Cell culture medium containing nano bubbles with ultra-small particle size as well as preparation method and application of cell culture medium

Abstract

The invention discloses a cell culture medium containing nano bubbles with ultra-small particle size as well as a preparation method and application of the cell culture medium. The cell culture medium contains the nano bubbles with the ultra-small particle size with the particle size smaller than 50nm, and the total concentration is gt; 1011 cells are taken per milliliter. The preparation method comprises the following steps: cooling a cell culture medium into which nano bubbles with ultra-small particle sizes need to be introduced to about-20 DEG C to-80 DEG C; putting the cooled cell culture medium into a pressurizing device; high-pressure gas is introduced into the pressurizing device or mechanical force is adopted for pressurizing, so that the internal pressure of the pressurizing device is increased; maintaining the high-pressure condition for a period of time; and slowly releasing the pressure to a normal pressure state. The cell culture medium prepared according to the invention not only can be used for delaying in-vitro replicative senescence of mesenchymal stem cells, but also is expected to be applied to the fields of biomedicine, medical cosmetology, health, life prolonging and the like.

Description

Technical Field

[0001] The invention relates to the fields of interface chemistry and cell biology, and in particular to a cell culture medium containing ultra-small particle size nanobubbles and a preparation method and application thereof. Background Art

[0002] Excessive reactive oxygen species (ROS) can cause oxidative damage to essential cellular components and lead to various human diseases, such as cancer as well as cardiovascular, ocular, and neurodegenerative diseases. ROS-related diseases are usually treated with natural or artificial chemical antioxidants. However, various antioxidants have failed to show clear clinical benefits against oxidative stress, especially for chronic diseases caused by oxidative damage. Possible reasons for the failure of chemical antioxidants include: 1) inability to enter tissues / organs or sites of oxidative damage, 2) interference with critical physiological functions of normal levels of ROS, and 3) toxic side effects. These aspects emphasize the urgent need for innovative antioxidant approaches.

[0003] Ultrasmall nanobubbles (UNBs) have unique physicochemical properties that may address these issues and provide a new approach to antioxidant activity. UNBs are nanoscale (less than 50 nanometers) bubbles suspended in water. Our recent studies have demonstrated that oxygen and nitrogen UNBs can protect organic molecules or metal surfaces in aqueous solutions from oxidative damage by chemically generated ROS. The ROS scavenging mechanisms of UNBs differ from those of chemical antioxidants, as categorized in a recent review. This approach has shown distinct advantages over conventional chemical antioxidants, including sustainability, stability, and biosafety. Therefore, if UNBs can exert antioxidant effects at the cellular level, these advantages could be exploited to alleviate oxidative stress in living organisms. As pointed out by Barry Halliwell, the in vitro and in vivo effects of many antioxidants are not always consistent. For example, hydrogen sulfide and melatonin exhibit ROS scavenging abilities in vitro but fail to achieve equivalent antioxidant effects in vivo. At the same time, while compounds such as ascorbic acid can scavenge ROS in vivo, they do not necessarily reduce oxidative damage, as factors such as nonlocal effects and potential toxic byproducts must be considered. Therefore, in vitro antioxidant effects often do not translate into in vivo effects. Mesenchymal stem cells (MSCs) have attracted much attention in the medical field due to their ability to differentiate into a variety of cell types and their potential in regenerative medicine. Their applications range from tissue engineering to the treatment of inflammatory and autoimmune diseases. Given their therapeutic potential, the demand for MSCs continues to increase. To meet this demand, large-scale expansion of MSCs in vitro is essential. However, achieving large-scale expansion while maintaining cell quality and function faces many challenges. Standard methods for increasing MSC expansion in vitro include the use of bioreactors, optimized culture media, and growth factors. Although these methods have achieved some success, they also have limitations. Bioreactors can be expensive and complex, optimized culture media may not always support long-term cell viability, and growth factors can introduce variability and potential safety issues. Addressing these shortcomings is critical to advancing MSC-based therapies. Continued research and innovation are needed to develop more efficient, cost-effective, and reliable large-scale MSC expansion methods. Overcoming these challenges will unlock the full potential of MSCs in medical applications, ultimately improving patient outcomes and advancing regenerative medicine.

