue, and have excellent safety against cytotoxicity. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is a flowchart for explaining the method for preparing human-derived acellular dermal matrix according to an embodiment of the present application.

[0015] Figure 2 is an image showing before and after removing residual fat with IPA.

[0016] Figure 3 a and Figure 3 b is an image showing the results according to Experimental Example 1.

[0017] Figure 4 and Figure 5 is a graph showing the results according to Experimental Example 2.

[0018] Figure 6 is a graph showing the results of 3-1 according to Experimental Example 3.

[0019] Figure 7 is an image showing the results of 3-2 according to Experimental Example 3.

[0020] Figures 8 to 11 is a graph showing the results according to Experimental Example 4.

[0021] Figures 12a to 12c is an image showing the results according to Experimental Example 5.

[0022] Figure 13a Table for showing the results according to Experimental Example 6. Figure 13b Table for showing the results according to Experimental Example 6.

[0023] Figures 14a to 14c Figure for showing the results according to Experimental Example 7.

[0024] Figure 15 Figure for showing the results according to Experimental Example 8. DETAILED DESCRIPTION

[0025] Hereinafter, the present application will be explained in detail by examples of the present application with reference to the accompanying drawings. However, the following examples are provided as examples of the present application, and detailed explanation of the technology or structure which is judged as being well known to one of ordinary skill in the art can be omitted when it is judged that the detailed explanation can unnecessarily obscure the main point of the present application, and the present application is not limited thereto. The present application can be modified and applied within the scope of the recitations of the claims and the equivalent scope interpreted therefrom.

[0026] Also, the terminology used in the present specification is used to appropriately express preferred embodiments of the present application, and can be changed according to the intention of a user, an operator, or a custom in the field to which the present application pertains, etc. Therefore, the terms should be defined based on the overall content of the present specification. Throughout the specification, when a certain part "comprises" a certain structural element, unless otherwise specifically recited contrarily, it means that other structural elements can also be included, rather than excluding other structural elements.

[0027] Throughout the present specification, unless otherwise specified, "%" used to express the concentration of a certain substance is % (w / w) for solid / solid, % (w / v) for solid / liquid, and % (v / v) for liquid / liquid.

[0028] In the human-derived acellular dermal matrix of the present application, the dermal matrix can be bone, ligament, tendon, or skin, and can include dermal tissue of xenogenic or allogenic origin.

[0029] Hereinafter, a method of preparing a human-derived acellular dermal matrix according to an embodiment of the present application will be explained in detail with reference to the accompanying drawings.

[0030] Figure 1 FIG. 1 is a flowchart for explaining a method of preparing a human-derived acellular dermal matrix according to an embodiment of the present application.

[0031] Reference Figure 1 First, a human-derived skin tissue which has not been subjected to a separate de-epidermization and de-fat process is prepared (step S10).

[0032] Then, the skin tissue is subjected to an immersion treatment in a hypotonic solution containing a surfactant (step S20).

[0033] The skin tissue prepared in the step S10 can be subjected to an immersion treatment in a hypotonic solution containing a surfactant for 2 to 24 hours, for example, 2 to 10 hours, further for example, 3 to 9 hours, further for example, 4 to 8 hours.

[0034] In the present embodiment, "hypotonic" means a state in which, when the osmotic pressures of two solutions separated by a semipermeable membrane are compared, the osmotic pressure inside is higher than that outside, that is, the concentration inside the membrane is higher than that outside, and thus the net movement of water is toward the inside of the membrane, the speed of the osmotic phenomenon as a passive diffusion phenomenon of such water increases or decreases in proportion to the concentration gradient between the two solutions, and when the concentration difference disappears, the net movement of water can disappear.

[0035] In the present embodiment, "hypotonic solution" can mean a solution having an osmolarity of about 270 mOsm / L or less, which is used for a treatment to affect cell lysis. For example, a hypotonic solution enters a cell by osmosis through a cell membrane, in which case, the cells of the tissue swell and rupture, and thus residual cells can be removed.

