A method for dynamically culturing 3D-printed single tissues and 3D-printing neuromuscular junctions in a bioreactor
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-29
- Publication Date
- 2026-08-14
Smart Images
Figure CN122563876A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedical engineering and tissue engineering technology, specifically to a method for dynamically culturing 3D-printed single tissue and 3D-printed neuromuscular junctions in a bioreactor, and the cultured 3D-printed neuromuscular junctions. Background Technology
[0002] The neuromuscular junction is a crucial signal transduction unit between motor neurons and skeletal muscle fibers. Abnormalities in its structure and function are directly linked to various diseases such as myasthenia gravis, amyotrophic lateral sclerosis (ALS), and neurodegenerative diseases. The normal physiological function of the neuromuscular junction depends on the synergistic action of nerve and muscle cells, as well as the microvascular network formed by endothelial cells providing nutrition and substance exchange. Therefore, the neuromuscular junction is essentially a complex tissue encompassing nerves, muscles, and blood vessels. Constructing functionally stable in vitro models of the neuromuscular junction is a core foundation for related disease research and drug development.
[0003] Currently, 3D printing technology can accurately construct single nerve tissues, single muscle tissues, single endothelial blood vessel tissues, and complex neuromuscular junction tissues. However, in vitro culture is mainly based on static culture systems, which cannot effectively simulate the dynamic mechanical microenvironment in vivo, including the mechanical stretching generated by muscle movement and the fluid shearing force brought about by body fluid circulation. This results in various cultured tissues having problems such as immature differentiation, loose structure, and poor functional stability, making it impossible to truly simulate physiological characteristics in vivo and meet the needs of precise research.
[0004] Bioreactors can simulate the dynamic physiological microenvironment in vivo by regulating parameters such as mechanical stretching and fluid perfusion, providing key signals for cell functional maturation. However, current technologies for dynamic culture of the neuromuscular junction are not yet mature, lacking standardized culture parameter systems and insufficient matching between dynamic mechanical stimulation and fluid shear force. This results in limited improvement in the maturation efficiency and functional integrity of the neuromuscular junction, restricting its application in scientific research and clinical practice. For example, Charoensook et al. (Integrative Biology, 2017, 9: 956-967) reported an in vitro model of the neuromuscular junction, co-culturing stem cell-derived motor neurons with muscle cells transdifferentiated from fibroblasts, and applying electrical stimulation (3 V / cm, 1 Hz, 2 ms pulse width, 6 hours daily) via carbon rod electrodes to promote the functional maturation of the neuromuscular synapse. While this approach preliminarily validates the promoting effect of electrical stimulation on the maturation of neurons and muscle cells, its construction method, a two-dimensional adherent co-culture system, cannot achieve the directional arrangement and structural formation of cells in three-dimensional space. Furthermore, the electrical stimulation parameters are not differentiated for different cell types, nor are endothelial vascularization components integrated, making it difficult to simulate the complex physiological microenvironment of multi-tissue coupling in vivo, involving nerves, muscles, and blood vessels. More critically, this approach lacks a standardized fluid shear force regulation mechanism, making it impossible to set mechanical parameters tailored to the physiological needs of individual neural, muscle, and endothelial vascular tissues, thus limiting its application in the precise construction and high-throughput screening of multiple tissue types.
[0005] Therefore, there is an urgent need to provide a dynamic culture method for 3D-printed neuromuscular junctions. By precisely controlling the dynamic culture conditions, the maturation and functional perfection of the neuromuscular junction can be significantly promoted, overcoming the technical shortcomings of static culture and providing new possibilities for research on neuromuscular injury repair and regenerative medicine. Summary of the Invention
[0006] The purpose of this invention is to provide a method for dynamically culturing 3D-printed single tissues and 3D-printed neuromuscular junctions within a bioreactor. The dynamic culture includes 3D-printing single nerve tissues, single muscle tissues, single endothelial vascular tissues, and complex neuromuscular junction tissues. By separately adjusting the matching parameters of the shaking speed of the bioreactor and the corresponding fluid shear force for each type of tissue, stable fluid shear force is generated to simulate the dynamic physiological microenvironment of different tissues in vivo. This significantly promotes the maturation and differentiation of various 3D-printed tissues, especially enhancing the synergistic effect of nerve cells, muscle cells, and endothelial cells in the 3D-printed neuromuscular junction, as well as the functional integrity of synaptic connections and vascular networks. This constructs various 3D-printed tissue models that more closely resemble in vivo physiological states, providing an ideal tool for related disease research and drug screening.
