Method for improving yield of endometrial stem cell exosome
By establishing a co-culture system between endometrial stem cells and fibroblasts, and using proliferating and stagnant fibroblasts to provide a growth environment for endometrial stem cells, the problems of low exosome yield and cell damage in the prior art are solved, and efficient and safe improvement of exosome yield is achieved.
Patent Information
- Application Number
- CN202411881260.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-14
AI Technical Summary
Existing methods have problems of cytotoxicity, reduced purity and cell damage when increasing the yield of endometrial stem cells, which is difficult to meet the needs of large-scale production and clinical applications.
Using a co-culture system, endometrial stem cells and mitomycin C-treated fibroblasts were used for non-contact co-culture through Transwell chambers and polyester membranes. The proliferation-stagnant fibroblasts were used to provide a growth environment for endometrial stem cells and improve exosome production.
It significantly improves the yield of exosomes of endometrial stem cells, avoids damage to target cells, improves the efficiency of exosome acquisition, and meets the needs of large-scale production and clinical applications.
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Figure CN119614487A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a method for increasing the yield of exosomes from endometrial stem cells. Background Art
[0002] Endometrial stem cells (EnSCs) are adult stem cells first discovered in 2004. They are mainly located in the stroma, around blood vessels and glands of the endometrium. During the menstrual cycle, the endometrium will regenerate and differentiate under the influence of hormones. The endometrial cell populations in different stages of the endometrium during the menstrual cycle (proliferative phase, early menstrual cycle, mid-secretory phase and late secretory phase) are very different. These cell populations with different molecular characteristics are responsible for different organ functions. Among them, the proliferative phase is the main stage of the endometrial proliferation and regeneration process. SFRP4 + The endometrial stem cell subpopulation is highly enriched specifically in this stage and is less abundant in other stages of the menstrual cycle. It is the main subpopulation that realizes the regenerative function of endometrial stem cells.
[0003] EnSCs have similar characteristics to bone marrow mesenchymal stem cells or adipose mesenchymal stem cells. They can proliferate in large numbers, restore and repair tissue cells, and promote cell regeneration. Compared with stem cells from other sources, EnSCs have higher in vitro and in vivo proliferation capacity and lower immunogenicity. They are non-tumorigenic in nude mice and can be passaged about 40 times in vitro. The donor age is negatively correlated with the cell proliferation capacity. EnSCs have high differentiation potential. Studies have shown that EnSCs can differentiate into a variety of cell lineages under in vitro induction conditions and express embryonic stem cell markers Oct-4, SSEA-4, and c-kit. They also have high telomerase activity, which suggests that EnSCs have multidirectional differentiation characteristics. In addition, the expression of matrix metalloproteins and the secretion of a large number of angiogenic and anti-apoptotic cytokines also show that EnSCs have advantages in angiogenesis and tissue repair and regeneration.
[0004] More and more studies have shown that the therapeutic benefits of stem cells are mainly achieved through paracrine effects, that is, the secretion of growth factors, chemokines and cytokines, rather than through their differentiation ability or cell replacement. In order to maintain and perform normal biochemical functions, cells release various types of extracellular vesicles. Exosomes are an important component of extracellular vesicles. They are vesicular vesicles secreted by living cells and have a lipid bilayer membrane structure. Exosomes can be released by various types of cells and exist in various body fluids of the body. Exosomes carry a variety of bioactive substances, including gDNA, mRNA, circRNA, lncRNA, miRNA, proteins and lipids. As a carrier for mediating intercellular communication, exosomes transport bioactive molecules from donor cells to nearby or distant recipient cells and participate in the pathophysiological regulation of recipient cells. In clinical applications, compared with cells, exosomes have lower tumorigenicity and allogeneic immune rejection risks, are less likely to cause ethical issues, and are easier to store and transport. Therefore, EnSCs-derived exosomes have important application advantages.
