Culture method of human placenta organoid in early pregnancy period
By improving the culture medium composition and culture steps, the problems of low spheroid formation rate and poor stability in human placental organoid culture have been solved, achieving efficient and stable culture of placental organoids, which is suitable for various molecular biology and cell biology research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-04
- Publication Date
- 2026-03-10
AI Technical Summary
Existing human placental organoid culture methods suffer from low spheroid formation rate, poor distribution, and difficulty in experimental observation. Furthermore, they are prone to empty apoptosis and premature disintegration during the culture process.
The modified culture medium composition, including the addition of Bovine serum albumin and increased concentrations of Y-27632 and Recombinant Human R-Spondin 1 Protein, was used to improve the purity of placental villus cells and the stability of the culture medium through specific culture steps such as cell digestion, centrifugation, and suspension culture.
This improved the spheroidization rate and culture distribution of placental organoids, reduced empty apoptosis and premature disintegration, ensured stable growth of organoids, and provided reliable experimental materials for subsequent scientific research.
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Figure CN121628816A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of biomedical technology, and relates to a culture method of human placenta organoids in early pregnancy. BACKGROUND
[0002] The human placenta is a channel for connecting and nutrient delivery between a female and a fetus after pregnancy, and poor development or abnormal differentiation of the placenta can cause serious pregnancy diseases such as preeclampsia and premature birth. In the research on the development of the placenta and related diseases, in vitro models such as animal disease models or passaged cell lines are often relied on. A common passaged trophoblast cell line cannot reproduce the in vivo situation due to its limitations, and the placenta organoid reproduces the structure of the placenta in the human body, which is of great significance for the scientific research on the development of the placenta and related diseases.
[0003] There are literatures reporting the culture method of human placenta organoids, but in actual culture operation, the culture distribution of the organoids is poor, which is not conducive to experimental observation and subsequent histological section identification. The culture method of the human placenta organoid is improved in the application, and the sphere formation rate and culture distribution of the placenta organoid are improved. At the same time, the human placenta organoid cannot be stably passaged in actual culture operation, and hollow apoptosis and early disintegration of the organoid occur during the culture process. The culture medium composition of the human placenta organoid is also improved in the application, and the growth state of the placenta organoid is improved and the passaging number is increased. SUMMARY
[0004] In order to solve the above problems, the application provides a culture method of human placenta organoids in early pregnancy.
[0005] The technical scheme of the application is as follows:
[0006] A culture method of human placenta organoids in early pregnancy, comprising the following steps:
[0007] (I) collecting 6-10 week placental villi for digestion, filtration and centrifugation to obtain a mixture of placental villi cells and blood cells;
[0008] (II) lysing red blood cells and centrifuging to obtain placental villi cells;
[0009] (III) after counting the placental villi cells, adding organoid culture medium ② to resuspend the cells, mixing the cells and matrigel at a ratio of 1:3 to make drops; after 2-5 min of culture in an inverted plate at 37 DEG C, placing the plate in a vertical position, continuing to place for 5-10 min, and then adding organoid culture medium ② for culture to obtain placenta organoids;
[0010] The composition of the organoid culture medium ② comprises:
[0011] The culture medium is prepared with DMEM / F12, with the addition of N-2 supplement; B-27 supplement; primary cell antibiotic; N-acetyl-L-cysteine; L-glutamine; recombinant human epidermal growth factor; CHIR99021; recombinant human R-spondin 1; recombinant human fibroblast growth factor; recombinant human hepatocyte growth factor; A83-01; prostaglandin E2; Y-27632; fetal bovine serum albumin.
[0012] Further, the specific steps for obtaining the placental villus cell and blood cell mixture in step (1) are as follows:
[0013] (1) Collect 6-10 week placental villi in culture medium ① containing DMEM / F12, N2, B27, Y-27632 cell factors, and store and transport at 0-4 degrees Celsius (preferably 4 degrees Celsius, which can be stored for 1-2 weeks at 4 degrees Celsius); low-temperature storage is conducive to preventing reagents from deteriorating or degrading.
[0014] (2) After scraping the placental villi cultured in step (1), digest them in 5-10 mL of 0.25% trypsin-0.02% EDTA in a metal bath at 37 degrees Celsius for 5-8 minutes.
