WO2023125531A1 - 一种灭菌剂及应用灭菌剂的杀菌方法 - Google Patents

一种灭菌剂及应用灭菌剂的杀菌方法 Download PDF

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WO2023125531A1
WO2023125531A1 PCT/CN2022/142313 CN2022142313W WO2023125531A1 WO 2023125531 A1 WO2023125531 A1 WO 2023125531A1 CN 2022142313 W CN2022142313 W CN 2022142313W WO 2023125531 A1 WO2023125531 A1 WO 2023125531A1
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sterilization
concentration
sterilizing agent
peracetic acid
hydrogen peroxide
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PCT/CN2022/142313
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English (en)
French (fr)
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舩本诚一
张永巍
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苏州昊本生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2101/00—Chemical composition of materials used in disinfecting, sterilising or deodorising

Definitions

  • This application relates to the field of decellularized tissue processing, more specifically, it relates to a sterilizing agent and a sterilizing method using the sterilizing agent.
  • Decellularized tissue is a material derived from a living body. Human or animal tissue with the same structure as human tissue is processed to remove the immunogenic components in the tissue to obtain acellular tissue. Acellular materials are often used in the fields of scaffold materials for regenerative medicine and materials for promoting wound repair, and acellular materials have attracted much attention due to their high efficiency.
  • decellularized tissue When producing decellularized tissue for research or clinical use, animal tissue is taken as a raw material in a clean environment, and then the animal tissue is decellularized. However, even animal tissues collected in a clean environment can carry bacteria and viruses themselves. Therefore, in the research and clinical use of decellularized tissues, they will be sterilized after the decellularized treatment to prevent Patients have symptoms such as fever or bacterial infection after transplantation of decellularized tissue.
  • the inventors found that in the steps before decellularization such as removal of remaining tissues, the growth of bacteria and the bacteria brought by external operations will increase the bacteria in biological tissues, thereby affecting subsequent operations.
  • the present application provides a sterilizing agent and a sterilizing method using the sterilizing agent.
  • the application provides a sterilizing agent, which adopts the following technical scheme:
  • a sterilizing agent comprising peracetic acid and hydrogen peroxide, the volume ratio of the peracetic acid and hydrogen peroxide is 1: (0.26-84.5); the sterilizing agent is used for the biological tissue in the process of decellularization Sterilize.
  • a sterilant includes peracetic acid, hydrogen peroxide and a solvent, the concentration of the peracetic acid is 0.098-1.844ml/L, and the concentration of the hydrogen peroxide is 0.484-8.281ml/L.
  • the concentration of the peracetic acid is 0.195-0.922ml/L.
  • the concentration of the hydrogen peroxide is 0.977-4.141ml/L.
  • the solvent includes at least one of physiological saline, MEM buffer, DMEM buffer and PBS buffer.
  • the application provides a sterilization method, which adopts the following technical solution:
  • a sterilization method comprising the steps of:
  • the sterilization temperature is 3-38°C.
  • the sterilization temperature is 4°C
  • the concentration of the peracetic acid is 0.391-0.922ml/L.
  • the concentration of the hydrogen peroxide is 1.953-4.141ml/L.
  • the sterilization temperature is 37°C
  • the concentration of peracetic acid is 0.195-0.461ml/L.
  • the concentration of the hydrogen peroxide is 0.977-2.07ml/L.
  • the sterilization time is 3-24 hours.
  • peracetic acid and hydrogen peroxide are configured into a sterilizing agent according to the specified dosage ratio, and then the sterilizing agent is applied to the sterilization of biological tissues in the exfoliated cell treatment process, and a good sterilizing effect is obtained;
  • This application controls the dosage ratio of peracetic acid and hydrogen peroxide, so that hydrogen peroxide can have a good auxiliary effect on the sterilization effect of peracetic acid; at the same time, this application controls the sterilization temperature and other conditions, so that the sterilization agent has a better sterilization effect.
  • This application utilizes hydrogen peroxide and normal saline to dilute peracetic acid, so that while the sterilant has a good sterilizing effect, it also reduces the damage of the sterilant to the collagen structure of biological tissues.
  • Figure 1 is a structural diagram of biological tissue collagen without sterilization.
  • Fig. 2 is a structural diagram of biological tissue collagen in Example 1 of the present application.
  • Fig. 3 is a structural diagram of biological tissue collagen in Example 14 of the present application.
  • Fig. 4 is a structural diagram of biological tissue collagen in Example 15 of the present application.
  • Fig. 5 is a structural diagram of biological tissue collagen in Example 23 of the present application.
  • FIG. 6 is a structural diagram of biological tissue collagen in Comparative Example 1 of the present application.
  • FIG. 7 is a structural diagram of biological tissue collagen in Comparative Example 2 of the present application.
  • FIG. 8 is a structural diagram of biological tissue collagen in Comparative Example 5 of the present application.
  • decellularized materials in the fields of scaffold materials for regenerative medicine and materials for promoting wound repair is of great significance and has been attracting attention.
