A method for culturing to obtain anterior foregut endoderm cell spheroids and applications thereof

Abstract

The present application relates to the technical field of biology, and particularly discloses a method for culturing and obtaining foregut endoderm cell spheroids and application thereof. The method for culturing and obtaining foregut endoderm cell spheroids of the present application obtains foregut endoderm cell spheroids by inducing differentiation of stem cells on a culture surface with a PDA-matrigel coating. The culture method of the present application can ensure the differentiation efficiency of DE and AFE adherent cells and spheroids, promote the generation of AFE cell spheroids and the separation from the adherent cell layer, reduce the edge rolling of AFE adherent cells, prolong the production time of AFE cell spheroids, significantly improve the yield of AFE cell spheroids, and provide a basis for subsequent differentiation and culture of lung organoids.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for culturing and obtaining spheroids of anterior foregut endoderm cells and its application. Background Technology

[0002] Human pluripotent stem cell (hPSC)-derived organoids offer new opportunities for research on human development and disease. The foregut endoderm (AFE) is a crucial transitional stage in embryonic development, the origin of organs such as the lung, thyroid, and esophagus. Current in vitro differentiation strategies primarily mimic the in vivo developmental process of the embryo, precisely regulating signaling pathways by sequentially adding specific growth factors and small molecule compounds to gradually differentiate stem cells into the defined endoderm (DE), and then into AFE. During AFE differentiation, spontaneous cell aggregation occurs in the 2D cultured adherent cell layer, producing AFE cell spheroids that detach from the adherent cells. These AFE cell spheroids are collected and embedded in the extracellular matrix (such as Matrigel), subsequently undergoing further growth and differentiation into lung organoids. The production of AFE cell spheroids directly determines the subsequent differentiation efficiency of lung organoids. However, even experienced technicians face instabilities such as low AFE cell spheroid yield and adherent cell curling, leading to insufficient AFE cell collection for subsequent lung organoid differentiation experiments.

[0003] Polydopamine (PDA) is a polymer with excellent bioadhesion properties and has wide applications in the field of biomaterials. In cell culture, PDA coatings can be used as an intermediate layer to modify cell culture plates before grafting extracellular matrix materials (ECMs, such as collagen, gelatin, and Matrigel) into the plates for cell culture, thereby improving cell adhesion in 2D cell culture. However, the formation of AFE cell spheroids is a dynamic in vitro differentiation process involving multiple cell differentiation stages. It presents challenges beyond simply addressing cell differentiation at each stage; it also involves managing the formation, detachment, and curling of remaining AFE adherent cells. Therefore, further research on AFE cell spheroid culture methods is necessary. Summary of the Invention

[0004] One of the objectives of this invention is to provide a method for increasing the yield of foregut endoderm cell spheroids in vitro culture.

[0005] This invention provides a method for culturing and obtaining foregut endoderm cell spheroids, which involves inducing stem cells to differentiate on a culture surface coated with PDA-Matrix gel to obtain foregut endoderm cell spheroids.

[0006] This invention has found that after human pluripotent stem cells (hPSCs) are further differentiated into foregut endoderm (AFE) cells through the definitive endoderm (DE) cell stage via in vitro culture, the yield of self-aggregating spheroids (AFE cell spheroids) of AFE cells is low. Furthermore, during the process of AFE cell spheroids detaching from the adherent AFE cell layer, the adherent cell layer is prone to curling and peeling off from the cell culture plate. This leads to abnormal cell accumulation, making it impossible to continue forming AFE cell spheroids, and ultimately failing to collect enough AFE cell spheroids for subsequent lung organoid differentiation.

[0007] Therefore, this invention has found that when the differentiation of human pluripotent stem cells begins from the differentiation stage, i.e., when cell differentiation is induced on a PDA-matrix coating, and the PDA-matrix participates in the entire differentiation process of hPSC, DE, AFE, and AFE cell spheroids, it can not only maintain the differentiation efficiency of DE and AFE adherent cells and spheroids, but also reduce the curling of AFE adherent cells and promote the spontaneous formation and detachment of AFE cell spheroids during AFE cell spheroid detachment, thus significantly increasing the yield of AFE cell spheroids.

[0008] Although there are existing teachings on using PDA coatings to promote cell adhesion, the culture object of this invention, AFE cell spheroids, needs to undergo multiple dynamic in vitro differentiation processes from the stem cell stage to endoderm differentiation, and then to AFE differentiation and spheroid formation. It is necessary to ensure that each differentiation stage can proceed smoothly and efficiently (without affecting differentiation efficiency), and also to ensure that AFE cell spheroids can still spontaneously form and detach from the cell adhesion layer. Therefore, only the adhesion-promoting effect of PDA is known, and it is impossible to predict whether it can be applied to solve multiple problems in the formation of AFE cell spheroids at the same time.

[0009] The method of this invention can enable cells to have appropriate adherence during the differentiation process before the AFE stage (stem cell ES and fixed endoderm (DE) stages), and during the AFE stage, while maintaining appropriate adhesion between the adherent cell layer and the cell culture plate, it allows the self-assembled spheres to detach smoothly from the adherent cell layer without causing the adherent layer to detach from the culture plate along with the spheres, resulting in curling and a sharp drop in sphere production. This ensures the efficient and large-scale production of AFE cell spheres to meet subsequent application needs.

