Skeletal muscle organoid and method of making same

Abstract

The application provides a skeletal muscle organoid and a preparation method thereof, takes muscle stem cells, neural stem cells and fibroblasts as primary cells for organoid culture, restores the microenvironment of skeletal muscle as much as possible, and improves the differentiation degree of the organoid; further, the mold and frame for constructing the organoid are improved, the batch production of the organoid is realized through the array design of the PDMS mold and the polyamide frame; finally, the calcium signal video of the organoid is analyzed by using the Organblink system independently developed by the application, and the accuracy of the function evaluation result of the organoid is improved. The method provided by the application realizes the high bionics and high batch production of the skeletal muscle organoid, has low cost, and greatly promotes the development of the medical research field.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering model manufacturing and application, specifically relating to a skeletal muscle organoid and its preparation method. Background Technology

[0002] Muscle tissue is the foundation of human movement. It is primarily composed of muscle cells, along with fibroblasts, endothelial cells, and immune cells, forming the tissue microenvironment. Cell-cell and cell-extracellular matrix interactions play crucial roles in physiological processes such as muscle function and skeletal muscle aging, and also participate in regulating physiological processes such as muscle tissue responses to exercise stimuli and drug toxicity. Therefore, 3D artificial muscle units with biomimetic muscle microenvironments have broad application prospects in both in vitro model construction and drug efficacy and toxicity testing.

[0003] Currently, most studies on the efficacy and toxicity of skeletal muscle drugs rely on traditional 2D cell culture and animal models. However, 2D cell models cannot simulate the three-dimensional structure and function of skeletal muscle, and the differentiated muscle fibers lack contractile ability, making it difficult to reflect the physiological state of muscle cells in vivo. Animal models, on the other hand, have limited operability and high-throughput screening capabilities. These factors collectively lead to biases in drug efficacy prediction (for example, in early sarcopenia studies, Enobosarm and Domagolzumab showed effective increases in muscle mass in 2D cells and animal models, but they failed to restore muscle function).

[0004] Tissue-engineered muscle aims to mimic the structure and functional characteristics of natural muscle through 3D culture. Bio-Artificial Muscle (BAM) constructed based on the mouse myoblast cell line C2C12 has been shown to exhibit higher differentiation and functionality compared to 2D cell cultures. Researchers have also constructed engineered skeletal muscle tissue using muscle stem cells (MuSCs) differentiated from induced pluripotent stem cells (iPSCs). These engineered muscles possess a certain degree of muscle function, and combined with sophisticated detection equipment, they can enable in vitro assessment of the effects of drugs on muscle function, such as the CUORE precision biomechanical testing device from Optics11.

[0005] However, existing BAM construction and detection methods still have three defects: insufficient biomimicry, high cost of detection equipment, and difficulty in large-scale production: (1) Insufficient biomimicry of cell composition: Most existing BAMs are constructed based on a single type of stem cell with myogenic potential, with C2C12 and other cell lines and MuSCs induced by iPSCs being the most common. The composition of a single cell makes it impossible for BAMs to realize the skeletal muscle microenvironment and microstructure. The lack of connective tissue and complex extracellular matrix (ECM) components makes it difficult for BAMs to obtain reliable results in drug screening. In addition, BAMs based on cell lines lack patient characteristics and cannot meet the needs of personalized drug screening and toxicity assessment; (2) High cost of detection equipment: The purchase cost of the CUORE equipment mentioned above is US$120,000, while the fiber optic force probe, as a precision consumable, needs to be replaced frequently. Its competitor, Mantarry, has a purchase cost as high as US$300,000. Foreign platforms are not only expensive, but their equipment and related software also have high learning costs and low compatibility, making the learning cost curve for researchers steep; (3) Difficulty in large-scale production: In most previous studies, in order to maintain the parallel arrangement of myotubes in BAM, the two ends of BAM must be surrounded by micropillar structures. The micropillar structure needs to maintain an appropriate size and elasticity, and be tightly bound to the cell-hydrogel mixture to prevent BAM from falling off. This leads to two problems: First, the design of the micropillar limits the minimum volume of BAM units. Most previous technical solutions required BAM units with a cell-hydrogel mixture volume of more than 60 μl (very few can be constructed in about 30 μl), which increases the consumption of cells and hydrogel, which is not conducive to the conversion efficiency of cells to organoids; Second, BAM is difficult to detach from the micropillar in a non-destructive manner, making it difficult to culture and detect freely in conventional cell culture well plates.

[0006] Therefore, there is an urgent need to provide a method for mass-producing biomimetic skeletal muscle models to address the functional evaluation bottleneck in current organoid model construction and drug screening platforms. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a skeletal muscle organoid and its preparation method. The aim is to utilize various primary cells to recreate the microenvironment and microstructure of skeletal muscle tissue as much as possible while maintaining individual genetic and phenotypic characteristics. Simultaneously, an immersion-type cell-scaffold combination method, combined with a mold array design, enables the mass production of bundled organoids. Finally, based on computer vision technology, an intelligent analysis process for batch processing calcium signal video data is developed, enabling batch detection and analysis of organoid functions.

[0008] To achieve the above objectives, the present invention employs the following technical solution:

[0009] On one hand, the present invention provides a method for preparing skeletal muscle organoids, comprising the following steps:

[0010] Step S1: Extraction and sorting of primary cells;

[0011] Step S2: Preparation of organoid molds and frameworks;

[0012] Step S3: Primary cell-hydrogel mixture is injected into the mold and induced to differentiate into skeletal muscle organoids;

[0013] In step S1, the primary cells include at least one of fibroblasts, neural stem cells, and muscle stem cells.

[0014] In order to replicate the cellular microenvironment of skeletal muscle in vivo as much as possible, achieve mass production of bundled skeletal muscle organoids, and accurately evaluate organoid function, this invention has studied and improved the primary cells for induced differentiation of organoids, organoid construction molds, and organoid function evaluation systems. Through multi-faceted optimization, the biomimicry and large-scale production of skeletal muscle organoids, as well as the accuracy of organoid function evaluation, have been improved.

[0015] The core objective of skeletal muscle tissue engineering is to mimic the developmental microenvironment of skeletal muscle in vivo, achieving the proliferation, differentiation, and orderly arrangement of seed cells, and forming myofibril bundles with contractile function, ultimately reconstructing physiological function and the microenvironment. Among various seed cells, muscle stem cells are the core functional cells for constructing skeletal muscle organoids. During culture, they can be activated and differentiate into myoblasts, which further fuse to form multinucleated myofibrils, serving as the direct source of the "skeletal muscle functional units" in organoids. Muscle stem cells express muscle-specific markers (such as MyoD and MyHC), ultimately forming myotube structures with contractile function, mimicking the core physiological functions of skeletal muscle in vivo. Furthermore, this invention has screened neural stem cells and fibroblasts through multiple experimental studies, resulting in skeletal muscle organoids composed of muscle stem cells, neural stem cells, and fibroblasts.

