A method and application of inducing organoids directly from IPS

Abstract

The present invention discloses a method and application of inducing the generation of organoids directly from IPS, and belongs to the field of cell engineering. The present invention provides a method for directly and quickly generating target organoids based on human IPS, and obtains lung organoids, placental organoids, and vascular organoids. The present invention induces organoids representing three germ layers, which shows that the method is suitable for the construction of various organoids. Compared with the prior art, the method does not require step-by-step induction, is successfully constructed in one step, is simple to operate, greatly reduces the construction time, has a repeatable process, and can be cultured and passaged for a long time under in vitro conditions, and has a high freezing survival rate; the cultured organoids have good uniformity, and the RNA-seq results show that high expression of vascular-related genes occurs in the organoids, and the organoids of the present invention can grow up to 400μm at most, indicating that the culture method of the present invention can spontaneously induce the expression of some vascular-related genes to support the growth of organoids. The present invention provides an important tool for studying the pathogenesis of the disease and subsequent drug development.

Description

Technical Field

[0001] The present invention belongs to the field of cell engineering, and specifically relates to a method and application of directly inducing organoids from IPS. Background Art

[0002] Organoids are self-organizing 3D in vitro micro-organ models formed by inducing differentiation of target organisms. They can simulate the complex structure and function of human organs more realistically under in vitro conditions, and can be used to conduct research on various issues such as human growth and development and physiological diseases. Compared with animal models, organoids can perform high-throughput screening and library construction, overcome problems caused by species differences, and play an important role in regeneration and repair, pathogenesis research, and drug screening.

[0003] Organoids generated from induced pluripotent stem cells (IPS) have been described for various tissues such as stomach, intestine, lung, brain, kidney, liver, etc. These organoids are attractive alternatives to other in vitro models; organoids retain many of the typical favorable characteristics of cell culture, such as the ability to maintain stable cultures for months, and the ability to cryopreserve and restore cultures. Organoids are also amenable to many standard laboratory techniques, including gene modification using CRISPR / Cas9, immunofluorescence staining, RNA expression quantification, western blotting, and toxicity assays. Organoid cultures have been used in both basic and translational research.

[0004] The culturing process of organoids is a process of precisely regulating the microenvironment of cell growth, simulating the microenvironment of cells in the body by providing personalized culture conditions. The 3D scaffold provides support for cell growth, self-organization of cell types, and spatial structure construction. Under the action of cell clusters, because different cells secrete different cell adhesion factors, different types of cells will move to different positions of the 3D scaffold, thereby achieving the separation of cell types. The classic preparation route of organoids is to culture the isolated stem cells on a 3D scaffold (such as matrix gel Matrigel), spread them in a culture dish or culture well plate, and add various growth factors including FGF and EGF to promote organoid formation. The current mainstream technical solutions are as follows: Lung organoids [1]Culture: IPS culture and seeding: First, IPS are cultured in culture medium until healthy cell clusters are formed, and then seeded and differentiated. Inducing the formation of definitive endoderm (DE): Adding factors such as activin A, CHIR9902, and Y27632, IPS gradually differentiates to form DE, which is the first step in the formation of lung organoids. Inducing the formation of anterior foregut endoderm (AFE): After the formation of DE, factors such as SB431542, Noggin 200, SAG, and FGF4 are added, and IPS further differentiates to form AFE. Inducing the formation of lung progenitors (Lung Progenitor Aggregates, LPAs): Adding factors such as BMP4, CHIR99021, and ATRA, AFE subsequently differentiates to form LPAs, and these cell clusters are formed on the 12th day. Formation of 3D lung organoids (LORGs) and re-embedding in Matrigel: On day 12, LPAs were mixed with Matrigel to form 3D structured LORGs that contained airway-like structures and distal and proximal mesenchymal and alveolar epithelial cell patterns. These LORGs could be maintained for more than 90 days by re-embedding in Matrigel.

[0005] Placental organoids [2] 3D culture of IPS: IPS were seeded on microcolumn chips and treated with BMP4 under 3D culture conditions to generate 3D cell clusters with trophoblast and mesodermal lineages. Hypoxic environment culture: IPS were cultured in a hypoxic environment from day 0 to day 12. Culture condition conversion: On day 12, the formed tissue was transferred to a 21% oxygen environment for continued culture. Growth factor treatment: 3D cell clusters gradually grew into millimeter-sized placental-like tissues over 2-3 weeks under the action of growth factors (such as VEGFA, bFGF, and R-spondin 1).

