Improved culture medium for enameloblastoma organs and application of improved culture medium

Abstract

The invention discloses an ameloblastoma organoid improved culture medium and application thereof, and belongs to the field of organoid culture. According to the improved culture medium for the ameloblastoma organoid, the L-cysteine is added on the basis of an organoid culture medium, and formation and stable growth of the ameloblastoma organoid can be promoted by adding the L-cysteine. The improved culture medium for the ameloblastoma organs, disclosed by the invention, can be specifically prepared from a basic culture medium, L-cysteine, a B27 supplement, N-acetyl-l-cysteine, nicotinamide, a human epidermal growth factor, A83-01, human FGF10 (Fibroblast Growth Factor), human FGF2 (Fibroblast Growth Factor 2), prostaglandin E2, Laduvillib, Y-27632, Forskolin, R-spondin-1 and Noggin. The ameloblastoma organ is successfully obtained by culturing the ameloblastoma by adopting the ameloblastoma organ improved culture medium disclosed by the invention, and the ameloblastoma organ improved culture medium has important significance in researching a heterogeneity mechanism of the ameloblastoma and developing a new treatment method.

Description

Technical Field

[0001] The invention belongs to the field of organoid culture, and particularly relates to an ameloblastoma organoid improved culture medium and application thereof. Background Art

[0002] Ameloblastoma (AM) is a benign tumor derived from odontogenic epithelium. Although it rarely undergoes malignant transformation, it has a high recurrence rate, causing great distress to patients. In recent years, the organoid model of ameloblastoma, as a three-dimensional (3D) cell cluster model, has become a powerful tool for studying ameloblastoma because of its ability to maintain self-renewal and its high similarity to the structure and cell composition of natural ameloblastoma epithelium. Compared with traditional two-dimensional cell culture models, it provides an in vitro research platform that is closer to the physiological state.

[0003] Although a variety of ameloblastoma organoid culture protocols have been reported, existing culture medium formulations often result in low survival rates of organoids, which limits their application in research and preclinical models. In addition, there are significant differences between the current culture medium formulations for ameloblastoma organoids and other types of organoids, which not only increases research costs but also increases the complexity of experimental operations. Therefore, the development of an efficient, low-cost and easy-to-operate culture medium formulation for ameloblastoma organoids is of great significance for promoting the research of ameloblastoma and the development of new therapies. By improving the culture medium formulation, the survival rate and biological relevance of ameloblastoma organoids can be improved, thereby providing a more effective tool for the research of ameloblastoma and the development of new therapies. Summary of the invention

[0004] Improved culture medium plays a vital role in the culture of organoids. In the existing technology, the culture of ameloblastoma organoids faces problems such as low success rate and slow growth. The purpose of the present invention is to solve these problems and provide an improved culture medium for ameloblastoma organoids and its application. The present invention can more effectively simulate the structure and physiological function of ameloblastoma by improving the culture medium, which is of great significance for studying the heterogeneity mechanism of ameloblastoma and developing new treatment methods.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides an ameloblastoma organoid improved culture medium, which adds L-cysteine ​​on the basis of the organoid culture medium. Wherein, the added concentration of the L-cysteine ​​is preferably 5-15mmol / L, and more preferably 10-15mmol / L. The organoid culture medium comprises: basal culture medium, B27 supplement, N-acetyl-l-cysteine, nicotinamide, human epidermal growth factor, A83-01 (TGF-β type I receptor inhibitor), human FGF10, human FGF2, prostaglandin E2, Laduviglusib, ROCK inhibitor Y-27632, Forskolin, recombinant human R-spondin-1 protein, recombinant human Noggin protein. The present invention adds L-cysteine ​​to the organoid culture medium to promote the formation and stable growth of ameloblastoma organoids.

[0007] In some embodiments, the ameloblastoma organoid improved culture medium comprises: basal culture medium, 5-15mmol / L L-cysteine, 1×B27 supplement, 1-1.5mmol / L N-acetyl-l-cysteine, 5-15mmol / L nicotinamide, 30-70ng / mL human epidermal growth factor, 300-700nmol / L A83-01, 5-15ng / mL human FGF10, 3-7ng / mL human FGF2, 0.5-1.5μmol / L prostaglandin E2, 0.1-0.5μmol / L Laduviglusib, 5-15nmol / L Y-27632, 0.5-1.5μmol / L Forskolin, 2-6% (w / v) R-spondin-1, 2-6% (w / v) Noggin.

