WO2022134434A1 - 一种外泌体冻存保护液及其制备方法 - Google Patents

一种外泌体冻存保护液及其制备方法 Download PDF

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WO2022134434A1
WO2022134434A1 PCT/CN2021/094288 CN2021094288W WO2022134434A1 WO 2022134434 A1 WO2022134434 A1 WO 2022134434A1 CN 2021094288 W CN2021094288 W CN 2021094288W WO 2022134434 A1 WO2022134434 A1 WO 2022134434A1
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exosome
kcl
tris
cryopreservation
solution
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PCT/CN2021/094288
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French (fr)
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陈海佳
王小燕
姜交华
刘志敏
陈东煌
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广州赛莱拉干细胞科技股份有限公司
广东国科细胞科技有限公司
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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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

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  • the application relates to the field of biotechnology, in particular to an exosome cryopreservation protection solution and a preparation method thereof.
  • the exosomes secreted by stem cells are vesicles with a diameter of 30-150 nm, which are wrapped by the same lipid bilayer as the cytoplasmic membrane and contain biologically active substances such as proteins and RNA.
  • Mesenchymal stem cells act in a paracrine manner, and the mechanism is that stem cells play a role in tissue repair by producing a large number of cytokines and growth factors, as well as producing exosomes.
  • stem cells are widely used in tissue repair and treatment of ischemic diseases. Studies have found that exosomes secreted by stem cells can mimic the biological functions of stem cells, and have gradually been proven to promote tissue repair and treat some intractable diseases, with broad application prospects.
  • exosomes can directly enter the target cells, and induce the proliferation, migration, vascularization and other biology of target cells by transporting specific proteins, lipids and RNA and other biologically active substances to the target cells. Changes, thereby changing the local microenvironment, stably and lastingly exert a variety of biological functions.
  • This "carrier-based" signal transduction method enables exosomes to have higher and more stable signal transduction efficiency, and will not be diluted by extracellular media in vivo.
  • stem cell exosomes as therapeutic products have certain characteristics that stem cell preparations do not have, for example, compared with living cells, exosomes have more definite components and are easier to quality control, so these characteristics make exosomes have great application value. . More and more studies have shown that stem cell exosomes can reduce apoptosis, reduce inflammation, promote angiogenesis, inhibit fibrosis, and improve tissue repair potential. These effects of exosomes make them have good application prospects in the treatment of myocardial infarction, skin trauma and other tissue injuries.
  • Exosomes have low stability and are prone to inactivation, and in order to maintain the structural integrity of exosomes, such agents must be maintained and delivered at relatively low temperatures in order to maintain their biological activity.
  • the loss of biological activity of exosomes mostly occurs in the storage and preservation stage.
  • liquid preparations are prepared and stored at ultra-low temperature (for example, not higher than -60 °C), transported under low-temperature freezing conditions, and thawed before use.
  • ultra-low temperature for example, not higher than -60 °C
  • One of the main challenges for storage stability at temperatures below the freezing point is to prevent physical destruction of structural and functional components during freezing and during storage.
  • the cryopreservation time is too long, repeated freezing and thawing during use or improper use, the biological activity of exosomes tends to decrease rapidly and greatly, thus limiting its normal use.
  • the activity of exosomes is usually preserved by adding cryopreservation agents, such as culture medium or bovine serum, but the activity of exosomes within 3 months of cryopreservation in culture medium drops sharply, and the preservation of bovine serum There are also potential pathogenic factors in exosomes and cause inconsistent stability between batches of exosomes.
  • cryopreservation agents such as culture medium or bovine serum
  • bovine serum There are also potential pathogenic factors in exosomes and cause inconsistent stability between batches of exosomes.
  • freeze-dried preparation methods designed for exosomes can preserve the activity of exosomes to a certain extent, the components of the reagents are complex and the preparation is cumbersome, which is not conducive to the quality control of exosome preparations.
  • the present application provides an exosome cryopreservation protection solution and a preparation method thereof.
  • the exosome cryopreservation solution is simple in composition, can preserve exosomes for a long time at low temperature, and does not affect its activity.
  • exosome cryopreservation solution which includes: Tris-HCl buffer solution containing NaCl and KCl, glucose, CD-Lipid concentrate, and human serum albumin (HSA) solution , polyvinyl alcohol (PVA).