[0006] Vascular Organoids [3]Culture: Day 0: Add CHIR99021 and BMP-4 to start inducing differentiation from human pluripotent stem cells to form embryoid bodies (EBs); Day 1: Mesoderm induction, which is the first step of vascular differentiation; Day 4: Add VEGF-A and forskolin to induce blood vessels and promote the formation of vascular networks; Day 6: Add VEGF-A, FGF-2, and FBS to embed the formed aggregates into a 3D collagen and soluble basement membrane matrix to support vascular maturation and network formation; After Day 7, a vascular network begins to form in the 3D matrix, and this process usually takes 2-3 weeks. After 2-3 weeks, the formed vascular organoids can be further grown in an expandable suspension culture medium.

[0007] It can be seen that at present, the differentiation from IPS to organoids usually requires step-by-step induction, first differentiating into three germ layers, and then inducing the three germ layers into related cells of the target organoid, and then 3D assembling into the target organoid, and the construction time is as long as more than a month. For example, the patent publication number CN 117887654 A discloses a method and application of lung organoid construction based on human pluripotent stem cells. The construction scheme of human IPS lung organoids is an integrated and optimized four-stage rapid induction scheme, which activates the lineage differentiation program of definitive endoderm, foregut endoderm, lung progenitor cells, and lung parenchymal cells by regulating TGF-β, BMP, Wnt, and FGF specific signaling pathways in sequence, and initiating the lineage differentiation program of definitive endoderm, foregut endoderm, lung progenitor cells, and lung parenchymal cells. On the 40th day, human pluripotent stem cells can be induced to differentiate into lung organoids containing 6 types of cells: AT2, AT1, basal cells, goblet cells, ciliated cells, and Club cells, with proliferation, differentiation, and secretory protein expression. Patent publication number CN109576211 A discloses a process for the directed differentiation of human pluripotent stem cells into lung organoids; the specific process includes: 1) human pluripotent stem cells are cultured on a culture plate coated with matrigel; 2) differentiation is induced when ESC / IPS grow to 95% confluence; 3) the second stage of differentiation is the abdominal foregut endoderm stage; 4) the third stage of differentiation is the lung differentiation stage; 5) 41 days of induction; the patent uses a 3D lung organoid directed differentiation platform with 5 cytokines / small molecule compounds. It can be seen that in the prior art, it takes 1-2 months or even longer to successfully construct organoids by inducing them from pluripotent stem cells.

[0008] It can be seen that the existing method of constructing organoids based on IPS is complex and time-consuming. This culture method requires too many steps and is highly complex. Different batches of reagents may lead to differences in cell growth, and the culture time of organoids is long and uneven. Although the cycle of organoids is already very short compared to adult research, using a more direct and simple method to build more organoids in a shorter time can greatly shorten research time, reduce experimental costs, and improve efficiency.

[0009] After searching, there are no documents or reports in the prior art that disclose a method for inducing organoids directly from IPS without stratification induction.

[0010] References:

[0011] [1]Tiwari SK, Rana TM. Generation of 3D lung organoids from humaninduced pluripotent stem cells for modeling of lung development and viralinfection. Heliyon. 2023 Aug 29;9(9):e19601. DOI: 10.1016 / j.heliyon.2023.e19601.

[0012] [2]Cui K, Chen T, Zhu Y, Shi Y, Guo Y, Qin J. Engineering placenta-like organoids containing endogenous vascular cells from human-induced pluripotent stem cells. Bioeng Transl Med. 2022 Sep 23;8(1):e10390. DOI:10.1002 / btm2.10390.

[0013] [3]Wimmer RA, Leopoldi A, Aichinger M, et al.Generation of bloodvessel organoids from human pluripotent stem cells[J].Nature Protocol, 2019,14(11).DOI:10.1038 / s41596-019-0213-z. Summary of the invention

[0014] In response to the above technical problems, the present invention intends to provide a method for inducing organoids directly from IPS.

[0015] The technical solution of the present invention is as follows:

[0016] A method for inducing organoids directly from IPs, comprising the following steps:

[0017] S1 Take the IPS cultured to 80-90% confluence, remove the culture medium, add ReLeSR digestion solution, and incubate at room temperature for 1 min; remove the digestion solution and incubate in the incubator for 4 min;

[0018] S2 was resuspended in mTeSR Plus medium to obtain a cell suspension and counted; the culture plate was preheated in a 37°C incubator;

[0019] S3: Use pre-cooled matrix gel with a concentration greater than 70% to adjust the volume of the cell suspension obtained in S2 to a mixture of mTeSR Plus medium and matrix gel; each well of the culture plate is incubated in a 37°C incubator for 5 minutes, then the culture plate is inverted and incubated for another 20 minutes;

[0020] S4 adds target organoid culture medium to each well of the culture plate, changes the medium every 2 days, and cultivates mature target organoids.

