Long-term Preservation Medium for Stem Cells Based on a Three-dimensional Bionic Microenvironment and Construction Method

Abstract

This application relates to the field of cell preservation, and discloses a long-term preservation medium for stem cells based on a three-dimensional biomimetic microenvironment, which comprises the following component materials in parts by mass: flexible skeleton, benzoyl peroxide, superoxide dismutase, retinoic acid, ferulic acid, mixed buffer, nucleic acid aptamer targeting stem cells, serum-free medium, bFGF and TGF-β. Its construction method comprises the following steps: S1, three-dimensional scaffold construction; S2, antioxidant loading; S3, buffer preparation; S4, aptamer modification; S5, medium integration. The three-dimensional scaffold structure constructed by natural polysaccharides and nanocomposites, combined with the biomimetic pore and flexible network design, effectively simulates the extracellular matrix microenvironment. At the same time, by improving the structural stability and chemical compatibility of the cell adhesion interface, the cell morphology maintenance and adhesion force maintenance during the freezing process are realized, so that the scaffold provides a dynamically adaptable stable environment for cells structurally.

Description

Technical Field

[0001] The present invention relates to the technical field of cell preservation, and specifically to a long-term preservation medium for stem cells based on a three-dimensional biomimetic microenvironment and a construction method thereof. Background Technique

[0002] Currently, for the three-dimensional culture and cryopreservation of stem cells, many studies have focused on the construction of scaffold materials, the optimization of cryoprotective systems, and the control of oxidative stress. Common solutions mostly use natural or synthetic polysaccharides as the base materials, and three-dimensional scaffolds with a certain pore structure are prepared by physical or chemical methods to simulate the extracellular matrix environment and support cell adhesion and proliferation. During the cryopreservation of stem cells, the traditional cryopreservation system uses DMSO as the core cryoprotectant, which can reduce cell structure damage by inhibiting ice crystal formation. At the same time, some studies have introduced antioxidants to control the generation of ROS during the resuscitation process and tried to use buffers to adjust the system stability. In addition, in terms of the targeted enrichment of stem cells, attempts have also been made to improve the adhesion selectivity through surface modification, affinity layer construction, etc.

[0003] With the increasing demand for the application of stem cells, there are still certain limitations in the existing technology during the specific application process. In terms of the scaffold structure, it is still difficult to simultaneously take into account the uniformity of pore distribution and the forming stability. Some methods have insufficient structural integrity under low-temperature treatment and are difficult to continuously provide effective support for cells. In the cryoprotective system, although a single cryoprotectant can partially relieve freeze-thaw stress, it is still limited in controlling the accumulation of cross-phase free radicals and maintaining membrane integrity. On the other hand, some buffer systems have pH fluctuations in the freeze-thaw dynamic environment, which may affect cell metabolic balance and functional expression. For stem cells positive for specific markers, how to achieve efficient and stable enrichment in a three-dimensional scaffold still faces problems such as rough operation methods and interference from non-target cells. Summary of the Invention

[0004] In view of the deficiencies of the existing technology, the present invention provides a long-term preservation medium for stem cells based on a three-dimensional biomimetic microenvironment and a construction method thereof, which solves the problems of decreased survival rate, increased oxidative damage, and low targeted enrichment efficiency of stem cells during cryopreservation and three-dimensional culture.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A long-term preservation medium for stem cells based on a three-dimensional biomimetic microenvironment, comprising the following component materials in parts by mass:

[0006] 9 - 17 parts of a flexible skeleton;

[0007] 0.01 - 0.1 part of benzoyl peroxide;

[0008] 1 - 2 parts of superoxide dismutase;

[0009] 1.5 - 2.5 parts of retinoic acid;

[0010] 0.5 - 1.5 parts of ferulic acid;

[0011] 90.5 - 130.5 parts of mixed buffer;

[0012] 0.1 - 1.0 part of nucleic acid aptamer targeting stem cells;

[0013] 1000 parts of serum - free medium;

[0014] 1 - 5 parts of bFGF;

[0015] 0.5 - 2.0 parts of TGF - β;