[0036] In the present embodiment, "osmolarity" means an expression of the osmotic concentration per unit volume of a solution (mOsm / L), and the osmotic concentration of blood plasma and other body fluids in the body can be 270 to 300 mOsm / L. If the osmolarity of a solution is about 270 mOsm / L or less, it can be defined as a hypotonic solution, if the osmolarity of a solution is in the range of about 270 to 300 mOsm / L, it can be defined as an isotonic solution, and if the osmolarity of a solution is about 300 mOsm / L or more, it can be defined as a hypertonic solution.

[0037] The low-osmolarity solution of the present embodiment has an osmolarity of 270 mOsm / L or less, and preferably, can contain 5 to 20 mM of Tris-HCl, 0.1 to 1.5% of ethylenediaminetetraacetic acid (EDTA), and 0.01 to 0.05 M of sodium hydroxide (NaOH), and more preferably, can contain 8 to 12 mM of Tris-HCl, 0.7 to 1.2% (w / v) of ethylenediaminetetraacetic acid (EDTA), and 0.01 to 0.02 M of sodium hydroxide (NaOH).

[0038] On the other hand, the surfactant can include an ionic surfactant, a non-ionic surfactant, or a mixture thereof, and preferably, can include an ionic surfactant. For example, the ionic surfactant can include one or more selected from the group consisting of alkyl benzene sulfonate, alkyl sulfate, alkyl ether sulfate, alkoxylated amide, olefin sulfonate, alkyl xylene sulfonate, dialkyl sulfosuccinate, fatty acid ester sulfonate, alcohol sulfate, glycerol fatty acid ester, glutamate, isethionate, phosphate such as lauryl phosphate or laureth-1-phosphate, taurate, alkoxylated alcohol, alkyl carboxylate, hydroxyalkyl, quaternary ammonium salt, and ethoxylated alkyl, and preferably, can include one or more selected from the group consisting of sodium dodecyl sulfate (SDS), sodium laureth sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium myreth sulfate (SMES), dioctyl sodium sulfosuccinate (DSS), perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate (PFBS), perfluorononanoate (PFNA), perfluorooctanoate (PFOA), and perfluorobutane sulfonate (PFBS).One or more of the group consisting of sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (SDBS), sodium dodecanedioate (SDD), sodium perfluorooctanoate (PFOA), sodium stearate, sodium lauroyl sarcosinate, cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride (DDAC), and dioctadecyldimethylammonium bromide (DODAB), more preferably, can be sodium dodecyl sulfate (SDS), but is not limited thereto.

[0039] In the past, a method of lysing a cell membrane by using a nonionic surfactant or a high concentration of an ionic surfactant has been used to remove cell components other than proteins in a tissue, but there are problems in that residual proteins in liposomes are denatured, the microstructure of an extracellular matrix is broken, and growth factors are excessively removed. Therefore, in the present embodiment, the above-described problems are solved by using a low concentration of an ionic surfactant. Specifically, a low concentration of an alkyl sulfate, more preferably, a low concentration of sodium dodecyl sulfate (SDS) is used as an ionic surfactant. The concentration of the sodium dodecyl sulfate (SDS) in a hypotonic solution can be 0.1 to 0.5% (w / v), more preferably, can be 0.2 to 0.4% (w / v), but is not limited thereto.

[0040] Also, the skin tissue is first washed with an isotonic solution (step S30).

[0041] The skin tissue subjected to the immersion treatment in the step S20 can be washed with an isotonic solution for 1 to 10 times for 1 to 24 hours each time, for example, can be washed for 1 to 10 times for 1 to 10 hours each time, and further, for example, can be first washed for 2 to 7 times for 2 to 7 hours each time.