[0010] In the method of the present invention, the preparation method of the culture surface with PDA-matrix coating includes: firstly, a PDA coating is laminated on the culture surface, and then a matrix adhesive coating is laminated on the PDA coating to obtain a culture surface with PDA-matrix coating; Preferably, dopamine monomers are first polymerized in situ on the culture surface to obtain a culture surface with a PDA coating; then, a mixture of matrix gel and basal culture medium is cured on the PDA coating to obtain a culture surface with a PDA-matrix gel coating. The culture surface is made of a material that can form a stable interface with the PDA.

[0011] In this invention, the PDA coating is preferably formed by the in-situ oxidative polymerization of dopamine monomers on the material surface. During the polymerization process, PDA, through various functional groups such as catechol, quinone, and amine groups, can form a stable bond with the substrate surface via covalent reactions (such as Schiff base reactions or Michael addition), coordination, π–π interactions, and hydrogen bonding. Therefore, the adhesion of the PDA coating to the culture surface is not simply physical adsorption, but a stable coating achieved through the synergistic effect of multiple interfacial interactions. Through these interfacial interactions, PDA can form a uniform and stable coating structure on the culture surface, providing a reliable interfacial basis for subsequent matrix gel curing and cell culture.

[0012] The proteins in the diluted matrix gel (the above mixture) will spontaneously settle onto the PDA coating. They can be cured onto the PDA coating by incubating at 37 degrees Celsius for 2 hours and then discarding the matrix gel dilution (mixture). They will react with the amino / hydroxyl / carbonyl groups in the PDA coating to form a stable and uniform bond.

[0013] The preferred preparation method of the PDA-matrix adhesive coating of the present invention enables uniform and stable adhesion between the culture surface, PDA, and matrix adhesive material, avoiding the shedding and separation of the culture substrate.

[0014] In the method of the present invention, the material of the culture surface is polystyrene, polycarbonate, polyethylene terephthalate, polymethyl methacrylate, glass, or a polymer material whose surface has been activated to introduce active groups; preferably, the material of the culture surface is polystyrene. Preferably, the activation treatment includes oxidation, plasma, or other conventional methods in the art.

[0015] The active group includes hydroxyl, amino, or other conventional active groups in the art.

[0016] In the method of the present invention, during the in-situ polymerization, the concentration of the dopamine monomer is 0.25-8 mg / mL, preferably 0.5-2 mg / mL, and more preferably 1 mg / mL.

[0017] Modifying the PDA layer using the preferred conditions of this invention can further improve the final culture effect (extend the production time of AFE cell spheres and increase the yield of AFE cell spheres).

[0018] The PDA coating antioxidant test revealed that the antioxidant performance was close to saturation when the concentration of dopamine monomer was 2 mg / mL. This indicates that the concentration of dopamine monomer may reach the reaction limit when it is higher than this concentration, and the performance is close to that of dopamine monomer at a concentration of 2 mg / mL.

[0019] In the method of the present invention, the pH value of the reaction system for in-situ polymerization is 8-10, preferably 8.5±0.2, and preferably, the reaction system includes Tris buffer. And / or, the reaction temperature of the in-situ polymerization is 20-60 degrees Celsius, preferably 20-37 degrees Celsius, and more preferably 22-25 degrees Celsius (room temperature); And / or, the reaction time of the in-situ polymerization is 0.5-24 hours, preferably 2-6 hours, more preferably 2-3 hours, and even more preferably 2 hours; And / or, the oxidant for the in-situ polymerization is one or more of oxygen, periodate (such as sodium periodate NaIO4), persulfate (such as ammonium persulfate (NH4)2S2O8), peroxide (such as hydrogen peroxide H2O2), permanganate (such as potassium permanganate KMnO4), chlorate (such as potassium chlorate KClO3), and high-valence transition metal ions (such as Ce(IV) and Fe(III)); preferably, the oxidant for the in-situ polymerization is one or more of oxygen, sodium periodate NaIO4, and hydrogen peroxide H2O2; more preferably, the oxidant is oxygen.

[0020] In this invention, the in-situ polymerization process of dopamine monomer can be carried out using methods known in the art. As a specific implementation, it includes: causing dopamine (DA) to undergo oxidative self-polymerization in an alkaline solution system and under the action of oxygen to generate polydopamine (PDA).

[0021] Preferably, the alkaline solution system includes Tris buffer and ultrapure water, with the ultrapure water primarily used to dissolve DA, thereby facilitating better dispersion of DA in the reaction system. For example, DA can be prepared as a 50 mg / mL solution before being mixed with Tris buffer. As long as the pH value and DA concentration of the final reaction system are within the limits defined by this invention, and the substances are uniformly mixed, the specific ratio of Tris buffer to ultrapure water in the alkaline solution system can be set according to common knowledge in the art.

[0022] Preferably, the contact between DA and oxygen is achieved by shaking the reaction system. For example, the reaction system can be placed on a shaker at 50-70 rpm for the reaction.

