Intrahepatic cholangiocarcinoma organoid-specific culture medium supplemented with p38 mapk inhibitor and ep2 / ep4 receptor agonist and standardized culture method

Abstract

The application provides an intrahepatic cholangiocarcinoma organoid special culture solution and a standardized culture method by adding a p38 MAPK inhibitor and an EP2 / EP4 receptor agonist. The p38 MAPK inhibitor and the EP2 / EP4 receptor agonist are added to specifically inhibit the p38 MAPK signal pathway, effectively relieve cell inflammatory stress, inhibit cell apoptosis and enhance the long-term culture stability of cells. At the same time, the EP2 / EP4 receptor promotes the proliferation of cholangiocytes, inhibits the excessive growth of fibroblasts, improves the purity of tumor organoids and enhances the antibacterial defense ability of cells. Finally, the success rate of iCCA organoid establishment using the culture solution and the standardized culture method of the application can reach more than 85%, which is much higher than the 50%-60% of the traditional culture medium. The organoids can be stably passaged for more than 40 generations, and the histological structure and genomic characteristics of the original tumor are maintained during the passage process, thereby providing an ideal in-vitro model for tumor research.

Description

Technical Field

[0001] This invention relates to the medical field, and more particularly to a culture medium for intrahepatic cholangiocarcinoma organoids containing p38 MAPK inhibitors and EP2 / EP4 receptor agonists, and a standardized culture method thereof. Background Technology

[0002] Intrahepatic cholangiocarcinoma (iCCA) is a malignant tumor originating from the epithelial cells of the intrahepatic bile ducts. Its incidence is increasing year by year, and its prognosis is extremely poor. Due to the high heterogeneity and complexity of iCCA, traditional 2D cell line models cannot accurately simulate the biological characteristics of tumors in vivo, resulting in slow progress in drug development and preclinical research.

[0003] Organoid technology is a three-dimensional in vitro culture technique that has developed in recent years. It can simulate the three-dimensional structure and physiological function of organs in vitro, providing an ideal in vitro model for tumor research. However, current iCCA organoid culture technology still has many shortcomings:

[0004] 1. Low success rate of cell line establishment: Traditional commercial culture media have limited support for primary tumor cells, and the success rate of cell line establishment is usually less than 50%, which is difficult to meet the research needs of clinical samples.

[0005] 2. Phenotypic drift during long-term passage: Tumor cells are prone to phenotypic drift during long-term passage, losing the subtype characteristics and drug sensitivity of the original tumor.

[0006] 3. Severe culture stress damage: Traditional commercial culture media often lead to cellular inflammatory stress, oxidative damage, hypoxia and necrosis, making organoids prone to dispersion and collapse, and resulting in poor long-term culture stability.

[0007] 4. Poor experimental reproducibility: The existing culture system lacks standardization, and the experimental results between different laboratories vary greatly, making it difficult to promote and apply.

[0008] Therefore, developing a high-efficiency, stable, and standardized culture medium specifically for iCCA organoids is of great significance for promoting basic research and clinical translation of iCCA. Summary of the Invention

[0009] To address the aforementioned problems, this invention aims to provide a specific culture medium for intrahepatic cholangiocarcinoma organoids containing p38 MAPK inhibitors and EP2 / EP4 receptor agonists, along with a standardized culture method, to solve the following technical issues:

[0010] 1. Improve the success rate of organoid establishment and solve the problem of difficulty in in vitro expansion of primary tumor cells.

[0011] 2. To prolong the stable passage time of organoids and maintain the histological, genomic, and drug response characteristics of their original tumors.

[0012] 3. Enhance the long-term culture stability of organoids and support continuous passage.

[0013] 4. Alleviate cellular stress damage during in vitro culture, including inflammatory stress, oxidative damage, and hypoxic necrosis, and improve the structural stability of organoids.

[0014] 5. Establish a standardized culture system to improve the reproducibility of experiments.

[0015] This invention provides a culture medium specifically for intrahepatic cholangiocarcinoma organoids containing p38 MAPK inhibitors and EP2 / EP4 receptor agonists, comprising the following components:

[0016] The basal culture medium and growth factors and small molecule additives; wherein, the basal culture medium is a basal culture medium suitable for epithelial cell culture; the growth factors and small molecule additives contain at least p38 MAPK inhibitors and EP2 / EP4 receptor agonists.

