Exosome 3D composite culture method
By using the 3D FloTrix miniSpin bioreactor method in exosome 3D composite culture, the problem of insufficient exosome production consistency and scale capability in the prior art is solved, and efficient, economical and high-quality cultivation of exosomes is achieved.
Patent Information
- Application Number
- CN202411836157.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-03-14
AI Technical Summary
The existing technology lacks a sound production process to enhance the consistency and scale-up capabilities of exosome production, making it difficult to achieve large-scale production.
The 3D FloTrix miniSpin bioreactor was used to realize scalable 3D dynamic culture based on microcarriers. The cell culture conditions were optimized through autoclave treatment, quantitative inoculation, and the medium composition and light conditions were adjusted.
The low-cost, high-yield and high-quality culture of exosomes is achieved, and the scalability and production consistency of cell culture are improved.
Smart Images
Figure CN119614477A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of 3D composite culture of exosomes, and specifically to a method for 3D composite culture of exosomes. Background Art
[0002] Exosomes (EVs) are lipid-encapsulated vesicles secreted by most eukaryotic and prokaryotic cells (including animal cells and bacteria). Studies have shown that exosomes can induce important immune responses, and with the advancement of research methods and technologies, a large number of studies in the past decade have shown that exosomes are important mediators of intercellular communication. Therefore, exosomes play a fundamental biological function in cellular interactions in health and disease.
[0003] Exosomes are heterogeneous vesicles classified according to their biogenesis, content, and size. Exosomes are small lipid bilayer-encapsulated exosomes that originate from the endosomal plasma membrane. As vesicles for intercellular communication, exosomes participate in many physiological processes. This property, as well as their molecular composition that reflects the state of the secreting cell, makes exosomes have great clinical potential. A large number of preclinical and clinical studies are currently underway to evaluate the safety and efficacy of exosomes as drug delivery vehicles, therapeutic agents, and novel diagnostic tools.
[0004] Exosomes have unique targeting capabilities, and many studies have demonstrated that they have both autocrine and paracrine effects. For example, cancer cells can promote metastasis through intercellular communication interactions supported by exosomes. In addition, exosomes can be engineered to change their targeting behavior (for example, using specific types of antibodies). Exosomes are less immunogenic than the cells from which they are derived, which provides a huge opportunity to improve current treatment systems. Exosomes are able to transport substances such as intercellular transfer proteins and messenger RNA (mRNA), and have been shown to have pro-angiogenic, pro-coagulant and anti-inflammatory effects, as well as the ability to regulate cardiomyocytes and endothelial cells, induce angiogenesis and restore oxygen supply to hypoxic areas.
[0005] Exosomes play a key role in cancer and tumor growth. Exosomes released by cancer cells have been shown to promote tumor growth by promoting cell proliferation, angiogenesis, increasing the migration and invasion ability of cancer cells, and increasing resistance to certain drugs. Therefore, they may become disease biomarkers suitable for non-invasive, detailed disease prognosis and diagnostic tools. In addition, due to their low toxicity, low immunogenicity and ability to penetrate dense boundaries such as the blood-brain barrier (BBB), exosomes are extremely attractive as drug delivery vehicles. The unique ability to cross the blood-brain barrier makes exosomes an attractive vehicle for delivering therapeutic drugs and gene therapies for neurological and psychiatric diseases.
[0006] Although exosomes (EVs) have shown great promise in therapeutic applications, there is currently a lack of robust production processes that can enhance the consistency and scalability of EV production. Therefore, in order to achieve large-scale production capabilities, it is urgent to develop robust and scalable production processes.
[0007] One of the key challenges is the selection of an appropriate cell culture medium. In the in vitro expansion of mesenchymal stem cells (MSCs), the most commonly used culture medium supplement is fetal bovine serum (FBS), which presents several deficiencies in the production of human cell therapy products due to its animal origin. As an alternative to animal-derived products, serum-free / xenogeneic (S / XF) culture supplements have been developed, such as human platelet lysate (hPL).
[0008] Another major challenge is to identify cell culture platforms that are suitable for scalable cell therapy production. To meet the large-scale production for clinical use, the culture platform needs to have scalability and the ability to monitor and control culture parameters, which is difficult to achieve in traditional static culture systems. Therefore, a variety of bioreactor configurations that operate under dynamic culture conditions have been developed. Stirred bioreactor configurations have been used to explore the expansion of MSCs immobilized on microcarriers. These bioreactors use a stirring system to keep the microcarriers suspended and the culture medium homogenous. However, the stirring process increases shear forces, which have an impact on cell physiology.