[0015] (3) Filter the digestate using a 40-100 μm filter screen, and terminate the digestion with 20% FBS-DMEM / F12; preferably a 100 μm filter screen.
[0016] (4) Centrifuge at 400-600 g for 5 minutes at room temperature to obtain a placental villus cell and blood cell mixture.
[0017] Further, the method for obtaining the placental villus cell and blood cell mixture further includes the following steps:
[0018] (5) Add the residual tissue on the 40-100 μm filter screen to 10 mL of 1.0 mg / mL collagenase V in a metal bath and digest for 5-10 minutes; collagenase V is prepared with 10% FBS-DMEM / F12; preferably a 100 μm filter screen.
[0019] (6) Filter the digestate obtained in step (5) using a 40-100 μm filter screen, and centrifuge at 400-600 g for 5 minutes at room temperature to obtain a placental villus cell and blood cell mixture. Preferably, a 100 μm filter screen is used.
[0020] Further, the specific steps for obtaining the placental villus cell and blood cell mixture in step (1) are as follows:
[0021] (1) Collect 6-10 week placental villi in culture medium ① containing DMEM / F12, N2, B27, Y-27632 cell factors, and store and transport at 0-4 degrees Celsius (preferably 4 degrees Celsius, which can be stored for 1-2 weeks at 4 degrees Celsius); low-temperature storage is conducive to preventing reagents from deteriorating or degrading.
[0022] (2) After centrifugation, add 3-5 times of red blood cell lysis solution to lyse the red blood cells for 1-3 min, and then centrifuge at 400-600 g for 5 min at room temperature.
[0023] (3) Discard the supernatant, wash and resuspend the cells with DMEM / F12, and then centrifuge at 400-600 g for 5 min at room temperature to obtain placental villus cells.
[0024] Further, the step (three) is specifically:
[0025] (1) After counting the placental villus cells, resuspend the cells with an appropriate amount of organoid culture medium ② according to the number of villus cells, and ensure that the cells are mixed with Matrigel at a ratio of 1:3 to make drops, 25-35 microliters per drop in a 24 or 48 well culture plate;
[0026] (2) After 2-5 min of inverted culture plate culture at 37 degrees Celsius, place the culture plate vertically, continue to place for 5-15 min, and then add 250 μL-750 μL of organoid culture medium ② for culture;
[0027] (3) After 7-10 days of culture, 100-200 μm diameter placental organoids can be observed.
[0028] Further, the specific components of the culture medium ① are: DMEM / F12 medium 90%; N-2 additive 1%; B-27 additive 2%; primary cell antibiotic 100 ug / ml; Y-27632 0.5 μg / mL. (The amount of N-2 additive and B-27 additive added will change according to the total amount of the prepared culture medium)
[0029] Further, the composition of the medium ② includes: medium DMEM / F12, 85-90% (the volume percentage of the DMEM / F12 medium in the total volume of the medium is based on the total volume percentage, and the other components are 10-15%. Similarly, the volume percentages of N-2 additives and B-27 additives are also volume percentages), wherein N-2 additives, 1%; B-27 additives, 2%; primary cell antibiotics, 100ug / ml; N-acetyl-L-cysteine, 150-250ug / mL; L-glutamine, 200-300ug / mL; recombinant human epidermal growth factor, 20-100ng / ml; CHIR99021, 650-750ug / mL; recombinant human R-spondin 1, 150-250ng / ml; recombinant human fibroblast growth factor, 50-150ng / ml; recombinant human hepatocyte growth factor, 20-100ng / ml; A83-01, 150-250ng / mL; prostaglandin E2, 800-900ng / mL; Y-27632, 2-5ug / mL; Bovine serum albumin, 0.5-2ug / mL.
