A method for preparing mesenchymal stem cells based on neural crest organoids and its application

Abstract

This invention discloses a method for preparing and applying mesenchymal stem cells based on neural crest organoids, comprising the following steps: Step 1: Adding human induced pluripotent stem cells to a digestive solution, digesting, and then diluting with culture medium; Step 2: Collecting cells, centrifuging to separate the cells, discarding the supernatant, and suspending them in culture medium, adjusting the cell block size to 50-80 micrometers; Step 3: Culturing the cells, and then inducing differentiation into neural crest organoids; Step 4: Digesting the neural crest organoids into single cells, transferring them to serum-free mesenchymal stem cell culture medium for adherent culture, and then expanding to obtain mesenchymal stem cells; This invention utilizes induced pluripotent stem cells (ihPSCs)3D to induce differentiation into neural crest organoids, obtaining neural crest-derived mesenchymal stem cells, and the obtained NCC-MSCs have a beneficial and repairing effect on pancreatitis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing mesenchymal stem cells based on neural crest organoids and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs), as a class of cells with multipotent differentiation potential and immunomodulatory properties, have shown great promise in the field of cell therapy in recent years. MSCs are widely derived, including bone marrow, adipose tissue, umbilical cord blood, and embryos. Although embryonic MSCs have excellent repair capabilities, ethical restrictions hinder their clinical application. Umbilical cord blood-derived MSCs are relatively easy to obtain, have low immunogenicity, and possess excellent tissue regeneration properties and immunomodulatory advantages, making them a hot topic in clinical research. However, the application of donor-derived MSCs is limited, including donor-to-donor variability, limited cell expansion efforts, low differentiation potential, and the inability to mass-produce them industrially, all of which restrict their widespread use. Therefore, developing a stable and high-quality MSC supply method is crucial for the application of MSCs.

[0003] Human induced pluripotent stem cells (hiPSCs) possess the ability to self-renew and generate all types of cells in the human body through induction. Mesenchymal stem cells (MSCs) differentiate from mesodermal cells or neural crest cells (NCCs). NCCs are pluripotent ectodermal cells that develop from the intermediate layer between the neuroectoderm and epidermal ectoderm during vertebrate development. The technology of inducing neural crest cells through hiPSCs to obtain MSCs with similar immunophenotypes and biological functions is of great significance. However, to date, a simple, efficient, and stable method for preparing neural crest-derived mesenchymal stem cells and its clinical application evaluation have not yet been found. Summary of the Invention

[0004] This invention addresses the problems existing in the prior art by providing a method for preparing mesenchymal stem cells based on neural crest organoids and its application.

[0005] The technical solution adopted in this invention is: a method for preparing mesenchymal stem cells based on neural crest organoids, comprising the following steps:

[0006] Step 1: Add human induced pluripotent stem cells to digestive fluid, digest them, and then dilute them with culture medium;

[0007] Step 2: Collect cells, centrifuge the cells, discard the supernatant and suspend them in the culture medium, adjust the cell block size until it becomes turbid like fine sand;

[0008] Step 3: Culture the cells and then induce differentiation;

[0009] On day 1 of embryoid formation, neural differentiation basal medium was added; from day 2 to 7, it was replaced with neural induction basal medium; and from day 8 to 21, it was replaced with neural differentiation basal medium.

[0010] Step 4: Transfer the cells to serum-free mesenchymal stem cell culture medium for adherent culture, and then expand them to obtain mesenchymal stem cells.

[0011] Furthermore, the basal culture medium for neural differentiation contains: 10 μM Y-27632, 10 μM SB-431542, 0.25 μM LDN193189, and 3 μM CHIR99021;

[0012] The basal culture media for neural differentiation included: DMEM / F12 medium 47% v / v, Gibco Neural Matrix™-A medium 47% v / v, N-2 additive 0.94% v / v, B-27 additive 1.9% v / v, GlutaMAX additive 0.94% v / v, penicillin-streptomycin mixture 0.94% v / v, MEM non-essential amino acids 0.94% v / v, 2-mercaptoethanol solution 0.094% v / v, and L-ascorbic acid solution 0.094% v / v; the concentration of 2-mercaptoethanol solution was 55 mM, and the concentration of L-ascorbic acid solution was 200 mM.

