A method for 3D culture and expansion of human mesenchymal stem cells

Abstract

The application discloses a kind of human mesenchymal stem cell 3D culture and amplification method, belong to stem cell culture amplification technical field.The application is prepared 3D cell culture matrix by structure design and component optimization, realize the accurate simulation of stem cell in-vivo microenvironment.The matrix is modified sodium alginate by amino hexene acid and is compounded with methacryl gelatin, and is crosslinked by lithium chloride and calcium chloride, and is synergistically formed by photocrosslinking, and the three-dimensional network has uniform pore size and excellent connectivity.The application significantly improves cell adhesion rate and proliferation efficiency, effectively maintains the biological characteristics of hMSC such as immune regulation and differentiation, the degradation rate of the matrix is dynamically matched with the cell proliferation cycle, the material has excellent biocompatibility and the degradation product is non-toxic, which solves the defects of traditional 2D culture and existing 3D technology, and provides an efficient and safe technical solution for hMSC scale-up and clinical translation.

Description

Technical Field

[0001] This invention belongs to the field of stem cell culture and expansion technology, and particularly relates to a method for 3D culture and expansion of human mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) possess migratory capabilities. When the body experiences inflammation or injury, damaged tissue can secrete chemokine receptors to guide their migration to the site of injury, further secreting cytokines to participate in tissue repair. MSCs exhibit low immunogenicity (they do not express MHC class I antigens, have low expression of MHC class II antigens, and do not express CD40, CD80, or CD86), and will not cause immune rejection when transplanted into a host. Furthermore, MSCs possess strong self-proliferation, immunomodulatory capabilities, and autocrine and paracrine functions. These characteristics make them highly promising for inhibiting the growth of various malignant tumor cells, treating joint injuries, autoimmune diseases, and ischemic diseases. They can also serve as vector cells for gene therapy in cancer treatment, making them a core seed cell in regenerative medicine and personalized medicine.

[0003] Cell growth depends on a specific microenvironment. Traditional two-dimensional (2D) planar culture methods differ fundamentally from the three-dimensional physiological environment in vivo, becoming a key bottleneck restricting the clinical translation of MSCs. 2D culture only provides planar growth space for cells, resulting in limited surface area that cannot meet the billions of cells required for clinical applications. It also leads to abnormal cell morphology, increased cell size, and significantly reduced migration ability and cytokine secretion, ultimately affecting therapeutic efficacy. A more prominent problem is that 2D-cultured MSCs are easily trapped in pulmonary microvessels and die rapidly after intravenous injection, greatly limiting their effectiveness in treating non-pulmonary diseases. Furthermore, long-term passages result in cell aging, loss of differentiation potential, and poor batch-to-batch quality stability, making it difficult to meet GMP requirements. In addition, traditional serum-containing culture media are complex in composition and exhibit significant batch-to-batch variations, increasing the difficulty of process control and introducing potential safety risks.

[0004] To overcome the limitations of 2D culture, 3D cell culture technology has emerged. It provides cells with a three-dimensional growth space through scaffold support, more closely mimicking the in vivo physiological microenvironment and reducing the impact on cell biological characteristics. However, existing 3D culture technologies still face several unresolved issues: First, scaffold performance is poor; some scaffolds lack sufficient mechanical strength to resist the contractile forces during cell growth, or their degradation rate does not match the cell proliferation cycle, affecting continuous cell growth. Second, cell-matrix interaction is weak; some scaffolds lack specific binding sites, leading to low cell adhesion and easy detachment. Furthermore, unreasonable pore structures can result in uneven nutrient permeation and obstructed metabolic waste removal, increasing cell population heterogeneity. Third, scaling and standardization are difficult; existing technologies often face low proliferation efficiency and high costs, and the lack of unified standards for scaffold material selection and process parameters makes it difficult to control cell product quality, failing to meet the stringent safety and consistency requirements for clinical translation.

[0005] The morphology, structure, mechanical force, and material properties of three-dimensional scaffolds are closely related to stem cell growth and directly affect cell adhesion, proliferation, and functional maintenance. Therefore, developing a 3D cell culture substrate and supporting culture method that can accurately simulate the in vivo growth microenvironment while taking into account high proliferation efficiency, stable cell characteristics, and clinical safety has become a core requirement for promoting the large-scale expansion and clinical application of MSCs. Summary of the Invention

[0006] In response to the above situation and to overcome the shortcomings of the existing technology, this invention achieves precise simulation of the in vivo growth microenvironment of stem cells by structural design and component optimization of 3D cell culture substrate, thereby improving cell adhesion rate, promoting proliferation efficiency, maintaining cell biological characteristics, and ensuring safety.

