Human-derived iPSC (induced pluripotent stem cell) induced liver organ and preparation method thereof

Abstract

The invention discloses a human-derived iPSC (induced pluripotent stem cell)-induced liver organ and a preparation method thereof. The method comprises the following steps: digesting human induced pluripotent stem cells obtained by culture into single cells; single cells are prepared into cell clusters through self-balling, or cell matrigel microspheres are prepared by using a T-type microfluidic technology and then are distributed one by one, so that one cell cluster is formed in each pore of the low-adsorption 96-pore plate; carrying out step-by-step induction culture on the obtained cell cluster, and culturing in a culture medium I for 5 hours; replacing with a culture medium II and culturing for 2 days; a culture medium III is used for culturing for 2 days; replacing with a culture medium IV and culturing for 5 days; replacing with a culture medium V, and culturing for 6 days; and replacing with a culture medium VI, and culturing for 10 days to obtain the mature human liver organ. According to the invention, liver organs with human normal liver cell characteristics and protein expression can be formed within 25 days, and the method can realize homogenization and high efficiency of iPSC induction.

Description

[0001] Field

[0002] The present invention belongs to the field of normal tissue organoid preparation, and specifically relates to a human iPSC-induced liver organoid and a preparation method thereof. Background Art

[0003] Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells that have similar characteristics to human embryonic stem cells and are cultured by artificially reprogramming somatic cells and reverse differentiation. iPSCs have multidirectional differentiation and strong self-replication potential. Under certain conditions, they can differentiate into a variety of functional cells, and a large number of clinically relevant phenotype cells can be cultured in vitro.

[0004] The liver is an important target organ for drug toxicity testing, among which hepatocytes and bile duct cells in the liver biliary system play a key role in drug metabolism and drug toxicity analysis, and are important components for building in vitro models. The traditional in vitro culture model is to inoculate hepatocytes in matrix gel, grow in layers on the formed basal layer, and easily lose the characteristics of hepatocytes.

[0005] The shortage of liver tissue resources is a major challenge in liver transplantation treatment research and liver drug screening. Liver organoids with metabolic functions obtained by induced pluripotent stem cells (iPSCs) provide a solution to this problem. iPSCs can differentiate into various liver cells during the induction process, such as hepatocytes, bile duct cells, endothelial cells, and Kupffer cells. The three-dimensional organoids formed by the self-assembly of these cells are a useful tool for clinical drug screening and also promote the development of liver cell transplantation.

[0006] At present, the technology of inducing iPSC cell clusters to form liver organoids still has limitations. The traditional 2D induction of iPSC has problems such as limited space for cell proliferation, low cell viability after induction and maturation, and difficulty in cell transfer. The artificial induction of liver organoids by wrapping iPSC with matrix gel requires a lot of time and effort, and there are easy differences between batches and low throughput, which cannot meet current research needs. At the same time, the current culture scheme requires 45 days or more to obtain mature liver organoids, which increases the time and cost of organoid-based drug screening.

[0007] Organoids are tissue analogs with a certain spatial structure and microenvironment formed by three-dimensional (3D) culture of adult stem cells or pluripotent stem cells in vitro. After iPSCs are prepared into microspheres through T-type microfluidics technology, they are induced to form mature liver organoids with high uniformity. The process is characterized by high efficiency, high throughput, short construction cycle, and easy transfer and preservation. Mature liver organoids can be widely used in drug screening, disease modeling, regenerative medicine, etc.

[0008] Although there have been many major breakthroughs in iPSC-derived liver organoids, they also face many shortcomings and challenges that need to be overcome. iPSC culture is demanding on temperature and environmental conditions, prone to non-directional differentiation, and has extremely strict requirements on production processes and quality control; iPSCs will have differences in morphology, growth, gene expression, and differentiation in different batches after culture and induction, and there are certain difficulties in reducing heterogeneity and formulating quality control methods; iPSC-induced liver organoids still differ from in vivo diseases in disease model construction, so the low degree of fit of the disease model is an urgent problem to be solved. Summary of the invention

[0009] In order to overcome the defects of the prior art, the purpose of the present invention is to provide a human iPSC-induced liver organoid and a preparation method thereof. The present invention uses iPSC induction technology to prepare a method for human liver organoids. The induction process involves a total of six culture media containing different induction factors, which are induced step by step. First, the proliferating stem cells are placed in culture medium I for several hours to complete the transition from proliferation to the beginning of induction, and then placed in culture medium II and culture medium III for two days each to obtain endoderm cells and mesoderm cells; the endoderm cells and mesoderm cells are placed in culture medium IV for differentiation and culture, and gradually differentiated into hepatocytes; the hepatocytes are placed in culture medium V for culture, and gradually differentiated into hepatocytes and bile duct epithelial cells, and finally placed in culture medium VI for culture for several days to form liver organoids. The present invention achieves the purpose of using iPSC to prepare human liver organoids in large quantities, realizes high-throughput preparation of mature liver organoids, and has the characteristics of high uniformity, rapid maturation, easy transfer and preservation, which can be used to study the molecular mechanism of liver development and metabolism in vitro, and provides a new method for liver disease modeling, drug screening and regenerative medicine.

