Preparation method of cell culture fluid containing nanobubbles and application of cell culture fluid in delaying cell senescence

Abstract

The invention belongs to the technical field of biology, and discloses a preparation method and application of a cell culture solution containing nano bubbles. The preparation method comprises the following steps: transferring a cell culture solution into a suitable container; the container is placed in a high-pressure cavity, and air in the high-pressure cavity is completely exhausted by introducing gas; continuously introducing gas to maintain the pressure state in the high-pressure cavity; the pressure in the high-pressure cavity is reduced by slowly releasing the gas, so that the cell culture fluid containing the nanobubbles is obtained. The invention further provides the cell culture fluid containing the nano bubbles prepared by the method and application of the cell culture fluid in delaying cell senescence. The method disclosed by the invention is simple and easy to implement, and the prepared cell culture fluid containing the nanobubbles can be used for remarkably reducing the aging positive cell rate and protecting the cell nucleus and cytoskeleton morphology of aging cells, and has a good anti-aging effect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for preparing a cell culture fluid containing nanobubbles and an application thereof in delaying cell aging. Background Art

[0002] The classic senescent cell theory states that human aging is the result of cells continuously reaching a state of senescence. Cellular senescence is a state of irreversible, permanent proliferation arrest in cells in response to endogenous and exogenous stressors. Reactive oxygen species (ROS) are a source of oxidative stress that contributes to aging, and regulating ROS levels can counteract cellular aging. Furthermore, antioxidants can reduce the rate of telomere shortening during aging. Therefore, various substances, including antioxidants, can be used to regulate cellular ROS levels to exert anti-aging effects.

[0003] Currently, antioxidants used to combat cellular aging primarily include endogenous antioxidants and exogenous antioxidants derived from chemical synthesis or natural sources, including coenzyme Q, glutathione, vitamin C, melatonin, resveratrol, metformin, etc. Delaying cellular aging can be achieved by adding appropriate amounts of inducers that slow cellular aging. However, due to the complex system of interactions within cells, the addition of exogenous substances can cause unknown and irreversible stress damage to cells.

[0004] The biocompatibility of nanobubbles facilitates drug delivery, improving the efficiency of disease treatment and finding widespread use in the medical field. However, there are no reports in the prior art on methods for preparing cell culture fluids containing nanobubbles or on their application in delaying cell aging. Summary of the Invention

[0005] In view of this, the present invention aims to provide a method for preparing a nanobubble-containing cell culture solution and its application in delaying cell aging. The nanobubble-containing cell culture solution provided by the present invention can reduce the positive rate of senescent cells and inhibit the enlargement of the cell nucleus and morphological changes of the cytoskeleton caused by aging.

[0006] In one aspect, the present invention provides a method for preparing a nanobubble-containing cell culture medium, the method comprising the following steps:

[0007] Step 1: Transfer the cell culture medium to a suitable container;

[0008] Step 2: Place the container in step 1 in a high-pressure chamber and introduce gas to exhaust the air in the high-pressure chamber;

[0009] Step 3: Continue to introduce gas to maintain the pressure state in the high-pressure chamber;

[0010] Step 4: The pressure in the high-pressure chamber is reduced by slowly releasing the gas, thereby obtaining a cell culture medium containing nanobubbles.

[0011] In a second aspect, the present invention provides a nanobubble-containing cell culture fluid prepared by the above method.

[0012] In a third aspect, the present invention provides use of a nanobubble-containing cell culture fluid in delaying cell aging.

[0013] As can be seen from the above technical solution, the method of the present invention is simple and easy to implement and operate. Moreover, experiments have shown that the nanobubble-containing cell culture medium prepared by the method of the present invention can reduce the rate of senescence-positive cells, inhibit the enlargement of the nuclei of senescent cells, and protect the cytoskeletal morphology of senescent cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the aging-related β-galactosidase staining of Example 10 of the present invention;

[0015] Among them: A and B are the senescence-related β-galactosidase staining images and the statistical results of senescence-positive cells;

[0016] Figure 2 is the cell nuclear staining of Example 10 of the present invention;

[0017] Among them: A, B, and C are the nuclear immunofluorescence staining images, the box plot and distribution histogram of the results of nuclear area statistics;

[0018] Figure 3 This is the cytoskeleton staining of Example 10 of the present invention. DETAILED DESCRIPTION

[0019] In order to clearly and completely describe the technical scheme of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the embodiments described below only serve for illustrative purposes and are not intended to limit the scope of protection of the present invention. Without departing from the purpose and spirit of the present invention, those skilled in the art can make various modifications and replacements to the present invention, and all of these modifications and replacements fall within the scope of the claims of the present invention.

