CN118879627A - A method for culturing umbilical cord mesenchymal stem cell exosomes and its application - Google Patents

Abstract

The application discloses a method for culturing umbilical mesenchymal stem cell exosomes and application thereof, relating to the technical field of exosomes, comprising the following steps: s1, resuscitating cryopreserved P3 generation umbilical cord mesenchymal stem cells, culturing the cells in a DMEM culture medium at the temperature of 5% CO ₂ and 37 ℃ for normoxic culture for 48 hours, and adding IL-1 beta and TNF-alpha; s2, performing high-oxygen culture for 12-16 hours after normal-oxygen culture; s3, performing hypoxia culture for 48-72 hours after the hypoxia culture; s4, after the culture is finished, harvesting cell culture supernatant, and performing ultracentrifugation separation and purification to obtain human umbilical cord mesenchymal stem cell exosomes; can greatly increase the number of cells; reducing unnecessary energy consumption while promoting secretion of exosomes again and gradually reducing possible damage to cells.

Description

A method for culturing umbilical cord mesenchymal stem cell exosomes and its application

Technical Field

The present invention relates to the field of exosomes, and in particular to a method for culturing umbilical cord mesenchymal stem cell exosomes and applications thereof.

Background Art

Umbilical cord mesenchymal stem cells refer to a type of multifunctional stem cells present in the umbilical cord tissue of newborns. They are derived from the mesoderm and have high self-renewal and multidirectional differentiation capabilities, and therefore have broad prospects for clinical application.

Exosomes refer to small vesicles with a saucer-shaped structure and a diameter between 30-150nm, which contain RNA, microRNA, proteins, DNA fragments, etc. Almost all cells in the human body can secrete exosomes, which exist naturally in body fluids, including saliva, urine, cerebrospinal fluid, blood, and breast milk. Umbilical cord mesenchymal stem cells are no exception. They can secrete exosomes under normal and pathological conditions. Exosomes are mainly derived from multivesicular bodies formed by the invagination of lysosomal microparticles in cells, and are released into the extracellular matrix after the fusion of the multivesicular outer membrane with the cell membrane. Compared with umbilical cord mesenchymal stem cells, exosomes contain proteins, lipids, and nucleic acids from maternal cells, and can act as signal molecules on other cells due to their small molecular weight and strong permeability.

For example, the Chinese patent, application number, CN202310651210.6, is a method for increasing the expression of exosomes of umbilical cord mesenchymal stem cells. Human umbilical cord mesenchymal stem cells are inoculated in a 3D bioreactor with variable dissolved oxygen conditions. The dissolved oxygen conditions are normoxia to hypoxia culture, the oxygen partial pressure under normoxia is controlled to 50%, and the oxygen partial pressure under hypoxia is controlled to 15%. The beneficial effects of this invention are: the expression of exosomes of umbilical cord mesenchymal stem cells cultured under normoxia to hypoxia conditions in a 3D bioreactor is significantly higher than that under normoxia, hypoxia, and hypoxia to normoxia conditions; it is suitable for batch production and is easy to promote the industrial production of umbilical cord-derived mesenchymal stem cell exosome biological products.

However, although the number of cell secretions increases under hypoxic conditions, the cell growth rate is greatly reduced, and the overall exosome content needs to be improved.

Summary of the invention

The embodiments of the present application provide a method for culturing exosomes of umbilical cord mesenchymal stem cells and its application, thereby solving the problem of insufficient number of exosomes secreted by cells in the prior art and achieving the extraction of more exosomes and higher purity exosomes.

The present application embodiment provides a method for culturing umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

S1. Resuscitated frozen P3 umbilical cord mesenchymal stem cells were cultured in DMEM medium at 5% CO ₂ and 37°C for 48 hours under normoxic conditions, with an initial concentration of 1×10 ⁵ cells/cm ² , and IL-1β and TNF-α were added; the concentration of IL-1β was 10 ng/mL, and the concentration of TNF-α was 10 ng/mL; the DMEM medium was kept warm at 45°C for 24 hours;

S2, hyperoxia culture for 12-16h after normoxic culture;

S3, hypoxic culture for 48-72h after hyperoxic culture;

S4. After the culture is completed, the cell culture supernatant is harvested and purified by ultracentrifugation to obtain human umbilical cord mesenchymal stem cell exosomes.

Furthermore, the normoxic condition is a 20% O ₂ concentration environment.

