A method for constructing high-throughput skeletal muscle organoids

Abstract

The present invention discloses a method for constructing high-throughput skeletal muscle organoids, which includes: mixing myogenic progenitor cells and rapidly adherent cells isolated from skeletal muscle with Matrigel and a CaCl2 solution to form a Matrigel cell suspension; then adding the suspension into a sodium alginate solution to obtain Matrigel cell spheres coated with calcium alginate capsules; then placing the spheres in an incubator to solidify Matrigel into a gel, and after gel formation, culturing the Matrigel cell spheres coated with calcium alginate capsules in a growth medium for 1 day; transferring the Matrigel cell mixture from the growth medium to a differentiation medium to induce myogenic differentiation into skeletal muscle organoids with contractile function. The method of the present invention is simple, highly operable, and can obtain a large number of skeletal muscle organoid precursors in a short time, and a large number of skeletal muscle organoids can be obtained after induced differentiation culture.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid models. Specifically, the present invention relates to a method for constructing high-throughput organoids, and more specifically, the present invention relates to a method for constructing high-throughput skeletal muscle organoids. Background Art

[0002] Organoid technology is a technology that uses the self-organizing characteristics of mammalian pluripotent stem cells or stem cells derived from adult tissues to construct cell aggregates with multiple cell types in a 3D in vitro microenvironment similar to that in vivo. In recent years, there have been reports on the research of organoids of various tissues and organs. These organoids with the complexity of multiple cell types are highly similar to the corresponding in vivo tissues and organs. This technology provides an ideal platform for studying the development, regeneration and pathology of tissues and organs.

[0003] Skeletal muscle organoids are an ideal drug screening model. Regarding the construction of skeletal muscle organoids, Patent CN108660107A discloses a method for constructing skeletal muscle organoids, which includes the step of co-culturing myogenic progenitor cells (MPCs) isolated from mouse skeletal muscle with rapidly adherent cells (RACs) in skeletal muscle in matrigel in 3D and obtaining a contractile muscle-like tissue mass after differentiation induction. The method is simple and highly operable. However, in this technology, the mouse skeletal muscle organoid technology platform is limited to a certain extent in the high-throughput production of skeletal muscle organoids due to the long gelation time of Matrigel and some steps requiring manual operation. Summary of the Invention

[0004] The present invention aims to solve at least to some extent the technical problems in the prior art. To this end, the present invention provides a method for constructing high-throughput skeletal muscle organoids.

[0005] The technical solution adopted by the present invention is as follows:

[0006] In the first aspect,

[0007] The present invention provides a method for constructing high-throughput skeletal muscle organoids, including the following steps:

[0008] (1) Mix myogenic progenitor cells (MPCs) and rapidly adherent cells (RACs) isolated from skeletal muscle with Matrigel and CaCl 2 solution to form a Matrigel cell suspension with a CaCl 2 concentration of 0.8 - 1.2% (w / v);

[0009] (2) Add the Matrigel cell suspension to a 4 - 6% (w / v) sodium alginate solution to obtain Matrigel cell spheres (precursors of skeletal muscle organoids) coated with calcium alginate capsules;

[0010] (3) Place the Matrigel cell spheres coated with calcium alginate capsules in an incubator to allow the Matrigel to solidify into a gel. After gel formation, place the Matrigel cell spheres coated with calcium alginate capsules in a growth medium and culture for 1 day (to remove the calcium alginate capsules).

[0011] (4) Transfer the Matrigel cell mixture from the growth medium to a differentiation medium and induce myogenic differentiation into skeletal muscle organoids with contractile function.