[0007] The technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a method for dynamically culturing 3D printed tissues in a bioreactor is provided. The 3D printed tissues include: (1) 3D printing a single nerve tissue; (2) 3D printing a single muscle tissue; (3) 3D printing a single endothelial vascular tissue; (4) 3D printing a neuromuscular junction (a complex tissue integrating nerve, muscle, and blood vessel); the method includes steps of dynamically culturing in a bioreactor and detecting and identifying the functions of various 3D printed tissues in sequence.
[0008] In some embodiments of the present invention, the various 3D printed tissues are all structures initially constructed by 3D printing technology: a single nerve tissue is formed by culturing neural stem cells (NSCs) alone; a single muscle tissue is formed by culturing muscle cells (C2C12) alone; a single endothelial vascular tissue is formed by culturing human umbilical vein endothelial cells (HUVECs) alone; a complex neuromuscular tissue is formed by co-culturing nerve cells, muscle cells, and endothelial cells; each tissue obtains a stable three-dimensional spatial structure through 3D printing technology and has a preliminary cell morphology and structural basis.
[0009] In some embodiments of the present invention, the steps of dynamically culturing in a bioreactor shaker include: transferring the 4 types of 3D printed tissues initially constructed in vitro into the culture wells of a six-well plate, adding the corresponding compatible culture medium to each well, then placing the six-well plate in a constant temperature CO2 compatible shaker supporting the bioreactor, respectively adjusting the shaker parameters to generate corresponding stable fluid shear forces for the culture medium, precisely maintaining the stability of the culture environment through the bioreactor, regularly observing the growth status and changing the culture medium; the shaker is a special constant temperature CO2 compatible type for the bioreactor, ensuring the stability of temperature and CO2 concentration during the culture process and avoiding the influence of environmental fluctuations on cell growth.
[0010] In some embodiments of the present invention, there is a clear matching relationship between the shaker rotation speed of the bioreactor supporting and the fluid shear forces of various tissues. Differentiated shear forces are set for the 4 types of tissues, specifically as follows: (l) For a single nerve tissue: the relative centrifugal force of the shaker is 0.015 - 0.027 g, and the corresponding generated fluid shear force is 0.3 - 0.6 dyne / cm 2 , and this shear force range can promote the axon extension, maturation and differentiation of nerve cells, and ensure the uniform distribution of nutrients and the clearance of metabolic wastes; (2) For a single muscle tissue: the relative centrifugal force of the shaker is 0.027 - 0.042 g, and the corresponding generated fluid shear force is 0.6 - 1.0 dyne / cm 2 , and this shear force range can promote the fusion of muscle cells and the maturation of myotubes; (3) Single endothelial vascular tissue: The relative centrifugal force of the shaker is 0.042~0.060 g, and the corresponding fluid shear force is 1.0~1.5 dyne / cm 2 This range of shear force can promote the arrangement of endothelial cells and the formation of complete vascular-like lumen structures; (4) Neuromuscular junction tissue: The relative centrifugal force of the shaker is 0.015~0.051 g, and the corresponding fluid shear force is 0.3~1.2 dyne / cm 2 This shear force range can take into account the growth needs of nerve, muscle and endothelial cells, promote their synergistic effect, and form complex tissues with complete structure and stable function. The above parameter ranges are all perfectly suited to the bioreactor's control capabilities, resulting in optimal dynamic stimulation effects.
[0011] In some embodiments of the present invention, the shaking mode of the shaker in the bioreactor is horizontal circular shaking with a shaking radius of 10-20 mm, which ensures uniform flow of the culture medium and avoids excessive local shear force that could cause cell damage or insufficient shear force that could not achieve the stimulation effect. The environmental conditions for dynamic culture are: temperature 37°C, CO2 volume fraction 5%, and culture humidity 95%, which are precisely maintained by the environmental control module of the bioreactor to simulate the physiological environmental parameters in vivo.
