Construction method of trophoblast organ-like model, trophoblast organ-like model and application of trophoblast organ-like model

Abstract

The invention discloses a construction method of a trophoblast organ-like model, the trophoblast organ-like model and application thereof, and relates to the technical field of cell model.The construction method comprises the steps that placenta villus tissue is aseptically separated, gradient separation and purification are conducted to obtain extraembryonic early cells, the extraembryonic early cells are mixed with matrigel, and the trophoblast organ-like model is obtained; and carrying out 3D culture by using a culture medium containing early-stage and later-stage differentiation compositions to obtain the organ-like model with various trophoblast cell lineages. Based on the model, placenta toxicity research is carried out on pregnancy drugs, and the research specifically comprises the degree of 3D organoid form destruction after drug treatment, organoid activity and the expression condition of trophoblast marker genes. Compared with a traditional two-dimensional cell model, the placental toxicity detection method has the advantages that the placental toxicity of the medicine is detected by taking the trophoblast-like organ as the model, the accuracy is higher, the predictability is higher, and the influence of the medicine on human placental tissues is accurately reflected.

Description

Technical Field

[0001] The present invention relates to the field of cell model application technology, and in particular to a method for constructing a trophoblast organoid model, a trophoblast organoid model and applications thereof. Background Art

[0002] The placenta is an important organ for maintaining the health of the fetus and the mother. It is a bridge connecting the mother and the fetus. It plays a key role in sensing the nutritional needs of the fetus, coordinating the maternal nutritional supply, and being responsible for the delivery of nutrients. Trophoblast cells are the most important cell type for the structure and function of the placenta. Chorionic trophoblast cells are the main component cells of the placenta and undergo complex differentiation during pregnancy: trophoblast stem cells located at the top of the chorion can produce chorionic cytotrophoblasts (CTBs), which further form syncytial trophoblasts (STBs) or differentiate into extravillous trophoblasts (EVTs) that invade the decidua through cell fusion. Trophoblast cells are also the main functional executors of the placenta. STBs cover the entire surface of the placental villi and are mainly responsible for secreting hormones and growth factors, regulating the blood supply from the mother to the placenta, and promoting the exchange of substances between the mother and the fetus. The EVTs located at the tip of the anchored villi invade the maternal endometrium and reshape the uterine spiral arteries to ensure that the placenta receives adequate blood perfusion during pregnancy.

[0003] There is a close link between placental dysfunction and adverse pregnancy outcomes. Studies have shown that abnormal placental development may lead to serious consequences such as fetal growth restriction, miscarriage, premature birth, and stillbirth. Reports have shown that the incidence of placental abruption is significantly increased in patients with hypertensive disorders complicating pregnancy, especially in patients with early-onset severe preeclampsia, where the incidence of placental abruption can reach 4.1% to 22.9%, and the perinatal mortality rate is between 4.4% and 67%. Placental dysfunction may also lead to fetal intrauterine growth restriction and low birth weight, which is related to the downregulation of placental CRH type I receptor expression caused by maternal stress. Abnormal placental development is an important factor leading to adverse pregnancy outcomes, which may be caused by a variety of factors such as genetics, environmental factors, or maternal health conditions.

[0004] Drug use during pregnancy is another important factor that affects the health and function of the placenta. Globally, approximately 25% to 99% of pregnant women use drugs during pregnancy, and up to 80% of pregnant women are exposed to insufficient drug safety information. The DEBC (Maternal Drug Exposure Birth Cohort) study showed that in early pregnancy, approximately 30.70% of pregnant women (34,690 cases) had taken one or more drugs, of which dydrogesterone and progesterone were the most commonly used drugs, with exposure rates of 11.97% and 10.82%, respectively. The study also found that the risk of adverse pregnancy outcomes associated with dydrogesterone exposure, such as stillbirth, premature birth, low birth weight, and birth defects, was significantly higher than that of non-exposed persons. In addition, in clinical practice, pregnant women often need to take medications to manage chronic diseases or pregnancy complications to ensure the health of mothers and babies. For example, the glucocorticoid-methylprednisolone, due to its anti-inflammatory and anti-allergic properties, is often used to treat rheumatoid arthritis, ankylosing spondylitis, and systemic lupus erythematosus. Although methylprednisolone can be converted to an inactive substance in the placenta, any drug that can cross the placenta during pregnancy may increase the risk of placental-mediated diseases. Drug treatment during pregnancy can affect the morphological and functional development of the fetus through multiple pathways, multiple organs, and multiple targets. Its mechanisms involve direct pathways such as oxidative stress, epigenetic modification, and metabolic activation, and may also be indirectly caused by placental dysfunction.

[0005] Due to ethical and technical constraints, pregnant women are often unable to participate in routine drug safety testing. Although existing drug evaluation system models provide us with some evaluation tools, they have limitations in simulating human placental development and drug response. Traditional in vitro evaluation methods cannot fully simulate the complex structure and function of the placenta, which limits the accuracy and reliability of drug evaluation. Animal models are difficult to fully simulate the drug response of the human placenta, so most drugs lack safety data for use during pregnancy.

[0006] In recent years, the development of 3D organoid technology has provided a new platform for simulating and studying human placental development. According to a study report, in 2018, Turco et al. purified CTB from placental tissue in early pregnancy, inoculated it into matrix gel, and added trophoblast organoid medium (TOM) composed of EGF, FGF2, CHIR99021, A83-01 and R-spondin1 for 3D culture, which can form placental trophoblast organoids with long-term expansion capacity. This organoid has a tissue structure similar to that of placental villi and contains a variety of trophoblast cell types; through whole-genome transcriptome and methylation analysis, trophoblast organoids are highly similar to early human placental tissue in gene expression and DNA methylation patterns. This shows that trophoblast organoids, as an in vitro model, not only simulate the human placenta in terms of cell composition and function, but also maintain a high degree of similarity with the early placenta at the molecular level. Compared with 2D cell models, trophoblast organoids are derived from placental tissue and are therefore more capable of simulating the developmental characteristics of the early human placenta.