[0007] To achieve the above objectives, the following technical solution is adopted: This invention provides a method for 3D culture and expansion of human mesenchymal stem cells, wherein the method utilizes a 3D cell culture substrate prepared in the following steps for the culture and expansion of human mesenchymal stem cells:

[0008] S1. Dissolve aminohexenoic acid and 4,4'-dihydroxy-3,3'-dialdehyde biphenyl in anhydrous ethanol, adjust the pH of the system to 4.5-5.0 with 1 mol / L hydrochloric acid, stir the reaction at 35-40℃ for 3-4 h, remove the solvent by vacuum distillation to obtain intermediate A;

[0009] S2. Dissolve intermediate A in anhydrous DMF, add DCC, stir at room temperature for 30-40 min, then add N-hydroxyethylimine diacetic acid and DMAP, and continue the reaction in the dark for 16 h to obtain intermediate B;

[0010] S3. Mix intermediate B with a 2% (w / w) sodium alginate DMSO solution, add hexamethylene diisocyanate and dibutyltin dilaurate, stir and react at 50°C for 3 h, dialyze to remove impurities, and freeze dry to obtain modified sodium alginate.

[0011] S4. Modified sodium alginate was mixed with sodium alginate and dissolved in deionized water to prepare a 1.5% mass fraction mixture. A mixed solution of 0.08 mol / L lithium chloride and 0.15 mol / L calcium chloride was added, and the mixture was stirred and crosslinked at room temperature for 45 min to form a gel precursor. Then, methacrylamide gelatin and I2959 photoinitiator were added, stirred evenly, and irradiated with 365 nm ultraviolet light for 8 min. After photocrosslinking, 3D cell culture substrate was obtained.

[0012] Furthermore, the method for 3D culture and expansion of human mesenchymal stem cells specifically includes the following steps:

[0013] (1) After cleaning and cutting the umbilical cord or placental tissue, the cells were digested with collagenase and dispersin to release them. After digestion, hMSCs were isolated and purified by differential adhesion. The obtained primary hMSCs were seeded into conventional 2D culture flasks and expanded in a 37°C, 5% CO2 saturated humidity incubator using α-MEM basal medium containing 10% fetal bovine serum. When the cell confluence reached 80%-90%, the cells were digested and passaged with trypsin, and cells in the third generation logarithmic growth phase were taken.

[0014] (2) The third-generation logarithmic growth phase hMSCs were washed twice with PBS buffer, and 0.25% trypsin-EDTA digestion solution was added. After incubation at 37°C for 2-3 min, α-MEM basal medium containing 10% fetal bovine serum was added to terminate digestion. The cells were centrifuged at 1000 r / min for 5 min to collect the cell pellet. The pellet was resuspended in fresh α-MEM complete medium, and the cells were counted by trypan blue staining. The cell concentration was adjusted to 1×10⁻⁶ cells / year. 6 -5×10 6 cells / mL, for later use;

[0015] (3) Take the 3D cell culture medium, cut it into three-dimensional carrier blocks with a diameter of 5-8 mm and a thickness of 2-3 mm, wash it 3 times with PBS buffer to remove lyophilized residues, and then soak it in α-MEM complete medium. Incubate it in a 37°C, 5% CO2 incubator for 2-4 h to fully hydrate the substrate and balance the osmotic pressure for later use.

[0016] (4) Transfer the pretreated 3D cell culture substrate to a 24-well cell culture plate, one carrier per well, and evenly drop 100-200 μL of the hMSC cell suspension prepared in step (2) onto the surface of each substrate to ensure that the cell suspension completely covers the surface of the substrate and penetrates into the internal pores; incubate at 37°C and 5% CO2 for 1-2 hours to allow the cells to fully adhere to the substrate scaffold;

[0017] (5) After the cells adhere, add α-MEM complete medium to each well until it covers the substrate surface by 1-2 mm, and place it in a 37℃, 5% CO2 saturated humidity incubator for continuous culture. Replace 50% of the volume of fresh complete medium daily for the first 3 days, and then replace the entire volume of fresh complete medium every 48 hours thereafter. The culture cycle is 7-14 days. Use the CCK-8 method to detect cell proliferation activity and monitor the amplification effect in real time. Collect cells after amplification.

