A medium, a culture method, a cell population of immune cells for anti-aging and applications thereof

Abstract

The application relates to an immune cell culture medium for anti-aging and a culture method thereof. The culture medium comprises a basic culture medium and a functional additive, and the functional additive is composed of resveratrol, nicotinamide mononucleotide (NMN), recombinant human IL-7, telomerase activator TA-65 and mitochondrial autophagy inducer urolithin A. The basic culture medium is a serum-free culture medium, which comprises DMEM / F12, human blood albumin, glutamine, sodium pyruvate and a chemically defined lipid concentrate. The culture method comprises the following steps: separating CD3+ T cells from peripheral blood, inoculating the CD3+ T cells into the culture medium under a low-oxygen condition, culturing the CD3+ T cells by using an intermittent dynamic culture system, and regularly supplementing a maintenance liquid and a telomerase activator. The immune cell group obtained by the method has a high CD28+CD57- subpopulation ratio, a prolonged telomere length and an enhanced mitochondrial membrane potential, can be used for preparing anti-aging drugs, can improve the thymus regeneration capacity, can reduce the level of aging-related secretory phenotype factors, and can restore the immune system's elimination function on aging cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an immune cell culture medium for anti-aging and its culture method, as well as the application of the immune cell population obtained by this method in anti-aging drugs. Background Technology

[0002] With the increasing aging of the global population, the incidence of age-related diseases, such as immune dysfunction and chronic inflammation, is rising year by year, seriously affecting human health and quality of life. Immune cells play a crucial role in maintaining homeostasis, resisting pathogen invasion, and clearing senescent cells. However, with age, the function and number of immune cells gradually decline, leading to a weakening of the body's immune surveillance function. This results in an inability to effectively identify and eliminate abnormal cells, thereby accelerating the aging process and increasing the risk of disease.

[0003] In recent years, research on anti-aging based on immune cells has gradually become a hot topic. Enhancing the function and vitality of immune cells through specific culture media and methods holds promise for providing new strategies and means to delay aging and improve the health of the elderly. Currently, there are some products and technologies on the market for immune cell culture, but most of them suffer from problems such as complex composition, high cost, and limited effectiveness in enhancing the anti-aging properties of immune cells, failing to meet the growing demand for anti-aging treatments. Summary of the Invention

[0004] The purpose of this invention is to provide a culture medium and its culture method that can effectively enhance the anti-aging properties of immune cells, as well as the application of the immune cell population obtained by this method in anti-aging drugs, so as to overcome the shortcomings of the prior art.

[0005] An immune cell culture medium for anti-aging

[0006] It includes a basal culture medium and functional additives, the functional additives being composed of the following components: resveratrol 5-20 μM, nicotinamide mononucleotide (NMN) 1-5 mM, recombinant human IL-7 10-50 ng / mL, telomerase activator TA-65 0.1-1 μg / mL, and urolithin A 10-50 μM, a mitophagy inducer. Among them, resveratrol is a natural polyphenol compound with various biological activities such as antioxidation, anti-inflammation, and anti-aging. It can activate the body's anti-aging pathways, such as the SIRT1 signaling pathway, thereby enhancing the cell's antioxidant capacity and survival ability. NMN, as a precursor of nicotinamide adenine dinucleotide (NAD+), plays an important role in cellular energy metabolism and DNA repair. Supplementing with NMN can increase intracellular NAD+ levels, enhance cellular energy metabolism and antioxidant capacity, and thus delay cellular aging. Recombinant human IL-7 is an important cytokine that can promote the proliferation, differentiation, and survival of T lymphocytes and enhance the function of the immune system. Especially in elderly individuals, the decline in IL-7 levels is closely related to the decline in immune function. Supplementing with IL-7 can effectively improve the function of immune cells. Telomerase activator TA-65 can activate telomerase activity, prolong the telomere length of cells, thereby delaying the cellular aging process. Mitochondrial autophagy inducer urolithin A can promote the clearance and renewal of damaged mitochondria in cells, maintain mitochondrial functional homeostasis, and improve the cell's energy supply efficiency and antioxidant capacity.

