A method and application of culturing stem cells / organoids in a trophoblast cell co-culture system

Abstract

The present invention belongs to the field of cell culture, and specifically discloses a method and application of culturing stem cells / organoids in a trophoblast cell co-culture system, the method comprising: infecting mouse embryonic fibroblasts with purified retrovirus to form an embryonic fibroblast cell line that stably expresses R-spodin1 and Noggin genes; irradiating the embryonic fibroblast cell line to screen out a monoclonal cell line, and using the monoclonal cell line as a trophoblast cell and a combination of multiple expression factors to culture stem cells or organoids as a co-culture system. Under this culture system, stem cells can be passaged stably for a long time, and the clonal formation, proliferation and differentiation potential of stem cells are not affected, and the 3D gas-liquid interface organoid culture is not affected in structure and function. The trophoblast cells obtained at the same time can save the purchase cost of R-spodin1 and Noggin, weakening the constraints of experimental funds on scientific research.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture, and particularly relates to a method and application of culturing stem cells / organoids in a trophoblast cell co-culture system. Background Art

[0002] Adult stem cell and organoid culture has become an indispensable technology in the field of basic research in regenerative medicine. It is mainly divided into two types: Matrigel culture method and trophoblast cell co-culture method. At present, 3D organoid technology using Matrigel culture is the most widely used method. By mixing primary stem cells with Matrigel and dropping them into the cell culture dish, after the Matrigel solidifies, the complete medium required for stem cell growth is added. The complete medium includes a certain proportion of conditioned medium and a series of components required for cell growth. The preparation of conditioned medium mainly utilizes modified cell lines (such as 293 cell lines). By overexpressing the two genes R-spodin1 and Noggin in the cell lines, the cells release R-spodin1 and Noggin proteins into the surrounding culture medium during growth. These two proteins are cytokines necessary to maintain the stemness of stem cells. By mixing 30%-50% of conditioned medium into the complete culture medium, R-spodin1 and Noggin, which are necessary for stem cell growth, are ensured. However, the production of conditioned medium requires cell culture, which takes several days. During this period, the number of cells, as well as the synthesis and release of R-spodin1 and Noggin proteins are extremely dynamic processes that cannot be accurately quantified, let alone accurately controlled. Therefore, the content of R-spodin1 and Noggin proteins in the prepared conditioned medium cannot be guaranteed, and there will be large differences between different batches. At the same time, during the preparation process, the cell line will continue to proliferate, consume the components in the culture medium, and accumulate a large amount of cell metabolites, some of which are toxic to the growth of stem cells. Therefore, a large number of uncontrollable factors in the preparation of conditioned medium will increase the error of the experimental results, introduce too many human factors, and reduce the repeatability of the experimental results.

[0003] The trophoblast cell co-culture method uses mouse embryonic fibroblasts as trophoblast cells, replacing matrix gel, to maintain the in vitro expansion of stem cells. Trophoblast cells provide a more superior microenvironment to maintain the stemness of stem cells, and more and purer stem cells can be obtained under this system. In addition, using the air-liquid interface differentiation technology of trophoblast cell co-culture, stem cells can spontaneously form 3D air-liquid interface organoids with complete epithelial barrier structure and function. Although embryonic fibroblasts were introduced for the culture of stem cells and organoids, the two cytokines R-spodin1 and Noggin are still indispensable in the culture medium. However, the high price of these two cytokines makes it difficult for most laboratories to continue.

[0004] The present invention aims to solve the problem of unstable conditioned medium, the errors and uncontrollable factors brought to the experiment, and seek a more stable, efficient and low-cost stem cell and organoid culture solution. Summary of the invention

[0005] In view of the above problems, in a first aspect, the present invention proposes a method for culturing stem cells / organoids in a trophoblast cell co-culture system, the method comprising:

[0006] Purified retrovirus was used to infect mouse embryonic fibroblasts to form an embryonic fibroblast cell line that stably expressed R-spodin1 and Noggin genes;

[0007] The embryonic fibroblast cell line is irradiated to select a monoclonal cell line, and the monoclonal cell line is used as a trophoblast cell in combination with a variety of expression factors as a co-culture system for culturing stem cells or organoids.

[0008] Furthermore, the mouse embryonic fibroblasts are 3T3-J2 cells.

