A culture method and culture medium for spinal cord organoids

Abstract

The present invention discloses a culture method and culture medium for spinal cord organoids. The present invention constructs spinal cord organoids based on a matrix gel-layered nanomaterial complex and a culture medium. The matrix gel-layered nanomaterial complex is obtained by blending layered nanomaterials and matrix gel, and the layered nanomaterial is a magnesium / iron layered double metal hydroxide. The matrix gel-layered nanomaterial complex in the present invention can effectively promote the efficient differentiation of induced pluripotent stem cells under the three-dimensional culture conditions of organoids to form spinal cord-like structural functions. After transplantation, the organoids of the present invention can significantly improve the motor ability of the hind limbs of animals with spinal cord injury, and develop precise spinal cord regional characteristics. The organoid culture method provided by the present invention has a simple process, has a significant intervention effect when applied to in vivo transplantation, and has potential application value in the field of organoid construction.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a culture method and culture medium for spinal cord organoids. Background Art

[0002] The construction of organoids plays an important role in promoting the development of biomedicine. It can serve as an effective means of drug efficacy testing and toxicity testing. It also has constructive significance for developmental biology, disease pathology research, and especially the field of regenerative medicine.

[0003] Since 2013, when the Lancaster MA team constructed a three-dimensional organic culture system for brain organoids[1], a large number of research teams have been committed to constructing central nervous system organoids in specific regions, such as spinal cord organoids. In 2018, Shi-Yan Ng's team used Matrigel combined with growth factors as the culture matrix and patient-induced pluripotent stem cells to construct spinal cord micro-organs to study the pathological mechanism of motor neuron damage in spinal muscular atrophy[2,3], but there was no intervention study on traumatic diseases of the nervous system. In 2020, Sergiu P. Pasxca's research team constructed a 3D culture assembly of cerebral cortex, hindbrain / spinal cord and skeletal muscle to form a neural circuit, which can simulate the control of cortex over muscle contraction in vitro for a long time[4]. However, this organoid composition cannot reproduce the specific spinal cord tissue morphology and development process, and is not suitable for the study of spinal cord-related diseases. In 2022, Woong Sun's research team reported a three-dimensional culture scheme for human spinal cord-like organs, which reproduced the morphogenesis of the neural tube of the early spinal cord. The organoids can differentiate into spinal cord neurons and glial cells with mature synaptic functions, and can be used to effectively screen anti-epileptic drugs that may cause neural tube development defects [5], but lack application as transplants and their role in neural circuit reconstruction. In the same year, Xiang Peng's research team at Sun Yat-sen University used neural mesodermal progenitor cells to construct a spine-spinal cord organoid that contained both cartilage tissue and neural tissue, providing an in vitro model that simulates the early development of the neural tube [6], but there are still certain limitations, such as the lack of motor neuron development, the formation of dorsal-ventral body axis patterning, and application in neurological diseases.

[0004] Functional biomaterials can create an adjustable central nervous system microenvironment during the in vitro development of organoids and guide the growth pattern of spinal cord organoids. In 2014, a study used laminin and PEG scaffolds to construct a neural tube model with controlled release of retinoic acid, inducing neuroepithelial tissue with a dorsal-ventral structure [7]. In 2018, Zeng Yuanshan's team simulated the white matter and gray matter of the spinal cord based on tissue engineering, and constructed a transplantable spinal cord micro-organ (SCLT) based on gelatin sponge and neural stem cells. Myelination, vesicle release and neuronal electrophysiological activity were found in the micro-organ, and after transplantation, signal transmission with the original dorsal root ganglia and muscle cells of rats was formed [8]. In 2022, the team constructed spinal cord-like white matter tissue based on the above scaffold materials and oligodendrocyte precursor cells. After transplantation, it solved the obstacles of directional axon regeneration and myelination in the process of spinal cord injury repair [9]. However, the spinal cord micro-organ constructed by this research group cannot simulate the development process of the neural tube and the organizational pattern of the spinal cord, and has certain limitations.

[0005] Although organoid technology can simulate some of the cell tissue structures of the central nervous system during development, it still has problems such as poor reproducibility, immature development, and disordered tissue structure

[10] . At the same time, it is difficult to construct complex neural circuits in in vitro central nervous system organoids, and it is difficult to achieve neural repair and regeneration in the damaged area in clinical transformation. The role of transplanted organoids as "relay stations" needs to be strengthened. Although the cell composition in the body can be simulated by changing the culture conditions, the maturity of its cell type is low, which affects subsequent applications.