[0020] A special gas state exists at the interface between gas and liquid. Nanobubbles are tiny bubbles with diameters between fifty micrometers (μm) and tens of nanometers (nm) generated when bubbles occur. At the cellular level, nanobubbles have higher drug loading efficiency and adhesion due to their particle size advantage, and can maintain longer contact with the cell's biomembrane interface. Drug-encapsulated nanobubbles often achieve drug delivery by manipulating the cellular molecular composition and extracellular environment to achieve therapeutic purposes. Even when the nanobubbles are not in contact with cells, the shear stress generated by the rupture of the nanobubbles is sufficient to penetrate the cells, thereby increasing drug absorption, without causing permanent damage to the cells. The inventors' latest research shows that nanobubbles exhibit sustainable antioxidant activity in the absence of exogenous additions. These advantages make nanobubbles theoretically suitable for delaying cell aging. Based on this, the present invention provides a method for preparing a cell culture medium containing nanobubbles, and the cell culture medium obtained thereby is used to delay cell aging.

[0021] In one aspect, the present invention provides a method for preparing a nanobubble-containing cell culture medium, the method comprising the following steps:

[0022] Step 1: Transfer the cell culture medium into a container;

[0023] Step 2: Place the container in step 1 in a high-pressure chamber and introduce gas to exhaust the air in the high-pressure chamber;

[0024] Step 3: Continue to introduce gas to maintain the pressure state in the high-pressure chamber in step 2;

[0025] Step 4: The pressure in the high-pressure chamber is reduced by slowly releasing the gas, thereby obtaining a cell culture medium containing nanobubbles.

[0026] In the embodiment of the present invention, in step 1, the cell culture medium can be a basic culture medium or a basic culture medium diluted with ultrapure water, or a basic culture medium supplemented with inorganic salts, that is, a complete culture medium.

[0027] Complete culture media are well-known in the art. They refer to various nutrient media created by adding natural substances such as amino acids, vitamins, and bases, or growth factors, to a basic culture medium, depending on the cell type or research needs. Complete culture media can be divided into cell growth medium and cell maintenance medium, depending on the amount of serum added, to accommodate different cell types and research needs.

[0028] In an embodiment of the present invention, the inorganic salt may be a salt of various metals and inorganic acids, such as lithium salts, magnesium salts, calcium salts, preferably lithium salts, such as LiCl.

[0029] In an embodiment of the present invention, in step 1, the container can be a glass reagent bottle, a centrifuge tube, a cell culture dish or a culture flask. Preferably, the container is a T25 cell culture flask.

[0030] In an embodiment of the present invention, in step 2, the gas introduced is oxygen, an inert gas, hydrogen or nitrogen, or a combination thereof, preferably hydrogen.

[0031] In an embodiment of the present invention, in step 3, the pressure in the high-pressure chamber is maintained at 0.01-10 MPa, for example, 0.01 MPa, 0.02 MPa, 0.05 MPa, 0.1 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa, 0.9 MPa, 1.0 MPa, 1.5 MPa, 2.0 MPa, 2.5 MPa, 3.0 MPa, 3.5 MPa, 4.0 MPa, 4.5 MPa, 5.0 MPa, 5.5 MPa, 6.0 MPa, 6.5 MPa, 7.0 MPa, 7.5 MPa, 8.0 MPa, 8.5 MPa, 9.0 MPa, 9.5 MPa, 10 MPa, preferably 1 MPa, and the duration of maintaining the pressure state may be 10-200 min, for example, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, 100 min, 110 min, 120 min, 130 min, 140 min, 150 min, 160 min, 170 min, 180 min, 185 min, 190 min, 195 min, 196 min, 197 min, 198 min, 199 min, 200 min, and values ​​between any two values, for example, 22 min, 23 min, 26 min, etc., preferably 30 min.

[0032] In an embodiment of the present invention, in step 4, the pressure in the high-pressure chamber is reduced to 0-0.5 MPa, for example, 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa, and any value between two values, for example, 0.15 MPa, 0.16 MPa, 0.22 MPa, 0.35 MPa, etc., by slowly releasing the gas in the high-pressure chamber. The duration of pressure release can be 0.5-5 h, for example, 1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 4.6 h, 4.7 h, 4.8 h, 4.9 h, 5 h, preferably 2.5 h.

