Sterilizing agents and sterilization methods using the sterilizing agents

Abstract

The present application relates to the field of decellularized tissue processing, and specifically discloses a sterilizing agent and a sterilization method using the sterilizing agent, the sterilizing agent containing peracetic acid and hydrogen peroxide, the volumetric dosage ratio of the peracetic acid and the hydrogen peroxide being 1:(0.26-84.5), and the sterilizing agent being used for sterilizing biological tissue during decellularization. The sterilization method includes a step of taking the biological tissue, and a step of sterilizing the biological tissue by placing it in a sterilizing agent, the sterilization temperature being 3-38°C. The present application can satisfactorily sterilize the biological tissue before decellularization processing.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION This application relates to the field of decellularized tissue processing, and more particularly to sterilizing agents and sterilization methods using sterilizing agents. [Background technology]

[0002] Decellularized tissue is a biological material obtained after processing human or animal-derived tissue with a similar structure to human tissue and removing immunogenic components in the tissue. Decellularized materials are frequently used in fields such as scaffolding materials for regenerative medicine and materials promoting wound repair, and have attracted attention due to their efficiency.

[0003] When preparing decellularized tissue for research or clinical use, animal tissue is collected as raw material in a clean environment, and then various decellularization processes are performed on the animal-derived tissue. However, even animal tissue collected in a clean environment may still contain bacteria and viruses. Therefore, in the case of decellularized tissue for research or clinical use, sterilization is performed after the final decellularization process to prevent symptoms such as fever and bacterial infection from occurring in patients after transplantation of the decellularized tissue.

[0004] The inventors have discovered that in the steps prior to decellularization, such as the removal of residual tissue, the proliferation of existing bacteria can be induced and bacterial contamination can occur due to external manipulation, resulting in the inability to suppress bacterial growth in living tissue during processing, which in turn affects subsequent manipulations. Summary of the Invention [Problem to be solved by the invention]

[0005] In order to effectively sterilize biological tissue before decellularization, the present application provides a sterilizing agent and a sterilization method using the sterilizing agent. [Means for solving the problem]

[0006] In a first aspect, the present application provides a sterilant, which uses the following technical solution:

[0007] The sterilizing agent includes peracetic acid and hydrogen peroxide, and the volumetric dosage ratio of the peracetic acid and the hydrogen peroxide is 1:(0.26-84.5), and the sterilizing agent is used for sterilizing biological tissue during decellularization.

[0008] Preferably, the sterilizing agent comprises peracetic acid, hydrogen peroxide, and a solvent, the peracetic acid having a concentration of 0.098 to 1.844 ml / L, and the hydrogen peroxide having a concentration of 0.484 to 8.281 ml / L.

[0009] Preferably, the concentration of the peracetic acid is 0.195 to 0.922 ml / L.

[0010] Preferably, the concentration of the hydrogen peroxide is 0.977 to 4.141 ml / L.

[0011] Preferably, the solvent includes at least one of physiological saline, MEM buffer, DMEM buffer, and PBS buffer.

[0012] In a second aspect, the present application provides a sterilization method, which uses the following technical solution:

[0013] 1. A sterilization method comprising: a step of taking biological tissue; and a step of sterilizing the biological tissue by placing it in a sterilizing agent, the sterilization temperature being 3 to 38°C.

[0014] Preferably, the sterilization temperature is 4°C and the concentration of the peracetic acid is 0.391 to 0.922 ml / L.

[0015] Preferably, the concentration of the hydrogen peroxide is 1.953 to 4.141 ml / L.

[0016] Preferably, the sterilization temperature is 37°C and the concentration of the peracetic acid is 0.195 to 0.461 ml / L.

[0017] Preferably, the concentration of the hydrogen peroxide is 0.977 to 2.07 ml / L.

[0018] Preferably, the sterilization time is 3 to 24 hours. [Effects of the Invention]

[0019] In summary, the present application has the following beneficial effects:

[0020] 1. In this application, peracetic acid and hydrogen peroxide are mixed as a sterilant according to a predetermined dosage ratio, and then the sterilant is used to sterilize biological tissues during the cell exfoliation process, achieving good sterilization effect.

