A method for efficiently preparing exosomes using a large-scale stem cell culture device

Abstract

This application provides a method for efficiently preparing exosomes using a large-scale stem cell culture device. The exosome preparation in this application utilizes a large-scale stem cell culture device, which includes a reaction vessel, a culture microenvironment control system, a real-time observation and imaging system, a gas delivery device, cell flasks, and storage bottles. Compared with conventional two-dimensional culture in an incubator, this method yields higher exosome production, thereby improving the efficiency of exosome preparation. Furthermore, it can be extended to the preparation of exosomes from mesenchymal stem cells and other cells derived from other tissues.

Description

Technical Field

[0001] This application relates to the field of stem cell technology, specifically to a method for efficiently preparing exosomes using a large-scale stem cell culture device. Background Technology

[0002] Exosomes are small vesicles secreted by cells, ranging from 30 to 100 nm in diameter, specifically disc-shaped vesicles with a diameter of 40 to 100 nm. They contain cell-derived bioactive molecules such as proteins and nucleotides. Exosomes were first discovered in sheep reticulocytes in 1983; Johnstone named them "exosomes" in 1987. Exosomes play a crucial role in intercellular communication. Recent studies have found that stem cell-derived exosomes effectively transport bioactive molecules such as mRNA, microRNA, and proteins, exerting various important biological functions, including anti-apoptosis, inhibition of inflammatory responses, promotion of angiogenesis, and inhibition of fibrosis, demonstrating promising clinical application prospects.

[0003] Mesenchymal stem cells (MSCs) possess anti-inflammatory, immunomodulatory, and angiogenesis-promoting functions, making them valuable for clinical applications. Meanwhile, numerous studies have shown that exosomes contained in MSC culture supernatants exhibit similar biological functions to MSCs, suggesting that exosomes may be important effector molecules for MSCs to exert their biological functions. Therefore, the large-scale preparation of clinical-grade MSC exosomes holds significant promise for clinical applications.

[0004] Currently, mesenchymal stem cell culture mostly uses conventional culture flask / dish culture methods. Under these culture conditions, cell proliferation is relatively slow, and the amount of culture supernatant harvested per unit culture area is limited, which greatly restricts the production of exosomes. Therefore, it is difficult to obtain a large number of exosomes that meet clinical usage doses. Summary of the Invention

[0005] This application provides a method for efficiently preparing exosomes using a stem cell large-scale culture device, enabling the method to efficiently and quickly prepare large doses of exosomes and achieve large-scale preparation.

[0006] A method for efficiently preparing exosomes using a large-scale stem cell culture device, wherein the preparation of exosomes is carried out using a large-scale stem cell culture device, the large-scale stem cell culture device including a reaction vessel, a culture microenvironment control system, a real-time observation and imaging system, a gas delivery device, cell flasks and storage bottles;

[0007] The reactor is divided into a top-connected culture chamber and a buffer chamber by a partition. A cell culture tray is installed in the culture chamber. Cell flasks and storage bottles are connected to the culture chamber via pipes. A real-time observation and imaging system is installed on the reactor to monitor cell growth. The reactor has one opening in each of the buffer and culture chamber areas (typically one opening in the top area of ​​the buffer chamber and one in the bottom area of ​​the culture chamber), connected to the outside of the reactor via pipes to form a self-circulating pipeline. Dissolved oxygen, temperature, and pH electrodes are placed in the buffer chamber of the reactor to monitor dissolved oxygen, temperature, and pH. A gas delivery device is connected to the buffer chamber via pipes and is controlled by the culture microenvironment control system.

[0008] The specific preparation process is as follows:

[0009] Step 1: Mesenchymal stem cell suspension is pumped from the cell culture flask into the reaction vessel using a peristaltic pump. Serum-free culture medium for culturing stem cells is also pumped from the culture medium reservoir into the reaction vessel using a peristaltic pump. The reaction vessel is placed in a constant temperature incubator. After settling, the peristaltic pump on the self-circulating pipeline is turned on to allow the culture medium in the reaction vessel to circulate. Cell growth is monitored in real time using a video observation system. The pH, temperature, and dissolved oxygen in the reaction vessel are monitored using a culture microenvironment control system. If dissolved oxygen and / or pH exceed the set range, the culture microenvironment control system activates the gas delivery device to deliver nitrogen and / or carbon dioxide into the reaction vessel through pipelines. The temperature is controlled by the constant temperature incubator. The culture parameters and cell growth status throughout the entire culture process can be automatically monitored in real time. The cells are cultured in a relatively uniform hydrodynamic microenvironment throughout the entire process.

