CN116889227A - Exosome freeze-drying protective agent, exosome freeze-drying method and exosome freeze-dried powder - Google Patents
Exosome freeze-drying protective agent, exosome freeze-drying method and exosome freeze-dried powder Download PDFInfo
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- CN116889227A CN116889227A CN202310732157.2A CN202310732157A CN116889227A CN 116889227 A CN116889227 A CN 116889227A CN 202310732157 A CN202310732157 A CN 202310732157A CN 116889227 A CN116889227 A CN 116889227A
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Classifications
-
- 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
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The application discloses an exosome freeze-drying protective agent, an exosome freeze-drying method and exosome freeze-drying powder. In a first aspect of the application, an exosome lyoprotectant is provided, comprising a non-reducing sugar, an alcohol, and an expectorant antioxidant. The exosome freeze-drying protective agent is suitable for freeze-drying preservation of exosomes of various cell sources, the tested freeze-dried powder is preserved for twelve months at normal temperature, the exosome integrity, the particle size concentration and the particle size distribution, the total protein and the membrane protein characterization, the total RNA of the inclusion and the like are stable, the cell proliferation promoting activity detection of the freeze-dried powder is also kept in a certain range, the normal temperature preservation after the freeze-drying of the exosomes of the cells has strong advantages, and the freeze-dried powder with good quality and long preservation period can be obtained after freeze-drying at a higher temperature.
Description
Technical Field
The application relates to the technical field of exosomes, in particular to an exosome freeze-drying protective agent, an exosome freeze-drying method and exosome freeze-drying powder.
Background
Exosomes (Exosomes) are vesicles (Extracellular Vesicles, EVs) secreted by cells to the outside of cells, having a size of 30-150 nm, a bilayer membrane structure and a tea-tray-like morphology, containing abundant contents (including nucleic acids, proteins, lipids, etc.), and involved in the transfer of molecules between cells. Exosomes are widely present in cell culture supernatants and in various body fluids, including blood, lymph, saliva, urine, semen, milk, etc., as well as in tissue samples, such as brain tissue, muscle tissue, adipose tissue, etc. Research has found that the exosomes secreted by stem cells mimic the biological function of stem cells and have also gradually been demonstrated to promote tissue repair and treat some refractory disorders. In addition, compared with stem cell preparations, the stem cell exosome has the characteristics of relatively determined components, easiness in quality control and the like, so that the stem cell exosome has wide application prospect and value.
The exosome double-layer lipid membrane structure ensures good stability, and can protect internal biomolecules from various enzymes in body fluid, thereby maintaining the integrity and bioactivity. However, the extracted exosomes are subject to a number of more complex storage environmental conditions, such as storage media, storage temperature and time. The temperature of-80℃is recognized as the optimum preservation temperature for preserving various biological specimens, such as semen, urine, milk, blood and bronchoalveolar lavage. The most common exosome storage method at present is-80 ℃ cryopreservation, but it is difficult to maintain such low temperature conditions during handling or transportation. Furthermore, cryopreservation may lead to changes in the shape and physical properties of the exosomes, and may also lead to the formation and aggregation of multiple vesicles. In addition, repeated freeze thawing can also result in changes in the biological properties, content and marker composition of the exosome surface molecules.
The freeze-drying technology provides a new choice for exosome preservation. The freeze-drying process is carried out at low temperature, and does not denature heat-sensitive substances such as proteins, microorganisms and the like or lose biological activity. Meanwhile, the growth of the microorganism and the action of the enzyme cannot be performed, so that the original properties can be maintained. In addition, the freeze-drying process is carried out in a frozen state, the volume is almost unchanged, the exosome can keep the original structure, and the concentration phenomenon can not occur. However, the existing exosome freeze-drying technology often requires pre-freezing for a long time at-80 ℃, freeze-drying is directly performed at a higher temperature, the quality of the freeze-dried powder is seriously reduced, and the storage life is obviously insufficient. Therefore, it is necessary to provide a more advantageous lyoprotectant, and use a more efficient lyophilization temperature parameter, so that the exosomes can be lyophilized at a higher temperature to obtain a lyophilized powder with good quality and long shelf life.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, the application provides the exosome freeze-drying protective agent, and the freeze-drying protective agent can be used for freeze-drying at a higher temperature to obtain freeze-dried powder with good quality and long storage life.