[0021] Furthermore, the IPS is cultured from human blood.

[0022] Preferably, the target organoids are lung organoids, placental organoids, and vascular organoids.

[0023] Preferably, the lung organoid culture medium is: human airway organoid culture medium; the placental organoid culture medium is: human trophoblast organoid isolation and culture kit / culture medium; the components of the vascular culture medium are: α-MEM basal culture medium 25-100ng / mLvEGF-A, 5-50ng / mLEGF, 50-100ng / mLbFGF, 10-15% FBS.

[0024] The present invention also provides for subculture of the organoid obtained in step S4. The subculture comprises the following steps:

[0025] S11: Remove the primary organoid culture medium, blow the matrigel pellet with 4°C pre-cooled PBS, and collect the organoids into a centrifuge tube; add pre-cooled PBS to the centrifuge tube;

[0026] S12: Place the centrifuge tube at 4°C for 10 min, and centrifuge at 300 x g for 5 min; remove the supernatant; resuspend the organoids in 4°C precooled matrix gel with a concentration greater than 70% to obtain an organoid-matrigel suspension;

[0027] S13: Plate the organoid-matrigel suspension on a culture plate preheated at 37°C, incubate in a 37°C incubator for 5 minutes, then invert the culture plate and incubate for another 20 minutes; add target organoid culture medium, change the medium every 2 days, and obtain the target organoid for subculture.

[0028] The present invention also protects the use of organoids cultured using the method of the present invention in related disease research or drug development and evaluation.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1) The method of constructing organoids in the present invention significantly reduces the difficulty of operation. It adopts the technology of rapid construction of organoids directly from IPS, does not require step-by-step induction, shortens the construction time, and has a repeatable process, long-term stable culture and passage under in vitro conditions, and a high freezing survival rate; the cultured organoids have good uniformity. Compared with the existing technology, the lung organoids are shortened by 20 days, the placenta is shortened by 10 days, and the vascular organoids are shortened by 5 days, making it possible to accurately, quickly, and high-throughput modeling of different organoids in vitro.

[0031] 2) RNA-seq results showed that high expression of vascular-related genes was observed in the organoids cultured by the method of the present invention, and that the organoids of the present invention could grow to a maximum size of 400 μm, indicating that the culture method of the present invention could spontaneously induce the expression of some vascular-related genes to support the growth of organoids.

[0032] 3) The organoid culture method of the present invention can construct a large number of organoids in a very short time, which is convenient for conducting research that requires a large number of organoids and quickly screening drugs with potential therapeutic effects. This is of great significance for accelerating the development of new drugs, reducing the cost of drug development, and is expected to play an important role in the fields of regenerative medicine, disease modeling and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 1 shows a cultured lung organoid, where Figure 1 A is a picture of lung organoids cultured for 2 days. Figure 1 B is a picture of lung organoids cultured for 16 days;

[0034] Figure 2 Figure 2 shows a cultured placental organoid, where Figure 2 A is a picture of placental organoids cultured for 2 days. Figure 2 B is a picture of placental organoids cultured for 9 days;

[0035] Figure 3 Figure 2 shows a cultured vascular organoid, where Figure 3 A is a vascular organoid cultured for 6 days. Figure 3 B is a picture of vascular organoids cultured for 10 days;

[0036] Figure 4 This is an immunofluorescence staining image of cultured lung organoids. Figure 4 A is a DAPI staining image of lung organoids; Figure 4 B is the EpCAM staining picture, Figure 4 C is the SCGB1A1 staining image, Figure 4 D is the merge image of fluorescent staining;

[0037] Figure 5 This is an immunofluorescence staining image of cultured placental organoids. Figure 5 A is a DAPI staining image of placental organoids; Figure 5 B is the CGA staining image of placental organoids. Figure 5 C is the HLA-G staining image of placental organoids. Figure 5 D is the merge image of fluorescent staining of placental organoids;

[0038] Figure 6 This is an immunofluorescence staining image of cultured vascular organoids. Figure 6 A is a DAPI staining image of vascular organoids; Figure 6 B is the CD31 staining image in vascular organoids. Figure 6 C is the PDGFRβ staining image of vascular organoids. Figure 6 D is the merge image of fluorescent staining of vascular organoids;

[0039] Figure 7 Schematic diagram of the state of organoids after passage, where: Figure 7 A is a picture of the second generation placental organoid cultured for 12 days. Figure 7 B is a picture of the second generation lung organoid cultured for 2 days;