[0005] However, one of the main challenges hindering the industrialization and clinical application of exosomes is their low yield, which makes it impossible to achieve large-scale and cost-effective production solutions required for clinical applications. To solve this problem, methods such as chemical stimulation, physical stimulation, and genetic manipulation of source cells have been explored to increase the yield of exosomes. For example, the use of monensin (MON) to induce a large amount of Ca2+ into the cell, or the use of the glycolysis inhibitor sodium iodoacetate combined with the oxidative phosphorylation inhibitor 2,4-dinitrophenol, can stimulate the release of exosomes in cells. There are also some small molecule drugs, such as fenoterol, norepinephrine, N-methyldopamine, mephenicol and forskolin, which can also stimulate the secretion of exosomes. Physical stimulation methods such as radiation, electrical stimulation, magnetic field stimulation, mechanical stimulation, etc. have been reported and proven to increase the yield of exosomes. In addition, methods such as hypoxia, heat stimulation, and pH change of source cells are also often used in exosome-related research.
[0006] Unfortunately, all of the above methods have shortcomings. Adding inducing agents and drugs will produce cytotoxicity, reduce cell viability, and introduce foreign substances to reduce the purity of exosomes. Strongly irritating physical methods will denature the surface proteins of exosomes, which may directly affect the morphology and performance of exosomes and damage their original capabilities. It can be seen that the existing methods for increasing the yield of cell-secreted exosomes are complicated to operate, cause great damage to cells, and are difficult to produce a large number of exosomes, which cannot meet actual research and production needs. Summary of the invention
[0007] To solve the above problems, the present invention discloses a method for increasing the production of EnSC exosomes using a co-culture system. Endometrial stem cells and fibroblasts treated with mitomycin C are co-cultured in a non-contact manner in a specific ratio using a Transwell chamber and a 12 mm polyester membrane with a pore size of 0.4 μm. When the cell confluence reaches 90%, the endometrial stem cells are taken out, the original culture medium is washed off, and the cells are transferred into a T75 bottle and cultured in a fresh exosome-specific serum-free culture medium. When the cell confluence reaches 90%, the cell supernatant is collected for exosome extraction. Compared with endometrial stem cells cultured alone, this method can significantly increase the production of endometrial stem cell exosomes.
[0008] To achieve the above object, the technical solution of the present invention is as follows:
[0009] The present invention provides a method for increasing the production of exosomes from endometrial stem cells, comprising the following steps:
[0010] S1, treat P3 fibroblasts with 4-8 μg / ml mitomycin C;
[0011] S2, co-culturing endometrial stem cells and fibroblasts indirectly through a Transwell chamber, separated by a semipermeable membrane; the endometrial stem cells are SFRP4 extracted from proliferative endometrial tissue + Endometrial stem cell subsets;
[0012] S3. The endometrial stem cells are removed and cultured separately in a serum-free culture medium dedicated to exosomes, and the cell supernatant is harvested; and the exosomes are extracted.
[0013] Furthermore, in step S1, the P3 generation fibroblasts are treated with mitomycin C for 2 to 6 hours, preferably for 4 hours. The P3 generation fibroblasts treated with mitomycin C are in normal culture, and the culture conditions are 37° C., 5% CO2, and saturated humidity.
[0014] Furthermore, in step S2, the endometrial stem cells are selected from the endometrium shed on the 5th to 10th day after the urine LH test paper test is strongly positive after the menstrual period without sexual intercourse.
[0015] Furthermore, in step S2, the semipermeable membrane is 12 mm polycarbonate (PC) or polyester / polyethylene terephthalate (PET) or polytetrafluoroethylene (PTFE), and the pore size is 0.4 μm.