[0017] Furthermore, the p38 MAPK inhibitor is SB202190, SB203580, etc.

[0018] Furthermore, the EP2 / EP4 receptor agonist is PGE2 (prostaglandin E2), Butaprost, etc.

[0019] Furthermore, the basal medium is DMEM / F12 basal medium or Advanced DMEM / F12 basal medium.

[0020] Furthermore, it also includes:

[0021] 1× B27 serum-free additive;

[0022] 1× N2 additive;

[0023] 1× GlutaMAX Glutamine Supplement;

[0024] 1× Penicillin-Streptomycin Bivalent Antibody;

[0025] 10 mM HEPES buffer;

[0026] 1 mM N-acetylcysteine;

[0027] Furthermore, the growth factors and small molecule additives also include:

[0028] EGF (epidermal growth factor): 40-60 ng / mL

[0029] FGF10 (fibroblast growth factor 10): 80-120 ng / mL

[0030] rhHGF (recombinant human hepatocyte growth factor): 20-30 ng / mL

[0031] R-spondin1 (R-spondin 1): 50-200 ng / mL

[0032] Noggin: 50-200 ng / mL

[0033] Wnt3a (Wnt family 3A): 50-150 ng / mL

[0034] Nicotinamide: 5-15 mM

[0035] Forskolin (adenylate cyclase activator): 5-20 μM

[0036] Gastrin I (CCK2 agonist): 5-20 nM

[0037] A83-01 (TGFβ inhibitor): 2-10 μM

[0038] Furthermore, SB202190 (p38 MAPK inhibitor): 5-20 μM

[0039] PGE2 (prostaglandin E2): 0.5-2 μM.

[0040] Compared with traditional iCCA organoid culture media, the optimized culture medium of this invention has the following key improvements:

[0041] (1) Adding SB202190: specifically inhibits the p38 MAPK signaling pathway, effectively alleviates cellular inflammatory stress, inhibits cell apoptosis, and enhances the long-term culture stability of cells.

[0042] (2) Adding PGE2: It promotes the proliferation of bile duct epithelial cells through EP2 / EP4 receptors, while inhibiting the excessive growth of fibroblasts, improving the purity of tumor organoids, and enhancing the antibacterial defense capabilities of cells.

[0043] This invention also provides a standardized culture method for culturing iCCA organoids using the above-mentioned culture medium, comprising the following steps:

[0044] (1) Tissue sample processing: The surgically removed iCCA tumor tissue was cut into 1 mm³ pieces and digested with a collagenase / hyaluronidase mixture for 30 minutes.

[0045] (2) Cell separation: The digested cell suspension was passed through a 70μm cell sieve to remove undigested tissue blocks, and the cells were collected by centrifugation.

[0046] (3) Matrix gel embedding: Mix cells with matrix gel at a ratio of 1:3 and seed them into 24-well plates, 50 μL per well.

[0047] (4) Adding culture medium: After the matrix gel solidifies, add the above optimized culture medium and place it in a 37℃, 5% CO2 incubator for incubation.

[0048] (5) Subculture: Subculture every 7-10 days. During subculture, organoids are digested into single cells using TrypLE digestive enzymes and then re-embedded in matrix gel.

[0049] The technical solution of the present invention has the following beneficial effects:

[0050] (1) High success rate of organoid establishment: Using the culture medium of the present invention, the success rate of iCCA organoid establishment can reach more than 85%, which is much higher than the 50%-60% of traditional culture medium.

[0051] (2) Good long-term culture stability: Organoids can be stably passaged for more than 40 generations, maintaining the histological structure and genomic characteristics of the original tumor during passage.

[0052] (3) Complete three-dimensional structure: The organoids can form a typical three-dimensional sac-like structure with complete cell polarity and normal tight connections, which simulates the physiological structure of the bile duct in the body.

[0053] (4) Strong applicability to drug screening: This culture system can be used for high-throughput drug screening, and the drug response of organoids is highly consistent with the treatment response of clinical patients.

[0054] (5) Low risk of contamination: The optimized culture medium of this invention adds antibacterial components, which significantly reduces the risk of bacterial contamination during long-term culture.