[0006] Nanobiomaterials are widely used in tissue engineering due to their excellent properties such as small size effect. Layered double hydroxides (LDH) are layered compounds that usually contain two metal cations and hydroxide ions, with a chemical composition of [M 2+ (1-x) M 3+ (x) (OH)2] x+ (A n- ) x / n ·mH2O. Among them, M 2+ is a divalent metal cation, such as Mg 2+ 、Ni 2+ 、Co 2+ ;M 3+ A trivalent metal cation, such as Al 3+ , Mn 3+ , Fe 3+ etc. n- Anions such as CO2-3, NO-3 and Cl -etc., usually existing between layers together with crystal water. In addition, studies have shown that nanomaterials can regulate the fate of stem cells and affect cell proliferation and differentiation

[11] . Our research group's previous studies have found that nanographene oxide can maintain the self-renewal of embryonic stem cells

[12] , and developed a layered nanomaterial Mg / Fe-LDH that can replace leukemia inhibitory factor (LIF)

[13] .

[0007] References:

[0008] [1]MALancaster,M.Renner,C.-A.Martin,D.Wenzel,LSBicknell,MEHurles,T.Homfray,JMPenninger,APJackson,JAKnoblich,Cerebralorganoids model human brain development and microcephaly,Nature 501(7467)(2013)373-+.

[0009] [2]JHHor,ES-Y.Soh,LYTan,VJWLim,MMSantosa,Winanto,BXHo,Y.Fan,B.-S.Soh,S.-Y.Ng,Cell cycle inhibitors protect motor neurons in anorganoid model of Spinal Muscular Atrophy,Cell Death&Disease 9(2018).

[0010] [3] Winanto, ZJKhong, B.-S.Soh, Y.Fan, S.-Y.Ng, Organoid cultures of MELAS neural cells reveal hyperactive Notch signaling that impacts neurodevelopment, Cell Death & Disease 11(3)(2020).

[0011] [4] J. Andersen, O. Revah, Y. Miura, N. Thom, NDAmin, KW Kelley, M. Singh,

[0012] [5]J.-H.Lee,H.Shin,MRShaker,HJKim,S.-H.Park,JHKim,N.Lee,M.Kang,S.Cho,THKwak,JWKim,M. -R.Song,S.-H.Kwon,DWHan,S.Lee,S.-Y.Choi,IJRhyu,H.Kim,D.Geum,I.-J.Cho,W.Sun,Production of human spinal-cord organoidsrecapitulating neural-tube morphogenesis,Nature Biomedical Engineering 6(4)(2022)435-+.

[0013] [6] Xiang Peng, Li Weiqiang, Li Dairui. A culture method and culture medium for spine-spinal cord organoids.

[0014] [7] A. Meinhardt, D. Eberle, A. Tazaki, A. Ranga, M. Niesche, M. Wilsch-Braeuninger, A. Stec, G. Schackert, M. Lutolf, EMTanaka, 3DReconstitution of the Patterned Neural Tube from Embryonic Stem Cells, Stem Cell Reports 3(6) (2014) 987-999.

[0015] [8]B.-Q.Lai,B.Feng,M.-T.Che,L.-J.Wang,S.Cai,M.-Y.Huang,H.-Y.Gu,B.Jiang,E.-A.Ling,M.Li,X.Zeng,Y.-S.Zeng,AModular Assembly of Spinal Cord-LikeTissue Allows Targeted Tissue Repair in the Transected Spinal Cord,AdvancedScience 5(9)(2018).

[0016] [9]B.-Q.Lai,Y.-R.Bai,W.-T.Han,B.Zhang,S.Liu,J.-H.Sun,J.-L.Liu,G.Li,X.Zeng,Y.Ding,Y.-H.Ma,L.Zhang,Z.-H.Chen,J.Wang,Y.Xiong,J.-H.Wu,Q.Quan,L.-Y.Xing,H.-B.Zhang,Y.-S.Zeng,Construction of a niche-specific spinal whitematter-like tissue to promote directional axon regeneration and myelinationfor rat spinal cord injury repair,Bioactive Materials 11(2022)15-31.

[0017]

[10] X.Qian,H.Song,G.-l.Ming,Brain organoids:advances,applications andchallenges,Development 146(8)(2019).

[0018]

[11] J.-H.Lee,J.Luo,H.K.Choi,S.-T.D.Chueng,K.-B.Lee,J.-W.Choi,Functional nanoarrays for investigating stem cell fate and function,Nanoscale12(17)(2020)9306-9326.

[0019]

[12] G.Jing, Z.Wang,

[0020]

[13] X.He, Y.Zhu, L.Yang, Z.Wang, Z.Wang, J.Feng, Summary of the invention

[0021] In view of the above background technology, the present invention provides a culture method and culture medium for spinal cord organoids, constructs motor neuron-rich organoids in which the fate of stem cells is regulated by Mg / Fe-LDH layered nanomaterials, simulates the development process and cell composition of the spinal cord, provides an in vitro research platform for the pathogenesis of human spinal cord-related diseases, and provides research tools for the prevention and treatment of related diseases.