[0012] The method of the present invention drops a Matrigel cell suspension (MPCs, RACs) mixed with CaCl 2 into a sodium alginate solution. Then, Ca 2+ combines with sodium alginate to form a layer of calcium alginate capsules on the surface of the Matrigel cell suspension to prevent the dissolution and diffusion of the non-gelled Matrigel cell suspension, forming a structure with an inner layer of Matrigel cell spheres coated with an outer layer of calcium alginate capsules, thereby achieving the purpose of rapidly and homogenously manufacturing Matrigel cell spheres. Then, place the Matrigel cell spheres coated with calcium alginate capsules (organoid precursors) in a 37 °C cell incubator to allow the Matrigel to solidify into a gel. After the Matrigel gels, place the Matrigel cell gel spheres coated with calcium alginate capsules in a growth medium and culture for 1 day. During the culture process, phosphate in the growth medium dissolves the calcium alginate capsule shell, thereby releasing the Matrigel cell spheres, which can continue to be amplified and cultured in the growth medium. Finally, culture in a differentiation medium for 5 - 7 days to obtain skeletal muscle organoids. The method of the present invention is simple, highly operable, and can obtain a large number (>100) of skeletal muscle organoid precursors within a short time (5 min). After induced differentiation and culture, a large number of skeletal muscle organoids are obtained, which is suitable for high-throughput drug screening research.

[0013] In some embodiments, the skeletal muscle is of human origin or non-human mammalian origin. In a specific embodiment, the skeletal muscle is of human or mouse origin.

[0014] In some preferred embodiments, in step (1), the concentration of CaCl 2 in the formed Matrigel cell suspension is 1% (w / v).

[0015] In some embodiments, step (1) is: in vitro amplify RACs and MPCs to 3 - 4 passages; digest RACs and MPCs into cell suspensions with trypsin and count them separately; take the required cell suspensions, mix RACs and MPCs in a number ratio of (1 - 2):(1 - 2), centrifuge to remove the supernatant; use 4% (w / v) concentration CaCl 2The solution and the growth medium are mixed evenly at a volume ratio of 1:1 to obtain Solution A (pre-cooled on ice). Then, Solution A (after pre-cooling on ice) and the Matrigel stock solution are mixed evenly on ice at a volume ratio of 1:1 to obtain Solution B. The cells are resuspended with Solution B and mixed evenly to obtain a 1% (w / v) CaCl 2 Matrigel cell suspension, and then it is placed on ice for later use.

[0016] As a preferred example, in step (1), the number ratio of RACs to MPCs is 1:1.

[0017] In some embodiments, the concentration of the sodium alginate solution in step (2) is 5% (w / v).

[0018] In some embodiments, the composition of the growth medium is: H-DMEM, 5-20% FBS (fetal bovine serum), 0.5-1.5% PS (penicillin-streptomycin), 5-50 ng / ml bFGF (basic fibroblast growth factor). Preferably, the composition of the growth medium is: H-DMEM, 10% FBS, 1% PS, 10 ng / ml bFGF.

[0019] In some embodiments, the composition of the differentiation induction medium is: H-DMEM, 1-3% HS (horse serum), 100-200 U / ml penicillin, 100-200 U / ml streptomycin. Preferably, the composition of the differentiation induction medium is: H-DMEM, 2% HS, 100 U / ml penicillin, 100 U / ml streptomycin.

[0020] In some embodiments, in step (3), the Matrigel cell spheres coated with calcium alginate capsules are placed in a CO 2 incubator at 37 °C for 15-40 min to solidify the Matrigel into a gel.

[0021] In some embodiments, in step (4), the culture time in the differentiation medium is 5-7 days. Further, the differentiation medium is changed every 24 h. It can be seen that under the method of the present invention, the Matrigel cell spheres can be induced to differentiate into skeletal muscle organoids with contractile function after being cultured in the differentiation medium for 5-7 days. Compared with the prior art, the method of the present invention greatly shortens the construction period of skeletal muscle organoids.

[0022] In some embodiments, the myogenic progenitor cells (MPCs) and rapidly adherent cells (RACs) are prepared by a method including the following steps:

[0023] 1) The muscle tissue with tendons, fat and fascia removed is cut into minced meat; centrifuged, and the supernatant is removed; washed with HBSS and centrifuged again;

[0024] 2) Discard the supernatant, resuspend the muscle with pre-warmed 0.2% Collagenase XI at 37°C to start digestion, incubate in a 37°C water bath for 1 hour, and invert and mix every 10 minutes;

[0025] 3) After centrifuging the meat slurry-enzyme mixture to discard the supernatant, resuspend it with dispase solution and incubate in a 37°C water bath for 1 hour;