  • the decellularization process usually includes steps such as removal of unclean tissue, tissue cleaning, and decellularization.
  • the decellularized tissue is generally sterilized after the decellularized tissue is completed.
  • the survival probability of microorganisms on the sterilized items should be less than one in one million.
  • Sterilization methods that can meet this standard usually include heating, irradiation, gas sterilization, filtration, and sterilant treatment.
  • the inventors used sterilizing agents to perform tissue sterilization before decellularization. The inventors have found in research that peracetic acid is more suitable for sterilizing biological tissues than other sterilizing agents, such as pentylene glycol, sodium hypochlorite, ethanol, etc.
  • the inventors found in the research that when using peracetic acid solution alone, once the concentration of peracetic acid is not well controlled, it is easy to affect the final sterilization effect, but as the concentration of peracetic acid increases, the collagen structure of biological tissues will also change. was destroyed. Therefore, the inventors choose hydrogen peroxide to cooperate with physiological saline or buffer solution to dilute peracetic acid, which can well control the degree of damage to the collagen structure of biological tissues.
  • the inventor tried to study the sterilization temperature and time; but after controlling the sterilization temperature, the inventor found that there was no rule to be found between the concentration of peracetic acid and the sterilization temperature.
  • the inventor has done a lot of research and finally found that when the sterilization temperature is 4°C, the concentration of peracetic acid is 0.391-0.922ml/L, and the concentration of hydrogen peroxide is 1.953-4.141ml/L, the sterilant has no effect on biological
  • the sterilization effect of the tissue is relatively high, and the collagen structure of the biological tissue is relatively complete.
  • the sterilization temperature is 37°C
  • the concentration of peracetic acid is 0.195-0.461ml/L
  • the concentration of hydrogen peroxide is 0.977-2.07ml/L
  • Embodiment 1 provides a kind of sterilizing agent, and sterilizing agent comprises peracetic acid, hydrogen peroxide and solvent;
  • sterilizing agent comprises peracetic acid, hydrogen peroxide and solvent;
  • concentration of peracetic acid is 1.563ml/L
  • concentration of hydrogen peroxide is 7.813ml/L
  • the solvent is saline.
  • the configuration method of a kind of sterilant is:
  • aqueous solution of peracetic acid and hydrogen peroxide as stock solution, wherein the concentration of peracetic acid is 50ml/L, and the concentration of hydrogen peroxide is 250ml/L.
  • the stock solution was diluted step by step with physiological saline, and the number of dilutions in this embodiment was 5 times, and the dilution factor was 32 times.
  • Secondary dilution Take 25ml of the primary mixture and mix with 25ml of normal saline to obtain the secondary mixture.
  • Tertiary dilution Take 25ml of the secondary mixed solution and mix with 25ml of normal saline to obtain the tertiary mixed solution.
  • Five-stage dilution Take 25ml of the four-stage mixture and mix it with 25ml of physiological saline to obtain a sterilizing agent. In addition, it should be noted that if further dilution is required, the above steps can be repeated.
  • Embodiment 1 also provides a kind of sterilization method, comprises the following steps:
  • Bio tissue was taken.
  • the raw material of the biological tissue in this embodiment was a commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.
  • the biological tissue is placed in a sterilizing agent and treated for 3 hours at 4°C to complete the sterilization.
  • Example 2-6 The difference between Examples 2-6 and Example 1 is that the temperature and time in the sterilization process are different, see Table 1 for details.
  • the difference between the sterilizing agent in Example 7 and the sterilizing agent in Example 1 is: the stock solution is diluted 6 times, and the dilution factor is 64 times to obtain the sterilizing agent.
  • the concentration of peracetic acid is 0.781ml/L
  • the concentration of hydrogen peroxide is 3.906ml/L.
  • Example 8-12 The difference between Examples 8-12 and Example 7 is that the temperature and time in the sterilization process are different, see Table 2 for details.
  • the stock solution is diluted 7 times, and the dilution factor is 128 times to obtain the sterilant.
  • the concentration of peracetic acid is 0.391ml/L
  • the concentration of hydrogen peroxide The concentration is 1.953ml/L.
  • Embodiment 14-18 The difference between Embodiment 14-18 and Embodiment 13 is: the temperature and time in the sterilization process are different, specifically see Table 3.
  • the stoste is diluted 8 times, and the dilution factor is 256 times to obtain the sterilizing agent.
  • the concentration of peracetic acid is 0.195ml/L
  • the concentration of hydrogen peroxide The concentration is 0.977ml/L.
  • Example 20-24 The difference between Examples 20-24 and Example 19 is that the temperature and time in the sterilization process are different, see Table 4 for details.
  • Example 25 The difference between Example 25 and Example 1 is that the stock solution is diluted 9 times, and the dilution factor is 512 times to obtain a sterilant.
  • the concentration of peracetic acid is 0.098ml/L
  • the concentration of hydrogen peroxide The concentration is 0.484ml/L.