[0018] Furthermore, in step S1, the molar ratio of aminohexenoic acid to 4,4'-dihydroxy-3,3'-dialdehyde biphenyl is 2.1-2.2:1.

[0019] Further, in step S2, the molar ratio of intermediate A, DCC, N-hydroxyethyliminodiacetic acid and DMAP is 1:2-2.1:2.4-2.8:0.05-0.1.

[0020] Further, in step S3, the mass ratio of intermediate B, sodium alginate DMSO solution, hexamethylene diisocyanate and dibutyltin dilaurate is 1:8-10:0.6-0.72:0.045-0.06.

[0021] Furthermore, in step S4, the mass ratio of modified sodium alginate to sodium alginate is 1:4-4.5.

[0022] Furthermore, in step S4, the mass ratio of the gel precursor, methacrylamide gelatin, and I2959 photoinitiator is 1:5-5.5:0.048-0.072.

[0023] Furthermore, the fresh α-MEM complete culture medium was supplemented with 5 ng / mL basic fibroblast growth factor, 2 ng / mL epidermal growth factor, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0024] The beneficial effects of this invention are:

[0025] The 3D cell culture medium of this invention achieves precise simulation of the in vivo growth microenvironment of stem cells through structural design and component optimization. It shows advantages in improving cell adhesion rate, promoting proliferation efficiency, maintaining cell biological characteristics and ensuring safety, and provides an efficient and feasible technical solution for the large-scale expansion and clinical translation of mesenchymal stem cells.

[0026] During the preparation process, the synergistic effect of secondary cross-linking of lithium chloride and calcium chloride and photo-initiated cross-linking enables the matrix to form a three-dimensional network structure with uniform pore size and excellent connectivity. This not only provides a three-dimensional growth space for cells, but also simulates the porous microenvironment of the extracellular matrix in vivo, which is conducive to the penetration of nutrients, the excretion of metabolic waste and the transmission of intercellular signals. This solves the problem of local nutrient deficiency caused by cell accumulation in traditional 2D culture and provides a stable microenvironment to support continuous cell proliferation.

[0027] This invention optimizes the mechanical strength of the matrix through hexamethylene diisocyanate-mediated cross-linking and the introduction of Schiff base structures, enabling it to resist contractile forces during cell growth and maintain structural stability. Simultaneously, the natural degradability of sodium alginate synergistically complements the enzymatic hydrolysis properties of gelatin, dynamically matching the matrix degradation rate with the cell proliferation cycle. Methacrylamide gelatin can specifically bind to integrin receptors on the surface of stem cells, significantly improving cell adhesion. The carboxyl, hydroxyl, and iminodiacetic acid groups retained in the modified sodium alginate possess excellent hydrophilicity, adsorbing extracellular matrix proteins and further enhancing cell-matrix interaction, reducing cell shedding.

[0028] In the matrix of this invention, the double bonds introduced by vigabatrin form a stable network with gelatin through photocrosslinking. The iminodiacetic acid groups, through coordination with metal ions such as calcium ions, not only enhance the crosslinking stability but also simulate the ion microenvironment in vivo, activate the mechanosensitive signaling pathways in stem cells, promote the expression of cell proliferation-related genes, and shorten the doubling time. In addition, the matrix materials are all natural polymers with excellent biocompatibility, and the degradation products have no toxic side effects, providing a safety guarantee for subsequent clinical applications of cells. Attached Figure Description

[0029] Figure 1 SEM images of 3D cell culture media of various embodiments and comparative examples of the present invention (a, b, c, d, and e correspond to Embodiment 1, Embodiment 2, Embodiment 3, Comparative Example 1, and Comparative Example 2, respectively).

[0030] Figure 2 Images showing the mechanical strength test results of 3D cell culture media in various embodiments and comparative examples of the present invention;

[0031] Figure 3 Images showing the swelling rate test results of 3D cell culture media in various embodiments and comparative examples of the present invention;

[0032] Figure 4 Images showing the porosity test results of 3D cell culture media in various embodiments and comparative examples of the present invention;

[0033] Figure 5 Images showing the cell compatibility test results of 3D cell culture media in various embodiments and comparative examples of the present invention;

[0034] Figure 6 This is a line graph showing the proliferation rate of the 3D cell culture medium and the 2D culture medium in Example 3 of the present invention.