In a first aspect of the application, an exosome lyoprotectant is provided, comprising an expectorant antioxidant, a non-reducing sugar and an alcohol.
In some embodiments of the application, the exosome lyoprotectant comprises 1 part by mass of an expectorant antioxidant, 50 to 15000 parts by mass of a non-reducing sugar, and 50 to 15000 parts by mass of an alcohol. The content of the non-reducing sugar may be, for example, 50 parts by mass, 80 parts by mass, 100 parts by mass, 120 parts by mass, 150 parts by mass, 200 parts by mass, 300 parts by mass, 500 parts by mass, 800 parts by mass, 1000 parts by mass, 1200 parts by mass, 1500 parts by mass, 2000 parts by mass, 3000 parts by mass, 5000 parts by mass, 8000 parts by mass, 10000 parts by mass, 12000 parts by mass, 15000 parts by mass. The content of the alcohols may be, for example, 50 parts by mass, 80 parts by mass, 100 parts by mass, 120 parts by mass, 150 parts by mass, 200 parts by mass, 300 parts by mass, 500 parts by mass, 800 parts by mass, 1000 parts by mass, 1200 parts by mass, 1500 parts by mass, 2000 parts by mass, 3000 parts by mass, 5000 parts by mass, 8000 parts by mass, 10000 parts by mass, 12000 parts by mass, 15000 parts by mass.
In some embodiments of the application, the exosome lyoprotectant comprises 5-15 wt% of a non-reducing sugar, 5-15 wt% of an alcohol, 0.001-0.1% of an expectorant antioxidant. The content of the non-reducing sugar may be, for example, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, the content of the alcohol may be, for example, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, and the content of the expectorant antioxidant may be, for example, 0.001wt%, 0.002wt%, 0.005wt%, 0.01wt%, 0.02wt%, 0.05wt%, 0.1wt%.
In some embodiments of the application, the exosome lyoprotectant is formulated such that the final concentration of the mixed solution after mixing with the exosome before lyophilization comprises 2.5 to 7.5wt% of the non-reducing sugar, 2.5 to 7.5wt% of the alcohol, 0.0005 to 0.05wt% of the expectorant antioxidant. Specifically, the non-reducing sugar in the exosome lyoprotectant may have a final concentration of 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, 5.5wt%, 6wt%, 6.5wt%, 7wt%, 7.5wt%, and the alcohol may have a final concentration of 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, 5.5wt%, 6wt%, 6.5wt%, 7wt%, 7.5wt%, and the expectorant antioxidant may have a final concentration of 0.0005wt%, 0.0008wt%, 0.001wt%, 0.002wt%, 0.005wt%, 0.008wt%, 0.01wt%, 0.02wt%, and 0.05wt% in the mixture before lyophilization.
In some embodiments of the application, the expectorant antioxidant comprises at least one of N-acetylcysteine, ambroxol hydrochloride, carbocisteine, and erdosteine.
Among them, N-acetylcysteine is a thiol-containing antioxidant whose principle of eliminating phlegm is to break disulfide bonds (-S-S-) of mucin, which is a main component of sputum, thereby diluting the sputum, and its antioxidant property is also based on thiol function. The phlegm eliminating principle of ambroxol hydrochloride is similar to that of ambroxol hydrochloride, and the viscous phlegm is promoted to be dissolved by breaking mucin fibers in the sputum, so that the viscosity of the sputum is reduced. Simultaneously, ambroxol hydrochloride also has good antioxidation effect on inhibiting and removing OH, HOCl, H 2 O 2 、O 2 -· And the like has better effect. Carbocisteine eliminates phlegm and increases the secretion of low-viscosity proteins and reduces the secretion of high-viscosity proteins by acting on the secretion of bronchus glands, so that the viscosity of sputum is reduced. In addition, carbocisteine contains a large amount of sulfhydryl groups in the molecular structure, so that the carbocisteine has high antioxidation effect. Erdosteine by augmentation of bronchiThe continuous secretion of the slurry reduces the viscosity of the sputum and accelerates the discharge of the sputum to realize the phlegm elimination. The antioxidant mechanism is also derived from the thiol character. Therefore, in the examples of the present application, these specific expectorant antioxidants are selected to prevent oxidation of the biologically active substances in the exosomes and to avoid deterioration of the exosomes during lyophilization and during storage. And the freeze-drying protection of exosomes is realized by combining non-reducing sugar and alcohols.
