Stable passage method of human induced pluripotent stem cell-derived small intestine organoid and application of stable passage method

Abstract

The invention belongs to the technical field of organoid culture, and particularly relates to a passage method of a human induced pluripotent stem cell-derived small intestine organoid and application of the passage method. According to the passage method disclosed by the invention, after matrigel is mildly removed by a dissociation reagent, long-range stable passage of the small intestine organoid can be maintained by a method for slightly breaking the small intestine organoid through a suction head, and the structure and cell type diversity of the small intestine organoid can be maintained. Meanwhile, enteroviruses are infected into the small intestine organoid obtained by adopting the passage method, and the enteroviruses can be effectively copied in the small intestine organoid and infect one or more cells including intestinal epithelial cells, proliferative cells, panning cells and intestinal endocrine cells; therefore, the method can be used for related research of enterovirus infection and host interaction.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and specifically relates to a stable passage method of small intestinal organoids derived from human induced pluripotent stem cells and an application thereof. Background Art

[0002] The advent of organoid technology has brought revolutionary changes to many fields. As the first technology proposed, small intestinal organoids have also received widespread attention and application in virology research. Traditional in vitro cell lines such as T84, Caco-2 and HT29 are derived from colorectal adenocarcinoma and have a single cell type. The consistency of the same cell lines from different sources is poor, and their genes, proteins and cell metabolism are very different from those of in vivo tissues. Therefore, there are great limitations in their application in virology research. Human intestinal organoids (HIOs) have a three-dimensional structure similar to crypts and contain a variety of small intestinal epithelial cell types such as stem cells, Paneth cells, intestinal epithelial cells, goblet cells and enteroendocrine cells. They can also be long-term passaged and frozen. Therefore, HIOs are closer to the physiological structure and function of human intestinal organs than traditional cell lines, providing a more realistic model for the study of the pathogenesis of viral infection in the intestine. HIOs can be established from adult stem cells (hASCs) or pluripotent stem cells (hPSCs). For example, some studies have isolated intestinal stem cells from surgically removed human intestinal tissue and embedded them in matrix gel, and constructed HIOs by three-dimensional culture in a culture system containing growth factors such as WNT 3A, R-spondin1, Noggin, and EGF. Other studies have also proposed methods for inducing hPSCs to differentiate into HIOs in vitro, treating hPSCs with activin A to differentiate them into FOXA2 + SOX17 + endoderm, followed by the addition of FGF4 and WNT 3A to form CDX2 + The mid / hindgut 3D spheroids were then embedded in Matrigel and further cultured with medium containing R-spondin1, Noggin and EGF to form HIOs. Since human small intestinal tissue is difficult to obtain, and human induced pluripotent stem cells (hiPSCs) have the advantages of commercial sources, easy access, and no ethical and moral issues and are widely used in many research fields, hiPSCs-derived HIOs provide researchers with more options.

[0003] HiPSC-derived HIOs are three-dimensional vesicle structures with multiple intestinal cell types that are differentiated by simulating the in vivo intestinal development process in vitro. They are widely used in the study of the infection characteristics and pathogenic mechanisms of various enteric viruses. The stable maintenance of HIOs structure and cell types is the premise and basis for its use as an experimental model. The passaging method is a key technical link for the long-term maintenance of the structural and functional stability of intestinal organoids. Currently, the commonly used passaging methods mainly include the fragmentation method. However, the fragmentation method will cause hiPSC-derived HIOs to gradually lose epithelial characteristics and transform into solid spheres during long-term passaging, and it is impossible to maintain the stable passage of HIOs and maintain the diversity of structure and cell types for a long time. Summary of the invention

[0004] The purpose of the present invention is to provide a stable passaging method for small intestinal organoids derived from human induced pluripotent stem cells and its application, wherein the passaging method can maintain the structure and stable passaging of small intestinal organoids and contain multiple cell types.

