Preparation method and application of mesenchymal stem cells based on neural crest organs

Abstract

The invention discloses a preparation method and application of mesenchymal stem cells based on neural crest organics.The preparation method comprises the following steps that 1, human induced pluripotent stem cells are added into digestive juice, and after digestion, a culture medium is added for dilution; 2, collecting cells, centrifugally separating the cells, removing supernatant, suspending the cells in a culture medium, and adjusting the size of a cell block to 50-80 microns; 3, culturing the cells, and then carrying out induced differentiation on neural crest organs; 4, digesting the neural crest organs into single cells, transferring the single cells into a serum-free mesenchymal stem cell culture medium for adherent culture, and then performing amplification to obtain the mesenchymal stem cells. According to the invention, the induced pluripotent stem cells ihPSCs3D are induced and differentiated to obtain neural crest organs, the neural crest-derived mesenchymal stem cells are obtained, and the obtained NCC-MSCs have improvement and repair effects on pancreatitis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing mesenchymal stem cells based on neural crest organoids and applications thereof. Background Art

[0002] Mesenchymal stem cells (MSCs), as a type of cell with multidirectional differentiation potential and immunomodulatory properties, have shown great application prospects in the field of cell therapy in recent years. MSCs are widely sourced, including bone marrow, adipose tissue, umbilical cord blood and embryos. Although embryonic-derived MSCs have excellent repair ability, they are difficult to enter the clinic due to ethical restrictions. Umbilical cord blood-derived MSCs are relatively easy to obtain, have low immunogenicity, and have good tissue regeneration properties and immunomodulatory advantages, making them a hot topic in clinical research. However, there are limitations in the application of donor-derived MSCs, including differences between donors, limited cell expansion efforts, low differentiation potential, and inability to industrialize production, which limit their widespread application. Therefore, developing a method for a stable and high-quality MSCs supply is crucial for the application of MSCs.

[0003] Human induced pluripotent stem cells (hiPSCs) have the ability to self-renew and generate all types of cells in the human body through induction. Mesenchymal stem cells are differentiated from mesoderm cells or neural crest cells (NCCs). NCCs are a type of multipotent ectoderm cell that develops from the intermediate layer between the neural ectoderm and the epidermal ectoderm during vertebrate development; the technology of inducing neural crest from hiPSCs to obtain MSCs with similar immune phenotypes and biological functions is of great significance. However, to date, there has been no simple, efficient and stable method for preparing neural crest-derived mesenchymal stem cells and evaluating their clinical therapeutic applications. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for preparing mesenchymal stem cells based on neural crest organoids and applications.

[0005] The technical solution adopted by the present 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 digestion solution, and after digestion, add culture medium to dilute;

[0007] Step 2: Collect the cells, centrifuge the cells, discard the supernatant and suspend them in the culture medium, and adjust the size of the cell mass to a fine sandy turbidity;

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

[0009] Neural differentiation basal medium was added on day 1 of embryoid body formation, replaced with neural induction basal medium on days 2 to 7, and replaced with neural differentiation basal medium on days 8 to 21;

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

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

[0012] The neural differentiation basal culture medium includes: DMEM / F12 culture medium 47% v / v, Gibco neural matrix TM-A culture 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, L-ascorbic acid solution 0.094% v / v; the concentration of 2-mercaptoethanol solution is 55 mM, and the concentration of L-ascorbic acid solution is 200 mM.

[0013] Furthermore, the neural induction basal medium includes 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; wherein the concentrations of each component are: 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 neural differentiation basal medium for inducing differentiation on days 8 to 14 includes retinoic acid, molecular compound, smoothened agonist, and growth differentiation factor 11; wherein the concentrations of the components are: retinoic acid 0.5 μM, molecular compound 0.5 μM, smoothened agonist 0.5 μM, and growth differentiation factor 11 20 ng / ml; the neural differentiation basal medium for inducing differentiation on days 15 to 21 includes γ-secretase inhibitor, CultureOne TM Additives, compound E, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor; the concentrations of each component are: γ-secretase inhibitor 10μM, 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, digestion solution is added and digestion is performed 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 were cultured at a density of 4 mL / 10 6 The cells were seeded into low-adhesion plates at a density of 1.54 μg / mL and cultured in a bioreactor adapter at 765 rpm for 24 h.

[0019] Furthermore, in step 4, the cells are expanded 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 a drug for treating pancreatitis.

[0021] The beneficial effects of the present invention are:

[0022] (1) The present invention utilizes induced pluripotent stem cells (ihPSCs) to induce 3D differentiation to obtain neural crest organoids and obtain neural crest-derived mesenchymal stem cells;

[0023] (2) NCC-MSCs prepared by the preparation method of the present invention have stronger proliferation and expansion ability than existing MSCs, and proteomics data show that they have immunomodulatory effects;

[0024] (3) The NCC-MSCs obtained in the present invention have an improving and repairing effect on pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the process of the present invention.