[0042] In the present embodiment, the osmolarity of the isotonic solution is about 270 to 300 mOsm / L, and preferably, can include 25 to 100 mM of Tris-HCl, 0.1 to 1.5% (w / v) of ethylenediaminetetraacetic acid (EDTA), 0.01 to 0.05 M of sodium hydroxide (NaOH), and 0.05 to 0.3 M of sodium chloride (NaCl), and more preferably, can include 45 to 55 mM of Tris-HCl, 0.8 to 1.2% (w / v) of ethylenediaminetetraacetic acid (EDTA), 0.01 to 0.02 M of sodium hydroxide (NaOH), and 0.05 to 0.3 M of sodium chloride (NaCl).

[0043] The concentration of NaCl in the isotonic solution described above can be 0.05 to 0.5 M, and preferably, the concentration can be 0.1 to 0.3 M, because, when the concentration of the isotonic solution exceeds the above range, the epidermis can not be well separated depending on the raw material of the skin.

[0044] In the present embodiment, "isotonic" can mean a state in which the osmotic pressure of two solutions separated by a semipermeable membrane is the same, i.e., a state in which there is no net movement of water due to the same concentration. If the concentrations of two solutions separated by a membrane are different, water moves from a low concentration to a high concentration, and the speed of the osmotic phenomenon, which is a passive diffusion phenomenon of such water, can be proportional to the concentration gradient between the two concentrations.

[0045] Between step S20 and step S30 and between step S30 and step S40, a step of washing with PBS for 6 to 24 hours or washing 1 to 10 times for 5 to 60 minutes each can be further included.

[0046] Then, the skin tissue subjected to the first washing is subjected to a second washing with a delipidation solution (step S40).

[0047] The skin tissue subjected to the first cleaning in the step S30 can be subjected to a second cleaning with a delipidization solution having a concentration of 50 to 100%, preferably 80 to 100% at a temperature of 4 to 40°C, for example, 20 to 40°C, further for example, 30 to 40°C for 2 to 24 hours, further for example, 3 to 24 hours, further for example, 3 to 5 hours. When the concentration of the delipidization solution is less than 50%, the fat removal rate can be very low, less than 20%, and thus is not desirable. When the cleaning time is less than 2 hours, the fat removal rate can be very low, less than 20%, and when more than 24 hours, the increase in the fat removal rate can not be large with respect to the increase in time. When the cleaning temperature is less than 4°C, the fat removal rate can be very low, less than 10%, and when more than 40°C, the skin tissue can be denatured.

[0048] When the preferred concentration of the delipidization solution, the preferred temperature and time conditions for the second cleaning are all satisfied, the fat removal rate can be very excellent, more than 58%.

[0049] For example, the delipidization solution can include a polar solvent, wherein the polar solvent can be an alcohol, and preferably, can include one or more selected from the group consisting of isopropanol (IPA), ethanol, methanol, butanol, octanol, and water, and preferably, can include isopropanol.

[0050] Finally, the skin tissue subjected to the second cleaning is subjected to a third cleaning with purified water (step S50).

[0051] The skin tissue subjected to the second cleaning in the step S40 can be subjected to a third cleaning with purified water for 4 to 24 hours, preferably 6 to 24 hours. When the cleaning time is less than 4 hours, toxicity can be generated due to the delipidization solution remaining in the skin tissue of more than 8000 ppm, and when more than 24 hours, the decrease in the remaining rate of the delipidization solution can not be large with respect to the increase in time. When the preferred time is satisfied, the remaining rate of the delipidization solution is 1500 ppm or less, and thus can be in a safe state.

[0052] In the present embodiment, the purified water can be sterile distilled water (DW).

[0053] Hereinafter, the present application will be described in more detail through examples. These examples are only for a more detailed description of the present application, and it will be apparent to those skilled in the art that the scope of the present application is not limited to these examples.

[0054] Example 1. Preparation of decellularized dermal matrix

[0055] The skin tissue was purchased from EURO skin bank, Allosource, CTS, and the tissue having a thickness of 1 mm or more was selected, and fat tissue attached to the skin tissue was removed using forceps, and then washed with sterile water 3 times. Then, immersion treatment was performed in a hypotonic solution (10 mM of Tris-HCl, 1 g of EDTA, 13.5 mL of 1M NaOH) containing 0.25% of SDS for 6 hours. And, the skin tissue was washed with PBS at a temperature of 4°C to remove fat, epidermis, cells, and hypotonic solution remaining in the skin tissue, overnight, and then first washing was performed with an isotonic solution (50 mM of Tris-HCl, 1 g of EDTA, 0.15 M of NaCl, 13.4 mL of NaOH) for 6 hours to prepare a human-derived acellular dermal matrix.