  • the consumption of each component in the exosome cryopreservation solution is:
  • the consumption of each component in the exosome cryopreservation solution is:
  • the molar concentration of Tris-HCl is 10-50 mM, the molar concentration of NaCl is 80-200 mM, and the molar concentration of KCl is 1-5 mM; Tris containing NaCl and KCl -
  • the pH of the HCl buffer solution is 7.0 to 8.0.
  • the molar concentration of Tris-HCl is 25 mM, the molar concentration of NaCl is 137 mM, and the molar concentration of KCl is 2.5 mM;
  • the pH is 7.4.
  • the application also provides a method for preparing the exosome cryopreservation solution, comprising the following steps:
  • Tris-HCl buffer solution containing NaCl and KCl
  • the exosome cryopreservation solution was prepared by mixing Tris-HCl buffer solution containing NaCl and KCl, glucose, polyvinyl alcohol, CD-Lipid concentrate and human serum albumin solution.
  • the operation of filtering each component through a filter membrane is also included before the mixing.
  • the pore size of the filter membrane is 0.2-0.3 ⁇ m.
  • the pore size of the filter membrane is 0.22 ⁇ m.
  • the present application provides an exosome cryopreservation protection solution and a preparation method thereof.
  • the exosome cryopreservation solution includes: Tris-HCl buffer solution containing NaCl and KCl, glucose, CD-Lipid concentrate, human serum albumin solution, and polyvinyl alcohol.
  • the exosome cryopreservation solution provided by this application has a reasonable proportion, simple ingredients and clear composition, and the exosomes added to the cryopreservation solution can be stored stably for 6-12 months at a low temperature of -20°C. Its biological activity can maintain 90% of its maximum activity;
  • composition of all components in the cryopreservation protection solution of the present application is clear, can be industrially produced, the raw materials are cheap and easy to obtain, and has the advantage of low cost.
  • Fig. 1 adopts the exosome particle size distribution and quantity of the exosome protection solution of the present application after storage for 12 months;
  • Fig. 2 adopts the exosome morphological diagram of the application's exosome protection solution after storage for 12 months;
  • the present application discloses an exosome cryopreservation solution and a preparation method thereof, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present application.
  • the method and application of the present application have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present application to achieve and Apply the technology of this application.
  • Exosome A tiny membrane vesicle that can be secreted by most cells, with a diameter of about 30-150nm, with a lipid bilayer membrane structure, carrying RNA and proteins, and is released by most cell types. part of the system. Almost all cells secrete extracellular vesicles, including mesenchymal stem cells. Mesenchymal stem cell-derived extracellular vesicles contain similar components to mother cells, such as cytokines, growth factors, lipids, mRNAs, and regulatory miRNAs. Wait. They are mainly involved in the communication between cells and change the microenvironment of the tissue, etc., and play an important role in immune regulation, tissue damage repair and so on.
  • Stem cell-derived exosomes Mesenchymal stem cells can not only secrete some soluble bioactive factors, but also secrete extracellular microvesicles, which can promote angiogenesis, inhibit cell apoptosis and fibrosis, and then promote the repair and regeneration of damaged tissues. More and more studies have shown that mesenchymal stem cell-derived extracellular vesicles play a very important role in tissue damage repair and organ function recovery. Therefore, extracellular vesicles are expected to replace cell therapy and become a safe Effective cell-free therapeutic agents.
  • the prepared exosomes are generally in liquid form, using PBS, culture medium, and serum as solvents, and stored at -20°C at low temperature, which has poor stability and is easy to inactivate.
  • exosome cryopreservation solution provided in this application and the raw materials or excipients used in the preparation method thereof can be purchased from the market.
  • CD-Lipid Concentrate Gibco, Cat. No. 11905031;
  • Polyvinyl alcohol sigma, Cat. No. P8136.