[0010] Step 2: Closely observe the cell growth status using a real-time observation and photography system connected to a computer. When the cell confluence reaches 80-90%, continue culturing for 24-48 hours. Use a peristaltic pump to pump all the cell supernatant back to the storage bottle for exosome collection, and then perform exosome extraction.

[0011] Preferably, the mesenchymal stem cell suspension is a serum-free culture medium for umbilical cord mesenchymal stem cells and cultured stem cells; the serum-free culture medium can be a conventional commercially available serum-free stem cell culture medium, such as StemPro. TM MSC SFMXenoFree medium.

[0012] In a specific embodiment of this application, the density of mesenchymal stem cells in the mesenchymal stem cell suspension is 1*103 4 / cm 2 .

[0013] Preferably, exosome extraction is performed using ultracentrifugation. In a specific embodiment of this application, the ultracentrifugation method is as follows: the cell supernatant is centrifuged at 300g for 10 min, the supernatant is collected, and then centrifuged at 10000g for 30 min. The supernatant is collected again and filtered through a 0.22-micron filter membrane. The filtrate is centrifuged at 100000g for 2 h, the supernatant is discarded, the precipitate is resuspended in PBS, centrifuged at 100000g for 2 h, the precipitate is resuspended in PBS, identified, and stored.

[0014] The device described in this application may be equipped with peristaltic pumps and valve switches on the pipeline according to actual conditions and needs, and the number and location are not limited.

[0015] To ensure the normal operation of the peristaltic pump, pipes are installed on the cell culture bottles and storage bottles to connect them to the outside air, and air filters are installed on these pipes. Similarly, an air filter is installed on the pipe between the gas delivery device and the reactor. Furthermore, a similar pressure balancing pipe is installed on the reactor. This pressure balancing pipe consists of two openings in two different areas of the reactor's culture chamber (generally the top and bottom areas), connected to the outside of the reactor via pipes. This connecting pipe is open to the atmosphere, and an air filter is installed at the point of connection to the atmosphere.

[0016] Compared with the conventional two-dimensional culture method using an incubator, the method of this application can obtain approximately 2.5 mg / L of exosomes in the culture supernatant under the same seeding amount and density of umbilical cord mesenchymal stem cells, while the conventional method only yields approximately 1.0 mg / L of exosomes in the culture supernatant. This indicates that the yield of exosomes per unit volume of culture supernatant in the method of this application is significantly higher than that of the conventional culture method, and the efficiency of exosome preparation is higher.

[0017] As can be seen from the above technical solutions, this application produces exosomes by using self-developed stem cell large-scale culture equipment. Compared with conventional two-dimensional culture in an incubator, this method has a higher yield of exosomes, thereby improving the efficiency of exosome preparation. It can also be extended to the preparation of exosomes from mesenchymal stem cells and other cells from other tissue sources. Attached Figure Description

[0018] Figure 1 The diagram shown is a schematic of the stem cell large-scale culture device of this application.

[0019] Figure 2 The diagram shows the process flow for exosome extraction using ultracentrifugation.

[0020] Figure 3 The figure shows the analytical results of exosomes prepared by the method of this application; the left figure shows the exosome concentration determination and particle size analysis; the right figure shows the exosome particle distribution, with arrows indicating exosome particles;

[0021] Figure 4The diagram shows a comparison of exosome yield between the method described in this application and the conventional two-dimensional culture method in an incubator. Detailed Implementation

[0022] This application discloses a method for efficiently preparing exosomes using a large-scale stem cell culture device. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this application. The method described in this application has been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the method described herein without departing from the content, spirit, and scope of this application to implement and apply the technology of this application.

[0023] In the comparative experiments of this application, the umbilical cord mesenchymal stem cells were of the same origin, the amount used to prepare exosomes was kept consistent in each group, and other experimental environments were kept consistent except for the differences in the methods themselves, unless otherwise specified.