[0008] Furthermore, the modified culture medium for ameloblastoma organoids comprises: basal culture medium, 10-15mmol / L L-cysteine, 1×B27 supplement, 1.25mmol / L N-acetyl-l-cysteine, 10mmol / L nicotinamide, 50ng / mL human epidermal growth factor, 500nmol / L A83-01, 10ng / mL human FGF10, 5ng / mL human FGF2, 1μmol / L prostaglandin E2, 0.3μmol / L Laduviglusib, 10nmol / L Y-27632, 1μmol / L Forskolin, 4% R-spondin-1, 4% Noggin.

[0009] In some embodiments, the basal culture medium is DMEM / F12 basal culture medium.

[0010] The present invention also provides the use of L-cysteine ​​in preparing an improved culture medium for ameloblastoma organoids.

[0011] The present invention also provides the use of L-cysteine ​​or the above-mentioned ameloblastoma organoid improved culture medium in constructing ameloblastoma organoid.

[0012] The present invention also provides a method for constructing ameloblastoma organoids, which uses L-cysteine ​​or the above-mentioned ameloblastoma organoid improved culture medium, and specifically comprises the following steps: washing the ameloblastoma tissue with PBS phosphate buffer containing penicillin-streptomycin; cutting the washed tissue into small pieces for digestion; filtering the single cell suspension obtained by digestion with a cell filter and then performing red lysis treatment; the obtained ameloblastoma single cell is implanted in Matrigel matrix gel, and the above-mentioned ameloblastoma organoid improved culture medium is added for culturing to construct ameloblastoma organoids.

[0013] The present invention has the following advantages and beneficial effects:

[0014] (1) Ameloblastoma cells were cultured using the improved ameloblastoma organoid culture medium of the present invention to successfully obtain ameloblastoma organoids, which is convenient for subsequent application research.

[0015] (2) The present invention adds L-cysteine ​​to the culture medium to promote the formation and stable growth of ameloblastoma organoids. The addition of L-cysteine ​​can increase the number of ameloblastoma organoids, make their morphology closer to a sphere, and increase their volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Typical HE staining images of ameloblastoma organoids constructed in the present invention. The left image is a cystic ameloblastoma organoid, and HE staining shows a cystic cavity-like structure in the middle, surrounded by ameloblasts, which is consistent with the pathological manifestations of cystic ameloblastoma; the right image is a solid ameloblastoma organoid, showing ameloblasts aggregated to form solid cell clusters, which is consistent with the pathological manifestations of solid ameloblastoma.

[0017] Figure 2 This is a representative immunofluorescence staining image of the ameloblastoma organoid (an example of cystic ameloblastoma) constructed in the present invention. DAPI is a nuclear dye with blue fluorescence; ODAM, AMB and AMG are all labeled with red fluorescence, and MERGE is co-localization staining. It can be seen from the figure that the ameloblastoma organoid constructed in the present invention expresses three classic ameloblastoma markers.

[0018] Figure 3The cell morphology of ameloblastoma organoids cultured for different periods of time in culture medium containing different concentrations of L-cysteine ​​was observed under a microscope. Compared with the culture medium without L-cysteine, the number and morphology of ameloblastoma organoids were improved by adding L-cysteine, as shown by the increase in the number of organoids (indicated by the red arrow) at the same time, and the morphology of the organoids was closer to a sphere and larger in size (indicated by the red arrow). When the concentration of L-cysteine ​​was 10-15mM, the number and morphology of ameloblastoma organoids were good.

[0019] Figure 4 This is a statistical line graph showing the changes in the number of ameloblastoma organoids cultured for different periods of time using culture medium containing different concentrations of L-cysteine.

