WO2022241633A1 - Procédé de préparation efficace d'exosomes à l'aide d'un dispositif de culture de cellules souches à grande échelle - Google Patents

Procédé de préparation efficace d'exosomes à l'aide d'un dispositif de culture de cellules souches à grande échelle Download PDF

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WO2022241633A1
WO2022241633A1 PCT/CN2021/094292 CN2021094292W WO2022241633A1 WO 2022241633 A1 WO2022241633 A1 WO 2022241633A1 CN 2021094292 W CN2021094292 W CN 2021094292W WO 2022241633 A1 WO2022241633 A1 WO 2022241633A1
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culture
exosomes
pipeline
reaction kettle
cells
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PCT/CN2021/094292
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English (en)
Chinese (zh)
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陈海佳
陈东煌
李学家
崔梓豪
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广州赛莱拉干细胞科技股份有限公司
广州赛莱拉生物基因工程有限公司
广东国科细胞科技有限公司
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Priority to CN202180046761.9A priority Critical patent/CN115803428A/zh
Priority to PCT/CN2021/094292 priority patent/WO2022241633A1/fr
Publication of WO2022241633A1 publication Critical patent/WO2022241633A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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 application relates to the field of stem cell technology, in particular to a method for efficiently preparing exosomes using a large-scale stem cell culture device.
  • Exosomes are small vesicles secreted by cells with a diameter of 30-100nm, specifically discoid vesicles with a diameter of 40-100nm, containing biologically active molecules such as proteins and nucleotides derived from cells .
  • exosomes were first discovered in sheep reticulocytes; in 1987, Johnstone named it "exosome". Exosomes play an important role in the communication between cells.
  • a series of studies have found that exosomes derived from stem cells can effectively transport biologically active molecules such as mRNA, microRNA and protein, play an anti-apoptotic, inhibit inflammatory response, promote It has many important biological functions such as angiogenesis and inhibition of fibrosis, showing good clinical application prospects.
  • Mesenchymal stem cells have functions such as anti-inflammation, immune regulation, and promotion of angiogenesis, and have important clinical application value in medicine.
  • exosomes contained in the culture supernatant of mesenchymal stem cells have similar biological functions to mesenchymal stem cells, suggesting that exosomes may be one of the important effector molecules for mesenchymal stem cells to exert biological functions . Therefore, large-scale preparation of clinical-grade mesenchymal stem cell exosomes has important clinical application prospects.
  • the culture of mesenchymal stem cells mostly adopts the conventional culture flask/dish culture method. Under this culture condition, the cell proliferation is relatively slow, and the culture supernatant harvested per unit culture area is limited, which leads to a relatively low yield of exosomes. Therefore, it is difficult to obtain a large amount of exosomes to reach the clinical use dose.
  • the present application provides a method for efficiently preparing exosomes using a large-scale stem cell culture device, so that the method can efficiently and quickly prepare large doses of exosomes and realize large-scale preparation.
  • a method for efficiently preparing exosomes using a large-scale stem cell culture device The preparation of exosomes is carried out using a large-scale stem cell culture device.
  • the large-scale stem cell culture device includes a reaction kettle, a culture microenvironment control system, and a real-time observation and photography system. , gas delivery device, cell bottle and liquid storage bottle;
  • the interior of the reaction kettle is provided with a partition to divide the reaction kettle into a culture room and a buffer room connected at the top, and a cell culture tray is arranged in the culture room, and the cell bottle and the liquid storage bottle are respectively communicated with the culture room of the reaction kettle through pipelines;
  • the reaction kettle is equipped with a real-time observation and photographing system that can observe the growth state of the internal cells;
  • the reaction kettle is provided with an opening in each of the two areas of the buffer room and the culture room (generally, one opening is provided at the top area of the buffer room and the bottom area of the culture room), And form a self-circulation pipeline through pipelines from the outside of the reactor;
  • the dissolved oxygen electrode, temperature electrode and pH electrode on the culture microenvironment control system are placed in the buffer chamber of the reactor to monitor dissolved oxygen, temperature and pH;
  • the gas transport The device communicates with the buffer chamber of the reactor through a pipeline, and is regulated by the culture microenvironment regulation system;
  • Step 1 The mesenchymal stem cell suspension is pumped from the cell bottle into the reaction kettle through the peristaltic pump, and the serum-free medium for cultivating stem cells is pumped into the reaction kettle from the medium storage bottle through the peristaltic pump, and the reaction kettle is placed at a constant temperature After standing still, turn on the peristaltic pump on the self-circulation pipeline to make the medium in the reactor self-circulate, monitor the growth of cells through the real-time observation and camera system, and monitor the pH, temperature and solution in the reactor through the culture microenvironment control system.
  • the culture microenvironment control system opens the gas delivery device, transports nitrogen and/or carbon dioxide to the reactor through the pipeline, and the temperature is regulated by the thermostat; the culture parameters of the whole culture process The cell growth status can be monitored automatically in real time, and the cells are in a relatively uniform hydrodynamic microenvironment during the entire culture process;