[0007] Currently, trophoblast organoids have shown great potential in studying placental development, viral infection, and the effects of environmental pollutants. In addition, trophoblast organoid models have also been used to evaluate the effects of environmental pollutants on the differentiation and function of human trophoblast lineages. Studies have found that exposure to bisphenol S (BPS) moderately reduced the expression of syncytial trophoblast marker genes and induced apoptosis of the trophoblast lineage in a dose-dependent manner, which may aggravate trophoblast cell apoptosis and moderately inhibit the trophoblast fusion pathway, thereby affecting placental development and pregnancy progression. Although the application of trophoblast organoids in multiple fields has been widely reported, their application in evaluating the effects of pregnancy drugs on trophoblast development is still a relatively new and underexplored field.

[0008] The present invention discloses a method for constructing a trophoblast organoid model, a trophoblast organoid model and its application, and relates to the field of cell model application; the trophoblast organoid model at least includes cytotrophoblast cells (CTBs), syncytiotrophoblast cells (STBs) and has the potential to differentiate into EVTs, and can simulate the tissue structure and physiological function of placental villi in vivo. The model is used to evaluate the effects of drugs on trophoblast development during pregnancy, including morphological changes of 3D organoids after drug treatment, the vitality of organoids and the expression of trophoblast marker genes. The present invention uses trophoblast organoids as a model to detect the placental toxicity of drugs, which is more scientific for evaluating the safety of medication during pregnancy and has reference value for rational clinical medication. Summary of the invention

[0009] In view of this, the present invention aims to address the deficiencies in the prior art, and its main purpose is to provide a method for constructing a trophoblast organoid model, a trophoblast organoid model and its application, which can provide a more accurate and predictive model than traditional two-dimensional cell culture when using trophoblast organoids to simulate exposure to drugs, and accurately reflect the effects of drugs on human placental tissue.

[0010] To achieve the above purpose, the present invention adopts the following technical solutions:

[0011] A method for constructing a trophoblast organoid model comprises the following steps:

[0012] S1. Isolation of trophoblast cells: Aseptically separate the placental villus tissue, add mixed digestive enzymes to the placental villus tissue to separate single cells, and centrifuge to obtain extraembryonic early cells;

[0013] S2, Trophoblast Organoid Differentiation Stage 1: Mix extraembryonic early cells with matrix gel, perform 3D culture using the hanging drop method, and culture using a culture medium containing a pre-differentiation composition to obtain trophoblast organoids containing cytotrophoblast cells and syncytial trophoblast cells;

[0014] S3. Trophoblast organoid differentiation stage 2: Digest the trophoblast organoids obtained in step S2, mix the digested organoids with matrix gel, re-prepare 3D organoids using the hanging drop method, and continue culturing using a culture medium containing a late differentiation composition to obtain a trophoblast organoid model containing extravillous trophoblast cells.

[0015] As a preferred embodiment: the pre-differentiation composition in step S2 includes a basal culture medium and additives, the basal culture medium is DMEM / F12 culture medium, the additives include 5%-20% fetal bovine serum, 1x N2 supplement, 1x B-27without vitamin A, 1x glutamine, 80-150μg / ml Primocin, 1μM-1mM N-acetylcysteine, 0.1-100μM A83-01, 1-10μM CHIR99021, 5-20uM prostaglandin-E2, 10-500ng / ml R-Spondin, 10-500ng / ml EGF, 10-500ng / ml FGF2, 10-500ng / ml HGF, and 1-200μM Y-27632.

[0016] As a preferred embodiment: the late differentiation composition in step S3 includes a basal culture medium and additives, the basal culture medium is DMEM / F12 culture medium, the additives include 1%-10% serum replacement, 0.1%-1% BSA, 1%-10% ITS-X additive, 1x penicillin / streptomycin, 0.1-1mM β-mercaptoethanol, 0.1-100μM A83-01 and 10-500ng / ml NRG1.

[0017] As a preferred solution: the mixed digestive enzyme in step S1 includes 0.25% pancreatin-EDTA and collagenase IV.

[0018] As a preferred solution: in step S2, the extraembryonic early cells are mixed with the matrix gel at a volume ratio of 1:10.

[0019] As a preferred embodiment: the trophoblast organoids obtained in step S3 are digested into organoids of 50-100 μm by digestive enzymes; the digested organoids are mixed with matrix gel having a concentration of 20%-100% at a volume ratio of 1:10.

[0020] As a preferred embodiment: the extraembryonic early cells in step S1 are selected from at least one of human trophoblast stem cells, human trophoblast precursor cells, human stem cells, cells isolated from human early embryonic tissue, and cell sublines that can be genetically edited or tissue engineered.

[0021] A method for constructing a trophoblast organoid model constructs a trophoblast organoid model, wherein the trophoblast organoid model includes at least one of cytotrophoblast cells, syncytial trophoblast cells and extravillous trophoblast cells.

[0022] A method for evaluating the effect of a drug on trophoblast development during pregnancy includes the following steps:

[0023] First, the trophoblast organoid model was treated with the clinical drug or drug metabolite to be evaluated, which was defined as the drug-treated group, and a negative control group was set up at the same time;

[0024] Second, data was collected from cell samples in the trophoblast organoid model to obtain the evaluation index results of the drug-treated group and the control group, and the results were analyzed to evaluate the effect of the drug on trophoblast development.

