Manufacturing method of acellular dermal matrix derived from human

Abstract

The present invention relates to a method for producing a human-derived acellular dermal matrix that not only has excellent safety against cytotoxicity but can also effectively remove residual fat by performing a second wash with an alcohol-based delipid solution.

Description

Technology Field

[0001] The present invention relates to a method for manufacturing a cell-free dermal matrix derived from the human body. Background Technology

[0003] Acellular dermal matrix (ADM) is obtained by removing the epidermis and cells within the dermis from skin tissue of a donated cadaver to eliminate immune rejection. It is widely used in the field for soft tissue reconstruction and is also widely used for allografts for burn treatment. Dermal tissue is composed of 80–90% collagen, elastin, and glycosaminoglycans.

[0004] Meanwhile, LifeCell had developed a process in which fragmented skin tissue was treated with 1 M NaCl and incubated at 37°C for 18–32 hours for human skin and 35–55 hours for porcine skin to remove the epidermal tissue, followed by decellularization using a decellularization solution containing 0.5% SDS HBSS and 1 mM EDTA at 40±5 RPM for 1 hour, and then treated with a cryopreservation solution. However, since removing the epidermis by applying NaCl requires a separate process solely for epidermal separation, there was a problem in that the process time was prolonged and the epidermis and dermis were not separated well.

[0005] To solve the aforementioned problems, L&C Bio replaced NaCl with neutral proteolytic enzymes such as dyspase, termolicin, and trypsin, and Decell Technologies Inc. replaced NaCl with a hypotonic solution to shorten the process time; however, if the concentration used and processing time were not controlled, it caused tissue damage and resulted in residual fat in the dermis.

[0006] Therefore, there is an urgent need to develop a method to remove residual fat without damaging the tissue. Prior art literature

[0008] Republic of Korea Registered Patent No. 10-1523878 (May 21, 2015) The problem to be solved

[0009] The objective of the present invention is to provide a method for manufacturing a human-derived acellular dermal matrix capable of effectively removing residual fat within the dermis, which is derived to solve the problem described above. means of solving the problem

[0011] To achieve the above objective, a method for preparing a human-derived acellular dermal matrix according to one embodiment of the present invention comprises: (i) a step of preparing human-derived skin tissue that has not undergone a separate deepidermal and defat removal process; (ii) a step of immersing the skin tissue in a hypotonic solution containing a surfactant; (iii) a step of washing the skin tissue immersed in step (ii) first with an isotonic solution; and (iv) a step of washing the skin tissue washed first in step (iii) second with a defat removal solution. Effects of the invention

[0013] According to one embodiment of the present invention, a method for manufacturing a human-derived acellular dermal matrix can effectively remove residual fat as well as have excellent safety against cytotoxicity by performing a second wash with an alcohol-based delipid solution.

[0014] In addition, safety can be improved by lowering the residual IPA amount to a low toxicity level through a third wash with purified water.

[0015] In addition, it minimizes tissue damage, reduces detergent residue in tissues, and offers excellent safety regarding cytotoxicity. Brief explanation of the drawing

[0017] FIG. 1 is a process diagram for explaining a method for manufacturing a human-derived cell-free dermal matrix according to an embodiment of the present invention. Figure 2 is a photograph showing the condition before and after removing residual fat with IPA. Figures 3a and 3b are photographs showing the results according to Experimental Example 1. Figures 4 and 5 are graphs showing the results according to Experimental Example 2. Figure 6 is a graph showing the results according to 3-1 in Experimental Example 3. Figure 7 is a photograph showing the results according to 3-2 in Experimental Example 3. Figures 8 to 11 are graphs showing the results according to Experimental Example 4. Figures 12a to 12c are photographs showing the results according to Experimental Example 5. Figure 13a is a table showing the samples, and Figure 13b is a table showing the results according to Experimental Example 6. Figures 14a to 14c are graphs showing the results according to Experimental Example 7. Figure 15 is a graph showing the results according to Experimental Example 8. Specific details for implementing the invention

[0018] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalent scope interpreted therefrom.

[0019] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as “comprising” a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0020] Throughout this specification, '%' used to indicate the concentration of a particular substance is %(w / w) for solid / solid, %(w / v) for solid / liquid, and %(v / v) for liquid / liquid, unless otherwise noted.

