Culture medium for adipose-derived stem cells
By using human platelet lysate and neurovascular niche factor, combined with dynamic mechanical stretching and a non-contact co-culture system, the safety and structural-functional issues in adipose stem cell culture were resolved, and efficient engineered adipose tissue construction was achieved.
Patent Information
- Application Number
- CN202511902698.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-17
- Publication Date
- 2026-01-16
AI Technical Summary
Existing adipose-derived stem cell culture technologies have problems such as the risk of immune rejection, the risk of contamination by animal-derived pathogens, lack of neural and vascular networks, weak lipid accumulation capacity, loose in vitro culture structure and insufficient mechanical strength, making it difficult to construct engineered adipose tissue with normal structure and function.
Human platelet lysate was used to replace fetal bovine serum. A non-contact co-culture system was constructed by combining neurovascular niche factors and programmed dynamic mechanical stretching to simulate the in vivo multicellular microenvironment. Through the synergistic effect of multifunctional factors, the formation of neurovascular networks and the maturation of adipose tissue were promoted.
It achieves efficient differentiation of adipose stem cells, forming engineered adipose tissue with a compact structure and mature function, exhibiting good lipid accumulation capacity and neurovascular network, making it suitable for clinical applications.
Smart Images
Figure CN121343894A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of stem cell culture, and relates to a culture medium for adipose-derived stem cells. BACKGROUND
[0002] In the frontier field of adipose tissue engineering and regenerative medicine, adipose-derived stem cells are widely considered to be the ideal seed cells for constructing engineered adipose tissue due to their multi-directional differentiation potential, wide source, easy acquisition through minimally invasive means, and less ethical controversy. However, despite its broad application prospects, the current in vitro culture and directional induction differentiation technology system of adipose-derived stem cells still has several significant limitations that need to be broken through.
[0003] First of all, and most importantly, the traditional culture system is heavily dependent on animal-derived additives such as fetal bovine serum. Fetal bovine serum can provide basic nutrients and growth factors required for cell growth, but its composition is complex and uncertain, and there are large differences between batches. More importantly, it carries potential xenogenic antigens, which can trigger immune rejection after transplantation. At the same time, animal-derived components also have difficulty in completely eliminating the risk of contamination by viruses, mycoplasma, and other pathogens. These safety hazards greatly restrict the conversion of adipose-derived stem cell technology to clinical treatment applications, and do not meet the strict requirements of regenerative medicine products for standardization and humanization.
[0004] Secondly, from the perspective of tissue function construction, the existing induction differentiation scheme is mostly too simple, mainly focusing on promoting adipose-derived stem cells to differentiate into mature adipocytes, i.e. simple adipogenic induction. However, natural adipose tissue is a highly structured and functional organ that contains a complex network of nerve fibers and abundant blood vessels for precise regulation of energy metabolism, sensing of external stimuli, and transport of oxygen and nutrients. The existing technology is difficult to reproduce this complex structure of nerve and blood vessel network symbiosis in an in vitro culture environment. Therefore, the generated engineered adipose tissue is often an isolated, lifeless adipocyte mass that lacks key innervation and effective blood supply. This structural defect directly leads to low survival rate of the engineered tissue after transplantation, making it difficult to effectively integrate with the host's original tissue and circulatory system, thereby failing to survive long-term and perform its normal physiological functions.
[0005] In addition, in the aspect of culture strategy, the conventional static culture mode cannot simulate the dynamic mechanical microenvironment in vivo, and the induction scheme with relatively unknown or single formula cannot provide precise biochemical signal guidance. The deficiencies in these two aspects jointly lead to the generally low efficiency of adipose stem cell differentiation in vitro: the formed lipid droplets are usually small and dispersed, and the cell lipid accumulation capacity is weak; the obtained engineered tissue structure is loose and has poor mechanical properties, and the key physical parameters such as elastic modulus are far from the natural fat tissue, which cannot provide the necessary three-dimensional structure support and suitable mechanical microenvironment for cells. These factors seriously restrict the possibility of constructing an engineered fat tissue with normal structure and function in vitro.