[0025] As a preferred embodiment: the clinical drug or drug metabolite is used to treat the trophoblast organoid model as follows: the trophoblast organoid model is digested at 37°C, filtered through a 100μm cell sieve to make the proportion of organoid size between 50μm and 100μm greater than 50%, the digested trophoblast organoid is mixed with matrix gel to make the organoid density 200-500 cells / ul, the mixed solution is added to the well plate, the well plate is allowed to stand at 37°C for 10-40min to allow the matrix gel to completely solidify, culture medium containing different drug concentrations is added to each well plate, and then cultured at 37°C and 5% CO2.

[0026] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical solution that:

[0027] First, more accurate and predictive: Trophoblast organoids have a three-dimensional structure and are highly similar to the placenta in structure and function: Trophoblast organoids contain multiple trophoblast cell types, have a microvilli structure, and can secrete placenta-specific hormones, reproducing the key functional characteristics of the placenta, and are more in line with the actual situation of the human placenta than traditional animal models; when trophoblast organoids are used to simulate exposure to drugs, they can provide a more accurate and predictive model than traditional two-dimensional cell culture, accurately reflecting the effects of drugs on human placental tissue.

[0028] Second, cost reduction: The application of trophoblast organoids in the evaluation of drug placental toxicity can reduce the use of animal testing, effectively alleviate the ethical controversies and high costs caused by animal testing, and can quickly and effectively screen the placental toxicity of drugs, improve resource utilization efficiency, and shorten the research cycle.

[0029] Third, the trophoblast organoid model can self-organize into a structure with syncytial trophoblast (ST) cells and secrete human chorionic gonadotropin (hCG), exhibiting placental barrier function, and is closer to human placental tissue in structure and function.

[0030] Fourth, trophoblast organoids can further differentiate into extravillous trophoblast cells with invasive characteristics, which can be further used in drug transport research and new drug development.

[0031] Fifth, the trophoblast organoid model is used to study the effects of different pregnancy drugs on trophoblast development. The cell morphology changes collected from drug-treated organoids, the activity changes of organoids, and the identification data of gene expression levels are all from the same sample, reducing batch differences; filling the gap between the current 2D / 3D cell models and animal models, helping to improve the understanding of drug-placental toxicity, and providing scientific basis and effective strategies for protecting maternal and child health and rational use of drugs.

[0032] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the H&E staining results of trophoblast organoids in Example 1 of the present invention;

[0034] Figure 2 This is a schematic diagram of the immunofluorescence staining results of the trophoblast organoids prepared in Example 1 of the present invention;

[0035] Figure 3 This is a schematic diagram of the expression level detection results of trophoblast marker genes of trophoblast organoids in Example 1 of the present invention;

[0036] Figure 4 This is a schematic diagram of the transmission electron microscopy scanning results of the trophoblast organoid in Example 1 of the present invention;

[0037] Figure 5 This is a schematic diagram of the qualitative results of detecting hCG-β in the supernatant of the culture medium after the trophoblast organoids were cultured in the culture medium for 7 days according to Example 1 of the present invention;

[0038] Figure 6 This is a schematic diagram of the observation of the morphological changes of the trophoblast organoids induced into extravillous trophoblast cells in Example 2 of the present invention;

[0039] Figure 7 This is a schematic diagram of the immunofluorescence staining results of the extravillous trophoblast cells induced in Example 2 of the present invention;

[0040] Figure 8 This is an optical microscopic morphological picture of the trophoblast organoids in Example 3 of the present invention after being treated with drugs for 48 hours;

[0041] Fig. 9 This is a schematic diagram of the results of viability detection of trophoblast organoids after 48 hours of drug treatment according to Example 3 of the present invention;

[0042] Fig.10 This is a schematic diagram of the qPCR test results of trophoblast organoids after drug treatment for 48 hours in Example 3 of the present invention;

[0043] Fig.11 This is an optical microscopic morphological image of the trophoblast organoids in Example 4 of the present invention after being treated with drugs for 8 days;

[0044] Fig.12 This is a graph showing the results of viability detection of trophoblastic organoids after 8 days of drug treatment in Example 4 of the present invention;

[0045] Fig.13This is a graph showing the qPCR test results of trophoblast organoids after 8 days of drug treatment in Example 4 of the present invention;

[0046] Fig.14 This is a flow chart of a method for constructing a trophoblast organoid model of the present invention. DETAILED DESCRIPTION

[0047] The present invention Figures 1 to 14 As shown, a method for constructing a trophoblast organoid model comprises the following steps:

[0048] S1. Isolation of trophoblast cells: Aseptically separate the placental villus tissue, add mixed digestive enzymes to the placental villus tissue to separate single cells, and centrifuge to obtain extraembryonic early cells;

[0049] S2, Trophoblast Organoid Differentiation Stage 1: Mix extraembryonic early cells with matrix gel, perform 3D culture using the hanging drop method, and culture using a culture medium containing a pre-differentiation composition to obtain trophoblast organoids containing cytotrophoblast cells and syncytial trophoblast cells;

[0050] S3. Trophoblast organoid differentiation stage 2: Digest the trophoblast organoids obtained in step S2, mix the digested organoids with matrix gel, re-prepare 3D organoids using the hanging drop method, and continue culturing using a culture medium containing a late differentiation composition to obtain a trophoblast organoid model containing extravillous trophoblast cells.

