WO2021114164A1 - Exosome, son procédé de préparation et son utilisation - Google Patents

Exosome, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2021114164A1
WO2021114164A1 PCT/CN2019/124773 CN2019124773W WO2021114164A1 WO 2021114164 A1 WO2021114164 A1 WO 2021114164A1 CN 2019124773 W CN2019124773 W CN 2019124773W WO 2021114164 A1 WO2021114164 A1 WO 2021114164A1
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Prior art keywords
exosomes
preparation
ultrasound
human astrocytes
cell culture
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PCT/CN2019/124773
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English (en)
Chinese (zh)
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邓志婷
郑海荣
严飞
肖杨
李飞
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中国科学院深圳先进技术研究院
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Priority to PCT/CN2019/124773 priority Critical patent/WO2021114164A1/fr
Publication of WO2021114164A1 publication Critical patent/WO2021114164A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to the field of biotechnology, in particular to an application of non-invasive ultrasound treatment of cells in the preparation of exosomes, exosomes and preparation methods and applications thereof.
  • Alzheimer's disease also known as Alzheimer’s disease
  • AD Alzheimer's disease
  • a ⁇ ⁇ -amyloid
  • NFT neurofibrillary tangles
  • Exosomes are round membranous bodies wrapped in lipid bilayers. They are a type of extracellular vesicles that contain a variety of proteins, RNA, miRNA, growth factors and their receptors. Wait. Exosomes can spread between the central nervous system and peripheral circulation1. A variety of neurodegenerative diseases have abnormal accumulation and aggregation of protein2. The accumulation of excess substances in neuronal endosomes and lysosomes is an important factor that leads to nerve fragility in Alzheimer's disease.
  • Extracellular vesicles (EVs) (exosomes and microvesicles) form an endogenous transport system that participates in the exchange of biomolecules (proteins and RNA) between cells. This makes EVs have great potential for drug delivery and regenerative medicine applications.
  • the current method for preparing exosomes has a complicated process, poor controllability and high cost.
  • the main technical problem solved by the present invention is to provide an exosomes with simple operation, low cost and strong controllability, and a preparation method and application thereof.
  • a technical solution adopted by the present invention is:
  • a preparation method of exosomes includes the following steps:
  • the human astrocytes stimulated by the ultrasonic cycle were cultured and the cell culture supernatant was collected, and the cell culture supernatant was centrifuged to obtain exosomes.
  • the cyclic stimulation conditions are: the probe frequency of the ultrasound device is 0.5-5MHz, the amplitude is 50-000mV, the pulse repetition frequency is 50-1000Hz, the duty cycle is 10%-70%, and the ultrasound device
  • the power amplifier amplifies 5%-45%, circulates 20-200k times, the pulse interval is 2 ⁇ s-2s, and the ultrasonic time is 1 minute to 10 minutes.
  • the medium is high-sugar DMEM without exosomes.
  • the human astrocytes stimulated by the ultrasound cycle are cultured for 24-72 hours and then the cell culture supernatant is collected.
  • a technical solution adopted by the present invention is to provide exosomes obtained by the above preparation method.
  • the average diameter of the exosomes is about 50 nm to 150 nm.
  • a technical solution adopted by the present invention is to use the exosomes prepared by the above preparation method in the preparation of drugs for the prevention and/or treatment of Alzheimer's disease.
  • a technical solution adopted by the present invention is to use the exosomes prepared by the above preparation method to degrade ⁇ -amyloid protein.
  • the beneficial effect of the present invention is that compared with the prior art, the present invention uses medical ultrasound technology to make the central nerve cells and glial cells at the stimulation site different through different intensities, frequencies, pulse repetition frequencies, pulse widths, and durations.
  • Astrocytes are the basis for the stability, defense and regeneration of the central nervous system.
  • the morphology and function of astrocytes can be adjusted by environmental stimuli or drugs.
  • the invention utilizes ultrasonic waves to stimulate human astrocytes, and the preparation method of the separated exosomes is simple in process, convenient in operation, strong in controllability and low in cost.
  • the obtained exosomes can slow down or even reverse the toxicity of ⁇ -amyloid (A ⁇ ) to nerve cells, reduce the deposition of A ⁇ plaques in the brain in mice, and can be used in new drugs for the treatment of Alzheimer's disease.
  • a ⁇ ⁇ -amyloid
  • Fig. 1 is a transmission electron micrograph of astrocyte exosomes without ultrasound stimulation according to an embodiment
  • Figure 2 is a schematic diagram of astrocyte exosomes stimulated by ultrasound
  • FIG. 3 is a schematic diagram of a nanoparticle tracking analysis (NTA) result of astrocyte exosomes without ultrasound stimulation according to an embodiment
  • NTA nanoparticle tracking analysis
  • Fig. 5 is a proteomic heatmap of astrocyte exosomes that have not undergone ultrasound stimulation and ultrasound-stimulated astrocyte exosomes according to an embodiment
  • Fig. 6 is a schematic diagram of an embodiment of the result of the ultrasound-stimulated exosomes cck-8 secreted by astrocytes;
  • Fig. 7 is a schematic diagram showing the in vivo therapeutic effect of exosomes secreted by ultrasound-stimulated astrocytes on APP/PSI transgenic Alzheimer’s disease mice according to an embodiment, wherein the diagram in A is stained with Thioflavin S The method detects the deposition of A ⁇ plaque, the green is the plaque, and the blue is the nucleus.
  • the ⁇ 1-42 antibody immunofluorescence staining detects the distribution of A ⁇ 1-42 in the brain, the green is A ⁇ , and the blue is the nucleus;
  • HA-EXO means astrocyte exosomes
  • US-HA-EXO means ultrasound-stimulated astrocyte exosomes.
  • a preparation method of exosomes includes the following steps:
  • the medium is high-sugar DMEM without exosomes.
  • human astrocytes (HA) cells are cultured in a cell culture flask, and exosomes-free high-sugar DMEM medium is added, and the cells are cultured in a 37° incubator. After 24 hours Stimulate the cell culture dish with an ultrasound device. The total time lasts 5 minutes. After the stimulation, the cell morphology was observed under a microscope, and the cell morphology was good, no difference from unstimulated cells.
  • the cyclic stimulation conditions are: the probe frequency of the ultrasound device is 0.5-5MHz, the amplitude is 50-000mV, the pulse repetition frequency is 50-1000Hz, the duty cycle is 10%-70%, and the ultrasound device
  • the power amplifier amplifies 5%-45%, circulates 20-200k times, the pulse interval is 2 ⁇ s-2s, and the ultrasonic time is 1 minute to 10 minutes. Circulating ultrasound can independently adjust multiple ultrasound parameters, which is helpful for the diversification of ultrasound parameters and the precise stimulation of cells.
  • S130 Continue culturing the human astrocytes stimulated by the ultrasonic cycle and collect the cell culture supernatant, and centrifuge the cell culture supernatant to obtain exosomes.
  • the average diameter of the exosomes is about 50 nm to 150 nm, and exhibits a typical exosomes particle size distribution.
  • HA cells Culture human astrocytes (HA) cells in a cell culture flask, add exosomes-free high-sugar DMEM medium, and wait for the cells to be cultured in a 37° incubator. After 24 hours, use an ultrasound device to culture the cells Dish for stimulation. The total time lasts 5 minutes. After being stimulated by ultrasound, HA cells were placed in a 37° incubator for continued cultivation. After 24 hours, 48 hours, and 72 hours, the cell culture supernatant was collected. The cell culture supernatant was subjected to a series of centrifugation treatments, 400g for 15 minutes, 2000g for 30 minutes, 10000g for 60 minutes, and 100,000g for 90 minutes, and the resulting pellet was exosomes. Refer to Figure 1 and Figure 2.
  • Figure 1 is a transmission electron microscope image of astrocyte exosomes that have not been stimulated by ultrasound
  • Figure 2 is a schematic diagram of astrocyte exosomes stimulated by ultrasound.
  • NTA Nanoparticle Tracking Analysis
  • exosomes secreted by astrocytes after ultrasound stimulation are compared with the exosomes secreted by the non-ultrasound group.
  • the exosomes are related to the proteasome function.
  • the increase in protein content of exosomes helps to degrade abnormally folded proteins.
  • the thioflavin S staining method is used to detect the deposition of A ⁇ plaques, the green is the plaque, and the blue is the cell nucleus.
  • a ⁇ 1-42 antibody immunofluorescence staining was used to detect the distribution of A ⁇ 1-42 in the brain. The green is A ⁇ and the blue is the nucleus.
  • the present invention uses medical ultrasound technology to produce different biological effects of central nerve cells and glial cells at the stimulation site through different intensities, frequencies, pulse repetition frequencies, pulse widths, and durations.
  • Astrocytes are the basis for the stability, defense and regeneration of the central nervous system.
  • the morphology and function of astrocytes can be adjusted by environmental stimuli or drugs.
  • the invention utilizes ultrasonic waves to stimulate human astrocytes, and the preparation method of the separated exosomes is simple in process, convenient in operation, strong in controllability and low in cost.
  • the obtained exosomes can slow down or even reverse the toxicity of ⁇ -amyloid (A ⁇ ) to nerve cells, reduce the deposition of A ⁇ plaques in the brain in mice, and can be used in new drugs for the treatment of Alzheimer's disease.
  • a ⁇ ⁇ -amyloid