  • the formula of the exosome preservation preparation in this example is:
  • Tris-HCl buffer solution containing NaCl and KCl 94.3 parts by weight of Tris-HCl buffer solution containing NaCl and KCl, 2.5 parts by weight of glucose, 1 part by weight of CD-Lipid concentrate, 2 parts by weight of human serum albumin (HSA) solution, 0.2 parts by weight of polyvinyl alcohol ; wherein, in the Tris-HCL buffer solution containing NaCl and KCl, the molar concentration of Tris-HCl is 25 mM, the molar concentration of NaCl is 137 mM, and the molar concentration of KCl is 2.5 mM; the Tris-HCl containing NaCl and KCl is 2.5 mM; The pH of the HCl buffer solution was 7.4.
  • the preparation method includes the following steps:
  • Tris-HCl buffer solution containing NaCl and KCl
  • Tris-HCl buffer solution containing NaCl and KCl, glucose, polyvinyl alcohol additive, CD-Lipid concentrate and human serum albumin solution are respectively filtered with a filter membrane with a pore size of 0.22 ⁇ m, and mixed to prepare exocytosis. Body cryopreservation solution.
  • Reference publication number CN107245472A patent "a preparation method and use method of human mesenchymal stem cell exosome freeze-dried powder" disclosed in the second embodiment of "adding excipient mannitol 6g per 100mL of exosome concentrate" technology Program.
  • exosomes were resuspended with Lactated Ringer's Injection to obtain an exosome suspension
  • Test Example 1 Extraction and identification of mesenchymal stem cell-derived exosomes
  • hUC-MSCs log-phase umbilical cord mesenchymal stem cells
  • centrifuge at 100,000 g for 70 min to discard the supernatant resuspend the pellet with 1 mL of PBS, and then centrifuge at 100,000 g for 70 min to discard the supernatant, resuspend with different cryoprotectants and store at -20°C.
  • Example 1 Exosome preservation preparation of the present application.
  • exosomes used for testing and comparison were 1.32x10 ⁇ 10particles (NTA detection). After using different cryopreservation solutions, they were stored at -20°C for 3, 6, and 12 months, and then the exosomes were identified ( NTA nanoparticle size), use the skin fibroblast viability model to detect its biological activity (CCK8), the specific test results are shown in Figure 1-3 and the following table:
  • cryopreservation solution of this application can better protect the biological activity of exosomes, maintain more than 90% of the exosome structure and the biological function of promoting cell proliferation (each solvent is used as a negative control, excluding the solvent formulation itself). Impact).

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Abstract

本申请涉及生物技术领域，特别涉及一种外泌体冻存保护液及其制备方法。该外泌体冻存保护液包括：含NaCl和KCl的Tris-HCl缓冲溶液、葡萄糖、CD-Lipid浓缩液、人血清白蛋白溶液、以及聚乙烯醇。本申请提供的外泌体冻存保护液的配比合理，成分简单，组成明确，加入所述冻存保护液的外泌体能在-20℃的低温条件下，稳定保存6-12个月，其生物学活性能维持其最大活性的90％；本申请冻存保护液中所有组分的构成明确，可以工业化生产，原材料价格便宜、易获取，具有低成本优势。

Description

一种外泌体冻存保护液及其制备方法 技术领域
本申请涉及生物技术领域，特别涉及一种外泌体冻存保护液及其制备方法。
背景技术
干细胞分泌的外泌体是直径在30-150nm的囊泡，由与细胞质膜相同的脂质双分子层包裹，内部含有蛋白质和RNA等生物活性物质。间充质干细胞(MSCs)以旁分泌方式发挥作用，其机制为干细胞通过产生大量细胞因子和生长因子，以及产生外泌体(exosomes)来起到组织修复的作用。目前干细胞广泛应用于组织修复及缺血性疾病治疗领域。研究发现，干细胞分泌的外泌体能模拟干细胞的生物学功能，也逐渐被证实能促进组织修复和治疗一些难治性疾病，具有广阔的应用前景。与干细胞分泌的可溶性细胞因子不同，外泌体可直接进入靶细胞，通过向靶细胞转运特异性蛋白、脂质及RNA等生物活性物质，来诱导靶细胞的增殖、迁移、血管化等生物学变化，从而改变局部微环境，稳定而持久地发挥多种生物学功能。这种“载体式”信号转导方式使外泌体具备更高更稳定的信号转导效率，且在生物体内不会被细胞外介质稀释。因为干细胞外泌体作为治疗产品具有干细胞制剂不具有的某些特性，例如外泌体相比活细胞而言，成分比较确定，易于质量控制，因此这些特性使得外泌体具有很大的应用价值。越来越多的研究表明，干细胞外泌体可减少细胞凋亡、减轻炎症反应、促进血管生成、抑制纤维化、提高组织修复潜力等重要生物学作用。外泌体的这些作用使得其在治疗心肌梗死、皮肤创伤等组织损伤中存在良好的应用前景。
外泌体的稳定性低、容易失活，为了维持外泌体的结构完整性必须在相对低温下维持和运送这类制剂以便维持其生物活性。外泌体生物活性的丧失多发生在贮存保藏阶段，一般制成液体制剂超低温(例如不高于-60℃)下保存，在低温冷冻条件下运输，并在使用前解冻。而在冷冻点以下的温度保存稳定的主要挑战之一就是防止结构性和功能性成分在冷冻期间和保存阶段的物理性破坏。另外，超低温保存的时间过长在使用过程中反复冻融或者使用不当，外泌体的生物活性往往会迅速大幅下降，从而限制了它的正常使用。
现有技术中通常通过添加冻存保护剂来保存外泌体的活性，例如培养基或 牛血清，但在培养基中冻存3个月内的外泌体的活性急剧下降，牛血清保存的外泌体中还存在潜在的致病因子，并造成外泌体批间稳定性不一致。也有针对外泌体设计的冻干制剂方法，这类试剂虽能一定程度上保存外泌体的活性，但试剂的成份复杂，制备较繁琐，并不利于外泌体制剂质量的控制。
因此亟需一种成分简单、能在较低温下长期保存外泌体且不影响其活性的冻存保护试剂及方法。
发明内容
本申请提供了一种外泌体冻存保护液及其制备方法。该外泌体冻存保护液成分简单、能在较低温下长期保存外泌体，且不影响其活性。
本申请提供了一种外泌体冻存保护液，外泌体冻存保护液包括：含NaCl和KCl的Tris-HCl缓冲溶液、葡萄糖、CD-Lipid浓缩液、人血清白蛋白(HSA)溶液、聚乙烯醇(PVA)。
作为优选，以重量份计，外泌体冻存保护液中各组分的用量为：
Figure PCTCN2021094288-appb-000001
优选地，以重量份计，外泌体冻存保护液中各组分的用量为：