[0051] The pre-differentiation composition in step S2 includes a basal culture medium and additives, the basal culture medium is a DMEM / F12 culture medium, and the additives include 5%-20% fetal bovine serum, 1x N2 supplement, 1xB-27without vitamin A, 1x glutamine, 80-150μg / ml Primocin, 1μM-1mM N-acetylcysteine, 0.1-100μM A83-01, 1-10μM CHIR99021, 5-20uM prostaglandin-E2, 10-500ng / ml R-Spondin, 10-500ng / ml EGF, 10-500ng / ml FGF2, 10-500ng / ml HGF, and 1-200μM Y-27632.

[0052] In step S3, the late differentiation composition includes a basal culture medium and additives, wherein the basal culture medium is DMEM / F12 culture medium, and the additives include 1%-10% serum replacement, 0.1%-1% BSA, 1%-10% ITS-X additive, 1x penicillin / streptomycin, 0.1-1mM β-mercaptoethanol, 0.1-100μM A83-01 and 10-500ng / ml NRG1.

[0053] The mixed digestive enzyme in step S1 includes 0.25% trypsin-EDTA and collagenase IV.

[0054] In step S2, the extraembryonic early cells are mixed with the matrix gel at a volume ratio of 1:10.

[0055] The trophoblast organoids obtained in step S3 are digested into organoids of 50-100 μm using digestive enzymes; the digested organoids are mixed with matrix gel having a concentration of 20%-100% at a volume ratio of 1:10.

[0056] In step S1, the extraembryonic early cells are selected from at least one of human trophoblast stem cells, human trophoblast precursor cells, human stem cells, cells isolated from human early embryonic tissues, and cell sublines that can be genetically edited or tissue engineered.

[0057] A method for constructing a trophoblast organoid model constructs a trophoblast organoid model, wherein the trophoblast organoid model includes at least one of cytotrophoblast cells, syncytial trophoblast cells and extravillous trophoblast cells.

[0058] A method for evaluating the effect of a drug on trophoblast development during pregnancy includes the following steps:

[0059] First, the trophoblast organoid model was treated with the clinical drug or drug metabolite to be evaluated, which was defined as the drug-treated group, and a negative control group was set up at the same time;

[0060] Second, data was collected from cell samples in the trophoblast organoid model to obtain the evaluation index results of the drug-treated group and the control group, and the results were analyzed to evaluate the effect of the drug on trophoblast development.

[0061] Clinical drugs and drug metabolites include one or more antihypertensive drugs, diabetes treatment drugs, immunomodulatory drugs, and other drugs for the treatment of pregnancy-related complications. These drugs include beta-blockers, vasodilators, diuretics, biguanides, and sulfonylureas.

[0062] The responses and changes of cells after drug treatment are evaluated by sampling and testing of cell culture medium, including cell viability reagents, morphological observation of trophoblast organoids, and identification of gene expression levels.

[0063] Testing is performed using cell viability detection instruments, microscopes, and organoid imaging equipment; evaluation indicators include the degree of organoid morphological destruction, organoid viability, and related indicators of trophoblast stemness and developmental differentiation.

[0064] The clinical drug or drug metabolite processes the trophoblast organoid model as follows: the trophoblast organoid model is digested at 37°C, filtered through a 100μm cell sieve so that the proportion of organoid size between 50μm and 100μm is greater than 50%, the digested trophoblast organoid is mixed with matrix gel to make the organoid density 200-500 / ul, the mixed solution is added to a well plate, the well plate is allowed to stand at 37°C for 10-40 minutes to allow the matrix gel to completely solidify, culture medium containing different drug concentrations is added to each well plate, and then the well plate is cultured at 37°C and 5% CO2.

[0065] Unless otherwise specified, the reagents and raw materials used in the present invention were purchased from commercial channels or are publicly available.

[0066] Example 1: Cultivation of trophoblast organoids

[0067] 1. Under sterile conditions, cut the chorionic tissue between the decidua and amniotic layers of the placenta tissue, wash it repeatedly 3 times with sterile isotonic saline, and process it within 1 hour.

[0068] 2. Transfer the placental villi sample to a new culture dish, remove any visible blood clots with forceps, be careful not to damage the villi, and then cut the tissue into 1-3mm pieces. 3 .

[0069] 3. Add 0.25% trypsin and 0.1 mg / ml collagenase IV to the chopped tissue in a volume ratio of 1:2 to form a digestion solution, and digest it in three times. Digest for 20 minutes each time in a constant temperature shaker at 37°C and 100 r / min.

[0070] 4. The digestion fluids from the first two times were filtered through medical sterile gauze and discarded, and the remaining tissues were digested for the third time. The serum digestion was terminated, and the digestion fluid was collected after filtration through a sieve. Centrifuged at 4°C, 500×g for 5 min. After centrifugation, the supernatant was discarded and the cells were resuspended with 3 ml of DMEM / F12 (Gibco#12634-010) containing 10% FBS, and filtered through a 100 μm cell sieve.

[0071] In a 5.15 ml sterile centrifuge tube, layer 3 ml of Percoll separation solutions of 70%, 50%, 30%, and 10% in descending order of concentration.

[0072] 6. Then slowly add 3 ml of cell suspension to the upper layer of Percoll separation solution, centrifuge at 4°C, 700×g for 30 min, and adjust the acceleration and deceleration speed to 1.

[0073] 7. Use a pipette to suck out 30%-50% of the cloudy cell layer (trophoblast cells), transfer it to a sterile centrifuge tube, resuspend the cells in 3 ml of DMEM / F12 containing 10% FBS, and centrifuge at 4°C, 500×g for 5 minutes.

[0074] 8. Observe whether the red color in the cell pellet is obvious after discarding the supernatant. If it is obvious, it means that there are many red blood cells mixed in. You need to add 1ml RBC Lysis Buffer (eBioscience TM #00-4333-57) were lysed on ice for 5-10 min, and lysis was terminated by adding 10% FBS in DMEM / F12 of 5x lysis system. The cells were centrifuged at 4°C, 500×g for 5 min, and the supernatant was discarded.