[0016] In this primary cell system, neural stem cells are evenly distributed alongside muscle stem cells, achieving neural innervation and improving the differentiation efficiency of muscle stem cells within the organoid. Simultaneously, neural cells can regulate muscle stem proliferation and differentiation by secreting neurotransmitters and fibroblast growth factors (FGFs). Fibroblasts, on the other hand, provide the organoid with a "structural scaffold and microenvironment regulation." Fibroblasts mimic the interstitial structure of skeletal muscle in vivo, guiding the directional alignment and differentiation of muscle stem cells, and maintaining the stability of neural stem cells within the organoid by secreting extracellular matrix (ECM) components such as collagen and fibronectin. Compared to single muscle stem cells or other combinations (such as muscle stem cells + fibroblasts), skeletal muscle organoids constructed through the co-culture and differentiation of these three cell types are functionally closer to in vivo skeletal muscle, and the myotubes and muscle fibers of the organoids exhibit more mature differentiation and more comprehensive microenvironmental signals. The co-culture of these three cell types has significant advantages in the preparation of skeletal muscle organoids.

[0017] Furthermore, in step S2, the organoid mold is constructed using PDMS, and the framework is made of polyamide (nylon).

[0018] The molds used to construct organoids serve as "microenvironment carriers" for cell growth, needing to mimic the three-dimensional structure, mechanical properties, and biological activity of skeletal muscle in vivo, providing physical support and signal regulation for primary cells. However, in most previous studies, to maintain the parallel arrangement of myotubes in skeletal muscle engineered tissue (BAM), the two ends of the BAM must be surrounded by micropillar structures. These micropillar structures need to maintain appropriate size and elasticity and be tightly bound to the cell-hydrogel mixture to prevent BAM detachment. This leads to two problems: firstly, the design of the micropillars limits the minimum volume of the BAM unit; most previous techniques required a cell-hydrogel mixture volume exceeding 60 μl (very few could be constructed in approximately 30 μl), increasing cell and hydrogel consumption and hindering cell-to-organoid conversion efficiency; secondly, BAMs cannot detach from the micropillars non-destructively, making free culture and detection in conventional well plates difficult. Therefore, addressing the bottlenecks of existing technologies, this invention proposes a construction scheme of "arrayed mold + immersion frame" using PDMS as the mold and polyamide as the framework. Polydimethylsiloxane (PDMS) is a commonly used mold material in organoid fabrication. Through its pre-fabricated microgrooved structure, it guides muscle stem cells to align, proliferate, and differentiate in a specific direction, mimicking the parallel arrangement of skeletal muscle fibers in vivo. Polyamide, acting as a framework, primarily serves as "structural support" and "mechanical simulation," ensuring the consistency of the organoid growth space. This invention utilizes the biocompatibility and structural properties of PDMS, along with the stability of polyamide, to synergistically improve the maturity and functionality of organoids. Furthermore, the immersion-type porous scaffold design significantly reduces the minimum volume of organoids (to 10 μl), enabling array-based batch construction and improving the cell-to-organoid conversion rate and cell differentiation efficiency within the organoids.

[0019] Furthermore, the primary cell-hydrogel mixture includes fibroblasts, neural stem cells, muscle stem cells, fibrinogen, matrix gel, and thrombin.

[0020] Optimizing the composition of hydrogels is crucial for improving cell differentiation and organoid maturation. Existing hydrogel materials used for culturing skeletal muscle organoids mainly include matrix gel and fibrinogen. Through multiple comparative experiments, this invention found that simultaneously adding thrombin, fibrinogen, and matrix gel to the hydrogel system, when co-cultured with fibroblasts, neural stem cells, and muscle stem cells, significantly promotes the differentiation of these cells and improves the maturation of myotubes and muscle fibers. Specifically, during differentiation culture, thrombin is activated at 37°C, catalyzing fibrinogen polymerization to form a fibrin network, providing an attachment scaffold for muscle stem cells and neural stem cells, thus providing greater support for organoid formation. Matrix gel not only provides nutrients to cells but also enhances cell-matrix interactions, promoting organoid maturation.

[0021] Furthermore, the primary cells are induced to grow and differentiate using growth and differentiation media. The growth media include fibroblast and muscle stem cell growth media and neural stem cell growth media. The fibroblast and muscle stem cell growth media include H-DMEM, fetal bovine serum, penicillin-streptomycin (PS), alanyl-glutamine supplement GlutaMAX, and fibroblast growth factor FGF-2. The neural stem cell growth media includes DMEM / F12, B27 supplement, fibroblast growth factor FGF-2, epidermal growth factor EGF, penicillin-streptomycin (PS), and alanyl-glutamine supplement GlutaMAX.

[0022] Preferably, the fibroblast and muscle stem cell growth medium, by volume percentage, comprises 83-88% H-DMEM, 10-15% fetal bovine serum, 1% penicillin-streptomycin PS, 1% alanyl-glutamine supplement GlutaMAX, and 5-20 ng / ml fibroblast growth factor FGF-2; and the neural stem cell growth medium comprises 96% DMEM / F12, 2% B27 supplement (without vitamin A), 10-20 ng / ml fibroblast growth factor FGF-2, 10-20 ng / ml epidermal growth factor EGF, 1% penicillin-streptomycin PS, and 1% alanyl-glutamine supplement GlutaMAX.

[0023] Preferably, the fibroblast and muscle stem cell growth medium, by volume percentage, comprises 88% H-DMEM, 10% fetal bovine serum, 1% PS, 1% GlutaMAX, and 10 ng / ml FGF-2, and the neural stem cell growth medium comprises 96% DMEM / F12, 2% B27 supplement (without vitamin A), 20 ng / ml FGF-2, 10 ng / ml EGF, 1% PS, and 1% GlutaMAX.

[0024] Furthermore, the differentiation medium includes DMEM / F12, horse serum, penicillin-streptomycin (PS), and alanyl-glutamine supplement GlutaMAX.

[0025] In some embodiments, the hydrogel cultured from a mixture of fibroblast and muscle stem cell growth medium and neural stem cell growth medium of the present invention has excellent effects in inducing the growth of fibroblasts, muscle stem cells and neural stem cells.

[0026] Preferably, the differentiation medium comprises, by volume percentage, 83-86% DMEM / F12, 2-5% horse serum, 1% penicillin-streptomycin PS, and 1% alanine-glutamine supplement GlutaMAX.

[0027] Preferably, the differentiation medium comprises, by volume percentage, 86% DMEM / F12, 2% horse serum, 1% PS, and 1% GlutaMAX.

[0028] In some implementations, when the differentiation medium is used to simultaneously induce the differentiation of muscle stem cells, fibroblasts and neural stem cells, the differentiation medium is also supplemented with 2% B27 supplement (containing vitamin A), 1% N2 supplement, 0.5 μM retinoic acid (RA) and 1 μM purmorphamine.

