Umbilical cord mesenchymal stem cell serum-free medium as well as use method and application thereof

Abstract

The invention discloses an umbilical cord mesenchymal stem cell serum-free culture medium, which is composed of a basic culture medium and additive components, and the additive components comprise, by final concentration, 5-20 [mu] g / mL of astragalus polysaccharide, 0.5-2.0 [mu] g / mL of salidroside, 0.1-0.5 [mu] g / mL of tanshinone IIA, 0.1-0.3 mg / mL of sodium pyruvate, 5-15 mM of D-ribose, 3-6 mg / mL of recombinant human albumin, and 5-10 [mu] g / mL of recombinant transferrin; the invention also discloses a method for culturing umbilical cord mesenchymal stem cells under the hypoxia condition by using the serum-free culture medium. The method comprises the steps of cell inoculation, culture under the hypoxia condition, subculture, cell passage and the like. The astragalus polysaccharide, the salidroside, the tanshinone IIA, the sodium pyruvate, the recombinant human albumin and the recombinant transferrin are added into the serum-free culture medium, so that nutrition supply and function maintenance of cells under a low-oxygen condition are guaranteed, the proliferation rate and the differentiation capacity of the cultured cells are improved, the apoptosis rate of the cells is reduced, and the survival rate of the cells is increased. Wide application prospects are realized in the fields of cell culture, regenerative medicine and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a serum-free culture medium for umbilical cord mesenchymal stem cells and a use method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs), a type of adult stem cell with self-renewal and multipotential differentiation potential, have demonstrated significant application value in tissue engineering, regenerative medicine, and cell therapy. Umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have become a promising area of ​​clinical research due to their unique advantages, including low immunogenicity, ease of isolation and harvest, high proliferation potential, and minimal ethical concerns. Umbilical cord-derived mesenchymal stem cells can differentiate into a variety of functional cell types, including osteoblasts, adipocytes, and chondrocytes. They also secrete bioactive molecules such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), which contribute to immune regulation, angiogenesis, and tissue repair. These cells hold broad application prospects in areas such as ischemic diseases, autoimmune disorders, and organ damage repair.

[0003] In the field of cell culture, the choice of culture medium is crucial. Traditional culture media containing animal serum, while providing rich nutrients for cell growth, have many drawbacks, such as complex and unclear composition, large batch-to-batch variability, and the easy introduction of pathogens and immunogenic substances. These drawbacks can lead to instability in the cell culture process and affect the repeatability and reliability of experimental results. In contrast, serum-free culture media have clear composition, stable properties, and controllable risks. They can effectively avoid the various problems caused by animal serum, provide a more reliable culture system for the research and clinical application of umbilical cord mesenchymal stem cells, and help achieve standardization and large-scale production of cell culture.

[0004] In recent years, studies have found that mesenchymal stem cells (MSCs) have enhanced survival, proliferation, and differentiation potential in a hypoxic environment (1%-5% O2). This discovery has opened up a new path for the large-scale culture of umbilical cord MSCs. However, in practical applications, traditional serum-free culture media for culturing MSCs under hypoxic conditions have significant drawbacks. For example, hypoxia can severely disrupt the normal metabolic balance within cells, leading to a significant increase in the production of reactive oxygen species (ROS) within the cells, significantly increasing the apoptosis rate of cultured UMBC MSCs and severely affecting cell quality and quantity. Furthermore, hypoxia can interfere with UMBC MSCs' energy metabolism, signal transduction pathways, and other processes, leading to cell cycle arrest and slowing cell proliferation. This makes it impossible to meet the stringent requirements for stem cell quality and function in regenerative medicine and cell therapy.

[0005] Therefore, the development of a culture medium and its use method that can adapt to hypoxic conditions, effectively reduce cell apoptosis rate, promote the proliferation of umbilical cord mesenchymal stem cells and maintain their stemness has become a key issue that needs to be urgently addressed in this field. It has extremely important practical significance for promoting the widespread application of umbilical cord mesenchymal stem cells in regenerative medicine and clinical treatment. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a serum-free culture medium for umbilical cord mesenchymal stem cells and a use method and application thereof.

[0007] In the first aspect of the present invention, the present invention provides a serum-free culture medium for umbilical cord mesenchymal stem cells, characterized in that it is composed of a basal culture medium and an additive, wherein the additive comprises, in terms of final concentration, 5-20 μg / mL of astragalus polysaccharide, 0.5-2.0 μg / mL of salidroside, 0.1-0.5 μg / mL of tanshinone IIA, 0.1-0.3 mg / mL of sodium pyruvate, 5-15 mM of D-ribose, 3-6 mg / mL of recombinant human albumin, and 5-10 μg / mL of recombinant transferrin. The serum-free culture medium of the present invention can culture umbilical cord mesenchymal stem cells under hypoxic conditions, thereby improving cell survival rate, proliferation rate, and differentiation ability. It is worth noting that the final concentration described in the present invention refers to the ratio of the mass or amount of each component in the solution to the total volume of the solution after the solution is prepared.