  • Example 26-30 The difference between Examples 26-30 and Example 25 is that the temperature and time in the sterilization process are different, see Table 5 for details.
  • embodiment 31-36 and embodiment 1 The difference of embodiment 31-36 and embodiment 1 is: the concentration of the concentration of peracetic acid and hydrogen peroxide is different, and sterilization condition is different.
  • the aqueous solution of peracetic acid and hydrogen peroxide is used as the stock solution, wherein the concentration of peracetic acid is 59ml/L, and the concentration of hydrogen peroxide is 265ml/L. See Table 6 for details.
  • Example 37 The difference between Example 37 and Example 13 is that the solvent is MEM buffer.
  • Example 38 The difference between Example 38 and Example 13 is that the solvent is DMEM buffer.
  • Example 39 The difference between Example 39 and Example 13 is that the solvent is PBS buffer.
  • a kind of sterilizing agent comprises peracetic acid, hydrogen peroxide and solvent, the concentration of peracetic acid is 0.391ml/L, the concentration of hydrogen peroxide is 3.125ml/L, The solvent is saline.
  • Example 41 The difference between Example 41 and Example 13 is: a sterilizing agent, including peracetic acid, hydrogen peroxide and solvent, the concentration of peracetic acid is 0.596ml/L, the concentration of hydrogen peroxide is 1.953ml/L, The solvent is saline.
  • a sterilizing agent including peracetic acid, hydrogen peroxide and solvent
  • the concentration of peracetic acid is 0.596ml/L
  • the concentration of hydrogen peroxide is 1.953ml/L
  • the solvent is saline.
  • Bio tissue was taken.
  • the raw material of the biological tissue in this embodiment was a commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.
  • a kind of sterilizing agent comprises peracetic acid, hydrogen peroxide and solvent, and the concentration of peracetic acid is 3.125ml/L, and the concentration of hydrogen peroxide is 15.625ml/L,
  • the solvent is saline.
  • the biological tissue is placed in a sterilizing agent and treated for 3 hours at 4°C to complete the sterilization.
  • the difference between comparative example 4 and embodiment 1 is: a kind of sterilant, comprises peracetic acid, hydrogen peroxide and solvent, and the concentration of peracetic acid is 50ml/L, and the concentration of hydrogen peroxide is 250ml/L, and solvent is saline.
  • Bio tissue was taken.
  • the raw material of the biological tissue in this embodiment was a commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.
  • the biological tissue is placed in a sterilizing agent at 37° C. for 3 hours to complete the sterilization.
  • the sterilization method of comparative example 6 comprises the following steps:
  • Bio tissue was taken.
  • the raw material of the biological tissue in this embodiment was a commercially available chicken heart, and the biological tissue was a small square piece of 1 cubic centimeter.
  • the biological tissue is placed in a sterilizing agent at 37° C. for 3 hours to complete the sterilization.
  • Comparative Example 7 The difference between Comparative Example 7 and Example 13 is: a sterilizing agent, including peracetic acid and a solvent, the concentration of peracetic acid is 0.391ml/L, and the solvent is physiological saline.
  • Comparative Example 8 The difference between Comparative Example 8 and Example 13 is: a sterilizing agent, including hydrogen peroxide and a solvent, the concentration of hydrogen peroxide is 1.953ml/L.
  • the sterilization effect test is carried out on the biological tissues after the sterilization of Examples 1-43 and Comparative Examples 1-8, and the specific operations are as follows:
  • the biological tissue was added to 4ml of E-MEM culture solution containing 10wt% fetal bovine serum (FBS), and cultured for 24 hours;
  • FBS fetal bovine serum
  • Table 7 Table of the number of bacteria after peracetic acid treatment
  • Example 10 0
  • Example 11 0
  • Example 12 0
  • Example 13 60
  • Example 14 0
  • Example 15 0
  • Example 16 20
  • Example 17 0
  • Example 18 90
  • Example 19 80
  • Example 20 30
  • Example 21 70
  • Example 22 55
  • Example 23 0
  • Example 24 120
  • Example 25 75
  • Example 26 60
  • Example 27 105
  • Example 28 90
  • Example 29 60
  • Example 30 140 Example 31 0
  • Example 32 0 Example 33 0 Example 34 0 Example 35 0 Example 36 0 Example 37 65 Example 38 63 Example 39 60 Example 40 90 Example 41 76 Example 42 142 Example 43 0 Comparative example 1 0 Comparative example 2 0 Comparative example 3 0 Comparative example 4 0 Comparative example 5 0 Comparative example 6 0 Comparative example 7 80 Comparative example 8 145
  • the biological tissue after embodiment 1-43 and comparative example 1-8 sterilization is carried out collagen tissue detection, concrete operation is as follows:
  • tissue sections were stained with HE (Hematoxylin-Cincinthin). After staining, the sections were sealed with neutral gum, and finally the sections were observed under a microscope to evaluate the changes in tissue collagen structure.