[0020] Figure 5 This is a statistical line graph showing the changes in the volume of ameloblastoma organoids cultured for different periods of time using culture medium containing different concentrations of L-cysteine. DETAILED DESCRIPTION

[0021] The present invention will be explained in detail below in conjunction with the following embodiments; it should be clear that the embodiments shown only represent several implementation methods of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should also be regarded as the scope of protection of the present invention.

[0022] Example 1

[0023] The present embodiment provides an ameloblastoma organoid improved culture medium, which is prepared by adding the following substances to a DMEM / F12 basal culture medium (HyClone, SH30023.01): 10 mmol / L L-cysteine ​​(Aladdin, C108238), 1×B27 supplement (Shanghai Yuanpei, S440J7), 1.25 mmol / L N-acetyl-l-cysteine ​​(Sigma-Aldrich, A9165), 10 mmol / L nicotinamide (Sigma-Aldrich, N0636), 50 ng / mL human epidermal growth factor (PeproTech, AF-100-15), and 500 nmol / mL LA83-01. TGF-β type I receptor inhibitor (MCE, HY-10432), 10 ng / mL human FGF10 (PeproTech, 100-26), 5 ng / mL human FGF2 (PeproTech, 100-18B), 1 μmol / L prostaglandin E2 (Tocris Bioscience, 2296), 0.3 μmol / L Laduviglusib (MCE, HY-10182), 10 nmol / L Y-27632 (MCE, HY-10071), 1 μmol / L Forskolin (MCE, HY-15371), 4% (w / v) R-spondin-1 and 4% (w / v) Noggin (both produced by the r-PEX protein expression platform of U-Protein Express BV).

[0024] Example 2

[0025] This embodiment provides a method for constructing ameloblastoma organoids, which comprises the following steps:

[0026] (1) The ameloblastoma pathological tissue samples were stored at four degrees C using MACS tissue preservation medium (Miltenyi Biotech, 130.100-008).

[0027] (2) The obtained ameloblastoma tissue was processed within 2 hours. First, the tissue sample was washed three times with PBS phosphate buffer (HyClone, SH40007.01) containing 5% penicillin-streptomycin solution (Gibco, 15070063), each time for 5 minutes. Then, the tissue was cut into small pieces and digested for 2 hours using a digestion solution prepared with a volume ratio of 20:1 of tumor tissue digestion solution A (Basal Medium) and solution B (Supplement 20×) in the organoid kit (organoid kit, BioGenous, K601003). The single cell suspension obtained after digestion was filtered through a 70-micron cell filter, and the red blood cells were lysed using red blood cell lysis solution (BioGenous, E238010), and then resuspended and washed with PBS buffer and centrifuged to obtain the ameloblastoma single cell pellet. Subsequently, 10 μL of the modified culture medium in Example 1 was first added to the cell pellet, the cells were blown evenly, and then 3 μL of cell suspension was sucked with a manual pipette and added to 300 μL of Matrigel matrix gel (Corning, 356231), and the precipitated cell mass was gently blown with a 200 μL gun tip to resuspend it to avoid blowing out bubbles; after fully mixing, the cell mass mixed with matrix gel was evenly dotted into a 24-well plate, 1 droplet per well, and the volume was about 40-50 μL. Put it in a cell culture incubator (37 ° C, 4% CO2) for 30 min until the matrix gel is fully solidified. After the matrix gel solidifies, 500-800 μL of the modified culture medium in Example 1 was added for culture, and the culture medium was submerged in the matrix gel and placed in a cell culture incubator. Fresh culture medium was replaced every 3 days, and organoid formation could be observed in 2-3 weeks, and then continued to be cultured for 1 month. Most of the organoid diameters measured by microscope reached 200-400 μm, and samples were collected to make frozen sections. Hematoxylin and eosin (HE) staining was performed on the frozen sections containing organoids to observe whether the organoid structure conformed to the histological morphology of ameloblastoma. The results are shown in Figure 1 Immunofluorescence staining was performed on frozen sections containing organoids to mark the expression of classic ameloblastoma markers, including odontoblastoma-associated protein (ODAM), amelogenin (AMG), and amelogenin (AMB). The results are shown in Figure 2 HE staining and immunofluorescence staining results showed that ameloblastoma organoids were successfully constructed, which were consistent with the pathological manifestations of ameloblastoma and expressed the classic markers of ameloblastoma, ODAM, AMG, and AMB.