[0030] Further, the specific composition of the medium ② includes: medium DMEM / F12, 90%; wherein N-2 additives, 1%; B-27 additives, 2%; primary cell antibiotics, 100ug / ml; N-acetyl-L-cysteine, 204ug / mL; L-glutamine, 292ug / mL; recombinant human epidermal growth factor, 50ng / ml; CHIR99021, 698ug / mL; recombinant human R-spondin 1, 200ng / ml; recombinant human fibroblast growth factor, 100ng / ml; recombinant human hepatocyte growth factor, 50ng / ml; prostaglandin E2, 882ng / mL; Y-27632, 2.5ug / mL; Bovine serum albumin, 1ug / mL. (The percentage involved is volume percentage)
[0031] The application also provides a preparation method of a medium for culturing early pregnancy human placenta organoids, specifically including the following steps:
[0032] The recombinant human epidermal growth factor, recombinant human R-spinal protein 1, cytokines, recombinant human fibroblast growth factor, recombinant human hepatocyte growth factor in the medium components are prepared into a thousand-fold concentrated solution and stored at-80℃, and the N-2 additive, B-27 additive, primary cell antibiotic, L-glutamine, prostaglandin E2, Y-27632, A83-01, fetal bovine serum albumin and other additives are stored at-20℃; when the medium is prepared, the medium components are added one by one in the DMEM / F12 basic medium according to the above-mentioned proportions, and the medium for the early pregnancy human placenta organoid is obtained.
[0033] Further, the prepared medium for the early pregnancy human placenta organoid can be stored at 4℃ for 1-2 weeks.
[0034] The application further provides an early pregnancy human placenta organoid cultured by the culture method of the early pregnancy human placenta organoid.
[0035] The application further provides an application of the medium for the early pregnancy human placenta organoid in culturing a human placenta organoid.
[0036] The medium of the application is prepared as follows: the cytokines and the like in the medium components are prepared into a thousand-fold concentrated solution and stored at a suitable temperature, and when the medium is prepared, the medium components are added one by one in the DMEM / F12 basic medium (the order of addition is not required), and the prepared complete organoid culture medium can be stored at 4℃ for 1-2 weeks.
[0037] The medium for the early pregnancy human placenta organoid has the following functions:
[0038]
[0039]
[0040] The improved culture medium of the application adds a Bovine serum albumin component and increases the concentration of Y-27632 and Recombinant Human R-Spondin 1 Protein, effectively reducing the empty apoptosis and early disintegration of human placental organoids. Due to the rapid degradation of cytokines in the organoid culture medium, it is recommended to use within 1-2 weeks after preparation, and the added Bovine serum albumin component plays a role in protecting cytokines from degradation; in addition, the added Y27632 is an anti-apoptotic inhibitor that reduces early apoptosis of organoids during subculture and culture; R-Spondin 1 is an enhancer of the WNT signaling pathway, which is reported to play an important role in maintaining cell stemness, and increasing the concentration of R-Spondin 1 is beneficial to the maintenance of placental organoid stemness and stable culture.
[0041] The application improves the medium components of human placental organoids, and the obtained human placental organoids have better growth state, better spheroid formation rate, and reduced apoptosis of organoids, laying a foundation for further application in various molecular biology and cell biology research.
[0042] Beneficial technical effects:
[0043] The application improves the culture method of human placental organoids, improves the spheroid formation rate of placental organoids and improves the culture distribution of organoids, which is beneficial to experimental observation and subsequent histological section identification. The application improves the culture medium and culture method of human placental organoids, which is simple to prepare, improves the success rate of placental organoid culture, reduces the empty apoptosis and early disintegration of organoids, and is beneficial to the growth and stability of organoids. Figure 1 ) The stable growth of organoids makes the subsequent scientific research results more reliable. BRIEF DESCRIPTION OF DRAWINGS
[0044] Figure 1 The application improves the culture medium of human placental organoids (bright field image).
[0045] Figure 2 The application improves the culture method of human placental organoids (bright field image).
[0046] Figure 3 The application improves the culture method of human placental organoids (bright field image).
[0047] Figure 4 The application improves the culture method of human placental organoids (bright field image).
[0048] Figure 5 The application improves the culture method of human placental organoids (bright field image). DETAILED DESCRIPTION
[0049] The application will be further described in detail below in combination with the drawings and specific examples:
[0050] A culture medium for early pregnancy human placenta organoids, the components of which include:
[0051] The culture medium is DMEM / F12, 90%, to which N-2 additive, 100x; B-27 additive, 50x; primary cell antibiotic, 100ug / ml; N-acetyl-L-cysteine, 204ug / mL; L-glutamine, 292ug / mL; recombinant human epidermal growth factor, 50ng / ml; CHIR99021, 698ug / mL; recombinant human R-spondin 1, 200ng / ml; recombinant human fibroblast growth factor, 100ng / ml; recombinant human hepatocyte growth factor, 50ng / ml; prostaglandin E2, 882ng / mL; Y-27632, 2.5ug / mL; A83-01, 50nM fetal bovine serum albumin, 1ug / mL are added.