[0055] (6) Clear mechanism: Through transcriptomics analysis, the mechanism of action of the optimized culture medium of the present invention was systematically elucidated, providing a theoretical basis for subsequent culture medium optimization. Attached Figure Description

[0056] Figure 1 Bright-field morphology images of 5th and 20th generation iCCA organoids in commercial culture medium (NC) and the optimized culture medium of this invention (PS): showing the typical morphology of iCCA organoids.

[0057] Figure 2This is a differential gene volcano plot, showing the distribution of differentially regulated genes between commercial culture medium (NC) and the optimized culture medium (PS) of this invention; in the figure, red dots represent significantly upregulated genes, blue dots represent significantly downregulated genes, and gray dots represent genes with no significant differences.

[0058] Figure 3 This is a heatmap of the top 100 differentially expressed genes, showing the expression patterns of differentially expressed genes and the clustering of samples; in the figure, red indicates high expression and blue indicates low expression.

[0059] Figure 4 This is a bubble chart of upregulated genes enriched through the GO biological process, showing the functional enrichment results of upregulated genes; in the chart, the size of the bubble represents the number of enriched genes, and the color represents the significance of the P-value.

[0060] Figure 5 This is a bar chart showing the enrichment results of the KEGG pathway for downregulated genes. In the chart, the horizontal axis represents the significance of enrichment (-log10 (P)) and the vertical axis represents the enriched pathway.

[0061] Figure 6 This is a Hallmark core pathway GSEA enrichment result graph, showing the GSEA enrichment results of the core pathway; in the graph, NES represents the standardized enrichment score, and the color represents the significance of the P-value. Detailed Implementation

[0062] The present invention will be further described in detail below with reference to specific embodiments.

[0063] Example 1: Establishment and Culture of Organoids

[0064] Surgically resected tumor tissue was collected from 15 patients with iCCA, and all patients signed informed consent forms.

[0065] Organoid culture was performed using the optimized culture medium (PS) of this invention, following standardized culture methods; simultaneously, parallel culture was conducted using a conventional commercial culture medium (NC) as a control. The standard culture method is as follows:

[0066] (1) Tissue sample processing: The surgically removed iCCA tumor tissue was cut into 1 mm³ pieces and digested with a collagenase / hyaluronidase mixture for 30 minutes.

[0067] (2) Cell separation: The digested cell suspension was passed through a 70μm cell sieve to remove undigested tissue blocks, and the cells were collected by centrifugation.

[0068] (3) Matrix gel embedding: Mix cells with matrix gel at a ratio of 1:3 and seed them into 24-well plates, 50 μL per well.

[0069] (4) Adding culture medium: After the matrix gel has solidified, add the culture medium and incubate in a 37°C, 5% CO2 incubator. The optimized culture medium (abbreviated as PS) of this invention includes the following components:

[0070] Advanced DMEM / F12 basal culture medium;

[0071] 1× B27 serum-free additive;

[0072] 1× N2 additive;

[0073] 1× GlutaMAX Glutamine Supplement;

[0074] 1× Penicillin-Streptomycin Bivalent Antibody;

[0075] 10 mM HEPES buffer;

[0076] 1 mM N-acetylcysteine;

[0077] EGF (epidermal growth factor): 50 ng / mL

[0078] FGF10 (fibroblast growth factor 10): 100 ng / mL

[0079] rhHGF (recombinant human hepatocyte growth factor): 25 ng / mL

[0080] R-spondin1 (R-spondin 1): 100 ng / mL

[0081] Noggin: 100 ng / mL

[0082] Wnt3a (Wnt family 3A): 100 ng / mL

[0083] Nicotinamide: 10 mM

[0084] Forskolin (adenylate cyclase activator): 10 μM

[0085] Gastrin I (CCK2 agonist): 10 nM

[0086] A83-01 (TGFβ inhibitor): 5 μM

[0087] SB202190 (p38 MAPK inhibitor): 10 μM

[0088] PGE2 (prostaglandin E2): 1 μM;

[0089] Commercial culture media (abbreviated as NC) components include:

[0090] Advanced DMEM / F12 basal culture medium;

[0091] 1× B27 serum-free additive;

[0092] 1× N2 additive;

[0093] 1× GlutaMAX Glutamine Supplement;

[0094] 1× Penicillin-Streptomycin Bivalent Antibody;

[0095] 10 mM HEPES buffer;

[0096] 1 mM N-acetylcysteine;

