A method for constructing a placenta organoid model

Abstract

The application provides a construction method of a placenta organoid model, belongs to the field of biology and medicine, and particularly relates to early placenta development and organoid research. The construction method comprises extracting and purifying bovine placenta trophoblast cells, obtaining an immortalized bovine placenta trophoblast cell line, and constructing an immortalized bovine placenta organoid. The application constructs the placenta organoid by using the immortalized bovine placenta trophoblast cell line, and the function is consistent compared with traditional primary cells, but the defects of difficulty in obtaining the placenta and insufficient amount of primary cells are overcome, the cell line used in the application can be taken without limit, and the construction cost is low, so that the application can be popularized and applied in most animal organoid constructions.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of biology and medicine, and in particular to a method for constructing a placenta organoid model. BACKGROUND

[0002] Recently, placental trophoblast organoids have been increasingly used in the study of maternal-fetal interaction in human placenta. Generally, the related researches use the cell clusters enriched with EPCAM+ cells prepared from primary trophoblast cells by enzymatic digestion as the cell clusters, which are inoculated into the Matrigel and can grow in the placental trophoblast organoid culture solution. After the first passage, the organoid structure appears, and the trophoblast organoid with uniform cell distribution can be established after 2 passages (10-14 days). Mononuclear cytotrophoblast cells (CTB cells) and multinucleated syncytiotrophoblast cells (STB) formed by the fusion of CTB cells can be detected in the organoid, and then the EVT cells are successfully differentiated using EVT differentiation medium (EVTM). The trophoblast organoid is highly similar to the placental villus tissue in structure and phenotype.

[0003] However, the culture of placental trophoblast cells is difficult. The placenta in the early stage of pregnancy is selected as the test material, at which time the trophoblast cells are strong in vitality and easy to grow. The placenta gradually ages with the increase of the gestational month, and at this time the trophoblast cells also age, and the cell number is small and the vitality is low, so it is difficult to obtain the primary trophoblast cells with stable properties required for the test. Moreover, the cells used to culture the placental organoid can only be the cell clusters rich in EPCAM+ cells from the placental primary cells, and the acquisition of the cells is difficult and the identification is also difficult.

[0004] Therefore, it is necessary to obtain a method for constructing an organoid, which is simple in cell acquisition operation, has a large number of cells, and can be widely applied in most animal organoid constructions. SUMMARY

[0005] The present application aims to provide a method for constructing a placental organoid model, which uses immortalized bovine placental trophoblast cell lines to construct an organoid similar to the placental tissue of a 3-month pregnant bovine, and the organoid has the same function as the organoid constructed by traditional primary cells, but the method can overcome the defects of difficulty in obtaining placenta and insufficient amount of primary cells.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a method for constructing a placental organoid model, which comprises the following steps:

[0008] (1) The purified bovine placental trophoblast cells are transfected by pCI-neo-hTERT plasmid and cultured to obtain an immortalized bovine placental trophoblast cell line;

[0009] (2) The obtained immortalized bovine placental trophoblast cell line was washed in culture medium, centrifuged, and resuspended in Matrigel;

[0010] (3) Adding TOM culture medium to the matrix gel mixed with the immortalized bovine placental trophoblast cell line for culturing, thereby obtaining immortalized bovine placental organoids.

[0011] Preferably, the culture medium in step (2) is Advanced DMEM / F12 culture medium; and the matrigel is a liquid matrigel.

[0012] Preferably, the volume ratio of the immortalized bovine placental trophoblast cell line to the matrix gel in step (2) is 1:(0.5-2).

[0013] Preferably, the matrix gel mixed with the immortalized bovine placental trophoblast cell line in step (3) further includes a coagulation treatment before adding TOM culture medium, the coagulation treatment temperature is 32-39° C., and the coagulation treatment time is 10-20 min.

[0014] Preferably, in step (3), the volume ratio of the matrix gel mixed with the immortalized bovine placental trophoblast cell line to the TOM culture medium is 1:(2-4).

[0015] Preferably, the temperature during the cultivation process in step (3) is 32-39° C., the relative saturated humidity during the cultivation process is 90-96%, and the CO 2 content during the cultivation process is 3-6%.

[0016] Preferably, the formula of the TOM culture medium in step (3) is:

[0017]

[0018] By adopting the above technical solution, the present invention has the following beneficial effects:

[0019] 1. In the present invention, cow placental trophoblast cells are used as host cells, transfected with the pCI-neo-hTERT eukaryotic expression vector, and the immortalized cow placental trophoblast cell line obtained by G418 screening has good homogeneity, strong stability and is easy to culture.

[0020] 2. The present invention uses an immortalized bovine placental trophoblast cell line to successfully construct organoids similar to bovine placental tissue at the third month of pregnancy, which have the same function as organoids constructed from traditional primary cells.

[0021] 3. The parameter conditions corresponding to the construction steps described in the present invention are obtained through research. Only under the specific conditions of the present invention can organoids grow normally and healthily.

[0022] 4. The placenta organoid construction method of the present application overcomes the defects of difficult access to placenta and insufficient amount of primary cells, and has low construction cost, and can be popularized and applied in most animal organoid construction. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Purified BTCs morphological characteristics (scale = 1 mm);

[0024] Figure 2 Immunofluorescence detection of CK-7, vimentin, E-cadherin and CD90 expression in primary BTCs (x40, scale = 50 μm);

[0025] Figure 3 Immunofluorescence detection of CK-7, vimentin, E-cadherin and CD90 expression in FBFs (x40, scale = 50 μm);

[0026] Figure 4 Giemsa staining results of bovine placental trophoblast cells;

[0027] Figure 5 Expression levels of TERT at protein and mRNA;

[0028] Figure 6 Expression of marker proteins in bovine placental trophoblast cells;

[0029] Figure 7 Growth curve of BTCs:

[0030] Figure 8 PL secretion ability of BTCs;

[0031] Figure 9 Migration ability of BTCs (x40, scale = 50 μm);