[0005] The present invention provides a method for stable propagation of small intestinal organoids derived from human induced pluripotent stem cells, comprising the following steps:

[0006] Mixing the Matrigel dissociation reagent with the small intestinal organoids embedded in the Matrigel to degel, thereby obtaining a small intestinal organoid suspension;

[0007] The small intestinal organoid suspension is blown with a pipette tip to obtain a small intestinal organoid fragment suspension;

[0008] washing the small intestinal organoid fragment suspension to obtain small intestinal organoid fragments;

[0009] The small intestinal organoid fragments were embedded in matrix gel and then subcultured.

[0010] Preferably, the degumming method comprises: mixing the matrix gel dissociation reagent with the small intestinal organoids embedded in the matrix gel and shaking the mixture horizontally at 4° C. for 1 hour.

[0011] Preferably, the matrix gel dissociation reagent includes a cell recovery solution.

[0012] Preferably, the blowing is performed 6 to 8 times.

[0013] Preferably, based on the number of small intestinal organoids, during the embedding, 50 to 70 small intestinal organoids are embedded in each gel droplet.

[0014] Preferably, the method for constructing small intestinal organoids comprises the following steps:

[0015] Inducing human induced pluripotent stem cells to differentiate into definitive endoderm cells;

[0016] Inducing the definitive endoderm cells to differentiate into mid / hindgut vesicle-like structures;

[0017] The mid / hindgut vesicle-like structure is embedded in matrix gel and then maintained in three-dimensional culture to obtain small intestinal organoids;

[0018] The small intestinal organoids were cultured using STEMdiff TM Intestinal Organoid Differentiation Culture Kit.

[0019] The present invention also provides the application of the passage method described in the above technical solution in the preparation of enterovirus infection related products.

[0020] Preferably, the enterovirus comprises EV-D68 virus.

[0021] Preferably, the enterovirus infection includes enterovirus infection of one or more of small intestinal epithelial cells, intestinal proliferative cells, Paneth cells and enteroendocrine cells.

[0022] Preferably, the application comprises: mixing the small intestinal organoid fragment suspension with a virus solution and then incubating the mixture, washing the mixture obtained by incubation to obtain virus-infected small intestinal organoid fragments;

[0023] The virus-infected small intestinal organoid fragments are embedded in matrix gel and then cultured to obtain virus-infected small intestinal organoids.

[0024] Beneficial effects:

[0025] The present invention provides a method for passage of small intestinal organoids derived from human induced pluripotent stem cells, comprising the following steps: mixing and degumming a matrix gel dissociation reagent with small intestinal organoids embedded in matrix gel to obtain a small intestinal organoid suspension; blowing the small intestinal organoid suspension with a pipette tip to obtain a small intestinal organoid fragment suspension; washing the small intestinal organoid fragment suspension to obtain small intestinal organoid fragments; embedding the small intestinal organoid fragments with matrix gel and then passage culturing. The passage method of the present invention, after the matrix gel is gently removed by the dissociation reagent, the small intestinal organoid is slightly broken by a large-aperture pipette tip, which can maintain the long-term stable passage of the small intestinal organoid and maintain the structure and cell type diversity of the small intestinal organoid.

[0026] At the same time, enterovirus is infected into the small intestinal organoids obtained by the above-mentioned passaging method. Enterovirus can effectively replicate in the small intestinal organoids and infect one or more cells including small intestinal proliferating cells, Paneth cells and enteroendocrine cells, and can then be used for related research on enterovirus infection and host interaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0028] Figure 1 This is a flow chart of the construction and passage method of small intestinal organoids derived from human induced pluripotent stem cells (hiPSCs) in Example 1;

[0029] Figure 2 The bright field, HE staining and immunofluorescence staining identification images of human small intestinal organoids obtained by different passage methods in Example 1, wherein the scale bars of A to B are 100 μm, and the scale bars of C to F are 25 μm;