[0009] PBS Biotech develops expandable vertical wheel to improve agitation in cell culture TM The vertically rotating wheel promotes radial and axial fluid flow, creating a more uniform hydrodynamic environment than traditional stirred bioreactors. TM The impeller is able to completely suspend the microcarriers at the lowest power input, thus minimizing the effects of shear forces. The scalability of the technology allows it to be expanded from 100 ml to 500 liters in working volume. Recently, VWBR has been successfully applied to the expansion of MSCs in microcarrier-based cell culture processes and the culture of human induced pluripotent stem cells. Summary of the invention
[0010] The purpose of the present invention is to provide a method for 3D composite culture of exosomes to solve the problems in the prior art.
[0011] To achieve the above object, the present invention provides the following technical solution: a method for 3D composite culture of exosomes, comprising the following steps:
[0012] S1. Scalable 3D dynamic culture based on microcarriers using 3D FloTrix miniSpin bioreactor, including miniSpin stirrer and spinner flask;
[0013] S2. Autoclave the spinner flask containing 320 mg of 3D TableTrix microcarriers.
[0014] S3, inoculate at a density of 5 × 10^4 cells / mL in basal medium containing 4% supplemented medium, set the speed of the miniSpin agitator at 25 rpm, and incubate the cells in 5% CO 2 Culture in an incubator at 37°C;
[0015] S4. Replace one quarter of the volume of medium with fresh medium every day;
[0016] S5. Starting from the second day, add 0.1 mM CaCl to the fresh culture medium. 2 Starting from the third day, 0.05 mM MgCl was added to the above culture medium. 2 ;
[0017] S6. 12 hours after inoculation, illuminate the rotating bottle at 265 nm for 2 hours; starting from the second day, illuminate at 265 nm for 2 hours each time, 4 times a day;
[0018] S7. Harvest after 4-7 days of cultivation.
[0019] Preferably, when sampling or extracting cells, the microcarriers carrying the cells are allowed to settle to the bottom of the sampling tube / EP tube, and the culture medium is aspirated to avoid movement of the microcarriers.
[0020] Preferably, when harvesting in the culture vessel, the sampling tube / EP tube is pipetted every 10 minutes.
[0021] Preferably, the culture medium is collected and centrifuged at 360×g for 10 minutes to remove residual microcarriers, cells and cell debris.
[0022] Preferably, after the culture medium is recovered, the cells attached to the microcarriers are resuspended in PBS and stored at -80°C.
[0023] Preferably, the preparation of extracellular vesicles comprises the following steps:
[0024] a. Centrifuge the culture medium at 2000 g for 30 minutes to remove cell debris, then incubate with the separation reagent at 4°C overnight;
[0025] b. The next day, the mixture was centrifuged again at 10,000 g and 4°C for 1 hour; the supernatant was discarded, and the extracellular vesicles were collected by thoroughly washing the centrifuge tube wall with 1x PBS containing nuclease-free purified distilled water;
[0026] c. Resuspend the extracellular vesicles in PBS at 20 to 70 times the volume of the original conditioned medium;
[0027] d. Aliquot the extracellular vesicles into 50 to 100 μl aliquots and store them in a -80°C freezer.
[0028] Compared with the prior art, the present invention has the following beneficial effects: using a 3D FloTrix miniSpin bioreactor to realize scalable 3D dynamic culture based on microcarriers, the equipment includes a miniSpin stirrer and a spinner bottle; the spinner bottle is sterilized by high pressure; the spinner bottle contains 320 mg of 3D TableTrix microcarriers; the cells are inoculated at a density of 5×10^4 cells / ml in a basic medium containing 4% supplementary medium, the speed of the miniSpin stirrer is set to 25 revolutions per minute, and the cells are heated at 5% CO 2 Cultured in an incubator at 37°C; replaced with a quarter of the volume of fresh culture medium every day; starting from the second day, 0.1 mM CaCl was added to the replaced fresh culture medium 2 Starting from the third day, 0.05 mM MgCl was added to the above culture medium. 2 ; The goal of low cost, high yield and high quality of exosome culture is achieved through 3D composite culture method. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:
[0030] Figure 1 is a graph showing changes in cell concentration over time under different culture conditions of the present invention;
[0031] Figure 2 It is a comparison chart of different culture methods of the present invention;
[0032] Figure 3 It is a graph of exosome yields of different culture methods of the present invention. DETAILED DESCRIPTION
[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.