[0040] Figure 8 The figure is a statistical chart of the frozen-thaw count and viability of the organoids of the present invention; wherein Figure 8 A is a chart showing the cryo-recovery count and viability of lung organoid cells. Figure 8 B is a chart showing the cryo-recovery count and viability of placental organoid cells;

[0041] Fig. 9 It is a four-parameter fitting curve diagram of MUC5AC expression in lung organoids; the X value is the concentration in ng / mL; the Y value is the absorbance value in AU;

[0042] Fig.10 This is the four-parameter fitting curve of HCG expression in placental organoids; X value is the concentration, in mIU / mL; Y value is the absorbance value, in AU;

[0043] Fig.11 is the expression of lung-specific markers in organoids. F represents lung organoids, and TP represents placental organoids.

[0044] Fig.12 is the expression of placenta-specific markers in organoids. F represents lung organoids, and TP represents placenta organoids.

[0045] Fig.13The expression of vascular-specific markers in organoids. F represents lung organoids, and TP represents placental organoids. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0047]

Experimental reagents and equipment

[0048] Biomatrix gel, mTeSR Plus culture medium, ReLeSR digestion solution, cell culture incubator, countstar cell counter, 12 and 24-well plates, 15mL centrifuge tubes, PBS, pipette tips, ice box. IPS are all constructed by the laboratory and derived from human blood. The three organoid culture media are: 1. Human Airway Organoid Kit Plus Model Bio Airway Organoid Culture Medium MA-0817H004LP; 2. Human Trophoblast (Villi) Organoid Kits / Medium OG50-HN; 3. Vascular Organoid Culture Medium: α-MEM basal medium, 25-100ng / mL vEGF-A, 5-50ng / mL EGF, 50-100ng / mL bFGF, 10-15% FBS.

[0049] Example 1 Method for constructing organoids induced by IPS

[0050] 1.1 Organoid Culture

[0051] Take human blood to culture IPS, take IPS when it grows to 80-90% confluence, remove the culture medium in the culture plate; add 500μL ReLeSR digestion solution to each well of the culture plate and incubate at room temperature for 1min; remove the ReLeSR digestion solution as much as possible, and then place the culture plate in a 37℃ incubator for 4min; use 1mL mTeSR for each three wells of cells Plus culture medium, and use Countstar cell counter to count the cells; preheat the culture plate in a 37-degree incubator; place the matrix gel on a precooled 4°C ice box and take it with a precooled pipette tip; take a cell suspension containing 5w cells and use matrix gel to make the volume into 50μL culture medium and matrix gel mixture, according to the ratio of 1w cells per 10μL mixture, and the matrix gel concentration must be greater than 70%; plate 50μL of cell suspension in each well of the culture plate, and after all the wells are plated, place the culture plate in a 37°C incubator for incubation for 5min, then invert the culture plate and incubate it for another 20min; add 500μL of target organoid culture medium to each well of the culture plate, and change the medium every two days to obtain the target organoid.

[0052] When the target organoid is lung organoid, the lung organoid culture medium is: airway organoid culture medium. When the target organoid is placental organoid, the placental organoid culture medium is: human trophoblast (villous) organoid isolation and culture kit / culture medium. When the target organoid is vascular organoid, the composition of vascular organoid culture medium is: α-MEM basal medium, 25-100ng / mL vEGF-A, 5-50ng / mL EGF, 50-100ng / mL bFGF, 10-15% FBS.

[0053] With daily observation, the initial organoid structure appeared in 2 to 3 days, and the expression of some markers of the target organoid could be detected in 5 days. After 16 days of culture, the lung organoids were mature. Figure 1 As shown; after 9 days of culture, the placental organoids were mature, as shown Figure 2 As shown, after 10 days of culture, the vascular organoids obtained were mature. Figure 3 shown.