[0052] The culture medium of the application is configured as follows: the recombinant human epidermal growth factor, recombinant human R-spondin 1, cytokine, recombinant human fibroblast growth factor, and recombinant human hepatocyte growth factor in the culture medium components are prepared into a thousand-fold concentrated stock and stored at -80℃, and the N-2 additive, B-27 additive, primary cell antibiotic, L-glutamine, prostaglandin E2, Y-27632, A83-01, and fetal bovine serum albumin are stored at -20℃; when the culture medium is configured, the culture medium components of the early pregnancy human placenta organoids are added one by one in the DMEM / F12 base medium (the order of addition is not required), and the culture medium for the early pregnancy human placenta organoids is obtained. The complete organoid culture medium after configuration can be stored at 4℃ for 1-2 weeks.
[0053] Comparison of the components of the culture medium of the application and the comparative culture medium:
[0054]
[0055]
[0056] The concentration of the working solution above is the concentration in the total amount of the culture medium.
[0057] The improved culture medium of the application adds a Bovine serum albumin component and increases the concentration of Y-27632 and Recombinant Human R-Spondin 1 Protein, effectively reducing the cases of apoptosis and early disintegration in human placental organoids. Due to the rapid degradation of cytokines in the organoid culture medium, it is recommended to use within 1-2 weeks after preparation. The Bovine serum albumin component added in the application plays a role in protecting cytokines from degradation. In addition, Y-27632, an anti-apoptotic inhibitor, reduces early apoptosis of organoids during subculture and culture. R-Spondin 1 is an enhancer of the WNT signaling pathway, which is reported to play an important role in maintaining cell stemness. Increasing the concentration of R-Spondin 1 is beneficial for the maintenance of placental organoid stemness and stable culture.
[0058] Example 1: A culture method for early pregnancy human placental organoids:
[0059] Steps:
[0060] 1. Collect 6-10 week placental villi (raw materials derived from placental villi tissue of women due to social factors induced abortion, which belongs to medical waste) in culture medium ① containing DMEM / F12, N2, B27, Y-27632 cytokines, and store and transport at 4 degrees.
[0061] The specific components of culture medium ① are: DMEM / F12 medium 90%; N-2 additive 1%; B-27 additive 2%; primary cell antibiotic 100 ug / ml; Y-27632 0.5 ug / mL.
[0062] 2. After scraping the placental villi, digest in 5-10 mL 0.25% Trypsin-0.02% EDTA in a 37°C metal bath at 810 rpm for 5-8 min.
[0063] 3. Filter the digest using a 100 um filter, and terminate the digestion with 20% FBS-DMEM / F12.
[0064] 4. Centrifuge at room temperature at 400g-600g for 5 min to obtain a mixture of placental villi cells and blood cells.
[0065] 5. Add the residual tissue on the 100 um filter to 10 ml 1.0 mg / ml collagenase V and digest in a metal bath at 810 rpm for 5-10 min. (Collagenase preparation: weigh the collagenase powder and prepare with 10% FBS-DMEM / F12)
[0066] 6. Filter using 100 μm filter screen, centrifuge at 400g-600g, 5 min at room temperature to obtain the mixture of placental villus cells and blood cells.
[0067] 7. Collect and combine the mixture of placental villus cells and blood cells obtained in step 4 and step 6, wash and resuspend with DMEM / F12, and centrifuge at 400g-600g, 5 min at room temperature.
[0068] 8. After centrifugation, add 3-5 times the volume of red blood cell lysis solution to lyse the red blood cells for 1-3 min, and centrifuge at 400g-600g, 5 min at room temperature.
[0069] 9. Discard the supernatant, wash and resuspend using DMEM / F12, and centrifuge at 400g-600g, 5 min at room temperature.
[0070] 10. Discard the supernatant, count the placental villus cells, and resuspend the cells with an appropriate amount of organoid culture medium II according to the number of villus cells, ensuring that the cells are mixed with Matrigel at a ratio of 1:3 to make drops, 25-35 μL per drop in a 24- or 48-well culture plate.