[0022] To achieve the above object, the technical solution adopted by the present invention is:

[0023] In one aspect, the present invention provides a culture medium for spinal cord organoids, comprising:

[0024] Medium 1: contains BMP signaling pathway inhibitor, WNT signaling pathway activator and L-ascorbic acid AA;

[0025] Medium 2: contains BMP signaling pathway inhibitor, WNT signaling pathway activator, L-ascorbic acid AA and retinoic acid RA;

[0026] Medium 3: contains L-ascorbic acid AA, retinoic acid RA and Purmorphamine;

[0027] Medium 4: contains L-ascorbic acid AA, retinoic acid RA and Purmorphamine;

[0028] Medium 5: contains L-ascorbic acid AA, glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF);

[0029] The concentration of retinoic acid RA in the culture medium 3 is lower than that in the culture medium 4, and the concentration of purmorphamine in the culture medium 3 is higher than that in the culture medium 4.

[0030] As a preferred embodiment, the culture medium comprises:

[0031] Culture medium 1: contains DMEM / F-12 culture medium, Neurobasal culture medium, N2 supplement (N2Supplement), B27 supplement (B27 Supplement), GlutaMAX, BMP signaling pathway inhibitor, WNT signaling pathway activator and L-ascorbic acid AA;

[0032] Medium 2: contains DMEM / F-12 medium, Neurobasal medium, N2 supplement (N2Supplement), B27 supplement (B27 Supplement), GlutaMAX, BMP signaling pathway inhibitor, WNT signaling pathway activator, L-ascorbic acid AA and retinoic acid RA;

[0033] Medium 3: DMEM / F-12 medium, Neurobasal medium, N2 supplement (N2Supplement), B27 supplement (B27 Supplement), GlutaMAX, L-ascorbic acid AA, retinoic acid RA and Purmorphamine;

[0034] Medium 4: contains DMEM / F-12 medium, Neurobasal medium, N2 supplement (N2Supplement), B27 supplement (B27 Supplement), GlutaMAX, L-ascorbic acid AA, retinoic acid RA and Purmorphamine;

[0035] Medium 5: DMEM / F-12 medium, Neurobasal medium, N2 supplement (N2Supplement), B27 supplement (B27 Supplement), GlutaMAX, L-ascorbic acid AA, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF);

[0036] Preferably, the BMP signaling pathway inhibitor is LDN-193189;

[0037] Preferably, the WNT signaling pathway activator is CHIR99021.

[0038] As a preferred embodiment, in the culture medium 1-5, the volume ratio of the DMEM / F-12 culture medium to the Neurobasal culture medium is 1:1;

[0039] Preferably, in the medium 1 or the medium 2, the concentration of LDN-193189 is 0.5 to 5 μM; for example, 0.5 μM, 1 μM, 3 μM, 4 μM, 5 μM or any concentration therebetween;

[0040] Preferably, in the culture medium 1 or the culture medium 2, the concentration of CHIR99021 is 1 to 5 μM; for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM or any concentration therebetween;

[0041] Preferably, in the culture media 2 to 4, the concentration of retinoic acid RA is 0.1 to 0.5 μM; for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM or any concentration therebetween;

[0042] Preferably, in the culture medium 3 or 4, the concentration of Purmorphamine is 0.1-1 μM; for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM or any concentration therebetween;

[0043] Preferably, in the culture medium 1-5, the concentration of the L-ascorbic acid AA is 0.05-0.5 mM; for example, 0.05 mM, 0.10 mM, 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM, 0.35 mM, 0.40 mM, 0.45 mM, 0.50 mM or any concentration therebetween;

[0044] Preferably, in the culture medium 5, the concentration of the brain-derived neurotrophic factor (BDNF) is 5 to 20 ng / mL; for example, 5 ng / mL, 7 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 17 ng / mL, 20 ng / mL or any concentration therebetween;

[0045] Preferably, in the culture medium 5, the concentration of the glial cell line-derived neurotrophic factor (GDNF) is 5 to 20 ng / mL; for example, 5 ng / mL, 7 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 17 ng / mL, 20 ng / mL or any concentration therebetween.

[0046] In another aspect, the present invention provides use of the culture medium in preparing biological implants, specifically in culturing spinal cord organoids.