In some embodiments of the application, the non-reducing sugar comprises at least one of trehalose, sucrose, and raffinose. Non-reducing sugars, one or more of which are utilized, provide non-specific protection to exosome proteins during lyophilization and drying.
In some embodiments of the application, the non-reducing sugar is a non-reducing disaccharide comprising at least one of trehalose and sucrose.
In some embodiments of the application, the alcohol comprises at least one of mannitol, sorbitol, ethylene glycol, polyethylene glycol, and glycerol. One or more of the alcohol substances are used for providing a solid structure for the freeze-dried powder cake, so that the appearance problems of shrinkage and collapse are prevented, and the temperature of a eutectic point is reduced.
In some embodiments of the application, the exosome lyoprotectant further comprises a solvent.
In some embodiments of the application, the exosome lyoprotectant comprises 5-15 wt% non-reducing sugar, 5-15 wt% alcohol, 0.001-0.1% expectorant antioxidant, and solvent.
In some embodiments of the present application, the solvent comprises at least one of an inorganic solvent, an organic solvent.
In some embodiments of the application, the inorganic solvent is water.
The exosome freeze-drying protective agent provided by the embodiment of the application has at least the following beneficial effects:
the exosome freeze-drying protective agent provided by the application is suitable for freeze-drying preservation of exosomes of various cell sources, can keep the original form and biological activity of exosomes for a long time in a freeze-drying state, has been tested that the freeze-dried powder is preserved for twelve months at normal temperature, has relatively stable exosome integrity, particle size concentration and particle size distribution, total protein and membrane protein characterization, total RNA of contents and the like, has relatively strong advantages in normal temperature preservation after freeze-drying of the exosomes, and has relatively strong cell proliferation promoting activity detection of the freeze-dried powder. In addition, the freeze-drying protective agent is basically harmless to cells, tissues and organs of a human body, so that the exosome freeze-dried powder achieves the effects of safety, non-toxicity, convenience and high efficiency.
In a second aspect of the present application, there is provided a method of exosome lyophilization comprising the steps of:
mixing the exosome with the pre-exosome freeze-drying protective agent, and freeze-drying at-35 to-28 ℃ to obtain the exosome freeze-dried powder.
In some embodiments of the application, the temperature of freeze-drying is-32 to-28 ℃.
In some embodiments of the application, the total protein of the exosomes after mixing has a final concentration of 1-5 mg/mL, which may be, for example, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL.
In some embodiments of the application, the total exosome protein is at a final concentration of 2-4 mg/mL after mixing.
In some embodiments of the application, the final concentration of the expectorant antioxidant after mixing is 0.0005 to 0.05wt%.
In some embodiments of the application, the total protein of the exosomes is at a final concentration of 1-5 mg/mL after mixing, and the expectorant antioxidant is at a final concentration of 0.0005-0.05 wt% after mixing.
In some embodiments of the application, the total protein of the exosomes is at a final concentration of 1-5 mg/mL after mixing, the expectorant antioxidant is at a final concentration of 0.0005-0.05 wt% after mixing, the non-reducing sugar is at a final concentration of 2.5-7.5 wt% after mixing, and the alcohol is at a final concentration of 2.5-7.5 wt% after mixing.
In some embodiments of the application, the lyophilization time is 15 to 30 hours.
In some embodiments of the application, the exosomes are mixed with the exosome lyoprotectant in the form of an exosome solution.
In some embodiments of the application, the total protein concentration of the exosomes in the exosome solution is 1-10 mg/mL, which may be, for example, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL.
In some embodiments of the application, the volume ratio of exosome solution to exosome lyoprotectant is 1: (0.5-2).
In some embodiments of the application, the exosomes comprise at least one of natural exosomes, engineered exosomes.
In some embodiments of the application, the cell source of the exosomes comprises at least one of a plurality of different types of cells, such as stem cells, immune cells, tumor cells, neural cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, endothelial cells, and the like.
In some embodiments of the application, the stem cells comprise at least one of hematopoietic stem cells, mesenchymal stem cells.
In some embodiments of the application, the hematopoietic stem cells are derived from at least one of bone marrow, peripheral blood, and the like.