[0009] Furthermore, the multiple expression factors include at least one or more of proteins, small molecule inhibitors, growth factors, vitamins and hormones or their analogs.

[0010] Furthermore, the protein includes: Jagged-1;

[0011] The small molecule inhibitors include: Rockinhibitor and SB431542;

[0012] The growth factors include: EGF;

[0013] The vitamins include: Nicotinamide;

[0014] The hormones include: Insulin and Hydrocortisone.

[0015] Furthermore, the multiple expression factors include 1 μM Jagged-1, 2.5 μM Lock inhibitor, 2 μM Nicotinamide and 10 ng / ml EGF.

[0016] In a second aspect, the present invention further proposes a trophoblast cell, which is 3T3-J2-RN, and has been deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms, with the deposit number: CGMCC NO.22310.

[0017] In a third aspect, the present invention provides a trophoblast cell co-culture system, wherein the co-culture system comprises the trophoblast cells and the multiple expression factors.

[0018] In a fourth aspect, the present invention proposes a stem cell cultured using the culture method.

[0019] Furthermore, the stem cells include normal stem cells derived from the stomach, duodenum, small intestine, colon and liver and / or stem cells derived from diseased tissues.

[0020] In a fifth aspect, the present invention provides an organoid cultured using the culture method.

[0021] Furthermore, the organoids include stomach, duodenum, small intestine, colon, liver and pancreas; and the organoids are derived from adult tissue, fetal tissue and / or adult tissue under various disease conditions.

[0022] The stem cells and / or organoids can be used in clinical transformation, specifically including applications in stem cell transplantation and cell therapy.

[0023] In a sixth aspect, the present invention proposes a biological model, which is constructed based on the aforementioned stem cells or organoids, or based on the aforementioned stem cells and organoids.

[0024] Furthermore, the biological models include bacterial models, viral infection models, cytokine models, toxin pathogenicity models, cancer occurrence models, immune cell interaction models and aging models.

[0025] The biological model can be used to detect and verify the absorption of small molecule drugs, macromolecule drugs, polypeptide drugs and nucleic acid drugs by gastrointestinal organoids.

[0026] Beneficial effects of the present invention:

[0027] The present invention utilizes gene modification technology to obtain 3T3-J2-RN cells that stably express R-spodin1 and Noggin proteins, so that the proliferation of the genetically modified 3T3-J2 cells will not be affected. The 3T3-J2-RN cells have the functions and characteristics of trophoblast cells and can be used as a sufficient source of the two cytokines R-spodin1 and Noggin to completely replace the R-spodin1 and Noggin proteins in the culture medium, thereby achieving the purpose of no longer needing to add the two factors to the culture medium.

[0028] The technical system of using 3T3-J2-RN cells as trophoblast cells to culture stem cells and organoids can save the purchase cost of R-spodin1 and Noggin, and weaken the constraints of experimental funds on scientific research. At the same time, there is no need to prepare conditioned medium, which will reduce the impact of batch differences in conditioned medium on the experiment. Under this culture system, stem cells can be passaged stably for a long time, and the cloning, proliferation and differentiation potential of stem cells are not affected, and the structure and function of 3D gas-liquid interface organoid culture are not affected.

[0029] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The cell morphology of gastric, duodenum, small intestine, colon, liver, colon cancer, and pancreatic cancer stem cells cultured using 3T3-J2-RN and 3T3-J2 cells as trophoblasts in Example 2 of the present invention is shown;

[0032] Figure 2A The cross-sectional view of epithelial tissue formed by colon stem cells cultured with 3T3-J2-RN and 3T3-J2 as trophoblasts in the air-liquid interface three-dimensional differentiation system in Example 3 of the present invention using H&E staining is shown;

[0033] Figure 2B The epithelial tissue staining diagram formed by colon stem cells in the three-dimensional differentiation system of air-liquid interface using immunofluorescence staining in Example 3 of the present invention is shown;

[0034] Figure 3 A comparison chart showing the ability of colon stem cell cloning cells cultured using 3T3-J2-RN and 3T3-J2 as trophoblasts in Example 4 of the present invention is shown;

[0035] Figure 4A A comparison of the expression levels of the gene MKI67 in colon stem cells cultured with 3T3-J2-RN and 3T3-J2 as trophoblasts in Example 5 of the present invention is shown;