[0023] In the method of the present invention, the matrix gel is one or more of Matrigel, Cultrex BME, dECM hydrogel, collagen hydrogel, fibrin gel, gelatin, hyaluronic acid, and polyethylene glycol hydrogel, preferably Matrigel.

[0024] In this invention, the gelatin may be methacrylamide gelatin.

[0025] The Matrigel mainly includes standard Matrigel, Growth Factor Reduced-Matrigel (GFR-Matrigel), Phenol Red-Free Matrigel, and LDEV-Free Matrigel.

[0026] The volume ratio of Matrigel to basal culture medium in the mixture is 1:(80-100), and the basal culture medium is preferably DMEM / F-12; The root mean square roughness Rq of the surface after the mixture is cured on the PDA coating is 50-150 nm.

[0027] After the mixture of the present invention cures, a thin film is deposited on the PDA coating to give the PDA-matrix adhesive coating surface a specific roughness Rq, thereby facilitating subsequent cultivation. The roughness can be adjusted by changing the curing time of the mixture using methods known in the art.

[0028] In the method of this invention, when stem cells are induced to differentiate on a culture surface coated with PDA-Matrix adhesive, the seeding amount of the stem cells is 0.5-1.25 × 10⁻⁶. 5 cells / 1.9cm 2 The preferred value is 0.8×10 5 cells / 1.9cm 2 .

[0029] In the method of the present invention, the stem cells successively pass through the differentiation stages of fixed endoderm cells and anterior foregut endoderm cells, and then obtain the anterior foregut endoderm cell spheres through the spontaneous aggregation of anterior foregut endoderm cells.

[0030] In the method of the present invention, the culture medium used to differentiate and form the fixed endoderm cells is a fixed endoderm complete culture medium, which includes a fixed endoderm basal culture medium, a TGF-β signaling pathway activator and a GSK3β signaling pathway inhibitor. The shaped endoderm basal culture medium includes one or more of the following: core basal culture medium, protein supplement, nutrient supplement, antibiotic, pH stabilizer, and glutamine supplement; Preferably, in the shaped endoderm basal culture medium, the core basal culture medium is MCDB131; Preferably, in the basal culture medium for shaping endoderm, the protein supplement is bovine serum albumin with a concentration of 0.4%-0.6%, more preferably 0.5%; Preferably, in the basal culture medium for shaping endoderm, the nutrient supplement is glucose with a concentration of 8-12 mM, more preferably 10 mM; Preferably, in the basal culture medium for shaping endoderm, the antibiotic is penicillin / streptomycin at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the basal culture medium for shaping endoderm, the pH stabilizer is sodium bicarbonate with a concentration of 1.5-2 mg / mL; more preferably, it is 1.7 mg / mL. Preferably, in the basal culture medium for shaping endoderm, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the complete endoderm culture medium, the TGF-β signaling pathway activator is selected from Activin A, Activin B, TGF-β1, TGF-β2 or TGF-β3; more preferably, the TGF-β signaling pathway activator is Activin A, with a concentration of 80-120 ng / mL, and even more preferably 100 ng / mL; Preferably, in the complete endoderm culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021, with a concentration of 0.1-3 µM. Preferably, the stem cells are induced to differentiate into defined endoderm cells after 2.5-4 days; more preferably, the differentiation time to form the defined endoderm cells is three days. The complete culture medium for the typed endoderm cells used on the first day of differentiation into the typed endoderm cells consisted of the typed endoderm basal culture medium, 100 ng / mL Activin A and 3 µM CHIR99021; The complete culture medium for the typed endoderm cells used on the second day of differentiation consisted of the typed endoderm basal medium, 100 ng / mL Activin A and 0.1 µM CHIR99021; The complete culture medium for the finalized endoderm cells, used on the third day of differentiation, consisted of the finalized endoderm basal culture medium and 100 ng / mL Activin A.

[0031] If the differentiation time to form the defined endoderm cells is four days, then the culture medium from day three is still used on day four.

[0032] In the method of the present invention, the anterior foregut endoderm culture medium used to differentiate and form the anterior foregut endoderm cells includes one or more of the following: basal culture medium, pH buffer, nutritional supplement, antibiotic, glutamine supplement, TGF-β signaling pathway inhibitor, BMP signaling pathway inhibitor, FGF4, SHH agonist and GSK3β signaling pathway inhibitor. Preferably, in the pre-endoderm culture medium, the basal culture medium is Advanced DMEM / F-12; Preferably, in the pre-endoderm culture medium, the pH buffer is HEPES buffer, and the concentration of the HEPES buffer is 0.5-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the nutrient supplement includes N-2 supplement and / or B-27 supplement; the concentration of the N-2 supplement is 0.5%-2%; more preferably 1%; the concentration of the B-27 supplement is 1%-3%; more preferably 2%. Preferably, in the pre-endoderm culture medium, the antibiotic is penicillin / streptomycin, with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the TGF-β signaling pathway inhibitor is selected from LY2109761, A83-01, SB-525334, SD-208, EW-7197, Disitertide, LY3200882, SM16, or SB431542; more preferably, the TGF-β signaling pathway inhibitor is SB431542, with a concentration of 5-15 μM; even more preferably, it is 10 μM; Preferably, in the pre-endoderm culture medium, the BMP signaling pathway inhibitor is selected from Noggin, Dorsomorphin, DMH1, or LDN-193189; more preferably, the BMP signaling pathway inhibitor is Noggin, with a concentration of 150 ng / mL to 250 ng / mL; even more preferably, it is 200 ng / mL. Preferably, the concentration of FGF4 in the pre-endoderm culture medium is 400-600 ng / mL; Preferably, in the pre-endoderm culture medium, the SHH agonist is selected from SHH or SAG; more preferably, the SHH agonist is SAG with a concentration of 0.5-1.5 μM; even more preferably, it is 1 μM. Preferably, in the pre-endoderm culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021 at a concentration of 1-3 μM; even more preferably, it is 2 μM.