In some embodiments of the application, the mesenchymal stem cells are derived from at least one of bone marrow, fat, umbilical cord blood, endometrium, amniotic membrane, placenta, muscle, corneal stroma, dental pulp, synovial membrane, bone, lung, liver, pancreas, peripheral blood, and the like.
In some embodiments of the application, the immune cells comprise at least one of T cells, B cells, NK cells, NKT cells, macrophages, or progenitor cells thereof.
In some embodiments of the application, the exosomes are derived from at least one of hematopoietic stem cells, mesenchymal stem cells, and natural killer cells.
In some embodiments of the application, the animal source of the exosomes comprises at least one vertebrate of reptiles, birds, mammals, etc.
In some embodiments of the application, mammals include animals of the order monocular, marsupials, food worm, jumping shrew, beast, winged, primates, barren, scaly, lagomorpha, rodentia, carnivora, beef, hoof and rabbit, pipe tooth, miracle, artiodactyla, whales, and the like.
In some embodiments of the application, the animal source of the exosomes comprises any of mice, rats, mice, guinea pigs, rabbits, horses, sheep, deer, monkeys, dogs, pigs, humans, etc.
In a third aspect of the present application, there is provided an exosome lyophilized powder prepared by any one of the exosome lyophilization methods described above.
In some embodiments of the application, the exosomes comprise at least one of natural exosomes, engineered exosomes.
In some embodiments of the application, the cell source of the exosomes comprises at least one of a plurality of different types of cells, such as stem cells, immune cells, tumor cells, neural cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, endothelial cells, and the like.
In some embodiments of the application, the stem cells comprise at least one of hematopoietic stem cells, mesenchymal stem cells.
In some embodiments of the application, the hematopoietic stem cells are derived from at least one of bone marrow, peripheral blood, and the like.
In some embodiments of the application, the mesenchymal stem cells are derived from at least one of bone marrow, fat, umbilical cord blood, endometrium, amniotic membrane, placenta, muscle, corneal stroma, dental pulp, synovial membrane, bone, lung, liver, pancreas, peripheral blood, and the like.
In some embodiments of the application, the immune cells comprise at least one of T cells, B cells, NK cells, NKT cells, macrophages, or progenitor cells thereof.
In some embodiments of the application, the exosomes are derived from at least one of hematopoietic stem cells, mesenchymal stem cells, and natural killer cells.
In some embodiments of the application, the animal source of the exosomes comprises at least one vertebrate of reptiles, birds, mammals, etc.
In some embodiments of the application, mammals include animals of the order monocular, marsupials, food worm, jumping shrew, beast, winged, primates, barren, scaly, lagomorpha, rodentia, carnivora, beef, hoof and rabbit, pipe tooth, miracle, artiodactyla, whales, and the like.
In some embodiments of the application, the animal source of the exosomes comprises any of mice, rats, mice, guinea pigs, rabbits, horses, sheep, deer, monkeys, dogs, pigs, humans, etc.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
Fig. 1 is a photograph of exosome lyophilized powder prepared in one embodiment of the present application. Wherein A is the state of freeze-dried powder in the penicillin bottle, and B is the state of freeze-dried powder after being taken out from the penicillin bottle.
FIG. 2 is an electron microscope image of morphological changes of exosomes after different storage times of the lyophilized premix and lyophilized powder according to an embodiment of the present application. Wherein the scale bar is 2 μm/100nm, and the magnification is 40000×.
FIG. 3 is a Western Blot results of lyophilized premix and lyophilized powder over different storage times for exosomes in one embodiment of the application.
FIG. 4 shows the results of testing the cell proliferation bioactivity of exosomes after various storage times of the lyophilized premix and lyophilized powder according to one embodiment of the application.
FIG. 5 is an electron micrograph of exosomes of different cell types in one embodiment of the application compared in morphology after lyophilization. Wherein A is mesenchymal stem cell exosomes (MSC-Exo), B is hematopoietic stem cell exosomes (HSC-Exo), and C is natural killer cell exosomes (NK-Exo).
Figure 6 is a comparison of the effect of exosome lyoprotectants obtained from different types of antioxidants in one embodiment of the application.
Detailed Description
The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.
The following detailed description of embodiments of the application is exemplary and is provided merely to illustrate the application and is not to be construed as limiting the application.
In the description of the present application, the meaning of a number is one or more, the meaning of a number is two or more, and greater than, less than, exceeding, etc. are understood to exclude the present number, and the meaning of a number is understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing embodiments of the application only and is not intended to be limiting of the application.