[0008] The serum-free culture medium of the present invention features astragalus polysaccharide, salidroside, and tanshinone IIA, which play important roles in culturing umbilical cord mesenchymal stem cells under hypoxic conditions. The three ingredients coordinate and complement each other's functions, collectively overcoming the challenges of oxidative damage, metabolic disruption, and stemness loss faced by cells during hypoxic culture. Astragalus polysaccharide plays a key role in activating signaling pathways and immune regulation in the culture medium. It activates the AMPK signaling pathway within cells, promoting cellular self-regulation of energy metabolism and enhancing mitochondrial function, thereby enhancing the cells' ability to produce ATP under hypoxic conditions. Furthermore, astragalus polysaccharide modulates the cellular microenvironment, inhibits inflammatory responses, and reduces cell damage caused by inflammatory factors. Salidroside in the culture medium induces the stable expression of hypoxia-inducible factor (HIF-1α) within cells, subsequently activating a series of genes associated with hypoxia adaptation and promoting the secretion of angiogenic factors such as vascular endothelial growth factor (VEGF). It also effectively scavenges reactive oxygen species (ROS) within cells, alleviating oxidative stress-induced cell damage and reducing the risk of apoptosis. Tanshinone IIA plays an important role in anti-apoptosis and regulating energy metabolism. It can target and inhibit the mitochondrial apoptosis pathway, stabilize the mitochondrial membrane potential, prevent the release of cytochrome C, and then inhibit the activation of apoptosis-related proteins such as Caspase-9, thereby blocking the occurrence of cell apoptosis from the source.

[0009] The serum-free culture medium of the present invention is characterized in that sodium pyruvate, D-ribose, recombinant human albumin, and recombinant human transferrin primarily play metabolic regulation and nutritional support roles during the culture of umbilical cord mesenchymal stem cells, providing additional energy and material support for cell metabolism under hypoxic conditions. As an energy supplement, sodium pyruvate can smoothly enter the tricarboxylic acid cycle of cells under hypoxic conditions, replenishing the insufficient energy supply caused by hypoxia and maintaining normal cell metabolism and physiological functions. It synergizes with astragalus polysaccharides and tanshinone IIA in energy metabolism. Astragalus polysaccharides enhance mitochondrial function, tanshinone IIA optimizes glycolysis, and sodium pyruvate directly supplements substrates for the tricarboxylic acid cycle, jointly ensuring the energy needs of cells under hypoxic conditions. Adding D-ribose to the culture medium can directly participate in the synthesis of purine nucleotides in cells, quickly replenishing the ATP consumed by cells under hypoxic stress, and providing immediate energy support for cell proliferation and metabolism. Together with sodium pyruvate, it quickly and continuously replenishes energy for cells from different angles and maintains cell vitality. Recombinant human albumin and recombinant human transferrin play an important role in the culture medium. As a nutrient carrier, recombinant human albumin can bind to and transport various nutrients in the basal culture medium, promote the uptake and utilization of these nutrients by cells, and also has certain antioxidant and cell protection functions, which can reduce oxidative damage to cells; recombinant human transferrin is responsible for binding and transporting iron ions, maintaining normal cell metabolism, DNA synthesis and cell proliferation. The two work synergistically to provide cells with a stable nutrient supply and ensure normal cell growth and proliferation.

[0010] Preferably, the serum-free medium for culturing mesenchymal stem cells under hypoxia has the following final concentrations of the following added ingredients: astragalus polysaccharide 10 μg / mL, salidroside 1.0 μg / mL, tanshinone IIA 0.3 μg / mL, sodium pyruvate 0.2 mg / mL, D-ribose 10 mM, recombinant human albumin 5 mg / mL, and recombinant transferrin 8 μg / mL. After adding the added ingredients at the above concentrations, the added ingredients cooperate with each other to enable the culture medium to effectively meet the growth requirements of umbilical cord mesenchymal stem cells under hypoxic conditions, significantly improving the cell proliferation capacity. The proliferation rate after 7 days of culture can reach 533.3±4.8%, while the cell apoptosis rate is significantly reduced, and the total apoptosis rate is reduced to 4.3±0.7%. The expression of stem cell-specific surface markers is stable, with the CD73 / CD90 / CD105 positive cell rate >99% and the Oct4 / Sox2 / Nanog positive cell rate >94%. The cultured umbilical cord mesenchymal stem cells exhibited high multidirectional differentiation potential, with osteogenic differentiation efficiencies reaching 49.8±1.9% and adipogenic differentiation efficiencies reaching 46.5±2.1%. Furthermore, elevated cytokine secretion levels enhanced their application value in cell therapy.

[0011] Preferably, the basal culture medium can be DMEM / F12 culture medium. DMEM / F12 culture medium does not contain serum components and contains a variety of basic nutrients, which can provide a comprehensive and necessary material basis for the growth and metabolism of umbilical cord mesenchymal stem cells under hypoxic conditions.

[0012] In a second aspect of the present invention, the present invention provides a method for culturing umbilical cord mesenchymal stem cells under hypoxic conditions using the above-mentioned serum-free medium, characterized in that it comprises the following steps: Step 1: Cell inoculation: Primary umbilical cord mesenchymal stem cells were cultured at a rate of 1×10 4 ~2×10 4 pieces / cm 2 The cells are inoculated into a culture container at a density of, and the serum-free culture medium is added to obtain an inoculated cell system.

[0013] Step 2, culture under hypoxic conditions: Place the inoculated cell system obtained in step 1 into a hypoxic incubator and culture at 37°C, 5% CO2, 2% O2, and saturated humidity. After 24 hours of culture, replace with fresh serum-free culture medium. Thereafter, replace the medium once every 3 days. Culture until the cell confluence reaches 80% to obtain a confluent cell system. When changing the medium, aspirate the old culture medium under a sterile environment and add new culture medium preheated to 37°C. The above statement of changing the medium once every 3 days means discarding all old serum-free culture medium and adding an equal amount of fresh serum-free culture medium every 3 days.

[0014] Step 3: Subculture: discard the culture medium in the confluent cell system obtained in step 2, wash with PBS, add trypsin for digestion, digest at 37℃ for 1~2min, add fresh serum-free medium to terminate digestion after the cells become round, obtain single cell suspension, collect cells by centrifugation, resuspend the collected cells in serum-free medium, and press 1×10 4 ~2×10 4 pieces / cm 2 The cells were seeded into a new culture container at a density of 100 μg / mL to obtain P1 umbilical cord mesenchymal stem cells.