  • Step 2 Closely observe the growth state of the cells through the real-time observation and camera system connected to the computer. When the confluence of the cells reaches 80-90%, continue to culture for 24-48 hours, and pump all the supernatant of the cultured cells back to collect exosomes through a peristaltic pump storage bottle, and then extract exosomes.
  • the mesenchymal stem cell suspension is a serum-free medium for umbilical cord mesenchymal stem cells and stem cells;
  • the serum-free medium can be a conventional commercially available stem cell serum-free medium, such as StemPro TM MSC SFM XenoFree Medium.
  • the density of the mesenchymal stem cells in the mesenchymal stem cell suspension is 1*10 4 /cm 2 .
  • exosomes are extracted by ultracentrifugation.
  • the ultracentrifugation method is: centrifuge the cell supernatant at 300g for 10min, collect the supernatant and centrifuge at 10000g for 30min, collect the supernatant again and filter it with a 0.22 micron filter, centrifuge the filtrate at 100000g for 2h, discard the supernatant PBS Resuspend the pellet, centrifuge at 100,000g for 2h, resuspend the pellet in PBS and store it for identification.
  • peristaltic pumps and valve switches can be installed on the pipeline according to the actual situation and needs, and the number and position are not limited.
  • pipes are provided on the cell bottle and the liquid storage bottle to communicate with the outside air, and an air filter element is provided on the pipes.
  • an air filter element is arranged on the pipeline between the gas conveying device and the reactor.
  • the reactor is also provided with an air pressure balance pipeline with a similar effect, and the air pressure balance pipeline is two openings arranged in two different areas (generally the top area and the bottom area) of the reactor cultivation chamber.
  • the connecting pipeline formed by connecting the pipeline from the outside of the reactor is connected with the atmosphere, and an air filter element is arranged at the position connected with the atmosphere.
  • the method of this application can obtain about 2.5 mg/L of exosomes in the culture supernatant under the premise of the same inoculation amount and inoculation density of umbilical cord mesenchymal stem cells, while the conventional The method only has about 1.0mg/L of exosomes in the culture supernatant, which shows that the yield of exosomes per unit volume of the culture supernatant of the method of this application is significantly higher than that of the conventional culture method, and the efficiency of exosome preparation is higher.
  • this application produces exosomes by using the self-developed large-scale stem cell culture equipment. Compared with the two-dimensional culture in conventional incubators, the yield of exosomes in this method is higher, thereby improving the efficiency of exosome preparation , and can also be extended to other tissue-derived mesenchymal stem cells and exosome preparation of other cells.
  • Figure 1 is a schematic diagram of the large-scale cultivation device for stem cells of the present application.
  • Figure 2 shows the flow chart of exosome extraction by ultracentrifugation
  • Figure 3 shows the analysis results of exosomes prepared by the method of the present application; the left picture shows the concentration measurement and particle size analysis of exosomes; the right picture shows the distribution of exosome particles, and the arrow shows the exosome particles;
  • Figure 4 shows the comparison of exosome production between the method of the present application and the two-dimensional culture method in a conventional incubator.
  • This application discloses a method for efficiently preparing exosomes using a large-scale stem cell culture device. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present application. The method described in this application has been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method described herein without departing from the content, spirit and scope of this application to realize and apply the technology of this application .
  • hUC-MSCs come from the Celera Umbilical Cord Mesenchymal Stem Cell Public Bank. The P4 generation cells are resuscitated. After 1-2 generations of culture, it is observed that the cells are in good condition and the proliferation rate is fast.
  • the large-scale stem cell culture device is mainly composed of a reaction kettle, a culture microenvironment control system, a real-time observation and camera system, a cell bottle and a liquid storage bottle.
  • a reaction kettle a culture microenvironment control system
  • a real-time observation and camera system a cell bottle and a liquid storage bottle.
  • the bioreactor is assembled as a whole, 121 Steam autoclave at °C for 1 hour; after the sterilization, when the temperature drops below 60°C, take out all parts of the device and place them in the ultra-clean bench of the research and development room, and irradiate with ultraviolet light for 1 hour for standby.
  • the structure diagram of the culture device is shown in Figure 1;
  • the culture microenvironment control system opens the gas delivery device, and delivers nitrogen and/or carbon dioxide to the reactor through the pipeline (nitrogen adjusts dissolved oxygen, carbon dioxide adjusts pH), the temperature is regulated by an incubator; the culture parameters and cell growth status of the whole culture process can be automatically monitored in real time, and the cells are in a relatively uniform hydrodynamic microenvironment during the whole culture process.
  • hUC-MSCs come from the Celera Umbilical Cord Mesenchymal Stem Cell Public Bank. P4 generation cells were resuscitated. After 1-2 generations of culture, it was observed that the cells were in good condition and the proliferation rate was fast. The same batch of cells as in Example 1 was used.
  • Example 2 Cells were inoculated on a 15 cm culture dish at 1 ⁇ 10 4 /cm 2 , cultured in the same hUC-MSCs serum-free medium as in Example 1 for 96 hours, and the culture supernatants were collected for exosome extraction and exocytosis Body identification, the specific method is the same as in Example 1.