[0016] Furthermore, in step S3, extracting exosomes comprises the following steps:
[0017] (1) Collect the cell supernatant into a centrifuge tube and centrifuge at 160-400g for 10 min;
[0018] (2) The supernatant after centrifugation was centrifuged again at 2000 g for 10 min;
[0019] (3) Collect the supernatant and centrifuge at 10,000 to 12,000 g for 10 to 30 min;
[0020] (4) Filter the supernatant after centrifugation with a 0.22 μm filter;
[0021] (5) The supernatant obtained in step (4) is purified using the EXODUS exosome extraction system by sine wave negative pressure oscillation and double coupled resonance technology to obtain exosomes.
[0022] Furthermore, in step S2, the ratio of the number of endometrial stem cells to the number of P3 fibroblasts treated with mitomycin C is 1:1 to 1:8, and preferably the ratio of the number of endometrial stem cells to the number of P3 fibroblasts treated with mitomycin C is 1:5.
[0023] The beneficial effects of the present invention are:
[0024] The present invention provides a co-culture system and a method for increasing the exosome production of endometrial stem cells. First, fibroblasts are pretreated with mitomycin C, and covalent cross-linking is formed between opposite DNA strands to avoid DNA double-strand separation during cell replication, so that the cells are arrested in the G1, S and G2 phases of the cell cycle, while RNA and protein synthesis continue, so that the fibroblasts remain in a non-proliferative but metabolically active state, becoming tool cells for promoting endometrial stem cells, and providing cytokines and chemicals for endometrial stem cells without increasing the consumption of nutrients. Then, the endometrial stem cells and fibroblasts are indirectly co-cultured through a Transwell chamber, separated by a semipermeable membrane in the middle, and a physical isolation is formed between the two cell types, so that the two cells do not contact each other, and stem cell differentiation caused by cell-to-cell contact reaction and fusion of stem cells with somatic cells is eliminated. Only the exchange of chemical substances such as cytokines secreted by cells and the influence of ion gradients are retained. After a certain period of co-culture, the endometrial stem cells are taken out and placed in a serum-free medium dedicated to exosomes for separate culture. After a certain period of time, the cell supernatant is harvested (by obtaining gradient centrifugation, filtration, sine wave negative pressure oscillation and double coupling resonance technology) to purify exosomes. Compared with endometrial stem cells cultured alone throughout the whole process, the co-culture system of the present invention significantly improves the proliferation rate of endometrial stem cells and the exosome yield per unit number of cells.
[0025] Compared with the current methods of directly treating target cells to increase the production of exosomes, the present invention avoids damage to target cells and adopts an indirect co-culture method to use fibroblasts with arrested proliferation to provide a growth environment for endometrial stem cells. On the one hand, the proliferation rate of endometrial stem cells is increased, and more source cells can be obtained per unit time; on the other hand, the exosome production of the same unit number of cells is also higher. The combination of these two aspects greatly improves the efficiency of obtaining exosomes.
[0026] The endometrial stem cells of the present invention are selected from SFRP4 extracted from proliferative endometrial tissue. + Endometrial stem cell subpopulation. This subpopulation has a strong regenerative capacity that other menstrual cycle subpopulations lack. Correspondingly, the exosomes it secretes also show more significant regenerative function, so it has higher scientific research value and broader clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a picture of co-culture of endometrial stem cells and fibroblasts;
[0028] Figure 2 Comparison of the average doubling time of EnSC experimental group co-culture and control group single culture;
[0029] Figure 3 The concentration of EnSC exosomes after co-culture with fibroblasts was 5 times higher;
[0030] Figure 4 Exosome concentration for EnSC culture alone;
[0031] Figure 5 Flow cytometry analysis of EnSCs. DETAILED DESCRIPTION
[0032] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.
[0033] Example 1
[0034] (I) Culture of endometrial stem cells
[0035] 1. Proliferative endometrial tissue acquisition
[0036] The endometrium shed on the 5th to 10th day after the self-test of urine LH test paper with a strong positive result after abstention from sexual intercourse in the current month was selected, the tissue was rinsed with sterile saline to remove blood stains, immediately placed in pre-cooled DMEM / F12 culture medium, refrigerated at 8°C, and sent to the laboratory for processing within 12 hours.