[0010] The specific technical solutions are as follows:

[0011] The present invention provides a method for preparing human iPSC-induced liver organoids, comprising the following steps:

[0012] 1) Digest the cultured human induced pluripotent stem cells into single cells;

[0013] 2) The single cells obtained in step 1) are separated by method 1 or method 2 to achieve one cell cluster per well of a low-adsorption 96-well plate;

[0014] Method 1: The single cells obtained in step 1) are inoculated into a low-adsorption 96-well plate and then self-spherized to prepare cell clusters, thereby achieving one cell cluster per well of the low-adsorption 96-well plate;

[0015] Method 2: The single cells obtained in step 1) are made into cell matrix gel microspheres using T-type microfluidics technology, and then distributed one by one into a low-adsorption 96-well plate, so that one cell cluster is achieved in each well of the low-adsorption 96-well plate;

[0016] 3) The cell clusters obtained in step 2) are subjected to step-by-step induction culture at 37°C and 5% CO2: the cell clusters obtained in step 2) are cultured in culture medium I for 5 h; then cultured in culture medium II for 2 days; then cultured in culture medium III for 2 days; then cultured in culture medium IV for 5 days; then cultured in culture medium V for 6 days; then cultured in culture medium VI for 10 days, thereby obtaining mature human liver organoids;

[0017] Medium I was RPMI1640 / B27 containing 100 ng / mL Activin A and 20 ng / mL BMP4;

[0018] Medium II was RPMI1640 / B27 containing 25% mTeSR, 100 ng / mL Activin A, and 20 ng / mL BMP4;

[0019] Medium III was RPMI1640 / B27 containing 25% mTeSR and 100 ng / mL Activin A;

[0020] Medium IV was RPMI1640 / B27 containing 25% mTeSR, 30 ng / mL FGF4, and 20 ng / mL BMP2;

[0021] Medium V was RPMI1640 / B27 containing 25% mTeSR, 20 ng / mL HGF, and 20 ng / mL KGF;

[0022] Culture medium VI is high-glucose DMEM containing 0.2% by mass of bovine serum albumin, 0.1% by volume of ITS, 0.1% by volume of gentamicin-amphotericin B, 10 ng / mL of OSM, and 15 μg / mL of cholesterol.

[0023] Furthermore, the step of digesting human induced pluripotent stem cells into single cells in step 1) includes: transferring the cell group obtained by subculture digestion into a centrifuge tube, discarding the culture medium, washing once with PBS, discarding PBS, adding TrypLE containing EDTA and digesting in a 37°C water bath for 5 minutes, and after taking out, gently blowing the cells 1-2 times, and tapping the tube wall with a finger until there is no obvious precipitation, terminating the digestion, centrifuging and discarding the supernatant, and collecting the precipitate.

[0024] Furthermore, method 1 specifically includes: adding the single cell obtained in step 1) to iPSC culture medium containing Y27632 to dilute, controlling the cell suspension density to 50,000-100,000 / mL, inoculating into a low-adsorption 96-well plate, culturing 100-120 μL per well to form a sphere.

[0025] Furthermore, in method 2, every 10 4 Add 1 μL of Matrigel to each single cell.

[0026] Furthermore, the method for preparing cell matrix glue microspheres in method 2 includes: uniformly mixing the single cell obtained in step 1) with matrix glue to obtain a cell-matrix glue mixture; using the obtained cell-matrix glue mixture as a dispersed phase and an oil phase reagent as a continuous phase, shearing the cell-matrix glue mixture into monodisperse droplets by using microfluidics technology, and fixing the obtained monodisperse droplets at 37° C. for 30 min to obtain cell matrix glue microspheres;

[0027] Preferably, the oil phase reagent is fluorinated oil;

[0028] Preferably, the flow rate of the cell-matrix gel mixture is controlled to be 10-15 μL / min, and the flow rate of the oil phase reagent is controlled to be 100-110 μL / min.

[0029] Furthermore, the diameter of the cell clusters prepared by self-sphering in method 1 is 800-1200 μm.

[0030] Furthermore, in method 2, the diameter of the cell matrix gel microspheres is 620-680 μm.

[0031] Furthermore, in step 3), before culturing the cell cluster obtained in step 2) in culture medium I, the step further includes: culturing the cell cluster obtained in step 2) in iPSC culture medium containing Y27632 for two days, and the culture conditions are 37° C. and 5% CO 2 .

[0032] Furthermore, the mature human liver organoids obtained in step 3) are further cultured using culture medium VI.

[0033] The present invention also provides a liver organoid prepared by the preparation method.