[0035] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0039] Example 1:

[0040] A human mesenchymal stem cell 3D cell culture medium is prepared through the following steps:

[0041] S1. Dissolve aminohexenoic acid and 4,4'-dihydroxy-3,3'-dialdehyde biphenyl in anhydrous ethanol, adjust the pH of the system to 4.5 with 1 mol / L hydrochloric acid, stir the reaction at 35℃ for 3 h, remove the solvent by vacuum distillation to obtain intermediate A;

[0042] S2. Dissolve intermediate A in anhydrous DMF, add DCC, stir at room temperature for 30 min, then add N-hydroxyethylimine diacetic acid and DMAP, and continue the reaction in the dark for 16 h to obtain intermediate B;

[0043] S3. Mix intermediate B with a 2% (w / w) sodium alginate DMSO solution, add hexamethylene diisocyanate and dibutyltin dilaurate, stir and react at 50°C for 3 h, dialyze to remove impurities, and freeze dry to obtain modified sodium alginate.

[0044] S4. Modified sodium alginate was mixed with sodium alginate and dissolved in deionized water to prepare a 1.5% mass fraction mixture. A mixed solution of 0.08 mol / L lithium chloride and 0.15 mol / L calcium chloride was added, and the mixture was stirred and crosslinked at room temperature for 45 min to form a gel precursor. Then, methacrylamide gelatin and I2959 photoinitiator were added, stirred evenly, and irradiated with 365 nm ultraviolet light for 8 min. After photocrosslinking, 3D cell culture substrate was obtained.

[0045] In step S1, the molar ratio of aminohexenoic acid to 4,4'-dihydroxy-3,3'-dialdehyde biphenyl is 2.1:1; in step S2, the molar ratio of intermediate A, DCC, N-hydroxyethylimine diacetic acid, and DMAP is 1:2:2.4:0.05; in step S3, the mass ratio of intermediate B, sodium alginate DMSO solution, hexamethylene diisocyanate, and dibutyltin dilaurate is 1:8:0.6:0.045; in step S4, the mass ratio of modified sodium alginate to sodium alginate is 1:4; in step S4, the mass ratio of gel precursor, methacrylamide gelatin, and I2959 photoinitiator is 1:5:0.048.

[0046] Example 2:

[0047] A human mesenchymal stem cell 3D cell culture medium is prepared through the following steps:

[0048] S1. Dissolve aminohexenoic acid and 4,4'-dihydroxy-3,3'-dialdehyde biphenyl in anhydrous ethanol, adjust the pH of the system to 5.0 with 1 mol / L hydrochloric acid, stir the reaction at 40℃ for 4 h, remove the solvent by vacuum distillation to obtain intermediate A;

[0049] S2. Dissolve intermediate A in anhydrous DMF, add DCC, stir at room temperature for 40 min, then add N-hydroxyethylimine diacetic acid and DMAP, and continue the reaction in the dark for 16 h to obtain intermediate B;

[0050] S3. Mix intermediate B with a 2% (w / w) sodium alginate DMSO solution, add hexamethylene diisocyanate and dibutyltin dilaurate, stir and react at 50°C for 3 h, dialyze to remove impurities, and freeze dry to obtain modified sodium alginate.

[0051] S4. Modified sodium alginate was mixed with sodium alginate and dissolved in deionized water to prepare a 1.5% mass fraction mixture. A mixed solution of 0.08 mol / L lithium chloride and 0.15 mol / L calcium chloride was added, and the mixture was stirred and crosslinked at room temperature for 45 min to form a gel precursor. Then, methacrylamide gelatin and I2959 photoinitiator were added, stirred evenly, and irradiated with 365 nm ultraviolet light for 8 min. After photocrosslinking, 3D cell culture substrate was obtained.

[0052] In step S1, the molar ratio of aminohexenoic acid to 4,4'-dihydroxy-3,3'-dialdehyde biphenyl is 2.2:1; in step S2, the molar ratio of intermediate A, DCC, N-hydroxyethylimine diacetic acid, and DMAP is 1:2.1:2.8:0.1; in step S3, the mass ratio of intermediate B, sodium alginate DMSO solution, hexamethylene diisocyanate, and dibutyltin dilaurate is 1:10:0.72:0.06; in step S4, the mass ratio of modified sodium alginate to sodium alginate is 1:4.5; in step S4, the mass ratio of gel precursor, methacrylamide gelatin, and I2959 photoinitiator is 1:5.5:0.072.