Furthermore, the ultracentrifugation method is specifically as follows: the cell culture supernatant is centrifuged at 300g for 10 minutes to remove cells and debris, centrifuged at 2000g for 20 minutes to remove cell debris and apoptotic bodies, centrifuged at 10000g for 30 minutes to remove large vesicles, and finally centrifuged at 100000g for 70 minutes to collect the exosome precipitate and resuspend it in PBS to make 10 ml of exosome solution.

Furthermore, the oxygen concentration of the hyperoxia culture is an _O2 concentration environment of 40%-60%.

Furthermore, the oxygen concentration of the hypoxic culture is an _O2 concentration environment of 5%-10%.

Furthermore, the order of the high oxygen culture step and the low oxygen culture step is reversed, that is:

S2, hypoxic culture for 48-72h after normoxic culture;

S3. Perform high oxygen culture for 12-16 hours after low oxygen culture.

Furthermore, as the oxygen concentration changes, CO ₂ changes synchronously.

Furthermore, the synchronous changes in CO ₂ are as follows: in normoxic culture, the CO ₂ concentration is 5%, in hypoxic culture, the CO ₂ concentration is 1-4%, and in hyperoxic culture, the CO ₂ concentration is 10-20%.

Furthermore, the inner surface of the culture vessel used for cell culture is also coated with a cell adhesion inhibitor.

The above-mentioned application of cultured umbilical cord mesenchymal stem cell exosomes is applied to knee arthritis.

One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

First, in a high-oxygen environment, cells will undergo oxidative stress, triggering intracellular signal transduction pathways, especially pathways related to antioxidant responses and cell survival, promoting cells to adapt to the upcoming low-oxygen environment and adjust metabolic pathways, thereby optimizing energy utilization, increasing the production of exosomes, and causing cells to proliferate in large quantities in the early stages, greatly increasing the number of cells; the subsequent low-oxygen environment induces cells to enter a protective metabolic state, reducing unnecessary energy consumption, while again promoting the secretion of exosomes and gradually reducing possible damage to cells.

Secondly, low oxygen first and then high oxygen, so that cells enter a protective metabolic state in a low oxygen environment, reduce unnecessary energy consumption, and promote the secretion of exosomes to cope with the hypoxic environment. The subsequent high oxygen environment further stimulates the cells, produces a stress response, and leads to the release of a large number of exosomes. A large number of exosomes will be released after the apoptosis program is initiated, but due to the low oxygen culture environment, the rate of cell apoptosis after stress slows down, so that the apoptotic bodies are quickly engulfed by other cells after they are produced, the number of dead cells and cell organs in the culture environment is reduced, the purity of exosomes is increased, and cells are better adapted to the alternation of hypoxia and oxygen-rich environments, thereby improving the cell's stress resistance and survival ability. It is also because exosomes are still stable in hypoxia or hyperoxia when used to load drugs or in vivo.

Third, the secretion of exosomes is affected by many factors in the intracellular and extracellular environment, including oxygen and carbon dioxide concentrations. Changes in oxygen concentration can affect the metabolic state of cells, and thus affect the production and secretion of exosomes. A low oxygen environment promotes the secretion of exosomes while increasing the cells' antioxidant capacity and reducing stress. A high oxygen environment increases cell stress, causing it to enter apoptosis and increase the output of exosomes.

Fourthly, by coating cell adhesion inhibitors, the stacking of cells on the wall of the culture container is reduced, thereby changing the growth environment and state of the cells, that is, the number of adherent cells is reduced and the number of suspended cells is increased. The reduction of adherent cells means the reduction of stacked cells, and the cells use oxygen more evenly, avoiding the situation in which the gas utilization of locally stacked cells is lower than that of other parts during the culture process; the increase of suspended cells increases the overall growth rate of cells, thereby increasing the total number of cells and the secretion capacity, and further increasing the production of exosomes.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "and/or" used herein includes any and all combinations of one or more related listed items.