[0034] The beneficial effects of the present invention are:

[0035] The present invention uses a low-adsorption 96-well plate with one cell cluster in each well, the induction culture medium is uniform, and the cell proliferation space is increased. Compared with traditional plating, the induction viability is improved and the induction cycle is short. It can achieve large-scale induction of iPSCs to form liver organoids, and is easy to dissociate and transfer, which is convenient for subsequent metabolism or drug screening experiments of liver organoids. Specifically: 1) The diameter of the cell clusters prepared by self-spheroidization in the present invention is about 800-1200 μm, and the diameter of the cell matrix gel microspheres is about 650 μm. Their morphology and structure remain uniform during the induction process; 2) The cell proliferation space of the present invention is increased. Compared with the traditional adherent cell culture, the cells expand during induction to form three-dimensional cell clusters with sufficient space, which can achieve large-scale cell proliferation and large-scale induction; 3) The induction cycle of iPSC to liver organoids is shortened in the present invention. Compared with the traditional 2D induction, the completion time of organoid culture induction is shortened from 45 days to about 25 days; 4) In the present invention, cell culture and induction are both carried out on low-adsorption well plates, and the cells grow non-adherently. After the induction is completed, they can be transferred to meet various characterization and detection needs; 5) The mature human liver organoids obtained by the present invention highly express liver endothelial cells, hepatocytes and mature bile duct cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Bright field of iPSC low-adsorption spheres and iPSC-DEO-induced liver organoids at different time points, Scale bar: 100μm;

[0037] Figure 2 HE staining of liver organoids at different time points after iPSC low-adsorption sphere induction;

[0038] Figure 3 HE staining of liver organoids at different time points induced by iPSC-DEO;

[0039] Figure 4 IF detection of stem cell marker OCT in the early stage of induction;

[0040] Figure 5 In the mid-induction period, IF was used to detect the hepatocyte marker HNF4α, the hepatic progenitor cell marker AFP, the cholangiocyte marker NOTCH2, and the biliary cell marker SOX9;

[0041] Figure 6 In the late induction period, IF was used to detect the cholangiocyte markers NOTCH2 and OPN, the hepatic endothelial cell marker RDX, and the hepatocyte nuclear cell marker HNF4α;

[0042] Figure 7 qPCR was performed to detect endoderm markers FOXA2, SOX17, GATA4, and GATA6 during the early induction period;

[0043] Figure 8 In the early stage of induction, qPCR was used to detect the ectoderm marker SOX1 and the pluripotency marker OCT4;

[0044] Fig. 9 Endothelial progenitor cell marker CD34 and mature vascular endothelial cell marker CD31 were detected by qPCR at different time points of induction;

[0045] Fig.10 qPCR was used to detect early liver differentiation markers HNF4α, AFP and mature hepatocyte markers CYP3A4, ALB at different induction time points;

[0046] Fig.11 qPCR was used to detect early bile duct differentiation markers NOTCH2 and CK19 and mature bile duct cell marker AE2 at different induction time points. DETAILED DESCRIPTION

[0047] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0048] Example 1: iPSC recovery, culture, passaging and cryopreservation

[0049] This embodiment provides a method for daily maintenance of iPSCs, comprising the following steps:

[0050] Step 1) Cell recovery

[0051] Prepare the culture dish three hours in advance, add 1 mL of basal medium containing 1% (volume percentage) Matrigel to each well of the 6-well plate, shake the well plate back and forth to completely cover the bottom of the well plate with the medium, and incubate it in the incubator for three hours, and lay the plate for later use. Clamp the cryotube with tweezers and quickly put it into a 37°C water bath, swirl it quickly and gently, and be careful not to immerse the tube cap in the liquid surface. When only ice cubes the size of rice grains are left in the tube, remove it from the water bath. Prepare a centrifuge tube containing 9 mL of room temperature PBS, slowly add the cell suspension drop by drop into PBS with a 1 mL pipette tip, centrifuge at 200xg for 3 min, obtain iPSC cell pellet, and gently resuspend the cell pellet with 1 mL of room temperature iPSC medium containing 5 μM Y27632. Aspirate the basal medium containing 1% (volume percentage) Matrigel on the plate, add 1.5-2 mL of iPSC medium containing 5 μM Y27632 to the well plate to keep the matrix gel at the bottom moist, add an appropriate amount of cell suspension, observe the cell density under a microscope, and place in a 37°C incubator for culture. The next day, remove the medium containing Y27632, wash once with PBS, and add fresh medium without Y27632.

[0052] Step 2) Cell culture

[0053] Thaw the iPSC culture medium and PBS at room temperature and return them to room temperature. Remove the culture dish, discard the original culture medium, add 1 mL of room temperature PBS to each well, tilt the dish back and forth twice, and discard the PBS. Add 1.5-2 mL of freshly prepared iPSC culture medium to each well along the wall.