[0053] Example 3:

[0054] A human mesenchymal stem cell 3D cell culture medium is prepared through the following steps:

[0055] S1. Dissolve aminohexenoic acid and 4,4'-dihydroxy-3,3'-dialdehyde biphenyl in anhydrous ethanol, adjust the pH of the system to 4.7 with 1 mol / L hydrochloric acid, stir the reaction at 38℃ for 3.5 h, remove the solvent by vacuum distillation to obtain intermediate A;

[0056] S2. Dissolve intermediate A in anhydrous DMF, add DCC, stir at room temperature for 35 min, then add N-hydroxyethylimine diacetic acid and DMAP, and continue the reaction in the dark for 16 h to obtain intermediate B;

[0057] S3. Mix intermediate B with a 2% (w / w) sodium alginate DMSO solution, add hexamethylene diisocyanate and dibutyltin dilaurate, stir and react at 50°C for 3 h, dialyze to remove impurities, and freeze dry to obtain modified sodium alginate.

[0058] S4. Modified sodium alginate was mixed with sodium alginate and dissolved in deionized water to prepare a 1.5% mass fraction mixture. A mixed solution of 0.08 mol / L lithium chloride and 0.15 mol / L calcium chloride was added, and the mixture was stirred and crosslinked at room temperature for 45 min to form a gel precursor. Then, methacrylamide gelatin and I2959 photoinitiator were added, stirred evenly, and irradiated with 365 nm ultraviolet light for 8 min. After photocrosslinking, 3D cell culture substrate was obtained.

[0059] In step S1, the molar ratio of aminohexenoic acid to 4,4'-dihydroxy-3,3'-dialdehyde biphenyl is 2.15:1; in step S2, the molar ratio of intermediate A, DCC, N-hydroxyethylimine diacetic acid, and DMAP is 1:2.05:2.6:0.075; in step S3, the mass ratio of intermediate B, sodium alginate DMSO solution, hexamethylene diisocyanate, and dibutyltin dilaurate is 1:9:0.66:0.0525; in step S4, the mass ratio of modified sodium alginate to sodium alginate is 1:4.25; in step S4, the mass ratio of gel precursor, methacrylamide gelatin, and I2959 photoinitiator is 1:5.25:0.06.

[0060] Comparative Example 1:

[0061] In this comparative example, sodium alginate was used for ionic cross-linking to prepare a 3D cell culture substrate. The preparation method is as follows: Sodium alginate was dissolved in deionized water to prepare a 1.5% (w / w) solution. Then, 0.15 mol / L calcium chloride solution was added, and the mixture was stirred at room temperature for 45 minutes to cross-link and form a hydrogel, which is the 3D cell culture substrate.

[0062] Comparative Example 2:

[0063] In this comparative example, sodium alginate was not chemically modified. Instead, sodium alginate and gelatin were physically mixed to prepare a 3D cell culture substrate. The preparation method is as follows: Sodium alginate and unmodified gelatin were mixed at the same mass ratio as in Example 3, dissolved in deionized water to prepare a 1.5% mass fraction mixture, and a mixed solution of lithium chloride and calcium chloride was added for ionic cross-linking to obtain the 3D cell culture substrate.

[0064] Test Example 1:

[0065] The 3D cell culture substrate prepared in the above embodiments was characterized as follows:

[0066] Microstructure: The surface and internal microstructure of the 3D cell culture substrates prepared in each example were observed using a scanning electron microscope (Hitachi SU8010). SEM images are shown below. Figure 1 Where a, b, c, d, and e correspond to Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2, respectively.

[0067] Figure 1 In the examples 1-3, the 3D cell culture medium has a more complete porous structure with well-developed pores, uniform pore size, and smaller pore size. In contrast, in the comparative examples 1-2, a collapse phenomenon occurred, the regularity of the porous structure was disrupted, and the pore size was slightly larger than that in the examples 1-3.

[0068] Mechanical strength testing: Mechanical strength testing was conducted using a computer-controlled electronic universal testing machine (RGM-4030). The specific steps were as follows: The 3D cell culture substrates prepared in each embodiment were cut into standard dumbbell shapes (12mm long, 3mm thick), with at least 5 samples per group. The clamp spacing was set to 15mm, and the test was performed at a tensile speed of 5mm / min. The maximum force value at sample breakage was recorded as the tensile strength. The substrate was also cut into cylindrical shapes (10mm in diameter, 50mm thick), with at least 5 samples per group. The test was performed at a compression speed of 1mm / min, and the force value when the sample was compressed to 50% of its original height was recorded as the compressive strength. Mechanical strength data images are shown below. Figure 2 .