[0021] 2. In this application, the dosage ratio of peracetic acid and hydrogen peroxide is controlled, so that hydrogen peroxide can play an excellent supporting role in the sterilization effect of peracetic acid. At the same time, the sterilization conditions such as the temperature are controlled, so that the sterilant has a better sterilization effect.

[0022] 3. In this application, hydrogen peroxide and saline are used to dilute peracetic acid, so that the sterilant has a good sterilization effect and at the same time reduces the destruction of collagen structure of living tissues by the sterilant. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 shows the collagen structure of living tissue that has not been sterilized. [Figure 2] FIG. 2 is a diagram showing the collagen structure of biological tissue in Example 1 of the present application. [Figure 3] FIG. 3 is a diagram showing the collagen structure of biological tissue in Example 14 of the present application. [Figure 4] FIG. 4 is a diagram of the collagen structure of biological tissue in Example 15 of the present application. [Figure 5] FIG. 5 is a diagram of collagen structure in biological tissue according to Example 23 of the present application. [Figure 6] FIG. 6 is a diagram showing the collagen structure of biological tissue in Comparative Example 1 of the present application. [Figure 7]FIG. 7 is a diagram showing the collagen structure of biological tissue in Comparative Example 2 of the present application. [Figure 8] FIG. 8 is a diagram showing the collagen structure of biological tissue in Comparative Example 5 of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be described in more detail below with reference to FIGS. 1 to 8 and examples.

[0025] Decellularized materials are used in fields such as scaffolding materials for regenerative medicine and materials for promoting wound repair, and are therefore attracting constant attention due to their significant significance. The decellularized manufacturing process typically includes steps such as removing impure tissue, cleaning the tissue, and decellularizing it. Furthermore, to reduce symptoms such as infection in patients due to damage to the decellularized tissue, the decellularized tissue is generally sterilized after the decellularization process is completed.

[0026] However, the inventors have found that before decellularization, environmental influences, handling techniques, and the tissue itself can all cause bacterial growth and proliferation, further affecting subsequent handling. Therefore, the inventors have determined that it is also important to sterilize the tissue before decellularization.

[0027] Sterilization standards for clinical use of implantable medical materials require that the probability of microbial survival in a sterilized item be less than one in a million. Sterilization methods that can meet this standard typically include heating, irradiation, gas sterilization, filtration, and sterilant treatment. Considering the characteristics of decellularized tissue and the features of various sterilization methods, the inventors used a sterilant to sterilize the tissue before decellularization. The inventors' research has shown that peracetic acid is more suitable for sterilizing biological tissue than other sterilants such as pentylene glycol, sodium hypochlorite, and ethanol.

[0028] However, in the research, the inventor found that when using a peracetic acid solution alone, if the concentration of peracetic acid is not properly controlled, it will affect the final sterilization effect, and as the concentration of peracetic acid increases, the collagen structure of biological tissue will also be destroyed. Therefore, the inventor selected hydrogen peroxide and mixed it with physiological saline or buffer solution to dilute the peracetic acid, which can effectively control the degree of destruction of the collagen structure of biological tissue.

[0029] In order to further improve the effectiveness of the sterilant, the inventors studied the sterilization temperature and time. When controlling the sterilization temperature, the inventors discovered that there was no regular pattern between the peracetic acid concentration and the sterilization temperature. After extensive research, the inventors discovered that when the sterilization temperature was 4°C, the peracetic acid concentration was 0.391-0.922 ml / L, and the hydrogen peroxide concentration was 1.953-4.141 ml / L, the sterilant produced a good sterilization effect on biological tissues and relatively intact collagen structure. When the sterilization temperature was 37°C, the peracetic acid concentration was 0.195-0.461 ml / L, and the hydrogen peroxide concentration was 0.977-2.07 ml / L, the sterilant produced a good sterilization effect on biological tissues and relatively intact collagen structure. Example Example 1

[0030] Example 1 provides a sterilant, which includes peracetic acid, hydrogen peroxide, and a solvent, in which the concentration of peracetic acid in the sterilant is 1.563 ml / L, the concentration of hydrogen peroxide is 7.813 ml / L, and the solvent is saline.