[0033] In the method of the present invention, the shaped endoderm cells are induced to differentiate into foregut endoderm cells after 2-7 days; preferably, the foregut endoderm cell spheroids are collected on days 2-7 of the foregut endoderm cell differentiation stage.

[0034] The methods and applications of this invention are not for disease diagnosis or treatment purposes, but are mainly used for the in vitro culture of foregut endoderm cell spheroids.

[0035] The methods of this invention do not include applications involving the destruction of human embryos for industrial or commercial purposes.

[0036] The present invention also provides the application of the above-described method for culturing and obtaining foregut endoderm cell spheroids in increasing the yield of foregut endoderm cell spheroids cultured in vitro.

[0037] The method of this invention can also be extended to the differentiation of midgut and hindgut organoids.

[0038] In this invention, "foregut endoderm" refers to the endoderm that forms the anterior portion of the liver endoderm. "Foregut endoderm" includes, for example, the pharyngeal endoderm and other more highly differentiated endoderm cell populations, and contains multiple cell types exhibiting different expression patterns of molecular markers. "Foregut endoderm" will develop into various tissues, such as the tonsils, tympanic membrane, thyroid gland, parathyroid glands, thymus, trachea, esophagus, stomach, lungs, larynx, and / or pharynx.

[0039] "Definitive endoderm cells (DE)" refer to cells that express one or more markers of a defined endoderm lineage. These markers include, but are not limited to, CXCR4, SOX17, GATA-4, FOXA2, AFP, CER1, C-KIT, EPCAM, SNAI1, GSC, E-Cad, and / or N-Cad. The defined endoderm is functionally defined by one or more cells that further differentiate from endoderm tissues. This can include the lung, thyroid, liver, pancreas, or intestine.

[0040] "Organoids" are three-dimensional, multicellular aggregates derived from stem cells that differentiate and self-organize, encapsulating the structural features and cell-cell interactions of mature tissues. These organoids are three-dimensional aggregates of one or more cell types that mimic the surface appearance, structure, or function of tissues or organs.

[0041] "Induction" or "induce" relates to processes or behaviors that cause a specific effect on a cell's phenotype. Such effects can take the form of causing a phenotypic change, such as differentiating to another cell phenotype; or they can take the form of maintaining a cell in a specific cellular state, such as preventing dedifferentiation or promoting cell survival.

[0042] Pluripotent stem cells are multipotent cells with the ability to self-renew and self-replicate, and can differentiate into various cell types under certain conditions.

[0043] The terms "precursor cell," "progenitor cell," and "stem cell" are used interchangeably in the art, and in this invention refer to pluripotent or lineage-determined progenitor cells that have the potential to undergo an unlimited number of mitotic divisions to renew themselves or differentiate into daughter cells of a desired cell type. Compared to pluripotent stem cells, lineage-determined progenitor cells are generally considered incapable of generating numerous cell types that are phenotypically different from each other. Instead, progenitor cells will be able to generate one or possibly two lineage-determined cell types.

[0044] In this invention, "differentiation" refers to the process by which less specialized cells, such as stem cells or induced pluripotent stem cells, become more specialized cell types so that they become specific lineages, including but not limited to certain progenitor cells and more specialized somatic cells. The conditions for stem cell differentiation are well known in the art.

[0045] In this invention, "differentiation medium" refers to a cell growth medium containing or lacking certain additives, so that when cultured in the medium, stem cells, induced pluripotent cells, or incompletely differentiated cells develop into differentiated cells or cells that exhibit some or all of the characteristics of cells that are more differentiated than stem cells, induced pluripotent cells, or other similar cells.

[0046] The DMEM / F-12 can also be replaced by one or more of the following: William's E cell culture medium, Neurobasal Medium, MEM cell culture medium, DMEM cell culture medium, 1640RPMI cell culture medium, or F-12 cell culture medium.

[0047] The DMEM / F-12 culture medium contains a 1:1 mixture of DMEM and Ham's F-12.

[0048] The DMEM / F-12 comprises a modified DMEM / F-12 culture medium with its composition adjusted according to the actual application.

[0049] The DMEM / F-12 modified culture medium includes, but is not limited to, DMEM-low-glucose-pyruvate-glutamine-free-phenol red-free, and DMEM / F-12-GlutaMAX. TM DMEM / F-12-HEPES (DMEM / F-12 with HEPES), DMEM-lowglucose-pyruvate-HEPES, Advanced DMEM / F-12 (containing glucose, non-essential amino acids, sodium pyruvate, and phenol red, but without L-glutamine and HEPES).