[0030] Figure 3 These are the replication kinetics curve of hiPSCs-derived intestinal organoids infected with EV-D68 in Example 1 and their cell-tropic immunofluorescence staining images, where the scale bar in B is 25 μm and the scale bar in C is 50 μm. DETAILED DESCRIPTION

[0031] The present invention provides a method for subculturing small intestinal organoids derived from human induced pluripotent stem cells, comprising the following steps:

[0032] Mixing the Matrigel dissociation reagent with the small intestinal organoids embedded in the Matrigel to degel, thereby obtaining a small intestinal organoid suspension;

[0033] The small intestinal organoid suspension is blown with a pipette tip to obtain a small intestinal organoid fragment suspension;

[0034] washing the small intestinal organoid fragment suspension to obtain small intestinal organoid fragments;

[0035] The small intestinal organoid fragments were embedded in matrix gel and then subcultured.

[0036] As an embodiment, the present invention constructs small intestinal organoids, comprising the following steps: inducing human induced pluripotent stem cells to differentiate into endoderm cells; inducing the endoderm cells to differentiate into mid-gut / hindgut vesicle-like structures; embedding the mid-gut / hindgut vesicle-like structures into matrix gel and then maintaining and culturing them in three dimensions to obtain small intestinal organoids; the small intestinal organoids are cultured using STEMdiff TM Intestinal Organoid Differentiation Culture Kit.

[0037] As an embodiment, the present invention inoculates human induced pluripotent stem cell clusters into a 24-well plate and cultures them until the confluence reaches 85%-90%. As an embodiment, the inoculation amount of human induced pluripotent stem cells described in the present invention is 6000 cell clusters / well. As an embodiment, the culture time described in the present invention is 24 to 48 hours; the temperature is 37°C, the carbon dioxide concentration is 5%, and the humidity is 95%. In a specific embodiment, the culture is carried out in a cell culture incubator at 37°C, 5% CO2, and 95% humidity. As an embodiment, the culture medium is a human pluripotent stem cell culture medium; in a specific embodiment, the culture medium is mTeSR TM Plus, brand STEMCELL TM , #100-0276.

[0038] After the confluence degree of the human induced pluripotent stem cells reaches 85% to 90%, the present invention induces the differentiation of the human induced pluripotent stem cells into definitive endoderm cells; specifically, definitive endoderm induction differentiation medium is added to the 24-well plate to induce differentiation of endoderm cells, and fresh medium is replaced every 24 hours to obtain definitive endoderm cells. As an embodiment, the amount of the definitive endoderm induction differentiation medium added in the present invention is 0.7 mL / well. As an embodiment, the definitive endoderm induction differentiation medium in the present invention is STEMdiff TM Definitive endoderm differentiation induction medium in the intestinal organoid differentiation culture kit. As an embodiment, the time for induction differentiation of endoderm cells described in the present invention is 2 days. As an embodiment, the conditions for induction differentiation of endoderm cells described in the present invention are a temperature of 37°C, a carbon dioxide concentration of 5%, and a humidity of 95%. In a specific embodiment, the culture is carried out in a cell culture incubator at 37°C, 5% CO2, and 95% humidity.

[0039] After obtaining the definitive endoderm cells, the present invention induces differentiation of the definitive endoderm cells into mid-gut / hindgut vesicle-like structures, specifically, adding a mid-gut / hindgut induction differentiation medium to a 24-well plate containing definitive endoderm cells to induce differentiation of mid-gut / hindgut vesicle-like structures, and replacing fresh medium every 24 hours to obtain mid-gut / hindgut vesicle-like structures. As an embodiment, the amount of mid-gut / hindgut induction differentiation medium added in the present invention is 0.5 mL / well. As an embodiment, the mid-gut / hindgut induction differentiation medium in the present invention is STEMdiff TMDefinitive endoderm induction differentiation medium in the intestinal organoid differentiation culture kit. As an embodiment, the time for inducing differentiation of the mid / hindgut vesicle-like structure of the present invention is 5 days. As an embodiment, the conditions for inducing differentiation of the mid / hindgut vesicle-like structure of the present invention are a temperature of 37°C, a carbon dioxide concentration of 5%, and a humidity of 95%. In a specific embodiment, the culture is carried out in a cell culture incubator at 37°C, 5% CO2, and 95% humidity.