Figure PCTCN2021094288-appb-000002
作为优选，含NaCl和KCl的Tris-HCl缓冲溶液中，Tris-HCl的摩尔浓度为10～50mM，NaCl的摩尔浓度为80～200mM，KCl的摩尔浓度为1～5mM；含NaCl和KCl的Tris-HCl缓冲溶液的pH值为7.0～8.0。
优选地，含NaCl和KCl的Tris-HCl缓冲溶液中，Tris-HCl的摩尔浓度为25mM，NaCl的摩尔浓度为137mM，KCl的摩尔浓度为2.5mM；含NaCl和 KCl的Tris-HCl缓冲溶液的pH值为7.4。
本申请还提供了该外泌体冻存保护液的制备方法，包括以下步骤：
将NaCl和KCl加入到Tris-HCl缓冲溶液中，得到含NaCl和KCl的Tris-HCl缓冲溶液；
将含NaCl和KCl的Tris-HCl缓冲溶液、葡萄糖、聚乙烯醇、CD-Lipid浓缩液和人血白蛋白溶液混合，制得外泌体冻存保护液。
作为优选，混合之前还包括将各组分采用滤膜进行过滤的操作。
作为优选，滤膜的孔径为0.2～0.3μm。
优选地，滤膜的孔径为0.22μm。
本申请提供了一种外泌体冻存保护液及其制备方法。该外泌体冻存保护液包括：含NaCl和KCl的Tris-HCl缓冲溶液、葡萄糖、CD-Lipid浓缩液、人血清白蛋白溶液、以及聚乙烯醇。本申请具有的技术效果为：
本申请提供的外泌体冻存保护液的配比合理，成分简单，组成明确，加入所述冻存保护液的外泌体能在-20℃的低温条件下，稳定保存6-12个月，其生物学活性能维持其最大活性的90％；
本申请冻存保护液中所有组分的构成明确，可以工业化生产，原材料价格便宜、易获取，具有低成本优势。
附图说明
图1采用本申请外泌体保护液保存12个月后的外泌体粒径分布与数量；
图2采用本申请外泌体保护液保存12个月后的外泌体形态图；
图3采用不同外泌体保护液保存12个月后外泌体促细胞增殖活力比较。
具体实施方式
本申请公开了一种外泌体冻存保护液及其制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本申请。本申请的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本申请内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本申请技术。
术语解释：
外泌体(Exosome)：一种能被大多数细胞分泌的微小膜泡，直径大约30-150nm，具有脂质双层膜结构，携带RNA和蛋白质，被大多数细胞类型释放，是细胞间通讯系统的一部分。几乎所有的细胞都会分泌胞外囊泡，包括间充质干细胞，间充质干细胞来源的胞外囊泡含有与母细胞相似的成分，如细胞因子、生长因子、脂类、mRNAs及调控型miRNAs等。他们主要参与细胞与细胞间的交流通讯及改变组织的微环境等，在免疫调控，组织损伤修复等方面起着不可低估的作用。
干细胞来源外泌体：间充质干细胞不仅能分泌一些可溶性生物活性因子，亦可分泌胞外微囊泡，促进血管再生，抑制细胞凋亡及纤维化，进而促进损伤组织修复再生。越来越多的研究表明，间充质干细胞来源的胞外囊泡在组织损伤修复和器官功能恢复过程中起着非常重要的作用，因此，胞外囊泡有望取代细胞治疗，成为一种安全有效的无细胞治疗药剂。
外泌体保存：制备好的外泌体一般以液体形式，使用PBS、培养基、血清作为溶剂，放在-20℃低温保存，稳定性差，容易失活。
本申请提供的外泌体冻存保护液及其制备方法中所用原料药或辅料均可由市场购得。
本申请中使用的部分试剂的来源如下：
CD-Lipid浓缩液：Gibco，货号11905031；
人血清白蛋白溶液：杰特贝林，货号S20140078；
聚乙烯醇：sigma，货号P8136。
下面结合实施例，进一步阐述本申请：
实施例1 外泌体保存制剂及其制备方法
本实施例外泌体保存制剂配方为：
94.3重量份含NaCl和KCl的Tris-HCl缓冲溶液，2.5重量份的葡萄糖，1重量份的CD-Lipid浓缩液，2重量份的人血清白蛋白(HSA)溶液，0.2重量份的聚乙烯醇；其中，所述含NaCl和KCl的Tris-HCL缓冲溶液中，Tris-HCl的摩尔浓度为25mM，NaCl的摩尔浓度为137mM，KCl的摩尔浓度为2.5mM；所述含NaCl和KCl的Tris-HCl缓冲溶液的pH值为7.4。
制备方法包括以下步骤：
将NaCl和KCl加入到Tris-HCl缓冲溶液中，得到含NaCl和KCl的Tris-HCl缓冲 溶液；
将所述含NaCl和KCl的Tris-HCl缓冲溶液、葡萄糖、聚乙烯醇添加剂、CD-Lipid浓缩液和人血白蛋白溶液分别采用孔径为0.22μm的滤膜进行过滤，混合，制得外泌体冻存保护液。
对比例1
参考公开号CN107245472A专利“一种人间充质干细胞外泌体冻干粉的制备方法、使用方法”实施例二中公开的“每100mL外泌体的浓缩液中添加赋形剂甘露醇6g”技术方案。