[0037] 2. Endometrial tissue digestion
[0038] Under sterile conditions, the tissue was repeatedly rinsed with phosphate buffer (containing 100U / ml penicillin and 100μg / ml streptomycin) to remove residual blood, and the tissue was cut into 1mm×1mm size with ophthalmic scissors and placed in a 10cm culture dish. 5ml of digestion solution (containing 0.25% trypsin + 0.02% EDTA + 0.25% type II collagenase) was added, and digested on a shaker at room temperature for 90min.
[0039] 3. Isolation and purification of endometrial stem cells
[0040] The collected cell digestion fluid was filtered through 100μm, 70μm, and 40μm sieves in sequence to remove undigested tissue, and the cells (cells on the surface of the 40μm filter) were collected. 1ml DMEM / F12 complete medium (DMEM / F12+10% FBS, 100U / ml penicillin and 100μg / ml streptomycin) was added to resuspend the cells, and 200 cells / ml were inoculated in a 10cm culture dish, and cultured in a saturated humidity cell culture incubator at 37°C and 5% CO2, and the medium was changed every 3-4 days.
[0041] Primary culture for clone screening: After 14 days, adherent cells with cloned growth were collected and transferred into 24-well plates for culture (the medium was changed every 3 days). When the cells grew to 80-90%, the cells were collected and transferred into 25 cm 2 Cell culture flasks were cultured and the medium was changed every 3 days.
[0042] Subculture: Cells were subcultured when they grew to nearly 80% confluence and inoculated in DMEM / F12 complete culture medium at a ratio of 1:2. The medium was changed every 3 days. The first subculture cells were labeled as P1, and the second subculture cells were labeled as P2. Cells were cultured to the fourth generation before subsequent experiments.
[0043] 4. Identification of Endometrial Stem Cells
[0044] Identification of universal surface markers of the endometrium: The cells obtained by the above separation method were detected for the expression of CD73, CD90 and SFRP4 by flow cytometry (instrument: NovoCyte 451141212168 software: NovoExpress1.4.3).
[0045] The results of the flow cytometry assay for cell surface protein expression are shown in Table 1. Figure 5 This is consistent with the strong regeneration ability of SFRP4 + Phenotypes of subpopulations of endometrial stem cells.
[0046] Table 1 Results of flow cytometry assay for cell surface protein expression
[0047]
[0048] (II) Fibroblast culture and pretreatment
[0049] Purchase primary human foreskin-derived dermal fibroblasts. After receiving the cells, take out the T25 cell culture flask and place it in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 3-4 hours to stabilize the cell state. Subculture when the cells reach 80% confluence. Aspirate the culture medium in the T25 cell culture flask and wash the cells once with PBS; add 1 mL of 0.25% trypsin digestion solution to the culture flask, gently rotate the culture flask until the digestion solution covers the entire bottom of the culture flask, aspirate the excess trypsin digestion solution, and warm it at 37°C for 1-3 minutes; observe under an inverted microscope, wait for the cells to shrink and become round, and then add 5 ml of complete culture medium to terminate digestion; gently blow and mix with a pipette, inoculate and subculture at a ratio of 1:2, and then add fresh complete culture medium to 5 mL, and place it in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for static culture; wait for the cells to fully adhere to the wall, culture and observe; then replace fresh complete culture medium every 3 days. The first passage cells were marked as P1, and the second passage cells were marked as P2. The cells were cultured to the third generation. When the confluence reached about 70-80%, they were treated with 6 μg / ml mitomycin C for 4 hours and then digested with trypsin to inhibit fibroblast DNA replication before use in the subsequent co-culture system construction.