[0040] After obtaining the mid / hindgut vesicle-like structure, the present invention embeds the mid / hindgut vesicle-like structure into matrix gel and then maintains and culture it in three dimensions to obtain small intestinal organoids. As an embodiment, the embedding step includes: mixing the mid / hindgut vesicle-like structure with the pre-cooled matrix gel evenly, and adding the gel droplets to the preheated 24-well plate to form a gel droplet in each well; incubating the 24-well plate inverted to solidify the gel droplets, and adding small intestinal organoid growth medium to the 24-well plate for three-dimensional maintenance culture to obtain small intestinal organoids, that is, small intestinal organoids embedded in matrix gel. As an embodiment, the temperature of the pre-cooled matrix gel in the present invention is 0°C. As an embodiment, the number of mid / hindgut vesicle-like structures in the present invention is 50 to 70 per well. As an embodiment, the temperature of the inverted incubation in the present invention is 37°C, and the time is 20 minutes. As an embodiment, the temperature of the preheated 24-well plate in the present invention is 37°C. As an embodiment, the small intestinal organoid growth medium in the present invention is STEMdiff TM Intestinal Organoid Growth Medium. As an embodiment, the three-dimensional maintenance culture conditions of the present invention are 37° C., 5% CO 2 , the culture medium is replaced every 4 days, and the cells are subcultured every 7 to 9 days.

[0041] After obtaining the small intestinal organoids embedded in the matrix gel, the present invention mixes the matrix gel dissociation reagent with the small intestinal organoids embedded in the matrix gel for degumming to obtain a small intestinal organoid suspension. As an embodiment, the matrix gel dissociation reagent of the present invention is a cell recovery solution (Cell Recovery Solution). As an embodiment, the degumming method of the present invention comprises: mixing the matrix gel dissociation reagent with the small intestinal organoids embedded in the matrix gel and shaking horizontally at 4°C for 1 hour. As an embodiment, the speed of the horizontal shaking is 120rpm.

[0042] After obtaining the small intestinal organoid suspension, the present invention uses a pipette to blow the small intestinal organoid suspension to obtain a small intestinal organoid fragment suspension. As an embodiment, the blowing method of the present invention is gentle blowing to avoid excessive breaking of the small intestinal organoids by violent blowing to produce a large number of single cells. The specific implementation method is to use a manual pipette to blow and aspirate for 4 seconds at a time. The specification of the manual pipette is 1mL, and the frequency of blowing and aspiration is 15 times / min. As an embodiment, the aperture of the pipette tip of the present invention is 1.4 to 1.6mm; as another embodiment, the aperture of the pipette tip is 1.5mm; in a specific embodiment, the pipette tip is a gun tip with a specification of 1mL. As an embodiment, the number of blowing and aspirating of the present invention is 6 to 8 times.

[0043] After obtaining the small intestinal organoid fragment suspension, the present invention washes the small intestinal organoid fragment suspension to obtain small intestinal organoid fragments. As an embodiment, the present invention uses a basal culture medium for the washing. As an embodiment, the basal culture medium of the present invention is a DMEM / F12 culture medium. In a specific embodiment, a DMEM / F12 culture medium of STEMCELL is used, and the product number is Cat#36254.

[0044] After obtaining the small intestinal organoid fragments, the present invention embeds the small intestinal organoid fragments with matrix gel and continues to culture. The embedding method described in the present invention is the same as the embedding method described in the above technical solution and will not be repeated. As an embodiment, based on the number of small intestinal organoids, when the present invention performs the embedding, 50 to 70 small intestinal organoids are embedded in each gel droplet.