[0032] Figure 10 Results of soft agar assay for cell tumorigenicity;

[0033] Figure 11 HE staining of bovine placental trophoblast tissue structure (A, B, C show HE staining results of placental trophoblast tissue of pregnant cows for 2, 3 and 4 months, respectively; black arrows mark trophoblast binucleate cells);

[0034] Figure 12 Comparison of Ki67 protein expression in bovine placental trophoblast tissue at 2, 3 and 4 months of pregnancy;

[0035] Figure 13 Growth morphology of primary cells to generate organoids;

[0036] Figure 14Generating organoid growth morphology for immortalized BTCs;

[0037] Figure 15 CSH1 protein expression and localization of bovine placental trophoblast tissue and organoids (A in the figure is the three-month pregnant trophoblast tissue; B in the figure is the primary cell-derived organoid; C in the figure is the BTCs-derived organoid);

[0038] Figure 16 CD71, CD46 protein expression and localization of bovine placental trophoblast tissue and organoids (A in the figure is the three-month pregnant trophoblast tissue; B in the figure is the primary cell-derived organoid; C in the figure is the BTCs-derived organoid);

[0039] Figure 17 Differential expression of receptivity-related factors before and after co-culture of EECs and organoids (A in the figure is the WB detection result of αVβ3, wnt7a, HOXA10, IFNAR-1 and IFNAR-2 before and after co-culture; B in the figure is the gray scale analysis result of the WB result figure, wherein Control represents the protein expression amount of EECs before co-culture, Co-culure1 represents the protein expression amount of EECs after co-culture with the primary cell-derived organoid, and Co-culure2 represents the protein expression amount of EECs after co-culture with the BTCs-derived organoid);

[0040] Figure 18 Placental organoid development and RNA sequencing analysis of placental tissue development in the second and third months of pregnancy. DETAILED DESCRIPTION

[0041] The application provides a construction method of a placental organoid model, comprising the following steps:

[0042] (1) The purified bovine placental trophoblast cells are transfected by pCI-neo-hTERT plasmid and cultured to obtain an immortalized bovine placental trophoblast cell line;

[0043] (2) The obtained immortalized bovine placental trophoblast cell line is washed in a culture medium, centrifuged and resuspended in Matrigel;

[0044] (3) The Matrigel mixed with the immortalized bovine placental trophoblast cell line is added with TOM culture solution for culture, and thus an immortalized placental organoid is obtained.

[0045] In the application, the bovine placental trophoblast cells are collected from the early pregnancy Holstein cow uterus in the 45-90d of pregnancy.

[0046] In the application, the purified bovine placental trophoblast cells are added at 1×10 5The cells are transferred into a six-well plate, and after 12 hours, when the cell density is 80%-90%, the cells can be transfected. The specific operation of the transfection is as follows: the plasmid and the lipofectamine 2000 transfection reagent are diluted with serum-free OPTI-MEM, then the diluted plasmid is added to the diluted transfection reagent, and then the mixed plasmid and transfection reagent are added to the six-well plate and placed in a CO2 incubator for further culture. The addition process is slow and dropwise. After 3-4 hours of culture, the liquid is replaced with DMEM / F12 complete medium containing 400 mg / mLG418 for screening, and the screening is continued for two weeks, so as to screen the immortalized bovine placental trophoblast cell line.

[0047] In the present application, the obtained immortalized bovine placental trophoblast cell line is washed once in Advanced DMEM / F12 medium, centrifuged at 200-350g for 2-8min, and resuspended in Matrigel on ice with a pre-cooled pipette tip. The further preferred centrifugal force is 250-320g, and the further preferred centrifugal force is 300g; the further preferred centrifugal time is 3-7min, and the further preferred centrifugal time is 5min. The volume ratio of the immortalized bovine placental trophoblast cell line and Matrigel is 1:(0.5-2), further preferably 1:(0.8-1.5), and more preferably 1:1. The Matrigel needs to be placed in a 4°C environment until it is in a liquid state; the pipette tip is pre-cooled on ice, and the pre-cooled Matrigel is liquid at low temperature and solidifies into a solid at high temperature, so as to ensure that the Matrigel is in a liquid state.

[0048] In the present application, the Matrigel mixed with the immortalized bovine placental trophoblast cell line is added dropwise to a pre-cooled 96-well plate, and preferably 40-60μL, further preferably 45-55μL, and more preferably 50μL is added to each well, and the cell amount in each well is controlled to be 2×10 2 Then, the plate is placed in a culture incubator for solidification treatment, and the temperature of the solidification treatment is preferably 32-39°C, further preferably 35-38°C, and more preferably 37°C; the time of the solidification treatment is preferably 10-20min, further preferably 12-17min, and more preferably 15min.

[0049] In the present application, the substrate gel mixed with immortalized bovine placental trophoblast cell line after coagulation is added with preheated TOM culture solution per hole, and the volume ratio of the substrate gel mixed with immortalized bovine placental trophoblast cell line to the TOM culture solution is preferably 1:(2-4), further preferably 1:(2.5-3.5), and more preferably 1:3. Then, it is placed in a culture box for culture, and the culture solution is replaced every 3 days, the temperature of the culture is preferably 32-39℃, further preferably 35-38℃, and more preferably 37℃; the relative saturated humidity of the culture is preferably 90-96%, further preferably 93-95.5%, and more preferably 95%; and the CO2 content during the culture is preferably 3-6%, further preferably 4-5.5%, and more preferably 5%.

[0050] In the present application, the formula of the TOM culture solution is as follows:

[0051]

[0052]

[0053] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the present application.