[0056] Examples 2 to 5. Preparation of decellularized dermal matrix with residual fat removed

[0057] The human-derived acellular dermal matrix prepared in Example 1 was second-washed according to the conditions shown in Table 1 below, and third-washed with purified water for 2 hours or more to prepare an acellular dermal matrix from which residual fat was removed, and images thereof before and after are shown in FIGS. 1 to 4. Figure 2

[0058] Table 1

[0059]

[0060]

[0061] Example 14. Preparation of a composition comprising fibrous decellularized dermal matrix

[0062] In Example 7, the acellular dermal matrix was third-washed with sterile purified water for 4 hours instead of 2 hours to prepare an acellular dermal matrix from which IPA was removed.

[0063] Example 15. Preparation of decellularized dermal matrix with IPA removed

[0064] In Example 7, the acellular dermal matrix was third-washed with sterile purified water overnight (O / N) instead of 2 hours to prepare an acellular dermal matrix from which IPA was removed.

[0065] Experimental Example 1. Acellularization evaluation (hematoxylin-eosin staining (H&E staining))

[0066] Acellularization evaluation was performed by histological examination of the human-derived dermal matrix prepared in Example 1 using H&E staining.

[0067] ​The paraffin block was cut into a section having a thickness of 4μ and dried to make a paraffin section. Then, the deparaffinization process was performed by reacting in xylene for 5 minutes 3 times, in 100% ethanol for 2 minutes 3 times, in 90% ethanol for 1 minute 1 time, in 80% ethanol for 1 minute 1 time, in 70% ethanol for 1 minute 1 time, and then washing with running water for 10 minutes. Hematoxylin staining was performed by reacting for 10 minutes, and then washing with running water for 3 minutes, Eosin staining was performed by reacting for 10 minutes, and then washing with running water until the Eosin staining disappeared. Ethanol was reacted for 1 second 10 times in 70% ethanol, for 1 second 10 times in 80% ethanol, for 1 second 10 times in 90% ethanol, for 1 minute 2 times in 100% ethanol, for 3 minutes 3 times in xylene, and then, the section was mounted with a mounting medium, and then, photographed using an optical microscope (Olympus BX51, Hematoxylin-Eosin staining (H&E staining)) and a scanning electron microscope (Hitachi S-4700, Japan), and the results thereof are shown in FIGS. 1 to 3. Figure 3

[0068] Referring to FIGS. 1 to 3, Figure 3 It can be confirmed that the epidermis was peeled off in Example 1 compared to Control 1 (Fresh skin). Also, comparing the results of the decellularization effects of 1% Triton X-100, 1% Tween 80, 0.1% SDS solution, and a hypotonic solution containing 0.25% SDS under the same treatment time conditions, it can be confirmed that when treated with 1% Triton X-100, 1% Tween 80, and 0.1% SDS solution, the cells in the skin tissue were not removed, and when treated with a hypotonic solution containing 0.25% SDS, most of the cells in the tissue were removed (data not shown).

[0069] Also, it can be confirmed that in the group of skin tissue treated with sonication for 1 hour and skin tissue treated with sonication and 1% Triton X-100 for 1 hour, the cells were not removed and the tissue was damaged (data not shown). Therefore, it can be known that when skin tissue not subjected to the epidermis removal (using NaCl or proteolytic enzyme) and fat removal (using HIPS) process is treated with a surfactant solution such as 1% Triton X-100, 1% Tween 80, and 0.1% SDS solution, decellularization is not performed, and when treated with a hypotonic solution containing 0.25% SDS under the same conditions as this, it is confirmed that the epidermis, fat, and cells can be removed in one step. ​

[0070] That is, it is known that even if a physical process combined with ultrasonic treatment and a surfactant is performed to remove the epidermis, fat, and cells at one time, the cells cannot be removed.