[0045] The present invention also provides the use of the passage method described in the above technical solution in the preparation of enterovirus infection related products. As an embodiment, the enterovirus of the present invention includes but is not limited to enterovirus EV-D68. As an embodiment, the enterovirus infection of the present invention includes enterovirus infection of one or more of small intestinal epithelial cells, intestinal proliferating cells, Paneth cells and enteroendocrine cells.

[0046] As an embodiment, the application includes: mixing the small intestinal organoid fragment suspension with a virus solution and incubating the mixture, washing the mixture obtained by incubation to obtain virus-infected small intestinal organoid fragments;

[0047] The virus-infected small intestinal organoid fragments are embedded in matrix gel and then cultured to obtain virus-infected small intestinal organoids. The present invention does not specifically limit the ratio of the small intestinal organoid fragment suspension to the virus solution, and it can be set according to the research routine. For example, in the embodiments of the present invention, based on the number of the small intestinal organoids, the number of the small intestinal organoids and the volume of the enterovirus EV-D68 virus solution are 70:500 μL.

[0048] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1

[0050] 1. Materials and Methods

[0051] 1. Cultivation of human induced pluripotent stem cells (hiPSCs) and construction of small intestinal organoids

[0052] (1) Culture and subculture of hiPSCs (purchased from Nanjing Baishou Biotechnology Co., Ltd., China): hiPSCs and 10 μg / μL embryonic stem cell culture matrix (Vitronectin XF TM , brand is STE MCELL TM , #07180) 37°C, 30 min coated six-well plate for later use;

[0053] Remove the original culture medium of the cells to be subcultured and replace with DPBS (without Ca 2+ Mg 2+ After washing with ion solution, add GCDR Gentle Cell Dissociation Reagent (STEMCELL TM , #100-0485) at room temperature for 4-5 minutes, tap the sides of the six-well plate several times until most of the cells fall off in the form of clumps, and add human pluripotent stem cell culture medium (mTeSR TM Plus, brand STEMCELL TM , #100-0276) to terminate digestion, and the cell clumps were inoculated into the coated six-well plate at a ratio of 1:6 for continued culture, that is, 1 mL of cell suspension containing cell clumps was added with 5 mL of culture medium, and fresh culture medium was replaced every 24 h, and passaged every 5 days;

[0054] (2) Construction of human small intestinal organoids: Before differentiation, hiPSCs were seeded into 24-well plates at a ratio of 6000 cell clusters / well and cultured in a cell culture incubator at 37°C, 5% CO2, and 95% humidity for 24-48 hours. Differentiation began when the confluence reached 85-90%. On days 0-2 of differentiation, 0.7 mL of definitive endoderm differentiation medium (STEMCELL ELECTRODEGRADATION SYSTEM) was added to each well of hiPSCs. TM , STEMdiff TMIntestinal Organoid Kit, #05140), fresh culture medium was replaced every 24 h; on differentiation days 3-7, 0.5 mL of midgut / hindgut differentiation medium (STEMCELL TM , STEMdiff TM Intestinal Organoid Kit, #05140), fresh culture medium was replaced every 24 h; on the 8th day of differentiation in a cell culture incubator at 37°C, 5% CO2, 95% humidity, the suspended vesicle-like structure was mixed with pre-cooled matrix gel ( Matrix, REF 356231) were mixed evenly, and 50-70 gel drops were added to the center of a 24-well plate preheated at 37°C to form a gel drop in each well. The 24-well plate was inverted and placed in a 37°C incubator for 20 minutes to solidify the gel drop, and then 0.6 mL of human small intestinal organoid growth medium (STEMCELL TM , STEMdiff TM Intestinal Organoid Growth Medium, #05145), cultured at 37°C with 5% CO2, medium completely replaced every 4 days, and passaged every 7-9 days;