  • HE Hematoxylin-Cincinthin
  • Example 1 Example 1, Example 14, Example 15, Example 23, Comparative Example 2, Comparative Example 3, Comparative Example 6 and the unsterilized biological tissue collagen structures are shown in Figures 1-8.
  • the collagen structure of the biological tissue of Comparative Example 1 was invented, and the sterilant of this concentration would destroy the collagen structure of the biological tissue to a certain extent.
  • the collagen structure of the biological tissue of Comparative Example 1 and Comparative Example 2 was compared and found that the comparative example The destruction of the collagen structure of biological tissues in 1 and Comparative Example 2 is more serious, indicating that although the sterilizing agent with a higher concentration has a better sterilization effect, it will affect the collagen structure of biological tissues; in addition, it can be found that Example 14 and Example 1 The collagen structure of the biological tissue in 15 was not destroyed, indicating that the concentration of the sterilant in Example 14 and Example 15 is more appropriate. Moreover, it can be seen from the comparison of the collagen structure of the biological tissue in Comparative Example 5 and Comparative Example 4 that when the temperature becomes higher, the damage to the collagen structure of the biological tissue is greater.
  • Example 10 Example 7, Example 16 and Example 13
  • Example 14 Comparing the test results of Example 8, Example 14 and Example 17, it can be seen that compared with the concentration and temperature drop alone, when the temperature and concentration both drop, the sterilizing effect does not change.
  • Example 9 Comparing the sterilizing effects of Example 9, Example 18 and Example 15, it was found that when the temperature and concentration both decreased, the sterilizing effect deteriorated compared to the concentration alone. It shows that the temperature and concentration will have compound effects on the sterilization effect of the sterilant at the same time.
  • the sterilization temperature is 37°C
  • the concentration of peracetic acid is 0.195-0.461ml/L
  • the concentration of hydrogen peroxide is 0.977-2.07ml/L