[0026] Figure 2A schematic diagram of the characteristics of the neural crest organoids and the mesenchymal stem cells derived therefrom in the present invention, A is a schematic diagram of the morphology of the neural crest organoids, B is a schematic diagram of the flow cytometric analysis results of surface markers of mesenchymal stem cells derived from neural crest organoids and existing mesenchymal stem cells, C is a schematic diagram of the morphology of primary MSCs induced after neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells adhere to the wall, and D is a schematic diagram comparing the cell numbers of mesenchymal stem cells derived from neural crest organoids and existing mesenchymal stem cells on the third day after induced passage.

[0027] Figure 3 Schematic diagram of the protein differences between the neural crest organoid-derived mesenchymal stem cells of the present invention and existing mesenchymal stem cells, A is the statistical result of protein expression, and B is the volcano diagram of the main differential proteins.

[0028] Figure 4 Schematic diagram of GO enrichment analysis results of up-regulated proteins of neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells in the present invention.

[0029] Figure 5 Schematic diagram of KEGG enrichment analysis results of up-regulated proteins of neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells in the present invention.

[0030] Figure 6 It is a schematic diagram of the test results of the neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells in the AP mouse model, A is the pancreatic pathological HE staining result on the 15th day, B is a schematic diagram of the changes in serum amylase after treatment with the neural crest organoid-derived mesenchymal stem cells in the present invention, and C is a schematic diagram of the changes in serum lipase levels after treatment with the neural crest organoid-derived mesenchymal stem cells in the present invention. DETAILED DESCRIPTION

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

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

[0033] Step 1: Add human induced pluripotent stem cells to digestion solution, and after digestion, add culture medium to dilute;

[0034] The specific process is as follows:

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

[0036] After digestion, remove the culture dish immediately and add 2 mL of ncTarget culture medium to dilute and terminate digestion.

[0037] Step 2: Collect the cells, centrifuge the cells, discard the supernatant and suspend them in the culture medium, and adjust the size of the cell mass to a fine sandy turbidity;

[0038] The specific process is as follows:

[0039] Collect the cells by gently blowing, and try not to generate bubbles during the blowing process. Transfer all the cells to a 15 mL centrifuge tube and centrifuge at 300 g for 5 min.

[0040] After discarding the supernatant, the cells were suspended in the culture medium (ncTarget + 10 μL Y-27632), and the cell mass size was adjusted by blowing with a Pasteur pipette (to the required size, adjusted according to the actual situation) until it was turbid with fine sand and the diameter of the cell mass was 50-80 μm.

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

[0042] On day 1, neural differentiation basal medium was added, replaced with neural induction basal medium on days 2 to 7, and replaced with neural differentiation basal medium on days 8 to 21;

[0043] The specific process is as follows:

[0044] The cell density per well was 4 mL / 10 6 The cells were inoculated 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 embryoid bodies were formed, differentiation induction medium was added and the speed of the 6-well plate bioreactor adapter was adjusted to 80 rpm.

[0046] Neural differentiation basal medium was added on the first day after embryoid body formation, and this stage was the first stage;

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

[0048] The neural differentiation basal medium includes: DMEM / F12 medium 47% v / v, Gibco NeurobasalTM-A Medium 47% v / v, N-2 supplement (100×N-2supplement) 0.94% v / v, B-27 supplement (50×B27 supplement) 1.9% v / v, GlutaMAX supplement (100×GlutaMAXsupplement) 0.94% v / v, penicillin-streptomycin 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 the 2-mercaptoethanol solution is 55 mM, and the concentration of the L-ascorbic acid solution is 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 respectively act on ALK2 and ALK3 in the BMP4 pathway, and ALK5 in the TGFBETA pathway to achieve the formation of transplanted endoderm and mesoderm, thereby inducing ectoderm development and neurogenesis. By inhibiting the BMP and TGFBETA pathways to simulate the signal pathways in the early embryonic development, the generation of neural stem cells is induced. In this way, induced neuronal cells can be obtained in the presence of serum-like components (Knockout Serum Replacement).

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

[0051] The neural induction basal medium includes 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 are: TGF-β receptor kinase inhibitor 10μM, BMP type I receptor inhibitor 0.25μM, GSK-3α / β inhibitor 3μM, CDK1 / cyclin B and GSK-3β inhibitor 2.5μM, FGF2 growth factor 20ng / ml, and FGF8 growth factor 25ng / 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. In addition, fibroblast growth factor 8 (FGF8) is a member of the fibroblast growth factor family and an important secretory regulatory signal molecule in the process of tissue development. Research results in recent years have shown that FGF8 plays an important role in the occurrence and development of the nervous system. FGF8 can change the fate of the midbrain to the cerebellum and promote the development of the midbrain.