[0031] Sterilant preparation method: The stock solution is an aqueous solution of peracetic acid and hydrogen peroxide, with the peracetic acid concentration being 50 ml / L and the hydrogen peroxide concentration being 250 ml / L. The stock solution is diluted stepwise with saline, with the dilution times being 5 in this example, resulting in a dilution ratio of 32.

[0032] Primary dilution: Mix 25 ml of the stock solution and 25 ml of saline to obtain the primary mixture.

[0033] Secondary dilution: Mix 25 ml of the primary mixture with 25 ml of saline to obtain the secondary mixture.

[0034] Tertiary dilution: Mix 25 ml of the secondary mixture with 25 ml of saline to obtain the tertiary mixture.

[0035] Quaternary dilution: Mix 25 ml of the tertiary mixture with 25 ml of saline to obtain a quaternary mixture.

[0036] Quinary dilution: Mix 25ml of the quaternary mixture with 25ml of saline to obtain the sterilant. If further dilution is required, repeat the above steps.

[0037] Example 1 further provides a sterilization method comprising the following steps:

[0038] The biological tissue was taken. The raw material for the biological tissue in this example was commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.

[0039] The biological tissue is placed in the sterilant and treated at 4°C for 3 hours to complete the sterilization. Examples 2 to 6

[0040] The differences between Examples 2 to 6 and Example 1 are the temperature and time during the sterilization process, and Table 1 is specifically referred to.

[0041] Table 1: Sterilization condition parameter table for Examples 1 to 6 JPEG0007763006000001.jpg92151Example 7

[0042] The sterilant in Example 7 differs from the sterilant in Example 1 in that the original solution is diluted six times, resulting in a dilution ratio of 64, and the sterilant contains peracetic acid at a concentration of 0.781 ml / L and hydrogen peroxide at a concentration of 3.906 ml / L. Examples 8 to 12

[0043] The differences between Examples 8 to 12 and Example 7 are the temperature and time during the sterilization process. See Table 2 for details.

[0044] Table 2: Sterilization condition parameter table for Examples 7 to 12 JPEG0007763006000002.jpg93151Example 13

[0045] The difference between Example 13 and Example 1 is that the original solution was diluted seven times, resulting in a dilution ratio of 128 times, to obtain a sterilant, in which the concentration of peracetic acid was 0.391 ml / L and the concentration of hydrogen peroxide was 1.953 ml / L. Examples 14 to 18

[0046] The differences between Examples 14 to 18 and Example 13 are the temperature and time during the sterilization process, and see Table 3 for details.

[0047] Table 3: Sterilization condition parameter table for Examples 13 to 18 JPEG0007763006000003.jpg93151 Example 19

[0048] The difference between Example 19 and Example 1 is that the original solution was diluted 8 times, resulting in a dilution ratio of 256 to obtain a sterilant, in which the concentration of peracetic acid was 0.195 ml / L and the concentration of hydrogen peroxide was 0.977 ml / L. Example 20

[0049] The differences between Examples 20 to 24 and Example 19 are the temperature and time during the sterilization process; see Table 4 for details.

[0050] Table 4: Sterilization condition parameters for Examples 19 to 24 JPEG0007763006000004.jpg82153 Example 25

[0051] The difference between Example 25 and Example 1 is that the stock solution was diluted 9 times, resulting in a dilution ratio of 512 to obtain a sterilant, in which the concentration of peracetic acid was 0.098 ml / L and the concentration of hydrogen peroxide was 0.484 ml / L. Example 26

[0052] The differences between Examples 26 to 30 and Example 25 are the temperature and time during the sterilization process; see Table 5 for details.