[0054] Example 1 Isolation and purification of bovine placental trophoblast primary cells

[0055] (I) Acquisition of bovine placental trophoblast primary cells

[0056] This experiment was conducted in accordance with the guidelines of the Animal Ethics Committee of Beijing Agricultural University. The uteruses of early pregnant Holstein cows at 45-90 days of pregnancy were collected, and the fetal bovine crown-hip length was 7 cm. The uterine surface was washed with calcium magnesium water containing 1% double-antibody, and the uterus was opened under sterile conditions and the placenta was separated from the uterine caruncle. The placental tissue was first soaked in sterile DPBS buffer containing 2% double-antibody, then soaked in 75% alcohol for 15-30 s, and immediately washed in DPBS buffer. The placental tissue was cut into 1 mm 3 tissue blocks with sterile surgical scissors, inoculated into 10 cm 2 culture dishes, and cultured in a 5% CO2, 37℃ incubator for 30 min until the tissue blocks adhered to the culture dishes. Then, DMEM / F12 culture medium containing 1% double-antibody and 10% EXO-FBS was added, and the culture was carried out in a 37℃, 5% CO2 incubator. Cell replacement was performed at 24 h and 48 h, and then the culture medium was replaced every 3 days until the cells were observed to crawl out under an inverted microscope.

[0057] (II) Purification of bovine placental trophoblast primary cells

[0058] Discard the culture supernatant, wash twice with DPBS, then add trypsin for digestion. Observe the cells to become round and partially float. Add complete DMEM / F12 medium to terminate the digestion, and use a pipette to blow the cells to make them all float. Collect the cells into a 15 mL centrifuge tube, centrifuge at 1500 r / min for 5 min, discard the supernatant, resuspend the cells with complete medium, reseed into a culture bottle, and culture for 20 min. Then aspirate the supernatant and seed into a new culture bottle. Repeat this subculture for 5 times to obtain a higher purity of bovine placental trophoblast cells (e.g. Figure 1 ).

[0059] Example 2: Identification of purified bovine placental trophoblast cells

[0060] (I) Immunofluorescence identification

[0061] Before plating, place the cell crawl sheet at the bottom of the 24-well plate. Take the cell crawl sheet out of 75% alcohol, and place it in the cell plate after the alcohol evaporates. Seed bovine placental trophoblast cells into the 24-well plate at 1 x 10 5 cells per well, and seed fetal bovine fibroblasts (FBF) into the 24-well plate as a positive control of fibroblast-like cells. Culture for 10-12 h, and observe the cell density under a microscope to be 80%-90%. Discard the culture supernatant and wash with PBS three times. Fix the cells with 4% paraformaldehyde for 40 min at room temperature, discard the fixing solution and wash with PBS three times. Add 0.1% Trition X-100 for 30 min at room temperature, wash with PBS three times, add blocking solution (1% BSA-PBS) for 1 h at room temperature, discard the blocking solution, and add 200 μL of CK-7 antibody (1:100), vimentin antibody (1:100), E-cadherin antibody (1:100), and CD90 antibody (1:100) per well according to the experimental design. Place the culture plate in a humid box, and incubate overnight at 4°C. Wash with PBS three times, add FITC-labeled secondary antibody (1:50), place in a humid box, and incubate at 37°C for 1 h in the dark. Wash with PBS three times, add DAPI, and incubate for 5 min in the dark. Wash with PBS three times. Take 5 μL of anti-fluorescence quenching mounting medium, and add it to a clean glass slide. Take the cell crawl sheet out of the cell plate, with the cell face downward, and observe under a fluorescence microscope and take a photo.

[0062] Use the same volume of PBS instead of the primary antibody to incubate the cells as a negative control, and the rest of the steps are the same as above. The results are shown in Figures 2-3It can be seen that the protein expression of CK-7, Vimentin, CD90 / Thy1 and E-Cadherin in BTCs and FBFs was detected respectively, and PBS was used as a negative control. It was found that the expression rates of CK-7, Vimentin and E-Cadherin proteins in BTCs were higher than 95%, and the fibroblast marker protein CD90 / Thy1 was not expressed. Vimentin, CD90 / Thy1 and E-Cadherin were expressed in FBFs, and the placental trophoblast marker protein CK-7 was not expressed.

[0063] (ii) Giemsa staining

[0064] Before plating, the cell crawl sheet was placed at the bottom of a 24-well plate, and the bovine placental trophoblast cells were subcultured into the 24-well plate at 1 x 10 4 cells per well. After 10-12 h of culture, the cell density was observed under a microscope to be 50%-60%, the culture supernatant was discarded, and the cells were washed with PBS three times. The cells were fixed with 4% paraformaldehyde for 40 min, the fixing solution was discarded, and the cells were washed with PBS three times. Giemsa staining solution I was diluted 10 times with PBS, and then was added dropwise to the crawl sheet. After 10 min of staining, the staining solution I was aspirated and washed with PBS three times. Then, Giemsa staining solution II was diluted 10 times with PBS, and then was added dropwise to the crawl sheet. After 10 min of staining, the staining solution II was aspirated and washed with PBS three times. The cell crawl sheet was taken out of the cell plate, and was placed with the cell side facing down. Then, the cell crawl sheet was observed under an inverted optical microscope.

[0065] From Figure 4 It can be seen that the primary cells have large nuclei, and a small number of cells are binucleated or multinucleated, which can communicate with each other through their extended pseudopods.

[0066] Example 3: Establishment of immortalized bovine placental trophoblast cell lines

[0067] (i) Determination of the optimal screening concentration of G418

[0068] The purified bovine placental trophoblast cells were inoculated in a 96-well plate, and when the cell confluence reached 80%, complete culture medium containing different concentrations of G418 (20 μM, 40 μM, 80 μM, 100 μM, 200 μM, 400 μM, 800 μM, 1000 μM) was added for culture, and the medium was replaced every 3 days. The cell state was observed. It was found that the cells completely died when treated with G418 at a concentration higher than 400 μM for 12-15 days, and the cell death rate gradually increased when treated with G418 at a concentration ranging from 20 μM to 400 μM for 12-15 days. Therefore, the G418 concentration corresponding to the complete death of the cells after 12-15 days of treatment was the optimal concentration of G418.

[0069] (2) pCI-neo-hTERT plasmid transfection of primary bovine placental trophoblast cells

[0070] The pCI-neo-hTERT plasmid was extracted according to the operation steps of the Endo-free plasmid midikit produced by OMEGA company.