[0029] On the other hand, the present invention provides skeletal muscle organoids prepared by the method described in any of the preceding claims.

[0030] In another aspect, the present invention provides the use of primary cells for preparing skeletal muscle organoids, wherein the primary cells include at least one of fibroblasts, neural stem cells, and muscle stem cells.

[0031] In another aspect, the present invention provides an analysis system for evaluating the function of skeletal muscle organoids as described above, the analysis system analyzing motion videos of skeletal muscle organoids based on calcium signals, wherein the calcium signals are indicated by any one of GCamp6, Cal-520, Fluo-4, and Rhod2.

[0032] Furthermore, the motion video of the skeletal muscle organoid is a motion video of the skeletal muscle organoid after being stimulated by pulses or chemicals.

[0033] Currently, 2D cell models or BAMs are mainly used for functional detection or evaluation through software like ImageJ or equipment such as CUORE. ImageJ analyzes differences between images using a continuous imaging method, but it requires manual identification and selection of target regions, resulting in significant subjectivity and analytical errors. Equipment like CUORE and Mantarry is expensive and difficult to widely use. This invention addresses organoid functional analysis by independently developing a calcium signal evaluation system for organoids—the Organblink system. Based on computer vision and image processing technology, the Organblink system automates and quantifies the analysis of calcium ion fluorescently labeled videos of organoids to characterize the dynamic physiological parameters of organoid functional status. It only requires a commonly available function generator and fluorescence microscope in the laboratory to complete motion induction and data acquisition, without relying on specific detection equipment. The Organblink system solves the problems of low analysis efficiency (through frame-by-frame calculation), high subjectivity, and ambiguous calcium signal selection found in existing analysis software such as ImageJ, greatly reducing experimental workload and improving the accuracy of evaluation results.

[0034] Furthermore, this invention provides the application of the skeletal muscle organoids described above in the testing of skeletal muscle drug efficacy.

[0035] In another aspect, the present invention provides a functional analysis system for skeletal muscle organoids, including a motion video data processing module for skeletal muscle organoids, a calcium signal change analysis module, and a data output module.

[0036] Furthermore, the skeletal muscle organoid is formed by the induced differentiation of at least one primary cell type selected from fibroblasts, neural stem cells, and muscle stem cells.

[0037] Furthermore, the calcium signal indication method includes any one of GCamp6, Cal-520, Fluo-4, and Rhod2.

[0038] Furthermore, the system analyzes the changes in calcium signals in the motion video of skeletal muscle organoids and outputs the organoids' response to pulsed or chemical stimuli to assess skeletal muscle organoid function.

[0039] In another aspect, the present invention provides a method for functional analysis of skeletal muscle organoids, which uses the Organblink system to analyze changes in calcium signals in skeletal muscle organoid motion videos. The calcium signal indication method includes any one of Cal-520, Fluo-4, Rhod2, or GCamp6 calcium signal indicator protein.

[0040] Furthermore, the analysis process of the Organblink system includes the following steps:

[0041] S1: Batch processing of motion video of skeletal muscle organoids;

[0042] S2: Automatic identification, selection, and region segmentation of fluorescent ROIs;

[0043] S3: Extraction of calcium signal intensity sequence within the ROI;

[0044] S4: Calculation of calcium signal characteristic parameters;

[0045] S5: Data visualization and output.

[0046] Furthermore, in step S1, the motion video of the batched skeletal muscle organoids is the motion video of the skeletal muscle organoids after pulse stimulation or chemical stimulation.

[0047] Further, in step S1, the batched skeletal muscle organoid motion videos are analyzed using a convolutional neural network defined by Keras and the VideoCapture method.

[0048] Furthermore, in step S1, the convolutional neural network transforms the grayscale image of the input video into a four-dimensional tensor containing the number of samples, height, width, and number of channels.

[0049] Furthermore, in step S2, the fluorescent ROI is obtained through a fusion algorithm of adaptive Gaussian threshold and global threshold, and a contour detection algorithm.

[0050] Furthermore, in step S4, the calcium signal characteristic parameters include relative fluorescence change rate, maximum calcium transient, and peak frequency.

[0051] Furthermore, in step S5, the data includes an average time series curve, a calcium signal distribution density curve, and a frequency distribution histogram.

[0052] The present invention has the following beneficial effects:

[0053] 1. Using three types of cells derived from skeletal muscle tissue—muscle stem cells, neural stem cells, and fibroblasts—for culture and differentiation can better reflect the microenvironment of skeletal muscle tissue in vivo, exhibiting strong biomimicry and a more mature degree of organoid differentiation.

[0054] 2. Organoids are constructed using a combination of PDMS molds and polyamide frames. The properties and material interactions of the molds and frames enhance the functionality of the organoids. Simultaneously, the organoid scaffold molds designed in this invention can form bundled skeletal muscle organoids with 10 μl of cell-hydrogel, greatly reducing the amount of hydrogel and cells used in organoid units. This improves the efficiency of constructing organoids from primary cells. Compared with previous pillar-type organoids, the productivity of equal-length (6 mm) skeletal muscle organoids is increased by 266.7%, and array-based batch construction is supported.

[0055] 3. A functional evaluation system for organoid calcium signal videos was developed. Based on computer vision and image processing technology, the system performs automated and quantitative analysis of organoid calcium ion fluorescently labeled videos, overcoming the bottlenecks of low analysis efficiency, strong subjectivity, and high cost of detection equipment in existing organoid functional analysis methods. Attached Figure Description

[0056] Figure 1 The flowchart for the preparation and functional analysis of skeletal muscle organoids provided by this invention.

[0057] Figure 2 The image shows the identification results of muscle stem cells, fibroblasts, and neural stem cells.

[0058] Figure 3 A schematic diagram of the organoid mold and framework designed for this invention.

[0059] Figure 4 The macroscopic morphology and bright-field microscopy of the bundle-like skeletal muscle organoid constructed in Example 1 are shown, where D1 and D7 represent the 1st and 7th day after organoid construction, respectively.

[0060] Figure 5 The images show the expression of the muscle marker gene MYHC in the bundle-shaped skeletal muscle organoid constructed in Example 1 (A) and the arrangement of myotube cells (B). DAPI is a nuclear dye used to stain and locate the cell nucleus, MYHC is myosin heavy chain, and Merge indicates the merged image. D3, D5, and D7 represent the 1st, 5th, and 7th days after organoid construction, respectively.

[0061] Figure 6A Analyze the logic flowchart of the Organblink system. Figure 6B This is a schematic diagram of the Organblink system (spherical). Figure 6CTo demonstrate the effectiveness of Organblink in automatically recognizing organoid ROIs of different sizes, shapes, and proportions, Figure 6D This is an example result of Organblink's evaluation of organoid function.

[0062] Figure 7A The results show the visualization of calcium signaling in organoids under resting and activated states. Figure 7B The graph shows the change of calcium signal over time and the frequency distribution of calcium signal intensity. Figure 7C This describes the response of skeletal muscle organoids to physical and chemical stimuli.