[0047] In another aspect, the present invention provides a method for culturing spinal cord organoids, wherein the method is to culture spinal cord organoids based on the above culture medium, comprising the following steps:

[0048] (1) culturing induced pluripotent stem cells (iPSCs) in culture medium 1-2 in sequence to induce differentiation;

[0049] (2) encapsulating the embryoid bodies obtained after culturing in step (1) in a matrix gel-layered nanomaterial complex to form cell spheres;

[0050] (3) continuing to culture the cell spheres obtained in step (2) in culture medium 3-5 to induce differentiation, thereby completing the spinal cord organoid culture;

[0051] In step (1), the matrix glue-layered nanomaterial composite is obtained by blending layered nanomaterial and matrix glue, the layered nanomaterial is magnesium / iron layered double hydroxide (Mg / Fe-LDH), and the matrix glue is preferably Matrigel matrix glue.

[0052] As a preferred embodiment, in step (1), the culture time in the culture medium 1 is 2 days;

[0053] Preferably, in step (1), the culture time in the culture medium 2 is 7 days;

[0054] Preferably, in step (3), the culture time in the culture medium 3 is 4 days;

[0055] Preferably, in step (3), the culture time in the culture medium 4 is 3 days;

[0056] Preferably, in step (3), the culture time in the culture medium 5 is 3 to 25 days.

[0057] As a preferred embodiment, in step (2), the cell sphere formation is performed by constant temperature incubation at 25 to 37° C.; the constant temperature incubation time is 20 to 30 min.

[0058] In certain specific embodiments, the method for obtaining induced pluripotent stem cells is to culture induced pluripotent stem cells (iPSCs) derived from wild-type fibroblasts in mTeSR1 medium, separate them into single cells using Versene digestion solution, and then resuspend them in mTeSR13 medium containing Y-27632 inhibitor.

[0059] In certain specific embodiments, the specific operation of step (1) is: culturing the induced pluripotent stem cells in culture medium 1, and after 2 days, adding retinoic acid RA to the culture medium 1 and continuing to culture for 7 days.

[0060] In certain specific embodiments, the specific operation of step (2) is: encapsulating the embryoid bodies obtained in step (1) in the matrix gel-layered nanomaterial complex, and incubating at a constant temperature of 25 to 37° C. for 20 to 30 minutes to solidify and form cell spheres.

[0061] In certain specific embodiments, the specific operation of step (3) is: resuspending the cell sphere obtained in step (2) in culture medium 3, culturing for 4 days, transferring to culture medium 4 for 3 days, and then transferring to culture medium 5 for 3 to 25 days.

[0062] As a preferred embodiment, the concentration of the layered nanomaterial in the matrix gel-layered nanomaterial complex is 1 to 500 μg / mL, and more preferably 10 μg / mL.

[0063] As a preferred embodiment, the method for preparing the matrix glue-layered nanomaterial composite comprises the following steps:

[0064] S1. In an inert atmosphere, add a mixed solution of magnesium nitrate and ferric nitrate to a NaOH solution and stir in a water bath at 25 to 60°C for 30 to 60 minutes;

[0065] S2. washing the product of step S1 and subjecting it to a hydrothermal reaction at 80 to 120° C. to obtain a magnesium / iron layered double hydroxide;

[0066] S3. Physically blending the magnesium / iron layered double hydroxide obtained in step S2 with the matrix glue.

[0067] Preferably, in step S1, the molar ratio of magnesium ions to iron ions in the mixed solution of magnesium nitrate and iron nitrate is 3 to 5:1;

[0068] Preferably, the concentration ratio of magnesium ions in the magnesium nitrate to the NaOH solution is 0.5 to 0.8:1;

[0069] Preferably, the mixed solution of magnesium nitrate and ferric nitrate is added to the NaOH solution, and the final molar concentration ratio of magnesium nitrate, ferric nitrate and sodium hydroxide is 5-5.2:1.0-1.3:1;

[0070] Preferably, step S3 is to perform physical blending at a low temperature of 0 to 10°C.

[0071] In another aspect, the present invention provides spinal cord organoids obtained by the above culture method.

[0072] The above technical solution has the following advantages or beneficial effects:

[0073] (1) The present invention provides a method and culture medium for culturing spinal cord organoids, and constructs spinal cord organoids based on the culture medium and a matrix gel-layered nanomaterial composite, wherein the composite is obtained by blending a layered nanomaterial and a matrix gel, and the layered nanomaterial is a magnesium / iron layered double metal hydroxide. The matrix gel-layered nanomaterial composite in the present invention can construct a spinal cord organoid rich in motor neuron precursor cells (HB9 + and Islet1 + In the technical solution of the present invention, the Mg / Fe-LDH layered nanomaterial combined with the matrix gel can effectively promote the efficient differentiation of induced pluripotent stem cells under the three-dimensional culture conditions of the organoids to form spinal cord-like structural functions.