[0016] Furthermore, sodium carboxymethyl chitosan is used as the basic material of the scaffold, which has good biodegradability and biocompatibility. The control of its degree of deacetylation and molecular weight enables the material to provide support and regulate the growth of stem cells during culture. Matched with it is nano - hydroxyapatite, which helps stem cells to attach and proliferate well in the three - dimensional scaffold by providing mineralization characteristics similar to bone tissue, thus simulating the microenvironment of natural biological tissues and promoting the long - term survival of stem cells. Benzoyl peroxide is a commonly used free - radical initiator, especially used in thermal - initiation and photo - initiation type free - radical polymerization reactions. In this invention, it is used in the ultraviolet - related reaction process between sodium carboxymethyl chitosan and nano - hydroxyapatite; superoxide dismutase, as a powerful antioxidant, can eliminate the oxidative stress that may occur during culture and protect cells from damage by free radicals. Retinoic acid helps to maintain the stemness of stem cells, prevent their premature differentiation, and ensure the regeneration potential of stem cells. Ferulic acid, as a natural antioxidant, can further enhance the antioxidant ability and protect stem cells from the influence of environmental changes. To adjust the pH value and ionic strength of the culture medium, components such as sodium chloride, trisodium citrate, trehalose, and hydroxyethyl starch are added to maintain a stable culture environment. The nucleic acid aptamer targeting stem cells is targeted - modified by specifically recognizing markers (such as CD73), enhancing the directional differentiation potential of stem cells. In addition, growth factors such as serum - free medium, bFGF, and TGF - β in the formula further support the proliferation and function maintenance of stem cells.

[0017] Preferably, the flexible skeleton includes sodium carboxymethyl chitosan and nano - hydroxyapatite, wherein the degree of deacetylation of sodium carboxymethyl chitosan is 85 - 95%, and the molecular weight is 50 - 100 kDa;

[0018] The particle size of the nano - hydroxyapatite is 20 - 50 nm, and the interlayer spacing of montmorillonite is 1.2 - 1.5 nm;

[0019] The mass ratio of the sodium carboxymethyl chitosan to the nano-hydroxyapatite is 8-12:2-4.

[0020] Preferably, the retinoic acid is retinoic acid palmitate with a purity of 98-99.5%.

[0021] Preferably, the mixed buffer solution includes sodium chloride, trisodium citrate, trehalose and hydroxyethyl starch, and their mass ratio is 3-5:1-1.5:0.05-0.1:0.02-0.05;

[0022] The pH value of the mixed buffer solution is 7.4±0.1.

[0023] Preferably, the nucleic acid aptamer sequence has a length of 15-30 nt and the target marker is CD73;

[0024] The serum-free medium is DMEM / F12, the pyruvate concentration is 1-5 mM, the L-glutamine concentration is 2-4 mM, and the dissociation constant ≤5 nm.

[0025] A method for constructing a long-term preservation medium for stem cells based on a three-dimensional biomimetic microenvironment includes the following steps:

[0026] S1. Three-dimensional scaffold construction: Mix the sodium carboxymethyl chitosan and the nano-hydroxyapatite in proportion, add deionized water to dissolve to form a uniformly dispersed solution, and then add benzoyl peroxide for ultraviolet cross-linking to obtain a porous scaffold;

[0027] S2. Antioxidant loading: Weigh superoxide dismutase, retinoic acid and ferulic acid in proportion, and then encapsulate them together in calcium alginate microspheres. After vacuum adsorption for 7-9 minutes, place them in the pores of the scaffold under a pressure of 0.1 MPa;

[0028] S3. Buffer solution preparation: Mix sodium chloride and trisodium citrate in proportion, and then add trehalose and hydroxyethyl starch;

[0029] S4. Aptamer modification: Fix the target aptamer on gold nanoparticles, and spray it onto the surface of the scaffold at a spraying amount of 15-25 μL / cm 2 under a pressure of 0.3 MPa;

[0030] S5. Medium integration: Immerse the scaffold in 1000 parts of serum-free medium, add bFGF and TGF-β, and let it stand at 4°C for 10-12 hours.