[0050] The beneficial effects of this invention are at least as follows: The culture method of the present invention can promote the generation of AFE cell spheres and their detachment from the adherent cell layer while ensuring the differentiation efficiency of DE and AFE adherent cells and spheres, as well as the quality of AFE cell spheres. It also reduces the curling of AFE adherent cells during differentiation, prolongs the production time of AFE cell spheres, and significantly increases the yield of AFE cell spheres, thus providing a foundation for subsequent lung organoid differentiation culture. Attached Figure Description

[0051] Figure 1 The results show the adhesion and antioxidant properties of the PDA coating. A is a schematic diagram of the PDA coating adhesion performance test method; B is a representative photograph of the PDA coating adhesion performance test results; C shows the quantitative results of the adhesion performance tests for the control group and the PDA group; and D shows the antioxidant efficacy of the PDA coating prepared with gradient concentrations of DA.

[0052] Figure 2 The effect of PDA coating on DE cell differentiation is shown in Figure 1. A represents the bright-field image of DE cell differentiation culture; B represents the qPCR results on day 3 of DE cell differentiation culture; C and D represent the staining and corresponding quantification (%) results on day 3 of DE cell differentiation culture, respectively.

[0053] Figure 3The results are qPCR results on day 4 of AFE cell differentiation culture. In the results, A represents the qPCR results of adherent AFE cells; B represents the qPCR results of AFE cell spheroids.

[0054] Figure 4 This paper illustrates the effect of PDA coating on the differentiation of AFE adherent cells. In the figure, A is a bright-field image of AFE adherent cells; B is a statistical analysis of the edge curling of adherent cells on day 5 of AFE cell differentiation culture; [The image shows...] This means P < 0.05.

[0055] Figure 5 The effect of PDA coating on AFE spheroid differentiation is shown. In the figure, A represents the bright-field image of spheroids from day 2 to day 7 of AFE cell differentiation culture; B represents the number of spheroids produced; and C represents the results of size statistical analysis.

[0056] Figure 6 The effect of PDA coatings prepared with different concentrations of DA on AFE cell differentiation. In the image, A shows bright-field images of adherent cells on days 3 and 5 of AFE cell differentiation culture; B shows bright-field images of AFE cell spheroids collected from days 2 to 7 of AFE cell differentiation culture. Detailed Implementation

[0057] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0059] Example 1 This embodiment provides a method for inducing and culturing AFE cell spheroids, as detailed below: 1. PDA-Matrigel Coating Construction 1.1 Preparation of PDA-coated modified perforated plates Dopamine (DA) powder was added to ultrapure water to prepare a 50 mg / mL DA solution. In a clean bench, Tris buffer (pH=8.5), ultrapure water, and DA solution were filtered through a 0.22 µm filter membrane. 490 µL of Tris buffer and 10 µL of DA solution were added to each well of a 24-well plate. After capping, the plate was shaken at 60 rpm for 2 hours at room temperature. After 2 hours, the plate was washed two to three times with sterile ultrapure water (500 µL / well), gently adding ultrapure water during washing and aspirating off any remaining liquid and reactants. Finally, the plate was left uncovered and air-dried in a clean bench, then sterilized by UV irradiation for 30 minutes. The PDA-coated plate was then sealed with sealing film and stored at room temperature for later use.

[0060] 1.2 Before cell culture, a Matrigel coating was added to the PDA-coated modified well plate to prepare a PDA-Matrigel-coated 24-well plate. Thaw Matrigel (previously stored at -20°C) on ice. Add 100 µL of the thawed Matrigel (Corning, membrane matrix, 354277) to 10 mL of pre-chilled DMEM / F-12 medium (Gibco). TM Resuspend the cells in DMEM / F-12 medium (11320033), mix thoroughly, and place on ice for later use. In a clean bench, open the PDA-coated 24-well plate (PDA) / the uncoated 24-well plate (control), add 300 µL of DMEM / F-12 medium containing Matrigel to each well, and incubate at 37°C for 2 h. After 2 h, discard the remaining DMEM / F-12 medium containing Matrigel (surface root mean square roughness Rq 100.27 ± 31.46 nm, n = 3) and immediately seed cells.

[0061] 2. Stem cell inoculation and differentiation With a density of 1.6 × 10 5 Human pluripotent stem cells (hPSCs) per cell / mL were seeded into 24-well culture plates with PDA-Matrix gel coating and control, 500 μL per well.

[0062] The medium was cultured at a constant temperature of 37 degrees Celsius and 5% CO2, with the medium changed every 24 hours. The medium used was mTeSR (Stemcell).

[0063] After 1-2 days, the stem cell proliferation was monitored under an optical microscope. When the spread area reached approximately 70-80% of the well area, DE medium was added to induce the stem cells to differentiate into DE cells. After 3 days, AFE medium was added to induce the DE cells into AFE cell spheroids, and the cells were cultured for a total of 7 days.

[0064] The cell culture medium should be changed every 24 hours.