[0075] 9. Add growth factor (-) Matrigel (Corning #356231) dissolved on ice at 10x volume based on the volume of the cell pellet.

[0076] 10. Pipette 10 μl / drop into the center of a well of a 96-well culture plate (Costar#3548).

[0077] 11. After standing in a 37°C, 5% CO2 incubator for 15 min, 100 μl of the culture medium of the early differentiation composition - trophoblast organoid medium (TOM) was added to each well to cover the cell suspension.

[0078] 12. Change the medium every 2-3 days, and organoids can be observed in 7 days.

[0079] 13. The trophoblast organoid model obtained by the above culture was subjected to H&E staining, immunofluorescence staining, qPCR and electron microscopy. Figure 1 As shown in Figure 2, the trophoblast organoid model has a tissue structure similar to that of placental villi; immunofluorescence staining is shown in Figure 2. Figure 2 As shown in Figure 2, the cell components include cytotrophoblast (EPCAM+ / Ki67+ cells) and syncytiotrophoblast (SDC1+ cells). Figure 3 As shown in Figure 2, the expression levels of trophoblast marker genes in trophoblast organoids are similar to those in placental villi. Figure 4As shown in Figure 2, trophoblast organoids can be observed to have multinucleated features consistent with the characteristics of syncytial trophoblasts and have microvilli. The culture supernatant was collected and identified by human chorionic gonadotropin diagnostic reagent (latex method). The results are shown in Figure 2. Figure 5 As shown, the trophoblast organoid culture supernatant contains a certain amount of hCG-β.

[0080] 14. Compared with 2D cell systems and animal models, trophoblast organoids are closer to placental tissue in structure and physiology, and are more suitable as an in vitro research model for the placenta.

[0081] Example 2: Induced differentiation of EVT

[0082] 1. Passage cells when at least 50% of the trophoblast organoids reach 200-300 μm in diameter (usually between 7-10 days).

[0083] 2. After removing the culture medium, add -100 μl of dispase with a working concentration of 1 mg / ml to the wells, place in a humidified incubator at 37°C, and incubate for 15-20 min.

[0084] 3. Adjust the range of the 100μl pipette to 80μl and gently blow the organoids to break down the organoids and gel, transfer them to an EP tube, and let them stand at room temperature for 5 minutes.

[0085] 4. Add an equal volume of 0.1 M EDTA solution to neutralize dispase, then add 5 times the volume of DMEM / F12, centrifuge at 500×g for 5 minutes at room temperature; discard the supernatant.

[0086] 5. Add 500 μl of preheated (37°C) TrypLE Express dissociation reagent, place in a 37°C humidified incubator, and incubate for 15-20 min.

[0087] 6. Use medium force with a pipette to blow 50-100 times, observe under a microscope to dissociate the organoids to 100μm, add 1ml DMEM / F12, centrifuge at 500×g for 5min at room temperature, and discard the supernatant.

[0088] 7. Passage at a ratio of 1:2, resuspend the organoid pellet with TOM, and add matrix gel to a final concentration of 50%, drop 10μl / drop into the wells of a 6-well culture plate, and after the gel drop solidifies, add 2ml of TOM for culture.

[0089] 8. After 2 days of TOM culture, the culture medium was replaced with a culture medium containing a late differentiation composition - EVTM#1 for induction culture for 2-3 days, and then replaced with EVTM#2 (EVTM#1 without NRG1) for another 2-3 days of induction. It can be observed that trophoblast spherical organoids form at the edges and extend outward, with invasive extravillous trophoblast cells (EVT).

[0090] 9. Identify the EVT induced above. Figure 6 As shown in the figure, after trophoblast organoids were induced and cultured, migrating cells were observed around the organoid spheres. Immunofluorescence staining further proved that after trophoblast organoids were exposed to EVTM, they could differentiate into HLA-G+ and HLA-A / B / C+ EVTs with invasive characteristics. The results are shown in Figure 7 shown.

[0091] 10. The process of trophoblast differentiation into EVT is crucial for correct placental formation. EVT expresses HLA-G and invades decidual tissue to transform spiral arteries, ensuring adequate blood supply during pregnancy. The construction of the EVT model helps to understand the drug transport mechanism in the placenta and can also evaluate the potential effects of drugs on the fetus.

[0092] Example 3: Effects of short-term drug treatment on trophoblast development

[0093] This embodiment provides a method for rapidly evaluating placental toxicity caused by short-term drug exposure using a trophoblast organoid model. The application in this embodiment 3 is based on the product obtained from the differentiation stage 1 of the trophoblast organoid, but the organoid model used in this application is not limited to using only the product of the differentiation stage 1. Specifically, the following steps are included:

[0094] 1. Observation of Trophoblast Organoid Morphology

[0095] (1) Referring to the steps of Example 2, after removing the matrigel from the passaged organoids, Tryple Express was added to digest the trophoblast organoids at 37°C for 3 min. Gently pipette up and down to lyse large organoids (so that the cell clusters with a diameter of more than 50 μm are more than 50%).

[0096] (2) After counting the organoids and centrifuging at 500 × g for 5 min, the undiluted digested organoids were resuspended in matrigel and seeded into a 96-well plate at 10 μl / drop, with 3,000 cell clusters per well. The plates were incubated upside down at 37°C for 20-30 min, and then 100 μM TOM was added to each well and cultured at 37°C in 5% CO2 for two days.