[0031] Furthermore, first, the three-dimensional scaffold is constructed by mixing sodium carboxymethyl chitosan and nano-hydroxyapatite in a specific ratio and forming a highly porous scaffold through ultraviolet crosslinking. Among them, the ultraviolet crosslinking process does not rely on the direct participation of sodium carboxymethyl chitosan or nano-hydroxyapatite itself in the free radical polymerization reaction, but by adding benzoyl peroxide as a free radical initiator. In this way, under ultraviolet light excitation, free radicals are generated on the carboxyl or hydroxyl groups of the sodium carboxymethyl chitosan molecular chain, and then crosslink with adjacent chain segments to form a stable scaffold network structure. At the same time, the Ca 2+ released by nano-hydroxyapatite can form an ionic chelate structure with the sodium carboxymethyl chitosan molecule, thereby jointly enhancing the formability and mechanical properties of the scaffold. The scaffold generated in this way provides the physical support required for stem cell growth and mimics the microstructure of bone tissue, which helps the attachment and growth of stem cells. Next, in the antioxidant loading step, superoxide dismutase, retinoic acid and ferulic acid are mixed in proportion and encapsulated in calcium alginate microspheres. Through vacuum adsorption and pressure treatment, these antioxidants and active substances are effectively loaded into the pores of the scaffold to ensure their continuous release during the culture process and improve the survival rate of stem cells. Subsequently, buffer solution preparation is carried out. Sodium chloride and trisodium citrate are mixed in proportion, and then trehalose and hydroxyethyl starch are added to form a stable mixed buffer solution, and its pH value is adjusted to 7.4±0.1 to ensure a suitable environment for the culture medium. Next, aptamer modification is achieved by immobilizing the targeting aptamer on gold nanoparticles. The specific operation of immobilization is as follows: select a nucleic acid aptamer modified with a thiol group at the 5' or 3' end, with a base length of 15–30 nt, which has specific recognition ability for the CD73 marker. Dissolve the aptamer in DEPC-treated RNase-free water to prepare a solution with a concentration of 5–20 μM; take another gold nanoparticle solution with a particle size range of 10–30 nm and a concentration of 0.01–0.05% mass fraction. Under dark conditions, slowly drop the aptamer solution into the gold nanoparticle solution while stirring magnetically, maintaining the reaction temperature at 4–8 °C and the reaction time at 6–12 hours to form an Au–S bond between the thiol group and the gold surface, realizing the stable and directional immobilization of the aptamer on the surface of the gold nanoparticles. After the reaction is completed, free aptamers can be removed by low-speed centrifugation or dialysis to obtain the aptamer-AuNP complex, which is sprayed onto the surface of the scaffold under a specific pressure to ensure that the aptamer can effectively bind to the CD73 marker on the surface of stem cells and achieve a directional targeting effect. Finally, in the culture medium integration step, the three-dimensional scaffold is soaked in a serum-free culture medium, growth factors such as bFGF and TGF-β are added, and it is left standing at a low temperature for a certain time to promote cell proliferation and differentiation while maintaining the stemness and function of the cells.

[0032] Preferably, in the step S1, the concentration of the prepolymer solution for ultraviolet crosslinking is 10%, the wavelength is 355-375 nm, and the intensity is 9-11 mW / cm2 , at a time of 8 - 12 min, the porosity of the crosslinked scaffold is 70 - 90%, and the elastic modulus is 3 - 5 kPa.

[0033] Preferably, in the step S2, the calcium alginate microspheres have a particle size of 20 - 40 μm, which are prepared by microfluidic technology, the flow rate is 5 μL / min, and the curing solution is 0.2 M CaCl2.

[0034] Preferably, in the step S4, the gold nanoparticles have a particle size of 8 - 12 nm, the density of the aptamers modified on their surface is 100 - 200 molecules / μm2, and the aptamer immobilization rate is ≥90%.

[0035] Preferably, in the step S5, the temperature during the soaking of the scaffold is 4 - 8 °C, and the dissolved oxygen content in the culture medium after soaking is maintained at 5 - 8 mg / L.

[0036] The present invention provides a long - term preservation culture medium for stem cells and a construction method based on a three - dimensional bionic microenvironment. It has the following beneficial effects:

[0037] 1. The three - dimensional scaffold structure constructed by the present invention through natural polysaccharides and nanocomposites, combined with the design of bionic pores and flexible networks, effectively simulates the extracellular matrix microenvironment. By enhancing the structural stability and chemical compatibility of the cell adhesion interface, the cell morphology and adhesion force during the freezing process are maintained. Compared with the conventional DMSO - free support system, the problems of cell drift, decreased adhesion rate, and irreversible morphological damage during freezing are solved, and the scaffold provides a dynamically adaptable stable environment for cells in terms of structure.

[0038] 2. The present invention constructs a ternary antioxidant system with both water - soluble and lipid - soluble free - radical scavenging capabilities, covering superoxide dismutase, retinoic acid, and ferulic acid components, which is suitable for the control of oxidative stress in a cross - phase environment. Through a multi - point action mechanism, the ability of cells to regulate their internal homeostasis under low - temperature and high - oxygen pressure conditions is guaranteed. Different from the existing single antioxidant or linear action mechanism, this system improves the applicable limitations of traditional antioxidant strategies in terms of interface permeability and oxidative chain break control.

[0039] 3. The present invention adopts a compound buffer system, which is composed of phosphate and citrate, to construct a microenvironment with better pH stability under freeze - thaw dynamic conditions. This scheme overcomes the problem of excessive acid - base fluctuations of traditional PBS under low - temperature conditions, and improves the physiological consistency of different - source stem cells during the same treatment process. The regulatory role of the buffer system further improves the repeatability and applicability of the system, especially suitable for the cross - source freezing application scenario of stem cells.