[0065] The DE medium formula is as follows: Basic culture medium formula: MCDB131 (Pronosai) + glucose (10 mM) + NaHCO3 (1.7 mg / mL) + BSA (0.5%) + GlutaMAX (1:100) + penicillin / streptomycin (1:100).

[0066] On day 1, replace the DE basal medium with Activin A (100 ng / mL) and CHIR99021 (3 µM). On the second day, the DE basal medium was replaced with Activin A (100 ng / mL) and CHIR99021 (0.1 µM). On day 3, replace the DE basal medium with Activin A (100 ng / mL).

[0067] The AFE medium formula is as follows: Basic culture medium formulation: Advanced DMEM / F-12 (Gibco) TM Advanced DMEM / F-12,12634010) + HEPES (1:100) + B-27 (1:50) + N-2 (1:100) + GlutaMAX (1:100) + Penicillin / Streptomycin (1:100).

[0068] Complete culture medium formula: basal medium + SB431542 (10µM) + CHIR99021 (2µM) + Noggin (200ng / mL) + SAG (1µM) + FGF4 (500ng / mL).

[0069] 3. Characterization Tests 3.1 Performance Characterization of PDA Coating PDA coating adhesion performance test: The precursor and curing agent of polydimethylsiloxane (PDMS) were prepared at a mass ratio of 10:1 and poured into a dish to a thickness of about 4 mm; the template with PDMS was placed in a vacuum desiccator and vacuumed for 2 min to remove air bubbles from the PDMS; the degassed PDMS was placed in an 80 ℃ oven for 2 h to cure the PDMS.

[0070] A PDA coating (PDA coating group) was prepared on PDMS according to the PDA coating preparation method described above (refer to 1.1). Untreated PDMS was used as a control group. Matrigel was dropped onto the surface of both the PDMS control group and the PDA coating group, and cured at 37°C for 15 minutes. Then, 15 mL of DPBS was added, and the mixture was shaken at 150 rpm for 10 hours. The number of remaining Matrigel droplets was recorded every two hours.

[0071] PDA coating antioxidant test: 24-well plates were treated according to the aforementioned PDA coating modification method (refer to 1.1). The final concentrations of DA in the 24-well plates before the reaction were set to 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, and 8 mg / mL, respectively. 200 mM DPPH was prepared, and 0.5 mL was added to each well. After standing in the dark for 25 min, the absorbance at 516 nm was measured and recorded as A1. An unmodified 24-well plate was used as a blank control, and its absorbance was recorded as A0. The free radical scavenging rate was A1 / A0 × 100%. The free radical scavenging rate of the PDA coatings prepared with gradient concentrations of DA against 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH·) was measured to evaluate the antioxidant properties of the PDA coatings.

[0072] See results Figure 1 The PDA coating provides good bioadhesion.

[0073] 3.2 Effects of PDA coating on cell differentiation To evaluate the effect of PDA coating on the development of DE and AFE cells, this invention employed a desktop binocular stereo microscope for bright-field imaging observation of the cells. Bright-field morphology of DE cells on the first and third days of differentiation culture was recorded (see...). Figure 2 (A) Bright-field morphology of adherent cells on days 3 and 5 of AFE cell differentiation culture (see A). Figure 4 (A in the text), and a statistical analysis of the curling of adherent cells on the fifth day of AFE cell differentiation culture (see A). Figure 4 (See section B). Additionally, AFE cell spheroids formed from AFE cell differentiation culture from day 2 to day 7 were collected from 6 wells of a 24-well plate, and their bright-field morphology was recorded (see section B). Figure 5 A) and perform statistical analysis on quantity and size (see A) Figure 5 (B and C in the middle).

[0074] To further investigate the effect of PDA coating on differentiation process, samples were taken on day 3 of DE cell differentiation culture and day 4 of AFE cell differentiation culture (AFE adherent cells and AFE cell spheroids). mRNA was extracted using an RNA purification kit and reverse transcribed into cDNA using the Maxima H Minus kit. Subsequently, qPCR was used to detect the expression levels of marker genes for DE and AFE cell development, including the endoderm marker genes SOX17 and FOXA2. The role of PDA coating in differentiation was analyzed by comparing the differences in expression of key genes among the groups. Each sample was prepared in triplicate, with GAPDH used as an internal control gene. The relative expression level was calculated using ΔCt relative to GAPDH to ensure the comparability and reliability of the results.

[0075] To further validate the qPCR results, immunofluorescence staining was used to collect AFE cell spheroids obtained on the third day of DE cell differentiation culture, digested into single cells using Accutase, resuspended, and then cultured at 2.5 × 10⁻⁶ cells / mL. 5 200 µL of cells / mL was added to a confocal dish pre-treated with Matrigel (1:100, diluted with DMEM / F-12) for 2 hours and incubated at 37°C with 5% CO2 for 2 hours. Subsequently, the slides were fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed three times with DPBS, and then placed in immunofluorescence permeation buffer containing 0.3% Triton X-100 for 1 hour at room temperature, followed by immunofluorescence blocking buffer for 30 minutes. After blocking, primary antibodies (SOX17, FOXA2) were added and incubated overnight at 4°C. The next day, secondary antibodies of the corresponding species and Hoechst 33342 were added and incubated at room temperature in the dark for 1 hour. Finally, microscopic imaging was performed using a 980 laser confocal microscope.