[0015] Step 4: Cell passaging: The P1 umbilical cord mesenchymal stem cells obtained in step 3 are cultured using the procedures of steps 1 to 3 to obtain P2 to Pn umbilical cord mesenchymal stem cells, where n is ≥ 3.

[0016] Preferably, the culture container is a T25 cell culture flask.

[0017] Preferably, after the trypsin digestion in step 3, the cells are collected by centrifugation at 1000 rpm for 5 minutes.

[0018] In the third aspect of the present invention, the present invention also discloses the use of the above serum-free culture medium in culturing umbilical cord mesenchymal stem cells. In summary, the beneficial effects of the present invention are: (1) The present invention achieves multi-faceted protection of umbilical cord mesenchymal stem cells under hypoxic conditions by adding astragalus polysaccharides, salidroside, and tanshinone IIA to serum-free culture medium. Astragalus polysaccharides activate the AMPK signaling pathway to enhance mitochondrial function and regulate immunity, salidroside induces stable expression of HIF-1α to promote angiogenesis and anti-oxidation, and tanshinone IIA inhibits the mitochondrial apoptosis pathway to block cell apoptosis. The three agents work together to overcome the problems of oxidative damage, metabolic interference, and stemness attenuation faced by cells when cultured under hypoxic conditions, maintain the normal physiological functions and stem cell characteristics of the cells, and solve the problems of cell vulnerability to damage, metabolic disorders, and difficulty in maintaining stemness under hypoxic conditions.

[0019] (2) The present invention achieves synergistic support for cellular energy metabolism by adding sodium pyruvate, D-ribose, astragalus polysaccharide and tanshinone IIA, recombinant human albumin, and recombinant transferrin to the serum-free culture medium. Sodium pyruvate directly supplements tricarboxylic acid cycle substrates, D-ribose participates in purine nucleotide synthesis and rapidly replenishes ATP, astragalus polysaccharide enhances mitochondrial function, tanshinone IIA optimizes glycolysis, and recombinant human albumin and recombinant transferrin promote nutrient uptake and iron ion transport. These three factors work together to ensure the energy supply of cells under hypoxic conditions, maintain the normal metabolism and physiological functions of cells, and solve the problems of insufficient cellular energy supply and metabolic imbalance under hypoxic conditions.

[0020] (3) The present invention adopts a hypoxic culture method and a serum-free culture medium with specific components to significantly optimize the culture effect of umbilical cord mesenchymal stem cells. Under constant hypoxic conditions, the cell proliferation rate is significantly improved, with the maximum proliferation multiple reaching 533.3±4.8% after 7 days of culture; the cell apoptosis rate is significantly reduced, and the anti-apoptosis ability is enhanced, with the total apoptosis rate reduced to 4.3±0.7%; the expression of stem cell-specific surface markers is stable, with the CD73 / CD90 / CD105 positive cell rate >99% and the Oct4 / Sox2 / Nanog positive cell rate >94%; the multidirectional differentiation potential, especially the osteogenic and adipogenic differentiation abilities, is maintained, with the osteogenic differentiation efficiency reaching 49.8±1.9% and the adipogenic differentiation efficiency reaching 46.5±2.1%; at the same time, the cytokine secretion level is improved, which enhances its application value in cell therapy.

[0021] (4) The serum-free culture medium used in the present invention has clear ingredients, stable properties, does not contain serum, and has no batch-to-batch differences. It meets the requirements of standardized production, is conducive to the industrial production and wide application of the culture medium, and can provide a reliable culture medium product for the culture of umbilical cord mesenchymal stem cells in scientific research and clinical applications.

[0022] (5) The present invention combines serum-free culture medium with hypoxic culture conditions, so that the cultured cells have a higher survival rate, stronger proliferation ability and more stable stemness, effectively meeting the demand for high-quality stem cells in regenerative medicine and cell therapy. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs.

[0024] In the present invention, the components and reagents involved are all conventional commercial products or can be obtained by conventional technical means in the art. Unless otherwise specified, the materials, methods and examples of the present invention are only illustrative and not restrictive.

[0025] Example 1: Serum-free culture medium for hypoxic culture of umbilical cord mesenchymal stem cells and its preparation This example provides a serum-free culture medium for culturing mesenchymal stem cells under hypoxia. The basal culture medium is DMEM / F12 medium (purchased from Wuhan Punosai Life Science Co., Ltd.). The final concentrations of the added components are as shown in Table 1. The resulting culture media are labeled as Medium 1, Medium 2, and Medium 3. The steps for preparing the culture medium are as follows: (1) Accurately measure DMEM / F12 basal medium, accurately weigh the corresponding mass of recombinant human albumin, sodium pyruvate and recombinant transferrin, add them to DMEM / F12 basal medium, and stir until completely dissolved to obtain fortified basal medium.

[0026] (2) Accurately weigh astragalus polysaccharide, salidroside and tanshinone IIA respectively, and dissolve them in an appropriate amount of dimethyl sulfoxide. Then slowly add them to the fortified basal medium obtained in step 1, stirring while adding to ensure that all components are evenly dispersed in the medium. D-ribose is directly added to the above mixture to obtain a serum-free medium stock solution.

[0027] (3) Filter the serum-free culture medium obtained in step 2 through a 0.22 μm filter membrane to sterilize, thereby obtaining a serum-free culture medium for umbilical cord mesenchymal stem cell culture under low oxygen conditions. Seal the prepared serum-free culture medium and store it in a refrigerator at 4°C.