[0022] In the human-derived acellular dermal matrix of the present invention, the dermal matrix may be bone, ligament, tendon, or skin, and may include dermal tissue of heterologous or homologous origin.

[0023] Hereinafter, a method for manufacturing a human-derived cell-free dermal matrix according to one embodiment of the present invention will be described in detail with reference to the drawings.

[0024] FIG. 1 is a process diagram for explaining a method for manufacturing a human-derived acellular dermal matrix according to one embodiment of the present invention.

[0025] Referring to FIG. 1, first, human-derived skin tissue that has not undergone a separate deepidermal and defat removal process is prepared (S10).

[0027] Next, the skin tissue is immersed in a hypotonic solution containing a surfactant (S20).

[0028] The skin tissue prepared in step S10 above can be immersed in a hypotonic solution containing a surfactant for 2 to 24 hours, for example, 2 to 10 hours, for another example, 3 to 9 hours, or for another example, 4 to 8 hours.

[0029] In this embodiment, “hypotonic” refers to a state in which, when comparing the osmotic pressures of two solutions separated by a semipermeable membrane, the inside has a higher osmotic pressure than the outside; that is, because the concentration inside the membrane is higher than the concentration outside, the net movement of water is directed toward the inside of the membrane. The rate of this osmotic phenomenon, which is a passive diffusion of water, increases or decreases in proportion to the concentration gradient between the two solutions, and if the concentration difference disappears, the net movement of water may cease.

[0030] In this embodiment, the “hypotonic solution” is used for treatment to affect cell lysis and may refer to a solution with an osmolarity of about 270 mOsm / L or less. For example, the hypotonic solution permeates the cell membrane through osmosis and enters the cell, at which time the cells of the tissue expand and rupture, thereby removing the remaining cells.

[0031] In this embodiment, “osmolarity” refers to the expression of osmotic concentration per volume of a solution (mOsm / L), and the osmotic concentration of plasma and other body fluids may be 270 to 300 mOsm / L. If the osmolarity of the solution is about 270 mOsm / L or less, it may be defined as a hypotonic solution; if it is in the range of about 270 to 300 mOsm / L, it may be defined as an isotonic solution; and if it is about 300 mOsm / L or more, it may be defined as a hypertonic solution.

[0032] The hypotonic solution of the present example has an osmolarity of 270 mOsm / L or less, preferably contains 5 to 20 mM Tris-HCl, 0.1 to 1.5% EDTA (ethylenediaminetetraacetic acid), and 0.01 to 0.05 M NaOH (sodium hydroxide), and more preferably contains 8 to 12 mM Tris-HCl, 0.7 to 1.2% (w / v) EDTA (ethylenediaminetetraacetic acid), and 0.01 to 0.02 M NaOH (sodium hydroxide).

[0033] Meanwhile, the surfactant may include an ionic surfactant, a nonionic surfactant, or a mixture thereof, and preferably may include an ionic surfactant. For example, ionic surfactants include glutamate-based compounds such as alkyl benzene sulfonate, alkyl sulfate, alkyl ether sulfate, alkoxylated amide, olefin sulfonate, alkyl xylene sulfonate, dialkyl sulfosuccinate, fatty acid ester sulfonate, alcohol sulfate, and glycerol fatty acid ester; isethionate-based compounds; phosphate-based compounds such as lauryl phosphate or laureth-1-phosphate; taurate-based compounds; and alkoxylated alcohol-based compounds. One or more selected from the group consisting of alkyl carboxylates, hydroxyalkyls, quaternary ammonium salts, and ethoxylated alkyls, preferably SDS (sodium dodecyl sulfate), SLS (sodium laureth sulfate), SLES (sodium lauryl ether sulfate), SMES (sodium myreth sulfate), DSS (dioctyl sodium sulfosuccinate), PFOS (perfluorooctanesulfonate), and PFBS (perfluorobutanesulfonate).It may include one or more selected from the group consisting of PFNA (perfluorononanoate), PFOA (perfluorooctanoate), sodium stearate, sodium lauroyl sarcosinate, CTAB (cetrimonium bromide), CPC (cetylpyridinium chloride), BAC (benzalkonium chloride), BZT (benzethonium chloride), DDAC (dimethyldioctadecylammonium chloride), and DODAB (dioctadecyldimethylammonium bromide), and more preferably may be SDS (sodium dodecyl sulfate), but is not limited thereto.