[0076] The qPCR results on day 3 of DE cell differentiation culture are shown below. Figure 2 B. The qPCR results of AFE adherent cells and AFE cell spheroids on the fourth day of AFE cell differentiation culture are shown in Figure B. Figure 3 The staining results of DE cell differentiation culture on the third day are shown in [the image / document]. Figure 2 The quantitative results of C-positive cells are shown in the figure. Figure 2 D in the middle.

[0077] The experimental results above show that the DE differentiation efficiency is not affected by the PDA coating.

[0078] The differentiation efficiency of adherent cells and spheroids in AFE is not affected by the PDA coating.

[0079] PDA coating can significantly reduce edge curling of adherent cells in AFE.

[0080] PDA coating can increase AFE cell spheroid production.

[0081] Example 2 This embodiment investigates the effect of PDA coatings prepared with different concentrations of DA on AFE cell differentiation. Specifically, 24-well plates were treated according to the PDA coating modification method in Example 1 (refer to 1.1), with the final concentrations of DA in the 24-well plates before the reaction set at 0.5 mg / mL and 2 mg / mL, respectively. Following the method described in Example 1, the obtained PDA-coated plates were further prepared into PDA-Matrix adhesive coated 24-well plates and used for stem cell culture.

[0082] The final culture results were compared with those of the control group and the PDA group (final concentration of DA in the 24-well plate before reaction was 1 mg / mL) in Example 1. The results are shown in [Figure 1]. Figure 6 In the figure, the control group without added DA is PDA-0, and the groups with final DA concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL in the 24-well plate before the reaction are PDA-0.5, PDA-1, and PDA-2, respectively.

[0083] Bright-field images of adherent cells on days 3 and 5 of AFE cell differentiation culture show that the application of PDA coating significantly improved the adhesion of adherent cells.

[0084] Bright-field images of AFE cell spheroids collected from day 2 to day 7 of AFE cell differentiation culture showed that the PDA group could prolong the spheroidization time from 3-4 days to 6-7 days. Among them, the PDA coating reaction concentration of 1 mg / mL showed the best overall performance.

[0085] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for culturing and obtaining spheroids of anterior foregut endoderm cells, characterized in that, Foregut endoderm cell spheroids were obtained by inducing differentiation of stem cells on a culture surface coated with PDA-Matrix gel.

2. The method for obtaining anterior foregut endoderm cell spheroids according to claim 1, characterized in that, The method for preparing the culture surface with the PDA-matrix coating includes: First, a PDA coating is laminated onto the culture surface, and then a matrix adhesive coating is laminated onto the PDA coating to obtain a culture surface with a PDA-matrix adhesive coating. Preferably, dopamine monomers are first polymerized in situ on the culture surface to obtain a culture surface with a PDA coating; then, a mixture of matrix gel and basal culture medium is cured on the PDA coating to obtain a culture surface with a PDA-matrix gel coating. The culture surface is made of a material that can form a stable interface with the PDA.

3. The method for obtaining anterior foregut endoderm cell spheroids according to claim 2, characterized in that, During the in-situ polymerization, the concentration of the dopamine monomer is 0.25-8 mg / mL, preferably 0.5-2 mg / mL, and more preferably 1 mg / mL; And / or, the pH of the reaction system for the in-situ polymerization is 8-10; preferably, the reaction system includes Tris buffer. And / or, the reaction temperature of the in-situ polymerization is 20-60 degrees Celsius, preferably 20-37 degrees Celsius, and more preferably 22-25 degrees Celsius; And / or, the reaction time of the in-situ polymerization is 0.5-24 hours, preferably 2-6 hours, more preferably 2-3 hours; And / or, the oxidant for the in-situ polymerization is one or more of oxygen, periodate, persulfate, peroxide, permanganate, chlorate, and high-valence transition metal ions; preferably, the oxidant for the in-situ polymerization is one or more of oxygen, sodium periodate, and hydrogen peroxide; more preferably, it is oxygen.

4. The method for obtaining anterior foregut endoderm cell spheroids according to claim 2 or 3, characterized in that, The matrix gel is one or more of Matrigel, Cultrex BME, dECM hydrogel, collagen hydrogel, fibrin gel, gelatin, hyaluronic acid, and polyethylene glycol hydrogel, preferably Matrigel; And / or, the volume ratio of Matrigel to basal medium in the mixture is 1:(80-100), preferably the basal medium is DMEM / F-12; And / or, the root mean square roughness Rq of the surface after the mixture is cured on the PDA coating is 50-150 nm.

5. The method for obtaining anterior foregut endoderm cell spheroids according to any one of claims 1-4, characterized in that, When stem cells are induced to differentiate on a culture surface coated with PDA-Matrix adhesive, the seeding density of the stem cells is 0.5-1.25 × 10⁻⁶. 5 cells / 1.9cm 2 .

6. The method for obtaining anterior foregut endoderm cell spheroids according to any one of claims 1-5, characterized in that, After undergoing the differentiation stages of fixed endoderm cells and anterior foregut endoderm cells, the stem cells spontaneously aggregate to obtain anterior foregut endoderm cell spheres.