[0028] Table 1 Main components of serum-free culture medium Comparative Example 1: A serum-free culture medium without astragalus polysaccharide and its preparation This comparative example provides a serum-free culture medium without astragalus polysaccharide. The medium is based on DMEM / F12 and contains the following additives at final concentrations: salidroside 1.0 μg / mL, tanshinone IIA 0.3 μg / mL, sodium pyruvate 0.2 mg / mL, D-ribose 10 mM, recombinant human albumin 5 mg / mL, and recombinant transferrin 8 μg / mL. The culture medium was prepared in the same manner as in Example 1, except that astragalus polysaccharide was omitted. The resulting culture medium is labeled Comparative Medium 1.

[0029] Comparative Example 2: A serum-free culture medium without salidroside and its preparation This comparative example provides a serum-free culture medium without salidroside and its preparation. The medium is based on DMEM / F12 and the final concentrations of the remaining additives are as follows: astragalus polysaccharide 10 μg / mL, tanshinone IIA 0.3 μg / mL, sodium pyruvate 0.2 mg / mL, D-ribose 10 mM, recombinant human albumin 5 mg / mL, and recombinant transferrin 8 μg / mL. The culture medium preparation method is the same as in Example 1, except that salidroside is not added. The resulting culture medium is labeled Comparative Medium 2.

[0030] Comparative Example 3: A serum-free culture medium without Tanshinone IIA and its preparation This comparative example provides a serum-free culture medium without tanshinone IIA and its preparation. The medium is based on DMEM / F12 and contains the following additives at final concentrations: astragalus polysaccharide 10 μg / mL, salidroside 1.0 μg / mL, sodium pyruvate 0.2 mg / mL, D-ribose 10 mM, recombinant human albumin 5 mg / mL, and recombinant transferrin 8 μg / mL. The culture medium was prepared in the same manner as in Example 1, except that tanshinone IIA was omitted. The resulting culture medium is labeled Comparative Medium 3.

[0031] Comparative Example 4: A serum-free culture medium without sodium pyruvate and D-ribose and its preparation This comparative example provides a serum-free culture medium without sodium pyruvate or D-ribose, and its preparation. The medium is based on DMEM / F12, and the final concentrations of the remaining supplemental ingredients are as follows: astragalus polysaccharide 10 μg / mL, salidroside 1.0 μg / mL, tanshinone IIA 0.3 μg / mL, recombinant human albumin 5 mg / mL, and recombinant transferrin 8 μg / mL. The culture medium preparation method is the same as in Example 1, except that sodium pyruvate and D-ribose are omitted. The resulting culture medium is labeled Comparative Medium 4.

[0032] Comparative Example 5: A serum-free culture medium containing no recombinant human albumin or recombinant transferrin and its preparation This comparative example provides a serum-free culture medium without recombinant human albumin or recombinant transferrin, and its preparation. The medium is based on DMEM / F12, and the final concentrations of the remaining supplemental ingredients are: astragalus polysaccharide 10 μg / mL, salidroside 1.0 μg / mL, tanshinone IIA 0.3 μg / mL, sodium pyruvate 0.2 mg / mL, and D-ribose 10 mM. The culture medium preparation method is the same as in Example 1, except that recombinant human albumin and recombinant transferrin are omitted. The resulting culture medium is labeled Comparative Medium 5.

[0033] Comparative Example 6: Serum-containing culture medium and its preparation This comparative example is based on DMEM / F12 basal culture medium, with 10% fetal bovine serum added, without any other additional components, as a traditional culture medium control, marked as comparative culture medium 6.

[0034] Example 2: Hypoxic culture of umbilical cord mesenchymal stem cells in serum-free medium In this example, mediums 1 to 3 and the medium described in comparative examples 1 to 6 were used to culture mesenchymal stem cells under hypoxic conditions. The specific operation was as follows: Step 1: Cell inoculation: After rapid recovery of frozen P0 cells in a 37°C water bath, wash them 1-2 times with PBS to remove the freezing solution and inoculate them at 1.5×10 4 pieces / cm 2 The cells were seeded at a high density in a T25 cell culture flask to obtain a seeded cell system.

[0035] Step 2: Hypoxic Culture: Place the inoculated cell system obtained in Step 1 into a hypoxic incubator and culture at 37°C, 5% CO2, 2% O2, and saturated humidity. After 24 hours of culture, replace with fresh serum-free culture medium. Thereafter, replace the medium completely every three days until the cell confluence reaches 80%. During the culture medium change, carefully aspirate the old culture medium under a sterile environment and add fresh culture medium preheated to 37°C.

[0036] Step 3: Subculture: Under sterile conditions, carefully remove the culture medium from the confluent cell system obtained in step 2, wash once with PBS, then add an appropriate amount of trypsin and digest at 37°C for 1-2 minutes. After the cells become round under microscopic examination, add the corresponding serum-free medium to terminate the digestion and obtain a single cell suspension. Collect the cells by centrifugation and resuspend the collected cells in serum-free medium at a concentration of 1.5×10 4 pieces / cm 2 The cells were seeded into new T25 culture flasks at a density of 1.5 to obtain the first generation (P1) umbilical cord mesenchymal stem cells.

[0037] Step 4: Cell passage: The P1 umbilical cord mesenchymal stem cells obtained in step 3 are cultured twice using steps 1 to 3 (step 1 starts from the seeding step) to obtain the second generation (P2) and third generation (P3) umbilical cord mesenchymal stem cells, respectively.

[0038] Example 3: Proliferation and activity detection of cells cultured under hypoxic conditions The P3 umbilical cord mesenchymal stem cells cultured in Example 2 were tested according to the following steps: Cell proliferation fold assay: On days 1, 3, 5, and 7 of P3 cell culture, pipette an appropriate amount of cell suspension into a centrifuge tube, centrifuge, and discard the supernatant. Mix a small amount of the cell suspension with trypan blue in a 1:1 ratio and count using a Thermo Fisher Countess3 automated cell counter to calculate cell density and cell proliferation fold. Perform three replicates for each sample.