对比例2
参考公开号CN108635372A专利“一种人间充质干细胞源外泌体的生物制剂的制备方法”实施例3中方案，对外泌体进行保护，具体的方案如下：
利用乳酸钠林格注射液对外泌体进行重悬处理，得到外泌体悬液；
在外泌体悬液中按质量比例加入氯化钠(1％)、氯化钾(1％)、乳酸钠(5％)、氯化钙(1％)、葡萄糖(5％)、必需氨基酸(10.01％)、非必需氨基酸(1％)、人血清白蛋白(10％)。
试验例1：间充质干细胞来源的外泌体的提取及鉴定
1)间充质干细胞来源的外泌体的提取(超高速离心或者超滤离心两种方法制备外泌体也能够达到同样的效果)
本实施例选取P3-P6代生长对数期的脐带间充质干细胞(hUC-MSCs)进行培养并提取外泌体，hUC-MSCs、无血清培养基由本申请单位提供。具体方法如下：
待细胞汇合度达到70％～80％时，收集第一批包含外泌体的条件性培养基，然后加入无血清的低糖DMEM培养基继续培养48h后收集第二批条件性培养基；然后利用超高速离心法或者超滤法分离提取外泌体。取1000mL细胞培养基以300g的离心力离心10min以去除细胞碎片，再以2000g离心力离心10min进一步去除细胞碎片和杂质，再以10,000g离心力离心30min，随后所得上清液经过0.22μm一次性滤器收集，最后以100,000g离心力离心70min弃上清， 沉淀以1mL PBS重悬，再以100,000g离心力离心70min弃上清，分别使用不同的冻存保护剂重悬后放置在-20℃保存。
2)间充质干细胞来源的外泌体的不同保存方式比较
分别将提取制备的外泌体分装使用不同的保护液进行保存，包括：
对比例1：PBS；
对比例2：DMEM；
对比例3：DMEM+10％；
对比例4：DMEM+10％+DMSO；
对比例5：CN107245472A实施例二专利配方
对比例6：CN108635372A实施例3专利配方
实施例1：本申请外泌体保存制剂。
保存条件：-20℃。
3)不同外泌体保护液对外泌体生物学活性的影响
用于测试比较的起始外泌体数量为1.32x10^10particles(NTA检测)，使用不同冻存液保存后分别置于-20℃保存3、6、12个月后，进行外泌体鉴定(NTA纳米粒径)，使用皮肤成纤维细胞活率模型检测其生物学活性(CCK8)，具体试验结果如图1-3和下表：
表1 不同外泌体保护液随时间对外泌体数量的影响
Figure PCTCN2021094288-appb-000003
结果发现，本申请冻存液能更好地保护好外泌体的生物学活性，维持90％以上的外泌体结构和促细胞增殖的生物学功能(各个溶剂作为阴性对照，排除溶剂配方本身的影响)。
以上所述仅是本申请的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。

Claims (9)

  1. 一种外泌体冻存保护液，其包括：含NaCl和KCl的Tris-HCl缓冲溶液、葡萄糖、CD-Lipid浓缩液、人血清白蛋白溶液、以及聚乙烯醇。
  2. 根据权利要求1所述的外泌体冻存保护液，其中，以重量份计，外泌体冻存保护液中各组分的用量为：
    Figure PCTCN2021094288-appb-100001
  3. 根据权利要求1所述的外泌体冻存保护液，其中，以重量份计，外泌体冻存保护液中各组分的用量为：
    Figure PCTCN2021094288-appb-100002
  4. 根据权利要求1所述的外泌体冻存保护液，其中，所述含NaCl和KCl的Tris-HCl缓冲溶液中，Tris-HCl的摩尔浓度为10～50mM，NaCl的摩尔浓度为80～200mM，KCl的摩尔浓度为1～5mM；并且所述含NaCl和KCl的Tris-HCl缓冲溶液的pH值为7.0～8.0。
  5. 根据权利要求1至4中任一项所述的外泌体冻存保护液，其中，所述含NaCl和KCl的Tris-HCl缓冲溶液中，Tris-HCl的摩尔浓度为25mM，NaCl的摩尔浓度为137mM，KCl的摩尔浓度为2.5mM；并且所述含NaCl和KCl的Tris-HCl缓冲溶液的pH值为7.4。
  6. 权利要求1至5中任一项所述外泌体冻存保护液的制备方法，其包括以下步骤：
    将NaCl和KCl加入到Tris-HCl缓冲溶液中，得到含NaCl和KCl的Tris-HCl缓冲溶液；
    将所述含NaCl和KCl的Tris-HCl缓冲溶液、葡萄糖、聚乙烯醇、CD-Lipid浓缩液和人血白蛋白溶液混合，制得外泌体冻存保护液。
  7. 根据权利要求6所述的制备方法，其中，所述混合之前还包括将各组分采用滤膜进行过滤的操作。
  8. 根据权利要求7所述的制备方法，其中，所述滤膜的孔径为0.2～0.3μm。
  9. 根据权利要求7或8所述的制备方法，其中，所述滤膜的孔径为0.22μm。
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