[0028] Example 3

[0029] A solid ameloblastoma pathological tissue sample was taken, and the above-mentioned culture medium without L-cysteine ​​(NC) and the above-mentioned culture medium with L-cysteine ​​concentrations of 5mM, 10mM, and 15mM were used to construct ameloblastoma organoids according to the method in Example 2. The samples were observed and photographed under a microscope on days 0, 10, 20, and 30, and the number and diameter of ameloblastoma organoids in the same field of view were counted, and the volume of the tumor sphere was calculated. The results are shown in Figure 3-5 Compared with the medium without L-cysteine, the number and morphology of ameloblastomas were improved by adding L-cysteine. At the same time, as the concentration of L-cysteine ​​increased, the number of organoids also increased, and the morphology of the organoids was closer to spherical and larger. When the concentration of L-cysteine ​​was 10-15mM, the number and morphology of ameloblastoma organoids were good.

[0030] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An improved culture medium for ameloblastoma organoids, characterized in that: The ameloblastoma organoid improved culture medium is prepared by adding L-cysteine ​​to the organoid culture medium.

2. The ameloblastoma organoid improved culture medium according to claim 1, characterized in that: The added concentration of the L-cysteine ​​is 5-15 mmol / L.

3. The ameloblastoma organoid improved culture medium according to claim 1, characterized in that: The organoid culture medium contains: basal culture medium, B27 supplement, N-acetyl-l-cysteine, nicotinamide, human epidermal growth factor, A83-01, human FGF10, human FGF2, prostaglandin E2, Laduviglusib, Y-27632, Forskolin, R-spondin-1, and Noggin.

4. The ameloblastoma organoid improved culture medium according to claim 1, characterized in that: The modified ameloblastoma organoid culture medium comprises: basal culture medium, 5-15mmol / L L-cysteine, 1×B27 supplement, 1-1.5mmol / L N-acetyl-l-cysteine, 5-15mmol / L nicotinamide, 30-70ng / mL human epidermal growth factor, 300-700nmol / L A83-01, 5-15ng / mL human FGF10, 3-7ng / mL human FGF2, 0.5-1.5μmol / L prostaglandin E2, 0.1-0.5μmol / L Laduviglusib, 5-15nmol / L Y-27632, 0.5-1.5μmol / L Forskolin, 2-6% R-spondin-1, and 2-6% Noggin.

5. The ameloblastoma organoid improved culture medium according to claim 1, characterized in that: The modified ameloblastoma organoid culture medium contains: basal culture medium, 10-15mmol / L L-cysteine, 1×B27 supplement, 1.25mmol / L N-acetyl-l-cysteine, 10mmol / L nicotinamide, 50ng / mL human epidermal growth factor, 500nmol / LA83-01, 10ng / mL human FGF10, 5ng / mL human FGF2, 1μmol / L prostaglandin E2, 0.3μmol / L Laduviglusib, 10nmol / L Y-27632, 1μmol / L Forskolin, 4% R-spondin-1, 4% Noggin.

6. The ameloblastoma organoid improved culture medium according to any one of claims 1 to 5, characterized in that: The basic culture medium is DMEM / F12 basic culture medium.

7. Application of L-cysteine ​​in the preparation of improved culture medium for ameloblastoma organoids.

8. Use of L-cysteine ​​or the ameloblastoma organoid improved culture medium according to any one of claims 1 to 6 in constructing ameloblastoma organoids.

9. A method for constructing ameloblastoma organoids, characterized in that: The method uses L-cysteine ​​or the ameloblastoma organoid modified culture medium according to any one of claims 1-6.

10. The method for constructing ameloblastoma organoids according to claim 9, characterized in that: The following steps are involved: The ameloblastoma tissue is washed with PBS containing penicillin-streptomycin; the washed tissue is cut into small pieces for digestion; the single cell suspension obtained by digestion is filtered and then subjected to red lysis treatment; the obtained ameloblastoma single cell is implanted in matrix gel, and the ameloblastoma organoid improved culture medium described in any one of claims 1 to 6 is added for culture to construct ameloblastoma organoids.

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