WO2021190187A1 - Method for constructing liver organs in vitro and use thereof - Google Patents

Abstract

Provided are a method for constructing liver organs in vitro and the use thereof. The method comprises extracting mouse hepatocytes to induce and construct liver organs; establishing an acute hepatic failure mouse model after a 70% hepatectomy; and evaluating the treatment effect of the liver organs on acute hepatic failure after the liver organs are transplanted. The liver organs have strong dryness and proliferation functions, can effectively relieve acute hepatic failure, and can promote liver regeneration and repair.

Description

An in vitro construction method and application of liver organoids Technical field

The invention relates to the technical field of biomedicine, in particular to an in vitro construction method and application of liver organoids.

Background technique

Acute liver failure (ALF) is a clinical syndrome in which a large number of liver cell necrosis and severe liver damage are caused by viruses, drugs, toxins or alcohol in a short period of time. It often causes jaundice, hepatic encephalopathy, and multiple organ functions. Complications such as exhaustion have a higher mortality rate. Liver transplantation is currently the only effective treatment method. However, the shortage of donor livers is far from meeting clinical needs. Many patients died while waiting for the donor liver. In addition, medical treatment is only symptomatic and supportive treatment and cannot improve the patient's prognosis. Therefore, there is an urgent need to develop an alternative treatment.

Stem cells have unique functions in terms of self-renewal and differentiation potential, and are regarded as the most promising therapeutic method for tissue repair and regeneration. Compared with liver transplantation, stem cell transplantation has the advantages of less invasiveness and repeatable transplantation treatment, but it also faces problems such as low implantation efficiency and short cell survival time after implantation. Studies have shown that 3D three-dimensional structure is conducive to cell proliferation and differentiation. 3D culture can simulate the extracellular environment in the body, and in long-term culture, it can achieve efficient energy transfer and various molecular signal exchanges between cells and the environment. Compared with traditional 2D cell culture, 3D cultured induced pluripotent stem cells (iPSCs) have significantly improved urea secretion and albumin synthesis capabilities. Organoids are cell clusters constructed in vitro based on a 3D culture system. They have the ability to automatically renew and assemble, and have partial functions of target organs in vivo. Existing studies have proved that the liver organoids derived from stem cells have a significantly enhanced ability to maintain liver function when implanted in pigs and mice. Huch et al. isolated EPCAM+ epithelial cells from human liver samples and constructed them into liver organoids in vitro. The chromosomal structure and gene composition of the liver organoids remained stable after amplification, and they had the potential to differentiate into hepatocytes and bile duct cells. Nude mice can successfully differentiate into functional hepatocytes in vivo. Through the 3D culture model, the construction of organ-like transplants in vitro is a development direction of the future replacement therapy for acute liver failure.

The treatment of liver diseases based on cell transplantation has made great progress, from embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells to hepatic progenitor cells. Although hepatic-like stem cells can be obtained from pluripotent stem cells, their clinical application is still limited due to the efficiency of cell differentiation, immune rejection, ethical issues, and teratogenic risks. And although pure cell transplantation has certain curative effects, it lacks an ideal cell source, the curative effect is inaccurate and unstable, and the clinical application has the risk of infiltration into other organs, which limits its large-scale promotion. At present, the combination of cell and tissue engineering technology is the current research hotspot. Organoids are cell clusters constructed in vitro based on 3D culture systems. Compared with the traditional culture mode, the signal effects between cells, cells and the environment are further enhanced, and they have automatic The ability to renew and assemble, so as to realize part of the functions of the original organs in the body. Current studies have proved that in chronic liver disease, liver progenitor cells can mediate liver regeneration, and liver organoids have the function of protecting liver cells and promoting liver regeneration. Then, after acute liver injury such as partial hepatectomy, whether exogenous implantation of liver organoids with properties similar to hepatic progenitor cells can promote liver regeneration has not been reported yet.

Summary of the invention

The purpose of the present invention is to provide an in vitro construction method and application of liver organoids in view of the problems in the prior art, extract mouse liver cells to induce the construction of liver organoids, observe its morphology under a microscope, and identify them by immunofluorescence and PCR. The properties of stem cells and epithelial cells were used to establish a 70% hepatectomy mouse model of acute liver failure. After liver organ transplantation, the liver function was evaluated on the first, fourth, and seventh days after the operation. Histochemical verification of the therapeutic effect of liver organoids on acute liver failure.