[0006] In summary, it is an urgent need to develop a new culture medium and supporting culture system that can simultaneously solve the three core problems of clinical safety, structural integrity and functional maturity, which is also the key direction of current technology research. SUMMARY
[0007] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present application provide a culture medium for adipose stem cells, which solves the problems of immune rejection risk, animal-derived pathogen contamination risk, lack of neural and vascular network, weak lipid accumulation capacity, loose structure and insufficient mechanical strength in the traditional adipose stem cell culture.
[0008] To achieve the above object, the present application provides the following technical solutions: A culture medium for adipose stem cells, specifically comprising the following components: Basic culture medium: DMEM / F (1:1), 10% (V / V) human platelet lysate, 1% (V / V) penicillin-streptomycin-amphotericin B solution; Neural-vascular niche: Slit3 recombinant protein, nerve growth factor-beta, glial cell-derived neurotrophic factor, lysyl oxidase inhibitor, dexamethasone, 3-isobutyl-1-methyl xanthine, insulin.
[0009] Preferably, the basic culture medium provides the necessary basic nutrients for cell survival and growth as a nutrient substrate and growth environment, including the required amino acids, vitamins, inorganic salts and glucose.
[0010] Preferably, the amino acids are selected from essential amino acids lysine, histidine, arginine, threonine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine and non-essential amino acids glutamine, asparagine, glycine, serine, proline, alanine, aspartic acid, glutamic acid, cysteine, tyrosine.
[0011] Preferably, the vitamins include choline, inositol, folic acid, nicotinamide, calcium pantothenate, vitamin B2, vitamin B1, pyridoxine hydrochloride, biotin, vitamin B12, p-aminobenzoic acid, lipoic acid.
[0012] Preferably, the inorganic salts include constant elements sodium chloride, potassium chloride, sodium dihydrogen phosphate, magnesium sulfate, calcium chloride.
[0013] Preferably, the Slit3 recombinant protein is a human Slit3 recombinant protein, and the working concentration is 50-200 ng / mL.
[0014] Preferably, the glial cell-derived neurotrophic factor is a human recombinant glial cell-derived neurotrophic factor, and the concentration is 10-50 ng / mL.
[0015] Preferably, the lysyl oxidase inhibitor is beta-aminopropionitrile, and the working concentration is 50-500 μM.
[0016] Preferably, the nerve growth factor-beta is a human recombinant nerve growth factor-beta, and the concentration is 20-100 ng / mL.
[0017] Preferably, the working concentration of dexamethasone is 0.1-1 μM, the working concentration of 3-isobutyl-1-methylxanthine is 0.1-1 mM, and the working concentration of insulin is 1-20 μg / mL.
[0018] The technical effects and advantages of the culture medium for fat stem cells are as follows: 1. In the application, 10% (V / V) human platelet lysate is used to provide a broad-spectrum growth factor, which is superior to fetal bovine serum and is more suitable for clinical transformation requirements; at the same time, multiple functional factors are used in cooperation, which surpasses the traditional single adipogenic induction; the preparation process of this method is standardized, has good stability, and is suitable for long-term storage and repeated use.
[0019] 2. In the application, the Slit3 recombinant protein in the nerve-vascular niche promotes the recruitment of perivascular cells and the maturation of blood vessels; the nerve growth factor-beta is used to guide the growth of sympathetic nerve axons into the differentiating adipose tissue; the glial cell-derived neurotrophic factor is used to support the survival and function of Schwann cells and promote innervation; the lysyl oxidase inhibitor is used to moderately inhibit excessive cross-linking of collagen and soften the early extracellular matrix; dexamethasone is a classic component of adipogenic induction, but the concentration is lower than that of the conventional formula; the 3-isobutyl-1-methylxanthine is a classic component of adipogenic induction; and the insulin is a classic component of adipogenic induction.