[0063] Figure 8A Morphological images of bundle-like skeletal organoids induced from different combinations of primary cells are shown. DAPI is a nuclear dye used to stain and locate cell nuclei; Myhc myotubes are myosin heavy chain staining for myotube cells; Tuji1 NSCs are β-III type microtubules staining for neural stem cells; DeepRed fibroblasts are far-infrared fluorescent dye staining for fibroblasts; and Merge indicates merged images. Figure 8B The relative angles of myotubes in bundle-shaped skeletal muscle organoids induced from different combinations of primary cells. Figure 8C Myotube fusion index of bundle-shaped skeletal muscle organoids induced to differentiate from different combinations of primary cells.

[0064] Figure 9 These are the fluorescent signals emitted after different calcium signaling indicators bind to calcium ions in cells.

[0065] Figure 10 The analytical workflow and results are presented for evaluating skeletal muscle organoid function using the Organblink system (A) and the Image J method (B).

[0066] Figure 11A The morphology of skeletal muscle organoids constructed with polyamide frameworks of different pore densities, where D1 and D6 represent day 1 and day 6 after organoid construction, respectively; Figure 11B Diameters of skeletal muscle organoids constructed from polyamide frameworks with different pore densities.

[0067] Figure 12 The structure of the mold used for screening the size of skeletal muscle organoids in the early stages of this invention is shown in (A), and the results of skeletal muscle organoids prepared from organoid construction slots of different sizes are compared (B). Detailed Implementation

[0068] The present invention will be further described in detail below with reference to embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding and application of the present invention. These embodiments are merely limited embodiments within the scope of the present invention and are intended to illustrate how the application of the present invention can be implemented. They do not constitute any limitation on the present invention or its application. The specific scope of protection of the present invention is embodied in the claims of the present invention.

[0069] The reagents used in this embodiment are all known products, obtained by purchasing commercially available products.

[0070] Example 1: Preparation and functional analysis of skeletal muscle organoids provided by the present invention

[0071] The preparation and functional analysis process of skeletal muscle organoids provided by this invention is as follows: Figure 1 As shown, the specific steps are as follows:

[0072] I. Obtaining Primary Cells

[0073] 1. Obtaining muscle stem cells and fibroblasts:

[0074] (1) Muscle tissue acquisition: gastrocnemius muscle biopsy samples were taken from male C57BL / 6 mice that were euthanized by cervical dislocation. During tissue acquisition and transportation, the temperature was kept as low as 4°C and aseptic conditions were maintained as much as possible.

[0075] (2) Pretreatment of muscle tissue: Wash with HBSS 3 times and remove tendons, fat and fascia. Then take 5g of muscle tissue and mince it into a paste with ophthalmic scissors in 1ml of HBSS. Centrifuge and discard the supernatant.

[0076] (3) Digesting muscle homogenate to obtain cell suspension: Add 5 ml of 0.2% collagenase II preheated at 37℃ to the muscle homogenate, mix thoroughly with a shaker, and then incubate in a cell culture incubator at 37℃ for 1 hour, mixing once every 10 minutes to ensure that the enzyme is in full contact with the homogenate. Then centrifuge at 900 g to obtain the precipitate.

[0077] (4) Digesting the precipitate to obtain a single-cell suspension: Resuspend the precipitate with 5 ml of dispase enzyme and incubate it in a cell culture incubator at 37°C for 1 hour, mixing it every 10 minutes to ensure that the enzyme and homogenate are in full contact. Then centrifuge at 900 g to obtain the precipitate.

[0078] (5) Filtering the single-cell suspension: The precipitate was resuspended in 3 ml of muscle stem cell culture medium. The suspension was observed under a microscope to confirm that it mainly contained single cells, and then filtered through a 40 μm cell sieve. The composition of the muscle stem cell culture medium by volume percentage was: 88% H-DMEM, 10% fetal bovine serum, 1% PS, 1% GlutaMAX, and 10 ng / ml FGF-2;

[0079] (6) The cell suspension that has passed through the cell sieve is seeded into cell culture dish Dish 1. After culturing for 24 hours, the cell culture medium is transferred to a new culture dish Dish 2, and an appropriate amount of muscle stem cell culture medium is added to Dish 1. The adherent cells in Dish 1 are mainly composed of fibroblasts, while the cells in Dish 2 are mainly composed of muscle stem cells. Both can be passaged and expanded, and can be further sorted by flow cytometry (α-Integrin). + Sorting muscle stem cells, Sca + High-purity cells were obtained using either fibroblast sorting or differential adhesion methods. The identification results of the sorted muscle stem cells and fibroblasts are as follows: Figure 2 As shown.

[0080] 2. Obtaining neural stem cells

[0081] (1) Obtaining embryonic spinal cord tissue: Take E14.5 fetal mice and carefully separate the embryonic spinal cord tissue under a microscope. During the tissue acquisition process, keep the temperature at 4℃ and aseptic as possible.

[0082] (2) Dissociation of spinal cord tissue to obtain single cells: The obtained spinal cord tissue was cut into pieces of about 1 mm. 3 The fragments were washed once with HBSS, and then an enzyme mixture (0.125% trypsin and 50 U / ml DNase I, using a solution containing Ca) was added. 2+ and Mg 2+ (Prepared with HBSS) Digested at 37°C for 20 min, during which the sample was thoroughly mixed with the enzyme mixture by pipetting with a 1 ml pipette every 5 min. Then, an equal volume of DMEM / F12 medium was added to terminate the digestion, followed by centrifugation at 400g for 5 min to obtain the precipitate. The precipitate was then resuspended in 3 ml of neural stem cell growth medium and seeded into culture dishes after passing through a 40 μm cell sieve.

[0083] (3) Culture of neural stem cells: In the dissociated single cells, neurons will adhere slightly to the culture dish, while neural stem cells will grow in suspension and form floating cell spheres within 3-5 days. Carefully collect the cell spheres using a pipette, keeping the culture dish stable as much as possible to reduce neuronal detachment. Transfer the collected neural stem cell spheres to a new culture dish, changing the culture medium every 2 days. The identification results of neural stem cells are as follows: Figure 2 As shown.