[0097] (3) After 2 days of culture, on the 3rd day of passage, considering that the effect of clinical drugs on the development and differentiation of trophoblast cells was evaluated, a series of concentration gradients were set based on the blood concentration of clinical drugs to ensure that the experimental results were consistent with the actual treatment situation (the blood concentration of dydrogesterone was 16nM, and the blood concentration of methylprednisolone was 1.2μM). Set the drug concentration gradient: prepare the dydrogesterone stock solution with DMSO and dilute it to 10nM, 20nM, 50nM, 100nM, 200nM with TOM medium; prepare the methylprednisolone stock solution with DMSO and dilute it to 0.1μM, 1μM, 10μM, 50μM, 100μM with TOM medium; add the TOM medium containing different concentrations to the trophoblast organoids and treat for 48h.

[0098] (4) Observe the changes in organoid morphology using the Countstar Castor S1 high-throughput intelligent 3D cell analyzer.

[0099] (5) Experimental results are as follows Fig. 9 As shown: After treatment with dydrogesterone and methylprednisolone for 48 hours respectively, with the increase of drugs, the organoids treated with dydrogesterone showed a normal state, while the degree of morphological damage of the organoids treated with methylprednisolone deepened with the increase of drug concentration.

[0100] 2. Detection of organoid viability

[0101] (1)Use bioGenous LivingCell-Fluo TM The Organoid Vitality Assay Kit detects cell proliferation and survival, and then evaluates the effects of exposure to pregnancy medications on trophoblast organoid cell viability and proliferation capacity.

[0102] (2) After 48 h of drug treatment, the cell plate was removed from the incubator, the original culture medium was removed, 100 μl of 1× organoid viability assay working solution was added to each well, and the cells were incubated in a light-proof 37°C cell culture incubator for 60-120 min.

[0103] (3) After the incubation, the fluorescence intensity of each well was measured using a multifunctional microplate reader (EnVision multi-label microplate reader) (excitation Ex / emission Em wavelength: 560 (±10) Ex / 590 (±10) Em).

[0104] (4) Analyze the relative viability of organoids based on the total fluorescence intensity value (minus the dye negative control).

[0105] (5) 48 hours after administration of the 3D organoids, cell viability was evaluated by detecting cell metabolic capacity. The fluorescent dye in the reagent can freely enter and exit living cells, react with metabolic reductases in living cells to be reduced to a red strong fluorescent product. The activity of the organoids is reflected by detecting the fluorescent signal, where the intensity of the fluorescent signal is proportional to the number of living cells in the system. The experimental results are as follows: Fig.10 As shown, compared with the negative control group, both dydrogesterone and methylprednisolone reduced the activity of trophoblast organoids, and methylprednisolone downregulated the activity of organoids in a dose-dependent manner. The results suggested that near the blood drug concentration, both dydrogesterone and methylprednisolone would damage the activity of trophoblast organoids.

[0106] 3. Expression of Trophoblast Marker Genes

[0107] (1) After 48 hours of drug treatment, after performing the organoid viability test in step 3.2, aspirate the organoid viability test working solution and then add dispase to dissociate the Matrigel.

[0108] (2) RNA was then extracted from organoids according to the procedures of the HiPure Total RNA Plus Micro Kit (MAGEN#R4122-02).

[0109] (3) The gene expression levels of trophoblast organoids were identified by qPCR: TP63, GATA3 (stemness marker genes of trophoblast cells), CDH1 (marker gene of epithelial cells), CGB, SDC-1, ERVW1, GCM1 (marker genes of different subtypes of trophoblast cells).

[0110] (4) Experimental results are as follows Fig.10 As shown in the data, after short-term drug treatment, compared with the control group, dydrogesterone specifically reduced the expression of the trophoblast fusion gene GCM1, and the results suggested that it might affect the development and function of syncytiotrophoblast cells; and the short-term treatment results of different concentrations of methylprednisolone also showed that the expression of trophoblast stemness gene TP63, epithelial cell marker gene CDH1 and trophoblast marker genes (CGB and SDC1) decreased in a dose-dependent manner, and the expression of ERVW1 gene, which plays a key role in the formation of placental syncytiotrophoblast, showed a phenomenon that low concentration promoted the expression and high concentration inhibited the expression.

[0111] Example 4: Effects of long-term drug treatment on trophoblast development

[0112] This embodiment provides a method for rapidly evaluating placental toxicity caused by long-term drug exposure using a trophoblast organoid model. The application in this embodiment 4 is based on the product obtained at the differentiation stage 1 of the trophoblast organoid, but the organoid model used in this application is not limited to using only the product at the differentiation stage 1. The following steps are included:

[0113] 1. Observation of Trophoblast Organoid Morphology

[0114] (1) Referring to the steps of Example 2, after removing the matrigel from the passaged organoids, Tryple Express was added to digest the trophoblast organoids at 37°C for 3 min. Gently pipette up and down to lyse large organoids (so that the cell clusters with a diameter of more than 50 μm are more than 50%).

[0115] (2) After counting the organoids and centrifuging at 500 × g for 5 min, the undiluted digested organoids were resuspended in matrigel and seeded into a 96-well plate at 10 μl / drop, with 3,000 cell clusters per well. The plates were incubated upside down at 37°C for 20-30 min, and then 100 μM TOM was added to each well and cultured at 37°C in 5% CO2 for two days.