[0054] It can be seen from the above culture medium that the lung organoid culture medium used in the present invention is used in the prior art to establish and maintain human tracheal organoids derived from human tracheal stem cells from adult lung tissue or stem cells. It was once believed that this culture medium could not be used to directly induce lung organoids from IPS. The human trophoblast (villous) organoid isolation and culture kit / culture medium is used to establish and maintain human trophoblast (villous) organoids from human cells (such as adult stem cells) such as clinical samples and primary tissues. It is also believed that it is impossible to directly induce adult trophoblast (villous) organoids from IPS. The inventor tried to use IPS cultured from human blood, and after treatment, used these two types of culture media to directly induce the target organoids, and succeeded. The vascular culture medium used in the present invention is self-prepared, and the present invention also uses the purchased STEMdiff™ Blood Vessel Organoid Maturation Medium for comparison. The results show that the self-prepared vascular culture medium is better than the purchased STEMdiff™ Blood Vessel Organoid Maturation Medium, the self-prepared culture medium has an earlier self-assembly time, and the efficiency is higher. ‌STEMdiff™ Blood Vessel Organoid Maturation Medium is a culture medium specifically used for the expansion and cultivation of mature vascular organoids. The prior art does not allow for the direct induction of vascular organoids from IPS. Through experimentation, it was found that the method and vascular culture medium of the present invention can be used to induce successful vascular organoids directly from IPS without the need for multi-embryonic layer induction.

[0055] 1.2 Organoid identification Whole-mount immunofluorescence staining of organoids

[0056] 1.2.1 Organoid collection and washing

[0057] 1) Organoid collection

[0058] Aspirate the culture medium with a pipette, collect the matrix gel in a 15 mL centrifuge tube, add PBS to make up to 10 mL (the more PBS, the more fully the matrix gel is diluted and the easier it is to remove), let it stand at 4°C for 10 min to soften the matrix gel, then centrifuge at 500 g for 5 min at 4°C and discard the supernatant.

[0059] 2) Organoid washing

[0060] Add 1 mL of PBS to resuspend, centrifuge at 500 g, 4°C for 5 min, and discard the supernatant.

[0061] 1.3.2 Immunofluorescence staining of organoids

[0062] First, fix with 4% paraformaldehyde for 15 minutes and wash with PBS three times for 2 minutes each time; then use 0.5% Triton to permeabilize the membrane for 20 minutes and wash with PBS three times for 2 minutes each time. Note: When washing with PBS, you can use the tip of the gun to gently blow up to avoid bubbles. Wait until the organoids settle to the bottom to start the next step. If the target protein to be detected is an intracellular protein or the antigen-antibody binding site is located in the cell Transmembrane protein, permeabilization treatment is required before antibody incubation; use 1% BSA to block at room temperature for 60 minutes and wash with PBS three times for 2 minutes each time; then use 5% BSA diluted primary antibody, incubate at 4°C overnight, wash with PBS three times for 2 minutes each time; use 5% BSA diluted fluorescent labeled secondary antibody, incubate at room temperature in the dark for 1 hour, wash with PBS three times for 2 minutes each time; incubate with 5ug / mLDAPI in the dark for 15 minutes and wash with PBS three times for 2 minutes each time; gently spread the organoids on a slide, and gently absorb excess residual liquid with absorbent paper; gently fix the slide with nail polish, observe and take pictures under a laser scanning confocal fluorescence microscope. The results are as follows Figure 4-6 shown.

[0063] Figure 4 A shows the distribution and morphology of cell nuclei in the lung organoids cultured in the present invention; Figure 4 B shows the expression of epithelial cell adhesion molecule (EpCAM) in lung organoids. In lung organoids, EpCAM staining can show the distribution and morphology of epithelial cells, which is of great significance for studying the epithelial structure of lung organoids. ‌ Figure 4 C shows the expression of secretory Clara cell 10kDa protein (SCGB1A1) in lung organoids. SCGB1A1 staining can identify the location and distribution of Clara cells in lung organoids, which is crucial for studying the development and function of specific cell types in lung organoids. ‌ Figure 4 D can simultaneously observe the relative position and distribution of cell nuclei, epithelial cells and Clara cells in lung organoids, thereby gaining a more comprehensive understanding of the structure and composition of lung organoids. Figure 4 This demonstrates that the lung organoids cultured using the method of the present invention are successful.

[0064] ‌ Figure 5 A shows the distribution of cell nuclei in placental organoids. ‌ Figure 5 The distribution of trophoblast cells in placental organoids is shown in B. mCGA (human chorionic gonadotropin alpha subunit) is one of the hormones secreted by placental trophoblast cells and is often used to mark placental cells. ‌ Figure 5 C shows the distribution of cells expressing HLA-G in placental organoids, an immunomodulatory molecule specifically expressed on placental trophoblast cells. ‌ Figure 5 D shows the comprehensive distribution of different cell types and structures in placental organoids. Figure 5 This demonstrates that the placental organoids cultured using the method of the present invention are successful.