[0071] Experimental Example 2: Detergent Residual Amount Evaluation

[0072] A methylene blue dye binding assay was performed to evaluate the detergent residual amount in the tissue.

[0073] The existing process (a sample in which the epidermis was removed using NaCl, the fat was removed, and the cells were removed using SDS) and the dermal tissue of Example 1 were dried, and then the eluate eluted with PBS was used to evaluate the detergent (SDS) residual amount.

[0074] A detergent (SDS) solution was diluted with methylene blue to different concentrations (1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0%), and then eluted with chloroform, and then the absorbance at 650 nm was measured to make a standard curve, and then the detergent residual amount was confirmed by measuring the absorbance at 650 nm of the eluate eluted from the existing process and Example 1 for a new process, and the results thereof are shown in Table 1 and Table 2. Also, the washing solution of each washing step of the dermal tissue of Example 1 was taken and the detergent residual amount was measured, and the results thereof are shown in Table 1 and Table 2. Figure 4 Figure 5

[0075] During the elution process, the dermal tissue having a thickness of about 3 mm and a weight of 4 g was selected, and eluted at a temperature of 37°C for 72 hours to obtain 20 mL of an eluate.

[0076] Referring to Table 1 and Table 2, it can be confirmed that the detergent residual amount in the eluate of Example 1 was about 32.87 ± 6.42 mg / L, which was lower than that of the existing process (data not shown). Figure 4 That is, it can be confirmed that the SDS detergent residual amount determined by measuring the absorbance at 650 nm of the eluate eluted with PBS of the human-derived acellular dermal matrix prepared according to the present application was 40 mg / L or less.

[0077] Table 2

[0078]

[0079] ​​​

[0080] Reference Figure 5 It was confirmed that the more the PBS was used for washing, the less the amount of detergent in the washing solution was reduced.

[0081] Experimental Example 3: Safety Evaluation (Cytotoxicity Analysis)

[0082] MTT assay and MEM Elution Test in ISO 10993-5: Cytotoxicity Test were performed to evaluate the safety through cell compatibility and toxicity.

[0083] 3-1. MTT assay (cell compatibility)

[0084] L929 cells were seeded in a 96-well plate at a concentration of 1 x 10 5 cells / mL (100 μL) per well and incubated for 24 hours. Then, the cells were treated with the eluate of the existing process and the eluate of Example 1 as a new process and incubated at a temperature of 37°C for 24 hours. The eluate was obtained by eluting 20 mL of the dermal tissue having a thickness of about 3 mm and a weight of 4 g using an animal cell culture medium at a temperature of 37°C for 24 hours after selecting the dermal tissue from the existing process and the new process.

[0085] After treating and incubating the cells with the eluate for 24 hours, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent was diluted at a ratio of 1:100 in a culture medium, and 0.1 mL thereof was added to each well and incubated for 5 hours. After 5 hours, the culture medium was removed from each well of the 96-well plate, and 0.1 mL of dimethylsulfoxide (DMSO) was added to each well to lyse the cells. Then, the 96-well plate was placed in a Spectrophotometer, and the absorbance was measured at a wavelength of 540 nm, the results of which are shown in Table 1. Figure 6

[0086] Reference Figure 6 As can be seen, the cell viability values of Example 1 as a new process were 96.26%, 101.49%, and 94.99%. This is higher than the cell viability values of the existing process, and thus the cell compatibility of Example 1 is higher than that of the existing process.​

[0087] That is, it can be confirmed that the cell viability determined by measuring the absorbance at 540 nm of the eluate of the human-derived acellular dermal matrix prepared according to the present application eluted with the MEM medium is very high, being 90% or more.