[0013] Furthermore, the neural induction basal medium comprises a TGF-β receptor kinase inhibitor, a BMP type I receptor inhibitor, a GSK-3α / β inhibitor, a CDK1 / cyclin B and GSK-3β inhibitor, an FGF2 growth factor, and an FGF8 growth factor; wherein the concentrations of each component are as follows: TGF-β receptor kinase inhibitor 10 μM, BMP type I receptor inhibitor 0.25 μM, GSK-3α / β inhibitor 3 μM, CDK1 / cyclin B and GSK-3β inhibitor 2.5 μM, FGF2 growth factor 20 ng / ml, and FGF8 growth factor 25 ng / ml.

[0014] Furthermore, the basal culture medium for neural differentiation during days 8-14 of induced differentiation includes retinoic acid, a molecular compound, a smoothing agonist, and growth differentiation factor 11; wherein the concentrations of each component are: retinoic acid 0.5 μM, molecular compound 0.5 μM, smoothing agonist 0.5 μM, and growth differentiation factor 11 20 ng / ml, respectively; the basal culture medium for neural differentiation during days 15-21 of induced differentiation includes a γ-secretase inhibitor and CultureOne. TM Additives, Compound E, Brain-derived neurotrophic factor, Glial cell line-derived neurotrophic factor; the concentrations of each component are as follows: γ-secretase inhibitor 10 μM, CultureOne TMAdditive 1 μM, compound E 0.1 μM, brain-derived neurotrophic factor 20 ng / ml, glial cell line-derived neurotrophic factor 20 ng / ml.

[0015] Furthermore, in step 1, after adding the digestive solution, digestion is carried out at 37°C for 4 minutes.

[0016] Furthermore, in step 2, a ROCK1 inhibitor is added to the culture medium, and the concentration of the ROCK1 inhibitor in the culture medium is 10 μM.

[0017] Furthermore, the centrifugation conditions in step 2 are: 300g, 15min.

[0018] Furthermore, in step 3, the cells are arranged at a density of 4 mL / 10 6 The samples were seeded at a density in low-adhesion plates and cultured in a bioreactor at 765 rpm for 24 hours.

[0019] Furthermore, in step 4, amplification is performed after 4 days of adherent culture.

[0020] An application of mesenchymal stem cells based on neural crest organoids, wherein the mesenchymal stem cells are used to prepare drugs for treating pancreatitis.

[0021] The beneficial effects of this invention are:

[0022] (1) This invention utilizes induced pluripotent stem cells (ihPSCs3D) to induce differentiation into neural crest organoids and obtain neural crest-derived mesenchymal stem cells.

[0023] (2) The NCC-MSCs prepared by the method of the present invention have stronger proliferation and amplification capabilities compared with existing MSCs, and proteomics data show that they have immunomodulatory effects.

[0024] (3) The NCC-MSCs obtained in this invention have the effect of improving and repairing pancreatitis. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the method flow of the present invention.

[0026] Figure 2The diagrams show the characteristics of neural crest organoids and their derived mesenchymal stem cells in this invention. A is a morphological diagram of neural crest organoids. B is a flow cytometry analysis result of surface markers of mesenchymal stem cells derived from neural crest organoids and existing mesenchymal stem cells. C is a morphological diagram of primary MSCs induced after adhesion of mesenchymal stem cells derived from neural crest organoids and existing mesenchymal stem cells. D is a comparison of cell numbers on the third day after induction and passage of mesenchymal stem cells derived from neural crest organoids and existing mesenchymal stem cells.

[0027] Figure 3 This is a schematic diagram showing the protein differences between neural crest organoid mesenchymal stem cells and existing mesenchymal stem cells in this invention. A represents the statistical results of protein expression, and B represents the volcano diagram of the main differentially expressed proteins.

[0028] Figure 4 This is a schematic diagram showing the enrichment results of GO protein upregulated by neural crest organoid mesenchymal stem cells and existing mesenchymal stem cells in this invention.

[0029] Figure 5 This is a schematic diagram showing the results of KEGG enrichment analysis of upregulated proteins in neural crest organoid mesenchymal stem cells and existing mesenchymal stem cells in this invention.