[0040] 4. The present invention realizes the precise capture and fixation of stem cells expressing specific surface markers by modifying the surface of the scaffold with gold nanocarriers conjugated with aptamers. This strategy constructs a highly selective adhesion interface within the three-dimensional scaffold, avoiding the non-specific damage to target cells caused by traditional physical enrichment methods. Compared with conventional separation methods such as centrifugation or screening, this technology improves the purity of the cell population and the stability of subsequent functional expression while maintaining cell viability. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a flowchart of the construction method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0043] Please refer to the attached Figure 1 , Example 1:

[0044] Composition and mass fraction of materials:

[0045] Flexible skeleton: 9 parts;

[0046] Benzoyl peroxide: 0.01 part;

[0047] Superoxide dismutase: 1.0 part;

[0048] Retinoic acid: 1.5 parts;

[0049] Ferulic acid: 0.5 part;

[0050] Mixed buffer: 90.5 parts;

[0051] Nucleic acid aptamer targeting stem cells: 0.1 part;

[0052] Serum-free medium: 1000 parts;

[0053] bFGF: 1 part;

[0054] TGF-β: 0.5 part;

[0055] Mass ratio of flexible skeleton components:

[0056] Sodium carboxymethyl chitosan: nano-hydroxyapatite = 8:2.

[0057] Mass ratio of buffer components:

[0058] Sodium chloride: trisodium citrate: trehalose: hydroxyethyl starch = 3:1:0.05:0.02.

[0059] Preparation steps:

[0060] S1. Mix sodium carboxymethyl chitosan with nano-hydroxyapatite, add deionized water and stir evenly, then add benzoyl peroxide and inject into a mold, and form a three-dimensional porous scaffold through ultraviolet cross-linking.

[0061] S2. Mix superoxide dismutase, retinoic acid and ferulic acid, then encapsulate them into calcium alginate microspheres, and load them into the pores of the three-dimensional scaffold by vacuum adsorption or pressure osmosis to obtain an antioxidant-loaded scaffold.

[0062] S3. Prepare a buffer solution containing sodium chloride, trisodium citrate, trehalose and hydroxyethyl starch, adjust the pH to an appropriate range and then compound it with the scaffold.

[0063] S4. Prepare an aptamer-gold nanoparticle complex with a targeting function and uniformly fix it on the surface of the three-dimensional scaffold by pressure spraying to form a targeted scaffold.

[0064] S5. Immerse the scaffold in a serum-free medium, add growth factors to the medium, and let it stand at low temperature to complete the integration of the medium and the three-dimensional scaffold, and obtain the target culture system.

[0065] Example 2

[0066] Material composition and parts by mass:

[0067] Flexible skeleton: 13 parts;

[0068] Benzoyl peroxide: 0.05 part;

[0069] Superoxide dismutase: 1.5 parts;

[0070] Retinoic acid: 2.0 parts;

[0071] Ferulic acid: 1.0 part;

[0072] Mixed buffer: 110 parts;

[0073] Nucleic acid aptamer targeting stem cells: 0.55 part;

[0074] Serum-free medium: 1000 parts;

[0075] bFGF: 3 parts;

[0076] TGF-β: 1.25 parts;

[0077] Mass ratio of flexible skeleton components:

[0078] Sodium carboxymethyl chitosan:nano-hydroxyapatite = 10:3.

[0079] Mass ratio of buffer components:

[0080] Sodium chloride: trisodium citrate: trehalose: hydroxyethyl starch = 4:1.2:0.08:0.04.

[0081] Preparation steps:

[0082] S1. Mix sodium carboxymethyl chitosan and nano-hydroxyapatite, add deionized water and stir into a homogeneous solution, then add benzoyl peroxide and perform injection molding, and use ultraviolet cross-linking technology to prepare a three-dimensional scaffold.

[0083] S2. Mix superoxide dismutase, retinoic acid and ferulic acid, then encapsulate them into calcium alginate microspheres, and load them into the three-dimensional scaffold by negative pressure adsorption or pressure infiltration to obtain an antioxidant sustained-release scaffold.

[0084] S3. Prepare a buffer solution composed of sodium chloride, trisodium citrate, trehalose and hydroxyethyl starch, adjust the pH to an appropriate value and then compound it with the scaffold.

[0085] S4. Fix the aptamer targeting stem cells on gold nanoparticles, and use pressure spraying to evenly distribute the aptamer-gold nanoparticle complex on the surface of the scaffold to form a targeted scaffold.

[0086] S5. Immerse the scaffold in a serum-free medium, add growth factors to the medium, and statically culture it under low-temperature conditions to obtain a three-dimensional stem cell culture system.