[0034] Conventionally, cellular components other than proteins were removed from tissues by dissolving cell membranes using nonionic surfactants or high concentrations of ionic surfactants; however, this method caused problems such as denaturation of residual proteins within the scaffold, collapse of the microstructure of the extracellular matrix, and excessive removal of growth factors. Therefore, in this embodiment, the problems described above were resolved by applying a low concentration of ionic surfactant. Specifically, a low concentration of alkyl sulfate, more preferably a low concentration of SDS (sodium dodecyl sulfate), was used as the ionic surfactant. The concentration of the SDS (sodium dodecyl sulfate) may be 0.1 to 0.5% (w / v), preferably 0.2 to 0.4% (w / v), in the hypotonic solution, but is not limited thereto.

[0036] Then, the skin tissue is washed first with an isotonic solution (S30).

[0037] The skin tissue immersed in the above S20 step can be washed 1 to 10 times for 1 to 24 hours each with an isotonic solution, for example, 1 to 10 times for 1 to 10 hours each, or for another example, 2 to 7 times for 2 to 7 hours each.

[0038] In this embodiment, the isotonic solution has an osmolarity of about 270 to 300 mOsm / L and preferably comprises 25 to 100 mM Tris-HCl, 0.1 to 1.5% (w / v) EDTA (ethylenediaminetetraacetic acid), 0.01 to 0.05 M NaOH (sodium hydroxide), and 0.05 to 0.3 M NaCl (sodium chloride), and more preferably may comprise 45 to 55 mM Tris-HCl, 0.8 to 1.2% (w / v) EDTA (ethylenediaminetetraacetic acid), 0.01 to 0.02 M NaOH (sodium hydroxide), and 0.05 to 0.3 M NaCl (sodium chloride).

[0039] The NaCl in the above-described isotonic solution can be applied at a concentration of 0.05 to 0.5 M, preferably 0.1 to 0.3 M, because if the isotonic solution falls outside the above-described range, the epidermis may not separate well depending on the raw material of the skin.

[0040] In this embodiment, “isotonic” refers to a state in which the osmotic pressure of solutions separated by a semipermeable membrane is the same, that is, a state in which the concentrations are equal and there is no net movement of water. If the concentrations of the solutions separated by the membrane are different, water will move from the area of ​​lower concentration to the area of ​​higher concentration, and the rate of osmosis, which is a passive diffusion phenomenon of water, can be proportional to the concentration gradient between the two solutions.

[0041] It may further include a step of washing for 6 to 24 hours using PBS between S20 and S30 and between S30 and S40, or washing 1 to 10 times for 5 to 60 minutes each.

[0043] After that, the skin tissue that was washed once is washed twice with a delipid solution (S40).

[0044] The skin tissue that was washed first in step S30 above may be washed a second time with a delipid solution at a concentration of 50 to 100%, preferably 80 to 100%, at a temperature of 4 to 40°C, for example, 20 to 40°C, or as another example, 30 to 40°C, for 2 to 24 hours, or as another example, 3 to 24 hours, or as another example, 3 to 5 hours. If the concentration of the delipid solution is less than 50%, the fat removal rate may be low, at 20% or less, which is undesirable. If the washing time is less than 2 hours, the fat removal rate may be low, at less than 20%, and if it exceeds 24 hours, the fat removal rate may not be very high compared to the increase in time. If the washing temperature is less than 4°C, the fat removal rate may be very low, at 10% or less, and if it exceeds 40°C, the skin tissue may be denatured.

[0045] When all of the above-mentioned desirable concentration of the delipid solution, desirable temperature and time conditions for the secondary wash are satisfied, the fat removal rate can be excellent at 58% or more.

[0046] For example, the delipid solution may contain a polar solvent, wherein the polar solvent is an alcohol-based solvent and preferably includes one or more selected from isopropyl alcohol (isopropanol, IPA), ethanol, methanol, butanol, octanol, and water, preferably isopropyl alcohol.

[0048] Finally, the skin tissue that has been washed twice is washed a third time with purified water (S50).