[0030] Figure 6 The diagram shows the test results of mesenchymal stem cells derived from neural crest organoids in this invention and existing mesenchymal stem cells in an AP mouse model. A shows the results of HE staining of pancreatic pathology on day 15. B shows the changes in serum amylase after treatment with mesenchymal stem cells derived from neural crest organoids in this invention. C shows the changes in serum lipase levels after treatment with mesenchymal stem cells derived from neural crest organoids in this invention. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] like Figure 1 As shown, a method for preparing mesenchymal stem cells based on neural crest organoids includes the following steps:

[0033] Step 1: Add human induced pluripotent stem cells to digestive fluid, digest them, and then dilute them with culture medium;

[0034] The specific process is as follows:

[0035] Take out 80% confluent hiPSCs, aspirate the culture medium from the culture dish with a Pasteur pipette, wash twice with 2 mL PBS, add 200 μL Relesa digestion solution, and digest at 37 °C for 4 min.

[0036] Immediately after digestion, remove the culture dish and add 2 mL of ncTarget culture medium to dilute and stop digestion.

[0037] Step 2: Collect cells, centrifuge the cells, discard the supernatant and suspend them in the culture medium, adjust the cell block size until it becomes turbid like fine sand;

[0038] The specific process is as follows:

[0039] Collect cells by gently pipetting, trying to avoid generating air bubbles during the process. Transfer all cells to a 15mL centrifuge tube and centrifuge at 300g for 5 minutes.

[0040] After discarding the supernatant, suspend the cells in the culture medium (ncTarget + 10 μL Y-27632) and use a Pasteur pipette to adjust the size of the cell blocks (to the desired size, adjust according to the actual situation) until the cells are turbid like fine sand and the diameter of the cell blocks is 50-80 μm.

[0041] Step 3: Culture the cells and then induce differentiation;

[0042] Add neural differentiation basal medium on day 1, replace with neural induction basal medium on days 2-7, and replace with neural differentiation basal medium on days 8-21.

[0043] The specific process is as follows:

[0044] Based on a cell density of 4 mL / 10 wells 6 One cell was seeded into a low-adhesion 6-well plate, and the 6-well plate was placed on a 6-well plate horizontal shaker with an amplitude of 24 mm and a rotation speed of 80 rpm.

[0045] After embryoids are formed, induction differentiation medium is added, and the rotation speed of the 6-well plate bioreactor is adjusted to 80 rpm.

[0046] The basal medium for neural differentiation was added on the first day after the formation of the embryoid body; this stage is the first stage.

[0047] The basal culture medium for neural differentiation contains: 10 μM Y-27632, 10 μM SB-431542, 0.25 μM LDN193189, and 3 μM CHIR99021;

[0048] The basal culture media for neural differentiation included: DMEM / F12 medium 47% v / v, Gibco Neurobasal™-A Medium 47% v / v, N-2 supplement (100×N-2 supplement) 0.94% v / v, B-27 supplement (50×B27 supplement) 1.9% v / v, GlutaMAX supplement (100×GlutaMAX supplement) 0.94% v / v, Penicillin-Strep mixture (100×Pen Strep) 0.94% v / v, MEM non-essential amino acids (100×MEM Non-Essential Amino Acids) 0.94% v / v, 2-mercaptoethanol solution 0.094% v / v, and L-ascorbic acid solution 0.094% v / v; the concentration of 2-mercaptoethanol solution was 55 mM, and the concentration of L-ascorbic acid solution was 200 mM.

[0049] Among them, Y-27632 is a selective ROCK1 and ROCK2 inhibitor; SB-431542 is a SMAD signaling inhibitor; LDN193189 is an effective selective BMP type I receptor inhibitor that inhibits the SMAD pathway, regulates the SMAD and WNT pathways, and achieves directed differentiation; CHIR99021 is a WNT pathway activator; LDN-193189 and SB431542 induce ectoderm development and neurogenesis by acting on ALK2 and ALK3 in the BMP4 pathway and ALK5 in the TGFBETA pathway, respectively, to achieve the formation of transplanted endoderm and mesoderm. By inhibiting the BMP and TGFBETA pathways to mimic the signaling pathways in early embryonic development, neural stem cell generation is induced. Induced neurons can be obtained in the presence of serum components (Knockout Serum Replacement) using this method.