[0069] Figure 2 Examples 1-3 all demonstrated excellent mechanical properties, illustrating that the modification and photocrosslinking steps of this invention enabled the 3D cell culture substrate to form a stable and dense three-dimensional network structure, thereby improving the tensile and compressive strength of the substrate. In Comparative Example 1, the network strength formed solely by the ionic crosslinking of sodium alginate was weak, and its tensile and compressive strengths were significantly lower than those of the embodiments of this invention. The substrate prepared in Comparative Example 2 was based on physical mixing and lacked strong chemical crosslinking, resulting in a relatively loose structure and therefore the lowest mechanical strength, failing to provide effective mechanical support for cell culture.

[0070] Swelling performance test: The 3D cell culture media prepared in each example were freeze-dried and then immersed in PBS buffer (pH=7.4) in a 37°C incubator for 24 hours. After removal, the surface moisture was gently blotted with filter paper, and the media were immediately weighed to calculate the swelling rate. The swelling rate test results are shown in […]. Figure 3 .

[0071] Figure 3 In Examples 1-3, the swelling ratios were moderate and controllable. The interpenetrating network formed by the modified sodium alginate and photocrosslinked gelatin limited excessive water absorption and maintained structural stability. In Comparative Example 1, the pure sodium alginate gel network was relatively uniform but highly hydrophilic and had a low degree of crosslinking, resulting in the highest swelling ratio. Excessive swelling could lead to smaller pore sizes, which is detrimental to cell migration and nutrient exchange. Comparative Example 2 lacked chemical crosslinking in water, resulting in an unstable structure, easy dissolution of gelatin, and an excessively high swelling ratio.

[0072] Porosity testing: Porosity was tested using the liquid displacement method. The 3D cell culture media prepared in each example were freeze-dried and then immersed in anhydrous ethanol. A vacuum was applied for 30 minutes to remove air from the pores, ensuring the ethanol fully filled the pores. After removal, the media were weighed, and the porosity was calculated. The porosity test results are shown below. Figure 4 .

[0073] Figure 4In the examples, Examples 1-3 all exhibited high porosity (>90%), with Example 2 showing the highest porosity. This indicates that the preparation method of the present invention can successfully construct a three-dimensional scaffold with a rich porous structure, which is beneficial for cell ingrowth and distribution. Although Comparative Examples 1 and 2 also possess a certain porosity, Comparative Example 2, due to the lack of stable cross-linking, may have experienced partial structural collapse during preparation and freeze-drying, resulting in lower and unevenly distributed porosity, consistent with the observed microstructure.

[0074] Cell compatibility test: The 3D cell culture media of each example and comparative example were sterilized with 60Co (dose 25kGy), then immersed in α-MEM complete medium at a ratio of 1g / 10mL, and extracted in a 37℃, 5% CO2 incubator for 24h. After filtration through a 0.22μm filter membrane for sterilization, the sample extract (recorded as 100% concentration) was obtained, and then diluted with complete medium to 50% and 25% concentrations for later use. Complete medium without matrix extract was used as a blank control group. hMSCs were cultured at 5×10 3 Cells were seeded at a density of 100 μL of complete culture medium per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. The old culture medium was discarded, and 100 μL of sample extract (20%, 50%, 100%) and blank control culture medium were added to each well, with 5 parallel wells per group. After 48 hours of incubation, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 h. The absorbance (OD450) was measured at 450 nm using a microplate reader, and the cell viability was calculated as (OD450 of experimental group - OD450 of blank group) / (OD450 of control group - OD450 of blank group) × 100%. The test results are shown below. Figure 5 .

[0075] Figure 5 In the study, both the example groups and the comparative groups showed a high survival rate advantage, indicating that the 3D cell culture substrate prepared by the present invention did not affect the biocompatibility of sodium alginate and gelatin-based materials, and could meet the non-toxic standard.

[0076] Test Example 2:

[0077] The method for 3D culture and expansion of human mesenchymal stem cells using the 3D cell culture substrate of Example 3 of this invention specifically includes the following steps:

[0078] (1) After cleaning and cutting the umbilical cord or placental tissue, the cells were digested with collagenase and dispersin to release them. After digestion, hMSCs were isolated and purified by differential adhesion. The obtained primary hMSCs were seeded into conventional 2D culture flasks and expanded in a 37°C, 5% CO2 saturated humidity incubator using α-MEM basal medium containing 10% fetal bovine serum. When the cell confluence reached 85%, the cells were digested and passaged with trypsin, and cells in the third generation logarithmic growth phase were taken.