[0026] 4) Centrifuge the meat slurry-enzyme mixture, discard the supernatant, resuspend it with 0.1% Trypsin, and incubate in a 37°C water bath for 45 minutes;

[0027] 5) Centrifuge, discard the supernatant, and resuspend with GM;

[0028] 6) Filter the resuspended matter through a 70-μm cell sieve;

[0029] 7) Aspirate the resuspended liquid with a syringe;

[0030] 8) Transfer the resuspended liquid to a culture dish coated with Collagen I and name it PP1;

[0031] 9) After placing it in a 37°C cell culture incubator for 2 hours, transfer the supernatant to a new 60-mm culture dish coated with Collagen I and name it PP2. Add fresh growth medium (GM) to PP1;

[0032] 10) After 24 hours, transfer the supernatant in PP2 to a new culture dish coated with Collagen I and name it PP3. Add fresh growth medium to PP2;

[0033] 11) Repeat step 10) until PP6 is obtained. Aspirate and discard the supernatant in the PP6 culture dish after 72 hours and replace it with a new medium;

[0034] The cells in PP1 and PP2 are rapidly adherent cells (RACs); the cells in PP3 and PP4 are myogenic progenitor cells (MPCs) after in vitro purification and amplification.

[0035] In the preparation of the myogenic progenitor cells (MPCs) and rapidly adherent cells (RACs), the composition of the growth medium is: H-DMEM, 5-20% FBS, 0.5-1.5% PS, 5-50 ng / ml bFGF. Preferably, the composition of the growth medium is: H-DMEM, 10% FBS, 1% PS, 10 ng / ml bFGF.

[0036] In the second aspect,

[0037] The present invention also provides a skeletal muscle organoid obtained by the above method, and the diameter of the skeletal muscle organoid is about 1.5-2 mm.

[0038] In some embodiments, the diameter of the skeletal muscle organoids is about 1.5 mm.

[0039] In a third aspect,

[0040] The present invention also provides the use of the above-mentioned skeletal muscle organoids for in vitro simulation of injury regeneration research, for screening therapeutic compounds that induce or promote injury regeneration, or as an in vitro model for reconstructing injury-related regeneration processes.

[0041] In a specific embodiment of the use, the skeletal muscle organoids are used for high-throughput screening of therapeutic compounds that induce or promote injury regeneration.

[0042] Advantages of the present invention:

[0043] (1) The method of the present invention is simple, highly operable, and a large number (e.g., >100) of skeletal muscle organoid precursors can be obtained within a short time (e.g., 5 min), and a large number of skeletal muscle organoids can be obtained after induced differentiation culture.

[0044] (2) The skeletal muscle organoids obtained by the present invention can contract, and the physiological functions of the skeletal muscle organoids can be quantitatively analyzed by combining the moveheat method, which is suitable for high-throughput drug screening research. Description of the Drawings

[0045] The above-mentioned and / or additional aspects and advantages of the present invention will become apparent and easily understood from the following description of the embodiments in conjunction with the drawings, where:

[0046] Figure 1 Shows the results of the isolation and identification of human muscle MPCs.

[0047] Among them, the left figure: PAX7 immunofluorescence (red) staining, the middle figure: DAPI staining, and the right figure: the merged figure of PAX7 immunofluorescence staining and DAPI staining.

[0048] Figure 2 Shows that the isolated human myoblasts can be induced to differentiate into myotubes under 2D culture conditions. Among them, the left figure: MHC (myosin heavy chain) immunofluorescence (green) staining, the middle figure: DAPI staining, and the right figure: the merged figure of MHC immunofluorescence staining and DAPI staining.

[0049] Figure 3 Shows the white field image (100×) of human skeletal muscle organoids.

[0050] Figure 4 Shows the white field image (40×) of human skeletal muscle organoids.

[0051] Figure 5 Shows the results of the isolation and identification of mouse muscle MPCs.

[0052] Among them, the left figure: PAX7 immunofluorescence (red) staining, the middle figure: DAPI staining, and the right figure: merged image of PAX7 immunofluorescence staining and DAPI staining.