[0071] (1) Cell plating: bovine placental trophoblast cells were added to a six-well plate at about 1 x 10 5 cells per well, and after 12 h, the cell density should be 80%-90% before transfection.

[0072] (2) Preparation of plasmid DNA and transfection reagent complex: in a 1.5 mL centrifuge tube, prepare a 1.5 mL sterile centrifuge tube, dilute the plasmid and transfection reagent respectively. Then add 250 μL of serum-free OPTI-MEM to each centrifuge tube. Then add 4 μg of plasmid and 10 μL of lipofectamine 2000 transfection reagent to the centrifuge tube. Add slowly, drop by drop. Incubate at room temperature for 5 min. After incubation, slowly drop the diluted plasmid into the diluted transfection reagent and incubate at room temperature for 20 min. Label the six-well plate with each experimental group, and slowly drop the mixed plasmid DNA and transfection reagent into the six-well plate. Shake gently, then place in a CO2 incubator for further culture. After 4 h, replace with DMEM / F12 complete medium containing 400 mg / mL G418 for selection, for two weeks. After two weeks, the cells that can survive are positive cells, which are digested and inoculated into a 6 mm culture dish with DMEM / F12 complete medium.

[0073] (3) Continuous passage for more than 50 times.

[0074] Example 4 Expression of exogenous hTERT gene in transfected bovine placental trophoblast cells

[0075] (1) Primer design

[0076] (1) Primer design

[0077] The sequence of the required gene was searched on NCBI, and the fluorescent quantitative primer was designed by using the NCBIPrimer-BLAST tool. The primer was synthesized by Shanghai Shengong. The primer sequence of the gene hTERT included SEQ ID NO. 1: 5'-TATGCCGTGGTCCAGAAGG-3' and SEQ ID NO. 2: 5'-CAAGAAATCATCCACCAAACG-3'; the primer sequence of the gene GAPDH included SEQ ID NO. 3: 5'-CGGCACAGTCAAGGCAGAGAAC-3' and SEQ ID NO. 4: 5'-CCACATACTCAGCACCAGCATCAC-3'. The specific information of the related primer was shown in Table 1.

[0078] Table 1 primer sequence

[0079]

[0080] (2) Synthesis of cDNA

[0081] a. Extraction of RNA

[0082] The TRIzol extraction reagent was used for extraction, and the specific steps were as follows:

[0083] The cells were cultured in a 6mm culture dish, the cell culture liquid in the culture dish was discarded, and the culture medium was washed twice with 2-3mL of pre-cooled PBS, and the PBS was finally sucked clean. 1mL of TRIzol Reagent was added to the culture dish, the cells were scraped from the culture dish with a cell scraper, and were simply blown and added to a 1.5mL centrifuge tube, and were placed at room temperature for 5min. 200μL of chloroform was added to each centrifuge tube, and was shaken up and down quickly, and was shaken vigorously for 15s, and was placed at room temperature for 5min;

[0084] 12000r / min, 4℃ centrifugation for 15min, after centrifugation, the layers were separated, the upper colorless transparent aqueous phase was sucked and placed in a clean 1.5mL centrifuge tube, 500μL of isopropanol was added, and after mixing, it was placed at room temperature for 10min;

[0085] 12000r / min, 4℃ centrifugation for 10min, after centrifugation, the bottom of the tube was observed, the supernatant was carefully discarded, 1mL of 75% ethanol was added, 7500r / min, 4℃ centrifugation for 5min, and the supernatant was discarded. The centrifuge tube was inverted on a clean bench, and was air-dried at room temperature for 15min, until the water vapor and ethanol at the bottom of the centrifuge tube were completely volatilized, 30-40μL of DEPC water was used to dissolve the RNA at the bottom of the tube, and the concentration and purity of the RNA were determined.

[0086] b. The reverse transcription is specifically operated as follows: according to the HiFi-MMMLV cDNA first strand synthesis kit instruction (the kit is purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), respectively take dNTP Mix 4 μL, Primer Mix 2 μL, RNA Template 3 μL, 5×RT Buffer 4 μL, 1×DTT 2 μL, HiFi-MmlV 1 μL, place in PCR instrument, RNase-Free Water 4 μL, supplement system to 20 μL, vortex mixing, centrifuge briefly, 42℃ 45 min, 85℃ 5 min, get mRNA reverse transcription product cDNA, -80℃ preservation.

[0087] (3) RT-PCR amplification

[0088] According to the UltraSYBR one-step fluorescence quantitative PCR kit instruction (the kit is purchased from Beijing Bai'ao Leibo Technology Co., Ltd.), take 2×UltraSYBR Mixture 12.5 μL, each 0.5 μL of upper and lower primers, Template DNA 2 μL, ddH2O 4.5 μL in an RT-qPCR tube, the reaction condition is 95℃ 30 s, 95℃ 5 s, 56℃ 30 s, 95℃ 15 s, 60℃ 1 min, 95℃ 15 s, a total of 30 cycles; Agilent Aria1.5 software is used to analyze data, and the relative expression of genes is calculated according to 2 -ΔΔCt Method.

[0089] (2) Western blot detection of TERT protein expression in transfected bovine placental trophoblast cells

[0090] The fifth generation of primary bovine placental trophoblast cells, the 30th and 50th generations of transfected bovine placental trophoblast cells and HeLa cells are collected respectively, and the cell lysate is mixed with PMSF at a ratio of 100:1 and placed on ice for standby; wash the cells with 1 mL of pre-cooled PBS for 2-3 times, and aspirate the last time; add 150 μL of cell lysate to each well to lyse the cells. After adding, place the cell culture plate on ice for 30 min, and use a pipette to transfer the protein sample to a pre-cooled 1.5 mL centrifuge tube; 12000 r / min, 4℃ centrifuge for 20 min. After centrifugation, carefully aspirate the supernatant and transfer it to another pre-cooled centrifuge tube. Take 50 μL of protein for BCA protein concentration determination. The rest of the protein is added to 5×protein loading buffer in proportion, mixed uniformly. Boil the protein in a metal bath for 10 min, place it on ice until it cools to room temperature, mix uniformly, and then dispense. Label, and the protein can be stored in a -80℃ refrigerator.