[0020] 3、The application, in the activation stage of 1-3 days, uses equiaxial stretching to simulate physiological mechanical fluctuation to activate the mechanical sensitive ion channel of the cell to start early differentiation; in the swelling growth stage of 4-10 days, equiaxial stretching is used to simulate the sustained stretching force suffered by the growth of adipose tissue to effectively promote lipid accumulation and cell swelling; in the functional maturation stage of 11-14 days, stretching is stopped to allow the cell to mature in a static state, so that the structure of the newly born adipose tissue is stable and the function is mature.
[0021] 4、The application, after result identification and effect verification, performs oil red O staining on the 7th day and the 14th day, a large number of red-stained lipid droplets with single morphology can be seen in the experimental group, and the cells are arranged closely to form a structure similar to a fat lobule; through qRT-PCR detection of lipogenic and functional related genes, the expression of PPARγ and aP2 in the experimental group is up-regulated by 3 times, and the lipogenic differentiation efficiency is higher; through immunofluorescence staining of cytoskeleton and specific markers on the 14th day, it can be observed that the tubular structure formed by CD31 positive cells penetrates between adipocytes, the fiber network positive for Tuj1 interweaves with the adipocyte area, and the α-SMA positive cells are wrapped around the CD31+ tubular structure.
[0022] 5、The application, among the neuro-vascular niche factors, Slit3 guides the recruitment of pericytes to promote vascular maturation; NGF-β attracts sympathetic axons to grow towards adipose tissue; GDNF is used to support the survival of Schwann cells to promote innervation, and the neuro-vascular niche factors actively guide and regulate the survival and maturation of the neuro-vascular network.
[0023] 6、The application, the construction of a non-contact co-culture system, uses a PDMS elastic film and a Transwell insert to construct a non-contact co-culture system, simulates the multi-cell microenvironment of adipose tissue in vivo, and promotes the preliminary formation of a neuro-vascular network through signal transmission between adipose stem cells, human umbilical vein endothelial cells and human Schwann cells; this method uses physical isolation but signal intercommunication, can effectively avoid cell mixing, and at the same time simulates the real tissue development environment to support multi-cell interaction.
[0024] 7、The application, using programmed dynamic mechanical stretching induction, uses mechanical stretching programs in 3 stages to control the adipose stem cells in a simulated physiological metabolic environment through a nutrition gradient; simulating a physiological mechanical environment significantly improves the adipogenic differentiation efficiency, and makes the PPARγ gene expression up-regulated by 2.8 times; biochemical and mechanical signals can promote tight structure and functional maturation.
[0025] 8、The application finally adopts a multi-dimensional detection method, including SEM observation of lipid droplets and ECM structure, qRT-PCR analysis of lipogenic and functional genes, oil red O staining for quantification of lipid droplet volume, 2-NBDG probe detection of glucose uptake, and immunofluorescence three-dimensional imaging analysis of neural-vascular network; the engineered adipose tissue is comprehensively, quantitatively and visually evaluated through multiple dimensions, and the structure, metabolism and neural-vascular function are systematically verified. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 is a flow chart of a culture medium for adipose stem cells according to the application. DETAILED DESCRIPTION
[0027] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments of the application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the application.
[0028] It should be noted that, in this document, relationship terms such as first and second are used only to distinguish one entity or operation from another, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations, and the terms “include”, “contain” or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such a process, method, article or device. Without more limitations, the elements defined by the statement “include” do not exclude the presence of other identical elements in the process, method, article or device that includes the elements. Embodiment 1
[0029] The embodiment provides a culture medium for adipose stem cells, and the specific implementation steps include: Experimental materials: Basic culture medium: DMEM / F (1:1), 10% (V / V) human platelet lysate, 1% (V / V) penicillin-streptomycin-amphotericin B solution; Neural-vascular niche: Slit3 recombinant protein, nerve growth factor-β, glial cell-derived neurotrophic factor, lysyl oxidase inhibitor, dexamethasone, 3-isobutyl-1-methyl xanthine, insulin.