[0039] Cell viability assay: On the 5th and 7th days of P3 cell culture, the cells in the cell culture flask were digested with trypsin and the cell density was adjusted to 5×10 3 Cells were plated at 100 μL per well in a 96-well plate, with five replicates per well. After a 3-hour incubation, 10 μL of CCK-8 solution was added to each well. The cells were incubated for another 3 hours, and the absorbance (OD) at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula. Three replicates were performed for each sample.

[0040] Cell proliferation fold = (cell density at the detection time point / cell density at the time of inoculation) × 100%.

[0041] Cell activity = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%, the blank group is the well with only culture medium and CCK-8 solution added, and the control group is the result of culture with culture medium in comparative example 6.

[0042] Table 2 shows the cell proliferation folds. As can be seen from the data in the table, the proliferation folds of experimental media 1-3 were significantly higher than those of most control groups at each time point, demonstrating the serum-free culture medium of the present invention's ability to promote umbilical cord mesenchymal stem cell proliferation. Among them, culture medium 2 performed the best, with proliferation folds reaching 200.0±2.8% and 533.3±4.8% on days 3 and 7, respectively, significantly higher than those of the other experimental and control groups. This indicates that the optimized concentration combination of astragalus polysaccharides (10 μg / mL) and salidroside (1.0 μg / mL) effectively accelerates cell division. The proliferation folds of control media 1-3 were all lower than those of the experimental groups, with control media 1 showing only 366.7±5.3% on day 7, confirming the key role of the three ingredients in synergistically promoting proliferation under hypoxic conditions. Control media 4 and 5 showed the lowest proliferation folds, demonstrating the essential role of energy metabolism components and nutrient carrier proteins in cellular energy supply and material transport under hypoxia. The proliferation rate of serum-containing control medium 6 was lower than that of medium 2, indicating the advantage of the serum-free system of the present invention in proliferation efficiency. It further proves that the added components jointly enhance the proliferation potential of cells under hypoxic conditions by activating the AMPK signaling pathway and optimizing glycolysis.

[0043] Table 2 Proliferation of cells cultured under hypoxic conditions Cell viability results are shown in Table 3. Experimental media 1–3 exhibited higher activity than the control group on days 5 and 7, with medium 2 performing best, reaching 95.3 ± 2.1% cell viability on day 7. This demonstrates the enhanced effect of the medium components on cellular metabolic activity. The antioxidant combination of astragalus polysaccharide and salidroside scavenged ROS and stabilized mitochondrial membrane potential, enhancing cell resistance to apoptosis. The apoptosis rate on day 7 was increased by up to 10.7% compared to control medium 1. Control medium 4 exhibited the lowest activity, confirming the critical role of energy metabolism components in maintaining cellular ATP levels and metabolic pathways under hypoxia. Serum-containing control medium 6 exhibited lower activity than the experimental group, indicating that the serum-free medium of the present invention, through synergistic composition, effectively reduced oxidative stress damage and maintained higher cellular integrity and metabolic activity. Across all experimental groups, the high activity of medium 2 was consistent with the proliferation rate trend, confirming the optimal regulatory effect of the core ingredient combination of "astragalus polysaccharide-salidroside-tanshinone IIA" proposed in this invention on cell survival and metabolism under hypoxic conditions, providing a reliable basis for high-quality stem cell culture.

[0044] Table 3 Cell viability assay under hypoxic conditions Example 4: Detection of apoptosis rate of cells cultured under hypoxic conditions The P3 generation umbilical cord mesenchymal stem cells cultured in Example 2 were detected, and the detection was carried out as follows: the P3 generation umbilical cord mesenchymal stem cells cultured to the 5th day were taken, the supernatant was discarded, and an appropriate amount of trypsin was added to digest the adherent cells. After microscopic observation of the cells starting to round and the intercellular space increasing, a single cell suspension was gently blown away and transferred to a centrifuge tube, centrifuged at 1000r / min for 5min, and the supernatant was discarded. The cultured cells were washed 2 times with pre-cooled PBS, 500μL Binding Buffer was added to resuspend the cells, and 5μL Annexin V-FITC and 5μL PI were then added respectively, mixed gently, and incubated in the dark at room temperature for 15-20min. The stained cell suspension was transferred to a flow tube, detected immediately by flow cytometry, and apoptosis data was recorded. The apoptosis rate was calculated according to the following formula. Three replicate experimental groups were set for each sample.

[0045] Early apoptotic cell rate (%) = number of early apoptotic cells / total number of cells × 100%.

[0046] Late apoptotic cell rate (%) = number of late apoptotic cells / total number of cells × 100%.

[0047] Total cell apoptosis rate (%) = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%.

[0048] Table 4 Apoptosis rate of mesenchymal cells cultured under hypoxic conditions As shown in Table 4, the early apoptosis rate, late apoptosis rate, and total apoptosis rate of experimental culture media 1 to 3 were significantly lower than those of the control group, demonstrating the effective inhibitory effect of the serum-free culture medium of the present invention on cell apoptosis under hypoxic conditions. Among them, culture medium 2 performed the best, with an early apoptosis rate of 3.2±0.5%, a late apoptosis rate of 1.1±0.3%, and a total apoptosis rate of only 4.3±0.7%, which was significantly lower than all control groups. This indicates that the synergistic effect of components such as astragalus polysaccharides, salidroside, and tanshinone IIA can effectively regulate the intracellular antioxidant enzyme system, inhibit ROS production and stabilize mitochondrial membrane potential, thereby blocking the Caspase-9-mediated apoptosis pathway from the source. The total apoptosis rate of control culture media 1 to 3 was significantly higher than that of the experimental group, verifying the indispensability of the added components in anti-apoptosis. The synergistic effect of the three reduced the apoptosis rate by 57.8% to 62.1% compared to the group lacking a single component. The total apoptosis rates of control cultures 4 and 5 were as high as 17.7±1.8% and 16.0±1.6%, respectively, indicating that the absence of energy metabolism components and nutrient carrier proteins can lead to decreased cellular ATP levels and impaired iron ion transport, exacerbating energy metabolism disorders under hypoxia and inducing apoptosis. The total apoptosis rate of serum-containing control culture 6 was 13.6±1.5%, higher than that of the experimental group, indicating that the serum-free system of the present invention, through its clear combination of components, is superior to traditional serum culture media in anti-apoptosis effects.

