Method for improving secretion and quality of stem cell exosome

Abstract

The invention relates to a method for improving secretion and quality of stem cell exosomes, and belongs to the technical field of cell culture.The method specifically comprises the following steps that S1, stem cells are resuspended in a basic culture medium and subjected to resuscitation treatment, and pretreated stem cells are obtained; s2, taking a basic culture medium, removing FBS of exosomes, and then adding ergothioneine and beta-nicotinamide mononucleotide according to the final concentration to obtain an improved culture medium; and S3, washing the pretreated stem cells, adding an improved culture medium, culturing, carrying out differential centrifugal precipitation on the exosome, and filtering and purifying through a filter membrane. According to the technical scheme, the ergothioneine and the NMN serve as components of an improved culture medium at the same time, ROS is removed by the ergothioneine, degradation of NAD < + > by the ergothioneine can be reduced, the NMN provides ATP support for the antioxidant process of the ergothioneine, and the ergothioneine and the NMN cooperate to improve the exosome secretion quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, more particularly, it relates to a method for improving the secretion and quality of stem cell exosomes. BACKGROUND

[0002] As a kind of nanoscale vesicle with important biological functions, stem cell exosomes show great application potential in cell communication, tissue repair, disease treatment and many other fields. However, there are still many problems to be solved in the current culture and preparation process of stem cell exosomes, which restricts its further application and development.

[0003] In terms of improving the secretion amount of exosomes, the existing methods have limited effect. Stem cells will produce reactive oxygen species (ROS) in the metabolic process, and oxidative stress will cause damage to cells, affect the expression of exosome biosynthesis related genes, and thus limit the basic secretion amount of exosomes. At present, there is no effective means to remove ROS, protect the stability of DNA and protein in stem cells, and maintain the normal physiological function of cells to promote exosome secretion.

[0004] In terms of exosome quality, stem cells will age during the culture process, the content of pro-inflammatory factors will increase, and other factors will cause the quality of exosomes to decrease, the contents of exosomes may be degraded, the content of functional molecules may be reduced, and the biological activity may be affected. Moreover, in the sorting process of the contents of exosomes, there is a lack of effective regulation mechanism, and it is difficult to realize the enrichment of functional components and the reduction of damaged components.

[0005] In addition, the existing technology mostly only focuses on the influence of a single factor on the secretion or quality of exosomes, and fails to comprehensively consider the synergistic effect between multiple factors. In fact, different substances may interact with each other in the physiological process of stem cells and the secretion of exosomes, and single factor regulation is difficult to comprehensively and effectively improve the secretion amount and quality of stem cell exosomes. Based on the above statement, the present application provides a method for improving the secretion and quality of stem cell exosomes. SUMMARY

[0006] In order to solve the problems proposed in the background art, the present application provides a method for improving the secretion and quality of stem cell exosomes.

[0007] The present application provides a method for improving the secretion and quality of stem cell exosomes, which adopts the following technical solution: A method for improving the secretion and quality of stem cell exosomes, comprising the following steps: S1, resuspend stem cells in a basic culture medium, perform resuscitation treatment, and obtain pretreated stem cells; S2. Take the basal culture medium, remove the FBS from the exosomes, and then add ergothionein and β-nicotinamide mononucleotide (hereinafter referred to as NMN) according to the final concentration to obtain the modified culture medium; S3. After washing the pretreated stem cells, add them to the modified culture medium for culture, then centrifuge at differential speed to precipitate exosomes and purify them by filtration through a filter membrane.

[0008] Furthermore, the stem cells are human mesenchymal stem cells or embryonic stem cells.

[0009] Furthermore, the stem cells are P3-P5 generation, and the positive rates of stem cell markers CD44, CD90, and CD105 are >95%.

[0010] Furthermore, the basal culture medium is prepared by the following steps: Centrifuge α-MEM medium containing 10% bovine serum at 100,000 xg and room temperature for 12-16 hours, then add penicillin-streptomycin 50-200 U / ml according to the final ratio to obtain the basal medium.

[0011] In the above scheme, a culture medium containing bovine serum is used as the base, and high-speed centrifugation is used to remove exosomes from FBS to avoid interference with the stem cell culture system.

[0012] Furthermore, in step S1, the resuscitation treatment specifically includes the following steps: The resuspended stem cells were seeded into T75 culture flasks and cultured in a 37°C, 5% CO2 incubator. After 24 hours of culture, the non-adherent cells were removed. The system was then digested with 0.25% trypsin and seeded into six-well plates. 2 ml of culture medium was added to each well and the system was cultured for 24-36 hours.