[0084] II. Design and fabrication of organoid molds and frameworks

[0085] (1) Organoid mold design: The anisotropic skeletal muscle organoid mold was constructed using PDMS, and the design was as follows: Figure 3The organoid construction mold unit shown;

[0086] (2) Organoid mold preparation: PDMS molds were prepared using PDMS Sylgard 184 (purchased from Dow Corning). The PDMS adhesive was prepared by mixing solutions A and B in a 1:10 ratio (solutions A and B are commercially available reagents, representing crosslinking agent and polymer, respectively). After thorough mixing, air bubbles were removed using a vacuum dryer. The prepared PDMS adhesive was poured into a polytetrafluoroethylene (Teflon) mold, placed in a 65℃ oven for 1.5 hours to cure, and then demolded to obtain the skeletal muscle organoid array construction mold. The mold dimensions, measured in length × width × height, were 6 mm × 2 mm × 0.75 mm (corresponding to...). Figure 3 The rectangular recess in the middle of each unit in the PDMS mold; the outer frame of each unit organoid is a ring-shaped polyamide mesh; the mold contains a recess for placing the mesh, with inner / outer diameters of 8mm / 10mm (corresponding to...). Figure 3 (The annular portion on the outside of each unit in the PDMS mold, the annular portion in the Nylon frame, and the annular portion in the pressure plate cover).

[0087] (3) Organoid framework design: The skeletal muscle organoid framework is obtained by laser precision cutting of polyamide (nylon) mesh. The polyamide mesh can be selected with pore sizes of 20 μm, 30 μm, and 40 μm according to actual stretching requirements.

[0088] (4) Pretreatment and assembly of the mold: After the PDMS mold is cured, it is sterilized by high-pressure sterilization. The polyamide frame is sterilized after being washed three times with sterile DPBS and then placed under a UV lamp for 30 minutes. After drying, the sterilized PDMS mold is coated with 0.4% Pluronic F-127 for 2 hours, then air-dried and sterilized by UV irradiation for 30 minutes. After that, the pretreated parts are assembled according to the instructions. Figure 3 Assemble as shown and store at 4°C for later use.

[0089] III. Construction, Culture, and Differentiation of Organoids

[0090] (1) Construction of skeletal muscle organoids based on primary cells

[0091] Prepare a primary cell-hydrogel mixture with the following composition: muscle stem cells, neural stem cells, and fibroblasts account for 60-70%, 25-35%, and 5-10% of the total cell number, respectively, with a total concentration of 1×10⁻⁶. 7 Cells / ml, H-DMEM, 20 mg / mL fibrinogen, Matrigel, Thrombin.

[0092] The hydrogel is configured with two solutions, A and B. Solution A contains H-DMEM, 20 mg / mL fibrinogen, and Matrigel matrix gel, with the contents of fibrinogen, H-DMEM, and Matrigel matrix gel being 35%, 30%, and 35% by volume, respectively. Solution B contains H-DMEM and thrombin, with the contents of H-DMEM and thrombin matrix gel being 95% and 5% by volume, respectively. Then, according to V... A :V B After thoroughly mixing solutions A and B in a 2:3 ratio, muscle stem cells, neural stem cells, and fibroblasts are added to obtain a primary cell-hydrogel mixture.

[0093] (2) Inject the above cell-hydrogel mixture into the assembled polyamide framework-PDMS mold (see assembly method). Figure 3 Polymerize at 37°C for 45 minutes. The formed hydrogel is then placed in a mixed culture medium (1:1 volume ratio) containing muscle stem cell growth medium and neural stem cell growth medium, along with the mold. 1 mg / mL of 6-aminocaproic acid is added to the mixed culture medium to inhibit fibrin degradation. After culturing at 37°C and 5% CO2 for one day, most organoids will detach spontaneously from the PDMS mold. Organoids that do not detach spontaneously can be gently detached from the PDMS mold by grasping the polyamide frame with forceps. The ends of the formed organoids should be tightly attached to the polyamide frame. The organoids can be moved into well plates containing differentiation medium by holding the polyamide frame to promote myogenic cell fusion and differentiation into myofibrils. Figure 4 As shown.

[0094] The growth media for fibroblasts and muscle stem cells, by volume percentage, consisted of: H-DMEM (88%), fetal bovine serum (10%), PS (1%), GlutaMAX (1%), and FGF-2 (10 ng / ml); the growth media for neural stem cells consisted of: DMEM / F12 (96%), B27 supplement (without vitamin A, 2%), FGF-2 (20 ng / ml), EGF (10 ng / ml), PS (1%), and GlutaMAX (1%); and the differentiation media consisted of: DMEM / F12 (86%), horse serum (2%), PS (1%), GlutaMAX (1%), B27 supplement (containing vitamin A, 2%), N2 supplement (1%), retinoic acid (RA, 0.5 μM), and Purmorphamine (1 μM).

[0095] IV. Organoid Morphology and Function Detection

[0096] (1) Organoid morphology detection

[0097] Morphological analysis of organoids was achieved by observing their morphology under a light microscope and the degree of differentiation and distribution of myotubes under a fluorescence microscope. The specific steps are as follows:

[0098] ① Within 7 days after organoid construction, observe the morphology of organoids under a microscope every day, measure and record the cross-sectional width of the thinnest part of the bundled organoids according to the scale, and measure the same organoid bundle 3 times independently and take the average value.

[0099] ② Immunofluorescence analysis was performed on days 3, 5, and 7 after organoid construction. The specific method was as follows: the organoids in differentiation were removed from the culture medium and washed with PBST three times for 5 minutes each time; then the organoids were fixed by immersing them in 4% PFA for 10-15 minutes, followed by washing with PBST three times for 5 minutes each time; 0.5% Triton X-100 was added for permeation for 10-15 minutes, followed by washing with PBST three times for 5 minutes each time; the organoids were blocked with 1% BSA at room temperature for 2 hours, then incubated overnight at 4°C with MYHC and TUJ1 primary antibodies diluted with 1% BSA, followed by washing with PBST three times for 5 minutes each time; the organoids were incubated with fluorescent secondary antibody at room temperature for 2 hours, followed by washing with PBST three times for 5 minutes each time; finally, the organoids were slightly submerged in a small amount of fluorescent mounting medium containing DAPI, and fluorescence microscopy was performed as soon as possible; the co-function was analyzed using ImageJ, the angle between 100 myotube cells and the overall direction of the organoid in the field of view was recorded, and the distribution map was drawn. Morphological analysis results of skeletal muscle organoids are as follows: Figure 5 As shown.

[0100] (2) Organoid function analysis

[0101] The functional analysis workflow for skeletal muscle organoids includes: first, infecting muscle stem cells (MuSCs) with GCamp6 lentivirus (GCamp6, a calcium signaling lentiviral plasmid used in this experiment, can bind to calcium ions in cells and emit green fluorescence) before organoid construction; then, stimulating the organoids with pulsed stimulation or chemical induction; and estimating the sensitivity of the skeletal muscle organoids to stimulation by analyzing the fluorescence values ​​of the organoid contraction videos using the Organblink system. The methods for pulsed stimulation and chemical induction stimulation are as follows:

[0102] 1) Pulse stimulation: A function signal transmitter was used as a physical stimulus source to detect whether skeletal muscle organoids could respond to rhythmic pulse signals. The contraction function of organoids was induced at a pulse intensity of 0-10 V / cm, a stimulation frequency of 1 Hz, and a pulse width of 10 ms. The contraction movement of the organoids was observed and recorded under a microscope.