[0071] 11. After inverting the culture plate at 37°C for 2-5 min, place it upright, continue to place it for 5-10 min, and then add 300 μL of organoid culture medium II for culture. The organoid culture medium II is the culture medium for early pregnancy human placental organoids described above.
[0072] 12. After 7-10 days of culture, 100-200 μm diameter placental organoids can be observed.
[0073] Specific embodiment 2: a culture method for early pregnancy human placental organoids
[0074] Steps:
[0075] 1. Collect an 8-week placental villus from a pregnant woman (the raw material is placental villus tissue from a woman who has had a miscarriage due to social factors, which is a medical waste) in culture medium I containing DMEM / F12, N2, B27, Y-27632 cell factors, and store and transport at 4°C.
[0076] The specific components of culture medium I are: DMEM / F12 medium 90%; N-2 additive 1%; B-27 additive 2%; primary cell antibiotic 100 ug / ml; Y-27632 0.5 μg / mL.
[0077] 2. After scraping the placental villus, digest it in 10 mL of 0.25% trypsin-0.02% EDTA in a 37°C metal bath at 810 rpm for 5-8 min.
[0078] 3. Filter the digestate using a 100μm filter screen, and terminate the digestion of the filtrate with 20% FBS-DMEM / F12.
[0079] 4. Centrifuge at room temperature for 600g for 5 minutes to obtain a mixture of placental villus cells and blood cells.
[0080] 5. Add the remaining tissue on the 100μm filter to 10ml of 1.0mg / ml collagenase V and digest in a metal bath at 810rpm for 8min. (Collagenase preparation: Weigh collagenase powder and prepare using 10% FBS-DMEM / F12)
[0081] 6. Filter using a 100μm filter, centrifuge at 600g for 5 minutes at room temperature to obtain a mixture of placental villus cells and blood cells.
[0082] 7. Collect and combine the placental villus cells and blood cell mixture obtained in steps 4 and 6, wash and resuspend with DMEM / F12, and centrifuge at 600g for 5min at room temperature.
[0083] 8. After centrifugation, add 5 times the amount of red blood cell lysis buffer to the precipitate, lyse the red blood cells for 2 minutes, and centrifuge at 600g for 5 minutes at room temperature.
[0084] 9. Discard the supernatant, wash and resuspend with DMEM / F12, and centrifuge at 600g for 5 min at room temperature.
[0085] 10. Discard the supernatant, count the placental villus cells, add an appropriate amount of organoid culture medium ② to resuspend the cells according to the number of villus cells, and make drops, 30 μL per drop in a 48-well culture plate, ensuring that the cells are mixed with the matrix gel at a ratio of 1:3.
[0086] After incubating the culture plate upside down at 11.37 degrees Celsius for 3 minutes, the plate was inverted and left to stand for another 10 minutes. Then, 300 μL of organoid culture medium ② was added for further incubation. Organoid culture medium ② is the culture medium for early pregnancy human placental organoids described in this invention.
[0087] 12. Placental organoids with a diameter of 200 μm were observed after 8 days of culture.
[0088] The organoids cultured by this invention can be used directly for scientific research, or they can be cryopreserved for future use or used after thawing.
[0089] like Figure 1The growth of human placental organoids in the modified culture medium is shown in the bright field image. As can be seen from the figure, organoids cultured in the original organoid culture medium (the original organoid culture method is described above, but without the use of a metal bath for digestion in steps 2 and 5, and without inverted culture in step 11, and using the aforementioned control culture medium. Therefore, this method will not be described in detail) are prone to apoptosis or premature disintegration in the later stages of culture. The modified human placental organoid culture medium reduces hollow apoptosis and premature disintegration in organoids, which is beneficial to the growth and stability of organoids.
[0090] like Figure 2 The comparison of the top and bottom cells of the human placental organoid matrix gel in the improved culture method shown indicates that inverted culture is beneficial for the suspension culture of organoids and reduces the adhesion of organoids at the bottom of the matrix gel.
[0091] When preparing cell droplets, inverting the culture for 3-5 minutes helps the organoids remain in suspension, making it less likely for the bottom cells to adhere to the cell wall, which is more conducive to the growth of the organoids into a spherical shape.
[0092] like Figure 3 As shown in Figures 4 and 5, the human placental organoids cultured using this culture medium and method exhibited normal morphology. The expression of placental trophoblast markers such as human leukocyte antigen (HLA), cytokeratin 7 (CK7), and E-cadherin was significant, demonstrating the successful culture of placental organoids.