[0036] Figure 4B A comparison of the expression levels of the gene SOX9 in colon stem cells cultured with 3T3-J2-RN and 3T3-J2 as trophoblasts in Example 5 of the present invention is shown;

[0037] Figure 4C The RNA-seq analysis diagram of colon stem cells cultured with two trophoblast cells in Example 5 of the present invention is shown;

[0038] Figure 5A The figure shows the distribution diagram of the absorption of saquinavir by the three-dimensional gastrointestinal organoid constructed with 3T3-J2-RN as the trophoblast in Example 6 of the present invention;

[0039] Figure 5B The distribution diagram of ibuprofen absorption by the three-dimensional gastrointestinal organoid constructed with 3T3-J2-RN as the trophoblast in Example 6 of the present invention is shown;

[0040] Fig. 6A The figure shows the distribution diagram of the absorption of fluorescein isothiocyanate-dextran (4KD, 40KD) by Caco-2 in Example 7 of the present invention;

[0041] Figure 6B The figure shows the distribution diagram of the absorption of fluorescein isothiocyanate-dextran (4KD, 40KD) by gastric organoids in Example 7 of the present invention;

[0042] Figure 6C The figure shows the distribution diagram of the absorption of fluorescein isothiocyanate-dextran (4KD, 40KD) by Small intestinal organoids in Example 7 of the present invention;

[0043] Figure 7 The absorption of polypeptide drugs by gastrointestinal organoids in Example 8 of the present invention is shown;

[0044] Figure 8 The absorption of nucleic acid drugs by gastrointestinal organoids in Example 9 of the present invention is shown. DETAILED DESCRIPTION

[0045] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. In addition, unless otherwise defined, the scientific terms mentioned in this disclosure have the same meaning as those generally understood by technicians in the field to which the subject to be protected by the present invention belongs. In addition, unless otherwise specified, the methods without specific recording of conditions or steps are all conventional methods, and the reagents and materials used can be obtained from commercial channels.

[0046] In the prior art, the R-spodin1 and Noggin proteins in the matrix gel culture 3D organoid technology cannot be accurately quantified and accurately controlled, resulting in the inability to guarantee the content of R-spodin1 and Noggin proteins in the prepared conditioned medium, and there will be large differences between different batches. It is not clear whether the use of 3T3 cells to express the two proteins R-spodin1 and Noggin can be used to maintain the growth of stem cells. At the same time, since 3T3-J2 cells need to be irradiated as trophoblast cells, the irradiated 3T3-J2 cells will enter the senescence stage due to genomic damage. It is also unknown whether the aged 3T3 cells can synthesize and synthesize sufficient amounts of R-spodin1 and Noggin. In addition, irradiation can cause DNA damage, and the genes expressing R-spodin1 and Noggin are integrated into the genome of 3T3-J2 cells. Therefore, it is inevitable that DNA sequence mutations will occur, which will cause protein mutations and fail to translate and synthesize functional R-spodin1 and Noggin proteins. It is unknown whether this will directly lead to the failure of 3T3-J2 cells as a technical solution for R-spodin1 and Noggin proteins. The most critical thing is that the quality and state of 3T3-J2 cells are particularly important for maintaining the self-renewal ability and differentiation potential of stem cells. Whether the genetic engineering operation process affects the function of trophoblast cells in maintaining stem cell characteristics is also the biggest difficulty faced.

[0047] 3T3-J2 mouse embryonic fibroblasts, as trophoblast cells, have been widely used in the culture of stem cells. The present invention is based on 3T3-J2 mouse embryonic fibroblasts, and considers the difficulties in the above-mentioned analysis process to conduct in-depth research and propose a culture method and co-culture system of trophoblast cells. By irradiating 3T3-J2 cells, the genome of the cells is damaged, resulting in cell senescence and inability to proliferate. Due to no longer proliferating, 3T3-J2 cells will maintain an extremely low basal metabolism, consume very few culture medium components, and a small amount of metabolites derived from 3T3-J2 cells are also accumulated in the culture medium. At the same time, due to the characteristics of embryonic fibroblasts themselves, the maintenance of stem cell stemness can be promoted, and therefore, 3T3-J2 cells are important supporting materials in stem cell culture. 3T3-J2 cells are cell lines that can be stably passaged, and genome operations can be performed on cells.