In order to achieve the above objective, the technical solution provided by the present invention is:

An in vitro construction method of liver organoids, including the following steps:

S1: Prepare a tissue suspension, take the liver of a normal mouse under aseptic conditions, rinse the liver and cut it into pieces to obtain the tissue suspension;

S2: prepare a cell suspension, add liver tissue digestion to the tissue suspension prepared in step S1, then transfer to a water bath for static digestion, perform mechanical pipetting after digestion, and collect the supernatant as the cell suspension after the cells settle;

S3: Seed the plate, filter, centrifuge, and lyse the cell suspension obtained in step S2 to obtain the cell pellet. Use Matrigel to resuspend the cell pellet and plant it in the center of the 24-well plate. After the matrigel is fixed, add it to the liver. The organoid induction medium is cultivated to obtain the initial organoid;

S4: Passage the initial organoids obtained in step S3, and select 3 to 5 generations as the final prepared liver organoids.

In order to optimize the above technical solutions, the specific measures taken also include:

In the above step S1, the liver is first rinsed in a pre-cooled 30 mL DMEM/F-12 medium to remove residual blood, and then cut to 1 mm in a new 30 mL DMEM/F-12 medium.

The specific steps of the above step S2 include: transfer the tissue suspension to a 50mL centrifuge tube at room temperature with a pipette, let stand for 1 minute to remove the supernatant, add 10mL liver tissue digestion solution, and transfer to a 37℃ water bath for static digestion. After the mechanical pipetting, let it stand for 1 minute. After the cells settle at the bottom and remove the supernatant, continue to add 10 mL liver tissue digestion solution, transfer to a 37℃ water bath and let stand for 20 minutes for digestion. After mechanical pipetting, let it stand for 1 minute. Precipitate at the bottom, collect the supernatant in a 50 mL pre-cooled centrifuge tube, continue to repeat the above digestion and collection steps 4 times, and collect 50 mL of the cell supernatant as the cell suspension.

The specific preparation of the above liver tissue digestion solution is: adding 7.5 mL of neutral protease and 7.5 mL of type IV collagenase to every 45 mL of DMEM/F12 medium.

The above step S3 specifically includes:

S31: The collected cell suspension is first filtered with a 100um cell sieve, leaving cells with a diameter less than 100um, and then the filtrate is filtered again with a 30um cell sieve, the filtrate is discarded, and the 30um filter is poured into 50mL On the centrifuge tube, rinse with DMEM/F-12 medium, and collect the cells trapped on the 30um filter;

S32: Centrifuge at 300g for 5 minutes at 4°C and remove the supernatant;

S33: Add 3 to 5 times the cell volume of red blood cell lysate, gently pipette, lyse for 4-5 minutes, centrifuge at 4°C, 100g for 5 minutes, discard the supernatant, and obtain a cell pellet;

S34: Use 240uL Matrigel to resuspend the cell pellet, pipe it up and down evenly to avoid air bubbles, and plant it in the center of the 24-well plate at the position of 8 holes;

S35: Let the cell incubator stand at 37°C for 10 minutes. After the matrigel is fixed, 750uL liver organoid induction medium is adhered to the wall, and the liver organoid induction medium is replaced every 2 to 3 days depending on the culture condition to obtain initial organoids.

The above step S4 specifically includes:

S41: Remove liver organoid induction medium, rinse gently with PBS, remove PBS, add DMEM/12 medium, stand for 1 minute for digestion, mechanically pipette the initial organoids;

S42: Centrifuge at 300g for 5 minutes at 4°C and remove the supernatant;

S43: The pipette tip is pre-cooled, and 240uL Matrigel is used to resuspend the cell pellet, pipetting up and down evenly to avoid air bubbles, planted in the center of the 24-well plate at the position of 8 holes;

S44: Let the cell culture incubator stand at 37°C for 10 minutes. After the matrigel is fixed, add 750uL liver organoid induction medium to the wall, and pass the culture at a ratio of 1:4.

The above-mentioned liver organoid induction medium is specifically formulated as follows: the liver organoid basal medium and the liver organoid basal medium additive are mixed in a volume ratio of 10:1, and the anti-mycoplasma reagent is added, and the mixture is divided into aliquots. Store at -20°C.

The invention also protects the application of the liver organoids prepared by the method in the treatment of acute liver failure.

The beneficial effects of the present invention are:

1. The mouse liver organoids prepared by the method of the present invention were cultured in vitro for 3 days from a cystic structure with a diameter of 20um to a cell mass of about 100um. The liver stemness genes EPCAM, SOX9 and CK19 were significantly up-regulated, and EPCAM, SOX9, CK19, TBX3, AFP, SOX17, FOXA2, HNF4A, CEBPA and CEBPB remained stable before and after passage.

2. The present invention shows that CK8, Desmin, AFP and PCNA are positive by immunofluorescence, the liver body ratio is significantly increased after liver organ transplantation, and the liver functions such as ALT, AST, ALB, TG, etc. are restored on the fourth day. Inflammation and proliferation indicators such as HE staining and ki-67 staining showed that the proliferation of liver organoids was significantly enhanced after transplantation.

3. The liver organoids of the present invention have strong dryness and proliferation functions; it can effectively alleviate the liver function of 70% of mice with acute liver failure after hepatectomy, increase the liver-to-body ratio, and reduce liver inflammation infiltration and promote 70% Liver regeneration and repair of acute liver failure after hepatectomy.