[0065] ‌ Figure 6 A shows the distribution of cell nuclei in vascular organoids. ‌ Figure 6 B shows the expression of CD31 in vascular organoids. CD31 is an endothelial cell marker that is widely distributed on the surface of vascular endothelial cells, indicating that the blood vessels have differentiated into tubes and have normal vascular functions. Figure 6 C shows the distribution of PDGFRβ in vascular organoids. PDGFRβ is a marker for perivascular cells. ‌ Figure 6 D shows the comprehensive distribution of different cell types and structures in vascular organoids. Figure 6 This indicates that the vascular organoids cultured using the method of the present invention are successful.

[0066] The organoids cultured by the present invention can be used to study the pathogenesis of diseases, drug development and drug evaluation. For example, the establishment of a human lung organoid injury model is used to study the molecular mechanism of high altitude hypoxia on lung injury, providing an important tool for pathogenic mechanism research and subsequent drug development, helping researchers to better understand the development process of the disease.

[0067] 1.3 Organoid subculture and cryopreservation recovery

[0068] 1.3.1 Organoid subculture

[0069] After the organoid culture matures, it can be subcultured. The specific steps are as follows: remove the organoid culture medium in the culture plate, use 4°C pre-cooled PBS to blow the matrix gel clumps, and collect the organoids into a 15mL centrifuge tube; add pre-cooled PBS to the centrifuge tube to 15mL. The more PBS, the easier it is to remove the matrix gel; after placing the centrifuge tube at 4°C for 10 minutes, add 5-10 times the volume of the organoid subculture digestion solution bioGenous TM Organoid Dissociation Solution, digest at 37℃ for 3-8 minutes, then centrifuge at 300g for 5 minutes; remove the supernatant and place the 15mL centrifuge tube on a pre-cooled ice box; calculate the subculture density, count all the matrix gel needed, use a 4℃ pre-cooled pipette tip to transfer the matrix gel to a 15mL centrifuge tube, and resuspend the organoids; plate the organoid-matrigel mixed suspension on a culture plate preheated at 37℃, 50μL per well, incubate the culture plate at 37℃ for 5 minutes, then invert the culture plate and incubate for another 20 minutes; add 500μL of new organoid culture medium to each well of the culture plate, and change the medium every two days to obtain the target organoids. According to experiments, the success rate of subculture of lung organoids and placental organoids is as high as 100%, and the success rate of subculture of vascular organoids is as high as 50%. The effect of subculture is shown in the figure below. Figure 7As shown, it is proved that the method of the present invention is simple and feasible for subculture of organoids.

[0070] 1.3.2 Organoid cryopreservation steps: Collect organoids in 15 ml centrifuge tubes; add 10 mL of 4°C pre-cooled PBS to the 15 ml centrifuge tubes and incubate at 4°C for 10 min; centrifuge at 300 g for 3 min; remove the supernatant and add 4°C pre-cooled organoid cryopreservation solution bioGenous TM Organoid Cryopreservation Medium (Serum Free); dispense into cryopreservation tubes according to the required cryopreservation quantity; place the cryopreservation tubes in a cell cryopreservation program cooling box, and immediately place the cooling box in a -80°C incubator. The next day or 12 hours later, transfer the cryopreservation tubes to liquid nitrogen for long-term storage under low temperature conditions (no higher than -70°C, dry ice or the original cooling box is recommended).

[0071] 1.3.3 Organoid recovery step: Preheat the basal culture medium required for organoids at 37°C in advance. Quickly thaw the cell cryovials in a 37°C water bath. When only a little ice residue remains in the cryovials, stop the water bath immediately and transfer to a clean operating table in time. Transfer the frozen suspension to a centrifuge tube, slowly add 5-10 times the volume of preheated basal culture medium, and mix gently. Centrifuge the organoid suspension obtained in the previous step at 300g for 3 minutes, discard the supernatant, and add the basal culture medium again to resuspend the organoid precipitate. Centrifuge the organoid suspension obtained in the previous step at 300g for 3 minutes. After discarding the supernatant, the organoids obtained can be used for subsequent organoid culture.

[0072] The cell viability of organoids recovered after cryopreservation by the method of the present invention is as high as over 80%. Figure 8 .

[0073] Example 2 Functional identification of cultured organoids

[0074] The present invention uses the Elisa method to identify the expression of specific proteins in organoids to illustrate the presence, concentration and possible biological function or disease state of the specific proteins in the organoids.