[0054] Step 3) Cell passaging

[0055] After 5-7 days of cell culture, the two cell clusters gradually approach or even connect, and then they need to be subcultured. Prepare the culture dish three hours in advance, add 1 mL of basal medium containing 1% (volume percentage) Matrigel to each well of the 6-well plate, shake the well plate back and forth to completely cover the bottom of the well plate with the medium, and incubate it in the incubator for three hours, and then lay the plate for use. Take out the cells to be subcultured, discard the original medium, wash once with 1 mL of room temperature PBS, add 1 mL of ReLeSR, gently shake the well plate to cover the entire bottom with liquid, wet it for a few seconds, move the ReLeSR to the next well, remove the ReLeSR after each well is wetted, and put it in a 37°C incubator for digestion for 3 minutes. If slight differentiation is observed at the edge of the stem cell cluster, the digestion time can be shortened to 2 min. Take out the well plate and gently tap the side wall of the well plate with your hand to let the cells fall off. Add 1 mL of iPSC medium containing 5 μM Y27632 to each well to terminate the digestion. Remove the plate, aspirate the basal medium of 1% (volume percentage) Matrigel, re-add 1.5-2 mL of iPSC medium containing 5 μM Y27632 to the plate to keep the matrix gel at the bottom moist, add the cell suspension after the digestion is terminated to the plate, gently rotate clockwise for 5 circles, and then rotate counterclockwise for 5 circles to shake the cells evenly. Observe the cell density under a microscope, and it is appropriate to have 3-5 cell clumps in a 4X field of view. The next day, remove the medium containing Y27632, wash once with PBS, and add fresh medium without Y27632.

[0056] Step 4) Cryopreservation of cells

[0057] Use a pipette to slowly transfer the cell suspension obtained after subculturing and digestion into a 15 mL centrifuge tube along the wall. Use your fingers to gently tap the bottom of the centrifuge tube to disperse the cell clumps. Centrifuge at 200 g for 3 min and discard the supernatant. Add 1 mL of cold CS10 cryopreservation solution (recommended cryopreservation density 1.2×10 6 cells / tube), pipette slowly 1-2 times, flick the bottom of the centrifuge tube to disperse the cells, and transfer them to the cryopreservation tube. Place them in a programmed cooling box, place them in a -80℃ refrigerator for 24 h, and then quickly transfer them to liquid nitrogen.

[0058] Example 2: Seeding iPSCs into low-adhesion 96-well plates

[0059] The iPSCs in Example 2 were seeded before induction, wherein the low-adsorption 96-well plate was purchased from Corning, with the catalog number 7007, and specifically included two methods:

[0060] Method 1) Count the iPSC cells obtained after subculture digestion, transfer them to the sample tank, add appropriate iPSC culture medium containing 5 μM Y27632 to dilute, control the cell suspension density to 50,000-100,000 / mL, inoculate them into a low-adsorption 96-well plate with a spray gun while shaking, let the cells settle or aggregate, inoculate 100-120 μL per well, and self-spherize. The cell microspheres obtained by method 1) are recorded as low-adsorption spheres.

[0061] Method 2) Transfer the cell mass obtained after subculture digestion to a centrifuge tube, discard the culture medium, wash once with PBS, add 1 mL TrypLE (containing EDTA) and digest in a 37°C water bath for 5 min. After taking out, slowly blow 1-2 times, and flick the outer wall of the centrifuge tube with your fingers until there is no obvious precipitation. Use iPSC culture medium containing 5 μM Y27632 to terminate digestion, centrifuge at 200xg for 3 min at room temperature, and discard the supernatant. The obtained cell pellet is resuspended in iPSC culture medium containing 5 μM Y27632, counted on a computer, and the amount of matrix gel is calculated according to the number of cells. 4 Cell matrix gel microspheres (DEO, Droplet-engineered organoid) were prepared by adding 1 μL of matrix gel to each cell. The prepared cell matrix gel microspheres were transferred one by one to each well of the low-adsorption well plate, and 100 μL of iPSC culture medium containing 5 μM Y27632 was added to each well for culture. The cell matrix gel microspheres obtained by method 2) were recorded as DEO microspheres.