[0055] 2. Passaging of Human Small Intestinal Organoids

[0056] (1) Degumming method

[0057] Add Cell Recovery Solution ( 354253) and then shake horizontally at 4°C for 1h to remove the matrix gel, and use a 1mL pipette tip (inner diameter 1.5mm) to gently blow (Eppendorf research plus manual pipette (1mL) single blow for 4s (blowing and aspiration frequency 15 times / min)) the organoid suspension 6 times, wash with basal culture medium and re-embed the matrix gel at a number of 50-70 organoids per well; after encapsulation, continue normal subculture.

[0058] (2) Subculture by fragmentation

[0059] Add Cell Recovery Solution ( 354253) and let it stand at room temperature for 1 min, then use a 1 mL pipette tip (inner diameter 0.8 mm) to directly blow the gel drop 8-12 times to form an organoid suspension, and after washing, re-embed the matrix gel with 50-70 organoids per well; after encapsulation, continue normal subculture.

[0060] 3. EV-D68 infection of intestinal organoids

[0061] Virus strain information (Enterovirus D68 isolate Beijing-R0132, GenBank: KP240936.1). After removing the matrix gel using Cell Recovery Solution, blow the organoids 8 times with a 1 mL pipette tip and mix with the virus solution at a rate of 70 cells / 500 μL (the titer of the virus is 10 5 TCID 50 / mL), incubated at 33°C for 2h, washed twice and then re-encapsulated and maintained at 33°C, 5% CO2, 100 μL of supernatant was collected every day to detect viral nucleic acid and an equal volume of fresh culture medium was added.

[0062] 4. Digital PCR detection of EV-D68 viral nucleic acid copy number

[0063] 100 μL of cell supernatant collected 7 days ago was used to extract nucleic acid using VAMNE Virus DNA RNA Reagents 3.0 (Vazyme, RM501) kit, and the viral nucleic acid copy number was detected using digital PCR reagents (Roche, 76640100) and a digital PCR instrument.

[0064] 5. HE staining of paraffin sections

[0065] The organoids fixed with 4% paraformaldehyde were embedded in CellGel (Beijing Zhuoding Biotechnology Co., Ltd., 0500-0072), embedded in paraffin (Leica, 39601006), and cut into 5 μm thick slices. After dewaxing, they were stained with hematoxylin-eosin (HE staining kit, Beijing Solebow Technology Co., Ltd., G1120-100) and imaged with an inverted microscope (LeicaDMi1).

[0066] 6. Immunofluorescence Staining

[0067] Using Cell Recovery Solution( 354253) After removing the matrix gel, the organoids were fixed and blocked, and then the primary antibodies (Rat E-Cadherin [1:400], Invitrogen, #13-1900; Rabbit Chromogranin A [1:400], NOVUS, NB120-15160; Rabbit Lysozyme [1:400], Invitrogen, #PA5-16668; Rabbit MUC 2 [1:400], NOVUS, NBP1-31231; Rabbit Ki-67 [1:400], Invitrogen, #MA5-14520; Mouse EV-D68 VP1 [1:200], GeneTex, GTX633770) were added. The cells were incubated at 4°C overnight with horizontal shaking. After washing three times, the secondary antibodies and dyes (Donkey anti-Rat Alexa Fluor 488 [1:1000], Invitrogen, #A-21208; Donkey anti-Rabbit Alexa Fluor 555 [1:1000], Invitrogen, #A-31572; Donkey anti-Mouse Alexa Fluor 647 [1:1000], Invitrogen, #A-31571; DAPI [1:1000], Invitrogen, D1306; Alexa Fluor 647 Phalloidin [1:1000], Invitrogen, A22287) were incubated at 4°C overnight with horizontal shaking, washed three times, and then transparent reagent was added and the slides were mounted. The images were taken using a laser confocal microscope (Leica SP8) (refer to the literature [van Ineveld RL, Ariese HCR, Wehrens EJ, Dekkers JF, Rios AC. Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids. VisExp.2020Jun 5;(160).doi:10.3791 / 60709.PMID:32568249.】conducted).