  • the sterilization effect of the sterilant on biological tissues is better, and the biological The collagen structure of the tissue is relatively complete.

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Abstract

本申请涉及脱细胞组织处理领域，具体公开了一种灭菌剂及应用灭菌剂的灭菌方法，所述灭菌剂包括过醋酸和过氧化氢，所述过醋酸和过氧化氢的体积用量比为1：(0.26-84.5)；所述灭菌剂用于脱细胞处理过程中的生物组织灭菌。所述灭菌方法包括以下步骤：取生物组织；将生物组织放入灭菌剂中进行灭菌；灭菌温度为3-38℃。本申请可以在脱细胞化处理前对生物组织进行良好灭菌。

Description

一种灭菌剂及应用灭菌剂的杀菌方法 技术领域
本申请涉及脱细胞组织处理领域，更具体地说，它涉及一种灭菌剂及应用灭菌剂的杀菌方法。
背景技术
脱细胞组织是一种生体由来材料，对人或者有着与人类组织同样结构的动物组织进行处理，将组织中的免疫原性成份去掉后，可以得到脱细胞组织。脱细胞材料经常被应用于再生医疗的支架材料以及促进创伤修复材料等领域，并且脱细胞材料由于其高效性而备受瞩目。
用于研究或临床的脱细胞组织在制作时，是在洁净的环境中采取作为原材料的动物组织，然后对动物组织进行脱细胞化处理。但是，即便是在洁净环境中采取的动物组织，其自身也会携带细菌和病毒，因此在进行脱细胞组织的研究和临床使用时，最终都会在脱细胞处理结束后进行灭菌处理，从而预防患者在移植脱细胞组织后而引起发烧或细菌感染等症状。
但是，发明人发现，在去除剩余组织等脱细胞化处理前的步骤中，细菌的滋生、外界操作带来的细菌，都会增加生物组织的细菌，从而会影响后续操作。
发明内容
为了在脱细胞化处理前对生物组织进行良好灭菌，本申请提供一种灭菌剂及应用灭菌剂的杀菌方法。
第一方面，本申请提供一种灭菌剂，采用如下的技术方案：
一种灭菌剂，包括过醋酸和过氧化氢，所述过醋酸和过氧化氢的体积用量比为1：(0.26-84.5)；所述灭菌剂用于脱细胞处理过程中的生物组织灭菌。
优选的，一种灭菌剂，包括过醋酸、过氧化氢和溶剂，所述过醋酸的浓度为0.098-1.844ml/L，所述过氧化氢的浓度为0.484-8.281ml/L。
优选的，所述过醋酸的浓度为0.195-0.922ml/L。
优选的，所述过氧化氢的浓度为0.977-4.141ml/L。
优选的，所述溶剂包括生理盐水、MEM缓冲液、DMEM缓冲液、PBS缓冲液中的至少一种。
第二方面，本申请提供一种灭菌方法，采用如下的技术方案：
一种灭菌方法，包括以下步骤：
取生物组织；
将生物组织放入灭菌剂中进行灭菌；灭菌温度为3-38℃。
优选的，所述灭菌温度为4℃，所述过醋酸的浓度为0.391-0.922ml/L。
优选的，所述过氧化氢的浓度为1.953-4.141ml/L。
优选的，所述灭菌温度为37℃，所述过醋酸的浓度为0.195-0.461ml/L。
优选的，所述过氧化氢的浓度为0.977-2.07ml/L。
优选的，所述灭菌时间为3-24小时。
综上所述，本申请具有以下有益效果：
1、本申请将过醋酸和过氧化氢，按照规定的用量比配置成灭菌剂，然后将灭菌剂应用于脱落细胞处理过程中的生物组织灭菌，得到了良好的灭菌效果；
2、本申请控制过醋酸和过氧化氢的用量比，使得过氧化氢可以对过醋酸的灭菌效果有很好的辅助作用；同时本申请对灭菌的温度等条件进行控制，使得灭菌剂具有更好地灭菌效果。
3、本申请利用过氧化氢和生理盐水对过醋酸形成稀释，使得灭菌剂具有良好灭菌效果的同时，也减小了灭菌剂对生物组织的胶原结构的破坏。
附图说明
图1是未进行灭菌操作的生物组织胶原结构图。
图2是本申请实施例1的生物组织胶原结构图。
图3是本申请实施例14的生物组织胶原结构图。
图4是本申请实施例15的生物组织胶原结构图。
图5是本申请实施例23的生物组织胶原结构图。
图6是本申请对比例1的生物组织胶原结构图。
图7是本申请对比例2的生物组织胶原结构图。
图8是本申请对比例5的生物组织胶原结构图。
具体实施方式
以下结合附图1-8和实施例对本申请作进一步详细说明。
脱细胞材料被用于再生医疗的支架材料以及促进创伤修复材料 等领域，意义十分重大，而且一直备受瞩目。脱细胞的制作流程通常包括去除不洁组织、组织清洗、脱细胞化处理等步骤。而且为了减少因脱细胞组织腐败而引起的患者感染等症状，一般会在脱细胞处理结束后，对脱细胞组织进行灭菌处理。
但是，发明人发现，在脱细胞处理前，环境影响、操作手法和生物组织本身都会造成细菌的滋生和增多，进而会影响到后续的操作。因此发明人认为在脱细胞化处理前对生物组织进行灭菌也十分的重要。
对于体内移植医用材料在临床使用时的灭菌标准要求，被灭菌物品上微生物的生存概率要低于100万分之一。能达到这一标准的灭菌法通常有加热法、照射法、气体灭菌法、过滤法以及灭菌剂处理法等。考虑到脱细胞组织的生体组织特性以及各种灭菌法的特点，发明人采用灭菌剂进行脱细胞化加工前的组织灭菌处理。发明人在研究中发现，过醋酸相比其他的灭菌剂，如戊二醇、次氯酸钠、乙醇等，更适合对生物组织进行灭菌。