[0062] The specific steps for preparing cell matrix gel microspheres include: placing a sterilized 100 μL pipette tip in a -20℃ refrigerator for precooling 1 day in advance, placing a 1 mL syringe containing 0.5 mL of fluorinated oil on ice or in a -20℃ refrigerator for precooling 30 min in advance, drawing 5 mL of fluorinated oil into a 10 mL syringe, connecting a needle and a polytetrafluoroethylene (PTFE) tube of about 20 cm, installing it in the right hole of the tee, installing a PTFE tube of about 10 m in the left hole of the tee, and installing it in a syringe pump outside the refrigerator (flow rate: 110 μL / min), adjusting the syringe pump so that the fluorinated oil in the 10 mL syringe fills the 20 cm PTFE tube connected to its needle. Transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 300 g for 3 min at room temperature, discard the supernatant, and count the cells according to the results of cell counting at 10 4The required volume of matrix gel was calculated by adding 1 μL matrix gel to each cell. The matrix gel was added to the cell pellet with a pre-cooled pipette tip. After gently stirring on ice several times, it was blown to ensure that no bubbles were generated. After mixing until there were no cell lumps, the cell-matrix gel mixture was injected into a pre-cooled 1mL syringe filled with fluorinated oil, connected to a needle and a PTFE tube of about 10 cm, and then installed in the syringe pump (flow rate: 15 μL / min) in the refrigerator, installed in the middle hole of the three-way, and the syringe pump was adjusted to make the cell-matrix gel mixture appear in the 10 cm PTFE tube. The syringe pump outside the refrigerator was started first, and then the syringe pump inside the refrigerator was started. The cell-matrix gel mixture was sheared into monodisperse droplets through the three-way tube, and then conducted to the PTFE tube of about 10 m. When the PTFE tube connected to the 1 mL syringe did not contain the cell-matrix gel mixture, the two syringe pumps were stopped. The PTFE tube carrying the dispersed droplets of the cell-matrix gel mixture was placed in a 37°C incubator for 30 min to obtain cell-matrix gel microspheres. Use a 1 mL syringe to blow the cell matrix glue microspheres into the dish containing 5 μM Y27632 iPSC culture medium, and use a 1 mL syringe with a needle to gently suck out the fluorine oil on the surface of the dish to obtain cell matrix glue microspheres with uniform size and uniform cells.

[0063] Example 3: Inducing iPSC cell clusters into liver organoids

[0064] This embodiment provides a method for inducing iPSC cell clusters into liver organoids, wherein the low-adsorption spheres obtained by method 1) in embodiment 2 and the DEO microspheres obtained by method 2) are respectively induced into liver organoids, comprising the following steps:

[0065] Step 1) Culture the cells in iPSC medium containing 5 μM Y27632 for two days. The cell mass obtained in this step is the induction time point D0 iPSC cells.

[0066] Step 2) Discard the original culture medium and start formal induction by switching to culture medium I for five hours. Culture medium I is RPMI1640 / B27 (RPMI1640, Gibco C11875500BT; B27 supplement, Gibco 17504044) containing 100 ng / mL Activin A and 20 ng / mL BMP4.

[0067] Step 3) Discard the original culture medium and replace it with culture medium II for two days. Culture medium II is RPMI1640 / B27 containing 100 ng / mL Activin A, 20 ng / mL BMP4 and 25% (volume percentage) mTeSR. The cell mass obtained in this step is the D2 germ layer cell at the induction time point.

[0068] Step 4) Discard the original culture medium and replace with culture medium III for two days. Culture medium III is RPMI1640 / B27 with 25% (volume percentage) mTeSR containing 100 ng / mL Activin A. The cell clusters obtained in this step are endoderm cells and mesoderm cells at the induction time point D4.

[0069] Step 5) Discard the original culture medium and replace with culture medium IV for five days. Culture medium IV is RPMI1640 / B27 containing 25% (volume percentage) mTeSR with 30 ng / mL FGF4 and 20 ng / mL BMP2. The cell mass obtained in this step is the hepatoblast at the induction time point D9.

[0070] Step 6) Discard the original culture medium and replace with culture medium V for six days. Culture medium V is RPMI1640 / B27 containing 25% (volume percentage) mTeSR with 20 ng / mL HGF and 20 ng / mL KGF. The cell mass obtained in this step is the hepatocytes and bile duct epithelial cells at the induction time point D15.

[0071] Step 7) Discard the original culture medium and replace it with culture medium VI for ten days. Culture medium VI is a high-glucose DMEM culture medium containing 0.2% (mass percentage) bovine serum albumin, 0.1% (volume percentage) insulin-transferrin-selenium (ITS) (Gibco, cat. no.41400-045), 0.1% (volume percentage) gentamicin-amphotericin B (Gibco, R01510), 10 ng / mL OSM, and 15 μg / mL cholesterol. The cell mass obtained in this step is the liver organoid at the induction time point D25.

[0072] Step 8) After induction is complete, you can continue to culture mature liver organoids using Medium VI.

[0073] The culture conditions in step 1) to step 8) are to place the culture in a 37°C, 5% CO2 incubator.

[0074] Example 4: HE staining at different stages of iPSC induction

[0075] HE staining was performed on the cell clusters induced to different stages in Example 3, specifically including:

[0076] Step 1) Organoid paraffin embedding

[0077] Turn on the embedding machine in advance and preheat it to 65℃.

[0078] Fixation: Add 4% PFA (about 200 μL) 10 times the volume of the organoid into the EP tube and fix at room temperature for 20 min.

[0079] Pre-staining of organoids: Carefully remove PFA with a 100 μL pipette, add 20 μL eosin solution, and stain for 5 minutes at room temperature.

[0080] Dehydration of organoids: add 10 times the volume of 75% ethanol and dehydrate at room temperature for 5 min; aspirate 75% ethanol and add 10 times the volume of 90% ethanol and dehydrate at room temperature for 5 min.