7. The method for obtaining anterior foregut endoderm cell spheroids according to claim 6, characterized in that, The culture medium used for differentiating and forming the defined endoderm cells is a defined endoderm complete culture medium, which includes a defined endoderm basal culture medium, a TGF-β signaling pathway activator and a GSK3β signaling pathway inhibitor. The shaped endoderm basal culture medium includes one or more of the following: core basal culture medium, protein supplement, nutrient supplement, antibiotic, pH stabilizer, and glutamine supplement; Preferably, in the shaped endoderm basal culture medium, the core basal culture medium is MCDB131; Preferably, in the basal culture medium for shaping endoderm, the protein supplement is bovine serum albumin with a concentration of 0.4%-0.6%, more preferably 0.5%; Preferably, in the basal culture medium for shaping endoderm, the nutrient supplement is glucose with a concentration of 8-12 mM, more preferably 10 mM; Preferably, in the basal culture medium for shaping endoderm, the antibiotic is penicillin / streptomycin at a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the basal culture medium for shaping endoderm, the pH stabilizer is sodium bicarbonate with a concentration of 1.5-2 mg / mL; more preferably, it is 1.7 mg / mL. Preferably, in the basal culture medium for shaping endoderm, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the complete endoderm culture medium, the TGF-β signaling pathway activator is selected from Activin A, Activin B, TGF-β1, TGF-β2 or TGF-β3; more preferably, the TGF-β signaling pathway activator is Activin A, with a concentration of 80-120 ng / mL, and even more preferably 100 ng / mL; Preferably, in the complete endoderm culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021, with a concentration of 0.1-3 µM. Preferably, the stem cells are induced to differentiate into defined endoderm cells after 2.5-4 days; more preferably, the differentiation time to form the defined endoderm cells is three days. The complete culture medium for the typed endoderm cells used on the first day of differentiation into the typed endoderm cells consisted of the typed endoderm basal culture medium, 100 ng / mL Activin A and 3 µM CHIR99021; The complete culture medium for the typed endoderm cells used on the second day of differentiation consisted of the typed endoderm basal medium, 100 ng / mL Activin A and 0.1 µM CHIR99021; The complete culture medium for the finalized endoderm cells, used on the third day of differentiation, consisted of the finalized endoderm basal culture medium and 100 ng / mL Activin A.

8. The method for obtaining anterior foregut endoderm cell spheroids according to claim 6, characterized in that, The anterior foregut endoderm culture medium used for differentiating and forming the anterior foregut endoderm cells includes: basal medium, pH buffer, nutritional supplement, antibiotic, glutamine supplement, TGF-β signaling pathway inhibitor, BMP signaling pathway inhibitor, FGF4, SHH agonist and GSK3β signaling pathway inhibitor, or one or more of these. Preferably, in the pre-endoderm culture medium, the basal culture medium is Advanced DMEM / F-12; Preferably, in the pre-endoderm culture medium, the pH buffer is HEPES buffer, and the concentration of the HEPES buffer is 0.5-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the nutrient supplement includes N-2 supplement and / or B-27 supplement; the concentration of the N-2 supplement is 0.5%-2%; more preferably 1%; the concentration of the B-27 supplement is 1%-3%; more preferably 2%. Preferably, in the pre-endoderm culture medium, the antibiotic is penicillin / streptomycin, with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the glutamine supplement is GlutaMAX with a concentration of 0.5%-2%; more preferably, it is 1%. Preferably, in the pre-endoderm culture medium, the TGF-β signaling pathway inhibitor is selected from LY2109761, A83-01, SB-525334, SD-208, EW-7197, Disitertide, LY3200882, SM16, or SB431542; more preferably, the TGF-β signaling pathway inhibitor is SB431542, with a concentration of 5-15 μM; even more preferably, it is 10 μM; Preferably, in the pre-endoderm culture medium, the BMP signaling pathway inhibitor is selected from Noggin, Dorsomorphin, DMH1, or LDN-193189; more preferably, the BMP signaling pathway inhibitor is Noggin, with a concentration of 150 ng / mL to 250 ng / mL; even more preferably, it is 200 ng / mL. Preferably, the concentration of FGF4 in the pre-endoderm culture medium is 400-600 ng / mL; Preferably, in the pre-endoderm culture medium, the SHH agonist is selected from SHH or SAG; more preferably, the SHH agonist is SAG with a concentration of 0.5-1.5 μM; even more preferably, it is 1 μM. Preferably, in the pre-endoderm culture medium, the GSK3β signaling pathway inhibitor is selected from CHIR99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, or AZD1080; more preferably, the GSK3β signaling pathway inhibitor is CHIR99021 at a concentration of 1-3 μM; even more preferably, it is 2 μM.

9. The method for obtaining anterior foregut endoderm cell spheroids according to any one of claims 6-8, characterized in that, The defined endoderm cells are induced to differentiate into foregut endoderm cells after 2-7 days; preferably, the foregut endoderm cell spheroids are collected on days 2-7 of the foregut endoderm cell differentiation stage.

10. The method for culturing and obtaining foregut endoderm cell spheroids according to any one of claims 1-9 is used to increase the yield of foregut endoderm cell spheroids cultured in vitro.

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