[0053] Figure 6 The isolated mouse myoblasts can be induced to differentiate into myotubes under 2D culture conditions. Among them, the left figure: MHC (myosin heavy chain) immunofluorescence (green) staining, the middle figure: DAPI staining, and the right figure: merged image of MHC immunofluorescence staining and DAPI staining.

[0054] Figure 7 White field image (100×) of mouse skeletal muscle organoids.

[0055] Figure 8 White field image (40×) of mouse skeletal muscle organoids

[0056] Figure 9 Morphological images of Matrigel cell spheres coated with calcium alginate capsules obtained in Example 3 and Comparative Example 1 and Comparative Example 2. Detailed implementation manners

[0057] The present invention illustrates how the present invention is implemented by way of examples. These are only limited listings under the essence of the present invention and do not constitute any limitation to the present invention.

[0058] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0059] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0060] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And the laboratory operation steps of molecular genetics, nucleic acid chemistry, and immunology used herein are all conventional steps widely used in the corresponding fields or are carried out according to the conditions recommended by the manufacturers.

[0061] The following are the term descriptions in this article:

[0062] In this article, the term "organoid" refers to a miniature organ constructed in a 3D culture environment.

[0063] In this text, in relation to "the first aspect", "the second aspect", "the third aspect", etc., the terms "first", "second", "third", etc. are only for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly specifying the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0064] In this text, in relation to "preferred", "more preferred", "better", etc., they are only for describing embodiments or examples with better effects and should be understood not to constitute a limitation on the protection scope of the present invention.

[0065] In this text, in relation to "further", "even further", etc., they are for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of the present invention.

[0066] In this text, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0067] In the case where a value is described as a range, it should be understood that such disclosure includes the disclosure of all possible sub-ranges within the range, as well as the specific values falling within the range, regardless of whether the specific values or specific sub-ranges are explicitly indicated.

[0068] An embodiment of the present invention provides a method for constructing high-throughput skeletal muscle organoids, comprising the following steps:

[0069] (1) Mix myogenic progenitor cells (MPCs) and rapidly adherent cells (RACs) isolated from skeletal muscle with Matrigel and CaCl 2 solution to form a Matrigel cell suspension with a CaCl 2 concentration of 0.8 - 1.2% (w / v);

[0070] (2) Add the Matrigel cell suspension to a 4 - 6% (w / v) sodium alginate solution to obtain Matrigel cell spheres (precursors of skeletal muscle organoids) coated with calcium alginate capsules;

[0071] (3) Place the Matrigel cell spheres coated with calcium alginate capsules in an incubator to allow the Matrigel to solidify into a gel. After gel formation, place the Matrigel cell spheres coated with calcium alginate capsules in a growth medium and culture for 1 day (to remove the calcium alginate capsules).

[0072] (4) Transfer the Matrigel cell mixture from the growth medium to a differentiation medium to induce myogenic differentiation into skeletal muscle organoids with contractile function.

[0073] Non-limiting examples are as follows: In step (1), the concentration of CaCl in the Matrigel cell suspension 2 can be 0.8% (w / v), 0.9% (w / v), 1.0% (w / v), 1.1% (w / v), 1.2% (w / v), etc., and the concentration of the sodium alginate solution in step (2) can be 4% (w / v), 4.5% (w / v), 5% (w / v), 5.5% (w / v), 6% (w / v), etc.

[0074] In some embodiments, in step (2), a pre-cooled 1 ml insulin syringe on ice is used to aspirate the Matrigel cell suspension, and the piston of the syringe is slowly pushed to extrude the Matrigel cell suspension to form droplets of about 8.5 μl, which are dropped into a 5% (w / v) sodium alginate solution to form a structure with an inner layer of Matrigel cell spheres coated with an outer layer of calcium alginate capsules.

[0075] In some embodiments, the skeletal muscle is of human or non-human mammalian origin. The non-human mammals include, for example, camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, mice, non-human primates, etc. The skeletal muscle is of human or mouse origin.