[0079] (2) The third-generation logarithmic growth phase hMSCs were washed twice with PBS buffer, and 0.25% trypsin-EDTA digestion solution was added. After incubation at 37°C for 2 min, α-MEM basal medium containing 10% fetal bovine serum was added to terminate digestion. The cells were centrifuged at 1000 r / min for 5 min to collect the cell pellet. The pellet was resuspended in fresh α-MEM complete medium, and the cells were counted by trypan blue staining. The cell concentration was adjusted to 3 × 10⁶ cells / year. 6 cells / mL, for later use;

[0080] (3) Take the 3D cell culture medium, cut it into three-dimensional carrier blocks with a diameter of 6 mm and a thickness of 2.5 mm, wash it three times with PBS buffer to remove residual impurities from lyophilization, and then soak it in α-MEM complete medium. Incubate it in a 37°C, 5% CO2 incubator for 3 h to fully hydrate the substrate and balance the osmotic pressure for later use.

[0081] (4) Transfer the pretreated 3D cell culture substrate to a 24-well cell culture plate, one carrier per well, and evenly drop 200 μL of the hMSC cell suspension prepared in step (2) onto the surface of each substrate to ensure that the cell suspension completely covers the surface of the substrate and penetrates into the internal pores; incubate at 37°C and 5% CO2 for 1-2 hours to allow the cells to fully adhere to the substrate scaffold;

[0082] (5) After the cells adhered, add α-MEM complete medium to each well until it covered the substrate surface by 1.5 mm, and place it in a 37℃, 5% CO2 saturated humidity incubator for continuous culture. Replace 50% of the volume of fresh complete medium daily for the first 3 days, and then replace the entire volume of fresh complete medium every 48 hours thereafter. The culture cycle is 7 days. Use the CCK-8 method to detect cell proliferation activity and monitor the amplification effect in real time. Collect cells after amplification.

[0083] The fresh α-MEM complete culture medium contains 5 ng / mL basic fibroblast growth factor, 2 ng / mL epidermal growth factor, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0084] Simultaneously, sterile polystyrene 2D cell culture dishes (6 mm in diameter, matching the size of the 3D carrier) were used for 2D cell culture and expansion. The cells were pre-incubated in α-MEM complete medium for 1 hour to simulate cell adhesion. The stem cells obtained in step (2) were used, and the remaining treatment steps were the same as those for 3D culture and expansion. The proliferation rate of stem cells in the 3D and 2D culture groups was measured using a CCK-8 assay kit for 1-7 days, measured daily. The results are shown in […]. Figure 6 .

[0085] from Figure 6 As can be seen, the proliferation difference between the two groups was small in the first two days of culture. From the third day onwards, the proliferation rate of the 3D culture group increased significantly, and the 2D culture group entered the plateau phase on the fifth day. The 3D cell culture substrate of the present invention showed a significant promoting effect on the proliferation of hMSCs.

[0086] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0087] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for 3D culture and expansion of human mesenchymal stem cells, characterized in that: The method utilizes the 3D cell culture substrate prepared in the following steps for the culture and expansion of human mesenchymal stem cells: S1. Dissolve aminohexenoic acid and 4,4'-dihydroxy-3,3'-dialdehyde biphenyl in anhydrous ethanol, adjust the pH of the system to 4.5-5.0 with 1 mol / L hydrochloric acid, stir the reaction at 35-40℃ for 3-4 h, remove the solvent by vacuum distillation to obtain intermediate A; S2. Dissolve intermediate A in anhydrous DMF, add DCC, stir at room temperature for 30-40 min, then add N-hydroxyethylimine diacetic acid and DMAP, and continue the reaction in the dark for 16 h to obtain intermediate B; S3. Mix intermediate B with a 2% (w / w) sodium alginate DMSO solution, add hexamethylene diisocyanate and dibutyltin dilaurate, stir and react at 50°C for 3 h, dialyze to remove impurities, and freeze dry to obtain modified sodium alginate. S4. Modified sodium alginate was mixed with sodium alginate and dissolved in deionized water to prepare a 1.5% mass fraction mixture. A mixed solution of 0.08 mol / L lithium chloride and 0.15 mol / L calcium chloride was added, and the mixture was stirred and crosslinked at room temperature for 45 min to form a gel precursor. Then, methacrylamide gelatin and I2959 photoinitiator were added, stirred evenly, and irradiated with 365 nm ultraviolet light for 8 min. After photocrosslinking, 3D cell culture substrate was obtained.