[0033] In a second aspect, the present invention provides a nanobubble-containing cell culture medium prepared by the above method, wherein the diameter of the nanobubbles contained therein is 30 nm-1000 nm.

[0034] In a third aspect, the present invention provides a use of the above-mentioned nanobubble-containing cell culture fluid in delaying cell aging.

[0035] In an embodiment of the present invention, cellular senescence includes cell senescence induced by replication, drug, radiation, and high glucose, or natural cellular senescence in vivo. In a specific embodiment, cellular senescence is mesenchymal stem cell senescence induced by serial passage or mesenchymal stem cell senescence induced by hydrogen peroxide.

[0036] This invention is the first to apply nanobubble-containing cell culture fluid to delay cell aging. Senescence-related β-galactosidase staining was used to characterize the anti-aging effect, and the results showed that the nanobubble-containing cell culture fluid can reduce the rate of senescence-positive cells. Furthermore, the present invention used nanobubble-containing cell culture fluid to delay cell aging and performed aging-related feature detection. The results showed that the nanobubble-containing cell culture fluid can inhibit the enlargement of the nucleus of senescent cells and protect the cytoskeletal morphology of senescent cells. Therefore, the nanobubble-containing cell culture fluid prepared by the present invention has application prospects in delaying cell aging, providing more clinical application value and research ideas for the development of anti-aging strategies.

[0037] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and all such modifications and substitutions fall within the scope of the claims of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0039] Example

[0040] In the examples, complete medium without any additives and complete medium supplemented with LiCl were used to prepare nanobubble-containing cell culture solutions, and mesenchymal stem cells induced by replication and hydrogen peroxide were used as senescent cell models. However, the nanobubbles described herein are not limited to these nanobubble-containing cell culture solutions, nor are their applications limited to these senescent cell models.

[0041] Establishment of a replicatively induced senescent mesenchymal stem cell model

[0042] Human umbilical cord-derived mesenchymal stem cells (purchased from Shanghai Biotechnology Co., Ltd.) were cultured in complete culture medium (see Microenvironment Influences on Human Umbilical Cord Mesenchymal Stem Cell-Based Bone Regeneration: Stem Cells Int. 2021 Aug 17:2021:4465022. doi:10.1155 / 2021 / 4465022). During continuous culture, cells were passaged when they reached approximately 90% confluency. By the eighth passage, a replicatively aged mesenchymal stem cell model was established.

[0043] Establishment of a hydrogen peroxide-induced senescent mesenchymal stem cell model

[0044] Human umbilical cord-derived mesenchymal stem cells were cultured in complete culture medium (as above) until passage 6. A 30% hydrogen peroxide solution was diluted to 200 μM in complete culture medium and incubated for another 2 hours. Fresh cell culture medium was then replaced and incubated for an additional 72 hours to establish chemically induced premature senescence of mesenchymal stem cells.

[0045] Example 1 Preparation of Nitrogen-Containing Nanobubble Cell Culture Fluid

[0046] (1) A certain volume (the volume is determined according to needs, and the number of nanobubbles finally formed is determined by measurement, the same below) of DMEM low-glucose complete medium (see Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro. Stem Cell Res Ther. 2018 May 11; 9(1): 131. doi: 10.1186 / s13287-018-0876-3) is transferred into a T25 cell culture flask;

[0047] (2) Place the T25 cell culture flask in a high-pressure chamber and introduce nitrogen to expel the air. Continue introducing nitrogen to 0.1-1 MPa and maintain the high-pressure state for 30 minutes.

[0048] (3) Slowly release the pressure to 0 MPa for about 2.5 hours;

[0049] (4) Rinse the glass syringe with ultrapure water three times and draw 2 mL of nanobubble culture solution. Slowly inject 0.5 mL of nanobubble solution into the Nanosight sample cell.

[0050] (5) Use NanosightNTA3.4 software to adjust CameraLevel and ScreenGain, adjust the focus of the Naosight instrument to select appropriate measurement conditions, and collect 5 times for each sample;

[0051] (6) Use Nanosight NTA 3.4software to analyze the particle size and concentration of the collected data.

[0052] It was observed that the particle size distribution range of nanobubbles was mainly concentrated in 100-200nm within 48h.