[0081] Re-staining of organoids: Add 20 μL of eosin solution and stain for 5 minutes at room temperature. (Re-staining can be performed if discoloration is severe).

[0082] Organoid dehydration: Add 10 times the volume of anhydrous ethanol and dehydrate at room temperature for 5 minutes. Repeat three times.

[0083] Organoid clearing: Add 10 times the volume of xylene, soak at room temperature for 5 min, repeat three times.

[0084] Wax immersion of organoids: Take a white embedding box, remove the lid, and mark the organoid batch number on the box. When the organoids sink to the bottom, carefully remove the upper layer of xylene (leave 50 μL), use a 1 mL pipette with the tip cut off to absorb the sample, transfer the sample to the paraffin mold, add paraffin, and soak at 65 °C for 10 min; tilt the mold to discard the paraffin, add paraffin again and soak for 10 min, gently place the white embedding box on the paraffin mold, let it stand at 65 °C for 10 min, wait for the paraffin to solidify, and store the wax block at room temperature.

[0085] Step 2) HE staining of sections

[0086] The paraffin-embedded blocks were sectioned on a microtome with a thickness of 5 μm.

[0087] Dewax the sections to water: sequentially place the sections in xylene I for 5 min, xylene II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and wash with distilled water.

[0088] Hematoxylin staining of cell nuclei: turn on the water bath in advance and preheat it to 60℃, soak the slices in hematoxylin at 60℃ for 3-5 min, soak in tap water for a while to remove excess hematoxylin, differentiate in 1% acidic ethanol differentiation solution for 20 seconds, soak in tap water for washing, blue in 1% ammonia water for 20 seconds, and soak in tap water for washing.

[0089] Eosin staining of cytoplasm: Immerse the sections in eosin staining solution for 30 seconds.

[0090] Dehydration and sealing: Put the slices into 95% alcohol I for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and make them transparent. Take the slices out of xylene, put a clean absorbent paper on the table, take the slide out of xylene and put it on the paper (with the slice side facing up), quickly drop a drop of gum in the center of the slice, hold the right side of the cover glass gently with tweezers, slightly tilt it so that the left side touches the sealing medium, and then slowly put the cover glass down to reduce or avoid bubbles. Then perform microscopic examination and image acquisition and analysis.

[0091] The results are as follows Figure 1-3 As shown in the figure. In the early stage of induction, the number of cell nuclei in the cell cluster increases, and the number of cells increases significantly. In the middle stage of induction, the cells proliferate and the overall morphology changes significantly, and a cavity structure appears. In the late stage of induction, the color of the cell nuclei deepens, the cavity structure gradually increases, and the liver organoids gradually mature. The HE structure diagram is basically consistent with the bright field structure diagram.

[0092] Example 5: IF detection at different stages during iPSC induction

[0093] The cell clusters induced to different stages in Example 3 were subjected to IF staining, specifically including:

[0094] Step 1) Organoid paraffin embedding

[0095] The specific steps are the same as step 1 of Example 4).

[0096] Step 2) Slice IF detection

[0097] The paraffin sections were placed in an oven at 65°C for 30 min-1 h until the wax dissolved.

[0098] Dewax the paraffin sections to water: sequentially place the sections in xylene I for 20 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol for 10 min, 95% ethanol for 10 min, 70% ethanol for 10 min, and wash with distilled water for 5 min.

[0099] Antigen repair: Place the slides in a repair box filled with EDTA antigen repair buffer (pH 8.0) in a microwave oven for antigen repair. Heat at high heat for 5 min, medium heat for 20 min, and then switch to low heat for 2 min. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After cooling naturally, place the slides in PBS (pH 7.4) and wash them twice on a decolorizing shaker, 5 min each time.

[0100] Blocking: After the slices are taken out of PBS and dried, use an immunohistochemistry pen to draw a circle around the tissue, add 100 μL of blocking solution (the blocking solution is PBS containing 5% BSA and 0.3% Triton X-100) into the circle and block for 1 hour.

[0101] Primary antibody incubation: dilute the primary antibody to an appropriate concentration with blocking solution, add 50 μL of primary antibody to each slice, place the slices flat in a humidified box, and incubate at 4°C overnight.

[0102] Secondary antibody incubation: Take the slices out of the refrigerator and rewarm them for 20-30 min, wash them 3 times with PBST, 10 min each time, dilute the fluorescent-labeled secondary antibody with PBS, add 50 μL of secondary antibody to each slice, and incubate at room temperature for half an hour.

[0103] Counterstaining with DAPI: Wash with PBST three times, 10 min each time, add diluted DAPI, incubate at room temperature for 10 min, and wash with PBS for 5 min.

[0104] Covering microscopy: Add about 30 μL of anti-fluorescence quencher to each slice and cover it with a coverslip. Observe under a fluorescence microscope and take photos for record.