[0034] See also Figure 1-3 In an embodiment of the present invention, a method for 3D composite culture of exosomes comprises the following steps:
[0035] S1. Scalable 3D dynamic culture based on microcarriers using 3D FloTrix miniSpin bioreactor, including miniSpin stirrer and spinner flask;
[0036] S2. Autoclave the spinner flask containing 320 mg of 3D TableTrix microcarriers.
[0037] S3, inoculate at a density of 5 × 10^4 cells / mL in basal medium containing 4% supplemented medium, set the speed of the miniSpin agitator at 25 rpm, and incubate the cells in 5% CO 2 Culture in an incubator at 37°C;
[0038] S4. Replace one quarter of the volume of medium with fresh medium every day;
[0039] S5. Starting from the second day, add 0.1 mM CaCl to the fresh culture medium. 2 Starting from the third day, 0.05 mM MgCl was added to the above culture medium. 2 ;
[0040] S6. 12 hours after inoculation, illuminate the rotating bottle at 265 nm for 2 hours; starting from the second day, illuminate at 265 nm for 2 hours each time, 4 times a day;
[0041] S7. Harvest after 4-7 days of cultivation.
[0042] When sampling or extracting cells, allow the microcarriers carrying cells to settle to the bottom of the sampling tube / EP tube and aspirate the culture medium to avoid movement of the microcarriers; when harvesting in the culture container, aspirate the sampling tube / EP tube every 10 minutes; collect the culture medium and centrifuge it at 360×g for 10 minutes to remove residual microcarriers, cells and cell debris; after the culture medium is recovered, resuspend the cells attached to the microcarriers in PBS and store at -80°C.
[0043] The preparation of extracellular vesicles includes the following steps:
[0044] a. Centrifuge the culture medium at 2000 g for 30 minutes to remove cell debris, then incubate with the separation reagent at 4°C overnight;
[0045] b. The next day, the mixture was centrifuged again at 10,000 g and 4°C for 1 hour; the supernatant was discarded, and the extracellular vesicles were collected by thoroughly washing the centrifuge tube wall with 1x PBS containing nuclease-free purified distilled water;
[0046] c. Resuspend the extracellular vesicles in PBS at 20 to 70 times the volume of the original conditioned medium;
[0047] d. Aliquot the extracellular vesicles into 50 to 100 μl aliquots and store them in a -80°C freezer.
[0048] The goal of low-cost, high-yield and high-quality exosome culture is achieved through the 3D composite culture method.
[0049] The cells were cultured in a basal medium containing 4% supplemented medium, which is a serum-free medium that helps promote the secretion of exosomes from cells. The experiments were conducted on cells from the 3rd to the 5th generation.
[0050] A 3D FloTrix miniSpin bioreactor (Beijing Saitonic Biotechnology Co., Ltd.) was used to realize scalable 3D dynamic culture based on microcarriers. The main equipment included a miniSpin stirrer and three sizes of spinner flasks.
[0051] Autoclaved spinner bottle (250 ml)
[0052] Contains 320 mg (5×10^4 cells / ml) of 3D TableTrix microcarriers (Cytoniche Biotechnology Co., Ltd.).
[0053] The cells were seeded at a density of 5×10^4 cells / ml in basal medium containing 4% supplemented medium, the speed of the miniSpin agitator was set at 25 rpm, and the cells were cultured at 37°C in a 5% CO2 incubator.
[0054] The cells were evenly distributed on the microcarriers, and a quarter of the volume of culture medium was replaced daily.
[0055] Starting from the second day, 0.1 mM CaCl was added to the fresh culture medium. 2 .
[0056] Starting from the third day, 0.05 mM MgCl was added to the above culture medium. 2 .
[0057] Twelve hours after inoculation, the flask was illuminated at 265 nm for 2 hours.
[0058] Starting from the second day, 265nm light was applied for 2 hours each time, 4 times a day, with even intervals.
[0059] The cells can be harvested in 4 to 7 days, depending on the cell line and needs.
[0060] During the culture period, cell viability was assessed using a calcein AM and propidium iodide (PI) kit (Wako, Japan) according to the manufacturer's instructions.