In the description of the present application, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Example 1
This example provides an exosome lyoprotectant using sterilized ddH 2 O and trehalose, mannitol and N-acetylcysteine are prepared into an exosome freeze-drying protective agent with 5wt% of trehalose, 5wt% of mannitol and 0.1wt% of N-acetylcysteine.
Example 2
The embodiment provides an exosome freeze-drying method, which comprises the following specific steps:
1. isolation and culture of umbilical cord mesenchymal stem cells
Collecting umbilical cord tissue of fetus with normal puerperal term mother body, thoroughly cleaning with PBS, shearing, adding appropriate amount of stem cell serum-free culture medium, and placing in 5% CO 2 Culturing in a 37 ℃ incubator, adding pancreatin for digestion when the fusion degree reaches 80%, harvesting to obtain primary umbilical cord mesenchymal stem cells (hUC-MSCs), and passaging.
2. Extraction of umbilical cord mesenchymal stem cell exosomes
Collecting hUC-MSCs of 3-8 generations with good growth, culturing in serum-free culture medium for 48h, collecting cell supernatant, concentrating and purifying the supernatant by tangential flow filtration system, collecting exosomes, re-suspending with PBS, and storing in a refrigerator at-80deg.C for use.
3. Stock solution for calibrating exosome
The total protein content of the exosome stock solution is tested by using a BCA method total protein test kit, and the specific operation steps are according to the instruction of the kit. Standard curve requirement R of test results 2 More than or equal to 0.95, according to the result of the total protein content of the exosome stock solution, using sterilized ddH 2 O the exosome stock solution was calibrated to be 6mg/mL exosome solution, and the calibrated exosome solution was used for subsequent testing.
4. Exosome freeze-drying
Exosome solution and exosome lyoprotectant in example 1 were prepared according to 1: mixing the materials according to the volume ratio of 1 to prepare a frozen intervention mixed solution, subpackaging each 2mL into 5mL of penicillin bottles made of high borosilicate glass materials, and freeze-drying at-30 ℃ for 20 hours to obtain the exosome frozen powder.
5. Freeze-dried powder detection
After the freeze-dried powder is qualified through routine inspection such as physical and chemical property inspection and safety inspection, stability tracking test is carried out, the freeze-dried powder is stored in an environment of 25+/-2 ℃/60%RH+/-5%R, and the change condition of exosome form, particle size and biological activity in the freeze-dried powder is tracked. Lyophilized powder in penicillin bottle using ddH equivalent to sterilization of pre-lyophilization volume (2 mL) 2 O is redissolved and then detected.
5.1 identification of lyophilized powder exosomes
Observing the morphology of exosomes in the freeze-dried powder by using a transmission electron microscope, performing particle size analysis on the freeze-dried powder by using a nano-particle tracking analyzer, detecting the expression conditions of CD9, CD63 and CD81 of the freeze-dried powder transmembrane protein by using Western blot, detecting the total protein content of the exosomes by using a BCA method, extracting the total RNA of the freeze-dried powder by using a Trizol method, and measuring the content of the total RNA. And comparing the detection results of the freeze-dried premixed liquid with the detection results of the freeze-dried premixed liquid by taking the initial freeze-dried powder and the freeze-dried powder stored for different times as experimental groups, and tracking and testing for 12 months.
5.2 detection of the biological Activity of the exosomes of the lyophilized powder
The effect of the exosome lyophilized powder on the growth of human fibroblasts for 0/48h was examined using CCK-8 method, and the bioactivity of exosome lyophilized powder for promoting cell proliferation was examined compared with a control group not treated with exosome.
5.3 data analysis
All assays were performed in triplicate using SPSS Statistics 25.0 software, and all experimental data were in mean.+ -. Standard deviation. The data comparison between the group and the control group adopts a double-sample t test, and the difference significance level is less than 0.05.
5.4 results
Referring to fig. 1, it can be seen that the lyophilized powder product is in the form of compact cake in penicillin bottle, and the cake is fine powder after pulverizing outside the bottle, and the cake is soluble (pharmacopoeia standard) and can be rapidly dissolved in ddH 2 O.
