Method for quickly constructing organoid based on horizontal shaking

Abstract

The invention belongs to the field of biological medicine, and particularly discloses a method for quickly constructing an organoid based on horizontal shaking, which is characterized by comprising the following steps: (1) inoculating a single liver cell into a culture dish; (2) putting the culture dish into a horizontal shaking table in an incubator, and carrying out shaking culture for 24 hours to obtain an organoid; and (3) continuously culturing for more than 8 days to obtain the large-volume liver organoid. Liver organs with uniform sizes can be formed on the second day. After the culture is performed for 8 days, the organoid becomes large. And carrying out immunofluorescence detection and expressing liver specific genes. The liver specific gene detected by qPCR is obviously improved compared with that of two-dimensional cultured cells. The medicine can be metabolized. The method can be used for rapidly producing the liver organs with the drug metabolism capability in batches.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for rapidly constructing organoids based on horizontal shaking. Background Art

[0002] Generally, organoids are constructed based on commercially available or self-developed hydrogels, which are made into the desired shape through biological 3D printing or molds. There is a common problem in both biological 3D printing and hydrogel encapsulation, that is, the dispersed single cells in the hydrogel need to be gradually expanded to further form organoids. The time required is relatively long, generally more than 2 weeks.

[0003] At present, the common method for constructing organoids is to add cells to low-adhesion 96-well plates for culture. After 4-5 days, the cells automatically aggregate into cell spheres without carrier scaffolds. However, this method has a low throughput and the speed of forming cell spheres is slightly slower.

[0004] Therefore, how to provide a method for rapidly constructing organoids is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides a method for rapidly constructing organoids based on horizontal shaking, which can cause scattered single cells to quickly aggregate into balls the next day, establish connections between cells, maintain cell functions and make them more mature.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for rapidly constructing organoids based on horizontal shaking, comprising the following steps:

[0008] (1) Inoculate a single organoid cell into a culture dish;

[0009] (2) Place the culture dish on a horizontal shaker in an incubator and shake for 24 to 36 hours to obtain organoids;

[0010] (3) Continue culturing for more than 8 days to obtain large-volume organoids.

[0011] Preferably, the organoid is a liver organoid or a tumor organoid, and the organoid cells are derived from humans or mice.

[0012] Preferably, in step (1), the cells are seeded into a culture medium at a density of 1 million cells per milliliter and placed in a culture dish with a hydrophobic surface.

[0013] Preferably, the culture medium is HEM.

[0014] Preferably, the culture is carried out at 37°C, 5% carbon dioxide, 95% humidity, and shaking at 50-100 rpm. Preferably, step (3) is cultured for more than 8 days, and the diameter of the organoid reaches more than 60 μm.

[0015] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following excellent effects:

[0016] The present invention can form organoids of uniform size on the second day. After 8 days of culture, the organoids become larger. Immunofluorescence detection shows the expression of liver-specific genes. qPCR detection of liver-specific genes is significantly higher than that of two-dimensional cultured cells. Drugs can be metabolized. The method can quickly mass-produce liver organoids with drug metabolism capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 This is a picture of liver organoids at 24h of mouse hepatocyte culture in Example 1 of the present invention;

[0019] Figure 2 These are pictures of liver organoids at different magnifications on the 2nd and 8th days of mouse hepatocyte culture in Example 1 of the present invention.

[0020] Figure 3 This is the verification of gene expression and drug metabolism ability of liver organoids on the 8th day of mouse hepatocyte culture in Example 1 of the present invention, wherein (A) is the immunofluorescence staining of liver marker genes ALBUMIN and HNF4a, and cytochrome p450 enzyme CYP2E1 of liver organoids; (B) is the qPCR detection of liver marker gene Albumin, cytochrome p450 enzymes Cyp2c29 and Cyp2b10 of liver organoids; Figure (C) is the detection of drug metabolism ability of liver organoids, which are acetaminophen (Phenacetin) metabolized by cytochrome p450 enzyme CYP1A2, nifedipine (Nifedipine) metabolized by CYP3A4, and chlorzoxalon (Chlorzoxalon) metabolized by CYP2E1.

[0021] Figure 4 This is a picture of liver organoids at 48 hours of culture of hepatocytes differentiated from human fibroblasts in Example 2 of the present invention.

[0022] Figure 5This is a picture of tumor organoids at 48 hours after culturing T47D cells in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] 1. Digest cultured mouse hepatocytes into single cells.

[0026] 2. Inoculate the cells into HEM medium at a density of 1 million cells per ml and culture them in a hydrophobic surface culture dish.

[0027] The formula of HEM medium can be found in the literature Revitalizing liver function in mice with liver failure through transplantation of 3D-bioprinted liver with expanded primary hepatocytes. HEM medium is based on DMEM / F12 medium and also contains ZnCl 2 (0.544mg / L), ZnSO 4 7H 2 O(0.75mg / L),CuSO 4 ·5H 2 O (0.2 mg / L), MnSO 4 (0.025 mg / L), bovine serum albumin (2 g / L), galactose (2 g / L), ornithine (0.1 g / L), proline (0.03 g / L), nicotinamide (0.61 g / L), insulin (10 mg / L), transferrin (5.5 mg / L), sodium selenite (6.7 μg / L), TGFα (50 ng / mL), EGF (40 ng / ml), IGFII (100 ng / mL), HGF (20 ng / ml), 1 μM sphingosine-1-phosphate, 5 μM forskolin, 10 μM dexamethasone, 10 μM Y-27632, 0.5 μM A-83-01, 3 μM CHIR99021, 1% FBS.