[0116] (3) After 2 days of culture, on the 3rd day of passage, considering that the effect of clinical drugs on the development and differentiation of trophoblast cells was evaluated, a series of concentration gradients were set based on the blood concentrations of clinical drugs to ensure that the experimental results were consistent with the actual treatment situation (the blood concentration of dydrogesterone was 16nM, and the blood concentration of methylprednisolone was 1.2μM). Set the drug concentration gradient: prepare the dydrogesterone stock solution with DMSO and dilute it to 10nM, 20nM, 50nM, 100nM, 200nM with TOM medium; prepare the methylprednisolone stock solution with DMSO and dilute it to 0.1μM, 1μM, 10μM, 50μM, 100μM with TOM medium; add the TOM medium containing different concentrations to the trophoblast organoids for 8 days, and replace it with fresh medium containing different concentrations of drugs every 2 days.

[0117] (4) Observe the changes in organoid morphology using the Countstar Castor S1 high-throughput intelligent 3D cell analyzer.

[0118] (5) The results are as follows Fig.11 As shown, after 8 days of treatment with dydrogesterone and methylprednisolone respectively, the organoids treated with different concentrations of dydrogesterone all showed normal growth, while the degree of morphological destruction and apoptosis of the organoids treated with methylprednisolone deepened with the increase of drug concentration.

[0119] 2. Detection of organoid viability

[0120] (1)Use bioGenous LivingCell-Fluo TM The Organoid Vitality Assay Kit detects cell proliferation and survival, and then evaluates the effects of exposure to pregnancy medications on trophoblast organoid cell viability and proliferation capacity.

[0121] (2) After 8 days of drug treatment, the cell plate was removed from the incubator, the original culture medium was removed, 100 μl of 1× organoid viability assay working solution was added to each well, and the cells were incubated in a light-proof 37°C cell culture incubator for 60-120 min.

[0122] (3) After the incubation, the fluorescence intensity of each well was measured using a multifunctional microplate reader (EnVision multi-label microplate reader) (excitation Ex / emission Em wavelength: 560 (±10) Ex / 590 (±10) Em).

[0123] (4) Analyze the relative viability of organoids based on the total fluorescence intensity value (minus the dye negative control).

[0124] (5) Experimental results are as follows Fig.12 As shown, 8 days after administration of 3D organoids, compared with the negative control group, dydrogesterone had a smaller effect on the activity of 3D organoids, while methylprednisolone induced apoptosis of trophoblast organoids in a dose-dependent manner.

[0125] 3. Expression of Trophoblast Marker Genes

[0126] (1) After 8 days of drug treatment, after performing the organoid viability test in step 4.2, aspirate the organoid viability test working solution and then add dispase to dissociate the Matrigel.

[0127] (2) RNA was then extracted from organoids according to the procedures of the HiPure Total RNA Plus Micro Kit (MAGEN#R4122-02).

[0128] (3) The gene expression levels of trophoblast organoids were identified by qPCR: TP63, GATA3 (stemness marker genes of trophoblast cells), CDH1 (marker gene of epithelial cells), CGB, SDC-1, ERVW1, GCM1 (marker genes of different subtypes of trophoblast cells).

[0129] (4) Experimental results are as follows Figure 8As shown in the figure: Long-term dydrogesterone treatment will lead to significant changes in the stemness genes and corresponding trophoblast marker genes of trophoblast organoids. At low concentrations, the trophoblast marker genes of organoids showed an up-regulated trend, while high-dose dydrogesterone treatment led to the down-regulation of trophoblast stemness genes and trophoblast marker genes; and the detection results of long-term treatment with different concentrations of methylprednisolone also showed that the expression of trophoblast stemness genes GATA3 and TP63, epithelial cell marker gene CDH1 and trophoblast marker gene (ERVW1) decreased in a dose-dependent manner, the expression levels of SDC1 and GCM1 increased in a dose-dependent manner, and the expression of CGB gene showed a phenomenon that low concentration promoted expression and high concentration inhibited expression.

[0130] comprehensive Figure 8-Figure 10 As shown, the present invention studied the effects of short-term and long-term treatments of dydrogesterone at different concentrations on trophoblast organoids. The results showed that the growth morphology of the organoids was normal, short-term treatment moderately reduced the activity, and long-term treatment had little effect on the activity. Short-term treatment specifically reduced GCM1 expression, while long-term treatment upregulated stemness genes and marker genes at low concentrations and downregulated them at high concentrations. This shows that the use of dydrogesterone during pregnancy has complex effects on trophoblast organoid function and gene expression, and its biological effects need to be carefully considered to ensure safety and effectiveness.

[0131] Figures 11 to 13 It is shown that the present invention studies the effects of short-term and long-term treatment with different concentrations of methylprednisolone on trophoblast organoids. The results show that at 10 μM, methylprednisolone causes organoid apoptosis and morphological disintegration, and the degree increases with concentration and time. Methylprednisolone induces apoptosis in a dose-dependent manner. Under short-term treatment, methylprednisolone causes a dose-dependent decrease in the expression of TP63, CDH1, CGB, and SDC1 genes, while the ERVW1 gene is promoted at low concentrations and inhibited at high concentrations. Long-term treatment shows that the expression of GATA3, TP63, CDH1, and ERVW1 decreases, the expression of SDC1 and GCM1 increases, and the expression of CGB is promoted at low concentrations and inhibited at high concentrations. This shows that the use of methylprednisolone during pregnancy significantly and complexly affects the function and gene expression of trophoblast organoids, and may affect placental development, so its biological effects should be carefully considered when used.

[0132] The design emphasis of the present invention is:

[0133] First, more accurate and predictive: Trophoblast organoids have a three-dimensional structure and are highly similar to the placenta in structure and function: Trophoblast organoids contain multiple trophoblast cell types, have a microvilli structure, and can secrete placenta-specific hormones, reproducing the key functional characteristics of the placenta, and are more in line with the actual situation of the human placenta than traditional animal models; when trophoblast organoids are used to simulate exposure to drugs, they can provide a more accurate and predictive model than traditional two-dimensional cell culture, accurately reflecting the effects of drugs on human placental tissue.