[0050] (III) Obtaining EnSC exosomes through co-culture system
[0051] 1. Construction of fibroblast co-culture system and comparison experiment of EnSC expansion rate
[0052] The experimental group adopted an indirect co-culture model, in which endometrial stem cells and fibroblasts were placed in the same culture system, and non-direct contact co-culture was performed in a 6-well plate using a Transwell chamber and a 12 mm polyester (PET) membrane (pore size 0.4 μm). The experimental group was divided into 3 groups ( Figure 1 :Co-culture of endometrial stem cells and fibroblasts (Figure 1), endometrial stem cells were inoculated on the upper layer of the 1x experimental group, 1×10 5 cells, 1×10 5 1×10 fibroblasts were seeded in the lower layer; endometrial stem cells were seeded in the upper layer of the 5-fold experimental group, with 1×10 5 cells, 5×10 5 1×10 fibroblasts were seeded in the lower layer; endometrial stem cells were seeded in the upper layer of the 8-fold experimental group, with 1×10 5 cells, 8×10 5 The control group cultured endometrial stem cells alone, and 1×10 fibroblasts were inoculated in the upper layer.5 Endometrial stem cells were cultured in a complete DMEM / F12 culture medium at 37°C, 5% CO2, and saturated humidity in a cell culture incubator. Cell counts were performed after 72 hours, and the doubling time of endometrial stem cells was calculated: DT = t*[lg2 / (lgNt-lgNo)] (t is the culture time, No is the number of cells recorded for the first time, and Nt is the number of cells after t time).
[0053] 2. Experimental results
[0054] EnSC cell expansion comparison experiment results:
[0055] Comparison of EnSC expansion rate in co-culture system:
[0056] Table 2: EnSC doubling time records for each group
[0057]
[0058] Figure 2 : Comparison of average doubling time between EnSC co-culture and single culture.
[0059] The experimental results showed that the average doubling time of EnSC co-culture was much shorter than that of single culture, with significant difference (P < 0.0001). There was a direct difference between the 1-fold experimental group and the 5-fold experimental group (P < 0.05), and there was no significant difference between the 5-fold experimental group and the 8-fold experimental group (ns).
[0060] Example 2
[0061] (I) Cultivation of endometrial stem cells is the same as in Example 1.
[0062] (II) Fibroblast culture and pretreatment were the same as in Example 1.
[0063] (III) Obtaining EnSC exosomes through co-culture system
[0064] 1. Comparative experiment on exosome acquisition
[0065] The experimental group adopted an indirect co-culture model, in which endometrial stem cells and fibroblasts were placed in the same culture system, and non-direct contact co-culture was performed in a 6-well plate using a Transwell chamber and a 12 mm polyester (PET) membrane (pore size 0.4 μm). The experimental group was divided into 3 groups ( Figure 1 :Co-culture of endometrial stem cells and fibroblasts (Figure 1), endometrial stem cells were inoculated on the upper layer of the 1x experimental group, 1×10 5 cells, 1×10 5 1×10 fibroblasts were seeded in the lower layer; endometrial stem cells were seeded in the upper layer of the 5-fold experimental group, with 1×10 5cells, 5×10 5 1×10 fibroblasts were seeded in the lower layer; endometrial stem cells were seeded in the upper layer of the 8-fold experimental group, with 1×10 5 cells, 8×10 5 The control group cultured endometrial stem cells alone, and 1×10 fibroblasts were inoculated in the upper layer. 5 Endometrial stem cells were added, and no cells were added to the lower layer. All were cultured in DMEM / F12 complete medium in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.
[0066] Based on the results of the fibroblast co-culture system construction and EnSC expansion rate comparison experiment in Example 1, cells in the 5-fold experimental group with the fastest expansion rate were selected and compared with the control group for subsequent exosome acquisition comparison experiments.
[0067] Control the same number of cells cultured in exosome-specific medium
[0068] The cells of the 5-fold experimental group with the fastest proliferation rate and the control group were selected for subsequent experiments. When the confluence of the endometrial stem cells in the experimental group and the control group reached 90%, the Transwell chamber was removed. The original complete culture medium was removed and the cells were rinsed with PBS for 3 times. The number of cells in the experimental group and the control group was controlled at 1×10 6 Each one is moved in 75cm 2 The cells were cultured in a cell culture flask, and fresh exosome-specific serum-free culture medium was added. When the cell confluence reached 90%, the cell supernatant was collected for exosome extraction.