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
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  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Neurology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Neurosurgery (AREA)
  • Epidemiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Psychiatry (AREA)
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Abstract

Procédé de préparation d'un exosome. Le procédé comprend les étapes suivantes : mise en culture d'astrocytes humains dans un milieu de culture ; réalisation d'une stimulation circulatoire sur les astrocytes humains par l'intermédiaire d'un appareil à ultrasons ; et poursuite de la culture des astrocytes humains, ayant été stimulés au moyen d'une circulation d'ultrasons, puis collecte du surnageant de culture cellulaire, et réalisation d'un traitement par centrifugation du surnageant de culture cellulaire pour obtenir l'exosome. L'invention concerne également l'exosome obtenu au moyen du procédé susmentionné et son utilisation. Le procédé de préparation susmentionné est fait d'opérations simples, est peu coûteux, et est hautement contrôlable.
PCT/CN2019/124773 2019-12-12 2019-12-12 Exosome, son procédé de préparation et son utilisation WO2021114164A1 (fr)

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Cited By (1)

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CN116716288A (zh) * 2023-04-28 2023-09-08 四川大学 一种声波振动提高外泌体产量的方法

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US20180273906A1 (en) * 2014-04-17 2018-09-27 Muhammad Ashraf Microvesicle and stem cell compositions for therapeutic applications
WO2017154951A1 (fr) * 2016-03-09 2017-09-14 国立大学法人名古屋大学 Procédé de récupération de vésicules extracellulaires
KR20190003399A (ko) * 2017-06-30 2019-01-09 주식회사 엑소코바이오 줄기세포 유래의 엑소좀을 유효성분으로 포함하는 조성물의 피부 섬유증 개선 용도
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116716288A (zh) * 2023-04-28 2023-09-08 四川大学 一种声波振动提高外泌体产量的方法
CN116716288B (zh) * 2023-04-28 2024-04-26 四川大学 一种声波振动提高外泌体产量的方法

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