不过发明人在研究中发现，单独使用过醋酸溶液时，过醋酸的浓度一旦控制不好，很容易影响到最后的灭菌效果，但是随着过醋酸的浓度增加，生物组织的胶原结构也会被发生破坏。因此发明人选择过氧化氢配合生理盐水或者缓冲液对过醋酸进行稀释，可以很好地控制对生物组织的胶原结构的破坏程度。
为了进一步提高灭菌剂的效果，发明人尝试对灭菌温度和时间进行研究；但是发明人在对灭菌温度进行控制后发现，过醋酸的浓 度和灭菌温度之间并没有可寻规律。发明人做了大量的研究最终发现，当灭菌温度为4℃，过醋酸的浓度为0.391-0.922ml/L，且过氧化氢的浓度为1.953-4.141ml/L时，灭菌剂对生物组织的灭菌效果较，且生物组织的胶原结构较为完整。当灭菌温度为37℃，过醋酸的浓度为0.195-0.461ml/L，且过氧化氢的浓度为0.977-2.07ml/L时，灭菌剂对生物组织的灭菌效果较好，且生物组织的胶原结构较为完整。
实施例
实施例1
实施例1提供了一种灭菌剂，灭菌剂包括过醋酸、过氧化氢和溶剂；灭菌剂中，过醋酸的浓度为1.563ml/L，过氧化氢的浓度为7.813ml/L，溶剂为生理盐水。
一种灭菌剂的配置方法为：
取过醋酸和过氧化氢的水溶液作为原液，其中过醋酸的浓度为50ml/L，过氧化氢的浓度为250ml/L。利用生理盐水对原液进行逐级稀释，本实施例的稀释次数为5次，稀释倍数为32倍。
一级稀释：取25ml的原液和25ml的生理盐水混合，得到一级混合液。
二级稀释：取25ml一级混合液和25ml的生理盐水混合，得到二级混合液。
三级稀释：取25ml二级混合液和25ml的生理盐水混合，得到三级混合液。
四级稀释：取25ml三级混合液和25ml的生理盐水混合，得到四级混合液。
五级稀释：取25ml四级混合液和25ml的生理盐水混合，得到灭菌剂。另外要要说明的是，需要进行进一步稀释时，重复上述步骤即可。
实施例1还提供了一种灭菌方法，包括以下步骤：
取生物组织，本实施例中的生物组织的原材料为市售鸡心，生物组织为1立方厘米的方形小块。
将生物组织置于灭菌剂中，4℃的环境下，处理3小时，完成灭菌。
实施例2-6
实施例2-6和实施例1的不同之处在于：灭菌过程中的温度和时间不同，具体见表1。
表1实施例1-6的灭菌条件参数表
Figure PCTCN2022142313-appb-000001
实施例7
实施例7中的灭菌剂和实施例1中的灭菌剂的不同之处在于：对原液进行6次稀释，稀释倍数为64倍，得到灭菌剂，灭菌剂中，过醋酸的浓度为0.781ml/L，过氧化氢的浓度为3.906ml/L。
实施例8-12
实施例8-12和实施例7的不同之处在于：灭菌过程中的温度和时间不同，具体见表2。
表2实施例8-12的灭菌条件参数表
Figure PCTCN2022142313-appb-000002
实施例13
实施例13和实施例1的不同之处在于：对原液进行7次稀释，稀释倍数为128倍，得到灭菌剂，灭菌剂中，过醋酸的浓度为0.391ml/L，过氧化氢的浓度为1.953ml/L。
实施例14-18
实施例14-18和实施例13的不同之处在于：灭菌过程中的温度 和时间不同，具体见表3。
表3实施例13-18的灭菌条件参数表
Figure PCTCN2022142313-appb-000003
实施例19
实施例19和实施例1的不同之处在于：对原液进行8次稀释，稀释倍数为256倍，得到灭菌剂，灭菌剂中，过醋酸的浓度为0.195ml/L，过氧化氢的浓度为0.977ml/L。
实施例20
实施例20-24和实施例19的不同之处在于：灭菌过程中的温度和时间不同，具体见表4。
表4实施例19-24的灭菌条件参数表
Figure PCTCN2022142313-appb-000004
Figure PCTCN2022142313-appb-000005
实施例25
实施例25和实施例1的不同之处在于：对原液进行9次稀释，稀释倍数为512倍，得到灭菌剂，灭菌剂中，过醋酸的浓度为0.098ml/L，过氧化氢的浓度为0.484ml/L。
实施例26
实施例26-30和实施例25的不同之处在于：灭菌过程中的温度和时间不同，具体见表5。
表5实施例25-30的灭菌条件参数表
Figure PCTCN2022142313-appb-000006
实施例31-36
实施例31-36和实施例1的不同之处在于：过醋酸和过氧化氢 的浓度的浓度不同，灭菌条件不同。同时，在灭菌剂的配置过程中，过醋酸和过氧化氢的水溶液作为原液，其中过醋酸的浓度为59ml/L，过氧化氢的浓度为265ml/L。具体见表6。
表6实施例31-36的灭菌剂组分浓度表
Figure PCTCN2022142313-appb-000007
实施例37
实施例37和实施例13的不同之处在于：溶剂为MEM缓冲液。
实施例38
实施例38和实施例13的不同之处在于：溶剂为DMEM缓冲液。
实施例39
实施例39和实施例13的不同之处在于：溶剂为PBS缓冲液。
实施例40