[0103] 2) Chemical induction stimulation: Chemical induction stimulation involves adding compounds with known muscle activity, including but not limited to acetylcholine chloride and caffeine, to the organoid culture medium at specific concentrations. In this example, 5-80 mM acetylcholine was added to the organoid differentiation medium, and then the organoid contraction movements were observed and recorded under a microscope.

[0104] After pulsed stimulation and chemical induction stimulation, the organoids imported video data into the testing program. Then, calcium signal analysis was performed on the videos using the Organblink system. The specific analysis logic and process of the Organblink system are as follows: Figure 6A and Figure 6B As shown, the code implementation is as follows:

[0105] ① Building a Convolutional Neural Network: Using Keras, a convolutional neural network (CNN) is defined to transform the grayscale image of the input video into a four-dimensional tensor containing Batch Size (number of samples), Height, Width, and Channels for subsequent sample feature extraction and analysis;

[0106] ② Video data processing and uniformity adjustment: The VideoCapture method of the OpenCV library is used to read the specific information of the video frame by frame to extract features. Spatial downsampling is performed on each frame and the brightness and contrast are adjusted based on the weight function to standardize the input signal and enhance the signal-to-noise ratio (SNR).

[0107] ③ Automatic segmentation of fluorescence ROI (Region of Interest): All frame sequences of the GCamp6 calcium signal in the video are extracted, and the average intensity projection of its fluorescence channel is calculated to obtain an integrated image representing the background and highlight areas. Based on the fusion algorithm of adaptive Gaussian thresholding and global thresholding, a binary image is generated and then morphological closure operation is performed to ensure the continuity of ROI. Finally, the contour detection algorithm is used to identify the connected components of the binary image and calculate its minimum circumcircle (spherical organoids) or minimum circumcircle rectangle (bundle organoids). This contour is defined as the ROI for subsequent analysis.

[0108] ④ Calcium signal intensity sequence extraction: For each frame of the video, apply the above-mentioned ROI to generate a mask, extract the grayscale values ​​of all pixels within the mask area in the green channel (for the Gcamp6 green fluorescent probe), and calculate their arithmetic mean as the fluorescence intensity F at that time point t. （t） F connecting all time points （t）This constitutes the original calcium signal intensity sequence of the video;

[0109] ⑤ Calculation of calcium signal characteristic parameters: Calcium signal characteristic parameters include the relative fluorescence change rate ΔF / F (t) Maximum calcium transient (Max Amplitude) max(ΔF / F) (t) ) and peak frequency.

[0110] Relative fluorescence change rate ΔF / F (t) = (F (t) - F0) / F0, this parameter calibrates the baseline fluorescence of different organoids, thus allowing for comparison of the magnitude of calcium signal changes between different samples. Here, the baseline fluorescence value F0 is the average fluorescence value of the ROI in the video sequence of frames where the calcium signal has not been pulsed or chemically activated;

[0111] Maximum calcium transient (ΔF / F) (t) This reflects the maximum calcium response of organoids to stimuli;

[0112] Peak frequency is ΔF / F per unit time. (t) The number of peak values ​​exceeding a specific threshold is used to assess the effective response of calcium signals in organelles. In this embodiment, the lowest threshold is set for the pulsed stimulation group (0V test group) and for the chemically induced stimulation group (0 mM test group).

[0113] ⑥ Data aggregation and multi-group analysis: Multiple video data under the same experimental conditions are aligned and aggregated, and then the average time series curve (i.e., the fluorescence intensity change curve over time) and the signal intensity frequency density analysis curve (used to evaluate the calcium signal distribution density) are plotted. The frequency polygon (by plotting the connection curve after smoothing the frequency distribution histogram) is analyzed to evaluate the differences between different treatment groups from multiple dimensions in the time and frequency domains.

[0114] like Figure 7CAs shown, the response of the organoids constructed in this invention to pulsed stimulation (voltage gradient) and chemical stimulation (acetylcholine gradient) was evaluated using the Organblink system. The results showed that under 1Hz pulse stimulation of various voltage intensities, the organoid calcium signal maintained a stable frequency change of 1 Hz, and the highest fluorescence signal increased with increasing voltage, similar to the response pattern of real muscle to electrical stimulation. However, under stimulation by the classic muscle agonist acetylcholine chloride, the organoid calcium signal was a single high-intensity peak. The slope and maximum value of the calcium signal were positively correlated with the administered acetylcholine concentration, and the calcium signal gradually decreased over time after reaching its peak, similar to the response pattern of muscle tissue to acetylcholine-based muscle agonists reported in previous literature. In summary, the organoids constructed in this invention possess a similar response capability to drugs and physical stimuli to in vivo muscle.

[0115] Example 2: Screening of primary cells

[0116] The composition of primary cells significantly influences the biomimicry of skeletal muscle organoids. A single cell composition or cell tissue component may make it difficult to achieve the skeletal muscle microenvironment or obtain reliable results in drug screening. Therefore, to obtain the optimal solution for skeletal muscle organoid construction (i.e., Example 1), this invention first compared and screened the composition of primary cells and conducted exploratory analysis. The specific experimental procedure is as follows:

[0117] Cell lines were screened using the same primary cell extraction and sorting methods as in Example 1. The final sorted cell composition included the following groups:

[0118] Combination 1: Muscle stem cells (MuSCs);

[0119] Combination 2: Muscle stem cells (MuSCs) and fibroblasts;

[0120] Combination 3: Muscle stem cells (MuSCs) and neural stem cells (NSCs);

[0121] Combination 4: Muscle stem cells (MuSCs), fibroblasts, and neural stem cells (NSCs);

[0122] The four groups of primary multicellular cells were used to construct, culture, and differentiate skeletal muscle organoids according to the method in Example 1. Immunofluorescence staining was then used to observe the degree of cell differentiation and the morphology of the organoids. The fusion index (number of nuclei in Myhc positive cells / total number of nuclei in the field of view) and the myotube angle distribution (mean ± standard deviation of the angle between myotube cells and the organoid direction) were evaluated. These two factors reflect the degree of differentiation and self-organization effect of skeletal muscle organoids, respectively. A higher fusion index indicates a higher degree of differentiation of skeletal muscle organoids, and a smaller myotube angle distribution indicates a better self-organization effect of the organoids.

[0123] like Figure 8B and Figure 8C As shown, the average muscle fusion index of the four combinations is 22.35%, 43.56%, 35.20%, and 61.54%, respectively; the myotube arrangement angle ranges are 15.92° ± 26.48°, 2.89° ± 4.19°, 5.45° ± 20.05°, and 3.49° ± 4.76°, respectively. It can be observed that the skeletal muscle organoids cultured from primary cells using combination four have the highest muscle fusion index and the lowest myotube arrangement angle range, indicating that organoids cultured and differentiated from muscle stem cells, fibroblasts, and neural stem cells have the best differentiation and self-organization efficiency. Furthermore, comparing the data from combinations one, two, and three reveals that fibroblasts and neural stem cells improve the differentiation and self-organization efficiency of skeletal muscle organoids to varying degrees, with fibroblasts playing a particularly important role in the organoid self-organization process; while fibroblasts and neural cells work synergistically to improve the differentiation efficiency of skeletal muscle organoids. In conclusion, combination four exhibits the best organoid construction effect.