[0012] In some embodiments of the present invention, the culture medium for dynamic culture is adapted and adjusted according to the tissue type, as follows: (1) Single nerve tissue culture medium: DMEM / F12 + 2% B27 + 1% FBS + 1% penicillin-streptomycin double antibiotic solution; (2) Single muscle tissue culture medium: DMEM + 2% horse serum + 1% penicillin-streptomycin double antibiotic solution; (3) Culture medium for single endothelial vascular tissue: ECM angiogenesis differentiation-specific culture medium: ECM + 5% FBS + 1% ECGS + 1% penicillin-streptomycin double antibiotic solution; (4) Neuromuscular junction tissue culture medium: DMEM + 10% horse serum + 1% penicillin-streptomycin double antibiotic solution; The culture medium for each type of 3D printed tissue is replaced every 24 hours, with a replacement volume of 70% of the total culture medium volume, to ensure nutrient supply and environmental stability. The continuous culture time is 7-14 days to ensure that each type of 3D printed tissue is fully matured. Automated culture monitoring can be achieved through the bioreactor's program control module.
[0013] In some embodiments of the present invention, the functional detection and identification of various 3D printed tissues are specifically adapted and adjusted according to the tissue type, including cell survival status detection, related gene and protein expression detection, to verify the differentiation maturity and functional integrity of various 3D printed tissues.
[0014] In some embodiments of the present invention, the cell viability detection utilizes the Calcein AM / PI cell live / dead staining method. Cultured 3D-printed tissues are collected, residual culture medium is washed with PBS, an appropriate volume of detection working solution is added, and the tissues are incubated at 37°C and 5% CO2 in the dark for 30 min. The staining effect is immediately observed under an inverted fluorescence microscope (Calcein AM is green fluorescence, indicating live cells; PI is red fluorescence, indicating dead cells) to assess cell viability and proliferation status.
[0015] In some embodiments of the present invention, the expression of the relevant genes is detected using real-time quantitative PCR. Total RNA is extracted from 3D-printed tissue and reverse transcribed to synthesize cDNA. Using the synthesized cDNA as a template, the mRNA expression levels of genes related to differentiation and maturation of various tissues are detected using real-time quantitative PCR (single muscle tissue detection). Myh4 , Myog , Myod Single endothelial vascular tissue detection VEGF , VWF , EGF2 , CD31 Neuromuscular junction tissue detection FOX3, DES, VEGF ); In some embodiments of the present invention, the detection of the relevant protein expression utilizes immunofluorescence staining. 3D-printed tissue samples are collected, fixed, perforated, and blocked. Then, corresponding primary antibodies (Tubb3, Map2, Syn, Chat, and D28K antibodies for single nerve tissue; Myh4 and Myog antibodies for single muscle tissue; VEGF and CD31 antibodies for single endothelial vascular tissue; and Myh3 antibody for neuromuscular junction tissue) are added and incubated overnight, followed by incubation with fluorescently labeled secondary antibodies. Finally, DAPI is added for nuclear staining, and the fluorescence intensity is observed using a laser confocal microscope to assess protein expression levels.
[0016] Another aspect of the present invention provides a 3D-printed neuromuscular junction obtained by the above-mentioned bioreactor dynamic culture method. The tissue simultaneously possesses mature nerve fibers, differentiated myotubes, and vascular-like structures. The degree of vascularization meets the physiological functional requirements, and the structure is complete and the function is stable. It can be directly applied to the study of disease mechanisms related to neuromuscular junctions, drug screening, and tissue repair-related experiments.
[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention simulates the dynamic physiological microenvironment of different tissues in vivo by precisely controlling the rotation speed of the shaker in the bioreactor and the fluid shear force of various tissues. Unlike existing static culture methods, this invention optimizes the growth and differentiation of nerve, muscle, and endothelial blood vessel cells through differentiated fluid shear force regulation, significantly improving cell maturity and synergistic effects. Especially in the neuromuscular junction tissue, it promotes effective collaboration among nerve cells, muscle cells, and endothelial cells, thereby improving the functional stability of the tissue model.
[0018] 2. This invention sets precise fluid shear force ranges based on the biological characteristics of different tissue types to promote the maturation and differentiation of various tissues. This differentiated shear force design includes shear forces of 0.3~0.6 dyne / cm² required for single nerve tissue, single muscle tissue, single endothelial vascular tissue, and neuromuscular junction tissue, respectively. 2 0.6~1.0 dyne / cm 2 1.0~1.5 dyne / cm 2 0.3~1.2 dyne / cm 2 This approach promotes physiological cell growth while avoiding damage to cells from excessively high or low shear forces. It effectively improves the precision and functionality of tissue construction.
[0019] 3. This invention combines 3D printing technology with a dynamic culture system to achieve effective culture of single and complex tissues such as nerves, muscles, and endothelial blood vessels. 3D printing technology provides a stable three-dimensional spatial structure for various tissues, enabling cells to grow and differentiate fully in a precisely controlled dynamic environment. Especially in the construction of neuromuscular junction tissues, it can realistically simulate the connection between nerves and muscles in vivo, ensuring the stability of tissue function and the integrity of its structure.