[0061] When sampling or extracting cells, the microcarriers loaded with cells must be allowed to settle to the bottom of the sampling tube / EP tube, and the culture medium must be carefully aspirated to avoid movement of the microcarriers.
[0062] Add 3D FloTrix at a ratio of 0.15 ml / mg TM Digest (CNR001-500, Cytonik Biotechnology, China) and incubated at 37°C for 30 min.
[0063] To assist in the lysis of the microcarriers and the detachment of the cells, gentle pipetting of the sampling tube / EP tube was performed every 10 minutes when harvesting in the culture vessel.
[0064] The number and activity of cells were counted by Trypan Blue exclusion test with the aid of an automatic cell counter (Countstar Biotech, ALIT Life Sciences, China).
[0065] The culture medium was collected and centrifuged at 360×g for 10 min to remove residual microcarriers, cells and cell debris.
[0066] After the culture medium was recovered, the cells attached to the microcarriers were resuspended in PBS and stored at -80°C for further analysis (eg Western blot detection).
[0067] The culture medium was first centrifuged at 2000 g for 30 minutes to remove cell debris and then incubated with the separation reagent at 4°C overnight.
[0068] The next day, the mixture was centrifuged again at 10,000 g and 4°C for 1 hour.
[0069] The supernatant was then discarded, and the extracellular vesicles were collected by thoroughly washing the centrifuge tube wall with 1xPBS containing nuclease-free purified distilled water produced by Invitrogen.
[0070] Next, the extracellular vesicles were resuspended in PBS at 20 to 70 times the volume of the original conditioned medium.
[0071] Finally, to reduce the impact of repeated freezing and thawing on the samples, the extracellular vesicles were divided into small portions of 50 to 100 μl and stored in a -80°C low-temperature freezer.
[0072] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for 3D composite culture of exosomes, characterized in that: The following steps are involved: S1. Scalable 3D dynamic culture based on microcarriers using 3D FloTrix miniSpin bioreactor, including miniSpin stirrer and spinner flask; S2. Autoclave the spinner flask containing 320 mg of 3D TableTrix microcarriers. S3, inoculated at a density of 5×10^4 cells / mL in basal medium containing 4% supplemented medium, the speed of the miniSpin stirrer was set to 25 rpm, and the cells were cultured in a 5% CO2 incubator at 37°C; S4. Replace one quarter of the volume of medium with fresh medium every day; S5. Starting from the second day, add 0.1 mM CaCl2 to the replaced fresh culture medium. Starting from the third day, add 0.05 mM MgCl2 to the above culture medium. S6. 12 hours after inoculation, illuminate the rotating bottle at 265 nm for 2 hours; starting from the second day, illuminate at 265 nm for 2 hours each time, 4 times a day; S7. Harvest after 4-7 days of cultivation.
2. The method for 3D composite culture of exosomes according to claim 1, characterized in that: When sampling or extracting cells, allow the microcarriers loaded with cells to settle to the bottom of the sampling tube / EP tube and aspirate the culture medium to avoid movement of the microcarriers.
3. The method for 3D composite culture of exosomes according to claim 2, characterized in that: When harvesting in culture vessels, pipette the sampling tube / EP tube every 10 minutes.
4. The method for 3D composite culture of exosomes according to claim 1, characterized in that: The culture medium was collected and centrifuged at 360×g for 10 min to remove residual microcarriers, cells and cell debris.
5. The method for 3D composite culture of exosomes according to claim 4, characterized in that: After the culture medium was recovered, the cells attached to the microcarriers were resuspended in PBS and stored at -80°C.
6. The method for 3D composite culture of exosomes according to claim 1, characterized in that: The preparation of extracellular vesicles includes the following steps: a. Centrifuge the culture medium at 2000 g for 30 minutes to remove cell debris, then incubate with the separation reagent at 4°C overnight; b. The next day, the mixture was centrifuged again at 10,000 g and 4°C for 1 hour; the supernatant was discarded, and the extracellular vesicles were collected by thoroughly washing the centrifuge tube wall with 1x PBS containing nuclease-free purified distilled water; c. Resuspend the extracellular vesicles in PBS at 20 to 70 times the volume of the original conditioned medium; d. Aliquot the extracellular vesicles into 50 to 100 μl aliquots and store them in a -80°C freezer.