[0074] (2) In the prior art, research on the cultivation of organoids generally lacks in vivo functional verification, while the humanized organoids prepared by the present invention can be transplanted into immunodeficient animals to form human-mouse chimeric spinal cord tissues, which can significantly improve and restore the motor ability of the hind limbs of animals with spinal cord injuries during transplantation therapy. After transplantation, it is affected by endogenous signals from the host and can develop precise regional characteristics of the spinal cord. The spatial transcriptome sequencing of the human-mouse chimeric tissues was compared with the human neural tube, and it was determined at the genetic level that it developed dorsal-ventral specific gene expression of the top plate (RP), bottom plate (FP), dorsal spinal cord (dl2, dl4) and ventral spinal cord (p3, MN).

[0075] (3) The Mg / Fe-LDH layered nanomaterial used in the present invention has good biocompatibility. The Mg / Fe-LDH prepared by hydrothermal synthesis has a regular morphology and uniform size. Its nanostructure characteristics and the metal ions released by degradation can regulate the fate of stem cells. The composite prepared by combining with the thermosensitive gel Matrigel has uniform dispersion of Mg / Fe-LDH and can continuously regulate the development of spinal cord organoids.

[0076] (4) The organoid culture method provided by the present invention has a simple process and has a significant intervention effect when applied to in vivo transplantation, and has potential application value in the field of organoid construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is the characterization result of the Mg / Fe-LDH layered nanomaterial in Example 1 of the present invention, wherein: Figure 1 (a) is the transmission electron microscopy result of Mg / Fe-LDH. Figure 1 (b) is the elemental analysis of Mg / Fe-LDH;

[0078] Figure 2 This is a flow chart of constructing spinal cord organoids in Example 3 of the present invention;

[0079] Figure 3 The cell composition and tissue structure of the spinal cord organoid constructed by Mg / Fe-LDH combined with Matrigel in Example 3 of the present invention; wherein: (a) is the distribution of Islet1-positive cells; (b) is the distribution of HB9-positive cells;

[0080] Figure 4 The spinal cord organoid transplantation scheme in Example 4;

[0081] Figure 5 The results of spatial transcriptome sequencing analysis of spinal cord chimeric tissue 7 weeks after spinal cord organoid transplantation in Example 5 of the present invention;

[0082] Figure 6 shows the recovery of motor ability of animals with spinal cord injury repaired by spinal cord organoid transplantation in Example 6 of the present invention; wherein, (a) the change in BMS score of animals 7 weeks after transplantation; (b) the gait of the hind limbs of animals 7 weeks after transplantation; (c) the MEP waveform diagram; and (d) the MEP quantitative result. DETAILED DESCRIPTION

[0083] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0084] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0085] Example 1: Preparation and characterization of magnesium / iron layered double hydroxide (Mg / Fe-LDH)

[0086] (1) heating double distilled water (ddH2O) to boiling, and then boiling for 40 min and then naturally drying to obtain ddH2O without CO2 for later use;

[0087] (2) Dissolve 0.544 g of sodium hydroxide (NaOH) in 80 mL of ddH2O to obtain a sodium hydroxide solution with a concentration of 6.8 mg / mL, and pour the solution into a three-necked flask, fix it on a constant speed stirrer, introduce nitrogen, and heat it in a 60°C water bath;

[0088] (3) Magnesium nitrate and ferric nitrate were weighed in a 50 mL centrifuge tube at a molar ratio of 4:1, and 20 mL of ddH2O was added and stirred to dissolve at a speed of about 500 rpm to obtain a nitrate solution with a magnesium ion concentration of 76.9 mg / mL and an iron ion concentration of 30.3 mg / mL;

[0089] (4) Add the nitrate solution obtained in step (3) dropwise into the NaOH solution filled with nitrogen in step (2); after vigorous stirring in a 60°C water bath for 30 min, centrifuge the obtained suspension at 4500 rpm for 5 min, wash three times with ddH2O and resuspend, place in a hydrothermal kettle, react in a 100°C drying oven overnight, collect the product, wash three times, turn it upside down to dry, and finally seal it and store it in a 4°C refrigerator for later use.

[0090] The Mg / Fe-LDH layered nanomaterials obtained by transmission electron microscopy, such as Figure 1 As shown in (a), it presents a hexagonal lamellar nanostructure with a particle size of about 100 nm. The Mg / Fe-LDH composed of two ions was characterized by an X-ray photoelectron spectrometer, as shown in Figure 1 As shown in (b), it has elemental characteristic peaks composed of two metal ions.

[0091] Example 2: Preparation of Matrigel-Layered Nanomaterial Composites:

[0092] Pre-cool PBS buffer, pipettes and centrifuge tubes on ice and thaw on ice Matrigel; the reserved Mg / Fe-LDH layered nanomaterials are dispersed in pre-cooled PBS buffer at a concentration of 10-5000 μg / mL; and then the suspension is mixed with the thawed Matrigel on ice at a volume ratio of 9:1.