[0053] Table 5: Sterilization condition parameters for Examples 25 to 30 JPEG0007763006000005.jpg82153 Examples 31 to 36

[0054] The differences between Examples 31 to 36 and Example 1 are the concentrations of peracetic acid and hydrogen peroxide and the sterilization conditions. At the same time, in the process of preparing the sterilant, an aqueous solution of peracetic acid and hydrogen peroxide was used as the stock solution, with a peracetic acid concentration of 59 ml / L and a hydrogen peroxide concentration of 265 ml / L. See Table 6 for details.

[0055] Table 6: Concentration of ingredients in sterilants for Examples 31 to 36 JPEG0007763006000006.jpg106151 Example 37

[0056] Example 37 differs from Example 13 in that the solvent is MEM buffer solution. Example 38

[0057] Example 38 differs from Example 13 in that the solvent is DMEM buffer solution. Example 39

[0058] Example 39 differs from Example 13 in that the solvent is PBS buffer solution. Example 40

[0059] Example 40 differs from Example 13 in that the sterilant contains peracetic acid, hydrogen peroxide, and a solvent, with the peracetic acid concentration being 0.391 ml / L, the hydrogen peroxide concentration being 3.125 ml / L, and the solvent being saline. Example 41

[0060] Example 41 differs from Example 13 in that the sterilant contains peracetic acid, hydrogen peroxide, and a solvent, with the peracetic acid concentration being 0.596 ml / L, the hydrogen peroxide concentration being 1.953 ml / L, and the solvent being saline. Example 42

[0061] Example 42 differs from Example 13 in that the sterilant contains peracetic acid, hydrogen peroxide, and a solvent, with the peracetic acid concentration being 0.098 ml / L, the hydrogen peroxide concentration being 8.281 ml / L, and the solvent being saline. Example 43

[0062] Example 43 differs from Example 13 in that the sterilant contains peracetic acid, hydrogen peroxide, and a solvent, with the peracetic acid concentration being 1.844 ml / L, the hydrogen peroxide concentration being 0.484 ml / L, and the solvent being saline. Comparative Example Comparative Example 1

[0063] The difference between Comparative Example 1 and Example 1 is that the sterilant contains peracetic acid, hydrogen peroxide, and a solvent, with the peracetic acid concentration being 50 ml / L, the hydrogen peroxide concentration being 250 ml / L, and the solvent being saline.

[0064] The sterilization method of Comparative Example 1 includes the following steps.

[0065] The biological tissue was taken. The raw material for the biological tissue in this example was commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.

[0066] The biological tissue is placed in the sterilant and treated at 4°C for 3 hours to complete the sterilization. Comparative Example 2

[0067] Comparative Example 2 differs from Comparative Example 1 in that the biological tissue is placed in a sterilizing agent and treated in an environment of 4°C for 12 hours to complete sterilization. Comparative Example 3

[0068] The difference between Comparative Example 3 and Example 1 is that the sterilant contains peracetic acid, hydrogen peroxide, and a solvent, with the peracetic acid concentration being 3.125 ml / L, the hydrogen peroxide concentration being 15.625 ml / L, and the solvent being saline.

[0069] The sterilization method of Comparative Example 3 includes the following steps.

[0070] The biological tissue was taken. The raw material for the biological tissue in this example was commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.

[0071] The biological tissue is placed in the sterilant and treated at 4°C for 3 hours to complete the sterilization. Comparative Example 4

[0072] The difference between Comparative Example 4 and Example 1 is that the sterilant contains peracetic acid, hydrogen peroxide, and a solvent, with the peracetic acid concentration being 50 ml / L, the hydrogen peroxide concentration being 250 ml / L, and the solvent being saline.

[0073] The sterilization method of Comparative Example 4 includes the following steps.

[0074] The biological tissue was taken. The raw material for the biological tissue in this example was commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.