[0004] A major challenge to large-scale MSC expansion is replicative senescence, which is the gradual decline in the ability of cells to proliferate and maintain stemness over successive generations. Among the factors that lead to replicative senescence, the accumulation of reactive oxygen species (ROS) is one of the important ones. ROS can cause oxidative stress and damage cellular components, which in turn affects cell function and accelerates aging. To mitigate the effects of ROS and extend the replicative lifespan of MSCs, researchers use exogenous antioxidants to neutralize ROS, thereby reducing oxidative stress and potentially increasing the number of cell divisions that MSCs can undergo in vitro. Multiple studies have reported that the use of antioxidants can successfully enhance the proliferation and lifespan of MSCs. However, chemical antioxidants sometimes interfere with normal cell signaling pathways, and the long-term effects of antioxidants on MSCs and their therapeutic potential remain unclear. Addressing these deficiencies is critical to optimizing in vitro MSC expansion. Summary of the invention

[0005] The purpose of the present invention is to provide a cell culture medium containing ultra-small particle size nanobubbles and a preparation method and application thereof, so as to solve the problem that the chemical antioxidants in the prior art have low efficacy and multiple risks.

[0006] According to a first aspect of the present invention, a cell culture medium containing ultra-small particle size nanobubbles is provided, wherein the cell culture medium contains ultra-small particle size nanobubbles with a particle size of less than 50 nm, and the total concentration of the ultra-small particle size nanobubbles in the cell culture medium is greater than 10 11 Pieces / mL.

[0007] Preferably, the ultra-small particle size nanobubbles show a particle size distribution range of 1 nm to 50 nm, more preferably 2 nm to 15 nm, in dynamic light scattering (DLS) measurement.

[0008] According to the ultra-small particle size nanobubble cell culture medium provided by the present invention, the ultra-small particle size nanobubbles are shown as having a particle size of less than 50 nm in the measurement of the nanoparticle tracking analysis system (NTA), wherein the number of bubbles around 10 nm accounts for the vast majority of the total number in the measurement of the dynamic light scattering technology (DLS). Preferably, the number of bubbles with a particle size distribution range of 1 nm to 15 nm in the measurement of the dynamic light scattering technology (DLS) accounts for more than 99% of the total number.

[0009] Preferably, the concentration of ultra-small-size nanobubbles is higher than the detection limit of dynamic light scattering (DLS), and the DLS number intensity of the nanobubbles accounts for >99%.

[0010] According to a second aspect of the present invention, a method for preparing a cell culture medium containing ultra-small particle size nanobubbles is provided, comprising the following steps: S1: cooling the cell culture medium to be introduced with ultra-small particle size nanobubbles to -20°C to -80°C; S2: placing the cooled cell culture medium into a pressurizing device; S3: introducing high-pressure gas into the pressurizing device or applying mechanical force to increase the internal pressure of the pressurizing device; S4: maintaining the high-pressure condition for a period of time; S5: slowly releasing the pressure to a normal pressure state, thereby preparing a cell culture medium containing ultra-small particle size nanobubbles.

[0011] Preferably, step S1 comprises: cooling the cell culture medium into which the ultra-small nanobubbles are to be introduced to -20°C to -80°C. Most preferably, cooling to -80°C. It should be understood that cooling the cell culture medium to -20°C to -80°C can better introduce the ultra-small nanobubbles.

[0012] The cell culture medium in step S1 can be selected as required, including but not limited to complete culture medium, maintenance culture medium, stem cell-specific culture medium, commercial brand cell culture medium, etc. It should be understood that the method of the present invention is applicable to any cell culture medium, not limited to stem cell culture medium, and does not have any limiting requirements on the formula of the cell culture medium.

[0013] Preferably, step S3 comprises: introducing high-pressure gas into the pressurizing device, and selecting different types of gas according to different needs, including nitrogen, oxygen, hydrogen, air, etc.