[0020] In summary, this invention achieves efficient culture and maturation of 3D-printed neuromuscular junction tissues by precisely controlling the culture environment of the bioreactor. It not only provides a highly realistic experimental platform for basic biological research, but also offers an effective technical solution for the study of the mechanisms of related diseases, drug screening, and tissue repair, and has broad application prospects. Attached Figure Description
[0021] Figure 1 This invention presents a schematic diagram of the process for dynamically culturing a single tissue and printing a neuromuscular junction in a bioreactor.
[0022] Figure 2Comparison of live / dead staining results of 3D-printed single neural tissue cells under static culture and dynamic culture in a bioreactor (A and B), as well as immunofluorescence staining and related fluorescence statistical analysis results (C and D).
[0023] Figure 3 Comparison of live / dead staining results of 3D printed single muscle tissue cells under static culture and dynamic culture in a bioreactor (A); Comparison of gene expression levels (B); Immunofluorescence staining results (C).
[0024] Figure 4 Comparison of 3D-printed endothelial cell live / dead staining results under static culture and dynamic culture in a bioreactor (A); Comparison of gene expression levels (B); Immunofluorescence staining results (C).
[0025] Figure 5 Comparison of live / dead staining results of 3D-printed neuromuscular junction cells under dynamic culture in a bioreactor (A); Comparison of gene expression levels (B); Immunofluorescence staining results (C). Detailed Implementation
[0026] The following detailed description of the invention's concept and technical effects, along with specific embodiments, will provide a clear and complete understanding of the invention's purpose, features, and effects. Obviously, the described embodiments are only a portion of the embodiments of the invention, not all of them, and the implementation of the invention is not limited thereto. Unless otherwise stated, the reagents, cells, instruments, etc., used in the embodiments of this application are all commercially available products.
[0027] Figure 1 This is a schematic diagram illustrating the process of dynamically culturing 3D-printed single tissues and 3D-printed neuromuscular junctions in a bioreactor according to the present invention. Utilizing 3D printing technology, preliminary single nerve tissues, single muscle tissues, single endothelial vascular tissues, and neuromuscular junction tissues are obtained. After dynamic culture in a bioreactor simulating the dynamic physiological microenvironment of different tissues in vivo, the maturation and differentiation of various 3D-printed tissues can be significantly promoted, resulting in 3D-printed tissue models that more closely resemble their in vivo physiological state and have more complete functions. This provides an ideal tool for related disease research and drug screening.
[0028] Example 1 Dynamic culture of 3D-printed single neural tissue in a bioreactor: Take the 3D printed single nerve tissue initially constructed in vitro and transfer it into a standard six-well cell culture plate. Add 2.5 mL of the corresponding dynamic culture medium to each well. Fix the six-well plate in a thermostatic CO2-compatible shaker supporting the bioreactor. Adjust the shaker parameters and the corresponding fluid shear force respectively and conduct dynamic culture. Take another 3D printed single nerve tissue, transfer it into a standard six-well cell culture plate, add 2.5 mL of the corresponding culture medium to each well, and place it in a conventional CO2 cell culture incubator for static culture. In the above embodiment, the dynamic culture conditions for the bioreactor are as follows: the relative centrifugal force is 0.020 g, horizontal circular shaking (shaking radius 15 mm), and the corresponding fluid shear force is 0.4 dyne / cm². The dynamic culture medium is: DMEM / F12 + 2% B27 + 1% FBS + 1% penicillin-streptomycin double antibody solution. In the above embodiment, the static culture conditions are as follows: place the 3D printed single nerve tissue in an incubator at 37°C, 5% CO2, and 95% humidity for static culture. The culture medium is the same as the dynamic culture medium.
[0029] In the above embodiment, for both the dynamic culture in the bioreactor and the static culture, the culture medium is changed every 24 h, and the changed volume is 70% of the total culture medium volume to ensure nutrient supply and environmental stability. Culture is continued for 7 days.