[0097] EGF (epidermal growth factor): 50 ng / mL

[0098] FGF10 (fibroblast growth factor 10): 100 ng / mL

[0099] rhHGF (recombinant human hepatocyte growth factor): 25 ng / mL

[0100] R-spondin1 (R-spondin 1): 100 ng / mL

[0101] Noggin: 100 ng / mL

[0102] Wnt3a (Wnt family 3A): 100 ng / mL

[0103] Nicotinamide: 10 mM

[0104] Forskolin (adenylate cyclase activator): 10 μM

[0105] Gastrin I (CCK2 agonist): 10 nM

[0106] A83-01 (TGFβ inhibitor): 5 μM

[0107] (5) Subculture: Subculture every 7-10 days. During subculture, organoids are digested into single cells using TrypLE digestive enzymes and then re-embedded in matrix gel.

[0108] The establishment success rate and passage stability of the two groups were statistically analyzed, and the results are as follows:

[0109] In the culture medium group of this invention, organoids were successfully established in 13 out of 15 samples, with a success rate of 86.7%; in the control group, organoids were successfully established in 8 out of 15 samples, with a success rate of 53.3%.

[0110] The organoids in the culture medium of this invention can be stably passaged for more than 40 generations, while the organoids in the control group usually experience growth arrest or phenotypic drift after about 20 passages. Figure 1 As shown.

[0111] Example 2: Mechanism verification at the transcriptome level

[0112] To systematically elucidate the mechanism of action of the optimized culture medium of this invention, transcriptome sequencing analysis was performed on organoids cultured in optimized medium (PS) and commercial conventional medium (NC). A total of 16,629 protein-coding genes were detected. Based on the criteria of |log2FoldChange|≥1 and pvalue<0.05, 1,350 significantly differentially expressed genes were identified, among which:

[0113] The optimized culture medium of this invention significantly upregulated 480 genes; the optimized culture medium of this invention significantly downregulated 870 genes; the overall distribution of differentially expressed genes is as follows. Figure 2 As shown in the figure. The clustering heatmap of the top 100 differentially expressed genes is as follows. Figure 3 As shown, the two groups of samples can be clearly separated, indicating that the optimized culture medium of the present invention can induce stable transcriptome changes.

[0114] Furthermore, GO functional enrichment and KEGG pathway enrichment analyses were performed on the differentially expressed genes, and the results showed that:

[0115] The functions of upregulated genes are enriched in:

[0116] Extracellular matrix tissue (P<0.001)

[0117] Redox process (P<0.01)

[0118] Cellular detoxification (P<0.01)

[0119] Ion transmembrane transport (P<0.001)

[0120] Antimicrobial humoral immune response (P<0.05)

[0121] The enrichment results of the GO biological process of upregulated genes are as follows: Figure 4 As shown.

[0122] The pathways that downregulate genes are enriched in:

[0123] Inflammatory response (P<1e-10)

[0124] Interferon-α response (P=7.65e-33)

[0125] Interferon-γ response (P=6.22e-35)

[0126] IL-17 signaling pathway (P<0.01)

[0127] NF-κB signaling pathway (P<0.01)

[0128] Cancer pathway (P<0.01)

[0129] KEGG pathway enrichment results for downregulated genes are as follows: Figure 5 As shown.

[0130] Further enrichment analysis of the GSEA gene set showed that the optimized culture medium of this invention can significantly regulate multiple core biological pathways:

[0131] EMT pathway: NES=1.89, P=0.00035, Adjusted P=0.0148, indicating that the optimized culture medium of the present invention can enhance extracellular matrix synthesis and stabilize the three-dimensional structure of organoids.

[0132] Inflammation and immune pathways: IFN-α, IFN-γ, inflammatory response, IL6 / JAK / STAT3 and other pathways were significantly inhibited, indicating that the optimized culture medium of the present invention can globally inhibit inflammatory stress.

[0133] Redox pathway: The glutathione metabolic pathway was significantly activated, indicating that the optimized culture medium of this invention can enhance the antioxidant capacity of cells.

[0134] Apoptosis pathway: The apoptosis pathway was significantly negatively regulated, indicating that the optimized culture medium of this invention can inhibit cell apoptosis and improve cell survival rate.