[0014] Preferably, steps S3 and S4 include: the pressure range of the high pressure condition is 0.2 MPa to 1.2 MPa, and the maintenance time is 20 min to 60 min.

[0015] More preferably, the pressure in step S4 is in the range of 0.4 MPa to 1.2 MPa, and the maintenance time is 40 min to 60 min.

[0016] Preferably, step S5 comprises: using a gas flow meter to control the gas release rate during the pressure release process.

[0017] Preferably, except step S1, no additional temperature increase or decrease operation is performed in steps S2 to S5, so that the temperature of the cell culture medium is kept to change normally.

[0018] However, it should be understood that the present invention prepares the cell culture medium containing ultra-small particle size nanobubbles by low temperature and pressure reduction method, and for the first time finds that nanobubbles below 50nm have anti-aging effects. Among them, the method for preparing ultra-small particle size nanobubbles is replaceable. In other words, the cell culture medium containing ultra-small particle size nanobubbles prepared by other methods should also have the same anti-aging effect.

[0019] According to a third aspect of the present invention, there is provided a use of a cell culture medium containing ultra-small-size nanobubbles in delaying the replicative senescence of mesenchymal stem cells in vitro.

[0020] According to a fourth aspect of the present invention, there is also provided a use of a cell culture medium containing ultra-small particle size nanobubbles in alleviating cell oxidative stress.

[0021] According to the research of the present invention, it is found that the ultra-small particle size nanobubbles have the effect of delaying the aging of mesenchymal stem cells, so the ultra-small particle size nanobubble cell culture medium containing the ultra-small particle nanobubbles also has the effect of delaying the aging of mesenchymal stem cells. According to the research of the present invention, it is also found that the ultra-small particle size nanobubbles have the effect of relieving cell oxidative stress, so the ultra-small particle size nanobubble cell culture medium containing the ultra-small particle nanobubbles also has the effect of relieving cell oxidative stress.

[0022] In view of the limitations of chemical antioxidants, ultra-small particle size nanobubbles have become a promising alternative for reducing intracellular ROS levels. These nanobubbles have unique properties that can directly reduce intracellular ROS, thereby protecting the stability of intracellular biomacromolecules and maintaining cell viability. In view of these advantages, the present invention prepares a cell culture medium with in situ introduction of ultra-small particle size nanobubbles, and uses it for in vitro expansion and culture of mouse mesenchymal stem cells. The results show that the presence of ultra-small particle size nanobubbles significantly delays the aging of MSCs and increases the number of cell divisions while maintaining their stem cell properties. This indicates that the cell culture medium containing ultra-small particle size nanobubbles prepared by the present invention can slow down the aging caused by in vitro expansion of MSCs. The present invention provides new insights and methods for optimizing stem cell culture conditions, ultimately contributing to the development of regenerative medicine and therapeutic applications.

[0023] Compared with the Chinese invention patent application "a small-size nanobubble water, preparation method and application thereof", the cell culture medium containing ultra-small-size nanobubbles provided by the present invention mainly has the following differences:

[0024] First, the medium in which ultra-small-sized nanobubbles exist is different. The present invention is the first to prepare a cell culture medium that introduces ultra-small-sized nanobubbles in situ. So far, the prior art has never disclosed a method of preparing a cell culture medium containing ultra-small-sized nanobubbles by low-temperature pressurization and decompression of a cell culture medium.

[0025] Secondly, the present invention also discovered for the first time that such a cell culture medium containing ultra-small particle size nanobubbles has the effect of delaying the aging of mesenchymal stem cells and alleviating cell oxidative stress;

[0026] Again, the total concentration of ultra-small particle size nanobubbles in the cell culture medium of the present invention is higher. The present invention requires that the total concentration of ultra-small particle size nanobubbles in the cell culture medium is greater than 10 11 / ml, while the small-particle nano bubble water disclosed in the previous patent application only requires a concentration of >10 7 The reason is that according to the research of the present invention, if the concentration is lower than 10 11 / ml (for example, 10 7 pcs / ml~10 11 / ml), may not produce the technical effect of delaying the replicative aging of mesenchymal stem cells in vitro, see the nNBs group in each embodiment of the present invention.