[0091] (1) SDS-PAGE electrophoresis: use a pipette to add the separation gel into the glass plate, add to 2 / 3, and use anhydrous ethanol for liquid sealing, use the 12% concentration of the separation gel formula in Table 2 to prepare 15 mL. After waiting for 20-30 min for the separation gel to solidify, prepare 5% concentrated gel (as shown in Table 3).

[0092] Table 2 12% separation gel formula

[0093]

[0094]

[0095] Table 3 5% concentrated gel formula

[0096]

[0097] (2) Transferring film: the film transfer instrument parameter is set to 200 mA, and the film transfer time is 1.5 h.

[0098] (3) Blocking: after film transfer, put the PVDF film into a plastic self-sealing bag with 5% skimmed milk, and place it on a shaker for 2 h, and adjust the rotation speed to 40 r / min.

[0099] (4) Primary antibody incubation: after blocking is completed, wash the PVDF film with PBST for 2 times, 5 min each time. Place it on a shaker for washing, and the rotation speed is about 60 r / min. According to the Marker band, cut the target protein of the corresponding size. In the antibody incubation box, incubate the primary antibody overnight with hTERT antibody (1:1000) and GAPDH (1:2000) antibody, respectively.

[0100] (5) Secondary antibody incubation: the next day, recover the primary antibody, and wash the PVDF film with PBST for 3 times, 30 min each time. After washing, incubate with HRP-labeled secondary antibody of the species of the primary antibody, and the dilution of the secondary antibody is 1:3000. Incubate on a shaker at room temperature for 2 h.

[0101] (6) Color development and exposure: after the secondary antibody incubation is completed, recover the secondary antibody, store it at 4°C, wash it with PBST for 3 times, 10 min each time. Prepare the exposure solution by mixing the ECL chemiluminescence kit, A and B liquids at a ratio of 1:1, and use it immediately. Place the protein band on the exposure instrument, add an appropriate amount of exposure solution to fully wet the PVDF film, and perform exposure. Save the picture and use ImageJ software for gray value analysis.

[0102] The BTC cells (placental trophoblast cells) were immortalized by introducing exogenous TERT into the cells, and the stable expression strains were screened by G418 for two weeks. The TERT expression in different generations of BTC cells was detected. No large-scale aging or death of the BTC cells with introduced TERT was observed after continuous subculture for more than 50 generations. Figure 5 As shown in FIG. 2, the TERT protein expression in the cells was detected by Western blot method. It was found that the TERT had only a very low level of expression in the cells without introduced exogenous TERT, while the TERT protein expression in the cells with introduced TERT and continuously subcultured for 30 generations or 50 generations was detected at a higher level, which was significantly different (P<0.01) from the primary BTCs (placental trophoblast cell line) and was higher than the known Hela immortal cells expressing TERT protein. The TERT mRNA expression in the cells was detected by RT-qPCR, and the results were consistent with the protein expression. The results showed that the BTC cells could stably express TERT protein at a high level by introducing exogenous TERT, and the BTC cells were immortalized.

[0103] (Three) Western blot detection of marker proteins in bovine placental trophoblast cells

[0104] The marker proteins in bovine placental trophoblast cells were detected by Western blot method using CK-7 antibody (1:1000), Vimentin antibody (1:1000), E-cadherin antibody (1:1000), and CD90 antibody (1:1000), and the specific operation method was the same as above (Two). The results are shown in FIG. 3. Figure 6

[0105] Example 5 Biological characteristics of bovine placental trophoblast cells

[0106] (One) Cell growth curve

[0107] The cell suspensions of the 5th generation of primary bovine trophoblast cells and the 50th generation of bovine placental trophoblast cells after transfection were prepared, and the cell counting was performed by a blood cell counter. 1×10 4 cells per well were inoculated into a 96-well plate, 3 parallel holes were set at each time point, and a blank control hole was set by adding only cell culture medium. PBS was added around the periphery of the hole with added culture medium to prevent liquid evaporation in the outermost circle of holes. After incubation in a 37°C incubator for 4h, the cell suspension was discarded, and 110μL of complete culture medium containing 10% CCK-8 was added. The 96-well plate was returned to the 37°C incubator for continuous culture for 1, 2, 4, 6, 8, and 10 days. The absorbance value at 450nm was measured at each time point. The calculation formula of relative cell viability is as follows:​

[0108]

[0109] Results show that, compared with primary BTCs, the 50th generation of BTCs after transfection significantly improves the growth rate of BTCs under the action of TERT (see Figure 7 ).

[0110] (II) ELISA detection of PL secretion of bovine placental trophoblast cells

[0111] Prepare cell suspensions of the 5th generation of primary bovine trophoblast cells and the 50th generation of bovine placental trophoblast cells after transfection, and count the cells with a blood cell counter. 1 x 10 5 cells per well were inoculated into a 6-well plate, and after 24 h the supernatant was discarded, washed once with PBS, and 500 μL of serum-free DMEM-F12 medium was added. The culture was continued at 37°C for 48 h, after which the cell culture supernatant was collected, and the relevant data OD was determined according to the instructions. Serum-free medium was used as a negative control, and the above test was repeated.

[0112] According to Figure 8 It can be seen that TERT-BTCs have similar PL hormone secretion ability as primary cells.

[0113] (III) Detection of the migration characteristics of bovine placental trophoblast cells

[0114] Transwell experiment was used to detect whether TERT-BTCs have migration ability. Cell suspensions of the 5th generation of primary bovine trophoblast cells and the 50th generation of bovine placental trophoblast cells and Hela cells were prepared, and the cells were counted with a blood cell counter. 1 x 10 4 cells per well were inoculated onto Matrigel-coated Transwell cell chambers, and 500 μL of complete medium was added to the lower chamber. The culture dish was incubated at 37°C in a 5% carbon dioxide atmosphere for 24 h. Non-invasive cells on the upper surface were removed with a cotton swab, and the lower surface of the basement membrane was fixed with 4% paraformaldehyde for 30 min. Then, the basement was washed with PBS three times and stained with crystal violet. The cells were observed and photographed under an inverted optical microscope.