[0048] Based on the above reasons, the present invention selects 3T3-J2 cells as host cells to construct a trophoblast cell co-culture system.

[0049] The specific method is as follows: First, construct eukaryotic expression vectors of the two genes R-spodin1 and Noggin, the two vectors carry Puromycin and Hygromycin resistance genes respectively. The insertion of the two genes is confirmed by sequencing, and the gene sequence is correct.

[0050] The eukaryotic expression vectors of R-spodin1 and Noggin genes were co-transfected into 293 cells with the retroviral packaging vectors pMDL and pVSVG by liposome transfection, the culture supernatant was collected, and the virus was concentrated and purified by ultracentrifugation.

[0051] The purified virus was used to infect 3T3 cells, and through the screening of resistant genes, several cell lines derived from single cell clones that stably expressed these two genes were obtained. These cell lines were irradiated and used as trophoblast cells to culture stem cells. Finally, a cell line was identified and screened, named 3T3-J2-RN. The proliferation of 3T3-J2-RN cells was not affected after gene editing, and they still had the functions and characteristics of trophoblast cells. The most important point is that 3T3-J2-RN cells can stably express R-spodin1 and Noggin proteins. When the two cytokines R-spodin1 and Noggin are completely removed from the culture medium, stem cells can still grow and can be stably passaged, and their differentiation potential is not affected. In addition, the air-liquid interface organoids obtained using 3T3-J2-RN cells as trophoblast cells were not affected in terms of cell composition, structure and function.

[0052] The trophoblast cell co-culture system and its application are described in detail below in conjunction with specific embodiments.

[0053] Example 1 Preparation of 3T3-J2-RN cells

[0054] 1.1 Construction of eukaryotic expression vectors of R-spodin1 and Noggin, namely pBABE-Rspondin1 and pBABE-Noggin, with Puromycin and Hygromycin resistance genes, respectively. The insertion of the two genes was confirmed by sequencing by Shanghai Sangon Biotechnology Co., Ltd., and the gene sequences were correct.

[0055] The specific construction method is: by high-fidelity PCR method, two restriction sites, EcoR1 and BamH1, are selected to amplify the CDS regions of the two genes, R-spondin1 and Noggin. The specific primers are shown in Table 1:

[0056] Table 1 PCR primers used

[0057]

[0058] Then, the full-length R-spodin1 and Noggin genes were cloned into the pBABE-puromycin and pBABE-Hygromycin vectors respectively by molecular cloning methods, and the recombinant vectors were transformed into DH5α. With ampicillin as the selection pressure, the recombinant strains were transferred to LB solid culture medium containing ampicillin selection pressure and cultured at 37°C for 12-16 hours. The recombinant transferred strains of single clones were selected and transferred to LB liquid culture medium containing ampicillin selection pressure and cultured at 37°C for 12-16 hours. The plasmids were extracted and sent for sequencing to confirm that the full-length R-spondin1 and Noggin genes were cloned into the pBABE-puromycin and pBABE-Hygromycin vectors respectively, and named pBABE-R-spondin1 and pBABE-Noggin respectively.

[0059] 1.2 Transfection of cells

[0060] By liposome transfection, the two eukaryotic expression vectors pBABE-Rspodin1 and pBABE-Noggin were co-transfected with the retroviral packaging vectors pMDL and pVSVG into 293 cells (293 cells were provided by other laboratories of the School of Pharmacy, Tsinghua University). The culture supernatant was collected, and the retrovirus was concentrated and purified by ultracentrifugation.

[0061] 1.3 Screening

[0062] 3T3-J2 cells (purchased from the ATCC cell bank) were infected with purified retrovirus, and cell lines derived from several single-cell clones that stably expressed the two genes R-spodin1 and Noggin were obtained by screening for resistance genes. These cell lines were irradiated and used as trophoblast cells to culture stem cells. A cell line was identified and screened and named 3T3-J2-RN. The 3T3-J2-RN cell line was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection on April 27, 2021. The address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101, and the deposit number is: CGMCC No. 22310.