[0030] Experimental purposes: A culture medium composition containing neural-vascular niche components is prepared.
[0031] Experimental steps: S1: Take a 1.5 mL sterile brown EP tube, accurately measure and add 10% human platelet lysate prepared with sterile PBS as a mixed solvent; S2: Add each specific component contained in the nerve-vascular niche to the above solvent in the specified order, one by one, accurately; S3: After adding each component, immediately use a vortex mixer to mix the solution for 30 seconds to ensure uniform dissolution and distribution of the components; S4: After all components are added and completely dissolved, filter the entire mixture using a sterile needle filter with a pore size of 0.22 μm to achieve complete sterilization; S5: Finally, accurately dispense the sterilized medium composition into 100 μL per tube, and immediately transfer it to a -80°C ultra-low temperature environment for long-term stable storage.
[0032] Experimental results: A special medium containing 10% human platelet lysate and nerve-vascular niche factors was successfully prepared. It was verified that each component was fully dissolved, the solution was clear, the sterile test was negative, and the storage stability was good after dispensing at -80°C; this example demonstrates the preparability and stability of the medium composition, providing a material basis for humanized and functional culture system. Example 2:
[0033] This example provides a medium for adipose stem cells, and the preparation of the medium used for cell inoculation after the establishment of a biological culture system. The specific implementation steps include: Experimental materials: PDMS elastic silicone membrane, type I collagen, Transwell insert.
[0034] Experimental purpose: To construct a biological culture system and inoculate cells, and to prepare and use the medium.
[0035] Experimental steps: S1: Place the PDMS elastic silicone membrane culture plate in an oxygen plasma treatment machine for surface activation treatment for 1 minute. Then, coat the activated PDMS membrane surface with a 50 μg / mL concentration of type I collagen solution. Next, transfer the entire system to a 37°C constant temperature environment and incubate for 2 hours to complete the protein coating fixation; S2: Take the 3rd passage of human adipose stem cells in the logarithmic growth phase and in good condition, and inoculate them into the Transwell insert at a density of 2.5 × 10 5S1: Resuspend the cells at a specific density of 5 x 105 cells / mL in 2 mL of basal medium, and then inoculate them evenly onto the surface of the PDMS membrane that has been pretreated as described above; S3: Inoculate the cell suspension prepared by mixing human umbilical vein endothelial cells and human Schwann cells at a specific ratio onto the polycarbonate membrane of the Transwell insert at a density of 5 x 105 cells per well; 4 S1: Resuspend the cells at a specific density of 5 x 105 cells / mL in 2 mL of basal medium, and then inoculate them evenly onto the surface of the PDMS membrane that has been pretreated as described above; S4: Place the Transwell insert that has completed the cell inoculation into the PDMS membrane culture plate inoculated with the adipose-derived stem cells, so as to construct a non-contact co-culture system that is physically isolated but allows signal molecules to exchange.
[0036] Experimental results: The non-contact co-culture system based on the PDMS membrane and the Transwell insert is successfully constructed; through microscopic observation, the human adipose-derived stem cells, the human umbilical vein endothelial cells and the human Schwann cells are all inoculated successfully, and the morphology is good, and no obvious cytotoxicity is observed after co-culturing for 24 hours. The system successfully simulates the multicellular microenvironment required for the development of adipose tissue in vivo, and lays a structural foundation for subsequent induced differentiation. Example 3:
[0037] The embodiment provides a culture medium for adipose-derived stem cells, which is cultured by a dynamic induced differentiation program. The specific implementation steps include: Experimental materials: Carbon dioxide, low-sugar DMEM culture solution, PBS, trypsin.
[0038] Experimental purposes: Culturing the adipose-derived stem cells by the dynamic induced differentiation program.