[0087] Example 3

[0088] Composition and mass parts of materials:

[0089] Flexible skeleton: 17 parts;

[0090] Benzoyl peroxide: 0.1 part;

[0091] Superoxide dismutase: 2.0 parts;

[0092] Retinoic acid: 2.5 parts;

[0093] Ferulic acid: 1.5 parts;

[0094] Mixed buffer: 130.5 parts;

[0095] Nucleic acid aptamer targeting stem cells: 1.0 part;

[0096] Serum-free medium: 1000 parts;

[0097] bFGF: 5 parts;

[0098] TGF-β: 2.0 parts;

[0099] Mass ratio of the flexible skeleton component:

[0100] Sodium carboxymethyl chitosan: nano-hydroxyapatite = 12:4.

[0101] Mass ratio of the buffer component:

[0102] Sodium chloride: trisodium citrate: trehalose: hydroxyethyl starch = 5:1.5:0.1:0.05.

[0103] Preparation steps:

[0104] S1. Mix sodium carboxymethyl chitosan and nano-hydroxyapatite to prepare a uniform solution, then add benzoyl peroxide and inject it into a mold, and use the ultraviolet cross-linking method to obtain a three-dimensional porous scaffold.

[0105] S2. Mix superoxide dismutase, retinoic acid and ferulic acid, and then encapsulate them into calcium alginate microspheres, and use the vacuum adsorption and pressure infiltration composite method to load them into the three-dimensional scaffold to form an antioxidant slow-release structure.

[0106] S3. Prepare a buffer solution containing sodium chloride, trisodium citrate, trehalose and hydroxyethyl starch, adjust the pH to the appropriate range, and compound it with the scaffold to form a microenvironment.

[0107] S4. Prepare an aptamer-gold nanoparticle complex, and fix it on the surface of the scaffold by pressure spraying to form a targeted scaffold.

[0108] S5. Immerse the functionalized scaffold in a serum-free medium, add growth factors to the medium, and let it stand at low temperature to complete the integration of the medium and the scaffold, and obtain a stem cell culture system with targeting and antioxidant capabilities.

[0109] Comparative example 1:

[0110] Based on Example 1, without superoxide dismutase, retinoic acid and ferulic acid, and the rest are the same;

[0111] Comparative example 2:

[0112] Based on Example 1, without ultraviolet cross-linking, and the rest are the same.

[0113] Comparative example 3:

[0114] Based on Example 2, the mixed buffer does not contain trehalose, and the rest are the same.

[0115] Comparative example 4:

[0116] Based on Example 2, without aptamer-gold nanoparticle modification, and the rest are the same.

[0117] Comparative Example 5:

[0118] Based on Example 3, bFGF and TGF-β were not added to the culture medium, and the rest were the same.

[0119] Comparative Example 6:

[0120] Based on Example 3, superoxide dismutase was not added, and the rest were the same.

[0121] Comparative experiment:

[0122] Experiment 1:

[0123] Experiment description:

[0124] This experiment aimed to verify the antioxidant protection effect of the synergistic action of superoxide dismutase, retinoic acid and ferulic acid on stem cells. The three-dimensional scaffolds prepared in Example 1 and Comparative Example 1 were used for comparison respectively.

[0125] The experimental steps were as follows: First, CD73+ human umbilical cord mesenchymal stem cells were inoculated into the scaffolds of Example 1 and Comparative Example 1. After 24 hours of culture, a normal culture group and an oxidative stress group were established respectively. After 48 hours of culture, the intracellular ROS level was detected using a DHE probe, and the cell viability was detected using a CCK-8 kit at the same time. Each experiment was repeated 3 times, and the average value was taken, and the cell survival rate and reactive oxygen species level were recorded.

[0126] To further verify the cell apoptosis under oxidative stress, cells from a parallel experimental group were subjected to AnnexinV / PI double staining detection, and the early and late apoptosis rates were analyzed by flow cytometry (the specific results are shown in Table 1).

[0127] Table 1

[0128]

[0129] Summary:

[0130] In the experiment, the three-dimensional scaffold prepared in Example 1 could effectively reduce the ROS level and apoptosis rate of stem cells under oxidative stress, and significantly improve the cell survival rate. Especially under the treatment condition of 200 μM H2O2, the activity of the stem cells in Example 1 was significantly better than that in Comparative Example 1, indicating that the antioxidants in the formula had an obvious protective effect on the cells.

[0131] This phenomenon was mainly attributed to the superoxide radical scavenging ability of superoxide dismutase, which could quickly convert superoxide anions into relatively mild hydrogen peroxide, and then be further degraded by the endogenous antioxidant system in the cells, significantly reducing the ROS level. While retinoic acid was antioxidant, it could also participate in gene regulation through RA receptors, maintain the self-renewal ability of stem cells, and inhibit the excessive differentiation or senescence induced by oxidative stress.