[0076] In some embodiments, the composition of the growth medium is: H-DMEM, 5-20% FBS, 0.5-1.5% PS, 5-50 ng / ml bFGF. Non-limiting examples are as follows: The concentration of FBS in the growth medium can be 5%, 8%, 10%, 15%, 20%, etc., the concentration of PS can be 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, etc., and the concentration of bFGF can be 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 50 ng / ml, etc. Preferably, the composition of the growth medium is: H-DMEM, 10% FBS, 1% PS, 10 ng / ml bFGF.

[0077] In some embodiments, the composition of the differentiation induction medium is: H-DMEM, 1-3% HS, 100-200 U / ml penicillin, 100-200 U / ml streptomycin. Non-limiting examples are as follows: in the differentiation induction medium, the concentration of HS can be 1%, 1.5%, 2%, 2.5%, 3%, etc., the concentration of penicillin can be 100 U / ml, 150 U / ml, 200 U / ml, etc., and the concentration of streptomycin can be 100 U / ml, 150 U / ml, 200 U / ml, etc. Preferably, the composition of the differentiation induction medium is: H-DMEM, 2% HS, 100 U / ml penicillin, 100 U / ml streptomycin.

[0078] In some embodiments, in step (3), the Matrigel cell spheres coated with calcium alginate capsules are placed in a CO 2 incubator at 37 °C for 15-40 min to solidify the Matrigel into a gel.

[0079] In some embodiments, in step (4), the culture time in the differentiation medium is 5-7 days. Non-limiting examples are as follows: the culture time can be 5 days, 6 days, 7 days, etc. Further, the differentiation medium is changed once every 24 h.

[0080] In the present invention, the physiological function of skeletal muscle organoids is detected by the Moveheat detection method.

[0081] Principle of Moveheat for detecting skeletal muscle organoids:

[0082] 1) Take out the matured skeletal muscle organoids from the incubator and take a video of their contraction (about 1 min) under a 40× microscope;

[0083] 2) After obtaining the video, use a 15*15 average operator to process each frame of the image in the video to blur the image;

[0084] 3) Calculate the gray difference between the corresponding pixels of the (n + 3)-th frame and the n-th frame;

[0085] Finally, calculate the average value of the gray-scale changes of each pixel of all frames as the heat of the image change in the corresponding area.

[0086] Example 1

[0087] I. Isolation of human skeletal muscle rapidly adherent cells (RACs) and myogenic progenitor cells (MPCs)

[0088] 1) Preheat collagenase XI (C7657, Sigma-Aldrich), dispase II ((D4693-1G, Sigma) and trypsin at 37 °C;

[0089] 2) Discarded human muscle tissue was obtained during orthopedic surgery (from the First Affiliated Hospital of Zhejiang University School of Medicine, obtained with informed consent), and it was washed 3 times with HBSS and the tendons, fat, and fascia were shaved off;

[0090] 3) The muscle was cut into minced meat with ophthalmic scissors (about 1 mm 3 );

[0091] 4) The minced meat was transferred to a 15 ml centrifuge tube; centrifuged;

[0092] 5) The supernatant was removed, and it was washed with HBSS (24020 - 117, Invitrogen) and centrifuged again;

[0093] 6) The supernatant was removed, 10 ml of 0.2% collagenase XI pre - warmed at 37°C was added to resuspend the muscle and digestion began. It was incubated in a 37°C water bath for 1 hour, and it was inverted and mixed evenly every 10 min;

[0094] 7) The minced meat - enzyme mixture was centrifuged at 930 g at 4°C for 5 min. After removing the supernatant, it was resuspended with 10 ml of dispase solution and incubated in a 37°C water bath for 1 hour, and it was inverted and mixed evenly every 10 min;

[0095] 8) The minced meat - enzyme mixture was centrifuged at 930 g at 4°C for 5 min. After removing the supernatant, it was resuspended with 10 ml of 0.1% trypsin and incubated in a 37°C water bath for 45 min, and it was mixed evenly (gently) every 10 min;

[0096] 9) Centrifuged at 930 g at 4°C for 5 min, and after removing the supernatant, it was resuspended with 10 ml of GM;