[0124] Example 3: Composition optimization of hydrogel

[0125] To promote better culture and differentiation of primary cells into organoids, this invention also investigated the effect of hydrogel composition during the culture process on organoid morphology. In this embodiment, organoid molds and frameworks were prepared according to the method of Example 1, and muscle stem cells, fibroblasts, and neural stem cells were extracted and sorted. Then, organoids were constructed and cultured according to the groups of the hydrogel mixture. The hydrogel mixture used during the culture process employed the following three combinations of components:

[0126] Combination 1: Fibrinogen, Matrigel, Thrombin

[0127] Combination 2: Matrigel matrix adhesive

[0128] Combination 3: Fibrinogen, thrombin

[0129] The remaining components, such as the growth medium and differentiation medium, were formulated the same as in Example 1. The morphology and differentiation degree of the organoids were evaluated using the same method as in Example 2, and the results are shown in Table 1.

[0130] Table 1. Effects of hydrogel composition on organoid structure

[0131] Group Muscle fusion index Myotube arrangement angle range Combination 1 63.3% 5.8° ± 6.5° Combination 2 NA NA Combination 3 24.3% 3.2° ± 3.5°

[0132] Note: NA indicates that the organoids decomposed during the culture process and no valid value could be measured; NA means no value.

[0133] The table shows that Combination 2, using only matrix gel, failed to maintain organoid morphology during differentiation. It lost its organoid structure on the second day of differentiation and dispersed into multiple cell aggregates, making it impossible to assess its effectiveness. Combination 3, which only added fibrinogen and thrombin, reduced the myotube arrangement area, but its myofusion index was significantly lower than Combination 1, which is detrimental to skeletal muscle organoid differentiation. In contrast, the hydrogel containing fibrinogen, matrix gel, and thrombin significantly promoted the differentiation and self-organization efficiency of skeletal muscle organoids. This is because thrombin is activated at 37°C, catalyzing fibrinogen polymerization and forming a fibrin network. Furthermore, fibrinogen's strong fibrinogenic properties provide more support for organoid formation. Matrix gel not only provides nutrients to cells but also enhances cell-matrix interactions, promoting organoid maturation. In conclusion, Combination 1 is the optimal solution.

[0134] Example 4: Optimization of an evaluation system for skeletal muscle organoid function analysis

[0135] I. Determination of the Indication Method of Calcium Signals

[0136] The Organblink system is a functional assessment algorithm for organoid calcium signals independently developed in this invention. Based on computer vision and image processing technology, it automates and quantifies the analysis of organoid calcium ion fluorescently labeled videos to extract dynamic physiological parameters characterizing the functional state of organoids. To improve the accuracy of the assessment results, this invention further investigates the impact of calcium signaling indicators on organoid functional assessment results.

[0137] In this embodiment, after constructing skeletal muscle organoids according to the method in Example 1, the sensitivity of the skeletal muscle organoids to stimulation was further evaluated based on calcium signaling methods. The calcium signaling methods employed included the GCamp6 transgenic method, the Cal-520, Fluo-4, and the Rhod2 indicator method. The GCamp6 protein was integrated into muscle stem cells via lentiviral infection before organoid construction. The Cal-520, Fluo-4, and Rhod2 probe indicators were added after organoid construction and before testing. The fluorescence signals emitted after the four calcium signaling methods bound to calcium ions in the cytoplasm are shown below. Figure 9 As shown.

[0138] according to Figure 9 The results show that all four calcium signaling methods can reflect changes in calcium signals in organoids. GCamp6 exhibits the best fluorescence effect and clarity, while Cal-520's fluorescence signal is slightly inferior to GCamp6. Fluo-4 and Rhod2, however, have poor fluorescence signal clarity. This indicates that GCamp6 performs best in video signal quality evaluation, followed by Cal-520, while Fluo-4 and Rhod2 are less effective. Therefore, GCamp6 is the preferred calcium signaling indicator for organoid functional assessment.

[0139] II. Comparison between the Organblink system and the ImageJ analysis method

[0140] This invention further evaluates the advantages of the developed Organblink system in assessing the response of organoids to pulsed and chemical stimuli. After successfully constructing skeletal muscle organoids based on the method of Example 1, the organoids' response to drugs and stimuli was assessed using both the Organblink system and ImageJ analysis. The two assessment methods are detailed below:

[0141] Organblink System: After contraction tests of skeletal muscle organoids involving pulsed and chemically induced stimulation, the test video files are imported into the Organblink system for analysis. The analysis process is the same as in Example 1. Taking the processing of calcium signal video data from a single organoid as an example, the Organblink processing workflow is as follows: Figure 10 As shown in A, after the video data is imported into the program, ROI selection, calcium signal intensity calculation, data standardization analysis, and chart output are automatically performed.

[0142] Image J Analysis: Taking the processing of calcium signal video data from a single organoid after pulse stimulation and chemically induced contraction tests of skeletal muscle organoids as an example, the Image J processing workflow is as follows: Figure 10As shown in B, the test video file is imported into ImageJ using the FFmpeg plugin. The video file is then converted into a time-series image set. The rectangular tool is then used to select the region of interest (ROI), and the fluorescence intensity within each ROI is calculated using ImageJ's measure function. Figure 10 In the approximately 10-second video (B in the example), 104 data measurements were required. The obtained data were exported to an Excel file, and the baseline calcium signal value F of the organoid was calculated based on the fluorescence intensity of the first 20 frames. （0） Then calculate the fluorescence signal F for each frame. (t) The ratio of the calcium signal baseline value to ΔF / F (t) = (F (t) – F (0) ) / F (0) These parameters are then imported into statistical software such as GraphPad for chart creation. Since ImageJ itself cannot currently automate the selection of multiple organoid ROIs and batch measurement data, batch output results are not displayed.

[0143] The chart analysis results of the two methods are as follows Figure 10 As shown, although the calcium signal intensity calculated manually by Organblink and ImageJ may differ slightly in the data results due to differences in ROI selection methods, absolute grayscale units, and graph curve smoothing, the magnitude and trend of calcium signal changes in the graphs obtained from the same organ type are consistent. This indicates that Organblink can effectively reflect the magnitude of calcium signal changes in muscle organoids to assess organoid function, and its assessment results are accurate and reliable. At the same time, Organblink can achieve automated and batch data processing, proving its advantages in operability and large-scale data analysis, while avoiding the problems of strong subjectivity and low efficiency caused by the need for manual identification and selection in traditional methods.