[0135] Tumor stem pathways: When tumor stem-related pathways such as KRAS and MYC are inhibited, the differences in proliferation and morphology among organoid populations are reduced, the intra-group replicate bias is reduced, and the experimental reproducibility is better.

[0136] GSEA enrichment results of the core pathway are as follows Figure 6 As shown.

[0137] These results systematically elucidate the mechanism of action of the optimized culture medium of this invention at the transcriptome level, demonstrating that it can effectively improve the culture microenvironment of organoids and enhance culture efficiency and stability through the synergistic regulation of multiple pathways.

[0138] This invention is the first to combine SB202190 and PGE2 in the classic cholangiocarcinoma organoid culture medium, which significantly improves the success rate of organoid establishment and long-term culture stability. At the same time, it verifies the systemic inhibitory effect of these two stress pathways on inflammation, oxidation and hypoxia at the transcriptome level.

[0139] For the first time, the mechanism of action of the optimized culture medium of this invention was elucidated through transcriptomics analysis, demonstrating that it can globally inhibit inflammatory stress, enhance antioxidant capacity, and optimize energy metabolism.

[0140] This invention establishes an efficient method for iCCA organoid culture, with a success rate of over 85%, far exceeding the approximately 50% of traditional culture media.

[0141] This culture system can be used for drug sensitivity testing, providing a reliable in vitro model for individualized clinical treatment.

[0142] Example 3:

[0143] This invention provides a culture medium specifically for intrahepatic cholangiocarcinoma organoids supplemented with PGE2 and SB202190, the components of which include:

[0144] Advanced DMEM / F12 basal culture medium;

[0145] 1× B27 serum-free additive;

[0146] 1× N2 additive;

[0147] 1× GlutaMAX Glutamine Supplement;

[0148] 1× Penicillin-Streptomycin Bivalent Antibody;

[0149] 10 mM HEPES buffer;

[0150] 1 mM N-acetylcysteine;

[0151] EGF (epidermal growth factor): 40 ng / mL

[0152] FGF10 (fibroblast growth factor 10): 120 ng / mL

[0153] rhHGF (recombinant human hepatocyte growth factor): 20 ng / mL

[0154] R-spondin1 (R-spondin 1): 50 ng / mL

[0155] Noggin: 50 ng / mL

[0156] Wnt3a (Wnt family 3A): 150 ng / mL

[0157] Nicotinamide: 15 mM

[0158] Forskolin (adenylate cyclase activator): 20 μM

[0159] Gastrin I (CCK2 agonist): 20 nM

[0160] A83-01 (TGFβ inhibitor): 10 μM

[0161] SB203580 (p38 MAPK inhibitor): 20 μM

[0162] Butaprost: 0.5 μM;

[0163] Experiments showed that this culture medium can specifically inhibit the p38 MAPK signaling pathway, effectively alleviating cellular inflammatory stress, inhibiting apoptosis, and enhancing the long-term culture stability of cells. Simultaneously, it promotes bile duct epithelial cell proliferation through EP2 / EP4 receptors, inhibits excessive fibroblast growth, improves the purity of tumor organoids, and enhances the cells' antibacterial defense capabilities. Ultimately, the success rate of iCCA organoid establishment reached over 85%, far exceeding the 50%-60% of traditional culture media. The organoids can be stably passaged for over 40 generations, maintaining the histological structure and genomic characteristics of the original tumor during passage.

[0164] Example 4:

[0165] This invention provides a culture medium specifically for intrahepatic cholangiocarcinoma organoids supplemented with PGE2 and SB202190, the components of which include:

[0166] Advanced DMEM / F12 basal culture medium;

[0167] 1× B27 serum-free additive;

[0168] 1× N2 additive;

[0169] 1× GlutaMAX Glutamine Supplement;

[0170] 1× Penicillin-Streptomycin Bivalent Antibody;

[0171] 10 mM HEPES buffer;

[0172] 1 mM N-acetylcysteine;

[0173] EGF (epidermal growth factor): 60 ng / mL

[0174] FGF10 (fibroblast growth factor 10): 80 ng / mL

[0175] rhHGF (recombinant human hepatocyte growth factor): 30 ng / mL

[0176] R-spondin1 (R-spondin 1): 200 ng / mL

[0177] Noggin: 200 ng / mL

[0178] Wnt3a (Wnt family 3A): 50 ng / mL

[0179] Nicotinamide: 5 mM

[0180] Forskolin (adenylate cyclase activator): 5 μM

[0181] Gastrin I (CCK2 agonist): 5 nM

[0182] A83-01 (TGFβ inhibitor): 2 μM

[0183] SB202190 (p38 MAPK inhibitor): 5 μM

[0184] PGE2 (prostaglandin E2): 2 μM;