The morphological results of the exosomes before lyophilization and in the lyophilized powder are shown in fig. 2, and the obvious tea tray (garland) like structure exists in the lyophilized premix, which indicates that the exosomes can be extracted by the preparation method. The garland structure of the classical exosomes is still obvious in the photos of each stage after freeze-drying and storage, the exosomes in the freeze-dried powder can be fully proved, and the exosomes still keep good integrity after the freeze-drying is finished and the storage is carried out for 12 months.
Particle sizes and concentrations of exosomes after different storage times of the lyophilized premix and lyophilized powder were measured, and the results are shown in table 1. Comparing the initial, 1 month, 3 months, 6 months and 12 months of freeze-dried powder with the premix liquid control detection results, and performing t-test to show that the average particle size and the concentration are not significantly different from those of the freeze-dried premix liquid in several groups of freeze-dried powder (the p value is more than 0.05).
TABLE 1 particle size and concentration of exosomes before and after lyophilization
And detecting the expression conditions of exosome transmembrane proteins CD9, CD63 and CD81 in the freeze-dried premix and the freeze-dried powder by using a Western blot technology and a nano-flow technology. The Western blot results are shown in fig. 3, wherein the ACTIN protein is an experimental negative control, the expression ratio flow results of the exosome transmembrane proteins are shown in table 2, and by combining fig. 3 and table 2, the exosome specific marker proteins, such as CD9, CD63 and CD81, in each sample are obviously expressed before and after lyophilization, but the ACTIN protein used as a control is not expressed. This result further demonstrates that the exosome properties are not substantially altered by the lyophilization process described above.
TABLE 2 expression of exosome transmembrane proteins before and after lyophilization
The total protein content in the freeze-dried premix and the freeze-dried powder was detected by BCA method, and the loss rate and retention rate of the total protein after the initial, storage for 1 month, 3 months, 6 months, and 12 months of the freeze-dried powder were calculated based on the freeze-dried premix, respectively, and the results are shown in table 3. The results in the table show that the loss rate of the total protein of the freeze-dried powder sample is low, the loss rate is within 2% in 3 months, and the loss rate is just over 3% in 1 year, so that the total protein is stable in the freeze-drying process and the freeze-dried powder storage process.
TABLE 3 variation of total protein concentration of samples
And extracting the freeze-dried premix and total RNA of the freeze-dried powder by using a Trizol method, measuring the content of the total RNA, and respectively calculating the loss rate and the retention rate of the total RNA after the freeze-dried powder is initially stored for 1 month, 3 months, 6 months and 12 months based on the freeze-dried premix, wherein the result is shown in Table 4, and the total RNA change is not obvious in the process of storing the exosome freeze-dried powder.
TABLE 4 variation of total RNA concentration of samples
The CCK-8 method is used for testing the growth condition of the human fibroblasts in 0/48h with and without the addition of the freeze-dried powder, the growth condition of the human fibroblasts in the absence of the freeze-dried powder (control group) is taken as a benchmark, and the proliferation promoting effect of the added freeze-dried powder (experimental group) on the cells is calculated according to the following calculation formula:
the results are shown in fig. 4, and it can be seen from the graph that the lyophilized premix, the lyophilized powder of exosomes in the product stored for a period of time have good cell proliferation promoting activity, and the biological activity is still maintained at a high level after 3 months to 12 months of storage, and the decrease is not obvious.
Example 3
Freeze-drying effect of different types of extracellular bodies
Referring to example 2, after preparing and purifying exosomes of Hematopoietic Stem Cells (HSCs) and natural killer cells (NK), the exosomes of the two cells were lyophilized using a lyophilization process therein, and compared with the Mesenchymal Stem Cell (MSC) exosomes of example 2, the results are shown in fig. 5. It can be seen from the figure that the exosomes of the three types of cells with larger difference still have obvious tea tray-like exosomes after being freeze-dried, and the exosomes of the various different types of cells are less influenced by the freeze-drying process of the embodiment of the application, so that the freeze-drying process is suitable for freeze-drying protection of a plurality of cell exosomes.
Example 4
Lyophilization time optimization
According to the lyophilization process in example 2, different lyophilization times were adjusted and the yield of lyophilized exosomes was calculated according to the following formula:
yield= (number of freeze-dried finished products/total freeze-dried) x 100%.