[0030] Functional identification of 3D printed single nerve tissue by dynamic culture in the bioreactor: Collect the 3D printed single nerve tissue obtained by dynamic culture in the bioreactor. Take the 3D printed single nerve tissue under static culture as a control, and perform cell viability / dead staining and immunofluorescence staining on the 3D printed single nerve tissue obtained by dynamic culture in the bioreactor. In the above embodiment, the cell viability / dead staining includes: prepare the Calcein AM / PI detection working solution, collect the 3D printed single nerve tissue, wash the residual culture medium with PBS, add an appropriate volume of the detection working solution, incubate in the dark at 37°C and 5% CO2 for 30 min, and then immediately observe the staining effect under an inverted fluorescence microscope (Calcein AM is green fluorescence, indicating live cells; PI is red fluorescence, indicating dead cells).
[0031] In the above embodiment, to determine the expression of related proteins in the 3D printed single nerve tissue obtained by dynamic culture in the bioreactor, the immunofluorescence staining method is used, which includes collecting the 3D printed single nerve tissue samples, fixing, permeabilizing, blocking, adding Tubb3, Map2, Syn, Chat, D28K antibodies and incubating overnight, then incubating with fluorescently labeled secondary antibodies, and staining the cell nuclei with DAPI. Finally, observe the staining situation under a laser confocal microscope.
[0032] The experimental results are as follows Figure 2 shown. In the 3D printed single nerve tissue under static culture and dynamic culture in a bioreactor, the cells grew well. Compared with static culture, the 3D printed single nerve tissue under dynamic culture in a bioreactor had higher expression of Tubb3 and Map2 proteins, and longer axon extension, indicating successful differentiation of neurons. Moreover, dynamic culture could significantly promote the expression of Syn, Chat, and D28K proteins, confirming that dynamic culture in a bioreactor could induce the formation of neural synapses and functional neurons.
[0033] Example 2 Dynamic culture of 3D printed single muscle tissue in a bioreactor: Take the 3D printed single muscle tissue preliminarily constructed in vitro, transfer it into a standard six-well cell culture plate, add 2.5 mL of the corresponding dynamic culture medium to each well, fix the six-well plate in a constant temperature CO2 compatible shaker supporting the bioreactor, adjust the shaker parameters and the corresponding fluid shear force respectively, and conduct dynamic culture; Take another 3D printed single muscle tissue, transfer it into a standard six-well cell culture plate, add 2.5 mL of the corresponding compatible culture medium to each well, and place it in a conventional CO2 cell culture incubator for static culture; In the above example, the conditions for dynamic culture in a bioreactor are as follows: relative centrifugal force is 0.034 g, horizontal circular shaking (shaking radius 15 mm), corresponding fluid shear force 0.8 dyne / cm², and the dynamic culture medium is DMEM + 2% horse serum + 1% penicillin-streptomycin double antibody solution; In the above example, the conditions for static culture are as follows: Place the 3D printed single muscle tissue in a culture incubator at 37 °C, 5% CO2, and 95% humidity for static culture; The culture medium is the same as the dynamic culture medium.
[0034] In the above example, the culture medium for both dynamic culture in a bioreactor and static culture was changed every 24 h, and the replacement volume was 70% of the total culture medium volume to ensure nutrient supply and environmental stability; The culture continued for 7 days.
[0035] Functional identification of 3D printed single muscle tissue cultured dynamically in a bioreactor: Collect the 3D printed single muscle tissue cultured dynamically in a bioreactor, use the 3D printed single muscle tissue under static culture as a control, conduct cell live / dead staining on the 3D printed single muscle tissue obtained by dynamic culture in a bioreactor, detect the mRNA expression levels of related genes by real-time fluorescence quantitative PCR, and perform immunofluorescence staining; In the above embodiments, the cell live / dead staining includes: preparing Calcein AM / PI detection working solution, collecting 3D printed single muscle tissue, washing residual culture medium with PBS, adding an appropriate volume of detection working solution, incubating at 37 ℃ and 5% CO2 in the dark for 30 min, and then immediately observing the staining effect under an inverted fluorescence microscope (Calcein AM is green fluorescence, indicating live cells; PI is red fluorescence, indicating dead cells).
[0036] In the above embodiments, the real-time quantitative PCR detection of related gene expression levels includes: extracting total RNA from a 3D-printed single muscle tissue and reverse transcribing it to synthesize cDNA, and then using the synthesized cDNA as a template to detect the mRNA expression level of related genes using real-time quantitative PCR.
[0037] In the above embodiments, the relevant genes include: Myh4 , Myog , Myod.