[0105] The results are as follows Figure 4-6 . In the early stage of induction, the pluripotency of iPSCs was detected by immunofluorescence. The statistical results showed that the nuclei of D0, D2, and D4 all expressed OCT4 (a marker of pluripotent stem cells), indicating that the cell clusters maintained the pluripotency of embryonic stem cells and the totipotency of differentiation. In the middle stage of differentiation, the results showed that HNF4α (a marker of hepatocytes) and AFP (a marker of hepatic progenitor cells) were significantly expressed, indicating that iPSCs differentiated into hepatic progenitor cells through culture in medium containing different induction factors, and some had differentiated into hepatocytes. At the same time, SOX9 (a marker of cholangiocytes) was significantly expressed, indicating that cholangiocytes were gradually formed at this stage. In the late stage of induction, the expression levels of NOCTH (a marker of cholangiocytes), HNF4α (a marker of hepatocytes), RDX (a marker of hepatic endothelial cells), and OPN (a marker of cholangiocytes) were all increased. This result shows that the NOTCH pathway promotes bile duct formation, and cholangiocytes and hepatocytes continue to increase and gradually mature. Immunofluorescence results showed that during the induced growth process, multiple proteins were co-expressed at different stages, and hepatocytes and bile duct cells were gradually formed and gradually matured with the induction time.

[0106] Example 6: qPCR detection at different stages during iPSC induction

[0107] The cell clusters induced to different stages in Example 3 were subjected to qPCR detection, specifically including:

[0108] Step 1) RNA extraction

[0109] Collect 30 cell clusters into a centrifuge tube, discard the culture medium, wash with 1 mL of pre-cooled PBS, discard the PBS, add 1 mL of TRIzol to resuspend, blow off the cell clusters vigorously, and incubate on ice for 3-5 min. Add 200 μL of chloroform and immediately shake vigorously for 15 s to mix the layered solutions thoroughly, and incubate on ice for 3 min. Centrifuge at 4℃ 12000 rpm for 15 min. After centrifugation, three layers can be observed. The lower layer is a pink organic layer, the middle layer is a white DNA precipitate, and the upper layer is a water layer in which RNA is dissolved. Take 400 μL of the top water layer and transfer it to a new 1.5 mL centrifuge tube. Be careful not to inhale the middle DNA layer. Add an equal volume (400 μL) of isopropanol, gently invert to mix, and incubate on ice for 10 min. Centrifuge at 4℃ 15000 rpm for 10 min. A small amount of white precipitate can be observed at the bottom. If the amount of RNA is low, centrifuge for 30 min. Gently remove the isopropanol, add 950 μL 75% ethanol, do not blow away the precipitate, gently invert to mix, centrifuge at 9500 rpm for 5 min at 4℃, and repeat the above steps three times. Aspirate the 75% ethanol and place the precipitated RNA in a fume hood. Place the centrifuge tube upside down on absorbent paper and air dry for 20 min. The air-drying standard is that the bottom precipitate becomes transparent. Add 30 μL enzyme-free water / sterile water, resuspend the precipitate, detect the absorbance value at 260 nm, and record the RNA concentration.

[0110] Step 2) cDNA synthesis

[0111] The system on the machine is 10 μL / well: 2 μL reverse transcriptase (5×) + a μL RNA + (8-a) μL sterile water. The RNA concentration is 500 ng / well, and the a value to be added is calculated based on the measured RNA concentration. PCR instrument parameters: 37℃ 15 min, 85℃ 5 s, 4℃∞. The synthesized cDNA is placed in a -20℃ refrigerator, which is relatively stable and can be stored for a long time.

[0112] Step 3) qPCR

[0113] The system on the machine is 20 μL / well: 10 μL MIX enzyme (2×) + 2 μL forward primer + 2 μL reverse primer + 2 μL cDNA + 4 μL sterile water. The qPCR instrument parameters are 40 cycles; 60℃-95℃, 0.1℃ / s to make a melting curve. The primer sequences involved are described in the following table:

[0114]

[0115] The results are as follows Figure 7-11. In the early stage of induction, qPCR results showed that the expression of OCT4 (pluripotency marker) decreased significantly, while FOXA2, SOX17, GATA4, GATA6 (endodermal markers) and SOX1 (ectoderm markers) all showed an upward trend. The low adsorption sphere formation trend was similar to that of IPS-DEO, because the differentiation of iPSC into endoderm, ectoderm and trophoblast cells is the first step in the directed differentiation into various types of cells. After iPSC differentiates into germ layers, it can continue to differentiate and develop in the induced direction. During the induction process, CD34 (endothelial progenitor cell marker) and CD31 (mature vascular endothelial marker) mainly showed a trend of first rising and then falling, reaching a peak before the completion of induction (D9 / D15). Endothelial cells can send signals to promote liver bud formation and guide bile duct morphogenesis, playing an important role in inducing differentiation into mature liver organoids. In the late stage of induction, the expression levels of HNF4α, AFP, ALB (early markers of liver differentiation) increased, and CYP3A4 (mature hepatocyte marker) also showed an upward trend, indicating that liver progenitor cells gradually differentiated into mature hepatocytes, and the expression of NOTCH2 and CK19 (early markers of bile duct differentiation) also increased with the increase of induction time. The expression level of AE2 (mature bile duct marker) increased after induction of maturation, indicating that some bile duct cells gradually matured.