[0024] The following provides a further explanation of the method for efficiently preparing exosomes using a large-scale stem cell culture device provided in this application.

[0025] Example 1: Preparation of exosomes using the culture method described in this application

[0026] 1) Preparation of human umbilical cord mesenchymal stem cells

[0027] hUC-MSCs are from the Cellera Umbilical Cord Mesenchymal Stem Cell Public Bank. P4 generation cells are revived and cultured for 1-2 generations. When the cells are observed to be in good condition and proliferating rapidly, they are used.

[0028] 2) Preparations before operation of large-scale stem cell culture device

[0029] The large-scale stem cell culture device mainly consists of a reactor, a culture microenvironment control system, a real-time observation and imaging system, cell flasks, and storage bottles. Before operation, all parts, as well as the tubing, connectors, and T-joints used to connect them, need to be disinfected, cleaned, and dried, and the airtightness of each part of the system must be tested. After the airtightness test, the bioreactor is assembled and autoclaved at 121°C for 1 hour. After sterilization, once the temperature drops below 60°C, all parts of the device are removed and placed in a clean bench in the research and development room for UV irradiation for 1 hour before use. A schematic diagram of the culture device is shown below. Figure 1 ;

[0030] 3) Large-scale culture of human umbilical cord mesenchymal stem cells

[0031] According to 1*10 4 / cm 2Cell suspension and 3L of serum-free hUC-MSCs culture medium were prepared. The cell suspension was pumped from the cell culture flask into the reactor using a peristaltic pump, and the serum-free culture medium was pumped from the storage bottle into the reactor using a peristaltic pump. After standing for 24 hours, the peristaltic pump on the self-circulating pipeline was turned on to allow the culture medium in the reactor to circulate. Cell growth was monitored in real time using a photographic system, and pH, temperature, and dissolved oxygen in the reactor were monitored using a culture microenvironment control system. If dissolved oxygen and / or pH exceeded the set range, the culture microenvironment control system activated the gas delivery device to deliver nitrogen and / or carbon dioxide into the reactor through pipeline (nitrogen regulates dissolved oxygen, and carbon dioxide regulates pH). Temperature was controlled by a constant temperature incubator. The culture parameters and cell growth status throughout the entire culture process can be automatically monitored in real time, and the cells are in a relatively uniform hydrodynamic microenvironment throughout the entire culture process.

[0032] 4) Large-scale preparation of stem cell exosomes

[0033] After 72 hours of culture, the cell growth status was closely observed through a real-time observation and photography system connected to a computer. When the cell confluence reached 80-90%, the cells were cultured for another 48 hours. The supernatant of the cultured cells was pumped back to a dedicated storage bottle using a peristaltic pump, and then exosomes were extracted by ultracentrifugation.

[0034] 5) See the exosome extraction procedure. Figure 2 ;

[0035] 6) Exosome identification, results are shown in [link to results]. Figure 3 The left figure shows the exosome concentration measurement and particle size analysis; the right figure shows the distribution of exosome particles, with arrows indicating exosome particles.

[0036] Example 2: Preparation of exosomes using conventional culture methods

[0037] 1) Preparation of human umbilical cord mesenchymal stem cells

[0038] hUC-MSCs were obtained from the Cellera Umbilical Cord Mesenchymal Stem Cell Public Bank. P4 generation cells were resuscitated and cultured for 1-2 generations. When the cells were observed to be in good condition and proliferating rapidly, they were used. The same batch of cells was used as in Example 1.

[0039] 2) Cells are processed at a rate of 1×10 4 / cm 2 The cells were inoculated onto 15cm culture dishes and cultured for 96 hours using the same hUC-MSCs serum-free medium as in Example 1. The culture supernatants were then collected for exosome extraction and identification, using the same methods as in Example 1.

[0040] Example 2: Comparison of exosome production

[0041] Exosomes were prepared according to the methods in Examples 1 and 2, and three parallel control experiments were conducted to prepare exosomes. Figure 4 The results show that the yield of exosomes per unit volume of culture supernatant using the culture method in this application is significantly higher than that using the conventional culture method, indicating that the culture method in this application is more efficient in exosome preparation.