[0049] Example 5: Detection of surface markers of cells cultured under hypoxic conditions The P3 umbilical cord mesenchymal stem cells cultured in Example 2 were tested according to the following steps: P3 umbilical cord mesenchymal stem cells were cultured to day 7. The adherent cells were digested with an appropriate amount of trypsin. After digestion for 1 min, the corresponding culture medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in the corresponding culture medium to adjust the cell concentration to 1×10 6 Cells were plated at 400 μg / mL. 100 μL of cell suspension was transferred to a flow cytometer. Appropriate amounts of fluorescently labeled anti-CD73, CD90, CD105, CD34, CD45, CD19, and HLA-DR antibodies were added. The cells were allowed to react in the dark for 30 minutes at room temperature. The cells were then washed once with PBS and resuspended in 500 μL of PBS. Cell surface marker expression was determined by flow cytometry, and the percentage of positive cells was analyzed. Three replicates were performed for each sample.

[0050] Table 5 Surface marker expression positive rate of cells cultured under hypoxic conditions Table 5 shows the results of surface marker testing. The positive rates for umbilical cord mesenchymal stem cell-specific markers (CD73, CD90, and CD105) in experimental culture media 1-3 all remained above 97.6%, with culture media 2 reaching 99.0% to 99.3%, significantly higher than those in the control group. This demonstrates the serum-free culture media's ability to effectively maintain stem cell stemness. In contrast, the positive rates for culture media 1-3 decreased to 95.5% to 96.8%, confirming that these three media play a key role in regulating the expression of stemness genes (Oct4 and Sox2) by activating the Wnt / β-catenin and Notch signaling pathways.

[0051] Negative marker detection results (CD34, CD45, CD19, and HLA-DR) showed that the positive rates in the experimental groups were all <1.5%, significantly lower than those in the control groups (4.1%-5.1%). This demonstrates that the culture medium of the present invention effectively inhibits hematopoietic cell contamination (CD34 / CD45) and antigen-presenting cell activation (CD19 / HLA-DR), maintaining the high purity of the cell culture system. The positive rates of negative markers in control culture media 4 and 5 increased to over 4%, reflecting residual non-mesenchymal cells due to energy metabolism disorders or nutrient carrier deficiency. This confirms the importance of sodium pyruvate supplementation of the tricarboxylic acid cycle and recombinant human transferrin in regulating iron metabolism for stabilizing cell phenotype. The positive rate in serum-containing control culture medium 6 was still higher than that in the experimental group, demonstrating that the serum-free system of the present invention, through the use of clearly defined additive factors, can more accurately eliminate the interference of exogenous components in serum on cell phenotype.

[0052] Example 6: Stemness gene detection of cells cultured under hypoxia conditions The P3 generation umbilical cord mesenchymal stem cells cultured in Example 2 were detected, and the detection was carried out according to the following steps: P3 generation umbilical cord mesenchymal stem cells cultured to the 7th day were taken, digested with an appropriate amount of trypsin, centrifuged and discarded the supernatant, washed twice with PBS, and centrifuged again and discarded the supernatant. Then, an appropriate amount of 4% paraformaldehyde solution was added to the cell pellet, fixed at room temperature for 15 to 20 minutes, centrifuged at 1000 rpm for 5 minutes, discarded the supernatant, and washed the cells twice with PBS. A PBS solution containing 0.1% TritonX-100 was added and permeabilized at room temperature for 10 to 15 minutes. The permeabilized cells were resuspended in PBS solutions containing Oct4 primary antibody, Sox2 primary antibody and Nanog primary antibody, respectively, and incubated overnight at 4°C in the dark. The next day, the cells were centrifuged and the supernatant was discarded. The cells were washed three times with PBS, and then the AlexaFluor488 fluorescently labeled secondary antibodies corresponding to the primary antibodies were added. The cells were incubated in the dark at room temperature for 1-2 hours. After washing three times with PBS, the washed cells were resuspended in an appropriate amount of PBS and the cell concentration was adjusted to 1×10 6 ~1×10 7Cells / mL were transferred to a sample tube dedicated to the flow cytometer. Detection was performed using a flow cytometer, with the excitation wavelength set to 488 nm and the emission wavelength set to 519 nm, collecting fluorescence signals with a wavelength of 510-530 nm. The fluorescence intensity of each cell was analyzed and recorded by flow cytometry, and unstained cells were set as negative controls. The number of positive cells and the total number of detected cells were counted by the flow cytometer software, and the positive cell rate was calculated according to the following formula: Oct4 positive rate (%) = number of cells expressing Oct4 gene positive / total number of cells detected × 100% Sox2 positive rate (%) = number of Sox2 gene positive cells / total number of cells detected × 100% Nanog positive rate (%) = number of Nanog gene expression positive cells / total number of detected cells × 100% Table 6 Positive expression rate of stemness genes in cells cultured under hypoxia conditions Table 6 shows the positive rates of stemness gene expression in umbilical cord mesenchymal stem cells (UCMSCs) cultured in experimental media 1–3. These rates were maintained above 89.7%, 10%–15% higher than those in control media 1–3. The positive rates for Oct4, Sox2, and Nanog in cultured media 2 were 96.7%, 95.9%, and 94.8%, respectively, significantly higher than those in the other groups. This suggests that astragalus polysaccharides, salidroside, and tanshinone IIA may synergistically promote stemness gene transcription by activating the Wnt / β-catenin and Notch signaling pathways. The positive rates in control media 1–3 decreased to 82%–85%, demonstrating the essential role of astragalus polysaccharides, salidroside, and tanshinone IIA. The positive rates in control media 4 and 5 were below 80%, reflecting the suppression of stemness gene expression by disrupted energy metabolism and impaired iron transport. This suggests that sodium pyruvate supplementation of the tricarboxylic acid cycle and recombinant human transferrin in regulating iron-dependent gene transcription are crucial for maintaining stemness. The positive rate of the comparative culture medium 6 was 85% to 88%, slightly lower than that of the experimental group, indicating that the serum-free culture medium of the present invention can more accurately maintain the expression of stemness genes by adding factors with clear components, and avoid the interference of growth factor fluctuations in serum on cell phenotype.