[0075] 2.1 Protein expression in lung organoids

[0076] The double antibody sandwich ELISA method was used: anti-human MUC5AC antibody was coated on the ELISA plate. During the experiment, human MUC5AC in the sample (or standard) would bind to the coated antibody. Then, biotinylated anti-human MUC5AC antibody and horseradish peroxidase-labeled avidin were added in sequence. The anti-human MUC5AC antibody bound to human MUC5AC bound to the coated antibody. Biotin and avidin specifically bound to form an immune complex, and the free components were washed away. The color substrate (TMB) was added. TMB showed blue under the catalysis of horseradish peroxidase, and turned yellow after adding the stop solution. The OD value was measured at a wavelength of 450nm using an ELISA instrument. The MUC5AC concentration was proportional to the OD450 value. The MUC5AC concentration in the sample was calculated by drawing a four-parameter fitting curve. The results are shown in Tables 1 and Fig. 9 shown.

[0077] Table 1 Corresponding values ​​of MUC5AC expression concentration and absorbance in lung organoids

[0078]

[0079] The regression equation obtained from the fitting curve is: Y=(7.612-0.027) / [1+(x / 25.334) -0.936 ]+0.027; X value is the concentration, in ng / mL; Y value is the absorbance value, in AU. The absorbance value of the sample well diluted 100 times is 0.054, which is calculated to be 0.0604ng / mL by the equation of the four-parameter fitting curve. The concentration of MUC5AC in the lung organoid can be calculated to be 6.04ng / mL. It can be seen that the lung organoid of the present invention expresses MUC5AC, indicating that the lung organoid constructed by the present invention has a function similar to that of the lung.

[0080] 2.2 Placental organoids

[0081] The double antibody sandwich ELISA method is used, and the anti-human HCG antibody is coated on the ELISA plate. During the experiment, the human HCG in the sample (or standard) will bind to the coated antibody, and then the biotinylated anti-human HCG antibody and horseradish peroxidase-labeled avidin are added in sequence. The anti-human HCG antibody binds to the human HCG bound to the coated antibody, and biotin and avidin specifically bind to form an immune complex, and the free components are washed away; the color substrate (TMB) is added, and TMB appears blue under the catalysis of horseradish peroxidase, and turns yellow after adding the stop solution; the OD value is measured at a wavelength of 450 nm using an enzyme reader. The HCG concentration is proportional to the OD450 value, and the HCG concentration in the sample is calculated by drawing a four-parameter fitting curve. The results are shown in Table 2 and Fig.10 shown.

[0082] The regression equation obtained from the fitting curve is: y=(2.94-0.056) / [1+(x / 169.813) -1.598 ]+0.056; X value is the concentration, in mIU / mL; Y value is the absorbance value, in AU. The absorbance value of the sample well diluted 20 times is 0.071, which is calculated to be 6.408mIU / mL by the equation of the four-parameter fitting curve. The concentration of HCG in the placental organoid can be calculated to be 128mIU / mL. This shows that the organoid constructed by the present invention expresses HCG and has a function similar to that of the placenta.

[0083] Table 2 Corresponding values ​​of HCG expression concentration and absorbance of placental organoids

[0084]

[0085] 2.3 Analysis of organoid RNA-seq experimental results

[0086] The present invention performs RNA-seq on organoids, and the results are as follows Figure 11-13 shown.

[0087] Fig.11 The difference in the expression of lung marker genes between the F and TP groups was revealed in the figure. Gene expression was generally higher in lung organoids (F), especially SOX2, which was highly expressed in lung organoids, indicating that it may be a key regulator of lung organoid development or function. This is consistent with known biological facts. SOX2 is an important transcription factor for epithelial cell development and maintenance. The moderate expression of others such as FGFR2 and BMPR1A supports the development and morphogenesis of lung organoids. The expression level was significantly reduced in placental organoids (TP), especially SOX2, which was almost absent, indicating that the cell type and function of placental organoids are completely different from those of lung organoids. The placenta may rely more on other genes related to the maternal-fetal interface or nutrient exchange rather than the lung development-related genes shown in the figure. Functional characteristics of lung organoids: The expressed genes (such as SOX2 and FGFR2) indicate that the active genes in lung organoids are mainly related to the development and differentiation of lung epithelial cells. These genes may be important factors for lung organoids to maintain tissue morphology, function and differentiation potential.

[0088] Fig.12The differences in the expression of placental marker genes between the F group and the TP group were revealed. In the placental organoids (TP group), highly expressed genes (such as CRH, HLA-G, CGA) are mainly related to placental function. Among them, CRH is for pressure regulation and hormone secretion at the maternal-fetal interface. HLA-G is for immune tolerance, protecting the fetus from maternal immune rejection. CGA is for hormone secretion to support pregnancy. Low-expressed genes (such as PLAC1, LEP) may be related to the limitations of the organoid development model. In the lung organoids (F), the expression levels of most placenta-related genes were extremely low, which is consistent with the functional characteristics of lung tissue, showing lower expression of immune regulation (such as HLA-G) and hormone regulation genes (such as CRH, CGA), indicating that lung organoids are mainly focused on the development of respiratory function. The gene expression of placental organoids is more focused on functions such as maternal-fetal immune balance, hormone regulation, and support for fetal development. Highly expressed genes such as CRH and HLA-G highlight the role of the placenta as an endocrine and immune regulatory organ.