[0053] Example 2 Preparation of oxygen-containing nanobubble cell culture medium

[0054] (1) Transfer a certain volume of DMEM low-glucose complete medium (same as above) into a T25 cell culture flask;

[0055] (2) Place the T25 cell culture flask in a high-pressure chamber and introduce oxygen to expel the air from the chamber. Continue introducing oxygen to 0.1-1 MPa and maintain the high-pressure state for 30 minutes.

[0056] (3) Slowly release the pressure to 0 MPa for about 2 hours;

[0057] (4) Rinse the glass syringe with ultrapure water three times and draw 2 mL of nanobubble culture solution. Slowly inject 0.5 mL of nanobubble solution into the Nanosight sample cell.

[0058] (5) Use NanosightNTA3.4 software to adjust CameraLevel and ScreenGain, adjust the focus of the Naosight instrument to select appropriate measurement conditions, and collect 5 times for each sample;

[0059] (6) Use Nanosight NTA 3.4software to analyze the particle size and concentration of the collected data.

[0060] It was observed that the particle size distribution range of nanobubbles was mainly concentrated in 80-300nm within 48h.

[0061] Example 3 Preparation of Hydrogen Nanobubble Cell Culture Fluid

[0062] (1) Transfer a certain volume of DMEM low-glucose complete medium (same as above) into a T25 cell culture flask;

[0063] (2) Place the T25 cell culture flask in a high-pressure chamber and introduce hydrogen to expel the air. Continue introducing hydrogen to 0.1-1 MPa and maintain the high pressure for 30 minutes.

[0064] (3) Slowly release the pressure to 0 MPa for about 2 hours;

[0065] (4) Rinse the glass syringe with ultrapure water three times and draw 2 mL of nanobubble culture solution; slowly inject 0.5 mL of nanobubble solution into the Nanosight sample cell;

[0066] (5) Use NanosightNTA3.4 software to adjust CameraLevel and ScreenGain, adjust the focus of the Naosight instrument to select appropriate measurement conditions, and collect 5 times for each sample;

[0067] (6) Use Nanosight NTA 3.4software to analyze the particle size and concentration of the collected data.

[0068] It was observed that the particle size distribution range of nanobubbles was mainly concentrated in 80-300nm within 48h.

[0069] Example 4 Preparation of Magnesium Ion-Containing Hydrogen Nanobubble Cell Culture Fluid

[0070] (1) A certain volume (the volume is determined according to needs, and the number of nanobubbles finally formed is determined by measurement, the same below) of DMEM low-glucose complete medium (same as above) is transferred to a T25 cell culture flask, and magnesium chloride (900 mM) solution is added, with the cell culture medium volume: magnesium chloride solution volume = 449:1;

[0071] (2) Place the T25 cell culture flask in a high-pressure chamber and introduce hydrogen to expel the air. Continue introducing hydrogen to 1 MPa and maintain the high pressure for 40 minutes.

[0072] (3) Slowly release the pressure to 0 MPa for about 2 hours;

[0073] (4) Rinse the glass syringe with ultrapure water three times and draw 2 mL of nanobubble culture solution. Slowly inject 0.5 mL of nanobubble solution into the Nanosight sample cell.

[0074] (5) Use NanosightNTA3.4 software to adjust CameraLevel and ScreenGain, adjust the focus of the Naosight instrument to select appropriate measurement conditions, and collect 5 times for each sample;

[0075] (5) Use Nanosight NTA 3.4software to analyze the particle size and concentration of the collected data.

[0076] It was observed that the particle size of the nanobubbles was distributed in the range of 100-200 nm in the first 12 hours, and after 24 hours the particle size distribution of the nanobubbles was mainly concentrated in the range of 50-200 nm.

[0077] Example 5 Preparation of Cell Culture Fluid Containing Lithium Ion Hydrogen Nanobubbles

[0078] (1) A certain volume (the volume is determined according to needs, and the number of nanobubbles finally formed is determined by measurement, the same below) of DMEM low-glucose complete medium (same as above) is transferred to a T25 cell culture flask, and lithium chloride (900 mM) solution is added, with the cell culture medium volume: lithium chloride solution volume = 449:1;

[0079] (2) Place the T25 cell culture flask in a high-pressure chamber and introduce hydrogen to expel the air. Continue introducing hydrogen to 0.1-1 MPa and maintain the high pressure for 30 minutes.