[0050] From day 2 to 7, the medium was replaced with the basal medium for neural induction; this stage is the second stage.

[0051] The basal medium for neural induction included TGF-β receptor kinase inhibitor, BMP type I receptor inhibitor, GSK-3α / β inhibitor, CDK1 / cyclin B and GSK-3β inhibitor, FGF2 growth factor, and FGF8 growth factor; the concentrations of each component were as follows: TGF-β receptor kinase inhibitor 10 μM, BMP type I receptor inhibitor 0.25 μM, GSK-3α / β inhibitor 3 μM, CDK1 / cyclin B and GSK-3β inhibitor 2.5 μM, FGF2 growth factor 20 ng / ml, and FGF8 growth factor 25 ng / ml.

[0052] The second stage achieves directed differentiation by regulating the SMAD and Wnt pathways, as well as small molecules of growth factors EGF and FGF2. Additionally, fibroblast growth factor 8 (FGF8), a member of the fibroblast growth factor family, is an important secretory regulatory signaling molecule in tissue development. Recent studies have shown that FGF8 plays a crucial role in the development and formation of the nervous system, and it can alter the fate of the midbrain to cerebellum and promote midbrain development.

[0053] From day 8 to 21, the medium is replaced with the basal medium for neural differentiation. Days 8 to 14 constitute the third stage, and days 15 to 21 constitute the fourth stage.

[0054] The basal culture medium for neural differentiation during days 8-14 of induced differentiation included retinoic acid, a molecular compound, a smoothing agonist, and growth differentiation factor 11; the concentrations of each component were as follows: retinoic acid 0.5 μM, molecular compound 0.5 μM, smoothing agonist 0.5 μM, and growth differentiation factor 11 20 ng / ml. The basal culture medium for neural differentiation during days 15-21 of induced differentiation included the γ-secretase inhibitor DAPT and CultureOne. TM Additives, compound E, brain-derived neurotrophic factor BDNF, and glial cell line-derived neurotrophic factor GDNF; the concentrations of each component are as follows: γ-secretase inhibitor 10 μM, CultureOne TM Additive 1 μM, compound E 0.1 μM, brain-derived neurotrophic factor 20 ng / ml, glial cell line-derived neurotrophic factor 20 ng / ml.

[0055] Retinoic acid (RA) is a derivative of vitamin A that plays a role in the neural development of vertebrates and the differentiation of many non-neuronal cells. It is frequently used in research on inducing stem cell differentiation into neural cells. RA's functions include the formation of the neural plate and neural tube during early embryonic development, neuronal differentiation, and the regeneration of adult neural axons. Together with SHH, FGF, and BMP, it determines the pattern specialization of neurons in different regions of the neural tube and the fate and development of sensory neurons, interneurons, and motor neurons.

[0056] To promote the differentiation of neural progenitor cells into neurons, brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) were added to the neural culture medium in the fourth stage. The addition of Gibco CultureOne additive can eliminate more than 75% of the mixed neural progenitor cells, achieving excellent results in uniform distribution and complete differentiation of neurons, thereby improving downstream detection, accelerating neuronal maturation, and stabilizing the culture system.

[0057] Step 4: Transfer the cells to serum-free mesenchymal stem cell culture medium for adherent culture, and then expand them to obtain mesenchymal stem cells.

[0058] After day 21, the cell spheres were digested and cultured in serum-free mesenchymal stem cell culture medium to induce NCC-derived mesenchymal stem cells.

[0059] The PRIME-XV MSC medium and reagents used for culturing mesenchymal stem cells allow for the maximum expansion of mesenchymal stem cells and mesenchymal stromal cells derived from human adipose tissue, umbilical cord, and other cells in a serum-free cell culture system.

[0060] After day 25, mesenchymal stem cells were expanded and cultured, and neural crest organoid mesenchymal stem cells (NCC-MSCs) were obtained through multiple passage inductions.

[0061] To illustrate the effects of the present invention, MSCs derived from umbilical cord blood UB were induced under the same conditions and are referred to as UB-MSCs.