[0069] Exosome extraction steps:
[0070] Collect the cell culture supernatants of the experimental group and the control group into 50 mL centrifuge tubes respectively:
[0071] (1) Centrifuge at 300 g for 10 min to separate live cells;
[0072] (2) The supernatant was centrifuged again at 2000 g for 10 min to precipitate dead cells;
[0073] (3) Collect the supernatant and centrifuge at 12000g for 30 min to remove cell debris;
[0074] (4) Filter the supernatant after centrifugation with a 0.22 μm filter to remove apoptotic bodies and microvesicles.
[0075] The preliminarily purified exosome solution was purified using the EXODUS exosome extraction system through sine wave negative pressure oscillation and double-coupled resonance technology. Finally, the separated exosomes were rinsed and resuspended with 200 μL of PBS, and the number of EnSC exosomes in the experimental and control groups was detected by NTA.
[0076] 2. Exosome acquisition and comparison experimental results:
[0077] Figure 3 : EnSC exosome concentration after co-culture (detected by NTA after 1000-fold dilution): 4.1E+10 / ml, average particle size 135.6nm.
[0078] Figure 4 : The exosome concentration of EnSC cultured alone (detected by NTA after 200-fold dilution): 5.9E+9 / ml, with an average particle size of 138.8nm.
[0079] The NTA test results showed that under the same initial cell number, the exosome production of endometrial stem cells cultured in a 5-fold ratio of fibroblasts co-culture system was 7 times that of single culture, and there was no significant difference in the average particle size of exosomes.
[0080] It should be noted that the above content only illustrates the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.
Claims
1. A method for increasing the production of endometrial stem cell exosomes, characterized in that: The following steps are involved: S1, treat P3 fibroblasts with 4-8 μg / ml mitomycin C; S2, co-culture endometrial stem cells and mitomycin C-treated fibroblasts indirectly through Transwell chambers, separated by a semipermeable membrane; S3. The endometrial stem cells are removed and cultured separately in a serum-free culture medium dedicated to exosomes, and the cell supernatant is harvested; and the exosomes are extracted.
2. The method for increasing the production of endometrial stem cell exosomes according to claim 1, characterized in that: In step S1, the P3 fibroblasts are treated with mitomycin C for 2 to 6 hours.
3. The method for increasing the production of endometrial stem cell exosomes according to claim 1, characterized in that: In step S2, the endometrial stem cells are selected from the endometrium shed on the 5th to 10th day after the urine LH test paper test is strongly positive after the menstrual period without sexual intercourse.
4. The method for increasing the production of endometrial stem cell exosomes according to claim 1, characterized in that: In step S2, the semipermeable membrane is 12 mm polycarbonate, or one of polyester / polyethylene terephthalate and polytetrafluoroethylene, and has a pore size of 0.4 μm.
5. The method for increasing the production of endometrial stem cell exosomes according to claim 1, characterized in that: In step S3, extracting exosomes includes the following steps: (1) Collect the cell supernatant into a centrifuge tube and centrifuge at 160-400g for 10 min; (2) The supernatant after centrifugation was centrifuged again at 2000 g for 10 min; (3) Collect the supernatant and centrifuge at 10,000 to 12,000 g for 10 to 30 min; (4) Filter the supernatant after centrifugation with a 0.22 μm filter; (5) The supernatant obtained in step (4) is purified using the EXODUS exosome extraction system by sine wave negative pressure oscillation and double coupled resonance technology to obtain exosomes.
6. The method for increasing the production of endometrial stem cell exosomes according to claim 1, characterized in that: In step S2, the ratio of the number of endometrial stem cells to the number of fibroblasts treated with mitomycin C is 1:1 to 1:8.