[0063] Example 2 2D culture of stem cells using 3T3-J2-RN and 3T3-J2 cells (mouse embryonic fibroblasts) as trophoblasts

[0064] Gastric, duodenal, jejunal, colon, liver, colon cancer, and pancreatic cancer stem cells were cultured using 3T3-J2-RN cells transfected with R-spodin1 and Noggin genes after irradiation as the trophoblast. Multiple factor components such as Jagged-1 (1 μM), Rockinhibitor (ROCK inhibitor, 2.5 μM), SB431542 (2 μM), Nicotinamide (10 mM), EGF (epidermal growth factor, 10 ng / ml), Insulin, and Hydrocortisone were added to the trophoblast. Gastric, duodenal, small intestinal, colon, liver, colon cancer, and pancreatic cancer stem cells were cultured without adding R-Sp ondin1 and Noggin. The cells were cultured every 2 days and subcultured every 7-10 days. During subculture, the cells were digested with 0.25% trypsin-EDTA (trypsin) at 37°C for 8-10 minutes. The digested cells were collected by centrifugation and inoculated on a new 3T3-J2-RN cell feeder layer. The cells were cultured every 2 days and subcultured every 7-10 days.

[0065] The morphology of the above stem cell clones is as follows Figure 1 As shown, Figure 1The left and right columns show the morphology of gastric, duodenum, colon, liver, cancer colon, and pancreatic cancer stem cells cultured using 3T3-J2-RN trophoblast cells (3T3-J2-RN Feeder) and 3T3-J2 trophoblast cells (3T3-J2Feeder) as feeder layers, respectively.

[0066] After multiple verifications, it can be confirmed that the above stem cells can be passaged stably for a long time, and compared with stem cells cultured with 3T3-J2 as the trophoblast, the cell clone state is more "stem". It can be seen that the 3T3-J2-RN trophoblast culture system can provide a more superior growth environment for stem cells and is more conducive to maintaining the good state of stem cells. It is a new and very stable and efficient stem cell culture system, which can be used not only for the culture of normal stem cells, but also for the culture of stem cells for various diseases.

[0067] Example 3 In vitro construction of epithelial organoid tissue

[0068] Differentiate colonic stem cells into three-dimensional colonic epithelial organoid tissue structures in an air-liquid interface differentiation system (ALI):

[0069] The irradiated 3T3-J2-RN cells in Example 1 were spread on the bottom of the upper chamber of a double-layer culture plate (transwell) and cultured overnight. The next day, colon stem cells were inoculated on the 3T3-J2-RN feeder layer cells in the chamber, and the culture medium inside and outside the chamber was updated every day (the culture medium was the stem cell culture medium mentioned above, and the culture medium inside and outside the chamber was the same), and cultured for 3-7 days to a fusion state, and the culture medium inside the chamber was removed, and the culture medium was only added and updated outside the chamber, thereby forming a gas-liquid interface culture condition, and a three-dimensional epithelial tissue was formed after 6-10 days. The epithelial tissue was fixed with formalin, and the three-dimensional epithelial tissue formed was embedded in paraffin and sliced.

[0070] H&E staining was used to identify the epithelial tissue morphology, and immunofluorescence staining was used to detect the expression of different cell markers. In this example, DAPI dye was used to identify the type of epithelial cells differentiated from stem cells and their spatial distribution and proportion. The main cell types of colon epithelial tissue and the corresponding detection markers included goblet cells (Muc2), enteroendocrine cells (CHGA), and epithelial cells (E-cadherin).

[0071] The results of H&E staining and immunofluorescence staining were shown in Figure 2A and Figure 2B As shown, Figure 2AThis is a cross-sectional view of the epithelial tissue formed by colon stem cells cultured with 3T3-J2-RN as trophoblast cells and 3T3-J2 as trophoblast cells in the air-liquid interface three-dimensional differentiation system, showing that the closely arranged cells constitute the epithelial cell layer barrier, HE staining; Figure 2B The main cell types of epithelial tissue formed by colon stem cells in the three-dimensional differentiation system of air-liquid interface. Immunofluorescence staining: Muc2 immunofluorescence (red) shows goblet cells, CHGA immunofluorescence (red) shows enteroendocrine cells, and E-cadherin immunofluorescence (green) shows epithelial cells. Figure 2B It can be seen that the morphological characteristics of the colon epithelial tissue constructed with 3T3-J2-RN as the trophoblast and 3T3-J2 as the trophoblast are highly similar to the in vivo tissue and have the main cell types of epithelial tissue.