[0049] The skin tissue that has been washed twice in step S40 above may be washed a third time with purified water for 4 to 24 hours, preferably 6 to 24 hours. If the washing time is less than 4 hours, the delipid solution remaining in the skin tissue may exceed 8000 ppm and be toxic, and if it exceeds 24 hours, the reduction in the residual rate of the delipid solution may not be very significant compared to the rate of increase in time. When the above-mentioned preferred time is satisfied, the residual rate of the delipid solution may be 1500 ppm or less, which is a safe state.

[0050] In this embodiment, the purified water may be sterile distilled water (DW).

[0052] The present invention will be described in more detail below using examples. These examples are solely for the purpose of explaining the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0054] Example 1. Preparation of acellular dermal matrix

[0055] Skin tissue was purchased from EURO skin bank, Allosource, CTS, and tissues with a thickness of 1 mm or more were selected. After removing the adipose tissue attached to the skin tissue using forceps, the tissue was washed three times with sterile water. Next, the tissue was immersed for 6 hours in a hypotonic solution containing 0.25% SDS (10 mM Tris-HCl, 1 g EDTA, 13.5 mL 1 M NaOH). Then, the skin tissue was washed with PBS at 4°C to remove residual fat, epidermis, cells, and hypotonic solution. After overnight washing, the tissue was washed once with an isotonic solution (50 mM Tris-HCl, 1 g EDTA, 0.15 M NaCl, 13.4 mL NaOH) for 6 hours to prepare a human-derived acellular dermal matrix.

[0057] Examples 2 to 5. Preparation of acellular dermal matrix with residual fat removed

[0058] The human-derived acellular dermal matrix prepared in Example 1 above was subjected to a second wash under the conditions listed in Table 1 below and a third wash with sterile purified water for more than 2 hours to prepare an acellular dermal matrix from which residual fat was removed, and the before and after photos are as shown in Fig. 2.

[0059] division IPA concentration (%) Second washing time Secondary washing temperature (°C) Tissue formulation Example 2 50 2h 20~25 slice(1~2mm) Example 3 70 2h 20~25 slice(1~2mm) Example 4 100 2h 20~25 slice(1~2mm) Example 5 100 4h 4 whole(2~3mm) Example 6 100 4h 20~25 whole(2~3mm) Example 7 100 4h 38 whole(2~3mm) Example 8 100 2h 38 slice(1~2mm) Example 9 100 4h 38 slice(1~2mm) Example 10 100 O / N 38 slice(1~2mm) Example 11 100 4h 38 whole(3~4mm) Example 12 100 O / N 38 whole(3~4mm) Example 13 100 4h 38 powder

[0061] Example 14. Preparation of a composition containing fibrotic acellular dermal matrix

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

[0064] Example 15. Preparation of IPA-removed acellular dermal matrix

[0065] In Example 7 above, the acellular dermal matrix was washed three times with sterile purified water for 0 / N instead of 2 hours to prepare an acellular dermal matrix from which IPA had been removed.

[0067] Experimental Example 1. Decellularization Evaluation (H&E staining)

[0068] The human-derived dermal matrix prepared in Example 1 above was evaluated for decellularization through histological examination using H&E staining.

[0069] Paraffin sections were prepared by sectioning a paraffin block to a thickness of 4 µm and drying it. Subsequently, for the deparaffinization process, the sections were reacted in xylene for 5 minutes three times, in 100% ethanol for 2 minutes three times, in 90% ethanol for 1 minute once, in 80% ethanol for 1 minute once, and in 70% ethanol for 1 minute once, followed by washing under running water for 10 minutes. After reacting with hematoxylin staining solution for 10 minutes, the sections were washed under running water for 3 minutes, and after reacting with eosin staining solution for 10 minutes, the sections were washed under running water until no eosin stain was detected. After reacting with 70% ethanol 10 times for 1 second, 80% ethanol 10 times for 1 second, 90% ethanol 10 times for 1 second, 100% ethanol 2 times for 1 minute, and xylene 3 times for 3 minutes, the sample was mounted with a mounting solution and then photographed using an optical microscope (Olympus BX51, H&E staining) and a scanning electron microscope (Hitachi S-4700, Japan), and the results are shown in Figure 3.