[0080] (3) Slowly release the pressure to 0.1 MPa for about 2.5 hours;

[0081] (4) Rinse the glass syringe with ultrapure water three times and draw 2 mL of nanobubble culture solution. Slowly inject 0.5 mL of nanobubble solution into the Nanosight sample cell.

[0082] (5) Use NanosightNTA3.4 software to adjust CameraLevel and ScreenGain, adjust the focus of the Naosight instrument to select appropriate measurement conditions, and collect 5 times for each sample;

[0083] (6) Use Nanosight NTA 3.4software to analyze the particle size and concentration of the collected data.

[0084] It was observed that the particle size of the nanobubbles was distributed in the range of 100-500 nm in the first 12 hours, and after 24 hours the particle size distribution of the nanobubbles was mainly concentrated in the range of 50-150 nm.

[0085] Example 6 Preparation of Lithium Ion-Containing Nitrogen Nanobubble Cell Culture Fluid

[0086] (1) A certain volume (the volume is determined according to needs, and the number of nanobubbles finally formed is determined by measurement, the same below) of DMEM low-glucose complete medium (same as above) is transferred to a T25 cell culture flask, and lithium chloride (900 mM) solution is added, with the cell culture medium volume: lithium chloride solution volume = 449:1;

[0087] (2) Place the T25 cell culture flask in a high-pressure chamber and introduce nitrogen to expel the air. Continue introducing nitrogen to 0.1-1 MPa and maintain the high-pressure state for 30 minutes.

[0088] (3) Slowly release the pressure to 0.1 MPa for about 2.5 hours;

[0089] (4) Rinse the glass syringe with ultrapure water three times and draw 2 mL of nanobubble culture solution. Slowly inject 0.5 mL of nanobubble solution into the Nanosight sample cell.

[0090] (5) Use NanosightNTA3.4 software to adjust CameraLevel and ScreenGain, adjust the focus of the Nanosight instrument to select appropriate measurement conditions, and collect 5 times for each sample;

[0091] (6) Use Nanosight NTA 3.4software to analyze the particle size and concentration of the collected data.

[0092] It was observed that the particle size of the nanobubbles was distributed in the range of 100-500 nm in the first 12 hours, and after 24 hours the particle size distribution of the nanobubbles was mainly concentrated in the range of 50-150 nm.

[0093] Example 7: Nitrogen-containing nanobubble cell culture medium delays aging of mesenchymal stem cells

[0094] The nitrogen-containing nanobubble culture medium prepared in Example 1 was used to culture hydrogen peroxide-induced senescence mesenchymal stem cells, and the proportion of positive cells was examined by β-galactosidase staining. The operation steps were as follows:

[0095] (1) When human umbilical cord mesenchymal stem cells were cultured to the sixth generation, 5×10 4 / cm 2 The cells were seeded at a high density in a cell culture dish and placed in a 37°C, 5% CO2 incubator for continued culture;

[0096] (2) After 24 h, the complete culture medium was aspirated and the cells were washed three times with 2 mL of 1× PBS. 30% H2O2 solution was diluted to 200 μM, and the same volume of H2O2 solution was added. The cells were then placed in a 37°C, 5% CO2 incubator for further culture.

[0097] (3) After 2 h, the complete culture medium was aspirated, and the nitrogen-containing nanobubble culture medium prepared in Example 1 was added, and the culture was continued in a 37°C, 5% CO2 incubator for 72 h;

[0098] (4) The proportion of positive cells was examined by β-galactosidase staining, and the results showed that hydrogen nanobubble culture medium could reduce the proportion of positive cells by more than 10%.

[0099] Example 8: Hydrogen-containing nanobubble cell culture medium delays aging of mesenchymal stem cells

[0100] The nitrogen-containing nanobubble culture medium prepared in Example 3 was used to culture hydrogen peroxide-induced senescence mesenchymal stem cells, and the proportion of positive cells was examined by β-galactosidase staining. The operation steps were as follows:

[0101] (1) When human umbilical cord mesenchymal stem cells were cultured to the sixth generation, 5×10 4 / cm 2 The cells were seeded at a high density in a cell culture dish and placed in a 37°C, 5% CO2 incubator for continued culture;

[0102] (2) After 24 h, the complete culture medium was aspirated and the cells were washed three times with 2 mL of 1× PBS. 30% H2O2 solution was diluted to 200 μM, and the same volume of H2O2 solution was added. The cells were then placed in a 37°C, 5% CO2 incubator for further culture.