[0027] The cell culture medium containing ultra-small nanobubbles and the preparation method and application thereof provided by the present invention have the advantages over the prior art in that:

[0028] 1) Without adding exogenous antioxidants, an ultra-small particle size nanobubble cell culture medium with anti-aging and antioxidant effects can be prepared, wherein most of the nanobubbles are around 10 nanometers in size;

[0029] 2) by first pressurizing and then depressurizing the cell culture medium after cryogenic freezing, a method for in situ introduction of ultra-small particle size nanobubbles into the cell culture medium is provided;

[0030] 3) The anti-aging effect of the ultra-small particle size nanobubble cell culture medium prepared by the present invention does not rely on the additional addition of a reducing agent, thus avoiding the defects of the above-mentioned chemical reducing agents;

[0031] 4) The method for preparing the ultra-small particle size nanobubble cell culture medium provided by the present invention can not only be used to delay the replicative aging of mesenchymal stem cells in vitro, but also has the potential to be applied in the fields of biomedicine, medical cosmetology, and health and longevity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The particle size distribution of the ultra-small-sized nanobubbles prepared in Example 1 is shown; wherein a is the particle size distribution of the ultra-small-sized nanobubbles in the complete cell culture medium measured using the dynamic light scattering technique (DLS); b is the particle size distribution of the ultra-small-sized nanobubbles in the basal cell culture medium measured using the dynamic light scattering technique (DLS); the abscissa represents the bubble particle size, and the higher the value of the ordinate, the greater the proportion of bubbles of this particle size;

[0033] Figure 2 It shows that the cell culture medium containing ultra-small particle size nanobubbles in Example 2 has the ability to delay the replicative senescence of mesenchymal stem cells; senescent cells are dyed blue, and the darker the blue, the higher the degree of cell senescence;

[0034] Figure 3 It shows that the cell culture medium containing ultra-small particle size nanobubbles in Example 3 has the ability to reduce the accumulation of ROS in mesenchymal stem cells; the darker the green color, the higher the ROS content in the cells;

[0035] Figure 4 It shows that the cell culture medium containing ultra-small particle size nanobubbles in Example 4 has the ability to improve the proliferation ability of mesenchymal stem cells;

[0036] Figure 5 It shows that the cell culture medium containing ultra-small particle size nanobubbles in Example 5 has the ability to reduce the ROS level of oxidative stress cells;

[0037] Figure 6 It is shown that the cell culture medium containing ultra-small particle size nanobubbles in Example 6 has the ability to enhance the viability and proliferation of oxidative stress cells. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the instrument and equipment manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0039] Example 1 Characterization of Ultra-Small Size Nanobubbles in Cell Culture Medium

[0040] Generation and analysis of ultra-small nanobubbles:

[0041] Experimental materials: pressure and pressure reduction device, high-purity nitrogen gas cylinder, high-purity oxygen gas cylinder, cell culture medium, cell culture bottle with 0.22μm filter membrane.

[0042] The ultra-small nanobubbles in the cell culture medium were prepared by pressurizing and decompressing the cell culture medium frozen at -80°C. The experiment was conducted in a custom-made metal chamber that can control the pressure. The frozen cell culture medium was placed in the chamber, nitrogen was introduced to a pressure of 0.6MPa for 30 minutes, and then slowly decompressed to normal pressure in about 4 hours.

[0043] Characterization of ultra-small nanobubbles:

[0044] The prepared nanobubble cell culture medium was subjected to particle number and size analysis using a nanoparticle tracking analysis (NTA) system (NS300, Malvern, UK) and a dynamic light scattering (DLS) system (nano-ZS90, Malvern, UK). Figure 1shown.