[0075] The biological tissue is placed in the sterilant and treated in a 37°C environment for 3 hours to complete the sterilization. Comparative Example 5

[0076] Comparative Example 5 differs from Comparative Example 4 in that the biological tissue is placed in a sterilizing agent and treated in an environment of 37°C for 12 hours to complete sterilization. Comparative Example 6

[0077] The difference between Comparative Example 6 and Example 1 is that the sterilant contains peracetic acid, hydrogen peroxide, and a solvent, with the peracetic acid concentration being 3.125 ml / L, the hydrogen peroxide concentration being 15.625 ml / L, and the solvent being saline.

[0078] The sterilization method of Comparative Example 6 includes the following steps.

[0079] The biological tissue was taken. The raw material for the biological tissue in this example was commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.

[0080] The biological tissue is placed in the sterilant and treated in a 37°C environment for 3 hours to complete the sterilization. Comparative Example 7

[0081] Comparative Example 7 differs from Example 13 in that the sterilant contains peracetic acid and a solvent, the concentration of the peracetic acid is 0.391 ml / L, and the solvent is physiological saline. Comparative Example 8

[0082] Comparative Example 8 differs from Example 13 in that the sterilant contains hydrogen peroxide and a solvent, and the concentration of hydrogen peroxide is 1.953 ml / L. Performance Detection Test

[0083] 1. Sterilization effect evaluation: The sterilization effect was examined for the sterilized biological tissues of Examples 1 to 43 and Comparative Examples 1 to 8, and the specific procedures were as follows. 1. After sterilization, the living tissue is washed to remove the sterilant. 2. After that, the tissue is washed in sterile saline for 24 hours, and then the saline is removed. 3. The tissue is placed in 4 ml of E-MEM culture medium containing 10 wt% fetal bovine serum (FBS) and cultured for 24 hours. 4. The dissolved agar medium is poured into a 10 cm cell culture dish, and after cooling, it is diluted 1,000,000 times with the E-MEM culture medium after cultivation. 100 μl of the diluted culture medium is poured into the agar medium and cultivated in an environment of 37 ° C for 48 hours. 5. After incubation, the strains on the agar gel are counted and evaluated.

[0084] The detection results are shown in Table 7. Table 7: Bacterial counts after peracetic acid treatment JPEG0007763006000007.jpg125151 JPEG0007763006000008.jpg236151 JPEG0007763006000009.jpg225151

[0085] Second, tissue structure evaluation after sterilization. Collagen tissue was detected from the sterilized biological tissues of Examples 1 to 43 and Comparative Examples 1 to 8, and the specific procedures were as follows. 1. After sterilization, the living tissue is washed to remove the sterilant. 2. After that, the tissue is washed in sterile saline for 24 hours, and then the saline is removed. 3. After treatment, the tissue was fixed in 4 wt% paraformaldehyde for 12 hours. 4. Dehydrate the tissue using alcohol in a stepwise manner according to the following schedule: 70 wt% alcohol for 5 hours, 80 wt% alcohol for 5 hours, 90 wt% alcohol for 5 hours, 100 wt% alcohol for 5 hours, and xylene for 5 hours. 5. Embed the tissue using paraffin, then cut the tissue into 7 μm paraffin sections. 6. Paraffin sections are stained with hematoxylin and eosin (HE), sealed with mounting medium, and finally, the sections are observed under a microscope to evaluate changes in tissue collagen structure.

[0086] Here, collagen structures of Examples 1, 14, 15, 23, Comparative Examples 2, 3, and 6, and non-sterilized biological tissues are shown in FIGS. Data analysis

[0087] The sterilization effect of the sterilant is good as can be seen from the detection results of Examples 1 to 6. At the same time, by comparing the detection results of Examples 7 to 9, it was found that when the concentration of peracetic acid was 0.781 ml / L and the concentration of hydrogen peroxide was 3.906 ml / L, bacteria still existed in the living tissue after sterilization in Example 7. Furthermore, as can be seen from the detection results of Examples 13 to 15, Examples 19 to 21, and Examples 25 to 27, the sterilization effect deteriorates as the concentrations of peracetic acid and hydrogen peroxide decrease.