[0088] 3-2. Minimum Essential Medium (MEM) elution test (toxicity)

[0089] L929 cells were seeded at a concentration of 1 x 10 5 cells / mL (4 ml) in a 6-well plate and cultured for 24 hours, and then the monolayer culture state of the cells was confirmed. Then, the cells were treated with the eluate according to the existing process and Example 1 as a new process and cultured for 48 hours. The eluate used 20 mL of the eluate obtained by selecting dermal tissue having a thickness of about 3 mm and a weight of 4 g from the dermal tissue according to the existing process and Example 1 as a new process and eluting the same with the MEM medium at a temperature of 37°C for 24 hours. After 24 hours, 48 hours of culture, DMSO and MEM were used instead of the positive control substance and the negative control substance, respectively.

[0090] After 24 hours, 48 hours of culture, the degree of growth and the degree of lysis of the cells were observed with a microscope, and the results were shown in Table 3 below. Figure 7 and Table 4. When the grade was determined to be 2 or less, it was judged that there was no cytotoxicity in the eluate of the test substance.

[0091] Table 3

[0092]

[0093]

[0094] Referring to Figure 7 and Table 4, it can be confirmed that Example 1 was also determined to be 0 grade after 48 hours, but the cytotoxicity of the existing process after 48 hours was 2 grade. Therefore, it can be known that the existing process is also applicable to cell compatibility and toxicity evaluation, and Example 1 corresponding to the new process exhibits higher applicability, i.e., higher safety, than the existing process.

[0095] Table 4

[0096]

[0097] Experimental Example 4: Measurement of fat removal rate according to IPA washing conditions

[0098] The fat removal conditions were roughly classified into the following 4 conditions, and the evaluation results under each test condition were plotted into a graph, and the results were shown inFigures 8 to 11 In the middle.

[0099] The sample subjected to the second IPA washing was freeze-dried for 24 to 72 hours, and then cut into a 0.5 x 0.5 cm size slice, and the weight of the sample and the empty bottle was measured. Also, the sample and the bottle were installed in a crude fat extraction device, and crude fat was extracted by a Soxhlet extraction method. Then, the weight of the extracted crude fat was measured, and the crude fat content was calculated according to the following equation (1), and then the fat removal rate of the IPA washing group with respect to the crude fat content of the control group sample was calculated according to the following equation (2). That is, since the thinner the thickness of the tissue, the more the fat removal rate increases, the fat removal rate was calculated as a difference from each control group so as to be able to offset the difference in results due to the difference in the thickness of the tissue applied in the examples.

[0100] Crude fat content (%) = [(residual bottle weight - empty bottle weight) / sample weight] x 100... Equation (1)

[0101] Fat removal rate (%) = [(control group crude fat content - experimental group crude fat content) / control group crude fat content] x 100... Equation (2)

[0102] As described below, the applicant of the present application can confirm that the fat removal rate is increased by 20% or more compared to the case where only the first washing is performed with an isotonic solution (control group).

[0103] 4-1. Determination of fat removal rate according to IPA washing concentration

[0104] In order to confirm the fat removal rate according to the IPA washing concentration, the fat removal rates of Examples 2 to 4 in which the second washing was performed with 50%, 70%, and 100% IPA, respectively, were measured, and the results thereof are shown in Table 1. Figure 8 In the middle.

[0105] Referring to Table 1, Figure 8 It can be confirmed that the fat removal efficiency is the highest when washing is performed with 100% IPA.

[0106] 4-2. Determination of fat removal rate according to washing temperature

[0107] In order to confirm the fat removal rate according to the washing temperature, the fat removal rates of Example 5 in which washing was performed at a temperature of 4°C, Example 6 in which measurement was performed at a temperature of 20 to 25°C, and Example 7 in which washing was performed at a temperature of 38°C were measured, respectively, and the results thereof are shown in Table 2. Figure 9 In the middle.

[0108] Referring to Table 2, Figure 9 The fat removal efficiency increases in the order of Example 5, Example 6, and Example 7, and thus it can be known that the higher the washing temperature, the higher the fat removal efficiency.