[0093] Figure 3 These are bright-field images of human placental organoids cultured for different days. It can be seen that the cultured placental organoids begin to take shape on day 3 and reach a diameter of 100-200 μm by day 12. Furthermore, their edges are clearly defined, and there is no evidence of apoptosis or disintegration.
[0094] Figures 4-5Human placental organoids were embedded and pretreated, and histological sections were prepared. Immunofluorescence staining was used to incubate placental trophoblast markers human leukocyte antigen-G (HLA, abcam, ab243581), cytokeratin 7 (proteintech, 17513-1-AP), CK7, and E-cadherin (Proteintech, 20874-1-AP). These antibodies were then labeled with fluorescent secondary antibodies. Finally, fluorescence microscopy was used to locate these markers at 488 nm (green) and 594 nm (red) fluorescence excitation wavelengths. The results showed that the placental trophoblast marker CK7 was fully expressed in the placental organoids, the cytotrophoblast marker E-cadherin was expressed in the outermost organoid cells, and the syncytiotrophoblast marker HLA-G was expressed in the innermost trophoblast cells, indicating that the placental organoids cultured in this invention are similar to the human placenta.
[0095] The method for culturing human placental organoids in early pregnancy, as described in this invention, achieves a success rate of 90-100%. Currently, 20 samples have been successfully cultured; the organoid passage success rate is approximately 80%. The placental organoids cultured using this invention have clear edges and are evenly distributed in the matrix gel, reducing adhesion during growth and promoting more efficient three-dimensional growth in the matrix gel. Figure 2 ).
[0096] Passage experiment: Of 10 samples passaged, 8 were successful. The success rate of organoid passage is 80%.
[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principle of the present invention are included within the scope of protection of the present invention.
Claims
1. A method of culturing a first trimester human placental organoid, characterized in that, It comprises the following steps: (I) Collect 6-10 weeks of placental villi for digestion, filtration, centrifugation to obtain a mixture of placental villi cells and blood cells; (II) Red blood cells are lysed again, and centrifugation is performed to obtain placental villi cells; (III) After counting the placental villi cells, add organoid culture medium ② to resuspend the cells, mix the cells and matrigel at a ratio of 1:3 to make drops; after 2-5 min of culture in an inverted plate at 37°C, place the plate upright, continue to place it for 5-10 min, and then add organoid culture medium ② for culture to obtain placental organoids; The composition of the organoid culture medium ② comprises: The culture medium is prepared with DMEM / F12, to which N-2 additive, B-27 additive, primary cell antibiotic, N-acetyl-L-cysteine, L-glutamine, recombinant human epidermal growth factor, CHIR99021, recombinant human R-spinal protein 1, recombinant human fibroblast growth factor, recombinant human hepatocyte growth factor, A83-01, prostaglandin E2, Y-27632, and fetal bovine serum albumin are added.
2. The method for culturing human placental organoids in early pregnancy according to claim 1, characterized in that, The specific steps for obtaining the mixture of placental villi cells and blood cells in step (I) are as follows: (1) Collect 6-10 weeks of placental villi in culture medium ① containing DMEM / F12, N2, B27, and Y-27632 cell factors, and store and transport at 0-4°C; (2) After scraping the placental villi cultured in step (1), digest them in 5-10 mL of 0.25% trypsin-0.02% EDTA at 37°C in a metal bath for 5-8 min; (3) Filter the digest using a 40-100 μm filter, and terminate the digestion with 20% FBS-DMEM / F12; (4) Centrifuge at 400-600 g for 5 min at room temperature to obtain the mixture of placental villi cells and blood cells.
3. The method for culturing human placental organoids in early pregnancy according to claim 2, characterized in that, The steps for obtaining the mixture of placental villi cells and blood cells also include the following steps: (5) Add the residual tissue on the 40-100 μm filter to 10 mL of 1.0 mg / mL collagenase V in a metal bath and digest for 5-10 min; collagenase V is prepared with 10% FBS-DMEM / F12; (6) Filter the digest obtained in step (5) using a 40-100 μm filter, and centrifuge at 400-600 g for 5 min at room temperature to obtain the mixture of placental villi cells and blood cells.