[0068] 2. Results and Analysis

[0069] 1. Continuous passaging with different passaging methods changes the morphology of small intestinal organoids derived from human induced pluripotent stem cells (hiPSCs)

[0070] hiPSCs can form tightly arranged endoderm cells 72 hours after adding definitive endoderm induction differentiation medium, followed by 3-4 days for mid / hindgut induction differentiation. At this stage, the production of three-dimensional cystic structures can be observed. Over time, self-assembled to form closed mid / hindgut vesicle-like structures and completely detached from the bottom of the cell culture plate and suspended. On the 8th day of differentiation, the suspended mid / hindgut vesicle-like structures were collected and embedded in matrix gel, and human small intestinal organoid growth medium was added to maintain three-dimensional culture for 7-10 days. It can be observed that the vesicle-like structure gradually becomes larger and the edges of the sphere are smooth. The construction of hiPSCs-derived small intestinal organoids is thus completed and marked as generation 0 ( Figure 1 (A).

[0071] Differentiated human small intestinal organoids can be passaged periodically, but different passage methods will have different effects on the stability of the organoids.

[0072] Degumming passaging: First, dissociate the matrix gel to release the human small intestinal organoids and gently blow them to break them into larger organoid fragments for passaging. This scheme maintains the typical epithelial structure of intestinal organoids during long-term passaging ( Figure 1 Medium B and Figure 2 Middle A), with lumen region and sprouting region.

[0073] The fragmentation method is to pass the organoids directly by blowing and percussing the matrix gel droplets. After 3-5 generations, the organoids are completely transformed from vesicle-like structures to solid cell spherical structures ( Figure 1 Medium B and Figure 2 (middle B).

[0074] 2. Continuous passage by degumming method can maintain the epithelial structure and main cell types of human induced pluripotent stem cell-derived small intestinal organoids for a long time

[0075] HE staining results showed that the human intestinal organoids obtained by continuous passage by degumming method had a hollow tubular structure, while the solid cell spheroid structure obtained by the crushing method ( Figure 2 (A~B).

[0076] The results of immunofluorescence staining showed that the human small intestinal organoids obtained by degumming expressed epithelial cell markers (E-Cadherin), proliferative cell markers (Ki-67), and intestinal epithelial cell microvilli structure markers (F-actin) ( Figure 2 C) and enteroendocrine cell markers (Chromogranin A), Paneth cell markers (Lysozyme) and goblet cell markers (MUC 2) ( Figure 2 However, the organoids obtained by long-term passage by the fragmentation method expressed no expression of other intestinal epithelial cell markers except for the proliferative cell marker (Ki-67) ( Figure 2 D and F).

[0077] It can be seen that although the fragmentation method of passaging by repeatedly blowing the matrix gel droplets directly with the pipette tip can break them up and liquefy them to release small intestinal organoids, HIOs will also be damaged by stronger mechanical forces, thus breaking them into small fragments and producing a large number of single cells. Long-term passaging using this method will lead to the loss of HIOs epithelial structure and conversion into solid spheres.

[0078] The degumming method, in which the matrix gel is gently removed with a dissociation reagent and then the HIOs are slightly broken up with a pipette tip, can maintain long-term stable passaging, ensuring that the HIOs structure is stable and contains a variety of cell types.

[0079] The above results indicate that degumming passaging is essential for maintaining the intestinal epithelial characteristics of hiPSCs-derived small intestinal organoids during long-term culture, while long-term passaging by the fragmentation method leads to the loss of the structure and cell composition of human small intestinal organoids.