[0028] 3. Place the shaker in the incubator and culture at 37°C, 5% carbon dioxide, 95% humidity, and 58 rpm.

[0029] 4. Take a picture on the first day of culture (24h). The organoids have formed into clusters with uniform size ( Figure 1 )

[0030] 5. After continuous culture until the 8th day (192h), most organoids reached a size of more than 60 microns ( Figure 2 ).

[0031] Organoids were collected on the 8th day and subjected to immunofluorescence staining, quantitative PCR detection and drug metabolism detection. Immunofluorescence results showed that organoids expressed liver marker genes ALBUMIN and HNF4a, and cytochrome p450 enzyme CYP2E1. Quantitative PCR results showed that compared with two-dimensional cultured cells (digested mouse hepatocytes, plated on collagen-coated 10 cm dishes and cultured with the same culture medium), the expression of liver marker genes Albumin, cytochrome p450 enzymes Cyp2c29 and Cyp2b10 in organoids were significantly increased. Among them, the expression level of Albumin in organoids was 3 times that of two-dimensional cultured cells, p = 0.0022. The expression level of Cyp2c29 in organoids was 11 times that of two-dimensional cultured cells, p < 0.0001. The expression level of Cyp2b10 in organoids was 311 times that of two-dimensional cultured cells, p = 0.0007. Results are shown in Figure 3 (B), Unpaired two-tailed t-test was used for statistical analysis. p<0.0001 is marked ****, p<0.001 is marked ***, and p<0.01 is marked **.

[0032] The organoids on the 8th day of culture were co-incubated with 100 μM acetaminophen, nifedipine, and chlorzoxazone to verify whether the organoids on the 8th day had the metabolic capacity of CYP1A2, CYP3A4, and CYP2E1. The control group did not add organoids on the 8th day. The culture supernatant was collected 24 hours after metabolism, and the standard curve of drug concentration was made by high-performance liquid chromatography to measure the concentration of drugs in the initial and post-metabolism culture supernatant. Figure 3 (C) The results of the drug metabolism test of liver organoids showed that they were able to metabolize acetaminophen, nifedipine, and chlorzoxalon, indicating that liver organoids have the metabolic capacity of cytochrome p450 enzymes CYP1A2, CYP3A4, and CYP2E1.

[0033] Example 2

[0034] 1. Digest the hepatocytes differentiated from cultured human fibroblasts into single cells.

[0035] 2. Inoculate the cells into a culture medium at a density of 1 million cells per milliliter and culture them in a hydrophobic surface culture dish. The culture medium is the same as that in Example 1.

[0036] 3. Place the shaker in an incubator and culture at 37°C, 5% carbon dioxide, 95% humidity, and 58 rpm.

[0037] 4. Take a picture on the second day of culture (48h). The organoids have formed into clusters with uniform size ( Figure 4 )

[0038] Example 3

[0039] 1. Digest cultured human breast ductal carcinoma T47D cells into single cells.

[0040] 2. Inoculate the cells at a density of 1 million cells per milliliter in a culture medium and culture them in a hydrophobic surface culture dish. The culture medium is RPMI 1640 containing 10% fetal bovine serum.

[0041] 3. Place the shaker in an incubator and culture at 37°C, 5% carbon dioxide, 95% humidity, and 58 rpm.

[0042] 4. Take a picture on the second day of culture (48h). The organoids have formed into clusters with uniform size ( Figure 5 )

[0043] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for rapidly constructing organoids based on horizontal shaking, characterized in that: The following steps are involved: (1) Inoculate a single cell for organoid construction into a culture dish; (2) Place the culture dish on a horizontal shaker in an incubator and shake for 24 to 36 hours to obtain organoids; (3) Continue culturing for more than 8 days to obtain large-volume organoids.

2. A method for rapidly constructing organoids based on horizontal shaking according to claim 1, characterized in that: The organoid is a liver organoid or a tumor organoid, and the organoid cells are derived from humans or mice.

3. A method for rapidly constructing organoids based on horizontal shaking according to claim 1, characterized in that: In step (1), the cells are seeded into culture medium at a density of 1 million cells per milliliter and placed in a hydrophobic surface culture dish.

4. A method for rapidly constructing organoids based on horizontal shaking according to claim 3, characterized in that: The culture medium was HEM.

5. The method for rapidly constructing organoids based on horizontal shaking according to claim 1, characterized in that: The culture was cultured at 37°C, 5% carbon dioxide, 95% humidity, and shaking at 50-100 rpm.

6. The method for rapidly constructing organoids based on horizontal shaking according to claim 1, characterized in that: Step (3) The organoids are cultured for more than 8 days until their diameter reaches more than 60 μm.

Images