[0038] In the above embodiments, the expression of relevant proteins in the 3D-printed single muscle tissue obtained by dynamic culture in a bioreactor was measured using immunofluorescence staining. This included collecting 3D-printed single muscle tissue samples, fixing, perforating, and blocking them, incubating with Myh4 and Myog antibodies, followed by incubation with fluorescently labeled secondary antibody, and staining the cell nuclei with DAPI. Finally, the staining was observed using a laser confocal microscope.
[0039] Experimental results are as follows Figure 3 As shown, the 3D-printed single muscle tissue obtained from dynamic culture in a bioreactor exhibited good cell growth and proliferation, with a viability exceeding 95%. Compared to static culture, early differentiation genes... Myod The expression is low, and Myog (Mid-term) and Myh4 The expression level of (late stage) was significantly increased, and dynamic culture could significantly induce the expression of Myh4 and Myog proteins, confirming that dynamic culture in a bioreactor can effectively promote the accelerated differentiation of muscle cells towards the functional maturity stage, and mature 3D printed single muscle tissue can be obtained.
[0040] Example 3 Dynamic culture of single endothelial blood vessel tissue printed dynamically in a bioreactor: Take the 3D printed single endothelial vascular tissue initially constructed in vitro, which is constructed by endothelial cells; transfer it into a standard six-well cell culture plate, add 2.5 mL of the corresponding dynamic culture medium to each well, fix the six-well plate in a constant temperature CO2 compatible shaker supporting the bioreactor, adjust the shaker parameters and the corresponding fluid shear force respectively, and perform dynamic culture; take another 3D printed single endothelial vascular tissue, transfer it into a standard six-well cell culture plate, add 2.5 mL of the corresponding culture medium to each well, and place it in a conventional CO2 cell culture incubator for static culture; In the above embodiment, the dynamic culture conditions for the bioreactor are: relative centrifugal force is 0.051 g, horizontal circular shaking (shaking radius 15 mm), and the corresponding fluid shear force is 1.2 dyne / cm 2 , and the dynamic culture medium is a special medium for ECM angiogenesis differentiation; In the above embodiment, the static culture conditions are: place the 3D printed single endothelial vascular tissue in an incubator at 37 °C, 5% CO2, and 95% humidity for static culture; the culture medium is the same as the dynamic culture medium.
[0041] In the above embodiment, the culture medium is changed every 24 h during both the dynamic culture in the bioreactor and the static culture, and the changed volume is 70% of the total culture medium volume to ensure nutrient supply and environmental stability; continuously culture for 7 days.
[0042] Functional identification of the 3D printed single endothelial vascular tissue by dynamic culture in the bioreactor: Collect the 3D printed single endothelial vascular tissue by dynamic culture in the bioreactor, take the 3D printed single endothelial vascular tissue by static culture as a control, perform cell live / dead staining on the 3D printed single endothelial vascular tissue obtained by dynamic culture in the bioreactor, detect the mRNA expression level of related genes by real-time fluorescence quantitative PCR, and perform immunofluorescence staining; In the above embodiment, the cell live / dead staining includes: prepare the Calcein AM / PI detection working solution, collect the 3D printed single nerve tissue, wash the residual culture medium with PBS, add an appropriate volume of the detection working solution, incubate in the dark at 37 °C and 5% CO2 for 30 min, and then immediately observe the staining effect under an inverted fluorescence microscope (Calcein AM is green fluorescence, indicating live cells; PI is red fluorescence, indicating dead cells).
[0043] In the above embodiment, the detection of the mRNA expression level of related genes by real-time fluorescence quantitative PCR includes: extract the total RNA of the 3D printed single nerve tissue and reverse transcription synthesize cDNA, and then use the synthesized cDNA as a template to detect the mRNA expression level of related genes by real-time fluorescence quantitative PCR.
[0044] In the above embodiments, the related genes include VEGF, VWF, EGF2, CD31 .
[0045] In the above embodiments, to determine the expression of related proteins in the 3D printed single endothelial vascular tissue obtained by dynamic culture of the bioreactor, an immunofluorescence staining method is used, which includes collecting 3D printed single endothelial vascular tissue samples, fixing, permeabilizing, and blocking them, then adding VEGF and CD31 antibodies for incubation, and then incubating with fluorescently labeled secondary antibodies, and staining the cell nuclei with DAPI. Finally, a laser confocal microscope is used to observe the staining situation.