[0093] In this example, the final concentration of Mg / Fe-LDH in the prepared matrix gel-layered nanomaterial composite is 1-500 μg / mL.

[0094] Example 3: Construction and culture of spinal cord organoids

[0095] like Figure 2 As shown, the construction and culture process of spinal cord organoids in this embodiment includes the following steps:

[0096] (1) Wild-type fibroblast-derived iPSCs were maintained and cultured in mTeSR1 medium and seeded on Matrigel-coated 6-well plates. iPSC clones were isolated into single cells using Versene, resuspended in mTeSR13 medium containing 50 μM Y-27632, and seeded at 30,000 cells / well on low-adsorption 96-well round-bottom plates.

[0097] (2) After 12 hours, the medium was changed to medium 1, which consisted of a 1:1 volume mixture of DMEM / F-12 medium and Neurobasal medium, plus N2 Supplement (100×), B27 Supplement (50×), GlutaMAX (100×), 1 μM LDN-193189, 3 μM CHIR99021, and 0.1 mM L-ascorbic acid AA;

[0098] (3) The initial step of step (1) is counted as day 0, and on day 3, medium 2 is used, specifically, retinoic acid RA is added to medium 1 at a final concentration of 0.1 μM; in steps (2) and (3), half of the medium is replaced with 0.1 mM L-ascorbic acid AA every 2 days;

[0099] (4) On the 10th day, the embryoid bodies were removed and wrapped in the matrix gel-layered nanomaterial complex prepared in Example 2, and incubated in a 37°C incubator for 30 min; then, the embryoid bodies were resuspended in culture medium 3 and inoculated in a 6-well plate with ultra-low adsorption; the composition of culture medium 3 was DMEM / F-12 culture medium and Neurobasal culture medium mixed in a volume ratio of 1:1, and N2Supplement (100×), B27Supplement (50×), GlutaMAX (100×), 0.1 μM retinoic acid RA and 1 μM Purmorphamine, and 0.1 mM L-ascorbic acid AA were added;

[0100] (5) On day 14, the 6-well plate was transferred to a shaker and the medium was replaced with medium 4, which consisted of a 1:1 volume mixture of DMEM / F-12 medium and Neurobasal medium, plus N2 Supplement (100×), B27 Supplement (50×), GlutaMAX (100×), 0.5 μM retinoic acid RA, 0.1-0.5 μM Purmorphamine, and 0.1 mM L-ascorbic acid AA;

[0101] (6) On day 17, the medium was changed to medium 5, which consisted of a 1:1 mixture of DMEM / F-12 medium and Neurobasal medium, and N2 Supplement (100×), B27 Supplement (50×), GlutaMAX (100×), 10 ng / mL BDNF and 10 ng / mL GDNF, and 0.1 mM L-ascorbic acid AA were added to promote the maturation of organoids.

[0102] The initial step (1) was counted as day 0, and organoid samples were collected on the 14th, 21st, and 35th days from the construction for immunofluorescence staining. The specific operations were as follows: the constructed organoids were fixed with 4% PFA solution overnight, then dehydrated with 15% and 30% sucrose solutions in a gradient manner, and then embedded in OCT cryosection embedding medium and frozen overnight; the samples were cut into 12 μm thickness using a freezing microtome; blocked with 5% donkey serum and 0.3% Triton-X PBS solution at room temperature for 1 hour, and then the primary antibody (Ilset1 and HB9) was diluted in the blocking solution and incubated at 4°C overnight; the secondary antibody was diluted with blocking solution and incubated at room temperature for 1 hour.

[0103] The above immunofluorescence staining Figure 3 The results in which Figure 3 (a) The Islet1 labeled cells are motor neuron precursor cells. Figure 3 (b) Labeled HB9 is a transcription factor for the development of spinal motor neurons. The results showed that the chamber structure developed in the early stage of organoid construction, indicating that the organoid constructed in vitro can simulate the development of the neural tube to a certain extent; in addition, Mg / Fe-LDH has a good promoting effect on the generation of spinal motor neurons, and at a concentration of 10μg / mL combined with Matrigel has a better regulatory effect on the cell fate in spinal cord organoids.