[0103] (3) After 2 h, the complete culture medium was aspirated, and the nitrogen-containing nanobubble culture medium prepared in Example 1 was added, and the culture was continued in a 37°C, 5% CO2 incubator for 72 h;

[0104] (4) The proportion of positive cells was examined by β-galactosidase staining, and the results showed that hydrogen nanobubble culture medium could reduce the proportion of positive cells by more than 10%.

[0105] Example 9: Lithium-ion-containing nitrogen nanobubble cell culture medium delays aging of mesenchymal stem cells

[0106] The lithium-ion nitrogen nanobubble culture medium prepared in Example 5 was used to culture hydrogen peroxide-induced senescence mesenchymal stem cells, and the proportion of positive cells was examined by β-galactosidase staining. The β-galactosidase staining method can be found in Progerin modulates the IGF-1R / Akt signaling involved in aging. Science advances vol. 8, 27(2022):eabo0322.doi:10.1126 / sciadv.abo0322. The operation steps are as follows:

[0107] (1) When human umbilical cord mesenchymal stem cells were cultured to the sixth generation, 5×10 4 / cm 2 The cells were seeded at a high density in a cell culture dish and placed in a 37°C, 5% CO2 incubator for continued culture;

[0108] (2) After 24 h, the complete culture medium was aspirated and the cells were washed three times with 2 mL of 1× PBS. 30% H2O2 solution was diluted to 200 μM, and the same volume of H2O2 solution was added. The cells were then placed in a 37°C, 5% CO2 incubator for further culture.

[0109] (3) After 2 h, the complete culture medium was aspirated, and the lithium ion hydrogen nanobubble culture medium prepared in Example 5 was added, and the culture was continued in a 37°C, 5% CO2 incubator for 72 h;

[0110] (4) The proportion of positive cells was examined by β-galactosidase staining, and the results showed that the culture medium containing lithium ion hydrogen nanobubbles could reduce the proportion of positive cells by more than 15%.

[0111] Example 10 Effect of Nanobubble-Containing Cell Culture Medium on Aging Mesenchymal Stem Cells

[0112] 1) Senescence-associated β-galactosidase staining

[0113] The senescence-associated β-galactosidase staining experiment was used to evaluate the effects of the nanobubble-containing cell culture solutions in Examples 1-6 on the senescent mesenchymal stem cells in Examples 7-9. After incubation with the nanobubble-containing cell culture solutions prepared in Examples 1-6, senescence-associated β-galactosidase staining was performed, and the cells were imaged using an ordinary optical microscope and counted to calculate the positive rate of senescent cells.

[0114] The experimental groups are as follows:

[0115] Control group (CK): without nanobubbles;

[0116] Positive control group (LiCl): cell culture medium containing lithium ions;

[0117] Treatment group I (N2 NBs): containing the nitrogen-containing nanobubble cell culture medium prepared in Example 1;

[0118] Treatment group II (H2 NBs): containing the cell culture medium containing hydrogen nanobubbles prepared in Example 3;

[0119] Treatment group III (LiCl+N2 NBs): contains the lithium ion-containing nitrogen nanobubble cell culture medium prepared in Example 6.

[0120] The specific steps include:

[0121] (1) Human umbilical cord-derived mesenchymal stem cells were treated with H2O2 for 2 hours to induce cell senescence;

[0122] (2) The cell culture medium containing H2O2 was removed, and the control group was replaced with fresh complete culture medium, and each treatment group was replaced with the corresponding complete culture medium containing nanobubbles and cultured for another 72 hours;

[0123] (3) For the senescent mesenchymal stem cells that have been cultured, the original cell culture medium was removed and 2 mL of PBS (1×) was added and washed three times;

[0124] (4) Add 1 mL of β-glucosidase staining fixative (Biyuntian, C0602) and fix at room temperature for 15 min; remove the fixative and add 2 mL of PBS to wash three times, each time for 3 min;

[0125] (5) Prepare the staining working solution by taking 10 μL of β-galactosidase staining solution A (Biyuntian, C0602), 10 μL of β-galactosidase staining solution B (Biyuntian, C0602), 930 μL of β-galactosidase staining solution C (Biyuntian, C0602), and 50 μL of X-gal solution (Biyuntian, C0602);

[0126] (6) Aspirate and discard PBS, add 1 mL of staining solution, and incubate in a 37°C incubator for 20 h.