[0097] 10) The resuspended matter was filtered through a 70 μm pore - size cell strainer;

[0098] 11) The resuspended liquid was aspirated successively with 10 ml syringes with 18G, 23G, and 27G needles (2 times each);

[0099] 12) The resuspended liquid was transferred to a 60 mm culture dish coated with collagen I and named PP1;

[0100] 13) After placing it in a 37°C cell culture incubator for 2 h, the supernatant was transferred to a new 60 mm culture dish coated with collagen I and named PP2, and 3 ml of fresh growth medium (Growth Medium, GM) was added to PP1;

[0101] The composition of the growth medium is: H - DMEM, 10% FBS, 1% PS, 10 ng / ml bFGF;

[0102] 14) After 24 h, transfer the supernatant in PP2 to a new collagen I-coated 60 mm culture dish named PP3, and add 3 ml of fresh growth medium to PP2;

[0103] 15) Repeat step 14) until PP6 is obtained. After 72 hours in the PP6 culture dish, the supernatant is discarded and replaced with new growth medium.

[0104] The cells in PP1 and PP2 are rapidly adherent cells (RACs), and their cell morphology is very similar to that of fibroblasts; most of the cells in PP3 and PP4 are muscle stem cells, which become myogenic progenitor cells, i.e., MPCs, after in vitro purification and expansion.

[0105] 2. MPCs in vitro purification steps:

[0106] 1) Aspirate the culture medium and wash with 2 ml 1X PBS;

[0107] 2) Digest with 0.5 ml 0.05% trypsin at 37°C for 30 s, then add 0.5 ml GM to terminate digestion;

[0108] 3) Transfer the digested cells to a 15 ml centrifuge tube and centrifuge at 930 g for 5 min;

[0109] 4) Aspirate the supernatant, add 3 ml GM to resuspend the cells and transfer the cell suspension to a new 60 mm collagen I-coated culture dish, then place the culture dish in the incubator to adhere to the wall for 30 minutes;

[0110] 5) After 30 minutes of adhesion treatment, the non-adherent cells in the supernatant were transferred to a new 60 mm collagen I-coated culture dish.

[0111] At this point, one purification is completed, and the number of times the purification is repeated depends on the degree of contamination of the fibroblast-like cells.

[0112] 3. Identification of MPCs

[0113] PAX7 is a classic marker for muscle stem cells, so this application uses this marker to identify the isolated muscle stem / progenitor cells.

[0114] Figure 1 The results of the isolation and identification of human muscle MPCs. Figure 1 Immunofluorescence results of PAX7 + The ratio of the cell number to the total cell number showed that myogenic progenitor cells with high purity (>95%) were isolated.

[0115] Figure 2 The results of the isolated human myogenic progenitor cells in 2D culture conditions were Figure 2It can be seen that the human myoblasts isolated by the method of Example 1 can be induced to differentiate into myotubes under 2D culture conditions.

[0116] Among them, the 2D culture conditions are as follows: growth medium (H-DMEM, 10% FBS, 1% PS, 10 ng / ml bFGF); the culture dish is coated with rat tail collagen I; cultured in a cell incubator at 37 °C with 5% CO 2 2.

[0117] Example 2

[0118] The difference from Example 1 is that Example 2 is directed to mouse skeletal muscle.

[0119] That is, in the first part, step 2) is as follows: Two 6-8-week-old male C57BL / 6 mice (Experimental Animal Center of Zhejiang University School of Medicine) are sacrificed by cervical dislocation and then placed in 75% alcohol for sterilization for 10 min; the hindlimb gastrocnemius and tibialis anterior muscles are taken, washed 3 times with HBSS, and the tendons, fat, and fascia are shaved off.

[0120] Other steps are the same as those in Example 1.

[0121] Figure 5 are the results of the isolation and identification of mouse muscle MPCs. By Figure 5 calculating the proportion of the number of PAX7 + cells in the total number of cells in the immunofluorescence results, it can be known that mouse myoblasts with a relatively high purity (>90%) are isolated.