[0045] a is the particle size distribution of ultra-small-sized bubbles in complete cell culture medium (DMEM) measured using dynamic light scattering technology (DLS). DLS can measure nanoparticles larger than 0.4nm. The horizontal axis in the figure represents the particle size of the nanobubbles, and the vertical axis represents the number ratio of nanobubbles of this particle size. 0d, 2d, and 4d in the figure refer to the distribution of ultra-small-sized nanobubbles measured on the 0th, 2nd, and 4th days of the prepared cell culture medium containing ultra-small-sized nanobubbles. Degassed refers to the distribution of ultra-small-sized nanobubbles after degassing. The results show that the ultra-small-sized nanobubble cell culture medium contains nanobubbles <10nm, accounting for nearly 100% of the total number and can be maintained for 4 days.

[0046] b is the particle size distribution of ultra-small-sized bubbles in the cell maintenance medium (DMEM) measured using dynamic light scattering technology (DLS). DLS can measure nanoparticles larger than 0.4nm. The horizontal axis in the figure represents the particle size of the nanobubbles, and the vertical axis represents the number ratio of nanobubbles of this particle size. 0d, 2d, and 4d in the figure refer to the distribution of ultra-small-sized nanobubbles measured on the 0th, 2nd, and 4th days of the prepared cell culture medium containing ultra-small-sized nanobubbles. Degassed refers to the distribution of ultra-small-sized nanobubbles after degassing. The results show that the ultra-small-sized nanobubble cell culture medium contains nanobubbles <10nm, accounting for nearly 100% of the total number and can be maintained for 2 days.

[0047] Example 2 Cell culture medium containing ultra-small nanobubbles delays replicative senescence of mesenchymal stem cells

[0048] Experimental materials: mouse placental mesenchymal stem cell complete culture medium (purchased from icell), mouse placental mesenchymal stem cells (purchased from icell), glutathione, β-galactosidase aging kit.

[0049] After culturing mouse placental mesenchymal stem cells for 9 generations using a cell culture medium containing ultra-small nanobubbles (complete culture medium for mouse placental mesenchymal stem cells) (cell culture incubator, 5% CO2, 37°C; 1:3 per passage), the intracellular β-galactosidase activity was measured to reflect the aging of mesenchymal stem cells. Figure 2 As shown in the figure, CK is the untreated control group, GSH is the 10 μm glutathione treated group, and nNBs is the ultra-small nanobubble with a concentration of less than 10 11 The nanobubble group is of pcs / mL, and UNBs is the prepared nanobubble group with ultra-small particle size.

[0050] The results showed that most of the stem cells cultured in normal culture medium underwent aging, while the stem cells cultured in cell culture medium containing ultra-small particle size nanobubbles showed no obvious aging.

[0051] Example 3 Cell culture medium containing ultra-small nanobubbles reduces the accumulation of ROS in mesenchymal stem cells

[0052] Experimental materials: mouse placental mesenchymal stem cell complete culture medium (purchased from icell), mouse placental mesenchymal stem cells (purchased from icell), glutathione, DCFH-DA fluorescent dye.

[0053] After culturing mouse placental mesenchymal stem cells for 9 generations using a cell culture medium containing ultra-small nanobubbles (the method is the same as in Example 2), the fluorescence intensity of DCFH in the cells was measured to reflect the amount of ROS accumulation in the stem cells.

[0054] The results are as follows Figure 3 As shown, most of the stem cells cultured in normal culture medium accumulated excessive ROS, while the stem cells cultured in the cell culture medium containing ultra-small particle nanobubbles did not have obvious ROS accumulation.

[0055] Example 4 Cell culture medium containing ultra-small nanobubbles increases the proliferation capacity of mesenchymal stem cells

[0056] Experimental materials: mouse placenta mesenchymal stem cell complete culture medium (purchased from icell), mouse placenta mesenchymal stem cells (purchased from icell), glutathione, MTT cell proliferation kit.

[0057] After culturing mouse placental mesenchymal stem cells for 9 generations using a cell culture medium containing ultra-small nanobubbles (the method is the same as in Example 2), the proliferation capacity of the cells was determined by the MTT method.

[0058] The results are as follows Figure 4 As shown, stem cells cultured in normal culture medium have no proliferation ability, while stem cells cultured in cell culture medium containing ultra-small particle size nanobubbles still maintain good proliferation ability.