[0007] Furthermore, the basal culture medium is a serum-free medium, comprising: DMEM / F12, 1-5% human serum albumin, 1-3mM glutamine, 0.5-2mM sodium pyruvate, and 0.1-0.5% chemically defined lipid concentrate. Serum-free culture medium avoids the complexity and batch-to-batch variations of serum components, providing a more stable and controllable culture environment, while reducing interference from exogenous factors, which is beneficial for the growth and function of immune cells. Specifically, DMEM / F12 provides the basic nutrients required for cell growth, including carbohydrates, amino acids, and vitamins; human serum albumin, as an important nutritional supplement, can maintain osmotic pressure balance in cells and promote cell adhesion and growth; glutamine is an important raw material for cellular energy metabolism and protein synthesis; sodium pyruvate can serve as an energy source for cells and promote cellular metabolic activities; and the chemically defined lipid concentrate provides the necessary lipid components for cell membrane synthesis and maintenance.

[0008] In some preferred embodiments, the functional additive further comprises a TGF-β inhibitor SB4315425-15 μM and / or a histone deacetylase inhibitor trichostatin A10-30 nM. The TGF-β inhibitor SB431542 can inhibit the TGF-β signaling pathway, which plays an important role in the senescence and functional suppression of immune cells. By inhibiting the TGF-β signaling pathway, the activity and proliferation capacity of immune cells can be enhanced. The histone deacetylase inhibitor trichostatin A can regulate gene expression, promote the transcription and expression of immune cell-related genes, and further enhance the function of immune cells.

[0009] 2. Method for culturing anti-aging immune cells using the above-mentioned culture medium

[0010] Includes the following steps:

[0011] a) Isolation of CD3+ T cells from peripheral blood: Mononuclear cells are isolated from peripheral blood using methods such as density gradient centrifugation, and then CD3+ T cells are further isolated using techniques such as magnetic bead sorting or flow cytometry. CD3+ T cells are an important subset of T lymphocytes in the immune system and play a core role in cellular immune responses. Enhancing their function is of great significance for improving the body's immune surveillance capabilities and anti-aging effects.

[0012] b) Under hypoxic conditions containing 5% CO2 and 3% O2, cells were incubated at a rate of 1×10⁻⁶. 6 Seedlings were placed at a density of / mL in the above-mentioned culture medium: The hypoxic environment can simulate the physiological oxygen concentration of tissues in vivo, which is beneficial to the survival and functional maintenance of immune cells, while avoiding the oxidative stress damage caused by the hyperoxic environment. Seeding cells at an appropriate density in the culture medium can ensure the nutrient supply and growth space for cells during the culture process, and promote cell adhesion and growth.

[0013] c) An intermittent dynamic culture system is used, with periodic oscillation culture at a tilt angle of 5-15° and a frequency of 0.1-0.5Hz. This intermittent dynamic culture system allows cells to be fully suspended and mixed in the culture medium through periodic oscillation, promoting the exchange of substances between cells and the medium, improving the efficiency of nutrient uptake and metabolic waste removal by cells, and enhancing cell-cell interactions, which is beneficial for the activation and proliferation of immune cells. The tilt angle and oscillation frequency are important parameters affecting the culture effect; appropriate tilt angles and frequencies ensure that cells receive uniform mechanical stimulation during oscillation, avoiding excessive cell aggregation or damage.

[0014] d) Replenish with maintenance medium containing NMN and IL-7 every 48 hours, and continue culturing for 7-14 days: Regularly replenishing the maintenance medium during culture ensures the concentration of key components in the culture medium, meets the nutritional needs of immune cells for long-term culture, and promotes continuous cell proliferation and functional maintenance. The concentrations of NMN and IL-7 in the maintenance medium should be optimized and adjusted according to the cell growth status and experimental requirements to ensure optimal cell growth and function during culture.

[0015] Furthermore, the oscillation parameters of the dynamic culture system in step c) are: tilt angle 10°±2°, frequency 0.2Hz, with each oscillation lasting 30 seconds followed by a 90-second pause. This specific combination of oscillation parameters was obtained through extensive experimental optimization, which can minimize mechanical damage and stress response to cells while ensuring thorough mixing and material exchange, thus promoting stable growth and function of immune cells.