[0080] 3.EV-D68 may infect intestinal epithelial cells, proliferating cells, Paneth cells, and enteroendocrine cells of human induced pluripotent stem cell-derived small intestinal organoids

[0081] After EV-D68 infected human intestinal organoids, dPCR results showed that the number of viral nucleic acid copies in the cell supernatant increased with the extension of inoculation time ( Figure 3 Middle A), immunofluorescence staining results on the 7th day of infection showed that the viral VP1 protein co-localized with intestinal epithelial cells (F-actin) and other cells in human small intestinal organoids ( Figure 3 Middle B), demonstrating that EV-D68 can infect small intestinal organoids and replicate efficiently.

[0082] In order to further prove the cell tropism of EV-D68 in intestinal organoids, the present example performed co-staining of virus and cell type markers. The results showed that EV-D68 VP1 protein co-localized with enteroendocrine cells (Chro mogranin A), Paneth cells (Lysozyme), and proliferating cells (Ki-67), suggesting that the virus may have the above-mentioned cell infection tropism ( Figure 3 Middle C).

[0083] The above results indicate that the passaging method of the present invention is not only conducive to the maintenance of the stability of iPSCs-derived HIOs in long-term passaging, but also successfully infects modern isolates of EV-D68, which can effectively replicate and infect intestinal epithelial cells, proliferating cells, Paneth cells and enteroendocrine cells, proving that the model can be applied to the study of enterovirus infection of human intestinal epithelium.

[0084] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for subculturing small intestinal organoids derived from human induced pluripotent stem cells, characterized in that: The steps include: Mixing the Matrigel dissociation reagent with the small intestinal organoids embedded in the Matrigel to degel, thereby obtaining a small intestinal organoid suspension; The small intestinal organoid suspension is blown with a pipette tip to obtain a small intestinal organoid fragment suspension; washing the small intestinal organoid fragment suspension to obtain small intestinal organoid fragments; The small intestinal organoid fragments were embedded in matrix gel and then subcultured.

2. The method for subculturing according to claim 1, characterized in that: The degumming method comprises: mixing the matrix gel dissociation reagent with the small intestinal organoids embedded in the matrix gel, and then shaking horizontally at 4° C. for 1 hour.

3. The method for subculturing according to claim 1 or 2, characterized in that: The matrix gel dissociation reagent includes a cell recovery solution.

4. The method for subculturing according to claim 1, characterized in that: The blowing and beating are performed 6 to 8 times.

5. The method for subculturing according to claim 1, characterized in that: In terms of the number of small intestinal organoids, during the embedding process, 50 to 70 small intestinal organoids were embedded in each gel droplet.

6. The method for subculturing according to claim 1, characterized in that: The method for constructing small intestinal organoids comprises the following steps: Inducing human induced pluripotent stem cells to differentiate into definitive endoderm cells; Inducing the definitive endoderm cells to differentiate into mid / hindgut vesicle-like structures; The mid / hindgut vesicle-like structure is embedded in matrix gel and then maintained in three-dimensional culture to obtain small intestinal organoids; The small intestinal organoids were cultured using STEMdiff TM Intestinal Organoid Differentiation Culture Kit.

7. Use of the passage method according to any one of claims 1 to 6 in the preparation of products related to enterovirus infection.

8. The use according to claim 7, characterized in that: The enterovirus includes EV-D68 virus.

9. The use according to claim 7, characterized in that: The enterovirus infection includes enterovirus infection of one or more of intestinal epithelial cells, intestinal proliferating cells, Paneth cells and enteroendocrine cells.

10. The use according to claim 7, characterized in that: The application comprises: mixing the small intestinal organoid fragment suspension with a virus solution and then incubating the mixture, washing the mixture obtained by incubation, and obtaining virus-infected small intestinal organoid fragments; The virus-infected small intestinal organoid fragments are embedded in matrix gel and then cultured to obtain virus-infected small intestinal organoids.

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