[0042] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A method for efficiently preparing exosomes using a stem cell scale-up culture device, wherein, The preparation of the exosome is carried out by using a stem cell scale culture device, and the stem cell scale culture device comprises a reaction kettle, a culture microenvironment regulation system, a real-time observation photographing system, a gas delivery device, a cell bottle and a liquid storage bottle. The reaction kettle is internally provided with a partition plate to divide the reaction kettle into a culture chamber and a buffer chamber which are in communication with each other, and a cell culture tower plate is arranged in the culture chamber, and the cell bottle and the liquid storage bottle are respectively in communication with the culture chamber of the reaction kettle through pipelines; the real-time observation photographing system for observing the internal cell growth state is arranged on the reaction kettle; the reaction kettle is respectively provided with an opening in the buffer chamber and the culture chamber, and a self-circulation pipeline is formed by connecting the two openings through pipelines outside the reaction kettle; the dissolved oxygen electrode, the temperature electrode and the pH electrode of the culture microenvironment regulation system are arranged in the buffer chamber of the reaction kettle to monitor the dissolved oxygen, the temperature and the pH; the gas delivery device is in communication with the buffer chamber of the reaction kettle through a pipeline and is regulated by the culture microenvironment regulation system. The specific preparation process is as follows: Step 1: The mesenchymal stem cell suspension is pumped into the reaction kettle from the cell bottle by using a peristaltic pump, and the serum-free culture medium for culturing stem cells is pumped into the reaction kettle from the culture medium storage bottle by using a peristaltic pump, and the reaction kettle is placed in a thermostat; after standing, the peristaltic pump on the self-circulation pipeline is started to make the culture medium in the reaction kettle self-circulate, the cell growth state is monitored by using the real-time observation photographing system, the pH, the temperature and the dissolved oxygen in the reaction kettle are monitored by using the culture microenvironment regulation system, when the dissolved oxygen and / or the pH exceed the set value range, the culture microenvironment regulation system starts the gas delivery device to deliver nitrogen and / or carbon dioxide into the reaction kettle through the pipeline, and the temperature is regulated by using the thermostat; the culture parameters and the cell growth state in the whole culture process can be automatically and real-timely monitored, and the cells are in a relatively uniform hydrodynamic microenvironment during the whole culture process; Step 2: The cell growth state is closely observed by using the real-time observation photographing system connected with a computer, when the cell confluence degree reaches 80-90%, the cells are continuously cultured for 24-48 h, the culture supernatant is pumped back to the liquid storage bottle for collecting the exosome by using a peristaltic pump, and then the exosome is extracted.

2. The method of claim 1, wherein, The mesenchymal stem cell suspension is umbilical cord mesenchymal stem cells and serum-free culture medium for culturing stem cells.

3. The method of claim 1 or 2, wherein, The serum-free medium for culturing stem cells is StemPro TM MSC SFM XenoFree medium.

4. The method of claim 1, wherein, The exosome is extracted by using a high-speed centrifugation method.

5. The method of claim 4, wherein, The high-speed centrifugation method is as follows: the cell supernatant is centrifuged at 300 g for 10 min, the supernatant is collected, then the supernatant is centrifuged at 10,000 g for 30 min, the supernatant is collected again, the collected supernatant is filtered through a 0.22-micron filter membrane, the filtrate is centrifuged at 100,000 g for 2 h, the supernatant is discarded, the precipitate is resuspended in PBS, the precipitate is centrifuged at 100,000 g for 2 h, the precipitate is resuspended in PBS and identified for storage.

6. The method of claim 1, wherein, Peristaltic pumps and valve switches are arranged on the pipelines of the device.

7. The method of claim 1, wherein, The pipelines of the cell bottle and the liquid storage bottle are in communication with the outside air, and air filters are arranged on the pipelines.

8. The method of claim 1, wherein, Air filters are arranged on the pipelines between the gas delivery device and the reaction kettle.

9. The method of claim 1, wherein, The reaction kettle is further provided with an air pressure balance pipeline, the air pressure balance pipeline is two openings arranged in two different areas of the reaction kettle culture chamber, a communication pipeline formed by pipeline communication from outside the reaction kettle, and the communication pipeline is in communication with the atmosphere, and an air filter is arranged at the position in communication with the atmosphere.

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