[0116] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing liver organoids induced by human iPSCs, characterized in that: The steps include: 1) Digest the cultured human induced pluripotent stem cells into single cells; 2) The single cells obtained in step 1) are separated by method 1 or method 2 to achieve one cell cluster per well of a low-adsorption 96-well plate; Method 1: The single cells obtained in step 1) are inoculated into a low-adsorption 96-well plate and then self-spherized to prepare cell clusters, thereby achieving one cell cluster per well of the low-adsorption 96-well plate; Method 2: The single cells obtained in step 1) are made into cell matrix gel microspheres using T-type microfluidics technology, and then distributed one by one into a low-adsorption 96-well plate, so that one cell cluster is achieved in each well of the low-adsorption 96-well plate; 3) The cell clusters obtained in step 2) are subjected to step-by-step induction culture at 37°C and 5% CO2: the cell clusters obtained in step 2) are cultured in culture medium I for 5 h; then cultured in culture medium II for 2 days; then cultured in culture medium III for 2 days; then cultured in culture medium IV for 5 days; then cultured in culture medium V for 6 days; then cultured in culture medium VI for 10 days, thereby obtaining mature human liver organoids; Medium I was RPMI1640 / B27 containing 100 ng / mL Activin A and 20 ng / mL BMP4; Medium II was RPMI1640 / B27 containing 25% mTeSR, 100 ng / mL Activin A, and 20 ng / mL BMP4; Medium III was RPMI1640 / B27 containing 25% mTeSR and 100 ng / mL Activin A; Medium IV was RPMI1640 / B27 containing 25% mTeSR, 30 ng / mL FGF4, and 20 ng / mL BMP2; Medium V was RPMI1640 / B27 containing 25% mTeSR, 20 ng / mL HGF, and 20 ng / mL KGF; Culture medium VI is high-glucose DMEM containing 0.2% by mass of bovine serum albumin, 0.1% by volume of ITS, 0.1% by volume of gentamicin-amphotericin B, 10 ng / mL of OSM, and 15 μg / mL of cholesterol.

2. The preparation method according to claim 1, characterized in that The step of digesting human induced pluripotent stem cells into single cells in step 1) includes: transferring the cell group obtained by subculture digestion into a centrifuge tube, discarding the culture medium, washing once with PBS, discarding PBS, adding TrypLE containing EDTA and digesting in a 37°C water bath for 5 minutes, and after taking out, gently blowing the cells 1-2 times, and flicking the tube wall with a finger until there is no obvious precipitation, terminating the digestion, centrifuging and discarding the supernatant, and collecting the precipitate.

3. The preparation method according to claim 1, characterized in that: Method 1 specifically includes: adding the single cell obtained in step 1) to iPSC culture medium containing Y27632 to dilute, controlling the cell suspension density to 50,000-100,000 / mL, inoculating into a low-adsorption 96-well plate, culturing 100-120 μL per well to allow self-sphere formation.

4. The preparation method according to claim 1, characterized in that: In method 2, every 10 4 Add 1 μL of Matrigel to each single cell.

5. The preparation method according to claim 1, characterized in that: The method for preparing cell matrix gel microspheres in method 2 includes: mixing the single cell obtained in step 1) with the matrix gel evenly to obtain a cell-matrix gel mixture; using the obtained cell-matrix gel mixture as a dispersed phase and an oil phase reagent as a continuous phase, using microfluidic technology to shear the cell-matrix gel mixture into monodisperse droplets, and fixing the obtained monodisperse droplets at 37°C for 30 min to obtain cell matrix gel microspheres.

6. The preparation method according to claim 5, characterized in that: The oil phase reagent is fluorinated oil; The flow rate of the cell-matrix gel mixture was controlled at 10-15 μL / min, and the flow rate of the oil phase reagent was controlled at 100-110 μL / min.

7. The preparation method according to claim 1, characterized in that: The diameter of the cell clusters prepared by self-spheroidization in method 1 was 800-1200 μm; In method 2, the diameter of the cell matrix gel microspheres was 620-680 μm.

8. The preparation method according to claim 1, characterized in that: Before culturing the cell cluster obtained in step 2) in culture medium I, step 3) further comprises: culturing the cell cluster obtained in step 2) in iPSC culture medium containing Y27632 for two days, and the culture conditions are 37° C. and 5% CO 2 .

9. The preparation method according to claim 1, characterized in that: Step 3) The obtained mature human liver organoids are continued to be cultured in culture medium VI.

10. The liver organoid prepared by the preparation method according to any one of claims 1 to 9.

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