[0070] Referring to Fig. 3, it was confirmed that the epidermis was exfoliated in Example 1 compared to Control 1 (Fresh skin). In addition, when comparing 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 was confirmed that no cells in the skin tissue were removed when treated with 1% Triton X-100, 1% Tween 80, and 0.1% SDS solution, while most of the cells in the tissue were removed when treated with the hypotonic solution containing 0.25% SDS (data not shown).

[0071] In addition, it was observed that in the group treated with sonication for 1 hour and the group treated with sonication and 1% Triton X-100 for 1 hour, cells were not removed and tissue damage occurred (data not shown). Accordingly, it was found that when surfactant solutions such as 1% Triton X-100, 1% Tween 80, and 0.1% SDS were applied to skin tissue that had not undergone epidermal removal (using NaCl or proteolytic enzymes) and fat removal (using HIPS), decellularization did not occur, and it was confirmed that when a hypotonic solution containing 0.25% SDS was applied under the same conditions, the epidermis, fat, and cells were removed in one step.

[0072] In other words, it was found that even when a physical process combining sonication and surfactants was performed to remove the epidermis, fat, and cells at once, the cells were not removed.

[0074] Experimental Example 2: Evaluation of Detergent Residue

[0075] A methylene blue dye binding assay was performed to evaluate the amount of detergent residue in the tissue.

[0076] After drying the dermal tissue of the conventional process (sample with epidermal removal using NaCl, fat removal, and cell removal using SDS) and Example 1, the residual amount of Detergent (SDS) was evaluated using the eluent eluted with PBS.

[0077] Detergent (SDS) solutions were diluted in methylene blue at different concentrations (1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0%) and eluted with chloroform. After measuring the absorbance at 650 nm, a standard curve was constructed. Subsequently, the 650 nm absorbance of the eluted solution from Example 1, corresponding to the existing and new processes, was measured to determine the residual detergent amount. The results are shown in Figure 4 and Table 2. Additionally, the washing solution from the dermal tissue of Example 1 was taken at each washing step to measure the residual detergent amount. The results are shown in Figure 5.

[0078] For the elution process, a dermal tissue with a thickness of approximately 3 mm and a weight of 4 g was selected, and 20 mL of the elution solution was obtained at 37°C for 72 hours.

[0079] Referring to Figure 4 and Table 2, the residual detergent in the eluent of Example 1 was approximately 32.87 ± 6.42 mg / L, which was lower than that of the conventional process (data not shown).

[0080] That is, it was confirmed that the human-derived acellular dermal matrix prepared according to the present invention had a residual amount of SDS detergent of 40 mg / L or less, measured by the absorbance at 650 nm for the eluent eluted with PBS.

[0081] division New process (72-hour dissolution) Residual amount (mg / L) 39.9566 27.4441 31.1979

[0082] Referring to Figure 5, it was confirmed that the amount of detergent in the washing solution decreases as washing with PBS increases.

[0084] Experimental Example 3: Safety Evaluation (Cytotoxicity Analysis)

[0085] To evaluate safety through cell compatibility and toxicity, the MTT assay and the MEM Elution Test from ISO10993-5: Cytotoxicity Test were performed.

[0087] 3-1. MTT assay (cell compatibility)

[0088] L929 cells in a 96-well plate, 1 x 10⁶ cells per well 5 After dispensing 100 µL at a concentration of cell / mL and culturing for 24 hours, the solution obtained by eluting the existing process and the novel process of Example 1 with MEM culture medium was treated and cultured at 37°C for 24 hours. For the above solution, dermal tissue with a thickness of approximately 3 mm and a weight of 4 g was selected in the existing process and the novel process, and then eluted with animal cell culture medium at 37°C for 24 hours to obtain 20 mL of the solution.

[0089] After 24 hours of incubation with the eluent, 5 mg / mL of MTT (3,(4,5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide) reagent was diluted 1:100 in the culture medium, and 0.1 mL of this 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 DMSO (dimethylsulfoxide) was added to each well to lyse the cells. Subsequently, the 96-well plate was placed in a spectrophotometer, and the absorbance was measured at a wavelength of 540 nm, and the results are shown in Figure 6.