[0053] Example 7: Differentiation ability of cells cultured under hypoxic conditions The differentiation ability of the P3 umbilical cord mesenchymal stem cells cultured in Example 2 was tested. The test was performed according to the following steps: P3 umbilical cord mesenchymal stem cells were taken and induced using osteogenic induction medium and adipogenic induction medium (the osteogenic induction medium was based on DMEM / F12 medium, and 1×10 -7mol / L dexamethasone, 10mmol / L β-glycerophosphate sodium, 50μg / mL vitamin C, 100U / mL penicillin, and 100μg / mL streptomycin; adipogenic induction medium was based on DMEM / F12 medium, with 1×10 -6 mol / L dexamethasone, 0.5mmol / L 3-isobutyl-1-methylxanthine (IBMX), 10μg / mL insulin, 200μmol / L indomethacin, 100U / mL penicillin, and 100μg / mL streptomycin). The cultured P3 umbilical cord mesenchymal stem cells were cultured at 1×10 4 pieces / cm 2 Cells were seeded at a density of 100 μg / mL in 6-well plates, and 2 mL of osteogenic induction medium or 2 mL of adipogenic induction medium was added to each well. Osteogenic induction culture was performed for 21 days, during which the medium was replaced every 3 days; adipogenic induction culture was performed for 14 days, during which the induction medium was replaced every 3 days. After induction, the culture medium was discarded, the cells were washed twice with PBS, and then 4% paraformaldehyde was added for fixation for 30 minutes, and then washed three times with PBS. Then, Alizarin Red stain was added to the osteogenic induction test group and stained for 30 minutes at room temperature in the dark. The cells were washed several times with distilled water to remove excess stain; Oil Red O stain was added to the adipogenic induction experimental group and stained for 60 minutes at room temperature in the dark. The cells were then washed several times with 60% isopropanol to remove excess stain. The 6-well plates were observed under an inverted microscope, and multiple fields of view were selected for counting. The cell differentiation efficiency was calculated according to the formula. Three replicate experimental groups were set up for each sample.

[0054] Osteoblast differentiation efficiency = (total number of mineralized nodule cells / total number of cells) × 100%.

[0055] Adipocyte differentiation efficiency = (total number of adipogenic staining / total number of cells) × 100%.

[0056] Table 7 Differentiation ability of cells cultured under hypoxic conditions in serum-free medium The results, as shown in Table 7, showed that compared to control media 1-5, media 1-3 exhibited higher osteoblast differentiation efficiencies. Culture 2 exhibited the highest osteoblast differentiation efficiency (49.8±1.9%), representing a 28%-35% increase compared to control media 1-3. This suggests that the addition of astragalus polysaccharides, salidroside, and tanshinone IIA to the culture media of the present invention may promote the expression of osteoblast-related genes (Runx2 and Osterix) by activating the BMP / Smad signaling pathway, thereby accelerating mineralized nodule formation. Culture 2 also exhibited the highest adipogenic differentiation efficiency, suggesting that its core components may promote lipid droplet accumulation by regulating the PPARγ pathway. D-ribose and sodium pyruvate provide energy precursors (such as acetyl CoA) for fatty acid synthesis. Control media 4 exhibited the lowest osteogenic / adipogenic efficiencies (31.5% / 27.1%), suggesting that disrupted energy metabolism may lead to insufficient ATP required for differentiation, impairing extracellular matrix synthesis in osteoblasts and lipid deposition in adipocytes. The differentiation efficiency of comparison cultures 1-3 decreased by 15%-20%, indicating that astragalus polysaccharides, salidroside, and tanshinone IIA are essential for initiating differentiation. The differentiation efficiency of comparison culture 6 was slightly lower than that of the experimental group, demonstrating that the serum-free culture medium of the present invention, through the precise addition of ingredients, can more effectively induce mesenchymal stem cells to differentiate into the mesodermal lineage, avoiding the interference of complex components in serum on differentiation signals, achieving an effect comparable to that of serum culture medium.

[0057] Example 8: Cytokine secretion of cells cultured under hypoxic conditions The cytokine secretion of the P3 generation umbilical cord mesenchymal stem cells cultured in Example 2 was detected, and the detection was performed according to the following steps: P3 generation umbilical cord mesenchymal stem cells cultured to day 5 were taken, and after centrifugation to remove cell debris, the VEGF, HGF, IL-10, and TGF-β cytokine concentrations therein were detected using an ELISA kit. The specific operation was to first prepare an ELISA kit, set up standard wells, sample wells, and blank wells in a 96-well plate, and sequentially add the standard, the treated supernatant, and related reagents. After incubation at 37°C and washing the plate (repeated 3 to 5 times), the detection antibody and enzyme-labeled secondary antibody were added and incubated and washed. The substrate solution was then added to protect from light for color development. Finally, the reaction was terminated with a stop solution, and the absorbance value (OD value) at the corresponding wavelength was detected by a microplate reader. The cytokine concentration was calculated according to the standard curve, and the data were statistically analyzed to evaluate the factor secretion of the cells under hypoxic conditions.