Embodiment 1

The present application provides a method for culturing umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

S1. Resuscitated frozen P3 umbilical cord mesenchymal stem cells were cultured in DMEM medium (containing 10% fetal bovine serum) at 5% CO ₂ and 37°C for 48 hours at an initial concentration of 1×10 ⁵ cells/cm ² , and IL-1β and TNF-α were added; the normoxic condition was 20% O ₂ concentration environment;

The concentration of IL-1β was 10 ng/mL, and the concentration of TNF-α was 10 ng/mL; the DMEM medium was kept at 45°C for 24 h;

S2. Perform hyperoxic culture for 12-16 hours after normoxic culture, with the hyperoxic oxygen concentration being 40%-60% O ₂ concentration environment;

S3. After the high oxygen culture, the hypoxic culture is performed for 48-72 hours, and the hypoxic oxygen concentration is 5%-10% _O2 concentration environment;

S4. After the culture is completed, the cell culture supernatant is harvested and purified by ultracentrifugation to obtain human umbilical cord mesenchymal stem cell exosomes;

The ultracentrifugation method is specifically as follows: the cell culture supernatant is centrifuged at 300g for 10 minutes to remove cells and debris, centrifuged at 2000g for 20 minutes to remove cell debris and apoptotic bodies, centrifuged at 10000g for 30 minutes to remove large vesicles, and finally centrifuged at 100000g for 70 minutes to collect the exosome precipitate, which is then resuspended in PBS to form 10 ml of exosome solution.

The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

In a high-oxygen environment, cells will undergo oxidative stress, triggering intracellular signal transduction pathways, especially pathways related to antioxidant responses and cell survival, promoting cells to adapt to the upcoming low-oxygen environment and adjust metabolic pathways, thereby optimizing energy utilization and increasing the production of exosomes. It also causes cells to proliferate in large quantities in the early stages, greatly increasing the number of cells and improving the extraction efficiency of exosomes. The subsequent low-oxygen environment induces cells to enter a protective metabolic state, reducing unnecessary energy consumption, while promoting the secretion of exosomes again and gradually reducing possible damage to cells.

By treating serum at low temperature, the impurities, cell vesicles and other possible hazards it brings are reduced; high oxygen pretreatment can enhance the antioxidant capacity and adaptive response of cells, thereby improving the survival rate of cells in a low oxygen environment, which is beneficial for large-scale cell culture and production of exosome biological products;

Due to the increase in exosome secretion, the production efficiency of exosomes is significantly improved and the production cost is reduced; by optimizing the culture conditions, the purity and activity of exosomes are improved, thereby improving the quality of the final product; high oxygen pretreatment enhances the adaptability of cells to environmental changes, allowing cells to maintain a more stable growth and metabolic state in a low oxygen environment.

Experiment on rat knee osteoarthritis model: 30 SD rats weighing 200±20g were randomly divided into 3 groups, 10 rats in each group, namely normal group, model group and experimental group. Except for the normal group, rats were injected with papain into the joint cavity to replicate the KOA model; the rats were skinned and disinfected, the hair at and below the knee joint of the right forelimb of the rats was shaved, the skin was disinfected with 75% ethanol, and 0.2mL of 4% papain solution was injected into the right forelimb knee joint cavity, once every 2 days, for 2 consecutive weeks. After 14 days, if the rats showed the following symptoms, it means that the replication was successful: redness, swelling, heat and pain in the right anterior knee joint, accompanied by swelling of soft tissue or effusion; stiffness and limited movement in the right anterior knee joint and the surrounding area; pathological examination of the right anterior knee joint found a large amount of exudate in the joint cavity; the cartilage surface was not smooth, and there were arthritis pathological changes such as synovial hyperplasia or adhesion.

On the second day after successful modeling, the normal group and the model group were gavaged with normal saline, and the rats in the experimental group were given the exosomes prepared in this example, and the tail vein was injected at a dose of 2 ml/kg/d. After 4 weeks of intervention, the femoral artery blood of the rats in each group was collected and centrifuged at 1500 rpm·min-1 for 10 min, and the serum was collected. The levels of IL-6, TNF-α, and IL-1β were detected by enzyme-linked immunosorbent assay. The results of the exosomes used for injection are shown in Table 1:

IL-6 (ng·L ^-1 ) TNF-α(ng·L-1) IL-1β (ng·L-1) Blank Group 18.24±2.58 22.54±5.24 6.24±0.98 Experimental Group 44.68±6.25 45.32±5.84 13.54±3.14 Experimental Group 20.54±4.31 24.18±2.97 6.85±0.67

Table 1

The BCA method was used to determine the concentration of exosome protein in the sample, and the number of exosome particles with a particle size range of 20-150 nm in the sample was statistically analyzed using nanoflow cytometry to calculate the purity of the exosomes. The results are shown in Table 2:

Table 2

Embodiment 2

Example 1 promotes cellular oxidative stress and increases the secretion of exosomes in cells under a high oxygen environment, thereby protecting cells and reducing cell apoptosis, and further improves Example 2.