[0122] Figure 6 are the results of the mouse myoblasts isolated under 2D culture conditions. By Figure 6 It can be seen that the mouse myoblasts isolated by the method of Example 2 can be induced to differentiate into myotubes under 2D culture conditions.

[0123] Among them, the 2D culture conditions are as follows: growth medium (H-DMEM, 10% FBS, 1% PS, 10 ng / ml bFGF); the culture dish is coated with rat tail collagen I; cultured in a cell incubator at 37 °C with 5% CO 2 2.

[0124] Example 3 Preparation of skeletal muscle organoids

[0125] 1) The growth medium and Matrigel are pre-cooled on ice 30 min in advance;

[0126] 2) Dilute Matrigel 1-fold with the growth medium according to the required usage amount and continue to pre-cool on ice;

[0127] 3) Expand the RACs and MPCs prepared in Example 1 in vitro to 3 - 4 passages, so that the RACs and MPCs can each reach at least 7*10 5 ;

[0128] 4) Digest the RACs and MPCs into cell suspensions with trypsin respectively and count them separately;

[0129] 5) Determine the dosages of RACs and MPCs according to the number of organoids to be made (RACs:MPCs = 1:1), then take the required cell suspensions, mix the RACs and MPCs at a cell number ratio of 1:1, with 1.4*10 5 cells each of RACs and MPCs, centrifuge at 200g for 5 min and then discard the supernatant.

[0130] 6) Mix a 4% (w / v) CaCl 2 solution with the growth medium at a volume ratio of 1:1 to obtain Solution A (pre-cooled on ice), then mix Solution A (after pre-cooling on ice) with the Matrigel stock solution at a volume ratio of 1:1 on ice to obtain Solution B, and resuspend the cells with Solution B and mix well to obtain a 1% (w / v) CaCl 2 -concentration Matrigel cell suspension, and then set it aside on ice for later use;

[0131] 7) Use a pre-cooled 1 ml insulin syringe on ice to aspirate the Matrigel cell resuspension, slowly push the syringe piston to extrude the Matrigel cell suspension to form droplet balls (about 8.5 μl), and drop the droplet balls into a 5% (w / v) sodium alginate solution to form a structure with an inner layer of Matrigel cell balls coated with an outer layer of calcium alginate capsules. Then place the Matrigel cell balls coated with calcium alginate capsules in a 37°C CO 2 incubator for 20 min to solidify the Matrigel into a gel;

[0132] 8) After the Matrigel solidifies into a gel, place the Matrigel cell balls coated with calcium alginate capsules in the growth medium (H-DMEM, 10% FBS, 1% PS, 10 ng / ml bFGF) and culture them in suspension for 24 h (automatically remove the calcium alginate capsules);

[0133] 9) Transfer the Matrigel cell mixture from the growth medium to the differentiation medium (H-DMEM, 2% HS, 100 U / ml penicillin, 100 U / ml streptomycin) and culture for 7 days (change the differentiation medium every 24 h) to obtain human skeletal muscle organoids.

[0134] By the method of Example 3, human skeletal muscle organoids that can twitch can be obtained. Figure 3 It is the bright field image (100×) of human skeletal muscle organoids.Figure 4 Bright field image of human skeletal muscle organoids (40×). Through Figure 3 , Figure 4 it can be seen that the diameter of the cultured human skeletal muscle organoids is about 1.5 mm.

[0135] Example 4

[0136] The difference from Example 3 is that in Example 4, the RACs and MPCs prepared in Example 2 were used to prepare mouse skeletal muscle organoids.

[0137] By the method of Example 4, contractile mouse skeletal muscle organoids can be obtained. Figure 7 is the bright field image of mouse skeletal muscle organoids (100×), Figure 8 is the bright field image of mouse skeletal muscle organoids (40×). Through Figure 7 , Figure 8 it can be seen that the diameter of the cultured mouse skeletal muscle organoids is about 1.5 mm.

[0138] Comparative Example 1

[0139] The difference from Example 3 is that the concentration of the sodium alginate solution is 1.25% (w / v).

[0140] Comparative Example 2

[0141] The difference from Example 3 is that the concentration of the sodium alginate solution is 10% (w / v).