[0134] Second, cost reduction: The application of trophoblast organoids in the evaluation of drug placental toxicity can reduce the use of animal testing, effectively alleviate the ethical controversies and high costs caused by animal testing, and can quickly and effectively screen the placental toxicity of drugs, improve resource utilization efficiency, and shorten the research cycle.

[0135] Third, the trophoblast organoid model can self-organize into a structure with syncytial trophoblast (ST) cells and secrete human chorionic gonadotropin (hCG), exhibiting placental barrier function, and is closer to human placental tissue in structure and function.

[0136] Fourth, trophoblast organoids can further differentiate into extravillous trophoblast cells with invasive characteristics, which can be further used in drug transport and new drug development.

[0137] Fifth, the trophoblast organoid model is used to study the effects of different pregnancy drugs on trophoblast development. The cell morphology changes, organoid activity and gene expression level identification collected from drug-treated organoids are all from the same sample, reducing batch differences; filling the gap between the current 2D / 3D cell models and animal models, helping to improve the understanding of drug-placental toxicity, and providing scientific basis and effective strategies for protecting maternal and child health and rational use of drugs.

[0138] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for constructing a trophoblast organoid model, characterized in that: The steps include: S1. Isolation of trophoblast cells: Aseptically separate the placental villus tissue, add mixed digestive enzymes to the placental villus tissue to separate single cells, and centrifuge to obtain extraembryonic early cells; S2, Trophoblast Organoid Differentiation Stage 1: Mix extraembryonic early cells with matrix gel, perform 3D culture using the hanging drop method, and culture using a culture medium containing a pre-differentiation composition to obtain trophoblast organoids containing cytotrophoblast cells and syncytial trophoblast cells; S3. Trophoblast organoid differentiation stage 2: Digest the trophoblast organoids obtained in step S2, mix the digested organoids with matrix gel, re-prepare 3D organoids using the hanging drop method, and continue culturing using a culture medium containing a late differentiation composition to obtain a trophoblast organoid model containing extravillous trophoblast cells.

2. The method for constructing a trophoblast organoid model according to claim 1, characterized in that: The pre-differentiation composition in step S2 includes a basal culture medium and additives, wherein the basal culture medium is a DMEM / F12 culture medium, and the additives include 5%-20% fetal bovine serum, 1x N2 supplement, 1xB-27without vitamin A, 1x glutamine, 80-150μg / ml Primocin, 1μM-1mM N-acetylcysteine, 0.1-100μM A83-01, 1-10μM CHIR99021, 5-20uM prostaglandin-E2, 10-500ng / ml R-Spondin, 10-500ng / ml EGF, 10-500ng / ml FGF2, 10-500ng / ml HGF, and 1-200μM Y-27632.

3. The method for constructing a trophoblast organoid model according to claim 1, characterized in that: The late differentiation composition in step S3 includes a basal culture medium and additives, wherein the basal culture medium is DMEM / F12 culture medium, and the additives include 1%-10% serum replacement, 0.1%-1% BSA, 1%-10% ITS-X additive, 1x penicillin / streptomycin, 0.1-1mM β-mercaptoethanol, 0.1-100μM A83-01 and 10-500ng / ml NRG1.

4. The method for constructing a trophoblast organoid model according to claim 1, characterized in that: The mixed digestive enzyme in step S1 includes 0.25% trypsin-EDTA and collagenase IV.

5. The method for constructing a trophoblast organoid model according to claim 1, characterized in that: In step S2, the extraembryonic early cells are mixed with the matrix gel at a volume ratio of 1:

10.

6. The method for constructing a trophoblast organoid model according to claim 1, characterized in that: The trophoblast organoids obtained in step S3 are digested into organoids of 50-100 μm using digestive enzymes; the digested organoids are mixed with matrix gel having a concentration of 20%-100% at a volume ratio of 1:

10.

7. The method for constructing a trophoblast organoid model according to claim 1, characterized in that: In step S1, the extraembryonic early cells are selected from at least one of human trophoblast stem cells, human trophoblast precursor cells, human stem cells, cells isolated from human early embryonic tissues, and cell sublines that can be genetically edited or tissue engineered.

8. A trophoblast organoid model constructed according to the method for constructing a trophoblast organoid model according to any one of claims 1 to 7, characterized in that: The trophoblast organoid model includes at least one of cytotrophoblast cells, syncytiotrophoblast cells and extravillous trophoblast cells.

9. Use of the trophoblast organoid model according to claim 8 in evaluating the effect of drugs on trophoblast development during pregnancy, characterized in that: The steps include: First, the trophoblast organoid model was treated with the clinical drug or drug metabolite to be evaluated, which was defined as the drug-treated group, and a negative control group was set up at the same time; Second, data was collected from cell samples in the trophoblast organoid model to obtain the evaluation index results of the drug-treated group and the control group, and the results were analyzed to evaluate the effect of the drug on trophoblast development.

10. The use according to claim 9, characterized in that: The clinical drug or drug metabolite is used to treat the trophoblast organoid model as follows: the trophoblast organoid model is digested at 37°C, filtered through a 100μm cell sieve to make the proportion of organoid size between 50μm and 100μm greater than 50%, the digested trophoblast organoid is mixed with matrix gel to make the organoid density 200-500 / ul, the mixed solution is added to a well plate, the well plate is allowed to stand at 37°C for 10-40min to allow the matrix gel to completely solidify, a culture medium containing different drug concentrations is added to each well plate, and then the well plate is cultured at 37°C and 5% CO2.

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