4. The method for culturing human placental organoids in early pregnancy according to claim 1, characterized in that, The specific steps for step (II) are as follows: (1) Collect and combine the mixture of placental villi cells and blood cells obtained in step (4) of claim 2 and step (6) of claim 3, wash and resuspend with DMEM / F12, and centrifuge at 400-600 g for 5 min at room temperature; (2) After centrifugation, add 3-5 times the volume of red blood cell lysis solution to the precipitate, lyse the red blood cells for 1-3 min, and centrifuge at 400-600 g for 5 min at room temperature; (3) Discard the supernatant, wash and resuspend with DMEM / F12, and centrifuge at 400-600 g for 5 min at room temperature to obtain placental villi cells.
5. The method for culturing human placental organoids in early pregnancy according to claim 1, characterized in that, The specific steps for step (III) are as follows: (1) After counting the placental villi cells, add appropriate amount of organoid culture medium ② to resuspend the cells, ensure the ratio of cells and matrigel 1:3, mix and drop, 25-35 microliters per drop in 24 or 48 well culture plates; (2) After 2-5 minutes of 37 degrees Celsius inverted culture plate culture, place the culture plate vertically, continue to place for 5-15 minutes, then add 250-750 μL of organoid culture medium ② for culture; (3) After 7-10 days of culture, 100-200 μm diameter placental organoids can be observed.
6. The method of culturing a first trimester human placental organoid according to claim 1, wherein, The specific components of the culture medium ① are: DMEM / F12 medium 90%; N-2 additive 1%; B-27 additive 2%; primary cell antibiotic 100 ug / ml; Y-27632 0.5 μg / mL.
7. The method for culturing human placental organoids in early pregnancy according to claim 1, characterized in that, The composition of the culture medium ② includes: Culture medium DMEM / F12 accounts for 85-90% of the total volume, wherein N-2 additive accounts for 1% of the total volume; B-27 additive accounts for 2% of the total volume; primary cell antibiotic 100 ug / ml; N-acetyl-L-cysteine 150-250 μg / mL; L-glutamine 200-300 μg / mL; recombinant human epidermal growth factor 20-100 ng / ml; CHIR99021 650-750 μg / mL; recombinant human R-spinal protein 1 150-250 ng / ml; recombinant human fibroblast growth factor 50-150 ng / ml; recombinant human hepatocyte growth factor 20-100 ng / ml; A83-01 150-250 ng / mL; prostaglandin E2 800-900 ng / mL; Y-27632 2-5 μg / mL; bovine serum albumin 0.5-2 μg / mL.
8. The method for culturing human placental organoids in early pregnancy according to claim 7, characterized in that, The composition of the culture medium ② includes: Culture medium DMEM / F12 accounts for 90% of the total volume; wherein N-2 additive accounts for 1% of the total volume; B-27 additive accounts for 2% of the total volume; primary cell antibiotic 100 ug / ml; N-acetyl-L-cysteine 204 μg / mL; L-glutamine 292 μg / mL; recombinant human epidermal growth factor 50 ng / ml; CHIR99021 698 μg / mL; recombinant human R-spinal protein 1 200 ng / ml; recombinant human fibroblast growth factor 100 ng / ml; recombinant human hepatocyte growth factor 50 ng / ml; prostaglandin E2 882 ng / mL; Y-27632 2.5 μg / mL; bovine serum albumin 1 μg / mL.
9. The culture method of claim 7 or 8, wherein the culture medium further comprises a Wnt pathway inhibitor. The culture medium ② is prepared by the following method: The recombinant human epidermal growth factor, recombinant human R-spondin 1, cytokine, recombinant human fibroblast growth factor, recombinant human hepatocyte growth factor in the medium component are prepared into a thousand-fold concentrated storage and are stored at-80℃, and the N-2 additive, B-27 additive, primary cell antibiotic, L-glutamine, prostaglandin E2, Y-27632, A83-01, fetal bovine serum albumin and other additives are divided and stored at-20℃; when the medium is prepared, the medium components of the early pregnancy human placental organoid are added one by one in the DMEM / F12 base medium to obtain the medium of the early pregnancy human placental organoid; the obtained medium of the early pregnancy human placental organoid after preparation can be stored at 4℃ for 1-2 weeks.
10. An early pregnancy human placental organoid cultured by the culture method of the early pregnancy human placental organoid according to any one of claims 1-9.