2. The method for 3D culture and expansion of human mesenchymal stem cells according to claim 1, characterized in that: Specifically, the following steps are included: (1) After cleaning and cutting the umbilical cord or placental tissue, the cells were digested with collagenase and dispersin to release them. After digestion, hMSCs were isolated and purified by differential adhesion. The obtained primary hMSCs were seeded into conventional 2D culture flasks and expanded in a 37°C, 5% CO2 saturated humidity incubator using α-MEM basal medium containing 10% fetal bovine serum. When the cell confluence reached 80%-90%, the cells were digested and passaged with trypsin, and cells in the third generation logarithmic growth phase were taken. (2) The third-generation logarithmic growth phase hMSCs were washed twice with PBS buffer, and 0.25% trypsin-EDTA digestion solution was added. After incubation at 37°C for 2-3 min, α-MEM basal medium containing 10% fetal bovine serum was added to terminate digestion. The cells were centrifuged at 1000 r / min for 5 min to collect the cell pellet. The pellet was resuspended in fresh α-MEM complete medium, and the cells were counted by trypan blue staining. The cell concentration was adjusted to 1×10⁻⁶ cells / year. 6 -5×10 6 cells / mL, for later use; (3) Take the 3D cell culture medium, cut it into three-dimensional carrier blocks with a diameter of 5-8 mm and a thickness of 2-3 mm, wash it 3 times with PBS buffer to remove lyophilized residues, and then soak it in α-MEM complete medium. Incubate it in a 37°C, 5% CO2 incubator for 2-4 h to fully hydrate the substrate and balance the osmotic pressure for later use. (4) Transfer the pretreated 3D cell culture substrate to a 24-well cell culture plate, one carrier per well, and evenly drop 100-200 μL of the hMSC cell suspension prepared in step (2) onto the surface of each substrate to ensure that the cell suspension completely covers the surface of the substrate and penetrates into the internal pores; incubate at 37°C and 5% CO2 for 1-2 hours to allow the cells to fully adhere to the substrate scaffold; (5) After the cells adhere, add α-MEM complete medium to each well until it covers the substrate surface by 1-2 mm, and place it in a 37℃, 5% CO2 saturated humidity incubator for continuous culture. Replace 50% of the volume of fresh complete medium daily for the first 3 days, and then replace the entire volume of fresh complete medium every 48 hours thereafter. The culture cycle is 7-14 days. Use the CCK-8 method to detect cell proliferation activity and monitor the amplification effect in real time. Collect cells after amplification.

3. The method for 3D culture and expansion of human mesenchymal stem cells according to claim 2, characterized in that: In step S1, the molar ratio of aminohexenoic acid to 4,4'-dihydroxy-3,3'-dialdehyde biphenyl is 2.1-2.2:

1.

4. The method for 3D culture and expansion of human mesenchymal stem cells according to claim 3, characterized in that: In step S2, the molar ratio of intermediate A, DCC, N-hydroxyethyliminodiacetic acid and DMAP is 1:2-2.1:2.4-2.8:0.05-0.

1.

5. The method for 3D culture and expansion of human mesenchymal stem cells according to claim 4, characterized in that: In step S3, the mass ratio of intermediate B, sodium alginate DMSO solution, hexamethylene diisocyanate, and dibutyltin dilaurate is 1:8-10:0.6-0.72:0.045-0.

06.

6. The method for 3D culture and expansion of human mesenchymal stem cells according to claim 5, characterized in that: In step S4, the mass ratio of modified sodium alginate to sodium alginate is 1:4-4.

5.

7. The method for 3D culture and expansion of human mesenchymal stem cells according to claim 6, characterized in that: In step S4, the mass ratio of the gel precursor, methacrylated gelatin, and I2959 photoinitiator is 1:5-5.5:0.048-0.

072.

8. The method for 3D culture and expansion of human mesenchymal stem cells according to claim 7, characterized in that: The fresh α-MEM complete culture medium was supplemented with 5 ng / mL basic fibroblast growth factor, 2 ng / mL epidermal growth factor, 100 U / mL penicillin and 100 μg / mL streptomycin.

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