[0046] The experimental results are as Figure 4 shown. In the 3D printed single endothelial vascular tissue dynamically cultured by the bioreactor, the cells are in good growth state and the viability is higher than 95%. Compared with static culture, the genes related to angiogenesis VEGF, VWF, EGF2, CD31 are significantly up-regulated ， and immunofluorescence staining shows that the fluorescent signals of CD31 and VEGF in the 3D printed single endothelial vascular tissue are stronger, indicating an increase in the protein expression level, which confirms that the dynamic culture of the bioreactor helps to form a 3D printed single endothelial vascular tissue with more mature structure and function.
[0047] Example 4 Dynamic culture of the bioreactor for 3D printing neuromuscular junctions: Take the 3D printed neuromuscular junctions preliminarily constructed in vitro, which are co-constructed by nerve cells, muscle cells, and endothelial cells, and transfer them to a standard six-well cell culture plate. Add 2.5 mL of the corresponding adapted dynamic culture medium to each well, fix the six-well plate in a thermostatic CO2-compatible shaker supporting the bioreactor, adjust the shaker parameters and the corresponding fluid shear force respectively, and perform dynamic culture for two weeks; Take another 3D printed nerve-muscle tissue (constructed by nerve cells and muscle cells), transfer it to a standard six-well cell culture plate, add 2.5 mL of the corresponding adapted culture medium to each well, fix the six-well plate in a thermostatic CO2-compatible shaker supporting the bioreactor, adjust the shaker parameters and the corresponding fluid shear force respectively, and perform dynamic culture for two weeks. The culture conditions of the 3D printed nerve-muscle tissue are the same as those of the 3D printed neuromuscular junctions.
[0048] In the above embodiments, the conditions for the dynamic culture of the 3D printed neuromuscular junctions by the bioreactor in the first week are: relative centrifugal force is 0.015 g, horizontal circular shaking (shaking radius 15 mm), and the corresponding fluid shear force is 0.3 dyne / cm 2 , and the dynamic culture medium is DMEM + 10% horse serum + 1% penicillin-streptomycin double antibody solution; The conditions for the dynamic culture of 3D-printed neuromuscular junctions in the bioreactor described in the second week were: a relative centrifugal force of 0.051 g, horizontal circular shaking (shaking radius 15 mm), and a corresponding fluid shear force of 1.2 dyne / cm. 2 The dynamic culture medium is ECM + 5% FBS + 1% ECGS + 1% penicillin-streptomycin double antibiotic solution; In the above embodiments, the conditions for dynamically culturing 3D-printed neuromuscular tissue in the bioreactor are the same as those for 3D-printed neuromuscular junctions, and the dynamic culture medium is the same.
[0049] Functional identification of 3D-printed neuromuscular junctions through dynamic culture in a bioreactor: 3D-printed neuromuscular junctions were collected from the bioreactor and used as a control. Cell live / dead staining was performed on the 3D-printed neuromuscular junctions obtained from the bioreactor dynamic culture, and the expression level of related gene mRNA was detected by real-time quantitative PCR and immunofluorescence staining. In the above embodiments, the cell live / dead staining includes: preparing Calcein AM / PI detection working solution, collecting 3D printed neuromuscular junctions, washing residual culture medium with PBS, adding an appropriate volume of detection working solution, incubating at 37°C and 5% CO2 in the dark for 30 min, and then immediately observing the staining effect under an inverted fluorescence microscope (Calcein AM is green fluorescence, indicating live cells; PI is red fluorescence, indicating dead cells).
[0050] In the above embodiments, the real-time quantitative PCR detection of related gene expression levels includes: extracting total RNA from a 3D-printed neuromuscular junction and reverse transcribing it to synthesize cDNA, and then using the synthesized cDNA as a template to detect the mRNA expression level of related genes using real-time quantitative PCR.
[0051] In the above embodiments, the relevant genes include: FOX3, DES, VEGF In the above embodiments, the expression of relevant proteins of the 3D-printed neuromuscular junction obtained by dynamic culture in the bioreactor was measured by immunofluorescence staining. The method included collecting 3D-printed neuromuscular junction tissue samples, fixing, perforating, and blocking them, adding Myh3 antibody and incubating overnight, then incubating with fluorescently labeled secondary antibody, staining cell nuclei with DAPI, and finally observing the staining using a laser confocal microscope.