[0039] Experimental steps: S1: Place the culture plate in a culture box connected with a linear stretching device controlled by a computer, and set it to an equal biaxial stretching mode. Activate it for 1-3 days, stretch it at a stretching amplitude of 5% and a frequency of 1 Hz for 30 minutes, and then rest for 30 minutes; S2: Place the cells treated in S1 in a growth stage, and set the mode to equal biaxial stretching. Stretch them at a stretching amplitude of 15% and a frequency of 0.1 Hz for 6 hours, and then rest for 18 hours; S3: Stop stretching to make the cells mature in a resting state, change the culture conditions, and gradually replace the culture medium with low-sugar DMEM. Then continue to culture them in a carbon dioxide incubator at a temperature of 37°C and a volume fraction of 5%; S4: In the medium of S3, low-sugar DMEM culture solution is added every 24 hours, so that the concentration of low-sugar DMEM is gradually reduced in a gradient of 30%, 15%, and 10%; then, after waiting for 3-5 days, the adipose-derived stem cells are observed to grow, and the adipose-derived stem cells are cultured in DMEM culture medium containing 10% fetal bovine serum, and the culture medium is completely replaced every 2-3 days, while the floating fat blocks in the culture medium are sucked and discarded; S5: When the adipose-derived stem cells completely grow and the fusion degree reaches 90%, the adipose-derived stem cells are subcultured, then the cells are washed twice with 3 mL of PBS, and the PBS is sucked and discarded; S6: 1 mL of 0.25% trypsin is added to S5 to completely cover the cells, then the cell suspension is collected into a 50 ml centrifuge tube, and centrifuged at 100xg for 5 minutes at room temperature, the supernatant is sucked and discarded, the cell precipitate is resuspended with a minimum volume of complete culture medium, the cells are counted, and 1x10 4 / cm 2 cell density, 4 ml / 25 cm 2 10% fetal bovine serum DMEM culture medium volume inoculation culture.
[0040] Experimental results: Through the programmed dynamic mechanical stretching, the adipose-derived stem cells are successfully induced and differentiated; qRT-PCR detection shows that the expression amount of the adipogenic key gene PPARγ of the cells in the experimental group is up-regulated by 2.8 times at the 7th day of induction compared with the static culture group; this shows that the three-stage stretching program designed in the application can effectively activate the mechanical sensitive signal pathway of the cells and synergistically promote the adipogenic differentiation process. Example 4:
[0041] The embodiment provides a culture medium for adipose-derived stem cells, and the function of the stem cells cultured in the culture medium is detected, and the specific implementation steps include: Experimental raw materials: 2.5% glutaraldehyde, oil red O staining, fluorescent probe 2-NBDG, insulin, glucose.
[0042] Experimental purposes: Detect the function of the stem cells obtained by culturing the culture medium.
[0043] Experimental steps: S1: The tissue formed by the adipose-derived stem cells is trimmed into particles with a diameter of 2.5-5 mm, the tissue particles are fixed with 2.5% glutaraldehyde, then critical point drying and gold spraying are performed, and the adipose droplet morphology, extracellular matrix fiber arrangement density, and porosity are observed under an SEM; finally, the cell skeleton dyed with a ghost pen annulet and the cell nucleus dyed with DAPI are three-dimensionally reconstructed under a confocal microscope, and the cell orientation consistency index is calculated. S2: qRT-PCR detection was performed to analyze the expression levels of lipogenic genes PPARy, aP2 and functional gene ADIPOQ; lipid droplet volume / nucleus number ratio was quantitatively analyzed by oil red O staining and high-content imaging to evaluate the lipid accumulation capacity; the glucose uptake rate of cells under insulin stimulation was detected by using fluorescent probe 2-NBDG and flow cytometry to evaluate the metabolic function; and the intracellular ATP content and LDH activity in the culture supernatant were determined to comprehensively evaluate the metabolic activity and cell health degree; S3: Multicolor immunofluorescence staining was performed to label endothelial cell marker CD31, pericyte marker a-SMA, and neuron markers Tuj1 and NF200; three-dimensional image acquisition was performed by confocal microscopy, and the blood vessel density, nerve density and nerve-blood vessel co-localization coefficient were quantified by image analysis software; the wrapping of a-SMA positive cells to CD31 positive tubular structure was analyzed in three-dimensional space, and the mature blood vessel ratio was calculated; the microfluidic chip model was used to verify the induction ability of the supernatant of the experimental group culture medium on the directional growth of nerve axons to the blood vessel area, and the functional correlation verification was performed.