[0013] Furthermore, the cell density was adjusted to 3 × 10⁻⁶ after digestion. 4 -5×10 4 pcs / cm 2 .

[0014] Furthermore, in step S2, the final concentration of ergothionein in the modified culture medium is 30-60 μM.

[0015] Furthermore, in step S2, the final concentration of NMN in the modified culture medium is 0.3-0.6 mM.

[0016] Furthermore, in step S3, the specific steps for adding the modified culture medium for culturing are as follows: Discard the original culture medium in the 6-well plate, wash twice with PBS buffer, then add the modified culture medium and incubate at 37°C and 5% CO2 for 36-48 hours, avoiding medium changes during the incubation.

[0017] Furthermore, the amount of modified culture medium added is 1.5-2 ml per well.

[0018] In summary, this application has the following beneficial effects: 1. In the technical solution of this invention, ergothioneine, as a natural antioxidant, can reduce oxidative stress damage to cells by scavenging reactive oxygen species produced by stem cell metabolism. It can protect the stability of DNA and proteins within stem cells, maintain normal cellular physiological functions, and thus promote the expression of genes related to exosome biosynthesis, such as Rab27a and Alix, increasing the basal secretion of exosomes. Simultaneously, ergothioneine can regulate the sorting process of exosome contents, promote the enrichment of functional miRNAs, reduce the degradation of exosome contents caused by oxidative damage, and enhance the biological activity of exosomes.

[0019] 2. In the technical solution of this invention, NMN is also used as a component of the modified culture medium, and NMN is used as NAD. + Its direct precursor can significantly enhance NAD in stem cells. + This enhances mitochondrial function and energy metabolism efficiency. This provides sufficient energy for exosome biosynthesis in stem cells, accelerates exocytosis, and improves secretion efficiency. Furthermore, NMN maintains stem cell stemness by regulating cellular senescence-related pathways, reducing the decline in exosome quality caused by cellular aging and increased pro-inflammatory cytokine levels, thus ensuring the continuous secretion of highly active functional molecules from exosomes.

[0020] 3. In the technical solution of this invention, ergothioneine and NMN are used simultaneously as components to improve the culture medium. Ergothioneine's ability to scavenge ROS can reduce its impact on NAD. + Degradation of NMN, maintaining NMN and enhancing NAD + The effects are multifaceted: NMN enhances mitochondrial function, providing ATP support for ergothioneine's antioxidant processes and strengthening its protective effect on stem cells. Simultaneously, ergothioneine promotes the expression of exosome synthesis genes, synergistically working with NMN's accelerated vesicle transport process to increase exosome production. At the quality level, both synergistically upregulate the sorting signals of anti-inflammatory factors and functional miRNAs, reducing damaging components such as oxidative proteins in exosomes and further improving the quality of exosome secretion. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] The human mesenchymal stem cells used in Examples 1-3 and Comparative Examples 1-5 below were derived from bone marrow, were P3 generation cells, and had a positive rate of >95% for stem cell markers CD44, CD90, and CD105; the purity of ergothioneine used was ≥98%, and the purity of NMN used was ≥99%.

[0023] Example 1 A method for improving the secretion and quality of stem cell exosomes includes the following steps: S1. Resuspend the stem cells in basal culture medium for resuscitation to obtain pretreated stem cells; wherein, the basal culture medium is prepared by the following steps: The α-MEM medium containing 10% bovine serum was centrifuged at 100,000 xg and room temperature for 12 hours, and then penicillin-streptomycin 100 U / ml was added according to the final ratio to obtain the basal medium.

[0024] The recovery process specifically includes the following steps: The resuspended stem cells were seeded into T75 culture flasks and cultured in a 37°C, 5% CO2 incubator. After 24 hours of culture, non-adherent cells were removed, and the system was then digested with 0.25% trypsin to adjust the cell density to 5 × 10⁶ cells / year. 4 pcs / cm 2 Then, the culture was inoculated into a six-well plate, and 2 ml of culture medium was added to each well for 24 hours of incubation.

[0025] S2. Take the basal culture medium (preparation steps are the same as in S1), remove the FBS from the exosomes, and then add ergothionein and β-nicotinamide mononucleotide according to the final concentration to obtain the modified culture medium; wherein, the final concentration of ergothionein in the modified culture medium is 30 μM and the final concentration of NMN is 0.3 mM. S3. After washing the pretreated stem cells, add modified culture medium and culture. Then, centrifuge at differential speed to precipitate exosomes and purify them by filtration through a filter membrane. The specific procedure is as follows: discard the original culture medium in the 6-well plate, wash twice with PBS buffer, then add modified culture medium and incubate at 37°C and 5% CO2 for 36 hours, avoiding medium changes during the incubation. The amount of modified culture medium added is 2 ml per well.