[0127] (7) Aspirate the staining solution and wash twice with 1 mL of PBS.

[0128] (8) Observe under an optical microscope and count the positive rate of senescent cells.

[0129] The results are as follows Figure 1 As shown, compared with the CK group, the senescence positive rate in the N2 NBs group decreased by about 3%, the senescence positive rate in the H2 NBs group decreased by about 5%, and the senescence positive rate in the LiCl+N2NBs group decreased by about 6%.

[0130] 2) Nuclear staining

[0131] To evaluate the effect of nanobubble-containing cell culture medium on the nuclei of senescent cells,

[0132] The experimental groups are as follows:

[0133] Control group (CK): cell culture medium without nanobubbles;

[0134] Positive control group (LiCl): cell culture medium containing lithium ions;

[0135] Treatment group I (N2 NBs): containing the nitrogen-containing nanobubble cell culture medium prepared in Example 1;

[0136] Treatment group II (H2 NBs): containing the cell culture medium containing hydrogen nanobubbles prepared in Example 3;

[0137] Treatment group III (LiCl+N2 NBs): contains the lithium ion hydrogen nanobubble cell culture medium prepared in Example 5.

[0138] The cell nuclei were stained by immunofluorescence (see SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation. Acta pharmaceutica Sinica. B, 11(5), 1261–1273. https: / / doi.org / 10.1016 / j.apsb.2020.12.010), and the cell nuclear area was statistically analyzed after fluorescence microscopy imaging.

[0139] The specific steps are as follows:

[0140] (1) Human umbilical cord-derived mesenchymal stem cells were treated with H2O2 for 2 hours to induce cell senescence;

[0141] (2) The cell culture medium containing H2O2 was removed, and the control group was replaced with fresh complete culture medium, and each treatment group was replaced with the corresponding complete culture medium containing nanobubbles and cultured for another 72 hours;

[0142] (3) For the senescent mesenchymal stem cells that have completed culture, the original culture medium was aspirated and 1 mL of PBS (1×) was added for washing three times. 1 mL of 4% PFA (Solerbo, P1110) was added for fixation at room temperature for 10 min.

[0143] (4) Aspirate and discard 4% PFA, add 1 mL PBS (1×) and wash three times for 5 min each time. Add 300 μL DAPI (3 μM) (Thermo Fisher Scientific, 2445405) and incubate at room temperature for 5 min.

[0144] (5) Aspirate and discard DAPI, add 1 mL PBS (1×) and wash three times. Add 2 mL PBS (1×) and examine under a fluorescence microscope. Use imageJ to calculate the nuclear area of ​​cells in 5 different regions of each group of cells, draw box plots and distribution histograms, and calculate the nuclear area.

[0145] (4) Fluorescence staining results ( Figure 2 ) showed that culture medium containing nanobubbles could reduce the nuclear volume of senescent cells.

[0146] The results are as follows Figure 2As shown in the figure, compared with the CK group, the number of cavitated nuclei in the LiCl group, N2 NBs group, H2 NBs group and LiCl+N2 NBs group was significantly reduced; the nuclear area distribution in the LiCl group, N2NBs group, H2 NBs group and LiCl+N2 NBs group was slightly lower than that in the control group, among which the nuclear area distribution in the H2 NBs group was the most concentrated.

[0147] 3) Cytoskeleton staining

[0148] In order to evaluate the protective effect of nanobubble-containing cell culture medium on the cytoskeleton morphology of senescent cells, the cytoskeleton was immunofluorescently stained (see Xiaodong Mu, et al. Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson-Gilford Progeria Syndrome Aging Cell. 2020 Aug; 19(8): e13152) and imaged using a laser confocal microscope.

[0149] The experimental groups are as follows:

[0150] Control group (CK): cell culture medium without nanobubbles;

[0151] Positive control group (LiCl): cell culture medium containing lithium ions;

[0152] Treatment group I (N2 NBs): containing the nitrogen-containing nanobubble cell culture medium prepared in Example 1;

[0153] Treatment group II (H2 NBs): containing the cell culture medium containing hydrogen nanobubbles prepared in Example 3;

[0154] Treatment group III (LiCl+N2 NBs): contains the lithium ion-containing nitrogen nanobubble cell culture medium prepared in Example 6.