[0059] Example 5 Cell culture medium containing ultra-small particle size nanobubbles reduces the ROS level in oxidatively stressed cells.

[0060] Experimental materials: DMEM cell culture medium (purchased from Gibico), human liver cancer cell HepG2, mouse embryonic fibroblast NIH-3T3, glutathione, 30% H2O2, DCFH-DA fluorescent dye.

[0061] Cells subjected to hydrogen peroxide-induced oxidative stress were cultured in a cell culture medium (DMEM) containing ultrasmall nanobubbles and treated with 0.2 mM H2O2 for 2 h. After DCFH-DA staining, the intracellular ROS level was detected by flow cytometry. Figure 5 Figure a is HepG2 cells, and figure b is NIH-3T3 cells. The Blank group is the blank group without H2O2 treatment, the MM group is the control group treated with H2O2, the GSH-MM group is the GSH treatment group after H2O2 treatment, and the UNBs-MM group is the ultra-small particle size nanobubble culture medium treatment group after H2O2 treatment.

[0062] The results are as follows Figure 5 As shown, the ROS level in cells induced by hydrogen peroxide-induced oxidative stress was significantly higher than that in cells of the normal group, while the ROS level in cells cultured in the cell culture medium containing ultra-small particle size nanobubbles was reduced by about 60%.

[0063] Example 6 Cell culture medium containing ultra-small particle size nanobubbles increases the viability and proliferation capacity of oxidative stress cells.

[0064] Cells induced by hydrogen peroxide oxidative stress (same as in Example 5) were cultured in a cell culture medium (DMEM) containing ultra-small-size nanobubbles, and cell viability was determined by MTT.

[0065] The results are as follows Figure 6 As shown, the cell viability and proliferation ability of cells in hydrogen peroxide-induced oxidative stress cells were significantly lower than those in the normal group, while the viability of cells cultured in the cell culture medium containing ultra-small particle size nanobubbles increased by 50% and the proliferation ability increased by 65%.

[0066] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A cell culture medium containing ultra-small particle size nanobubbles, characterized in that: The cell culture medium contains ultra-small particle size nanobubbles with a particle size of less than 50 nm, and the total concentration of the ultra-small particle size nanobubbles in the cell culture medium is greater than 10 11 Pieces / mL.

2. The cell culture medium according to claim 1, characterized in that The particle size distribution range of the ultra-small particle size nanobubbles is 2nm to 15nm.

3. The cell culture medium according to claim 1, characterized in that The DLS number intensity of the ultra-small particle size nanobubbles accounts for >99%.

4. A method for preparing a cell culture medium containing ultra-small nanobubbles according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: Cooling the cell culture medium into which ultra-small nanobubbles are to be introduced to -20°C to -80°C; S2: placing the cooled cell culture medium into a pressurizing device; S3: introducing high-pressure gas into the pressurizing device or applying mechanical force to increase the internal pressure of the pressurizing device; S4: Maintain high pressure conditions for a period of time; S5: Slowly release the pressure to normal pressure to prepare a cell culture medium containing ultra-small particle size nanobubbles.

5. The preparation method according to claim 4, characterized in that: Step S3 includes: introducing high-pressure gas into the pressurizing device, and selecting different types of gas according to different needs.

6. The preparation method according to claim 4, characterized in that: In steps S3 and S4, the pressure range of the high pressure condition is 0.2 MPa to 1.2 MPa.

7. The preparation method according to claim 4, characterized in that: In step S4, the high pressure condition is maintained for a duration of 20 min to 60 min.

8. The preparation method according to claim 4, characterized in that: In steps S2 to S5, the temperature of the cell culture medium is kept at a normal level without performing additional temperature increase or decrease operations.

9. Use of the cell culture medium containing ultra-small-size nanobubbles according to any one of claims 1 to 3 in in vitro expansion of mesenchymal stem cells.

10. Use of a cell culture medium containing ultra-small-size nanobubbles according to any one of claims 1 to 3 in alleviating cell oxidative stress.

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