[0026] After culture, collect the supernatant into a sterile centrifuge tube and centrifuge at 4°C and 300xg for 10 minutes. Then, aspirate the supernatant again and centrifuge at 4°C and 2000xg for 15 minutes. Next, aspirate the supernatant and change the centrifugation conditions to 4°C and 10000xg for another 30 minutes. Aspirate the supernatant again and change the centrifugation conditions to 4°C and 100000xg for 70 minutes, retaining the precipitate at the bottom of the tube. Resuspend the precipitate with sterile PBS buffer and let the system stand at 4°C for 30 minutes. Then, take the resuspended exosome solution and filter it through a 0.22μm sterile polyethersulfone filter membrane at a filtration pressure of 0.1MPa. Collect the filtered solution, which is the purified exosome.

[0027] Example 2 A method for improving the secretion and quality of stem cell exosomes includes the following steps: S1. Resuspend the stem cells in basal culture medium for resuscitation to obtain pretreated stem cells; wherein, the basal culture medium is prepared by the following steps: The α-MEM medium containing 10% bovine serum was centrifuged at 100,000 xg and room temperature for 12 hours, and then penicillin-streptomycin 100 U / ml was added according to the final ratio to obtain the basal medium.

[0028] The recovery process specifically includes the following steps: The resuspended stem cells were seeded into T75 culture flasks and cultured in a 37°C, 5% CO2 incubator. After 24 hours of culture, non-adherent cells were removed, and the system was then digested with 0.25% trypsin to adjust the cell density to 5 × 10⁶ cells / year. 4 pcs / cm 2 Then, the culture was inoculated into a six-well plate, and 2 ml of culture medium was added to each well for 30 hours of incubation.

[0029] S2. Take the basal culture medium (preparation steps are the same as S1), remove the FBS of the exosomes, and then add ergothionein and β-nicotinamide mononucleotide according to the final concentration to obtain the modified culture medium; wherein, the final concentration of ergothionein in the modified culture medium is 45 μM and the final concentration of NMN is 0.45 mM. S3. After washing the pretreated stem cells, add modified culture medium for culture. The specific procedure is as follows: discard the original culture medium in the 6-well plate, wash twice with PBS buffer, then add modified culture medium and incubate at 37°C in a 5% CO2 incubator for 40 hours, avoiding medium changes during the incubation. The amount of modified culture medium added is 2 ml per well.

[0030] After culture, collect the supernatant into a sterile centrifuge tube and centrifuge at 4°C and 300xg for 10 minutes. Then, aspirate the supernatant again and centrifuge at 4°C and 2000xg for 15 minutes. Next, aspirate the supernatant and change the centrifugation conditions to 4°C and 10000xg for another 30 minutes. Aspirate the supernatant again and change the centrifugation conditions to 4°C and 100000xg for 70 minutes, retaining the precipitate at the bottom of the tube. Resuspend the precipitate with sterile PBS buffer and let the system stand at 4°C for 30 minutes. Then, take the resuspended exosome solution and filter it through a 0.22μm sterile polyethersulfone filter membrane at a filtration pressure of 0.1MPa. Collect the filtered solution, which is the purified exosome.

[0031] Example 3 A method for improving the secretion and quality of stem cell exosomes includes the following steps: S1. Resuspend the stem cells in basal culture medium for resuscitation to obtain pretreated stem cells; wherein, the basal culture medium is prepared by the following steps: The α-MEM medium containing 10% bovine serum was centrifuged at 100,000 xg and room temperature for 12 hours, and then penicillin-streptomycin 200 U / ml was added according to the final ratio to obtain the basal medium.

[0032] The recovery process specifically includes the following steps: The resuspended stem cells were seeded into T75 culture flasks and cultured in a 37°C, 5% CO2 incubator. After 24 hours of culture, non-adherent cells were removed, and the system was then digested with 0.25% trypsin to adjust the cell density to 5 × 10⁶ cells / year. 4 pcs / cm 2 Then, the culture was inoculated into a six-well plate, and 2 ml of culture medium was added to each well for 36 hours of incubation.