[0155] The specific steps are:

[0156] (1) Human umbilical cord-derived mesenchymal stem cells were treated with H2O2 for 2 hours to induce cell senescence;

[0157] (2) The cell culture medium containing H2O2 was removed, and the control group was replaced with fresh complete culture medium, and each treatment group was replaced with the corresponding complete culture medium containing nanobubbles. The complete culture medium containing bubbles was replaced and cultured for 72 hours;

[0158] (3) For the senescent mesenchymal stem cells that have completed culture, the original culture medium was aspirated and washed three times with 1 mL of PBS (1×). 1 mL of 4% PFA (Solerbo, P1110) was added and fixed at room temperature for 10 min.

[0159] (2) Aspirate and discard 4% PFA, add 1 mL PBS (1×) and wash three times, 5 min each time. Add 1 mL 0.1% Triton X-100 (Coolaber, CT11451) and treat for 5 min.

[0160] (3) Aspirate 0.1% Triton X-100 and wash three times with 1 mL of 1× PBS for 5 min each. Add 300 μL of phalloidin (1:400) (Thermo Fisher Scientific, 2486570) and incubate at room temperature for 20 min.

[0161] (4) Aspirate phalloidin and wash three times with 1 mL of 1× PBS for 5 min each. Add 300 μL of LDAPI (3 μM) (Thermo Fisher Scientific, 2445405) and incubate at room temperature for 5 min.

[0162] (5) Aspirate and discard DAPI, add 1 mL of PBS (1×) and wash three times. Add 2 mL of PBS (1×) and examine under a laser confocal microscope.

[0163] The results are as follows Figure 3 As shown in the figure, compared with the CK group, the LiCl group showed a certain degree of increase in cytoskeletal actin filaments, and the cells were arranged more neatly. In the N2 NBs group, H2 NBs group, and LiCl+N2NBs group, the cells recovered to an orderly state, with clear cell morphology, neatly arranged actin filaments, and a significant increase in number.

[0164] In summary, the nanobubble-containing cell culture medium prepared in the present invention can protect cells from aging-induced morphological variations of the cell nucleus and cytoskeleton, and has a good anti-aging effect.

[0165] Finally, it should be noted that the preferred embodiments of the present invention described above are only intended to illustrate the specific technical solutions of the present invention and are not intended to limit the present invention. All other implementations obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a nanobubble-containing cell culture medium, characterized in that: The method comprises the following steps: Step 1: Transfer the cell culture medium to a suitable container; Step 2: placing the container in the high-pressure chamber and introducing gas to exhaust the air in the high-pressure chamber; Step 3: Continue to introduce gas to maintain the pressure state in the high-pressure chamber; Step 4: Slowly releasing gas to reduce the pressure in the high-pressure chamber, thereby obtaining the nanobubble-containing cell culture fluid.

2. The preparation method according to claim 1, characterized in that The cell culture medium can be a basic culture medium or a basic culture medium diluted with ultrapure water or a complete culture medium to which one or more inorganic salts are added. Preferably, the inorganic salt is a lithium salt, a magnesium salt or a calcium salt, and more preferably, the lithium salt is, for example, LiCl.

3. The preparation method according to claim 1, characterized in that The container is a glass reagent bottle, a centrifuge tube, a cell culture dish or a culture bottle.

4. The preparation method according to claim 1, characterized in that The gas is oxygen, an inert gas, hydrogen, nitrogen or a combination thereof, preferably hydrogen.

5. The method for preparing the nanobubble-containing cell culture fluid according to claim 1, wherein: In step (3), the pressure in the high-pressure chamber is maintained at 0.01-10 MPa, preferably 1 MPa.

6. The method for preparing the nanobubble-containing cell culture fluid according to claim 5, wherein: The duration of maintaining the pressure in the high-pressure chamber is 10-200 minutes, preferably 30 minutes.

7. The method for preparing a nanobubble-containing cell culture fluid according to claim 1, wherein: In step (4), the pressure in the high-pressure chamber is reduced to 0-0.5 MPa, and the decompression time is continued for 0.5-5 hours, preferably 2.5 hours.

8. A cell culture medium containing nanobubbles, prepared by the method according to any one of claims 1 to 7.

9. Use of the nanobubble-containing cell culture medium according to claim 8 in delaying cell aging.

10. The use according to claim 9, characterized in that The cell senescence includes cell senescence induced by replication, drug, radiation and high glucose or natural cell senescence in vivo. Preferably, the cell senescence is mesenchymal stem cell senescence induced by continuous passage or mesenchymal stem cell senescence induced by hydrogen peroxide.

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