[0072] Example 4 Comparison of clone formation ability

[0073] Colon stem cells were cultured with 3T3-J2-RN and 3T3-J2 as trophoblasts, and their clone-forming ability was identified. First, the cells in the logarithmic growth phase were counted and 1,000 cells were inoculated into a 12-well plate. The culture medium was replaced every two days. After 10-12 days of growth, the culture medium was removed, the cells were washed twice with PBS, fixed with 4% formaldehyde, stained with rhodamine for 20 minutes, washed with water to remove excess stain, and dried. The number of cell clones formed was counted. The results of the cell clone number are as follows: Figure 3 As shown in the figure, the horizontal axis corresponds to 3T3-J2-RN and 3T3-J2 as the trophoblast layer, and the vertical axis represents the cell cloning ability. The results show that the colon stem cells cultured with 3T3-J2-RN as the trophoblast layer have stronger cloning ability than the colon stem cells cultured with 3T3-J2 as the trophoblast layer.

[0074] Example 5 Transcriptome Sequencing Analysis

[0075] To identify the effect of 3T3-J2-RN trophoblast on stem cell gene expression, RNA-seq analysis was performed on colon stem cells cultured with 3T3-J2-RN as trophoblast and 3T3-J2 as trophoblast. Figure 4CAs shown in the figure, from the results of RNA-seq analysis, colon stem cells cultured with the above two trophoblast cells are basically similar at the transcriptome level, with only a small number of differentially expressed genes. However, the expression levels of genes MKI67 and SOX9 related to cell proliferation and stemness maintenance are shown in 4A and 4B. The ordinate in the figure represents the standardized count, from which it can be seen that the expression levels of genes MKI67 and SOX9 of colon stem cells cultured with 3T3-J2-RN as the trophoblast are higher than those of colon stem cells cultured with 3T3-J2 as the trophoblast. Therefore, it can be seen that 3T3-J2-RN as the trophoblast cell has a better effect on maintaining the stemness and proliferation ability of stem cells.

[0076] The characteristics of gastrointestinal organoids constructed in vitro are highly similar to those of in vivo tissues, and can theoretically be used to simulate and detect the absorption of drugs in gastrointestinal epithelial tissues. This mainly includes detecting and verifying the absorption of small molecule drugs, peptide macromolecule drugs, and nucleic acid drugs by gastrointestinal organoids. The following examples verify their practicality and effectiveness as drug absorption and screening models one by one.

[0077] Example 6 Detection and verification of gastrointestinal organoids' absorption of small molecule drugs

[0078] Small molecule drugs saquinavir and ibuprofen were added to a transwell of a three-dimensional gastrointestinal organoid constructed with 3T3-J2-RN as a trophoblast. In this embodiment, the stomach and small intestine were selected for construction. After a period of time (4 hours), the culture medium at the bottom of the chamber was taken for liquid chromatography / mass spectrometry analysis to detect the drug absorption in the culture medium and compare it with the absorption of the drug in the Caco-2 (human colorectal adenocarcinoma cell) model. The absorption of saquinavir and ibuprofen by Caco-2, Gastric organoids and Small intesti ne organoids is shown in the figure below. Figure 5A and 5B As shown in the figure, the results show that the permeability of small molecule drugs such as saquinavir and ibuprofen in Caco-2 and three-dimensional gastrointestinal organoids constructed with 3T3-J2-RN as the trophoblast layer is basically similar. Therefore, the three-dimensional gastrointestinal organoids constructed with 3T3-J2-RN as the trophoblast layer can be used as a research model to detect the absorption of small molecule drugs in the gastrointestinal epithelium.

[0079] Example 7 Detection and verification of gastrointestinal organoids' absorption of macromolecules

[0080] Fluorescein isothiocyanate-dextran (4KD, 40KD) was added to the transwell of Caco-2 and 3D gastrointestinal organoids constructed with 3T3-J2-RN as the trophoblast layer, and the culture medium at the bottom of the chamber was taken after a period of time (4h) to detect the fluorescence intensity. In addition, in order to further fully verify the drug absorption function of this model, sodium decanoate (C10), a substance that increases the permeability of the gastrointestinal epithelium and promotes drug absorption, was added to detect the changes in fluorescence intensity. The absorption of macromolecules by Caco-2, Gastric organoids and Small intestinal organoids is shown in Figures 6A, 6B and 6C, respectively. It can be seen from the figure that compared with Caco-2, gastrointestinal organoids have complete epithelial barrier function, and the addition of C10 significantly increases the permeability of fluorescein isothiocyanate-dextran (4KD, 40KD) in Caco-2, Gastric organoids and Small intestinal organoids.