[0109] On the other hand, in this invention, when measuring the fat removal rate based on the washing temperature, even for the same batch (Lot), if the fat content measured in the control group tissue is too high or too low, there may be errors in the fat removal efficiency measured under all experimental group conditions being too high or too low. Taking this into consideration, the fat removal rate is measured based on the result calculated based on the average fat content of the control group tissue.

[0110] 4-3. Determination of fat removal rate according to washing time

[0111] To confirm the fat removal rate based on washing time, the fat removal rates of sliced ​​samples (Examples 8 to 10) and whole samples (Examples 11 and 12) were measured, and the results are shown below. Figure 10a and Figure 10b middle.

[0112] refer to Figure 10a and Figure 10b In the case of slices, the fat removal efficiency increases sequentially from Example 8, Example 9, and Example 10, indicating that the longer the washing time, the higher the fat removal efficiency. In the case of whole form, similar to slices, Example 12, with its longer washing time, exhibits higher fat removal efficiency.

[0113] 4-4. Determination of fat removal rate according to tissue morphology

[0114] To confirm the fat removal rate based on tissue morphology, the fat removal rates of Example 7 (whole morphology), Example 9 (slice morphology), and Example 13 (powder morphology) were measured, and the results are shown below. Figure 11 middle.

[0115] refer to Figure 11 The fat removal efficiency increases sequentially from Example 7, Example 9 and Example 13, but it can be seen that the fat removal efficiency is excellent in almost all tissue morphologies.

[0116] Experimental Example 5: Determining whether residual fat was removed based on tissue morphology

[0117] To confirm whether residual fat had been removed based on tissue morphology, residual fat in the tissues of the control group (Example 1), Example 7, and Example 9 was stained and photographed using an optical microscope. The results are shown below. Figures 12a to 12c middle.

[0118] refer to Figures 12a to 12cResidual fat was confirmed in the control group before IPA washing, but no residual fat was confirmed at all in Example 7 and Example 9 after IPA washing. Thus, it was known that most of the residual fat was removed by IPA washing.

[0119] Experimental Example 6: Measurement of IPA Residual Amount

[0120] To confirm the IPA residual amount according to the DW washing time, the IPA residual amounts of Example 14 washed for 4 hours and Example 15 washed overnight were measured according to the GC-FID test method, and the results thereof are shown in Table 2. Figure 13a Figure 13b

[0121] Referring to Table 2, it can be confirmed that the IPA residual amount of Example 14 is 7975 ppm, and the IPA residual amount of Example 15 is 1248 ppm. Figure 13b

[0122] Experimental Example 7: ADM powder particle distribution uniformity evaluation

[0123] To confirm the particle distribution uniformity according to whether residual fat is removed, the human-derived acellular dermal matrix prepared according to Example 1, Example 7, and Example 9 was freeze-dried for 24 to 72 hours, then pulverized into a μm unit using a freeze pulverizer, and the particle size and distribution were measured using the Mie theory of the laser diffraction method of the particle size analyzer, and the results thereof are shown in Table 3. Figures 14a to 14c

[0124] The span value is the distribution width of the sample, and the wider the distribution of the particles, the greater the value, and the narrower the distribution, the value approaches 0.

[0125] Referring to Table 3, it was measured that the average particle size of the control group (Example 1), Example 7, and Example 9 was 100 μm or less, and there was no significant difference, but the span value measured in the control group was high, and it can be confirmed that the particle size was relatively non-uniform. Thus, it was known that Example 7 and Example 9 are suitable for use as injectables such as an injectable ADM product prepared using an ADM powder. Figures 14a to 14c Experimental Example 8: Measurement of change in amino acid content after second washing

[0126]

[0127] ​​​​​To confirm the change in the amino acid content according to the IPA treatment, the amino acid content of the control group (Example 1) and Example 7 was measured using an amino acid autoanalyzer (S433D, Sykam GmbH Co., Germany), and the results are shown in Table 3. Figure 15

[0128] Referring to Table 3, Figure 15 It can be confirmed that the amino acid content in Example 7 is not reduced compared to the control group, and thus it can be known that the IPA treatment does not affect the constituent amino acids of the tissue.