[0104] Example 4: Transplantation of Spinal Cord Organoids

[0105] (1) Construction of spinal cord complete transection injury model mice:

[0106] like Figure 4 As shown: Spinal cord organoids were transplanted using Shanghai model organism NOD-PrkdcscidIl2rgem1 / Smoc (M-NSG) severely immunodeficient mice, and a complete spinal cord transection injury model was established using female M-NSG mice aged 8-9 weeks and weighing 20±2g: After the mice were anesthetized with isoflurane, the dorsal spine was exposed by laminectomy, and transection was performed at the T8-9 segment, and then a 2.0mm spinal cord segment was removed, and the residual nerve fibers were sucked out by a suction pump to ensure complete transection of the ventral and lateral sides;

[0107] (2) The model animals were randomly divided into four groups. The organoids constructed on the 21st day in Example 2 were collected for transplantation. The transplants were spheres with a diameter of 1.5 to 2 mm. Among them, the organoids prepared by using a matrix gel-layered nanomaterial complex containing 10 μg / mL Mg / Fe-LDH layered nanomaterials were implanted into the lesion area, and the matrix gel-layered nanomaterial complex of the same concentration was added to fill the transplantation gap (LDH-Organoid group (n=25)); the Matrigel group (n=25) was implanted with solidified Matrigel of appropriate size into the lesion area; the spinal cord injury group (SCI group (n=25)) did not receive any treatment after surgery;

[0108] The muscles and skin tissues were then sutured, and the mice were placed in a warm sterile environment after surgery, with the bladders massaged twice a day to facilitate urination.

[0109] Example 5: Spatial transcriptome sequencing of chimeric spinal cord tissue

[0110] Fresh spinal cord tissue was taken from mice 7 weeks after transplantation intervention and quickly frozen (isopentane liquid nitrogen bath) to avoid RNA degradation and ice crystal formation. After quick freezing, the tissue was embedded in OCT on dry ice and frozen into sections of 10 μm. Tissue sections were captured by spatial transcriptome sequencing chips, and the library was generated by Visium Spatial Gene Expression Slide & Reagent kit (10X Genomics) and sequenced using an Illumina NextSeq sequencer.

[0111] Figure 5 The results of cell clustering of spatial transcriptome sequencing of spinal cord chimeric tissue are shown. It can be seen that the LDH-Organoid transplanted area developed the top plate (RP), floor plate (FP), dorsal spinal cord tissue characteristics (dl2, dl4) and ventral spinal cord tissue characteristics (p3, MN).

[0112] Example 6: Effects of spinal cord organoid transplantation intervention on animal motor ability and neural circuit reconstruction

[0113] After the transplantation intervention, the BMS score was recorded weekly to evaluate the motor recovery of the mouse hind limbs. The BMS assessment was conducted in an open field (1m×1m×0.5m) and recorded for 7 weeks. The left and right hind limb movements were scored separately and the average value was recorded. As shown in Figure 6(a), the LDH-Organoid transplantation treatment group began to show significant improvement in motor ability in the 4th week, and showed a continuous upward trend in the 7th week.

[0114] The DigiGait system was used to record the footprint information: 7 weeks after surgery, multi-speed gait analysis (GAMS) was performed at a predetermined speed of 6.0 m / s. The video was taken from the ventral side of the mouse using a camera under the running belt. The mice were pre-trained on the treadmill to adapt to the speed. The gait data was generated by Mouse specific analysis software. At least 3 mice in each group participated in the gait recording. Figure 6(b) shows the landing of the animal's hind limbs. It can be seen that the hind limbs of the animals in the LDH-Organoid transplantation group had obvious retraction and pushing off the ground, suggesting that LDH-Organoid can improve the animal's hind limb movement ability.