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Abstract

La présente invention concerne un procédé de préparation efficace d'exosomes à l'aide d'un dispositif de culture de cellules souches à grande échelle. La préparation des exosomes de la présente invention est réalisée à l'aide du dispositif de culture de cellules souches à grande échelle. Le dispositif de culture de cellules souches à grande échelle comprend une cuve de réaction, un système de régulation du microenvironnement de culture, un système d'observation et de photographie en temps réel, un dispositif de distribution de gaz, une bouteille pour cellules et une bouteille de stockage de liquide. Par comparaison avec la culture bidimensionnelle dans des incubateurs classiques, le procédé produit un rendement plus élevé en exosomes, améliorant ainsi l'efficacité de la préparation des exosomes, et peut être appliqué de manière extensible à la préparation des exosomes d'autres cellules souches mésenchymateuses dérivées de tissus et d'autres cellules.
PCT/CN2021/094292 2021-05-18 2021-05-18 Procédé de préparation efficace d'exosomes à l'aide d'un dispositif de culture de cellules souches à grande échelle WO2022241633A1 (fr)

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Application Number Priority Date Filing Date Title
CN202180046761.9A CN115803428A (zh) 2021-05-18 2021-05-18 一种利用干细胞规模化培养装置高效制备外泌体的方法
PCT/CN2021/094292 WO2022241633A1 (fr) 2021-05-18 2021-05-18 Procédé de préparation efficace d'exosomes à l'aide d'un dispositif de culture de cellules souches à grande échelle

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PCT/CN2021/094292 WO2022241633A1 (fr) 2021-05-18 2021-05-18 Procédé de préparation efficace d'exosomes à l'aide d'un dispositif de culture de cellules souches à grande échelle

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115786107A (zh) * 2022-12-14 2023-03-14 深圳市寰宇生物科技有限公司 一种用于外泌体的高效制备装置及其方法

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CN102061260A (zh) * 2010-12-01 2011-05-18 汪华 一种医用细胞自动化生产装置及其生产方法
CN106232801A (zh) * 2014-04-28 2016-12-14 维瓦生物细胞股份公司 自动化细胞培养和收获装置
CN205874455U (zh) * 2016-08-03 2017-01-11 苏州大学附属第二医院 细胞间接共培养装置
CN109468274A (zh) * 2018-12-29 2019-03-15 深圳市旷逸生物科技有限公司 一种利用细胞工厂制备临床级脐带间充质干细胞的工艺
CN111363680A (zh) * 2018-12-26 2020-07-03 浙江大学 一种用于外泌体分泌、分离和收集的脉冲搅拌式生物反应器

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102061260A (zh) * 2010-12-01 2011-05-18 汪华 一种医用细胞自动化生产装置及其生产方法
CN106232801A (zh) * 2014-04-28 2016-12-14 维瓦生物细胞股份公司 自动化细胞培养和收获装置
CN205874455U (zh) * 2016-08-03 2017-01-11 苏州大学附属第二医院 细胞间接共培养装置
CN111363680A (zh) * 2018-12-26 2020-07-03 浙江大学 一种用于外泌体分泌、分离和收集的脉冲搅拌式生物反应器
CN109468274A (zh) * 2018-12-29 2019-03-15 深圳市旷逸生物科技有限公司 一种利用细胞工厂制备临床级脐带间充质干细胞的工艺

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115786107A (zh) * 2022-12-14 2023-03-14 深圳市寰宇生物科技有限公司 一种用于外泌体的高效制备装置及其方法

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