[0052] Experimental results are as follows Figure 5As shown, the cells in the 3D-printed neuromuscular junction, dynamically cultured in a bioreactor, exhibited good growth and proliferation. Compared to the 3D-printed neuromuscular tissue dynamically cultured in a bioreactor, the genes involved in angiogenesis... VEGF Expression significantly upregulated ， Furthermore, immunofluorescence staining showed that the green Myh3 fluorescence signal in the 3D-printed neuromuscular junction was stronger than that in the 3D-printed neuromuscular tissue, indicating that the protein expression level of Myh3 was increased. This suggests that the dynamic culture of "introducing blood vessels" can promote the maturation of vascular networks and the differentiation and maturation of muscle cells, confirming that dynamic culture in a bioreactor helps to form a more structurally and functionally mature 3D-printed neuromuscular junction.
[0053] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.
Claims
1. A method for dynamically culturing 3D-printed tissues in a bioreactor, characterized in that, The 3D printed tissues include: 3D printed single nerve tissue; 3D printed single muscle tissue; 3D printed single endothelial vascular tissue; 3D printed nerve-muscle junction tissue; The method includes the following steps: Transfer the above 4 types of 3D printed tissues in vitro to a bioreactor respectively. By adjusting the rotation speed of the thermostatic CO2 compatible shaker supporting the bioreactor, induce the growth medium to generate corresponding fluid shear forces, and perform dynamic culture by simulating the in vivo dynamic mechanical microenvironment to obtain mature various 3D printed tissues.
2. The method according to claim 1, characterized in that, The 3D-printed single neural tissue, during dynamic culture, had a fluid shear force corresponding to the shaking speed of the bioreactor-equipped shaker at a speed of 0.3~0.6 dyne / cm. 2 .
3. The method according to claim 1, characterized in that, The 3D-printed single muscle tissue, during dynamic culture, exhibited a fluid shear force corresponding to the shaking speed of the bioreactor-equipped shaker at a rate of 0.6~1.0 dyne / cm. 2 .
4. The method according to claim 1, characterized in that, The 3D-printed single endothelial blood vessel tissue, during dynamic culture, had a fluid shear force corresponding to the shaking speed of the bioreactor-equipped shaker at 1.0~1.5 dyne / cm. 2 .
5. The method according to claim 1, characterized in that, The 3D-printed neuromuscular junction tissue exhibited a fluid shear force of 0.3~1.2 dyne / cm² during dynamic culture on the shaker in the bioreactor. 2 .
6. The method according to claim 1, characterized in that, The shaker supporting the bioreactor is in a horizontal circular shaking mode, with a shaking radius of 10 - 20 mm, adapting to the regulation range of the bioreactor; The shaker is a special thermostatic CO2 compatible type for the bioreactor. The environmental conditions for the dynamic culture are: temperature 37 °C, CO2 volume fraction 5%, and culture humidity 95%.
7. The method according to claim 1, characterized in that, There is a clear matching relationship between the rotation speed of the shaker supporting the bioreactor and the fluid shear forces of various tissues: for single nerve tissue, the relative centrifugal force of the shaker is 0.015 - 0.027 g; for single muscle tissue, the relative centrifugal force of the shaker is 0.027 - 0.042 g; for single endothelial vascular tissue, the relative centrifugal force of the shaker is 0.042 - 0.060 g; for nerve-muscle junction tissue, the relative centrifugal force of the shaker is 0.015 - 0.051 g.
8. The method according to claim 1, characterized in that, The culture medium for the dynamic culture is adjusted according to the tissue type: the culture medium for single nerve tissue is DMEM / F12 + 2% B27 + 1% FBS + 1% penicillin-streptomycin double antibody solution; the culture medium for single muscle tissue is DMEM + 2% horse serum + 1% penicillin-streptomycin double antibody solution; the culture for single endothelial vascular tissue is ECM + 5% FBS + 1% ECGS + 1% penicillin-streptomycin double antibody solution; the culture medium for nerve-muscle junction tissue contains DMEM + 10% horse serum + 1% penicillin-streptomycin double antibody solution; the culture time for each type of tissue is 7 - 14 days, and 70% of the volume of the culture medium is replaced every 24 h. The liquid change reminder can be achieved through the program control module of the bioreactor.
9. The method according to claim 1, characterized in that, Perform functional detection and identification on various 3D printed tissues cultured in the bioreactor, including detection of cell survival status, detection of related gene and protein expression, to verify the differentiation maturity and functional integrity of various 3D printed tissues.
10. A 3D-printed neuromuscular junction obtained by the method according to any one of claims 1 to 9, characterized in that, The 3D printed nerve-muscle junction simultaneously has mature nerve fibers, differentiated myotubes and vascular-like structures, meeting the physiological function requirements.