[0081] Example 8 Detection and verification of gastrointestinal organoids' absorption of polypeptide drugs

[0082] In subsequent studies, the absorption of the peptide drug Leuprorelin by gastrointestinal organoids was tested. Leuprorelin was added to the transwell of the formed three-dimensional gastrointestinal epithelial tissue. After a period of time, the culture medium at the bottom of the chamber was taken for liquid chromatography / mass spectrometry analysis to detect the drug absorption in the culture medium. Figure 7 As shown. The results showed that the permeability of the macromolecular drug leuprorelin in the three-dimensional gastrointestinal organoids constructed with 3T3-J2-RN as the trophoblast layer was closer to the permeability (Rat) detected in rat animal experiments reported in the current literature, and statistical analysis in the literature found that compared with the Caco-2 model, the permeability of the drug measured in rat experiments was more correlated with the absorption of the drug by the human body. Therefore, the data show that the three-dimensional gastrointestinal organoids constructed with 3T3-J2-RN as the trophoblast layer can be used as a more appropriate research model for detecting the absorption of macromolecular drugs in the gastrointestinal epithelium.

[0083] Example 9 Detection and verification of gastrointestinal organoids' absorption of nucleic acid drugs

[0084] In recent years, mRNA vaccines have become known to more people due to their outstanding performance in the COVID-19 pandemic. Correspondingly, the "third medical revolution" led by nucleic acid drugs has become increasingly popular. Since the oral administration route has the advantages of convenience, high safety, and good patient compliance, oral nucleic acid drugs have also become a hot topic of research at home and abroad. This example attempts to add fluorescently labeled nucleic acid drugs encapsulated in nanocapsules to the transwell of a three-dimensional gastrointestinal organoid constructed with 3T3-J2-RN as the nutrient layer. After a certain period of time, the culture medium at the bottom of the chamber is taken to detect the fluorescence intensity and identify the absorption of the nucleic acid drug. Specifically, Figure 8 The results showed that nucleic acid drugs were not significantly absorbed by the three-dimensional gastrointestinal organoid model due to their large molecular weight.

[0085] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing stem cells / organoids in a trophoblast cell co-culture system, characterized in that: The method comprises: The purified retrovirus was used to infect mouse embryonic fibroblasts to form an embryonic fibroblast cell line that stably expressed R-spodin1 and Noggin genes; The embryonic fibroblast cell line is irradiated to screen out a monoclonal cell line, which is 3T3-J2-RN, and has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCCNO.22310. The monoclonal cell line is used as a trophoblast cell in combination with a variety of expression factors as a co-culture system for culturing stem cells or organoids.

2. The method for culturing stem cells / organoids using a trophoblast cell co-culture system according to claim 1, characterized in that: The mouse embryonic fibroblasts are 3T3-J2 cells.

3. The method for culturing stem cells / organoids using a trophoblast cell co-culture system according to claim 1, characterized in that: The multiple expression factors include at least one or more of proteins, small molecule inhibitors, growth factors, vitamins and hormones or their analogs.

4. The method for culturing stem cells / organoids using a trophoblast cell co-culture system according to claim 3, characterized in that: The proteins include: Jagged-1; The small molecule inhibitors include: Rock inhibitor and SB431542; The growth factors include: EGF; The vitamins include: Nicotinamide; The hormones include: Insulin and Hydrocortisone.

5. The method for culturing stem cells / organoids using a trophoblast cell co-culture system according to claim 1, characterized in that: The multiple expression factors included 1 μM Jagged-1, 2.5 μM Rock inhibitor, 2 μM Nicotinamide and 10 ng / ml EGF.

6. A trophoblast cell co-culture system, characterized in that: The co-culture system comprises the trophoblast cells described in claim 1 and the multiple expression factors described in any one of claims 3-5.

7. Use of the trophoblast cell co-culture system according to claim 6 in clinical transformation, wherein the use is in the preparation of stem cell transplantation and cell therapy drugs.

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