Replace the high oxygen culture step with the low oxygen culture step, specifically:

S2. After normoxic culture, hypoxic culture is performed for 48-72 hours, and the hypoxic oxygen concentration is 5%-10% _O2 concentration environment;

S3. Perform hyperoxia culture for 12-16 hours after hypoxia culture, with the hyperoxia oxygen concentration being 40%-60% O ₂ concentration environment;

The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

A hypoxic environment can simulate the hypoxic state of tissues or organs in the body, triggering the hypoxia sensing mechanism in cells and the activation of HIF (hypoxia-inducible factor). The activation of HIF can regulate the expression of a series of genes, promote cells to adapt to the hypoxic environment, and promote the secretion of exosomes to transmit hypoxia signals or participate in intercellular communication under hypoxic conditions.

The high oxygen environment produces a certain amount of oxidative stress on cells, activates the antioxidant response and stress response pathways in cells, promotes the release of exosomes by cells to remove oxidative damage substances accumulated in cells or transmit stress signals to other cells, and initiates the cell apoptosis program, causing cells to gradually apoptosis and produce and release a large number of exosomes;

First hypoxia and then hyperoxia, so that cells enter a protective metabolic state in a hypoxic environment, reduce unnecessary energy consumption, and promote the secretion of exosomes to cope with the hypoxic environment. The subsequent high oxygen environment further stimulates the cells, produces a stress response, and leads to the release of a large number of exosomes. A large number of exosomes will be released after the apoptosis program is initiated, but due to the hypoxic culture environment, the rate of cell apoptosis after stress slows down, so that the apoptotic bodies are quickly phagocytosed by other cells after they are produced, the number of dead cells and cell organs in the culture environment is reduced, the purity of exosomes is increased, and cells are better adapted to the alternation of hypoxia and oxygen-rich environments, improving the cell's stress resistance and survival ability. It is also the reason why exosomes are still stable in hypoxic or hyperoxic environments when used to load drugs or in vivo.

The concentration and purity of exosomes were detected by the method of Example 1, and the results are shown in Table 3:

Table 3

Embodiment 3

In Example 2, the purity of exosomes is increased by first cultivating the culture environment in low oxygen and then in high oxygen. In order to increase the secretion amount of exosomes in cells, further improvements are made on the basis of Example 2.

As the oxygen concentration changes, CO ₂ changes synchronously, that is:

In normoxic culture, the CO ₂ concentration is 5%, in hypoxic culture, the CO ₂ concentration is 1-4%, and in hyperoxic culture, the CO ₂ concentration is 10-20%;

The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

The secretion of exosomes is affected by many factors in the intracellular and extracellular environment, including oxygen and carbon dioxide concentrations. Changes in oxygen concentration can affect the metabolic state of cells, and thus affect the production and secretion of exosomes. A low oxygen environment promotes the secretion of exosomes while increasing the antioxidant capacity of cells and reducing stress, while a high oxygen environment increases cell stress, causing them to enter the apoptosis process and increase the output of exosomes.

Carbon dioxide is an important regulator of cell metabolism. Changes in its concentration also affect the secretion of exosomes. Under low _CO2 concentrations, cells are more able to maintain stable cell growth. Under high _CO2 concentrations, carbon dioxide inhibits the activity of enzymes related to aerobic respiration, reduces the utilization of oxygen by cells, causes oxygen accumulation, increases the speed and degree of oxidative stress, and increases the number of apoptotic cells.

Therefore, when the concentrations of oxygen and carbon dioxide change synchronously, these two factors work together on cells to increase the number of exosomes secreted. By optimizing the concentration combination of oxygen and carbon dioxide, the secretion amount and composition of exosomes can be regulated to meet specific research or application needs. In the field of biomanufacturing, this regulation method is used to produce exosome products with specific functions.

Using the oxygen introduction strategy of Example 3 in Example 2, the concentration and purity of exosomes were detected by the method of Example 1. The results are shown in Table 4:

Table 4

Embodiment 4

Example 3 The secretion of exosomes is increased by changing the combination of carbon dioxide and oxygen. In order to optimize the culture method and reduce the uneven gas concentration in the culture system caused by the increased cell adhesion ability due to high concentration of carbon dioxide, further improvements are made on the basis of Example 3.