[0016] The maintenance solution in step d) contains: NMN 2mM, IL - 725ng / mL, and urolithiasis A 25μM, with the replenishment amount being 20-30% of the original culture medium volume. The concentration and replenishment amount of each component in the maintenance solution are determined based on the metabolic needs and component consumption of immune cells during long-term culture. By replenishing an appropriate amount of maintenance solution, the concentration of each component in the culture medium can be maintained within a suitable range, promoting continuous cell proliferation and functional maintenance.

[0017] During the culture process, telomerase activator TA-65 was added every 72 hours to a final concentration of 0.5 μg / mL, with the stimulation time not exceeding 48 hours. Regularly adding TA-65 can further activate the telomerase activity of immune cells, prolonging telomere length and thus delaying cellular aging, improving cell proliferation and survival. Simultaneously, controlling the TA-65 stimulation time to no more than 48 hours can avoid potential risks from over-activation of telomerase, such as unlimited cell proliferation or carcinogenesis.

[0018] 3. Immune cell populations obtained by the above methods and their application in anti-aging drugs.

[0019] The immune cell population obtained through the above culture method has the following characteristics: the proportion of the CD28+CD57- subset is ≥85%, telomere length is 15-30% longer than that of the initial cells, and mitochondrial membrane potential is 2-3 times higher. The CD28+CD57- subset is a T cell subset with high proliferative capacity and functional activity. The increased proportion of this subset indicates that the cultured immune cell population has a stronger immune response and anti-aging potential. The increase in telomere length means that the cellular aging process is effectively delayed, the cell lifespan is extended, and its immune function can be better exerted. The increase in mitochondrial membrane potential reflects the enhancement of mitochondrial function, the more sufficient energy supply to the cells, and the better the cells' metabolic activities and functional maintenance.

[0020] The application of this immune cell population in the preparation of anti-aging drugs, which are used to: enhance thymus regeneration capacity, reduce the level of aging-associated secretory phenotype (SASP) factors, and restore the immune system's function of clearing senescent cells. The thymus is the site of T lymphocyte development and maturation. With age, the thymus gradually atrophies, and the production and output of T cells decrease, leading to a decline in immune system function. The anti-aging drugs prepared using the immune cell population obtained in this invention can promote thymus regeneration and repair, increase T cell production, and thus enhance the body's immune function. Simultaneously, the drug can reduce the level of aging-associated secretory phenotype factors, which can trigger chronic inflammation and tissue damage during aging. By reducing their secretion, the inflammatory state of the body can be improved, and the aging process can be slowed down. Furthermore, the drug can restore the immune system's function of clearing senescent cells, promptly removing accumulated senescent cells from the body and preventing them from damaging surrounding tissues and organs, thereby achieving an anti-aging effect.

[0021] 4. The beneficial effects of the present invention:

[0022] 1) The anti-aging immune cell culture medium provided by the present invention has clearly defined components and a reasonable formula, which can provide good nutritional support and microenvironment for the growth and function maintenance of immune cells, and effectively enhance the anti-aging properties of immune cells.

[0023] 2) By employing low-oxygen conditions and an intermittent dynamic culture system, the in vivo physiological environment was simulated, which promoted the activation and proliferation of immune cells and improved cell vitality and function.

[0024] 3) Regularly replenishing maintenance medium and telomerase activator ensured the concentration of key components during culture, further slowing down the aging process of cells and improving the quality and quantity of immune cell populations.

[0025] 4) The immune cell population obtained by the method of the present invention has a higher proportion of CD28+CD57- subsets, longer telomere length and enhanced mitochondrial membrane potential, indicating that it has stronger immune response capacity and anti-aging potential.

[0026] 5) The application of this immune cell population in the preparation of anti-aging drugs can effectively enhance the regenerative capacity of the thymus, reduce the level of aging-related secretory phenotypic factors, and restore the immune system's function of clearing senescent cells, providing new means and strategies for anti-aging treatment and having broad application prospects. Detailed Implementation

[0027] The present invention will be further described in detail below through specific embodiments, but these embodiments are not intended to limit the scope of protection of the present invention.