[0142] The morphological images of the Matrigel cell spheres coated with calcium alginate capsules obtained in Example 3, Comparative Example 1 and Comparative Example 2 are as shown in Figure 9 . Through Figure 9 it can be seen that the homogeneity of the shape of the Matrigel cell spheres coated with calcium alginate capsules obtained by dropping the Matrigel cell suspension with a concentration of 1% (w / v) CaCl 2 into 1.25% (w / v) and 10% (w / v) sodium alginate solutions is not as good as that of the Matrigel cell spheres coated with calcium alginate capsules obtained by dropping into 5% (w / v) sodium alginate solution. Moreover, the shape of the Matrigel cell spheres coated with calcium alginate capsules obtained in 5% (w / v) sodium alginate solution is hemispherical, while the shape of the Matrigel cell spheres coated with calcium alginate capsules obtained in 1.25% (w / v) and 10% (w / v) sodium alginate solutions is flaky, which is not conducive to the subsequent self-organization of cells to generate functional muscle organoids.

[0143] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing high-throughput skeletal muscle organoids, characterized in that: The steps include: (1) Myoblast progenitor cells and fast-attaching cells separated from skeletal muscle are mixed with Matrigel and CaCl2 solution to form a Matrigel cell suspension with a CaCl2 concentration of 0.8-1.2% w / v; myoblast progenitor cells are named MPCs, and fast-attaching cells are named RACs; the skeletal muscle is derived from human or non-human mammals; (2) Add the Matrigel cell suspension to a 5% w / v sodium alginate solution to obtain a Matrigel cell sphere coated with calcium alginate capsules; (3) Placing the Matrigel cell spheres coated with calcium alginate capsules in a CO2 incubator at 37°C for 15-40 minutes to allow the Matrigel to solidify into gel, and then culturing the Matrigel cell spheres coated with calcium alginate capsules in a growth medium for 1 day to remove the calcium alginate capsules; the composition of the growth medium is: H-DMEM, 5-20% FBS, 0.5-1.5% PS, 5-50 ng / ml bFGF; (4) Transferring the Matrigel cell mixture from the growth medium to the differentiation medium and culturing for 5 to 7 days to induce myogenic differentiation into skeletal muscle organoids with contractile function; the composition of the differentiation medium is: H-DMEM, 1 to 3% HS, 100 to 200 U / ml penicillin, and 100 to 200 U / ml streptomycin.

2. A method for constructing a high-throughput skeletal muscle organoid according to claim 1, characterized in that: The skeletal muscle is derived from human or mouse.

3. A method for constructing a high-throughput skeletal muscle organoid according to claim 1, characterized in that: In step (1), the CaCl2 concentration of the formed Matrigel cell suspension is 1% w / v.

4. A method for constructing a high-throughput skeletal muscle organoid according to claim 3, characterized in that: Step (1) is: amplifying RACs and MPCs in vitro to the 3rd to 4th generation; digesting RACs and MPCs with trypsin to form cell suspensions and counting them respectively; taking the required cell suspension, mixing RACs and MPCs in a number ratio of (1 to 2): (1 to 2), centrifuging and removing the supernatant; mixing 4% w / v CaCl2 solution with growth medium in a volume ratio of 1:1 to obtain solution A, and then mixing solution A with Matrigel stock solution in a volume ratio of 1:1 on ice to obtain solution B, resuspending the cells with solution B and mixing to obtain a 1% w / v CaCl2 Matrigel cell suspension, and then placing on ice for later use.

5. A method for constructing a high-throughput skeletal muscle organoid according to claim 4, characterized in that: In step (1), the ratio of the number of RACs to MPCs is 1:

1.

6. A method for constructing a high-throughput skeletal muscle organoid according to claim 1, characterized in that: The composition of the growth medium is: H-DMEM, 10% FBS, 1% PS, 10 ng / ml bFGF.

7. A method for constructing a high-throughput skeletal muscle organoid according to claim 1, characterized in that: The composition of the differentiation medium is: H-DMEM, 2% HS, 100 U / ml penicillin, and 100 U / ml streptomycin.

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