[0062] Figure 2 The morphology and test results of the mesenchymal stem cells obtained from the above embodiments and comparative examples (UB-MSCs) are as follows: Figure 2 As shown in the diagram. A is a morphological schematic of neural crest organoids; B is a flow cytometry analysis result of surface markers of mesenchymal stem cells (MSCs) derived from neural crest organoids and existing MSCs; C is a morphological schematic of primary MSCs induced after adhesion of MSCs derived from neural crest organoids and existing MSCs; D is a comparison of cell numbers on day 3 after induction and passage of MSCs derived from neural crest organoids and existing MSCs (the initial passage number for both was 1×10⁻⁶). 5 ).

[0063] from Figure 2 As can be seen from the results of cell counting on the third day, the number of NCC-MSCs cells was twice that of UB-MSCs, indicating that NCC-MSCs have a strong amplification capacity.

[0064] Figure 3This is a schematic diagram showing the protein differences between neural crest organoid mesenchymal stem cells and existing mesenchymal stem cells in this invention. A represents the statistical results of protein expression, and B represents the volcano diagram of the main differentially expressed proteins.

[0065] from Figure 3 As can be seen from the data, compared with umbilical cord blood-derived mesenchymal stem cells (UB-MSCs), proteomics data analysis shows that NCC-MSCs highly express 3557 proteins, upregulated 1910 proteins, and downregulated 1647 proteins. Proteins such as EPHA3, ADRA2A, PIK3R1, and SOX2, which are upregulated in NCC-MSCs compared to UB-MCSs, are related to maintaining pancreatic glucose homeostasis, insulin sensitivity, and pancreatic protection. C7, EIF1AY, and APOE are related to immune responses.

[0066] Figure 4 This is a schematic diagram showing the enrichment results of GO protein upregulated by neural crest organoid mesenchymal stem cells and existing mesenchymal stem cells in this invention. Figure 5 This is a schematic diagram showing the results of KEGG enrichment analysis of upregulated protein in neural crest organoid mesenchymal stem cells and existing mesenchymal stem cells in this invention.

[0067] As shown in the figure, the KEGG signaling pathway enrichment results indicate that NCC-MSCs have a stronger immunomodulatory effect. The upregulated genes are mainly enriched in the Hippo signaling pathway (related to inflammation signal regulation), the TGF-β signaling pathway (related to in vivo immune regulation efficacy), and the regulation of TRP channels by inflammatory mediators. Among them, the proteins EPHA3, ADRA2A, PIK3R1, and SOX2, which are upregulated by NCC-MSCs compared to UB-MCSs, are related to maintaining pancreatic glucose homeostasis, insulin sensitivity, and pancreatic protection. C7, EIF1AY, and APOE are related to the regulation of immune responses.

[0068] To verify the ameliorative and reparative effects of NCC-MSCs on inflammation, an acute pancreatitis (AP) mouse model was used to evaluate its therapeutic effects on acute pancreatitis. An AP mouse model was established, and MSCs were transplanted. The therapeutic efficacy of NCC-MSCs was assessed by observing pathological changes on day 15, measuring serum amylase and lipase levels, and comparing the mortality rate of AP mice. Results are as follows: Figure 6 As shown.

[0069] As can be seen from the figure, serum amylase and lipase levels decreased after mesenchymal stem cell transplantation in the mouse model. Compared with the UB-MSCs treatment group, the pancreatic pathological changes and serum amylase and lipase levels in the NCC-MSCs treatment group were significantly improved. Furthermore, the mortality rate of AP mice was significantly reduced after NCC-MSCs transplantation treatment.

[0070] This invention provides a stable and efficient method for obtaining neural crest organoids using small-molecule 3D-induced human induced pluripotent stem cells (ihPSCs), resulting in neural crest cells that more closely resemble human physiological conditions. Furthermore, 2D culture can effectively yield neural crest-derived mesenchymal stem cells (NCC-MSCs). The NCC-MSCs obtained by this invention exhibit stronger proliferation and expansion capabilities compared to existing UB-MSCs. Proteomic data shows that NCC-MSCs possess stronger immunomodulatory functions compared to UB-MSCs, demonstrating an upregulation trend in pancreatic glucose homeostasis and pancreatic protection-related proteins. Moreover, NCC-MSCs showed significant repair and improvement effects compared to UB-MSCs in an acute pancreatitis mouse model. The proteins from the obtained neural crest organoid-derived mesenchymal stem cells (NCC-MSCs) have greater value in immunomodulation.