[0058] Table 8 Cytokine secretion of cells cultured under hypoxia conditions in serum-free medium The results, as shown in Table 8, show that experimental cultures 1-3 secreted significantly more cytokines than control cultures 1-5. Culture 2 achieved the highest VEGF and HGF concentrations, reflecting a synergistic effect between hypoxia-induced HIF-1α and astragalus polysaccharides and salidroside, promoting vascular endothelial growth factor synthesis by 35%-50% compared to control cultures 1-3. Furthermore, after 5 days of culture, the VEGF secretion concentration of P3 cells reached ≥1800 pg / mL, demonstrating its significance for the treatment of ischemic diseases. Culture 2 also produced significantly higher levels of IL-10 and TGF-β than the control culture, indicating that tanshinone IIA inhibits the NF-κB inflammatory pathway and astragalus polysaccharides promote regulatory T cell polarization, jointly enhancing immune regulation. Control cultures 4 and 5 produced the lowest cytokine secretion concentrations, likely due to insufficient ATP production and impaired amino acid transport, which impair cytokine synthesis and secretion. Cytokine secretion decreased by 20%-30% in control cultures 1-3, demonstrating the necessity of astragalus polysaccharides, salidroside, and tanshinone IIA.

[0059] In summary, the present invention relates to a serum-free culture medium for culturing umbilical cord mesenchymal stem cells under hypoxic conditions. By adding ingredients such as astragalus polysaccharide, salidroside, tanshinone IIA, sodium pyruvate, D-ribose, recombinant human albumin, and recombinant transferrin, the medium significantly improves the proliferation efficiency of cells under hypoxic conditions, reduces the total apoptosis rate, effectively maintains the stemness of stem cells, and enhances osteogenic and adipogenic differentiation capabilities and the secretion level of immunomodulatory factors. This culture medium has clear ingredients and optimized ratios, solving the key issues of cell survival, proliferation, and functional maintenance in hypoxic culture. It also eliminates the risk of animal serum, has high batch stability, and meets the standardized production requirements for large-scale stem cell culture. It provides an efficient and safe solution for the scientific research transformation and clinical application of umbilical cord mesenchymal stem cells in the field of regenerative medicine.

[0060] At the same time, the present invention provides scientific ideas and methods for culturing umbilical cord mesenchymal stem cells using the serum-free culture medium. The technical solutions described provide ideas and methods for technicians in this field. For ordinary technicians in this technical field, technical solutions such as component optimization, concentration adjustment and culture condition improvement based on the core principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A serum-free culture medium for umbilical cord mesenchymal stem cells, characterized in that: The product is composed of a basal culture medium and supplementary ingredients. The supplementary ingredients include, by final concentration, astragalus polysaccharide 5-20 μg / mL, salidroside 0.5-2.0 μg / mL, tanshinone IIA 0.1-0.5 μg / mL, sodium pyruvate 0.1-0.3 mg / mL, D-ribose 5-15 mM, recombinant human albumin 3-6 mg / mL, and recombinant transferrin 5-10 μg / mL.

2. The serum-free culture medium according to claim 1, wherein The final concentrations of the added components were as follows: astragalus polysaccharide 10 μg / mL, salidroside 1.0 μg / mL, tanshinone IIA 0.3 μg / mL, sodium pyruvate 0.2 mg / mL, D-ribose 10 mM, recombinant human albumin 5 mg / mL, and recombinant transferrin 8 μg / mL.

3. The serum-free culture medium according to claim 1, wherein The basic culture medium is DMEM / F12 culture medium.

4. A method for culturing umbilical cord mesenchymal stem cells under hypoxic conditions using the serum-free medium according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1: Cell inoculation: Primary umbilical cord mesenchymal stem cells were cultured at a rate of 1×10 4 ~2×10 4 pieces / cm 2 inoculating the cells at a density of 100 μg / ml in a culture container, adding the serum-free culture medium, and obtaining an inoculated cell system; Step 2: Hypoxic culture: Place the inoculated cell system obtained in step 1 into a hypoxic incubator and culture at 37°C, 5% CO2, 2% O2, and saturated humidity. After 24 hours of culture, replace with fresh serum-free culture medium. Thereafter, replace the medium completely every 3 days until the cell confluence reaches 80%, thereby obtaining a post-confluent cell system. Step 3: Subculture: discard the culture medium in the confluent cell system obtained in step 2, wash with PBS, add trypsin for digestion, digest at 37℃ for 1-2 min, add serum-free medium to terminate digestion after the cells become round, obtain single cell suspension, collect cells by centrifugation, resuspend the collected cells in fresh serum-free medium, and press 1×10 4 ~2×10 4 pieces / cm 2 The cells were seeded into a new culture container at a density of 100 μg / mL to obtain P1 umbilical cord mesenchymal stem cells; Step 4: Cell passaging: The P1 generation umbilical cord mesenchymal stem cells obtained in step 3 are cultured using the operations of steps 1 to 3 to obtain P2 to Pn generation umbilical cord mesenchymal stem cells in sequence.

5. The method according to claim 4, characterized in that The culture container is a T25 cell culture flask.

6. The method according to claim 4, characterized in that After trypsin digestion as described in step 3, cells were collected by centrifugation at 1000 rpm for 5 min.

7. Use of the serum-free culture medium according to any one of claims 1 to 3 in culturing umbilical cord mesenchymal stem cells.

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