[0033] S2. Take the basal culture medium (preparation steps are the same as S1), remove the FBS of the exosomes, and then add ergothionein and β-nicotinamide mononucleotide according to the final concentration to obtain the modified culture medium; wherein, the final concentration of ergothionein in the modified culture medium is 60 μM and the final concentration of NMN is 0.6 mM. S3. After washing the pretreated stem cells, add modified culture medium for culture. The specific procedure is as follows: discard the original culture medium in the 6-well plate, wash twice with PBS buffer, then add modified culture medium and incubate at 37°C in a 5% CO2 incubator for 48 hours, avoiding medium changes during the incubation. The amount of modified culture medium added is 2 ml per well.

[0034] After culture, collect the supernatant into a sterile centrifuge tube and centrifuge at 4°C and 300xg for 10 minutes. Then, aspirate the supernatant again and centrifuge at 4°C and 2000xg for 15 minutes. Next, aspirate the supernatant and change the centrifugation conditions to 4°C and 10000xg for another 30 minutes. Aspirate the supernatant again and change the centrifugation conditions to 4°C and 100000xg for 70 minutes, retaining the precipitate at the bottom of the tube. Resuspend the precipitate with sterile PBS buffer and let the system stand at 4°C for 30 minutes. Then, take the resuspended exosome solution and filter it through a 0.22μm sterile polyethersulfone filter membrane at a filtration pressure of 0.1MPa. Collect the filtered solution, which is the purified exosome.

[0035] Comparative Example 1 The difference between this comparative example and Example 2 is that in step S2, ergothioneine was used to replace NMN in the modified culture medium, that is, the final concentration of ergothioneine in the modified culture medium was 495 μM.

[0036] Comparative Example 2 The difference between this comparative example and Example 2 is that in step S2, NMN is used to replace ergothioneine in the modified culture medium, that is, the final concentration of NMN in the modified culture medium is 495 μM.

[0037] Comparative Example 3 The difference between this comparative example and Example 2 is that, in step S2, the final concentration of ergothioneine in the modified culture medium is 200 μM.

[0038] Comparative Example 4 The difference between this comparative example and Example 2 is that, in step S2, the modified culture medium is specifically derived from the following steps: Take the basal culture medium (preparation steps are the same as S1), remove the FBS of the exosomes, and then add resveratrol and β-nicotinamide mononucleotide according to the final concentration to obtain the modified culture medium; wherein, the final concentration of resveratrol in the modified culture medium is 60 μM and the final concentration of NMN is 0.6 mM.

[0039] Comparative Example 5 The difference between this comparative example and Example 2 is that, in step S2, the modified culture medium is specifically derived from the following steps: Take the basal culture medium (preparation steps are the same as in S1), remove the FBS from the exosomes, and then add pyruvate and β-nicotinamide mononucleotide to the final concentration to obtain the modified culture medium; wherein, the final concentration of pyruvate in the modified culture medium is 60 μM and the final concentration of NMN is 0.6 mM.

[0040] Performance testing The exosome secretion quality of the systems prepared in Examples 1-4 and Comparative Examples 1-5 of this application is now tested. The specific test method is as follows: Total RNA was extracted from the culture systems in Examples 1-3 and Comparative Examples 1-5, respectively. Cells were collected from each culture system and washed three times with pre-cooled PBS buffer to remove impurities from the culture medium. Then, an appropriate amount of TRIzol reagent was added, at a concentration of 1 × 10⁻⁶ per cell. 6 Add 1 mL of TRIzol reagent to each cell, gently pipette to lyse the cells, and incubate at room temperature for 5 minutes. Add 0.2 mL of chloroform to the lysis buffer, tighten the centrifuge tube cap, and vigorously vortex for 15 seconds. Incubate at room temperature for 3 minutes. Then centrifuge at 12000 x g for 15 minutes at 4°C. After centrifugation, transfer the upper aqueous phase to a new centrifuge tube, add 0.5 mL of isopropanol, gently invert the tube to mix, and incubate at room temperature for 10 minutes. Centrifuge at 12000 x g for 10 minutes at 4°C, discard the supernatant, and wash the RNA precipitate with 1 mL of 75% ethanol (prepared with RNase-free water). Gently vortex the tube to resuspend the precipitate. Then centrifuge at 7500 x g for 5 minutes at 4°C, discard the supernatant, and incubate at room temperature for 10 minutes. Add 50 μL of RNase-free water and gently pipette to dissolve the RNA precipitate. Subsequently, 5 μg of total RNA was added to 10×RNase III reaction buffer, RNase III enzyme, and RNase-free water to a total volume of 20 μL. The reaction mixture was incubated at 37°C for 60 minutes. EDTA solution was added to a final concentration of 20 mM, and the mixture was then incubated at 65°C for 10 minutes to inactivate RNase III. After reverse transcription to generate a cDNA library, sequencing was performed. The reverse transcription system consisted of 500 ng of RNase III-treated RNA, 100 pmol of random primers, a dNTP mixture (10 mM for each dNTP), RNase-free water, and reverse transcriptase to a total volume of 20 μL. The reaction mixture was incubated at 25°C for 10 minutes to allow primer binding to the RNA template. Then, the mixture was incubated at 42°C for 60 minutes to perform reverse transcription and synthesize the first strand of cDNA. Finally, the mixture was incubated at 70°C for 15 minutes to inactivate the reverse transcriptase and complete the reverse transcription process. Sequencing was then performed, and the expression levels of functional miRNAs were calculated to evaluate the quality of exosome secretion. Among them, functional miRNA1-miRNA8 refer to hsa-miR-183-5p, hsa-miR-451a, hsa-miR-107, hsa-miR-199a-3p, hsa-miR-145-3p, hsa-miR-27b-3p, hsa-miR-223-3p, and hsa-miR-200a-3p, respectively. The specific test results are shown in Table 1 below.