[0129] The above, the exemplary embodiments of the present application are described in detail, but the scope of the present application is not limited thereto, the person of ordinary skill in the art with the basic concept of the present application defined by the following claims to make various modifications and improvements also fall within the scope of the present application.

[0130] Unless otherwise defined, all technical terms used in the present application have the same meanings as commonly understood by one of ordinary skill in the art to which the present application pertains. All publications cited in the specification are incorporated by reference herein.​

Claims

1. A method for preparing acellular dermal matrix derived from human tissue, characterized in that, Includes the following steps: Step (i) is to prepare human skin tissue that has not undergone separate descaling and degreasing processes. Step (ii) involves immersing the skin tissue in a hypotonic solution containing a surfactant. Step (iii) involves first rinsing the skin tissue that underwent immersion treatment in step (ii) with an isotonic solution; as well as In step (iv), the skin tissue that was first cleaned in step (iii) is cleaned a second time with a delipinizing solution.

2. The method for preparing human-derived decellularized dermal matrix according to claim 1, characterized in that, Following step (iv), there is also step (v), in which the skin tissue that was second-washed in step (iv) is washed a third time with purified water.

3. The method for preparing human-derived acellular dermal matrix according to claim 1, characterized in that, In step (ii), the surfactant includes ionic surfactants, nonionic surfactants, or mixtures thereof.

4. The method for preparing human-derived decellularized dermal matrix according to claim 1, characterized in that, In step (ii), the hypotonic solution contains 5–20 mM Tris-HCl, 0.1–1.5% (w / v) ethylenediaminetetraacetic acid, and 0.01–0.05 M sodium hydroxide.

5. The method for preparing human-derived decellularized dermal matrix according to claim 1, characterized in that, In step (ii), the concentration of surfactant in the hypotonic solution is 0.1 to 0.5% (w / v).

6. The method for preparing human-derived decellularized dermal matrix according to claim 1, characterized in that, In step (iii), the isotonic solution contains 25–100 mM Tris-HCl, 0.1–1.5% (w / v) ethylenediaminetetraacetic acid, 0.01–0.05 M sodium hydroxide, and 0.05–0.3 M sodium chloride.

7. The method for preparing human-derived decellularized dermal matrix according to claim 1, characterized in that, In step (iv), the second washing is performed at a temperature of 4–40°C with a 50–100% delipidation solution for 2–24 hours.

8. The method for preparing human-derived decellularized dermal matrix according to claim 1, characterized in that, In step (iv), the delipidation solution contains a polar solvent.

9. The method for preparing human-derived decellularized dermal matrix according to claim 2, characterized in that, In step (v), the third cleaning takes 4 to 24 hours.

10. The method for preparing human-derived decellularized dermal matrix according to claim 1, characterized in that, Compared with the control group, which was prepared by omitting step (iv) and proceeding through steps (i), (ii), and (iii) using human-derived decellularized dermal matrix, the fat removal rate, as determined by formulas (1) and (2) below, was increased by more than 20%. Crude fat content (%) = [(weight of residue bottle - weight of empty bottle) / weight of sample] × 100………Equation (1) Fat removal rate (%) = [(crude fat content of control group - crude fat content of experimental group) / crude fat content of control group] × 100 ………Equation (2).

11. A human-derived acellular dermal matrix, characterized in that, It was prepared by repeatedly washing human-derived skin tissue that had not undergone separate descaling and degreasing processes. The cleaning includes cleaning with an isotonic solution and cleaning with a degreasing solution. The delipidated solution contains a polar solvent.

12. The human-derived acellular dermal matrix according to claim 11, characterized in that, The residual amount of SDS detergent was 40 mg / L, as determined by measuring the absorbance of the eluent eluted with MEM culture medium at 650 nm.

13. The human-derived acellular dermal matrix according to claim 11, characterized in that, Cell viability was over 90% when the absorbance of the eluent eluted with MEM culture medium was measured at 540 nm.

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