[0132] As a natural small molecule antioxidant, ferulic acid can further synergistically relieve cellular oxidative stress with superoxide dismutase and retinoic acid through direct free radical scavenging and metal chelation. The antioxidant protection system constructed by the three not only effectively reduces the intracellular ROS level and decreases the apoptosis rate of cells, but also ensures the survival and function maintenance of stem cells in the three-dimensional scaffold, providing a good microenvironment for the long-term preservation and application of stem cells.

[0133] Experiment 2:

[0134] Experiment description:

[0135] To verify the influence of ultraviolet cross-linking on the formability and mechanical properties of the three-dimensional scaffold, a comparative test was carried out using the scaffolds prepared in Example 1 (including ultraviolet cross-linking) and Comparative Example 2 (without ultraviolet cross-linking).

[0136] The experimental steps are as follows: First, the pore structures of the two prepared scaffolds were observed by scanning electron microscopy (SEM), and the porosity of the scaffolds was calculated using image analysis software. At the same time, the compression mechanical properties of the scaffolds were tested using a material testing machine to determine the compression elastic modulus and the maximum compression stress. The experiments were repeated 3 times and the average values were taken. In terms of formability, the integrity, mechanical stability, and hydration swelling stability of the formed scaffolds were evaluated (the specific results are shown in Table 2).

[0137] Table 2

[0138]

[0139] Summary:

[0140] The experimental results show that the scaffold prepared in Example 1 formed a more uniform and dense porous network structure after ultraviolet cross-linking, with a moderate porosity and good pore continuity, which is beneficial to cell adhesion and proliferation. In Comparative Example 2, due to the lack of ultraviolet cross-linking, the scaffold structure was loose, the porosity was high, and the local structure collapsed, resulting in poor mechanical properties.

[0141] Ultraviolet cross-linking introduces benzoyl peroxide as a free radical initiator, which decomposes to generate free radicals under ultraviolet light irradiation, and then initiates the free radical cross-linking reaction between carboxymethyl chitosan molecules to form a stable chemical cross-linking network in the three-dimensional framework, thus significantly improving the mechanical strength and forming stability of the scaffold. The cross-linking network can effectively improve the load-bearing capacity and deformation recovery ability of the scaffold, meeting the support requirements of three-dimensional cell culture for the scaffold.

[0142] The good mechanical properties of the scaffold can not only provide a bionic physical microenvironment for stem cells, but also avoid structural damage during the culture process, providing stable and reliable growth support for stem cells during three-dimensional culture, transplantation, and long-term preservation, and ensuring the homeostasis and functional exertion of cells in the three-dimensional environment.

[0143] Experiment 3:

[0144] Experiment description:

[0145] To verify the cryoprotective effect of trehalose in the buffer on stem cells, a comparative experiment was carried out using the three-dimensional scaffolds prepared in Example 2 (buffer containing trehalose) and Comparative Example 3 (buffer without trehalose).

[0146] The experimental steps are as follows: hUC-MSCs were respectively inoculated into the scaffolds of Example 2 and Comparative Example 3, and after conventional culture for 48 h, they were cryopreserved in liquid nitrogen (programmed cooling to -80 °C and then transferred to liquid nitrogen), thawed after 7 days, and continued to be cultured for 24 h. Subsequently, CCK-8 was used to detect cell viability, and Calcein-AM / PI double staining was used to detect cell survival rate to observe the survival status and membrane integrity of cells after cryopreservation-thawing, and to evaluate the effect of the scaffold system on the cryopreservation-thawing protection of cells (the specific results are shown in Table 3).

[0147] Table 3

[0148]

[0149] Summary:

[0150] The experimental results show that after cryopreservation-thawing, the cell viability and survival rate of Example 2 are significantly better than those of Comparative Example 3, and the cell morphology is closer to normal, showing a typical long spindle shape or star shape and adhering well. In Comparative Example 3, due to the absence of trehalose in the buffer, the cell viability decreased and the survival rate decreased after thawing, and obvious cell shrinkage, detachment, and cell debris appeared.

[0151] As a natural non-permeating low molecular weight cryoprotectant, trehalose can effectively stabilize the cell membrane structure during cell cryopreservation, inhibit the physical damage of ice crystals to the cell membrane and cell organelles, and at the same time reduce the osmotic shock inside and outside the cell. During the cryopreservation and thawing of stem cells, trehalose can assist in maintaining the integrity of the cell membrane and the stability of the intracellular environment, and significantly improve the survival rate and recovery activity of stem cells.