The results are shown in Table 5:
TABLE 5 influence of different lyophilization times on lyophilized powder
The freeze-drying temperature curve in the embodiment is friendly to exosome freeze-drying, and the freeze-drying temperature of about-30 ℃ and the freeze-drying time of about 20 hours can be selected, so that compared with the prior art, the freeze-drying temperature curve is more energy-saving in the process of pre-freezing at the low temperature of-80 ℃, and the morphological structure or the biological activity of the exosome are not obviously influenced.
Comparative example
Freeze-drying effect comparison of different types of antioxidants
Common antioxidants include vitamins (T1), detoxication (T2) and expectorant (T3), in this example, vitamin a as T1, sodium thiosulfate as T2, N-acetylcysteine as T3, and a control group (C) without antioxidant, and the total protein of each group was tested with reference to example 2, and the results are shown in fig. 6. As can be seen from the graph, the total protein amount of the T3 group sample is higher than that of the control group, and the total protein amounts of the T1 and T2 group samples are significantly lower than that of the control group, so that the T3 group antioxidant has better protection effect on exosomes in the freeze-drying process.
Example 5
The present example provides an exosome lyoprotectant, which differs from example 1 only in that N-acetylcysteine is replaced with ambroxol hydrochloride of equal mass.
Example 6
This example provides an exosome lyoprotectant which differs from example 1 only in that the N-acetylcysteine is replaced with equal mass of carbocisteine.
Example 7
This example provides an exosome lyoprotectant, which differs from example 1 in that the N-acetylcysteine is replaced with equal mass of erdosteine.
Example 8
This example provides an exosome lyoprotectant which differs from example 1 in that trehalose at a final concentration of 5wt% is replaced with sucrose at a final concentration of 15 wt%.
Example 9
This example provides an exosome lyoprotectant that differs from example 1 in that mannitol at a final concentration of 5wt% is replaced with sorbitol at a final concentration of 15 wt%.
Example 10
This example provides an exosome lyoprotectant, which differs from example 1 in that the final concentration of N-acetylcysteine is 0.001wt%.
Examples 11 to 16
Examples 11 to 16 were lyophilized according to the method of example 2 using the exosome lyoprotectants of examples 5 to 10, respectively, and the results showed that the lyophilized powder obtained in these examples showed a complete tea-like exosome structure even after 12 months of storage, no significant changes in particle size and concentration, significant expression of exosome-specific marker proteins CD9, CD63, and CD81, a total protein loss of about 3%, an RNA loss of about 6%, and no significant decrease in cell proliferation promoting activity. Therefore, the exosome freeze-drying protective agent and the freeze-drying process provided by the embodiment can obtain the exosome freeze-dried powder with higher quality and good storage stability at higher freeze-drying temperature and shorter freeze-drying time.
The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The exosome freeze-drying protective agent is characterized by comprising an phlegm eliminating antioxidant, non-reducing sugar and alcohols.
2. The exosome lyoprotectant of claim 1, wherein the exosome lyoprotectant comprises 1 part by mass of the expectorant antioxidant, 50-15000 parts by mass of the non-reducing sugar, and 50-15000 parts by mass of the alcohol.
3. The exosome lyoprotectant of claim 1 or 2, wherein the expectorant antioxidant comprises at least one of N-acetylcysteine, ambroxol hydrochloride, carbocisteine, and erdosteine.
4. The exosome lyoprotectant of claim 1 or 2, wherein the non-reducing sugar comprises at least one of trehalose, sucrose, and raffinose.
5. The exosome lyoprotectant of claim 1 or 2, wherein the alcohol comprises at least one of mannitol, sorbitol, glycerol, ethylene glycol, and polyethylene glycol.
6. The exosome freeze-drying method is characterized by comprising the following steps:
mixing the exosome with the exosome freeze-drying protective agent according to any one of claims 1 to 5, and freeze-drying at-35 to-28 ℃ to obtain exosome freeze-dried powder.
7. The method of claim 6, wherein the total protein of the exosome has a final concentration of 1-5 mg/mL after mixing.
8. The method according to claim 6, wherein the final concentration of the expectorant antioxidant after mixing is 0.0005-0.05 wt%.
9. An exosome lyophilized powder prepared by the exosome lyophilization process of any one of claims 6 to 8.
10. The exosome lyophilized powder of claim 9, wherein the cell source of the exosome comprises at least one of hematopoietic stem cells, mesenchymal stem cells, and natural killer cells.
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