实施例40和实施例13的不同之处在于：一种灭菌剂，包括过醋酸、过氧化氢和溶剂，过醋酸的浓度为0.391ml/L，过氧化氢的浓 度为3.125ml/L，溶剂为生理盐水。
实施例41
实施例41和实施例13的不同之处在于：一种灭菌剂，包括过醋酸、过氧化氢和溶剂，过醋酸的浓度为0.596ml/L，过氧化氢的浓度为1.953ml/L，溶剂为生理盐水。
实施例42
实施例42和实施例13的不同之处在于：一种灭菌剂，包括过醋酸、过氧化氢和溶剂，过醋酸的浓度为0.098ml/L，过氧化氢的浓度为8.281ml/L，溶剂为生理盐水。
实施例43
实施例43和实施例13的不同之处在于：一种灭菌剂，包括过醋酸、过氧化氢和溶剂，过醋酸的浓度为1.844ml/L，过氧化氢的浓度为0.484ml/L，溶剂为生理盐水。
对比例
对比例1
对比例1和实施例1的不同之处在于：一种灭菌剂，包括过醋酸、过氧化氢和溶剂，过醋酸的浓度为50ml/L，过氧化氢的浓度为250ml/L，溶剂为生理盐水。
对比例1的灭菌方法，包括以下步骤：
取生物组织，本实施例中的生物组织的原材料为市售鸡心，生物组织为1立方厘米的方形小块。
将生物组织置于灭菌剂中，4℃的环境下，处理3小时，完成灭 菌。
对比例2
对比例2和对比例1的不同之处在于：将生物组织置于灭菌剂中，4℃的环境下，处理12小时，完成灭菌。
对比例3
对比例3和实施例1的不同之处在于：一种灭菌剂，包括过醋酸、过氧化氢和溶剂，过醋酸的浓度为3.125ml/L，过氧化氢的浓度为15.625ml/L，溶剂为生理盐水。
对比例3的灭菌方法，包括以下步骤：
取生物组织，本实施例中的生物组织的原材料为市售鸡心，生物组织为1立方厘米的方形小块。
将生物组织置于灭菌剂中，4℃的环境下，处理3小时，完成灭菌。
对比例4
对比例4和实施例1的不同之处在于：一种灭菌剂，包括过醋酸、过氧化氢和溶剂，过醋酸的浓度为50ml/L，过氧化氢的浓度为250ml/L，溶剂为生理盐水。
对比例4的灭菌方法，包括以下步骤：
取生物组织，本实施例中的生物组织的原材料为市售鸡心，生物组织为1立方厘米的方形小块。
将生物组织置于灭菌剂中，37℃的环境下，处理3小时，完成灭菌。
对比例5
对比例5和对比例4的不同之处在于：将生物组织置于灭菌剂中，37℃的环境下，处理12小时，完成灭菌。
对比例6
对比例6和实施例1的不同之处在于：一种灭菌剂，包括过醋酸、过氧化氢和溶剂，过醋酸的浓度为3.125ml/L，过氧化氢的浓度为15.625ml/L，溶剂为生理盐水。
对比例6的灭菌方法，包括以下步骤：
取生物组织，本实施例中的生物组织的原材料为市售鸡心，生物组织为1立方厘米的方形小块。
将生物组织置于灭菌剂中，37℃的环境下，处理3小时，完成灭菌。
对比例7
对比例7和实施例13的不同之处在于：一种灭菌剂，包括过醋酸和溶剂，过醋酸的浓度为0.391ml/L，溶剂为生理盐水。
对比例8
对比例8和实施例13的不同之处在于：一种灭菌剂，包括过氧化氢和溶剂，过氧化氢的浓度为1.953ml/L。
性能检测试验
一、灭菌效果评价：
对实施例1-43和对比例1-8灭菌后的生物组织进行灭菌效果检测，具体操作如下：
1.对灭菌后的生物组织进行清洗，除去灭菌剂；
2.再将生物组织加入灭菌生理盐水进行24小时洗净，洗净后除去生理盐水；
3.将生物组织加入4ml含有10wt％的牛胎血清(FBS)的E-MEM培养液，进行24h培养；
4.将琼脂培养基高温融化后取注入到10cm的培养皿中，然后对培养后的E-MEM培养液进行100万倍稀释，并取100μl稀释后的培养液加入培养皿中，然后在37℃环境下培养48小时；
5.培养后查数琼脂凝胶上的菌株来进行评价。
检测结果表7所示。
表7：过醋酸处理后的细菌数量表
项目 细菌数量(×10 6cfu/ml)
实施例1 0
实施例2 0
实施例3 0
实施例4 0
实施例5 0
实施例6 0
实施例7 10
实施例8 0
实施例9 0
实施例10 0
实施例11 0
实施例12 0
实施例13 60
实施例14 0
实施例15 0
实施例16 20
实施例17 0
实施例18 90
实施例19 80
实施例20 30
实施例21 70
实施例22 55
实施例23 0
实施例24 120
实施例25 75
实施例26 60
实施例27 105
实施例28 90
实施例29 60
实施例30 140
实施例31 0
实施例32 0
实施例33 0
实施例34 0
实施例35 0
实施例36 0
实施例37 65
实施例38 63
实施例39 60
实施例40 90
实施例41 76
实施例42 142
实施例43 0
对比例1 0
对比例2 0
对比例3 0
对比例4 0
对比例5 0
对比例6 0
对比例7 80
对比例8 145
二、灭菌后组织结构评价。
对实施例1-43和对比例1-8灭菌后的生物组织进行胶原组织检 测，具体操作如下：
1.对灭菌后的生物组织进行清洗，除去灭菌剂；
2.再将生物组织加入灭菌生理盐水进行24小时洗净，洗净后除去生理盐水；
3.将处理后的生物组织进入4wt％的多聚甲醛中12小时进行组织固定；
4.按照以下流程使用酒精对组织进行阶段性脱水置换；
70wt％的酒精，处理5小时；80wt％的酒精，处理5小时；90wt％的酒精，处理5小时；100wt％的酒精，处理5小时；二甲苯，处理5小时。