[0088] A comparison of the sterilization effects of Examples 7-9 and 10-12, the sterilization effects of Examples 13 and 16, and the detection results of Examples 19-20 and 22-23 shows that the sterilization effect at 37°C is better than that at 4°C, indicating that an increase in temperature contributes to sterilization. However, a comparison of the detection results of Examples 16-18, 22-24, and 28-30 shows that the number of bacteria conversely increases with an increase in sterilization time, indicating that there is no relationship between sterilization effect and time.

[0089] In the invention of the collagen structure of biological tissue in Comparative Example 1, this concentration of sterilant causes a certain degree of destruction of the collagen structure of biological tissue. A comparison of the collagen structure of biological tissues in Comparative Examples 1 and 2 revealed that the destruction of the collagen structure of biological tissues in Comparative Examples 1 and 2 was more severe, indicating that higher concentrations of sterilant have a better sterilization effect but affect the collagen structure of biological tissues. Furthermore, it was found that the collagen structure of biological tissues in Examples 14 and 15 was not destroyed, indicating that the sterilant concentrations in Examples 14 and 15 are more appropriate. Furthermore, a comparison of the collagen structure of biological tissues in Comparative Examples 5 and 4 shows that higher temperatures result in greater destruction of the collagen structure of biological tissues.

[0090] However, comparing the sterilization effects of Examples 10, 7, 16, and 13, it was found that the sterilization effect decreased more when both the concentration and temperature were decreased than when either the concentration or temperature was decreased alone. A comparison of the detection results of Examples 8, 14, and 17 revealed that the sterilization effect was unchanged when both the temperature and concentration were decreased than when either the concentration or temperature was decreased alone. A comparison of the sterilization effects of Examples 9, 18, and 15 revealed that the sterilization effect was worse when both the temperature and concentration were decreased than when either the concentration or temperature was decreased alone. It was also found that temperature and concentration simultaneously have a complex effect on the sterilization effect of a sterilant. Furthermore, the inventors discovered that when the sterilization temperature was 4°C, the peracetic acid concentration was 0.391-0.922 ml / L, and the hydrogen peroxide concentration was 1.953-4.141 ml / L, the sterilization effect of the sterilant on biological tissue was excellent and the collagen structure of the biological tissue was relatively intact. When the sterilization temperature is 37°C, the peracetic acid concentration is 0.195-0.461 ml / L, and the hydrogen peroxide concentration is 0.977-2.07 ml / L, the sterilant has an excellent sterilization effect on biological tissues and the collagen structure of the biological tissues is relatively intact.

[0091] As can be seen from the test results of Examples 37 to 38, the choice of buffer solution has little effect on the sterilization effect, and the buffer solution is mainly used to reduce swelling of cell tissues. Also, as can be seen from the test results of Examples 40 and 41, the dosage ratio of peracetic acid and hydrogen peroxide affects the sterilization effect, but as can be seen from the test results of Examples 42, 43 and Comparative Examples 7 and 8, it is peracetic acid that mainly plays the bactericidal role, and hydrogen peroxide mainly plays a supporting role to peracetic acid.

[0092] The specific examples are merely illustrative of the present application and do not limit the present application. After reading this specification, a person skilled in the art may make amendments to the present examples as necessary without making any creative contributions, but all such amendments within the scope of the claims of the present application shall be protected by the Patent Law.

Claims

1. A sterilant for use in sterilizing biological tissue before decellularization treatment, The solution contains peracetic acid, hydrogen peroxide, and a solvent, wherein the concentration of the peracetic acid is 0.391 to 0.461 ml / L and the concentration of the hydrogen peroxide is 1.953 to 2.07 ml / L. When using the sterilizing agent, the sterilization temperature is 4°C, the sterilization time is 12 to 24 hours, and the solvent includes at least one of physiological saline, MEM buffer solution, DMEM buffer solution, and PBS buffer solution. A sterilizing agent characterized by:

2. 1. A sterilization method comprising: a step of taking biological tissue; and a step of sterilizing the biological tissue by placing it in the sterilizing agent according to claim 1, wherein the sterilization temperature is 4°C and the sterilization time is 12 to 24 hours. A sterilization method characterized by:

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