[0090] Referring to Figure 6, the novel process in Example 1 showed cell viability values ​​of 96.26%, 101.49%, and 94.99%. These are higher viability values ​​compared to the conventional process, indicating that Example 1 has higher cell compatibility than the conventional process.

[0091] That is, it was confirmed that the human-derived cell-free dermal matrix produced according to the present invention showed a very high cell viability of over 90%, measured by the absorbance at 540 nm for the eluent eluted with MEM culture medium.

[0093] 3-2. MEM (Minimum Essential Medium) Elution Test (Toxicity)

[0094] L929 cells in a 6-well plate, 1 x 10⁶ cells per well 5 After dispensing 4 mL aliquots at a cell / mL concentration and culturing for 24 hours, the monolayer culture status of the cells was checked. Then, the cells were treated with the eluent of Example 1, which is the conventional and novel process, and cultured for 48 hours. For the eluent, 20 mL of the eluent was prepared by selecting a dermal tissue of approximately 3 mm thickness and a weight of 4 g from Example 1, which is the conventional and novel process, and eluting it with MEM culture medium at 37°C for 24 hours. After 24 and 48 hours of culture, the positive and negative control substances were replaced with DMSO and MEM, respectively.

[0095] After 24 and 48 hours of culture, the degree of cell growth and lysis was observed using a microscope, and the grade was determined according to Table 3 below. The results are shown in Figure 7 and Table 4. If the grade was 2 or lower, it was determined that the test substance eluent had no cytotoxicity.

[0096] rating Reactivity Condition of cultured cells 0 doesn't exist Separation of intracytoplasmic granules, no cell lysis, no inhibition of cell growth 1 very weak The cells are rounded and loosely attached, and no more than 20% of the cells show loss of cytoplasmic granules or morphological changes. Lysed cells are occasionally present, and slight growth inhibition is observed. 2 weak Cells become round, cytoplasmic granules are lost, cells do not exceed 50%, and extensive cell lysis is not observed. Cell growth inhibition does not exceed 50%. 3 Severity Cells became rounded, and the percentage of lysed cells did not exceed 70%. The cell layer was not completely destroyed, but showed growth inhibition of more than 50%. 4 Severe Cell layer is almost or completely destroyed

[0097] Referring to Figure 7 and Table 4, it was confirmed that Example 1 had a judgment grade of 0 even after 48 hours, whereas the conventional process had a cytotoxicity grade of 2 after 48 hours. Therefore, it was found that the conventional process was also suitable for cell compatibility and toxicity evaluation, and that Example 1, which corresponds to the novel process, showed higher suitability, that is, higher safety, than the conventional process.

[0098] division Cytotoxicity grade beginning 24hr 48hr Existing processes 0 0 2 Example 1 (New Process) 0 0 0 Negative control group 0 0 0 positive control group 0 4 4

[0100] Experimental Example 4: Measurement of fat removal rate according to IPA washing conditions

[0101] The fat removal conditions were broadly divided into four categories as follows, and the evaluation results for each test condition were graphed. The results are shown in Figures 8 to 11.

[0102] After the second IPA wash was completed, the sample was freeze-dried for 24 to 72 hours, cut into 0.5 x 0.5 cm pieces, and the weight of the sample and the empty container were measured. Then, the sample and container were installed in a crude fat extraction device, and crude fat was extracted using the Soxhlet extraction method. Next, the weight of the extracted crude fat was measured, and the crude fat content was calculated according to the following formula (1). Then, the fat removal rate according to the IPA washing group relative to the crude fat content of the control sample was calculated according to the following formula (2). That is, since the fat removal rate increases as the tissue thickness decreases, the fat removal rate was calculated as the difference value compared to each control group to offset the difference in results caused by the different tissue thicknesses applied in the above examples.

[0103] Crude fat content (%) = [(Weight of residue container - Weight of empty container) / Weight of sample] x 100… … … Equation (1)

[0104] Fat removal rate (%) = [(Control group crude fat content - Experimental group crude fat content) / Control group crude fat content] x 100… … … Formula (2)

[0105] As described below, the applicant of the present invention was able to confirm that the fat removal rate was improved by more than 20% compared to the case where only a first wash was performed with an isotonic solution (control group).