[0115] The results are shown in Figure 9 Hela cervical cancer cells with migration ability were used as a positive control, and it was found that both primary BTCs and immortal TERT-BTCs had a certain migration ability.

[0116] (IV) Detection of malignant transformation characteristics of bovine placental trophoblast cells after transfection

[0117] Soft agar colony formation test: 2 mL of DMEM / F12 medium containing 1.2% soft agar was added to a six-well plate as the bottom layer and then placed at 4°C until the agar solidified. Subsequently, 5×10 4 Bovine placental trophoblast cells and HeLa cells at passage 50 post-transfection were resuspended and plated into the wells. The cells were incubated overnight at 37°C in a 5% CO2 incubator. They were treated with complete medium once a week and colony growth was assessed over the next two weeks. Cell clumps larger than 100 μm were considered a single colony and photographed using an inverted light microscope.

[0118] from Figure 10 It can be seen that immortalized TERT-BTCs are not tumorigenic in vitro.

[0119] Example 6 Construction of cow placental organoids using primary placental trophoblast cells and immortalized cell lines

[0120] The following experimental reagents are required in this example, as shown in Table 4:

[0121] Table 4 Experimental reagents

[0122]

[0123]

[0124] (1) Acquisition and culture of primary placental trophoblast cells

[0125] 1. Extraction of bovine placenta in the second, third and fourth months of pregnancy

[0126] This was also conducted in accordance with the guidance of the Animal Ethics Committee of Beijing Agricultural University. All dairy cows were from slaughterhouses and confirmed to be free of pregnancy-related diseases such as mastitis, placental development disorders, intrauterine growth retardation, and preeclampsia before slaughter. The uteri of pregnant cows were then collected from the slaughtered cows. The uteri of the 2nd month (55-65 days, fetal body length 2-3 cm), 3rd month (85-95 days, fetal body length 7-8 cm), and 4th month (115-125 days, fetal body length 15-18 cm) of pregnancy were selected based on the fetal head-neck ratio and body length. They were sent to the laboratory as soon as possible for follow-up work and kept fresh on ice during transportation. The uterus was first rinsed with clean water until there was no blood or foreign matter, then rinsed with calcium magnesium water (with 1% double antibody) 4 times, rinsed with 75% alcohol once, and then rinsed with calcium magnesium water again to control the moisture. The uterus was then carefully opened along the posterior center to expose the fetal membranes, and the integrity of the chorion was maintained as much as possible. Carefully peel off the placental villi, trim them, and place them in 4% paraformaldehyde for later use.

[0127] 2. Preparation of Bovine Placenta Paraffin Sections

[0128] (1) Trimming: The fixed placental trophoblast tissue was taken out from 4% paraformaldehyde and trimmed into small pieces of 0.5 cm with scissors and surgical blades.

[0129] (2) Dehydration: Ethanol gradient dehydration was performed.

[0130] (3) Transparency: The tissue pieces were taken out from alcohol and put into xylene for 10 min x 2 times.

[0131] (4) Wax immersion and embedding: The trophoblast tissue pieces were completely immersed into melted paraffin and then placed in a melting paraffin box to prevent paraffin solidification. Then the melted paraffin was poured into an embedding box, and the paraffin- immersed tissue pieces were quickly picked up with forceps and placed in liquid paraffin. Finally, the embedding box was placed on a quick-freezing table to solidify the paraffin.

[0132] (5) Sectioning: First, the wax block was trimmed to expose the tissue to the cutting surface, and then the trimmed wax block was fixed on a microtome for sectioning with a thickness of 5 μm.

[0133] (6) Flopping: The cut sections were gently spread and picked up with forceps and then floated in a flopper with 40 °C warm water to flatten the tissue, and the glass slide picked up the tissue sections (note that the sections should not be wrinkled).

[0134] (7) Baking: The sections were baked in a 60 °C oven. After the wax was baked, the sections were taken out and placed at room temperature.

[0135] 3. HE Staining

[0136] (1) De-waxing: The sections were placed in xylene for 10 min and then taken out to remove the paraffin in the sections.

[0137] (2) Rehydration: After alcohol gradient treatment, the sections were washed with distilled water for 2 min.

[0138] (3) Nucleus staining: The sections were placed in hematoxylin staining solution for 6-8 min and then washed with tap water. Then the sections were immersed in 1% hydrochloric acid alcohol for 25-30 s and finally washed with running water to blue.

[0139] (4) Cytoplasm staining: The sections were placed in 1% eosin staining solution for 1-2 min.

[0140] (5) Dehydration and transparency: The same as the dehydration and transparency steps in the preparation of paraffin sections.

[0141] (6) Mounting: The melted neutral resin was added to the sections, and then a cover glass was placed to fix it.

[0142] (7) Observation under a microscope and taking pictures.

[0143] By Figure 11It can be seen that the trophoblast tissue of bovine placenta in the early pregnancy contains mononuclear cells and binucleate cells. There is no obvious stratification boundary between mononuclear cells and binucleate cells, and they are arranged alternately in a ratio of about 8:2, which is not affected by the length of pregnancy. The mononuclear cell nucleus is short columnar, spherical or irregular. There is a large nucleolus in the nucleus, and the chromatin is dispersed. In the binucleate cells, the immature binucleate cell nucleus is large, the nuclear-cytoplasmic ratio is large, contains multiple nucleoli and is lightly colored, and purple-red granules can be seen in the cytoplasm; the mature binucleate cell nucleus is small, round or oval and darkly colored, and a large number of deep purple granules can be seen in the cytoplasm.

[0144] 4. Immunohistochemical staining

[0145] (1) Dewaxing and rehydration: the test steps are the same as those in the HE staining part.