Claims

1. An application of mesenchymal stem cells based on neural crest organoids, characterized in that, The application of the mesenchymal stem cells in the preparation of drugs for treating pancreatitis; The method for preparing mesenchymal stem cells includes the following steps: Step 1: Add human induced pluripotent stem cells to digestive fluid, digest them, and then dilute them with culture medium; Step 2: Collect cells, centrifuge the cells, discard the supernatant and suspend them in the culture medium, and adjust the cell block size to 50-80 micrometers; Step 3: Culture the cell clumps into embryoid bodies, and then induce differentiation of neural crest organoids; add neural differentiation basal medium on day 1 of embryoid body formation, replace with neural induction basal medium on days 2-7, and replace with neural differentiation basal medium on days 8-21. The basal culture media for neural differentiation included: DMEM / F12 medium 47% v / v, Gibco Neural Matrix™-A medium 47% v / v, N-2 additive 0.94% v / v, B-27 additive 1.9% v / v, GlutaMAX additive 0.94% v / v, penicillin-streptomycin mixture 0.94% v / v, MEM non-essential amino acids 0.94% v / v, 2-mercaptoethanol solution 0.094% v / v, and L-ascorbic acid solution 0.094% v / v; the concentration of 2-mercaptoethanol solution was 55 mM, and the concentration of L-ascorbic acid solution was 200 mM. The basal culture medium for neural differentiation on day 1 of the induced differentiation contained: 10 μM Y-27632, 10 μM SB-431542, 0.25 μM LDN193189, and 3 μM CHIR99021. The basal medium for neural induction included a TGF-β receptor kinase inhibitor, a BMP type I receptor inhibitor, a GSK-3α / β inhibitor, a CDK1 / cyclin B and GSK-3β inhibitor, FGF2 growth factor, and FGF8 growth factor; the concentrations of each component were as follows: TGF-β receptor kinase inhibitor 10 μM, BMP type I receptor inhibitor 0.25 μM, GSK-3α / β inhibitor 3 μM, CDK1 / cyclin B and GSK-3β inhibitor 2.5 μM, FGF2 growth factor 20 ng / ml, and FGF8 growth factor 25 ng / ml. The basal culture medium for neural differentiation during days 8–14 of induced differentiation included retinoic acid, a molecular compound, a smoothing agent agonist, and growth differentiation factor 11; the concentrations of each component were: retinoic acid 0.5 μM, molecular compound 0.5 μM, smoothing agent agonist 0.5 μM, and growth differentiation factor 11 20 ng / ml. The basal culture medium for neural differentiation during days 15–21 of induced differentiation included the γ-secretase inhibitor DAPT, CultureOne™ additive, compound E, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor; the concentrations of each component were: γ-secretase inhibitor DAPT 10 μM, CultureOne™ additive 1 μM, compound E 0.1 μM, brain-derived neurotrophic factor 20 ng / ml, and glial cell line-derived neurotrophic factor 20 ng / ml. The molecular compound is the SHH agonist PMF, and the smoothing agonist is the SHH agonist SAG; Step 4: After digesting and dispersing the neural crest organoids into a single-cell suspension, the suspension is transferred to a serum-free mesenchymal stem cell culture medium for adherent culture, and then expanded to obtain mesenchymal stem cells; the mesenchymal stem cells show upregulated expression of EPHA3, ADRA2A, PIK3R1, SOX2, C7, EIF1AY and APOE proteins.

2. The application of mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that, In step 1, after adding the digestive solution, digestion is carried out at 37 °C for 4 min.

3. The application of mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that, In step 2, a ROCK1 inhibitor is added to the culture medium at a concentration of 10 μM.

4. The application of mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that, The centrifugation conditions in step 2 are: 300 g, 15 min.

5. The application of mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that, In step 3, the cells are arranged at a density of 4 mL / 10 6 The samples were seeded at a density in low-adhesion plates and cultured in a bioreactor at 765 rpm for 24 hours.

6. The application of mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that, In step 4, amplification is performed after 4 days of adherent culture.

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