[0152] In addition, hydroxyethyl starch, as a high molecular weight thickener, synergizes with trehalose to further improve the cryoprotective performance of the extracellular fluid and weaken the mechanical damage of cells during cryopreservation. Therefore, the buffer system containing trehalose can effectively improve the cryopreservation-thawing protection ability of stem cells, maintain cell viability, and provide guarantee for the long-term preservation of stem cells.

[0153] Experiment 4:

[0154] Experiment description:

[0155] To verify the effect of aptamer modification on the targeted enrichment ability of stem cells, a comparative experiment was carried out using the scaffolds prepared in Example 2 (the scaffold surface contains CD73 aptamer) and Comparative Example 4 (without aptamer modification).

[0156] The experimental steps are as follows: After co-culturing CD73+ hUC-MSCs with the two groups of scaffolds for 24 h respectively, the distribution of cells on the scaffolds was observed by laser confocal microscopy. At the same time, by the method of counting after desorption, the number of cells adhered to the surface of the scaffold per unit area was counted to evaluate the targeted enrichment ability of the scaffold for stem cells (the specific results are shown in Table 4).

[0157] Table 4

[0158]

[0159] The results showed that significantly more stem cells could be enriched on the surface of the scaffold in the Example 2 group, and the cells were evenly distributed and had a good adherent state; the cells in the Comparative Example 4 group were sparsely distributed and the adhesion amount was significantly reduced.

[0160] The CD73 aptamer can specifically recognize the CD73 marker and is stably fixed on the surface of the scaffold through gold nanoparticles, which can effectively improve the targeted adsorption ability of the scaffold for stem cells and significantly enhance the interaction between cells and the scaffold. The targeted binding mechanism is helpful for the spatial distribution and functional localization of cells during three-dimensional culture, providing a stable basis for the subsequent directional differentiation and function maintenance of stem cells.

[0161] Experiment 5:

[0162] Experiment description:

[0163] To verify the role of growth factors (bFGF, TGF-β) in the culture medium, a comparison was made between Example 3 (containing bFGF and TGF-β) and Comparative Example 5 (without adding bFGF and TGF-β).

[0164] The experimental steps are as follows: CD73+ stem cells were respectively inoculated into the two groups of scaffolds and continuously cultured for 7 days. During this period, the cell proliferation ability (EdU incorporation method) was regularly detected, and osteogenic and adipogenic differentiation inductions were carried out on the 7th day. The differentiation ability was observed by alizarin red S staining and oil red O staining (the specific results are shown in Table 5).

[0165] Table 5

[0166]

[0167] The results showed that the cell proliferation activity in Example 3 was significantly higher than that in Comparative Example 5, and the osteogenic and adipogenic differentiation abilities were better than those in the comparative example. bFGF can effectively stimulate the proliferation of stem cells and maintain their self-renewal ability, while TGF-β helps to maintain cell stemness and promote the interaction between cells and scaffolds, thus jointly enhancing the amplification and multi-directional differentiation potential of stem cells in a three-dimensional culture system.

[0168] The continuous supply of growth factors in the microenvironment helps to simulate the growth factor concentration gradient in vivo, significantly improving the biological function performance of stem cells and providing a good cell basis for the application value of the scaffold.

[0169] Experiment 6:

[0170] Experiment description:

[0171] To further confirm the protective effect of superoxide dismutase (SOD) under oxidative stress conditions, a comparative experiment was conducted using the scaffolds prepared in Example 3 (containing superoxide dismutase) and Comparative Example 6 (without superoxide dismutase).

[0172] The experimental steps were as follows: After culturing for 48 h, 200 μM H2O2 was added to both groups of systems to establish an oxidative stress model. After continuing to culture for 24 h, the intracellular ROS level was detected using a ROS fluorescent probe (DHE), and the apoptosis rate was detected using AnnexinV / PI staining (the specific results are shown in Table 6).

[0173] Table 6

[0174]

[0175] Summary:

[0176] The results showed that Example 3 could significantly reduce the intracellular ROS level of stem cells under oxidative stress, inhibit apoptosis, and protect stem cells from oxidative damage. Superoxide dismutase can effectively maintain the activity of stem cells and the integrity of the cell membrane by efficiently scavenging superoxide radicals and cooperating with the synergistic effect of other antioxidants in the culture system.

[0177] The reduction of ROS level not only reduces cell apoptosis but also improves the stability of various oxidation-sensitive signaling pathways in cells, ensuring the functional expression and biological activity of stem cells in a stress environment.