5.使用石蜡对组织进行包埋，然后将组织切割成7μm的石蜡切片；
6.组织切片进行HE(苏木精-衣红)染色，染色后使用中性树胶封片，最后通过显微镜观察切片来评价组织胶原结构变化。
其中实施例1、实施例14、实施例15、实施例23、对比例2、对比例3、对比例6以及未灭菌的生物组织胶原结构如图1-8所示。
数据分析
由实施例1-6的检测结果可知，灭菌剂的灭菌效果良好。同时对比实施例7-9的检测结果发现，当过醋酸的浓度为0.781ml/L，过氧化氢的浓度为3.906ml/L的时候，实施例7灭菌后的生物组织上仍有细菌；并且结合实施例13-15、实施例19-21、实施例25-27的检测结果可知，过醋酸和过氧化氢的浓度降低，会导致灭菌效果变 差。
对比实施例7-9和实施例10-12的灭菌效果、实施例13和实施例16的灭菌效果、实施例19-20和实施例22-23的检测结果可知，37℃的灭菌效果比4℃的灭菌效果要更好，说明提高温度有助于灭菌。但是，对比实施例16-18的检测结果、对比实施例22-24的检测结果以及对比实施例28-30的检测结果发现，随着灭菌时间的增加，细菌反而增多，说明灭菌效果和时间并没有关系。
对比例实施例1的生物组织的胶原结构发明，此浓度的灭菌剂会对生物组织的胶原结构有一定的破坏，同时比较对比例1和对比例2的生物组织的胶原结构发现，对比例1和对比例2的生物组织的胶原结构破坏更为严重，说明较高浓度的灭菌剂虽然灭菌效果比较好，但是会影响生物组织的胶原结构；另外可以发现，实施例14和实施例15的生物组织的胶原结构没有破坏，说明实施例14和实施例15的灭菌剂浓度较为合适。而且由对比例5和对比例4的生物组织的胶原结构对比可知，温度变高的时候，对生物组织的胶原结构破坏更大。
然而通过对比实施例10、实施例7、实施例16和实施例13的灭菌效果发现，相比于浓度和温度单独下降，当浓度和温度均下降的时候，灭菌效果下降更多；通过对比实施例8、实施例14和实施例17的检测结果可知，相比于浓度和温度单独下降，当温度和浓度均下降的时候，灭菌效果没有改变。通过对比实施例9、实施例18和实施例15的灭菌效果发现，相比于浓度单独下降，当温度和浓度 均下降的时候，灭菌效果变差。说明温度和浓度会同时对灭菌剂的灭菌效果产生复配的影响。并且发明人发现当灭菌温度为4℃，过醋酸的浓度为0.391-0.922ml/L，且过氧化氢的浓度为1.953-4.141ml/L时，灭菌剂对生物组织的灭菌效果较，且生物组织的胶原结构较为完整。当灭菌温度为37℃，过醋酸的浓度为0.195-0.461ml/L，且过氧化氢的浓度为0.977-2.07ml/L时，灭菌剂对生物组织的灭菌效果较好，且生物组织的胶原结构较为完整。
由实施例37-38的检测结果可知，缓冲液的选择对灭菌效果影响不大，缓冲液主要是减少细胞组织的溶胀。另外由实施例40和实施例41的检测结果可知，过醋酸和过氧化氢的用量比会对灭菌效果有影响，但是经过实施例42、实施例43和对比例7、对比例8的检测结果可知，主要起到杀菌作用的是过醋酸，过氧化氢主要对过醋酸有辅助作用。
本具体实施例仅仅是对本申请的解释，其并不是对本申请的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本申请的权利要求范围内都受到专利法的保护。

Claims (10)

  1. 一种灭菌剂，其特征在于：包括过醋酸和过氧化氢，所述过醋酸和过氧化氢的体积用量比为1：(0.26-84.5)；所述灭菌剂用于脱细胞处理过程中的生物组织灭菌。
  2. 根据权利要求1所述的一种灭菌剂，其特征在于：包括过醋酸、过氧化氢和溶剂；所述灭菌剂中，所述过醋酸的浓度为0.098-1.844ml/L，所述过氧化氢的浓度为0.484-8.281ml/L。
  3. 根据权利要求2所述的一种灭菌剂，其特征在于：所述灭菌剂中，所述过醋酸的浓度为0.195-0.922ml/L。
  4. 根据权利要求3所述的一种灭菌剂，其特征在于：所述灭菌剂中，所述过氧化氢的浓度为0.977-4.141ml/L。
  5. 根据权利要求2所述的一种灭菌剂，其特征在于：所述溶剂包括生理盐水、MEM缓冲液、DMEM缓冲液、PBS缓冲液中的至少一种。
  6. 一种灭菌方法，其特征在于：包括以下步骤：
    取生物组织；
    将生物组织放入灭菌剂中进行灭菌；灭菌温度为3-38℃。
  7. 根据权利要求6所述的一种灭菌方法，其特征在于：所述灭菌温度为4℃；所述灭菌剂中，所述过醋酸的浓度为0.391-0.922ml/L。
  8. 根据权利要求7所述的一种灭菌方法，其特征在于：所述灭菌剂中，所述过氧化氢的浓度为1.953-4.141ml/L。
  9. 根据权利要求6所述的一种灭菌方法，其特征在于：所述灭菌温度为37℃；所述灭菌剂中，所述过醋酸的浓度为0.195-0.461ml/L。
  10. 根据权利要求9所述的一种灭菌方法，其特征在于：所述灭菌剂中，所述过氧化氢的浓度为0.977-2.07ml/L。
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