[0053] From day 8 to day 21, the medium is replaced with neural differentiation basal medium. During this stage, from day 8 to day 14 is the third stage, and from day 15 to day 21 is the fourth stage:

[0054] The neural differentiation basal medium for inducing differentiation on the 8th to 14th day includes retinoic acid, molecular compound, smoothened agonist, and growth differentiation factor 11; wherein the concentrations of the components are: retinoic acid 0.5 μM, molecular compound 0.5 μM, smoothened agonist 0.5 μM, and growth differentiation factor 11 20 ng / ml; the neural differentiation basal medium for inducing differentiation on the 15th to 21st day includes γ-secretase inhibitor DAPT, CultureOne TM Additives, compound E, brain-derived neurotrophic factor BDNF, glial cell line-derived neurotrophic factor GDNF; the concentrations of each component are: γ-secretase inhibitor 10μM, 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. It has effects on the neural development of vertebrates and the differentiation of many non-neural cells. It is often used in the study of inducing differentiation of stem cells into neural cells. The effects of RA include the formation of neural plate and neural tube during early embryonic development, the differentiation of neurons 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 determination and occurrence of sensory neurons, interneurons and motor neurons.

[0056] In order to promote the differentiation of neural progenitor cells into neurons, brain-derived neurotrophic factor BDNF and glial cell-derived neurotrophic factor GDNF are 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 to achieve the excellent effect of uniform distribution and complete differentiation of neuronal cells, thereby improving downstream detection, accelerating neuronal maturation, and stably maintaining the culture system.

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

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

[0059] PRIME-XV MSC Medium and Reagents used for the culture of mesenchymal stem cells allow for the limited expansion of mesenchymal stem cells and mesenchymal stromal cells from human adipose tissue, umbilical cord, and other cell sources in a serum-free cell culture system.

[0060] After the 25th day, the mesenchymal stem cells were expanded and cultured, and neural crest organoid-derived mesenchymal stem cells (NCC-MSCs) were obtained through multiple passages and induction.

[0061] In order to illustrate the effect of the present invention, MSCs derived from umbilical cord blood UB were induced under the same conditions and were recorded as UB-MSCs.

[0062] Figure 2 The morphology and test results of the mesenchymal stem cells obtained in the above examples and comparative examples (UB-MSCs) are as follows: Figure 2 A is a schematic diagram of the morphology of neural crest organoids, B is a schematic diagram of the flow cytometric analysis results of surface markers of neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells, C is a schematic diagram of the morphology of primary MSCs induced by neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells after attachment, and D is a schematic diagram of the comparison of the number of cells on the third day after induced passage of neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells (the starting number of passage is 1×10 5 ).

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

[0064] Figure 3Schematic diagram of the protein differences between the neural crest organoid-derived mesenchymal stem cells of the present invention and existing mesenchymal stem cells, A is the statistical result of protein expression, and B is the volcano diagram of the main differential proteins.

[0065] from Figure 3 It can be seen that compared with umbilical cord blood-derived mesenchymal stem cells (UB-MSCs), proteomic data analysis showed that NCC-MSCs highly expressed 3557 proteins, upregulated 1910 proteins, and downregulated 1647 proteins relative to UB-MCS. Proteins such as EPHA3, ADRA2A, PIK3R1, and SOX2 that were upregulated in NCC-MSCs relative to UB-MCSs are related to maintaining pancreatic glucose homeostasis, insulin sensitivity, and protecting the pancreas. C7, EIF1AY, and APOE are related to immune response.

[0066] Figure 4 Schematic diagram of GO enrichment analysis results of up-regulated proteins of neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells in the present invention. Figure 5 Schematic diagram of KEGG enrichment analysis results of up-regulated proteins of neural crest organoid-derived mesenchymal stem cells and existing mesenchymal stem cells in the present invention.

[0067] As can be seen from the figure, the KEGG signaling pathway enrichment results show that NCC-MSCs have stronger immunoregulatory effects, and the upregulated genes are mainly enriched in the Hippo signaling pathway (related to inflammatory signal regulation), TGF-B signaling pathway (related to the immune regulation effect in vivo), and the regulation of TRP channels by inflammatory mediators. Among them, the upregulated expression of EPHA3, ADRA2A, PIK3R1, SOX2 and other proteins in NCC-MSCs relative to UB-MCSs are related to maintaining pancreatic glucose homeostasis, insulin sensitivity and protecting the pancreas, and C7, EIF1AY and APOE are related to immune response regulation.