[0044] Experimental results: see Table 1 in detail.
[0045] Table 1: Test results of Example 4 The results of multifunctional quantitative evaluation of mature engineered adipose tissue are shown in Table 1; the data show that the experimental group shows the best level in key indicators such as lipid accumulation, elastic modulus, lipogenic specific PPARy expression and nerve-vascular coupling co-localization coefficient; which systematically proves the synergistic effect of the combination of programmed stretching + specific culture medium + co-culture system, which can guide the generation of engineered constructs that are more similar to natural adipose tissue in structure, metabolic function and nerve-vascular network. Comparative Example 1:
[0046] This example provides a traditional fetal bovine serum culture medium, and the specific implementation steps include: Experimental materials: High-sugar DMEM, fetal bovine serum FBS, 1% (V / V) penicillin-streptomycin solution, phosphate buffer, 0.25% trypsin-EDTA digestion solution.
[0047] Experimental purpose: Prepare a traditional fetal bovine serum culture medium.
[0048] Experimental steps: S1: In a sterile clean bench, take an appropriate amount of high-sugar DMEM basic culture medium into a sterile container; S2: Add fetal bovine serum to the culture medium to make the final volume concentration reach 10%; S3: Continue adding penicillin-streptomycin solution to S2 to achieve a final volume concentration of 1% to provide antibiotic protection; S4: Gently shake or vortex the medium to ensure that all components are thoroughly mixed to avoid generating too many air bubbles; S5: Use a vacuum filter bottle or needle filter with a pore size of 0.22μm to filter and sterilize the mixed culture medium to obtain the culture medium; store the prepared culture medium in a refrigerator at 4℃ for later use. S6: Take well-grown third-generation human adipose stem cells, wash with PBS, add 0.25% trypsin-EDTA digestion solution for digestion, and terminate digestion with DMEM complete medium containing 10% FBS. After centrifugation and resuspending, count the cells. S7: with 1×10 4 pcs / cm 2 To achieve the desired cell density, adipose-derived stem cells were seeded into cell culture plates, and the complete culture medium prepared in step S5 was added. The plates were then placed in an incubator at 37°C and 5% CO2 for static culture. S8: Replace with fresh culture medium every 2-3 days until the cell confluence reaches 90% or more; S9: Once the cell confluence reaches 100%, replace the medium with the same DMEM complete medium containing 10% FBS as described above, and continue static culture to induce differentiation for 14 days, changing the medium every 3 days during this period.
[0049] Experimental results: See Table 2 for details.
[0050] Table 2: Test Results of Comparative Example 1 Using traditional fetal bovine serum culture medium and static culture methods, adipose stem cells can undergo basic adipogenic differentiation, but the differentiation efficiency is low, the formed lipid droplets are small and dispersed, the tissue structure is loose, the mechanical properties are poor, gene expression is not significantly upregulated, and it is completely unable to support the symbiosis and formation of neural and vascular networks.
[0051] Example 1 describes a method for preparing a specialized culture medium using human platelet lysate combined with neurovascular niche factors. The mechanism involves using human platelet lysate to provide a broad spectrum of growth factors, replacing traditional fetal bovine serum and avoiding the risks of immune rejection and pathogen contamination. Simultaneously, the neurovascular niche factors synergistically promote perivascular cell recruitment, vascular maturation, and nerve innervation, soften the extracellular matrix, and optimize the adipogenic induction environment. Specific steps include using 10% human platelet lysate PBS as a solvent, sequentially adding the neurovascular niche components, vortexing, sterilizing through a 0.22 μm filter, aliquoting, and storing at -80°C. The method successfully prepared a clear, sterile, and stable culture medium, laying the foundation for a humanized and functionalized culture system.