[0144] Example 5: Optimization of organoid molds and frameworks

[0145] Traditional methods for producing skeletal muscle engineered tissue (BAM) often employ anchoring structures with micropillars at both ends, resulting in large volumes (typically >30 μl), difficulty in mass production, and cumbersome operations. To address the challenges of large-scale BAM production, this invention also researches and optimizes organoid construction molds. On one hand, it considers optimizing the framework's structure or material to improve the conversion rate of primary cells to organoids; on the other hand, it aims to achieve mass production of bundled organoids by improving the mold's size or specifications. Therefore, this embodiment designs and explores a framework for organoid construction.

[0146] The framework materials for organoid construction included polyamide (Nylon) with different pore sizes and PDMS columns, a material commonly used in previous studies. Other preparation and assembly methods, as well as the extraction and sorting of primary cells, were the same as in Example 1. The morphological and differentiation differences of organoids constructed from different framework materials were also observed. Table 2 shows that the myofusion index of organoids constructed with polyamide frameworks was generally higher than that of the PDMS column group. This is because the polyamide framework has a smaller adhesion area and better hydrogel adhesion, resulting in a more uniform distribution of cells in the central region of the organoid and improving the myocell fusion rate. In the polyamide framework, the myofusion index gradually increased with decreasing pore diameter, but the range of myotube arrangement angles widened. This is related to the different elastic effects provided by different frameworks. However, considering both the fusion index and the range of arrangement angles, the 30 μm pore size polyamide framework showed better organoid construction results. Figure 11A and Figure 11B As shown, skeletal muscle organoids constructed from 30 μm pore size polyamide frameworks exhibit the best appearance uniformity and stability. Therefore, 30 μm polyamide is preferred as the framework material.

[0147] Table 2. Effects of different framework materials on organoid structure

[0148] Frame materials Myocyte fusion index Myotube arrangement angle range 20 μm Nylon 66.3% 13.8° ± 8.9° 30 μm Nylon 61.5% 4.1° ± 5.9° 40 μm Nylon 52.1% 3.1° ± 7.7° PDMS column 46.2% 9.1° ± 8.3°

[0149] like Figure 12 As shown in Figure A, the circular red area represents the region where organoid culture medium is added and replaced, and the central yellow groove represents the region where the cell-hydrogel mixture is injected, i.e., the organoid construction groove (corresponding to...). Figure 3 The size of the cuboid groove in the middle of each unit in the PDMS mold directly determines the shape and size of the organoid construct. If the organoid construct groove is too small, hydrogel injection becomes more difficult, air bubbles easily form during operation, and the small organoid volume makes the connection with the support too fragile, making it difficult for the hydrogel to maintain the mechanical strength for cell contraction, and the organoid is prone to breakage during culture. If the organoid volume is too large, the distance between the organoid center and the culture medium is too far, making it difficult for nutrients to diffuse to the central region of the organoid, leading to insufficient central differentiation or even cell apoptosis. Therefore, in the mold design of this invention, the influence of construct grooves of different sizes on the morphology of the organoid was also evaluated. According to the organoid construction method provided in Example 1, the construct groove ( Figure 12 The dimensions of the yellow rectangular groove in the center of each unit are designed in four different ways according to the specifications of length × width × thickness:

[0150] Specification 1: 6 mm × 2 mm × 0.75 mm;

[0151] Specification 2: 8 mm × 3 mm × 1.5 mm;

[0152] Specification 3: 10 mm × 5 mm × 2 mm;

[0153] Specification 4: 12 mm × 7 mm × 2 mm;

[0154] Cell activity and differentiation efficiency of skeletal muscle organoids of different sizes were examined using light microscopy. The results are as follows: Figure 12 As shown in B in the figure. Based on cell morphology under a light microscope, the cells in the 6 mm × 2 mm × 0.75 mm group showed more intact morphology and uniform cell distribution, while aggregates of apoptotic cells were clearly observed in groups three and four. Simultaneously, CFDA-SE staining of viable organoid cells showed that the 6 mm × 2 mm × 0.75 mm cells exhibited the strongest green fluorescence and the best cell viability, followed by the 8 mm × 3 mm × 1.5 mm cells. Groups three and four showed poorer cell viability, indicating that the 6 mm long, 2 mm wide, and 0.75 mm thick mold best ensures cell viability and differentiation efficiency of skeletal muscle organoids, making it the preferred design. Furthermore, the results of this embodiment also demonstrate that the immersion-type porous scaffold designed in this invention significantly reduces the minimum volume of organoids (to 10 μl), enabling array-based batch construction and improving the cell-to-organoid conversion rate and the cell differentiation efficiency within the organoids.

[0155] Obviously, the embodiments described above are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

Claims

1. A method for preparing skeletal muscle organoids, characterized in that, Includes the following steps: Step S1: Extraction and sorting of primary cells; Step S2: Preparation of organoid molds and frameworks; Step S3: Primary cell-hydrogel mixture is injected into the mold and induced to differentiate into skeletal muscle organoids; In step S1, the primary cells include fibroblasts, neural stem cells, and muscle stem cells; in step S2, the organoid mold is constructed using PDMS, and the framework is made of polyamide; in step S3, the primary cell-hydrogel mixture includes fibroblasts, neural stem cells, muscle stem cells, fibrinogen, matrix gel, and thrombin; the primary cells are induced to grow and differentiate using growth and differentiation media, the growth media including fibroblast and muscle stem cell growth media and neural stem cell growth media; the fibroblast and muscle stem cell growth media includes H-DMEM, fetal bovine serum, penicillin-streptomycin PS, alanyl-glutamine supplement GlutaMAX, and fibroblast growth factor FGF-2, the neural stem cell growth media including DMEM / F12, B27 supplement, fibroblast growth factor FGF-2, epidermal growth factor EGF, penicillin-streptomycin PS, and alanyl-glutamine supplement GlutaMAX; the differentiation media includes 86%... DMEM / F12, 2% horse serum, 1% penicillin-streptomycin PS and 1% alanine-glutamine supplement GlutaMAX, 2% B27 supplement, 1% N2 supplement, 0.5 μM retinoic acid and 1 μM Purmorphamine.

2. The skeletal muscle organoid prepared by the method as described in claim 1.

3. The use of primary cells in the preparation of skeletal muscle organoids, characterized in that, The primary cells include fibroblasts, neural stem cells, and muscle stem cells; the skeletal muscle organoids are prepared by the method described in claim 1.

4. The use of an analytical system for evaluating the function of the skeletal muscle organoid as described in claim 2, characterized in that, The analysis system analyzes the motion video of skeletal muscle organoids based on calcium signals, wherein the calcium signals are indicated by at least one of GCamp6, Cal-520, Fluo-4, and Rhod2.

5. The use as described in claim 4, characterized in that, The motion video of the skeletal muscle organoid is a motion video of the skeletal muscle organoid after being stimulated by pulse or chemical stimulation.

6. The application of the skeletal muscle organoids as described in claim 2 in the testing of skeletal muscle drug efficacy.

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