[0068] In order to verify the improvement and repair effect of NCC-MSCs on inflammation, the AP mouse model was used to evaluate the repair and improvement effect of NCC-MSCs on acute pancreatitis. The AP mouse model was established and MSCs transplantation was performed. The therapeutic effect of NCC-MSCs was evaluated by observing pathological changes on the 15th day, measuring serum amylase and lipase levels, and comparing the mortality rate of AP mice. Figure 6 shown.

[0069] As can be seen from the figure, serum amylase and lipase levels decreased in the mouse model after mesenchymal stem cell transplantation. 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, and the mortality rate of AP mice was significantly reduced after NCC-MSCs transplantation.

[0070] The present invention stably and efficiently utilizes small molecule 3D to induce human induced pluripotent stem cells (ihPSCs) to obtain neural crest organoids, and obtains neural crest cells that are closer to the real physiological conditions of humans. And neural crest-derived mesenchymal stem cells (NCC-MSCs) can be effectively obtained through 2D culture. The NCC-MSCs obtained by the present invention have stronger proliferation and expansion capabilities than the existing UB-MSCs; its proteomics data show that NCC-MSCs have stronger immunomodulatory effects in terms of function compared with UB-MSCs, and show an upregulation trend in pancreatic glucose homeostasis and pancreatic protection-related proteins. And NCC-MSCs show significant repair and improvement effects compared to UB-MSCs in an acute pancreatitis mouse model. The obtained neural crest organoid-derived mesenchymal stem cell (NCC-MSCs) protein has greater value in immunomodulation.

Claims

1. A method for preparing mesenchymal stem cells based on neural crest organoids, characterized in that: The following steps are involved: Step 1: Add human induced pluripotent stem cells to digestion solution, and after digestion, add culture medium to dilute; Step 2: Collect the cells, centrifuge the cells, discard the supernatant and suspend them in culture medium, and adjust the size of the cell mass to 50-80 microns; Step 3: Culture the cell clumps into embryoid bodies and then induce differentiation into neural crest organoids; Neural differentiation basal medium was added on day 1 of embryoid body formation, replaced with neural induction basal medium on days 2 to 7, and replaced with neural differentiation basal medium on days 8 to 21; Step 4: After the neural crest organoids are digested and dispersed into a single-cell suspension, they are transferred to a serum-free mesenchymal stem cell culture medium for adherent culture, and then expanded to obtain mesenchymal stem cells.

2. The method for preparing mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that: The neural differentiation basal medium contains: 10 μM Y-27632, 10 μM SB-431542, 0.25 μM LDN193189, and 3 μM CHIR99021; The neural differentiation basal culture medium includes: DMEM / F12 culture medium 47% v / v, Gibco neural matrix TM-A culture 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, L-ascorbic acid solution 0.094% v / v; the concentration of 2-mercaptoethanol solution is 55 mM, and the concentration of L-ascorbic acid solution is 200 mM.

3. The method for preparing mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that: The neural induction basal culture 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 the components are: 10 μM TGF-β receptor kinase inhibitor, 0.25 μM BMP type I receptor inhibitor, 3 μM GSK-3α / β inhibitor, 2.5 μM CDK1 / cyclin B and GSK-3β inhibitor, 20 ng / ml FGF2 growth factor, and 25 ng / ml FGF8 growth factor.

4. The method for preparing mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that: The neural differentiation basal medium for inducing differentiation on the 8th to 14th day includes retinoic acid, molecular compound, smoothened agonist, and growth differentiation factor 11; wherein the concentrations of the components are: retinoic acid 0.5 μM, molecular compound 0.5 μM, smoothened agonist 0.5 μM, and growth differentiation factor 11 20 ng / ml; the neural differentiation basal medium for inducing differentiation on the 15th to 21st day includes γ-secretase inhibitor, CultureOne TM Additives, compound E, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor; the concentrations of each component are: γ-secretase inhibitor 10μM, 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.

5. The method for preparing mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that: After adding the digestion solution in step 1, digestion was carried out at 37° C. for 4 minutes.

6. The method for preparing mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that: In the 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.

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

8. The method for preparing mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that: In step 3, the cells were cultured at a density of 4 mL / 10 6 The cells were seeded into low-adhesion plates at a density of 1.54 μg / mL and cultured in a bioreactor adapter at 765 rpm for 24 h.

9. The method for preparing mesenchymal stem cells based on neural crest organoids according to claim 1, characterized in that: In the step 4, the cells are expanded after 4 days of adherent culture.

10. Use of mesenchymal stem cells obtained by any one of the preparation methods of claims 1 to 9, characterized in that: The mesenchymal stem cells are used for preparing medicine for treating pancreatitis.

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