[0146] (2) Antigen repair: immerse the trophoblast tissue section in 0.01 mol of sodium citrate buffer, place it in a microwave oven and heat it to boiling, then switch to medium heat and heat it for another 10 min, then take it out and cool it to room temperature.

[0147] (4) Staining: use the immunohistochemical kit for subsequent staining.

[0148] ① Add 3% hydrogen peroxide to the tissue section and incubate for 5 min, then pour it off to eliminate endogenous peroxidase activity, and wash it with PBS for 5 min x 3 times.

[0149] ② Add reagent A (normal goat serum working solution for blocking) and incubate at room temperature for 15-20 min, then pour it off. Add the first antibody Ki67 (diluted at a ratio of 1:100) and incubate overnight at 4°C, then wash it with PBS for 3 min x 3 times.

[0150] ③ Add reagent B (biotin-labeled goat anti-rabbit, rat, mouse and guinea pig lgG) and incubate at room temperature for 15-20 min. Wash with PBS for 3 min x 3 times.

[0151] ④ Add reagent C (S-A / HRP) and incubate at room temperature for 15-20 min, then wash with PBS for 3 min x 3 times.

[0152] ⑤ Add DAB color developing agent (5% solution A + 5% solution B + 90% PBS) and stain for 30 s-2 min, then immerse the section in tap water.

[0153] ⑥ Add safranine to the section for 5 min, then wash the stain with tap water, then differentiate it with hydrochloric acid alcohol for 5 s, then immerse it in tap water for 5 min, change the water and immerse it for another 10 min, then observe the results under a microscope.

[0154] ⑦ Finally, dehydrate it with gradient alcohol, make it transparent with xylene, and then use neutral resin to mount it.

[0155] The results show that the proportion of the marker Ki67 in the trophoblast tissue of the 3-month placenta is significantly higher than that of the 4-month placenta and slightly higher than that of the 2-month placenta (Ki67 marks proliferative cells), as shown in Figure 12 .

[0156] 5. Primary culture of the trophoblast cells of the 3-month placenta

[0157] (1) Extraction of the trophoblast tissue of the bovine placenta: the steps are the same as in 1, but after the placental villus tissue is peeled off, instead of being put into 4% paraformaldehyde, it is put into DPBS (with 10% double-antibiotic) for 1 min, DPBS (with 5% double-antibiotic) for 1 min and DPBS (with 2% double-antibiotic) for 1 min, respectively. After washing, the trophoblast villus is scraped into small particles using a sterile scalpel.

[0158] (2) Adhesion culture of the tissue: the scraped trophoblast villus tissue is clamped using a sterilized forceps and placed on the bottom wall of a cell culture bottle for adhesion culture, with a 5-mm gap between the tissue blocks. After adhesion for 30 min, the tissue blocks are adhered to the bottom wall of the culture bottle without falling off, and preheated 10% serum-added DMEM / F12 and TOM culture medium are carefully added for culture, with the culture medium being replaced every three days.

[0159] (3) Observation and photographic analysis of the results: the cell culture results are observed and photographed under an inverted microscope.

[0160] (II) Immunofluorescence staining of the primary cells and immortalized BTCs

[0161] (1) Fixation of the primary cells: the coverslips are washed with pure water in advance and soaked in 75% alcohol. After the cells are suspended in DMEM+10% FBS culture medium, the coverslips are clamped out of the alcohol using a sterilized forceps, dried by the outer flame of an alcohol lamp and then put into a 24-well plate, with 1 ml of the cell suspension being added to each well, with the amount of cells in each well being controlled at about 2×10 5

[0162] (2) Fixation of the immortalized BTCs: the same as above.

[0163] (3) Permeation: the washed primary cells and BTCs are respectively dropped with 0.2% Trion-X100 for permeation for 10 min, and washed with PBS for 3 min×5 times.

[0164] (4) Blocking: blocking liquid (1% BSA-PBS) is respectively dropped for blocking for 30 min, and the liquid is discarded without washing.

[0165] ​(5) First antibody incubation: After dilution of the first antibody 1:200 (diluent is 0.1% BSA-PBS), add separately, incubate in wet box at 4°C overnight. Wash with PBS for 5 min x 5 times.

[0166] (6) Second antibody incubation: After dilution of the anti-rabbit FITC 1:200 (diluent is 0.1% BSA-PBS), add separately, incubate at room temperature for 1 h in the dark, wash with PBS for 5 min x 5 times.

[0167] (7) Nuclei staining: Add Hoechst 33342 separately, stain in the dark for 10 min, wash with PBS for 5 min x 3 times. Keep the last PBS, store in the dark.

[0168] (8) Photography: After adding the anti-fluorescence quencher to the primary cells and immortalized BTCs, and mounting the coverslips, take pictures using a confocal microscope.

[0169] (Three) Culture of placental trophoblast organoids

[0170] (1) Place the 96-well culture plate and the pipette tip on ice in advance, and take the Matrigel from -80°C and place it at 4°C until it becomes liquid.

[0171] (2) Wash the cultured trophoblast primary cells and immortalized BTCs in the Advanced DMEM / F12 medium, then centrifuge at 300g for 5 min using a low-speed centrifuge, and resuspend in Matrigel on ice using the pre-cooled tip, 0.5 mL of Matrigel is needed for every 0.5 mL of trophoblast primary cells or immortalized BTCs.

[0172] (3) Add 50 μL of mixed cell Matrigel (cell amount controlled at 2 x 10 2 per well) to each well of the 96-well plate. Place in a 37°C incubator for 15 min, then cover each well with 150 μL of preheated TOM medium after the Matrigel solidifies, and place in a 37°C humidified incubator with 5% CO2, change the culture medium every 3 days.

[0173] (4) Take pictures of the growth of the organoids on the 3rd, 7th, and 15th days of culture (see Figures 13-14 ).

[0174] Meanwhile, it was also detected that the trophoblast tissue at three months of pregnancy and the primary cell-derived organoids and the immortalized BTC-derived organoids all expressed CSH1 protein, and also expressed CD71 and CD46, two syncytial marker proteins, and the results are shown in Figures 15-16 .