[0052] Example 2 employs a non-contact co-culture system based on a PDMS elastic silicone membrane and a Transwell insert. The mechanism simulates the multicellular microenvironment of adipose tissue development in vivo. By co-culturing human adipose stem cells, human umbilical vein endothelial cells, and human Schwann cells, intercellular paracrine signal transduction is promoted, enhancing the potential for neurovascular network formation. Specific steps include oxygen plasma treatment of the PDMS membrane, coating with type I collagen, followed by seeding adipose stem cells onto the PDMS membrane, and simultaneously seeding endothelial cells and Schwann cells into the Transwell insert to form a non-contact co-culture system. Results confirmed good cell morphology, no toxic reactions, and successful construction of a microenvironmental structure supporting multicellular interactions.
[0053] Example 3 employed a programmed dynamic mechanical stretching-induced differentiation method. The mechanism involves activating mechanosensitive channels and signaling pathways in cells through a three-stage mechanical stretching process: the first stage uses iso-biaxial stretching to simulate physiological mechanical fluctuations, initiating early differentiation; the second stage uses high-intensity stretching to simulate tissue growth tension, promoting lipid accumulation and cell expansion; and the third stage involves static maturation to stabilize the newly formed tissue structure. Specific steps include placing the culture plate in a computer-controlled stretching device, setting different stretching amplitudes and frequencies, and combining this with culture medium replacement and gradient reduction of nutrient concentrations. qRT-PCR results showed that PPARγ expression was upregulated by 2.8-fold, confirming that mechanical stretching effectively synergistically promotes adipogenic differentiation through biochemical signals.
[0054] Example 4 employed a comprehensive functional quantitative assessment method. The mechanism involved using a multi-index detection system to verify the lipid accumulation capacity, mechanical properties, metabolic function, and neurovascular coupling efficiency of engineered adipose tissue. Specific steps included SEM observation of lipid droplet morphology and extracellular matrix arrangement, qRT-PCR analysis of PPARγ, aP2, and ADIPOQ gene expression, Oil Red O staining to quantify the lipid droplet volume / nucleus ratio, detection of glucose uptake using the fluorescent probe 2-NBDG, and multicolor immunofluorescence staining combined with confocal microscopy three-dimensional imaging analysis of vascular density, nerve density, and colocalization coefficient. The final data are shown in Table 1: lipid droplet volume / nucleus ratio reached 2850±320 μm³, elastic modulus 12.5±1.8 kPa, PPARγ expression upregulated by 3.8±0.5 times, and vascular-neural colocalization coefficient 0.67±0.08, comprehensively demonstrating the superiority of this culture medium in terms of structure, metabolism, and neurovascular function.
[0055] Comparative Example 1 used traditional fetal bovine serum culture medium and static culture method. Its mechanism relies on animal-derived serum to provide basic growth factors, but it lacks specific neurovascular signals and mechanical stimulation, resulting in low differentiation efficiency and functional loss. The specific steps included preparing DMEM medium containing 10% fetal bovine serum, inoculating adipose stem cells and then statically culturing them for 14 days with regular medium changes. The results are shown in Table 2: the lipid droplet volume / nucleus ratio was only 620±105 μm³, the elastic modulus was 3.2±0.9 kPa, PPARγ expression was 1.2±0.3 times, and the vascular-neural colocalization coefficient was 0.05±0.02, which fully exposed the serious deficiencies of the traditional method in lipid accumulation, tissue mechanical properties, adipogenic gene expression, and neurovascular network formation.