[0107] 4-1. Measurement of fat removal rate according to IPA washing concentration

[0108] To determine the fat removal rate according to the IPA washing concentration, the fat removal rates of Examples 2 to 4, which were washed a second time with IPA 50%, 70%, and 100%, were measured, and the results are shown in Fig. 8.

[0109] Referring to Figure 8, it was confirmed that the fat removal efficiency was highest when washing was performed with 100% IPA.

[0111] 4-2. Measurement of fat removal rate according to washing temperature

[0112] To determine the fat removal rate according to the washing temperature, the fat removal rates of Example 5, which was washed at 4°C, Example 6, which was washed at 20-25°C, and Example 7, which was washed at 38°C were measured, and the results are shown in Fig. 9.

[0113] Referring to Figure 9, the fat removal efficiency was found to be higher in Examples 5, 6, and 7, indicating that the fat removal efficiency is higher as the washing temperature increases.

[0114] Meanwhile, considering that when measuring the fat removal rate according to washing temperature in this invention, errors may occur in which the fat removal efficiency is measured higher or lower under all experimental group conditions if the fat content in the control tissue area is measured higher or lower even within the same Lot, the fat removal rate was measured from the result calculated by reflecting the average fat content of the control tissue.

[0116] 4-3. Measurement of fat removal rate according to washing time

[0117] To determine the fat removal rate according to washing time, the fat removal rates of Examples 8 to 10, which are slice formulations, and Examples 11 and 12, which are whole formulations were measured, and the results are shown in Figures 10a and 10b.

[0118] Referring to Figures 10a and 10b, in the case of slice, the fat removal efficiency was found to be higher in Examples 8, 9, and 10, indicating that the longer the washing time, the higher the fat removal efficiency. In the case of Whole, similar to slice, Example 12, which had a longer washing time, also showed a higher fat removal efficiency.

[0120] 4-4. Measurement of fat removal rate according to tissue formulation

[0121] To determine the fat removal rate according to the tissue formulation, the fat removal rates of Example 7 (whole formulation), Example 9 (slice formulation), and Example 13 (powder formulation) were measured, and the results are shown in Figure 11.

[0122] Referring to Figure 11, although the fat removal efficiency was higher in Examples 7, 9, and 13, it was found that the fat removal efficiency was generally excellent in all tissue formulations.

[0124] Experimental Example 5: Measurement of Residual Fat Removal According to Tissue Formulation

[0125] To determine whether residual fat was removed according to the tissue formulation, residual fat in the tissues of the control group (Example 1), Example 7, and Example 9 was stained and photographed using an optical microscope, and the results are shown in Figures 12a to 12c.

[0126] Referring to Figures 12a to 12c, residual fat was detected in the control group before IPA washing, but no residual fat was detected in Examples 7 and 9 after IPA washing. Therefore, it was found that most of the residual fat was removed by IPA washing.

[0128] Experimental Example 6: Measurement of IPA Residue

[0129] To determine the amount of residual IPA according to the DW washing time, the amount of residual IPA was measured according to the GC-FID test method for Example 14, which was washed for 4 hours as shown in Fig. 13a, and Example 15, which was washed overnight, and the results are shown in Fig. 13b.

[0130] Referring to Fig. 13b, it was confirmed that for Example 14 it was 7975 ppm and for Example 15 it was 1248 ppm.

[0132] Experimental Example 7: Evaluation of ADM powder particle distribution uniformity

[0133] To confirm the uniformity of particle distribution depending on whether residual fat is removed, human-derived acellular dermal matrix prepared according to Examples 1, 7, and 9 was freeze-dried for 24 to 72 hours, then ground to the μm level using a freeze grinder, and the particle size and distribution were measured by applying the Mie theory of the laser diffraction method of a particle size analyzer, and the results are shown in Figures 14a to 14c.

[0134] The Span value is the distribution width of the sample; the wider the particle distribution, the larger the value appears, and the narrower the distribution, the closer it approaches 0.

[0135] Referring to FIGS. 14a to 14c, the average particle size was measured to be 100 µm or less for the control group (Example 1), Example 7, and Example 9, showing no significant difference; however, the Span value was measured to be higher in the control group, confirming that the particle size was relatively uneven. Therefore, it was found that Examples 7 and 9 are suitable for use in injectable drugs, such as injectable ADM products made using ADM powder.