[0175] (Four) Establishment of a co-culture system of trophoblast organoids and EECs

[0176] (1) EECs cell resuscitation: EECs were taken out from liquid nitrogen tank and placed in 37 °C water bath for thawing, and centrifuged at 1000 rpm for 5 min. After carefully discarding the supernatant, add DMEM / F12 (add 10% FBS) to resuspend the cells.

[0177] (2) EECs plating: remove the chamber in the Transwell nest (Corning, 3421), and plate the EECs suspension in the well plate, controlling the cell amount in each well to be about 2 x 10 5 .

[0178] (3) Organoid processing: remove the TOM culture solution in the primary culture organoids and BTCs culture organoids, respectively, and then add PBS and place at 4 °C for 3 min to dissolve the Matrigel. Take out the organoids and wash with PBS for 3 times.

[0179] (4) Organoid and EECs co-culture: place the primary culture organoids and BTCs organoids in the Transwell chamber above the EECs small holes, and add appropriate amount of culture solution in the chamber. Then place in the incubator for 36 h.

[0180] After co-culturing the two kinds of organoids, it was found that the protein expression of EECs receptivity regulating factors Integrin avb3, Wnt7a, HOXA10 and IFNAR-2 were significantly increased, with the same regulating effect (see Figure 17 ).

[0181] (Three) RNA sequencing analysis of placental organoids and placental tissue of different gestational months

[0182] (1) Extraction and quality control of total RNA: add Trizol to the placental organoids and gestational month 2 and 3 trophoblast tissues and shake for 3 min, and stand for 5 min on ice. Then add chloroform (5:1 ratio with Trizol) and shake for 15 s, and stand for 3 min on ice. Start the centrifuge in advance, set at 4 °C, 12000 r / min, 15 min. After aspirating the supernatant, place it in a new tube, add 0.5 mL isopropanol, mix well, and stand for 4 °C centrifugation, 14000 r / min, 10 min. After discarding the supernatant, wash twice with 85% ice ethanol, and then add 40 pL RNase-free Water and mix well, and then perform quality control. The results show that the absorbance ratio OD260 / 280 at 260 nm and 280 nm is between 1.8 and 2.0.

[0183] (2) Construction of sequencing library:

[0184] a. Isolation and purification of mRNA: use oligodT magnetic beads and poly(A) tail of mRNA to specifically bind to purify mRNA.

[0185] b. RNA fragmentation: the purified mRNA is fragmented using fragmentation reagent.

[0186] c. Synthesis of cDNA: using mRNA as a template, adding a six-base random primer, and synthesizing cDNA under the action of reverse transcriptase.

[0187] d. Double-stranded cDNA is end-repaired using Taq polymerase, then base A is added to the 3' end, a sequencing adapter is used to connect the two ends of the DNA fragment, and finally high-fidelity polymerase is used to amplify the library.

[0188] (3) Clustered DNA amplification: the DNA library is seeded onto a flowcell, and then PCR amplification is performed.

[0189] (4) Sequencing on machine

[0190] a. The double-stranded amplified DNA is changed into single-stranded, that is, a primer that can cut a specific group is added, and then an alkaline solution is added to separate the double-stranded to obtain single-stranded.

[0191] b. Start sequencing by adding sequencing primers. After preprocessing the sequencing result data, subsequent data analysis is performed.

[0192] It was found that the immortalized placental trophoblast cell organoid expressed common genes with the placental tissue of 3-month pregnancy, such as Figure 18 .

[0193] From the above examples, it can be seen that the present application provides a kind of placental organoid constructed by immortalized bovine placental trophoblast cell line, which is consistent with the function of the organoid obtained by traditional primary cell construction of placental organoid. However, the cell line described in the present application is very rich, which overcomes the defects of insufficient amount of primary cells and difficulty in obtaining, and the construction process is relatively simple and low in cost, which can be popularized and applied in most animal organoid construction.

[0194] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. A method of constructing a placental organoid model, characterized by, The method comprises the following steps: (1) transfecting and culturing the purified bovine placental trophoblast cells by pCI-neo-hTERT plasmid to obtain an immortalized bovine placental trophoblast cell line; (2) washing and centrifuging the obtained immortalized bovine placental trophoblast cell line in a culture medium, and resuspending it in Matrigel; (3) adding TOM culture solution to the Matrigel mixed with the immortalized bovine placental trophoblast cell line for culture, thereby obtaining an immortalized bovine placental organoid; The volume ratio of the Matrigel mixed with the immortalized bovine placental trophoblast cell line to the TOM culture solution in step (3) is 1:(2-4); The formula of the TOM culture solution in step (3) is: 1X Advanced DMEM / F12; 1X N2 supplement; 1X B27 supplement minus vitamin A; 1.25 mM N-Acetyl-L-cysteine; 2mM L-glutamine; 500 nM ALK-4,-5,-7inhibitor, A83-01; 1.5uM CHIR99021; 50ng / mL Recombinant human EGF; 80ng / mL ProDots Recombinant Human R-Spondin 1 Protein; 100 ng / mL bFGF2; 10 uL / mL Pen Strep.

2. The method of claim 1, wherein, The culture medium in step (2) is Advanced DMEM / F12 culture medium; and the Matrigel is Matrigel in a liquid state.

3. The method of claim 1, wherein, The volume ratio of the immortalized bovine placental trophoblast cell line to Matrigel in step (2) is 1:(0.5-2).

4. The method of claim 1, wherein, The Matrigel mixed with the immortalized bovine placental trophoblast cell line in step (3) further comprises a coagulation treatment before the addition of the TOM culture solution, the temperature of the coagulation treatment is 32-39℃, and the time of the coagulation treatment is 10-20min.

5. The method of claim 1, wherein, The temperature in the culture process in step (3) is 32-39℃, the relative saturated humidity in the culture process is 90-96%, and the CO2 content in the culture process is 3-6%.

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