[0178] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A long-term preservation medium for stem cells based on a three-dimensional bionic microenvironment, characterized in that, Comprising the following component materials in parts by mass: Flexible skeleton 9 - 17 parts; Benzoyl peroxide 0.01–0.1 part; Superoxide dismutase 1 - 2 parts; Retinoic acid 1.5 - 2.5 parts; Ferulic acid 0.5 - 1.5 parts; Mixed buffer 90.5 - 130.5 parts; Nucleic acid aptamer targeting stem cells 0.1 - 1.0 part; Serum-free medium 1000 parts; bFGF 1 - 5 parts; TGF-β 0.5 - 2.0 parts; The mixed buffer comprises sodium chloride, trisodium citrate, trehalose and hydroxyethyl starch, and their mass ratio is 3 - 5:1 - 1.5:0.05 - 0.1:0.02 - 0.

05.

2. The long-term preservation culture medium for stem cells based on a three-dimensional bionic microenvironment according to claim 1, wherein The flexible skeleton comprises sodium carboxymethyl chitosan and nano-hydroxyapatite, wherein the deacetylation degree of sodium carboxymethyl chitosan is 85 - 95%, and the molecular weight is 50 - 100 kDa; The particle size of the nano-hydroxyapatite is 20 - 50 nm, and the interlayer spacing of montmorillonite is 1.2 - 1.5 nm; The mass ratio of the sodium carboxymethyl chitosan to the nano-hydroxyapatite is 8 - 12:2 - 4.

3. The long-term preservation medium for stem cells based on a three-dimensional bionic microenvironment according to claim 1, wherein The retinoic acid is retinoic acid palmitate, and the purity is 98 - 99.5%.

4. The long-term preservation culture medium for stem cells based on a three-dimensional bionic microenvironment according to claim 1, wherein The pH value of the mixed buffer is 7.4 ± 0.

1.

5. The long-term preservation culture medium for stem cells based on a three-dimensional bionic microenvironment according to claim 1, wherein The nucleic acid aptamer sequence length is 15 - 30 nt, and the targeting marker is CD73; The serum-free medium is DMEM / F12, the pyruvate concentration is 1 - 5 mM, the L-glutamine concentration is 2 - 4 mM, and the dissociation constant ≤ 5 nm.

6. A method for constructing a long-term preservation medium for stem cells based on a three-dimensional bionic microenvironment, according to the long-term preservation medium for stem cells based on a three-dimensional bionic microenvironment described in any one of claims 1-5, characterized in that, Comprising the following steps: S1. Three-dimensional scaffold construction: Mix sodium carboxymethyl chitosan and nano-hydroxyapatite in proportion, add deionized water to dissolve to form a uniformly dispersed solution, and then add benzoyl peroxide, and carry out ultraviolet cross-linking to obtain a porous scaffold; S2. Antioxidant loading: Weigh superoxide dismutase, retinoic acid and ferulic acid in proportion, and then encapsulate them together in calcium alginate microspheres. After vacuum adsorption for 7 - 9 minutes, place them in the pores of the scaffold under a pressure of 0.1 MPa; S3. Buffer preparation: Mix sodium chloride and trisodium citrate in proportion, and then add trehalose and hydroxyethyl starch; S4. Aptamer modification: Fix the targeting aptamer on gold nanoparticles and spray it onto the surface of the stent at a spraying volume of 15 - 25 μL / cm under a pressure of 0.3 MPa; 2 ​ S5. Medium integration: Immerse the scaffold in 1000 parts of serum-free medium, add bFGF and TGF-β, and let it stand at 4°C for 10 - 12 hours.

7. The construction method of the long-term preservation culture medium for stem cells based on a three-dimensional bionic microenvironment according to claim 6, characterized in that In the step S1, the concentration of the prepolymer solution for ultraviolet crosslinking is 10%, the wavelength is 355 - 375 nm, the intensity is 9 - 11 mW / cm 2 , the time is 8 - 12 min, the porosity of the crosslinked scaffold is 70 - 90%, and the elastic modulus is 3 - 5 kPa.

8. The construction method of the long-term preservation culture medium for stem cells based on a three-dimensional biomimetic microenvironment according to claim 6, wherein, In the step S2, the particle size of the calcium alginate microspheres is 20 - 40 μm, which is prepared by microfluidic technology, the flow rate is 5 μL / min, and the solidifying solution is 0.2 M CaCl2.

9. The construction method of the long-term preservation culture medium for stem cells based on a three-dimensional bionic microenvironment according to claim 6, characterized in that, In the step S4, the particle size of the gold nanoparticles is 8-12 nm, and the density of the aptamer modified on the surface thereof is 100-200 molecules / μm 2 , and the aptamer immobilization rate is ≥90%.

10. The construction method of the long-term preservation culture medium for stem cells based on a three-dimensional biomimetic microenvironment according to claim 6, wherein In the step S5, the temperature during the immersion of the scaffold is 4 - 8°C, and the dissolved oxygen content of the medium after immersion is maintained at 5 - 8 mg / L.

Images