[0185] Experiments showed that this culture medium can specifically inhibit the p38 MAPK signaling pathway, effectively alleviating cellular inflammatory stress, inhibiting apoptosis, and enhancing the long-term culture stability of cells. Simultaneously, it promotes bile duct epithelial cell proliferation through EP2 / EP4 receptors, inhibits excessive fibroblast growth, improves the purity of tumor organoids, and enhances the cells' antibacterial defense capabilities. Ultimately, the success rate of iCCA organoid establishment reached over 85%, far exceeding the 50%-60% of traditional culture media. The organoids can be stably passaged for over 40 generations, maintaining the histological structure and genomic characteristics of the original tumor during passage.

[0186] The preferred embodiments of the present invention disclosed above are merely for illustrating the present invention, and the present invention is not limited thereto. Those skilled in the art will understand that, within the scope of the present invention's concept, modifications can be made to the technical solutions of the present invention, or some technical features can be combined in any other way. Such modifications or combinations do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the various embodiments of the present invention, and should be considered as the content disclosed in the present invention, all of which fall within the protection scope of the present invention.

Claims

1. A culture medium specifically for intrahepatic cholangiocarcinoma organoids supplemented with p38 MAPK inhibitors and EP2 / EP4 receptor agonists, characterized in that, Include: The basal culture medium and growth factors and small molecule additives; wherein, the basal culture medium is a basal culture medium suitable for epithelial cell culture; the growth factors and small molecule additives contain at least p38 MAPK inhibitors and EP2 / EP4 receptor agonists.

2. The culture medium according to claim 1, characterized in that, The p38 MAPK inhibitors are SB202190 and SB203580.

3. The culture medium according to claim 1, characterized in that, The EP2 / EP4 receptor agonists are PGE2 and Butaprost.

4. The culture medium according to claim 1, characterized in that, The basal medium is either DMEM / F12 basal medium or Advanced DMEM / F12 basal medium.

5. The culture medium according to claim 1, characterized in that, Also includes: 1× B27 serum-free additive; 1× N2 additive; 1× GlutaMAX Glutamine Supplement; 1× Penicillin-Streptomycin Bivalent Antibody; 10 mM HEPES buffer; 1 mM N-acetylcysteine.

6. The culture medium according to claim 1, characterized in that, Also includes: EGF (epidermal growth factor): 40-60 ng / mL FGF10 (fibroblast growth factor 10): 80-120 ng / mL rhHGF (recombinant human hepatocyte growth factor): 20-30 ng / mL R-spondin1 (R-spondin 1): 50-200 ng / mL Noggin: 50-200 ng / mL Wnt3a (Wnt family 3A): 50-150 ng / mL Nicotinamide: 5-15 mM Forskolin (adenylate cyclase activator): 5-20 μM Gastrin I (CCK2 agonist): 5-20 nM A83-01 (TGFβ inhibitor): 2-10 μM.

7. The culture medium according to claim 1, characterized in that, The p38 MAPK inhibitor concentration was 5-20 μM; the EP2 / EP4 receptor agonist concentration was 0.5-2 μM.

8. A standardized culture method for culturing iCCA organoids using the intrahepatic cholangiocarcinoma organoid culture medium containing p38 MAPK inhibitors and EP2 / EP4 receptor agonists as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) Cut the surgically removed iCCA tumor tissue into small pieces and digest it with a collagenase / hyaluronidase mixture; (2) After digestion, the cell suspension is passed through a cell sieve to remove undigested tissue blocks, and the cells are collected by centrifugation; (3) Mix the cells with the matrix gel and seed them into well plates; (4) After the matrix gel solidifies, add the intrahepatic cholangiocarcinoma organoid culture medium containing p38 MAPK inhibitor and EP2 / EP4 receptor agonist as described in any one of claims 1-7, and place it in an incubator for culture; (5) Passage every 7-10 days. During passage, use TrypLE digestive enzyme to digest organoids into single cells and re-embed them in matrix gel.

Images