[0089] Fig.13 The differences in the expression of vascular marker genes between the F and TP groups were revealed. The expression of VEGFB and VEGFD in lung organoids (F) was significantly higher than that in placental organoids (TP), especially VEGFB. PECAM1 and VEGFA were highly expressed in placental organoids (TP), especially PECAM1, which reached significantly high levels in the placenta, while almost no expression was found in lung organoids. ACTA2 was highly expressed in lung organoids, while almost no expression was found in the placenta. PDGFRB was expressed to some extent in both the placenta and lung, but slightly higher in the placenta. VEGFC was expressed higher in lung organoids than in placental organoids, but the difference was not as significant as that of VEGFB or VEGFD. Differential expression of angiogenesis-related genes: The figure shows significant differences in the expression of different genes in the lung and placenta, which may be related to the angiogenesis, growth, and functional requirements of the two organs. Higher expression of VEGFB and VEGFD in lung (F) organoids may support the maintenance of their complex vascular network. High expression of PECAM1 and VEGFA in the placenta (TP) may be associated with angiogenesis in placental tissue and maternal-fetal circulation.

[0090] The above-mentioned organoids cultured by the present invention show high expression of vascular-related genes, and the organoids can grow to a maximum diameter of 400 μm, indicating that some vascular-related gene expression can be spontaneously induced to support organoid growth. After searching the patents in the prior art, the current vascularized organoids need to be mixed with branched lung organoids and vascular progenitor cell spheres to achieve, and cannot be spontaneously induced (human vascularized lung organoids based on iPSC and their construction method, CN 117384824 A).

Claims

1. A method for inducing organoids directly from IPS, comprising the following steps: S1 Take the IPS cultured to 80-90% confluence, remove the culture medium, add ReLeSR digestion solution, and incubate at room temperature for 1 minute; remove the digestion solution, and incubate in an incubator for 4 minutes; the IPS is cultured from human blood; S2 was resuspended in mTeSR Plus medium to obtain a cell suspension and counted; the culture plate was preheated in a 37°C incubator; S3 uses pre-cooled matrix gel with a concentration greater than 70% to adjust the volume of the cell suspension obtained in S2 to a mixture of mTeSR Plus culture medium and matrix gel; each well of the culture plate is incubated in a 37°C incubator for 5 minutes, then the culture plate is inverted and incubated for another 20 minutes; S4 adds target organoid culture medium to each well of the culture plate, changes the medium every 2 days, and cultivates mature target organoids.

2. The method for inducing organoids directly from IPS according to claim 1, characterized in that: The target organoids are lung organoids, placental organoids, and vascular organoids.

3. The method for inducing organoids directly from IPS according to claim 2, characterized in that: The lung organoid culture medium is: human airway organoid culture medium; the placental organoid culture medium is: human trophoblast organoid isolation and culture kit / culture medium; the components of the vascular culture medium are: α-MEM basal culture medium, 25-100ng / mL vEGF-A, 5-50ng / mL EGF, 50-100ng / mL bFGF, 10-15% FBS.

4. The method for inducing organoids directly from IPS according to claim 1, characterized in that: The organoids obtained in step S4 are used for subculture.

5. The method for inducing organoids directly from IPS according to claim 4, characterized in that: The subculture comprises the following steps: S11: Remove the primary organoid culture medium, blow the matrigel pellet with 4°C pre-cooled PBS, and collect the organoids into a centrifuge tube; add pre-cooled PBS to the centrifuge tube; S12: Place the centrifuge tube at 4°C for 10 min, and centrifuge at 300 x g for 5 min; remove the supernatant; resuspend the organoids in 4°C precooled matrix gel with a concentration greater than 70% to obtain an organoid-matrigel suspension; S13: Plate the organoid-matrigel suspension on a culture plate preheated at 37°C, incubate in a 37°C incubator for 5 minutes, then invert the culture plate and incubate for another 20 minutes; add target organoid culture medium, change the medium every 2 days, and obtain the target organoid for subculture.

6. Use of the organoid cultured according to the method of any one of claims 1 to 5 in related disease research or drug development and evaluation.

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