[0028] Example 1: Preparation of Anti-aging Immune Cell Culture Medium

[0029] 1) Preparation of basal culture medium

[0030] Weigh an appropriate amount of DMEM / F12 powder, add an appropriate amount of deionized water according to the instructions, stir to dissolve, then add 1-5% human serum albumin (the specific ratio can be adjusted according to experimental needs), and continue stirring to mix thoroughly.

[0031] Then add 1-3mM glutamine, 0.5-2mM sodium pyruvate, and 0.1-0.5% chemically defined lipid concentrate (the specific concentration can be adjusted according to experimental needs) in sequence. After stirring evenly, filter with a 0.22μm filter membrane for sterilization, dispense into containers, and store at -20℃ for later use.

[0032] 2) Formulation of functional additives

[0033] Weigh an appropriate amount of resveratrol, dissolve it in an appropriate amount of dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution, and then dilute it to a working concentration of 5-20 μM.

[0034] Weigh an appropriate amount of nicotinamide mononucleotide (NMN), dissolve it in an appropriate amount of physiological saline to prepare a 10 mM stock solution, and then dilute it to a working concentration of 1-5 mM.

[0035] Take recombinant human IL-7, dissolve it in an appropriate amount of physiological saline to prepare a stock solution of 100 ng / mL, and then dilute it to a working concentration of 10-50 ng / mL.

[0036] Weigh an appropriate amount of telomerase activator TA-65, dissolve it in an appropriate amount of DMSO to prepare a stock solution of 1 mg / mL, and then dilute it to a working concentration of 0.1-1 μg / mL.

[0037] Weigh an appropriate amount of the mitochondrial autophagy inducer urolithin A, dissolve it in an appropriate amount of DMSO to prepare a 10 mM stock solution, and then dilute it to a working concentration of 10-50 μM.

[0038] Mix the above components evenly according to the working concentration to obtain a functional additive solution, which is then dispensed and stored at -20℃ for later use.

[0039] 3) Preparation of culture medium

[0040] Take an appropriate amount of basal culture medium and add an appropriate amount of functional additive solution per liter of basal culture medium to make the concentration of each component meet the above requirements. After mixing evenly, filter it with a 0.22μm filter membrane to remove bacteria, and you will get the immune cell culture medium for anti-aging. After dispensing, store it at 4℃ for later use.

[0041] Example 2: Culture of anti-aging immune cells

[0042] 1) Isolation of CD3+ T cells

[0043] Peripheral blood was collected from healthy volunteers, and mononuclear cells were separated using density gradient centrifugation. CD3+ T cells were then separated using magnetic bead sorting. The specific procedures are as follows:

[0044] Peripheral blood was mixed with an equal volume of lymphocyte separation medium and centrifuged at 800g for 30 minutes. Mononuclear cells at the interface were collected and washed twice with Hank's balanced salt solution.

[0045] Following the instructions of the magnetic bead sorting kit, mononuclear cells were incubated with CD3 antibody-labeled magnetic beads, and then CD3+ T cells were separated by a magnetic separation column. The purity was then detected by flow cytometry to ensure that the purity was ≥95%.

[0046] 2) Cell Culture

[0047] The isolated CD3+ T cells were used at a concentration of 1×10⁻⁶. 6 The inoculum was inoculated at a density of / mL in the culture medium prepared in Example 1 and placed in a hypoxic incubator containing 5% CO2 and 3% O2.

[0048] An intermittent dynamic culture system was used, with an inclination angle of 10°±2° and a frequency of 0.2Hz. Each oscillation lasted for 30 seconds and was followed by a 90-second pause, for periodic oscillation culture.

[0049] Replenish with maintenance medium containing 2mM NMN, 25ng / mL IL-7, and 25μM urolithiasis A every 48 hours, at a volume of 20-30% of the original culture medium, and continue culturing for 7-14 days.

[0050] Add telomerase activator TA-65 every 72 hours to a final concentration of 0.5 μg / mL, with continuous stimulation for no more than 48 hours.

[0051] 3) Cell detection

[0052] During the culture process, samples were taken periodically, and the expression of cell surface markers CD28 and CD57 was detected by flow cytometry to calculate the proportion of the CD28+CD57- subset.