[0056] By comprehensively comparing the various embodiments and comparative examples, Example 1 achieved the optimal balance in providing humanized growth factors and specific neurovascular niche signals, laying a biochemical foundation for subsequent induction; the co-culture system constructed in Example 2 ingeniously simulated the in vivo microenvironment, enhancing paracrine signal transduction among various cells; the programmed mechanical stretching used in Example 3 directly activated the cell's mechanosensitive pathways, synergistically promoting differentiation with biochemical signals; the multi-dimensional identification method used in Example 4 systematically confirmed the structural integrity and functional maturity of the final product; Comparative Example 1, using traditional fetal bovine serum culture medium and static culture, highlighted the limitations of traditional methods in constructing neurovascular networks and achieving insufficient functional maturity. Therefore, this invention, through the organic integration of biochemical factors, physical stimulation, and the cellular microenvironment, generates a synergistic effect that surpasses existing technologies, successfully solving the key technical bottleneck of in vitro generation of functionalized adipose tissue.
[0057] refer to Figure 1 The flowchart systematically demonstrates the complete technical path for constructing engineered adipose tissue in this invention: First, a functional culture medium based on human platelet lysate and containing specific neurovascular niche factors is prepared. Then, a non-contact co-culture system is constructed using a PDMS membrane and Transwell. Subsequently, a programmed three-stage mechanical stretching is implemented—passing through a patterned activation, expansive growth, and functional maturation stage in sequence, simulating a physiological mechanical environment. Finally, multi-dimensional identification verifies that the obtained tissue possesses large lipid droplets, high adipogenic gene expression, good mechanical properties, and a mature neurovascular network, fully embodying the core innovative process of in vitro construction of functional adipose tissue driven by the synergistic effect of culture medium, culture system, and mechanical stimulation.
[0058] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.
[0059] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A medium for adipose-derived stem cells, characterized by, Specifically comprising the following components: Basic medium: DMEM / F (1:1), 10% (V / V) human platelet lysate, 1% (V / V) penicillin-streptomycin-amphotericin B solution; Neuro-vascular niche: Slit3 recombinant protein, nerve growth factor-β, glial cell-derived neurotrophic factor, lysyl oxidase inhibitor, dexamethasone, 3-isobutyl-1-methyl xanthine, insulin.
2. The medium for adipose-derived stem cells according to claim 1, wherein The basic medium serves as a nutrient substrate and growth environment, providing the necessary basic nutrients for cell survival and growth, including required amino acids, vitamins, inorganic salts, and glucose.
3. The medium for adipose-derived stem cells according to claim 2, wherein The amino acids are selected from essential amino acids lysine, histidine, arginine, threonine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, and non-essential amino acids glutamine, asparagine, glycine, serine, proline, alanine, aspartic acid, glutamic acid, cysteine, and tyrosine.
4. The medium for adipose-derived stem cells according to claim 2, wherein The vitamins include choline, myo-inositol, folic acid, nicotinamide, calcium pantothenate, vitamin B2, vitamin B1, vitamin B6 hydrochloride, biotin, vitamin B12, p-aminobenzoic acid, and lipoic acid.
5. The medium for adipose-derived stem cells according to claim 2, wherein The inorganic salts include constant elements sodium chloride, potassium chloride, sodium dihydrogen phosphate, magnesium sulfate, and calcium chloride.
6. The medium for adipose-derived stem cells according to claim 1, wherein The Slit3 recombinant protein is a human Slit3 recombinant protein, with a working concentration of 50-200 ng / mL.
7. The medium for adipose-derived stem cells according to claim 1, wherein The glial cell-derived neurotrophic factor is a human recombinant glial cell-derived neurotrophic factor, with a concentration of 10-50 ng / mL.
8. The medium for adipose tissue stem cells according to claim 1, wherein The lysyl oxidase inhibitor is β-aminopropionitrile, with a working concentration of 50-500 μM.
9. The medium for adipose tissue stem cells according to claim 1, wherein The nerve growth factor-β is a human recombinant nerve growth factor-β, with a concentration of 20-100 ng / mL.
10. The medium for adipose tissue stem cells according to claim 1, wherein The working concentration of dexamethasone is 0.1-1 μM, the working concentration of 3-isobutyl-1-methyl xanthine is 0.1-1 mM, and the working concentration of insulin is 1-20 μg / mL.