The inner surface of the culture container used in the present application is also coated with a cell adhesion inhibitor;

The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

By coating cell adhesion inhibitors, the stacking of cells on the wall of the culture container is reduced, thereby changing the growth environment and state of the cells, that is, the number of adherent cells decreases and the number of suspended cells increases. The reduction of adherent cells means the reduction of stacked cells, and the cells use oxygen more evenly, avoiding the situation in which the gas utilization of locally stacked cells is lower than that of other parts during the culture process; the increase of suspended cells increases the overall growth rate of the cells, thereby increasing the total number of cells and the secretion capacity, and further increasing the production of exosomes.

The principle of action of cell adhesion inhibitors is mainly to inhibit the adhesion of cells to vessels and change the growth environment and state of cells, which may affect cell signal transduction, gene expression, etc., thereby regulating the secretion of exosomes; in addition, combining cell adhesion inhibitors with changes in oxygen and carbon dioxide concentrations can also optimize cell culture conditions and improve cell growth efficiency and product quality.

Using the oxygen introduction strategy of Example 3 in Example 3, the concentration and purity of exosomes were detected by the method of Example 1, and the dihydrochloride cell adhesion inhibitor (K-7174 dihydrochloride) was used for coating. The result showed that after concentration 25 times, the exosome protein concentration was 29.4±1.8 mg/ml, and the concentration was 90.5%.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. 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 (10)

1. A method for culturing exosomes of umbilical cord mesenchymal stem cells, comprising the following steps: S1. Resuscitated frozen P3 umbilical cord mesenchymal stem cells were cultured in DMEM medium at 5% CO ₂ and 37°C for 48 hours under normoxic conditions, with an initial concentration of 1×10 ⁵ cells/cm ² , and IL-1β and TNF-α were added; the concentration of IL-1β was 10 ng/mL, and the concentration of TNF-α was 10 ng/mL; the DMEM medium was kept warm at 45°C for 24 hours; S2, hyperoxia culture for 12-16h after normoxic culture; S3, hypoxic culture for 48-72h after hyperoxic culture; S4. After the culture is completed, the cell culture supernatant is harvested and purified by ultracentrifugation to obtain human umbilical cord mesenchymal stem cell exosomes. 2. A method for culturing umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that the normoxic condition is a 20% O ₂ concentration environment. 3. A method for culturing umbilical cord mesenchymal stem cell exosomes as described in claim 1, characterized in that the ultracentrifugation method is specifically: the cell culture supernatant is centrifuged at 300g for 10 minutes to remove cells and debris, centrifuged at 2000g for 20 minutes to remove cell debris and apoptotic bodies, centrifuged at 10000g for 30 minutes to remove large vesicles, and finally centrifuged at 100000g for 70 minutes to collect the exosome precipitate and resuspend it in PBS to make 10 ml of exosome solution. 4. A method for culturing umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that the oxygen concentration of the hyperoxia culture is an _O2 concentration environment of 40%-60%. 5. A method for culturing umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that the oxygen concentration of the hypoxic culture is an _O2 concentration environment of 5%-10%. 6. A method for culturing exosomes of umbilical cord mesenchymal stem cells according to claim 1, characterized in that the order of the high oxygen culture step and the low oxygen culture step is reversed, that is: S2, hypoxic culture for 48-72h after normoxic culture; S3. Perform high oxygen culture for 12-16 hours after low oxygen culture. 7. A method for culturing umbilical cord mesenchymal stem cell exosomes as claimed in claim 6, characterized in that when the oxygen concentration changes, _CO2 changes synchronously. 8. A method for culturing exosomes of umbilical cord mesenchymal stem cells as described in claim 7, characterized in that the synchronous change of _CO2 is specifically: during normoxic culture, the _CO2 concentration is 5%, during hypoxic culture, the _CO2 concentration is 1-4%, and during hyperoxic culture, the _CO2 concentration is 10-20%. 9 . The method for culturing exosomes of umbilical cord mesenchymal stem cells according to claim 8 , wherein the inner surface of the culture vessel used for cell culture is also coated with a cell adhesion inhibitor. 10. A use of the cultured umbilical cord mesenchymal stem cell exosomes according to any one of claims 1 to 9, characterized in that it is used for knee arthritis.