[0041] Table 1

[0042] As shown in Table 1, the functional miRNAs presented in Examples 1-3 exhibited superior expression levels compared to the preparation systems in Comparative Examples 1-5. Higher TPM values ​​of the miRNAs indicated better anti-inflammatory and repair-promoting exosome functions. The results from Comparative Examples 1-3 demonstrate that only with a modified culture medium and appropriate concentrations of NMN and ergothioneine can the secretion quality of exosomes be significantly improved. The results from Comparative Example 4 show that, at the same concentration, the synergistic effect of NMN with other molecules that can activate stem cell energy metabolism and aging pathways, such as resveratrol, is not as significant as that with ergothioneine. The results from Comparative Example 5 indicate that pyruvate can also compensate for energy consumption under high exosome secretion conditions, but its effect is not as pronounced as the effect produced by the combination of ergothioneine and NMN in this application.

[0043] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0044] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for improving the secretion and quality of stem cell exosomes, characterized in that, Includes the following steps: S1. Resuspend the stem cells in basal culture medium and perform resuscitation treatment to obtain pretreated stem cells; S2. Take the basal culture medium, and then add ergothionein and β-nicotinamide mononucleotide according to the final concentration to obtain the modified culture medium; S3. After washing the pretreated stem cells, add them to the modified culture medium for culture, then centrifuge at differential speed to precipitate exosomes and purify them by filtration through a filter membrane.

2. The method for improving stem cell exosome secretion and quality according to claim 1, characterized in that, The stem cells mentioned are human mesenchymal stem cells or embryonic stem cells.

3. The method for improving stem cell exosome secretion and quality according to claim 1, characterized in that, The stem cells were P3-P5 generation, and the positive rates of stem cell markers CD44, CD90, and CD105 were >95%.

4. The method for improving stem cell exosome secretion and quality according to claim 1, characterized in that, The basal culture medium is prepared by the following steps: Centrifuge the α-MEM medium at 100,000 xg and room temperature for 12-16 hours, then add penicillin-streptomycin 50-200 U / ml according to the final ratio to obtain the basal medium.

5. The method for improving stem cell exosome secretion and quality according to claim 1, characterized in that, In step S1, the resuscitation treatment specifically includes the following steps: The resuspended stem cells were seeded into culture flasks for culture. After culture, the non-adherent cells were removed, the system was then digested, and then seeded into six-well plates. 2 ml of culture medium was added to each well, and the system was cultured for 24-36 hours.

6. The method for improving stem cell exosome secretion and quality according to claim 5, characterized in that, Digestion to adjust cell density to 3×10 4 -5×10 4 pcs / cm 2 .

7. The method for improving stem cell exosome secretion and quality according to claim 1, characterized in that, In step S2, the final concentration of ergothionein in the modified culture medium is 30-60 μM.

8. The method for improving stem cell exosome secretion and quality according to claim 1, characterized in that, In step S2, the final concentration of NMN in the modified culture medium is 0.3-0.6 mM.

9. The method for improving stem cell exosome secretion and quality according to claim 1, characterized in that, In step S3, the specific steps for adding the modified culture medium for culturing are as follows: Discard the original culture medium in the 6-well plate, wash with buffer, then add the modified culture medium and incubate at 37°C in a 5% CO2 incubator for 36-48 hours, avoiding medium changes during the incubation.

10. A method for improving the secretion and quality of stem cell exosomes according to claim 9, characterized in that, The amount of modified culture medium added is 1.5-2 ml per well.

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