[0053] The length of telomeres in cells was detected using fluorescence in situ hybridization (FISH) and compared with the initial cells to calculate the percentage of telomere length extension.

[0054] Mitochondrial membrane potential was detected by flow cytometry. The JC-1 staining kit was used for staining and detection according to the instructions. The fold increase in mitochondrial membrane potential was calculated.

[0055] Example 3: Application of immune cell populations in anti-aging drugs

[0056] 1) Drug preparation

[0057] The immune cell population obtained from Example 2 was collected, washed 2-3 times with physiological saline, and the cell concentration was adjusted to an appropriate range. It was then mixed with pharmaceutical excipients to prepare an injectable anti-aging drug, which was packaged and stored in accordance with Good Manufacturing Practice (GMP).

[0058] 2) Drug efficacy evaluation

[0059] Aged animal models (such as aged mice) were selected and randomly divided into a control group and a treatment group, with 10 mice in each group. The control group was given an equal volume of physiological saline, while the treatment group was given the prepared anti-aging drug, administered according to a specific dosage and dosing cycle.

[0060] The anti-aging effects of drugs are evaluated by detecting indicators such as thymus index, peripheral blood T cell count and function, and levels of aging-related secretory phenotypic factors. For example, after euthanizing the animals, the thymus is weighed and the thymus index is calculated; flow cytometry is used to detect the proportion and proliferation capacity of peripheral blood T cell subsets; and ELISA is used to detect the levels of aging-related secretory phenotypic factors (such as IL-6 and TNF-α) in serum.

[0061] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium for anti-aging immune cells, characterized in that, It includes a basal culture medium and functional additives, the functional additives being composed of the following components: Resveratrol 5-20 μM Nicotinamide mononucleotide (NMN) 1-5 mM Recombinant human IL-7 10-50 ng / mL Telomerase activator TA-65 0.1-1 μg / mL Mitochondrial autophagy inducer urolithin A 10-50 μM.

2. The culture medium according to claim 1, characterized in that, The basal culture medium is a serum-free culture medium, which contains: DMEM / F12, 1-5% human serum albumin, 1-3 mM glutamine, 0.5-2 mM sodium pyruvate, 0.1-0.5% chemically defined lipid concentrate.

3. The culture medium according to claim 1, characterized in that, The functional additive comprises: TGF-β inhibitor SB431542 5-15 μM, and / or histone deacetylase inhibitor trichostatin A 10-30 nM.

4. A method for culturing anti-aging immune cells using the culture medium according to any one of claims 1-3, characterized in that, Includes the following steps: a) Isolate CD3+ T cells from peripheral blood; b) Under hypoxic conditions containing 5% CO2 and 3% O2, cells were incubated at a rate of 1×10⁻⁶. 6 / mL inoculated into the culture medium described in claims 1-3; c) An intermittent dynamic culture system was used to perform periodic oscillation culture at a tilt angle of 5-15° and a frequency of 0.1-0.5 Hz; d) Replenish with maintenance medium containing NMN and IL-7 every 48 hours and continue culturing for 7-14 days.

5. The method according to claim 4, characterized in that, The oscillation parameters of the dynamic culture system in step c) are: The tilt angle is 10°±2°, the frequency is 0.2 Hz, and each oscillation lasts for 30 seconds and then stops for 90 seconds.

6. The method according to claim 4, characterized in that, The maintenance solution in step d) contains: NMN 2 mM, IL-7 25 ng / mL, and urolithiasis A 25 μM, supplemented at 20-30% of the original culture medium volume.

7. The method according to claim 4, characterized in that, During the culture process, telomerase activator TA-65 was added every 72 hours until the final concentration was 0.5 μg / mL, and the stimulation time did not exceed 48 hours.

8. An immune cell population obtained by any one of claims 4-7, characterized in that: CD28+CD57 subgroup ratio ≥ 85% Telomere length is 15-30% longer than in the initial cell. The mitochondrial membrane potential increases by 2-3 times.

9. The application of the immune cell population according to claim 8 in the preparation of anti-aging drugs, characterized in that, The drug is used to: enhance thymus regeneration capacity, reduce the level of aging-associated secretory phenotype (SASP) factor, and restore the immune system's ability to clear senescent cells.