[0137] Experimental Example 8: Measurement of change in amino acid content after secondary washing

[0138] To confirm the change in amino acid content before and after IPA, the amino acid content of the control group (Example 1) and Example 7 was measured using an automatic amino acid analyzer (S433D, Sykam GmbH Co., Germany), and the results are shown in Fig. 15.

[0139] Referring to Figure 15, it was confirmed that the amino acid content in Example 7 did not decrease compared to the control group, and thus it was found that the presence or absence of IPA treatment did not affect the constituent amino acids of the tissue.

[0141] Although exemplary embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims also fall within the scope of the present invention.

[0142] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

Claims

Claim 1 (i) a step of preparing human-derived skin tissue that has not undergone a separate deepidermal and defat removal process; (ii) a step of immersing the skin tissue in a hypotonic solution containing a surfactant; (iii) a step of washing the skin tissue immersed in step (ii) first with an isotonic solution; and (iv) a step of washing the skin tissue washed first in step (iii) second with a defat removal solution; wherein the isotonic solution in step (iii) comprises Tris-HCl, EDTA, NaOH, and NaCl. Claim 2 A method for manufacturing a human-derived acellular dermal matrix, comprising, in claim 1, (v) a step of washing the skin tissue that was washed twice in step (iv) a third time with purified water after step (iv). Claim 3 A method for preparing a human-derived acellular dermal matrix, wherein, in step (ii) above, the surfactant comprises an ionic surfactant, a nonionic surfactant, or a mixture thereof. Claim 4 A method for preparing a human-derived acellular dermal matrix, wherein, in step (ii), the hypotonic solution comprises 5-20 mM Tris-HCl, 0.1-1.5% (w / v) EDTA (ethylenediaminetetraacetic acid), and 0.01-0.05 M NaOH (sodium hydroxide). Claim 5 A method for preparing a human-derived acellular dermal matrix, wherein, in step (ii) above, the concentration of the surfactant is 0.1 to 0.5% (w / v) in a hypotonic solution. Claim 6 A method for preparing a human-derived acellular dermal matrix, wherein, in step (iii) above, the isotonic solution comprises 25–100 mM Tris-HCl, 0.1–1.5% (w / v) EDTA (ethylenediaminetetraacetic acid), 0.01–0.05 M NaOH (sodium hydroxide), and 0.05–0.3 M NaCl (sodium chloride). Claim 7 A method for preparing a human-derived acellular dermal matrix, wherein, in step (iv) above, the secondary washing is performed with a delipidening solution of 50 to 100% concentration at 4 to 40°C for 2 to 24 hours. Claim 8 A method for preparing a human-derived acellular dermal matrix, wherein, in step (iv) above, the delipid solution comprises a polar solvent. Claim 9 A method for preparing a human-derived acellular dermal matrix, wherein, in step (v) of paragraph 2, the third wash is performed for 4 to 24 hours. Claim 10 A method for manufacturing a human-derived acellular dermal matrix according to claim 1, wherein step (iv) is omitted and the fat removal rate measured by the following formulas (1) and (2) is improved by 20% or more compared to a control group, which is a human-derived acellular dermal matrix manufactured by steps (i), (ii), and (iii). Crude fat content (%) = [(Weight of residue - Weight of empty container) / Weight of sample] x 100… … … Formula (1) Fat removal rate (%) = [(Crude fat content of control group - Crude fat content of experimental group) / Crude fat content of control group] x 100… … … Formula (2) Claim 11 A human-derived acellular dermal matrix prepared by washing human-derived skin tissue that has not undergone separate deepidermal and delipidification processes multiple times, wherein the washing includes washing with an isotonic solution and washing with a delipidification solution, wherein the isotonic solution includes Tris-HCl, EDTA, NaOH, and NaCl, and the delipidification solution includes a polar solvent. Claim 12 A human-derived acellular dermal matrix according to claim 11, wherein the residual amount of sodium dodecyl sulfate, measured by absorbance at 650 nm for the eluent eluted with phosphate-buffered saline, is 40 mg / L or less. Claim 13 In claim 11, a human-derived acellular dermal matrix having a cell viability of 90% or more, measured by absorbance at 540 nm for an eluent eluted with Minimum Essential Medium.

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