[0115] Seven weeks after surgery, three mice were randomly selected from each group for motor evoked potential (MEP) test: the anesthetized mice were placed on a stereotaxic device (RWD), the meninges were exposed in the sensorimotor cortex area of ​​the hind limbs, electrodes were attached to the meninges to give 1.5 mA stimulation, and the MEP signal was recorded with a bipolar disc electrode in the sciatic nerve. The results are shown in Figure 6(c). The latency and amplitude of MEP were quantified, and the results are shown in Figure 6(d). It can be seen that the animals in the LDH-Organoid transplantation intervention group had a short latency and a large waveform amplitude, proving that their neural pathways were effectively connected.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for culturing spinal cord organoids, characterized in that: The following steps are involved: (1) Inducing differentiation of induced pluripotent stem cells by culturing in culture medium 1-2 in sequence; (2) encapsulating the embryoid bodies obtained after culturing in step (1) in a matrix gel-layered nanomaterial complex to form cell spheres; (3) The cell spheres obtained in step (2) are cultured in culture medium 3-5 to induce differentiation, thereby completing the spinal cord organoid culture; In step (2), the matrix glue-layered nanomaterial composite is obtained by blending layered nanomaterial and matrix glue, the layered nanomaterial is magnesium / iron layered double metal hydroxide, and the matrix glue is Matrigel matrix glue; Medium 1 consisted of DMEM / F-12 medium, Neurobasal medium, N2 supplement, B27 supplement, GlutaMAX, BMP signaling pathway inhibitor, WNT signaling pathway activator, and L-ascorbic acid AA; Medium 2 consisted of DMEM / F-12 medium, Neurobasal medium, N2 supplement, B27 supplement, GlutaMAX, BMP signaling pathway inhibitor, WNT signaling pathway activator, L-ascorbic acid AA, and retinoic acid RA; Medium 3 consisted of DMEM / F-12 medium, Neurobasal medium, N2 supplement, B27 supplement, GlutaMAX, L-ascorbic acid AA, retinoic acid RA, and Purmorphamine; Medium 4 consisted of DMEM / F-12 medium, Neurobasal medium, N2 supplement, B27 supplement, GlutaMAX, L-ascorbic acid AA, retinoic acid RA, and Purmorphamine; Medium 5 consisted of DMEM / F-12 medium, Neurobasal medium, N2 supplement, B27 supplement, GlutaMAX, L-ascorbic acid AA, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor; The concentration of retinoic acid RA in the culture medium 3 is lower than that in the culture medium 4, and the concentration of purmorphamine in the culture medium 3 is higher than that in the culture medium 4; The BMP signaling pathway inhibitor is LDN-193189; the WNT signaling pathway activator is CHIR99021; In the culture medium 3-4, the concentration of retinoic acid RA is 0.1-0.5 μM; In the culture medium 3 or 4, the concentration of Purmorphamine is 0.1-1 μM; In the culture medium 3-5, the concentration of L-ascorbic acid AA is 0.05-0.5 mM; In the culture medium 5, the concentration of the brain-derived neurotrophic factor is 5-20 ng / mL; the concentration of the glial cell-derived neurotrophic factor is 5-20 ng / mL.

2. The culture method according to claim 1, characterized in that In the culture medium 1-5, the volume ratio of the DMEM / F-12 culture medium to the Neurobasal culture medium is 1:

1.

3. The culture method according to claim 1, characterized in that In the culture medium 1 or culture medium 2, the concentration of LDN-193189 is 0.5~5 μM.

4. The culture method according to claim 1, characterized in that In the culture medium 1 or culture medium 2, the concentration of CHIR99021 is 1-5 μM.

5. The culture method according to claim 1, characterized in that In the culture medium 2, the concentration of retinoic acid RA is 0.1-0.5 μM.

6. The culture method according to claim 1, characterized in that In the culture medium 1-2, the concentration of L-ascorbic acid AA is 0.05~0.5 mM.

7. The culture method according to claim 1, characterized in that: In step (1), the culture time in the culture medium 1 is 2 days.

8. The culture method according to claim 1, characterized in that: In step (1), the culture time in the culture medium 2 is 7 days.

9. The culture method according to claim 1, characterized in that: In step (3), the culture time in the culture medium 3 is 4 days.

10. The culture method according to claim 1, characterized in that: In step (3), the culture time in the culture medium 4 is 3 days.

11. The culture method according to claim 1, characterized in that: In step (3), the culture time in the culture medium 5 is 3 to 25 days.

12. The culture method according to claim 1, characterized in that In step (2), the cell spheres are formed by incubating at a constant temperature of 25-37° C.; the time of the constant temperature incubation is 20-30 min.

13. The culture method according to claim 1, characterized in that The concentration of the layered nanomaterial in the matrix gel-layered nanomaterial complex is 1-500 μg / mL.

14. The culture method according to claim 13, characterized in that The concentration of the layered nanomaterial in the matrix gel-layered nanomaterial complex is 10 μg / mL.

15. The culture method according to claim 1, characterized in that: The preparation method of the matrix glue-layered nanomaterial composite comprises the following steps: S1. In an inert atmosphere, add the mixed solution of magnesium nitrate and ferric nitrate to the NaOH solution and stir in a water bath at 25-60°C for 30-60 min. S2. washing the product of step S1 and subjecting it to a hydrothermal reaction at 80 to 120° C. to obtain a magnesium / iron layered double hydroxide; S3. Physically blending the magnesium / iron layered double hydroxide obtained in step S2 with the matrix glue.

16. The culture method according to claim 15, characterized in that: In step S1, the molar ratio of magnesium ions to iron ions in the mixed solution of magnesium nitrate and iron nitrate is 3-5:

1.

17. The culture method according to claim 15, characterized in that: In step S1, the concentration ratio of magnesium ions in the magnesium nitrate to the NaOH solution is 0.5-0.8:

1.

18. The culture method according to claim 15, characterized in that: In step S1, the mixed solution of magnesium nitrate and ferric nitrate is added to the NaOH solution, and the final molar concentration ratio of magnesium nitrate, ferric nitrate and sodium hydroxide is 5-5.2:1.0-1.3:

1.

19. The culture method according to claim 15, characterized in that: Step S3 is to perform physical blending at a low temperature of 0 to 10°C.

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