Description of the drawings

Figure 1 shows the morphology of organoids cultured for 1, 2, and 3 days under an optical microscope.

Fig. 2 is a statistical diagram of the diameter of the organoid shown in Fig. 1.

Figure 3 is a comparison diagram of the first-generation liver organoids, the third-generation liver organoids, and liver tissue gene detection.

Figure 4 is a schematic diagram of organoid value-added ability detection.

Figure 5 is a schematic diagram of the liver-to-body ratio between the control group and the treatment group in Application Example 1.

Figure 6 is a comparison diagram of alanine aminotransferase changes between the control group and the treatment group in Application Example 1.

Figure 7 is a comparison diagram of aspartate aminotransferase changes between the control group and the treatment group in Application Example 1.

Figure 8 is a comparison diagram of liver albumin changes between the control group and the treatment group in Application Example 1.

Figure 9 is a graph showing the results of liver organ transplantation reducing 70% of acute liver failure inflammation and promoting liver regeneration after hepatectomy.

Detailed ways

The present invention will be further described below with reference to the drawings and embodiments. Where specific conditions are not indicated in the examples, it is carried out in accordance with the conventional conditions well known in the art or the conditions recommended by the manufacturer. The instruments or reagents used are all conventional products that can be purchased commercially. For details, see the following table.

Advanced DMEM/F12	Gibco (United States)
DMEM F12/50	Gibco (United States)
HepatiCuLt TM OGM Mouse Basal Medium	StemCell Corporation (U.S.)
HepatiCuLt TM OGM Mouse Supplement	StemCell Corporation (U.S.)
Dispase	StemCell Corporation (U.S.)
Collagenase Type IV	StemCell Corporation (U.S.)
100um cell sieve	BD Corporation (U.S.)
30um cell sieve	Miltenyi Biotec (Germany)
Anti-Adherence Rinsing Solution	StemCell Corporation (U.S.)
PlasmocinTM treatment	InvivoGen (China)
PBS	KGI Corporation (China)
AFP antibody	R&D Company (U.S.)

PCNA antibody	Abcam (UK)
CK8 antibody	Mitaka Corporation (China)
Desmin antibody	Novus Biologicals Company (U.S.)
Goat anti-mouse IgG H&L	Abcam (UK)
Goat anti-rabbit IgG H&L	Abcam (UK)
Hoest cell nuclear stain	Biyuntian Biological Company (China)
Gene primer	King Cary Corporation (China)
Trizol	Taraka Corporation (Japan)
Trichloromethane (analytical grade)	Sinopharm Chemical Reagent Co., Ltd.
Anhydrous ethanol (analytical pure)	Hangzhou Changzheng Chemical Plant (China)
Isopropyl alcohol (analytical grade)	Hangzhou Changzheng Chemical Plant (China)
DEPC water	KGI Corporation (China)
Reverse transcription kit	Yisheng Biotechnology Company (China)
SYBR Premix	Yisheng Biotechnology Company (China)
5×SDS protein loading buffer	Biyuntian Biological Company (China)
Ki67 antibody	Mitaka Biotech (China)

In this example, 6-week-old, C57B/6 mice were selected as experimental animals, with a 12h/12h light/dark light cycle (06:00-18:00), free food and water, and a temperature of 22°C. It was purchased from the Laboratory Animal Center of Nanjing Medical University by the agent of the Laboratory Animal Center of Gulou Hospital Affiliated to Nanjing University School of Medicine. The breeding site is managed by the Experimental Animal Center of Gulou Hospital Affiliated to Nanjing University School of Medicine. All animal experiments are carried out in the Laboratory Animal Center of Gulou Hospital Affiliated to Nanjing University School of Medicine. All animal experiments were approved by the Animal Experiment Ethics Committee of Gulou Hospital Affiliated to Nanjing University School of Medicine (approval number: 2018010017).

Example 1

Organoids in vitro construction

1. Formulation preparation

1) Organoid induction medium: HepatiCuLt ^TM OGM Mouse Basal Medium is used as the liver organoid basal medium, HepatiCuLt ^TM OGM Mouse Supplement is used as the liver organoid basal medium additive, and Plasmocin ^TM treatment is used as the anti-mycoplasma reagent. Mix HepatiCuLt ^TM OGM Mouse Basal Medium and HepatiCuLt ^TM OGM Mouse Supplement at a volume ratio of 10:1, then add 200uL of Plasmocin ^TM treatment, mix well and distribute them and store at -20°C.

2) Liver tissue digestion solution: Take 45mL of Advanced DMEM/F12, add 7.5mL of Dispase and 7.5mL of Collagenase type IV, mix well, and prepare for immediate use.

2. Take material

In the ultra-clean platform, 6-8 weeks old C57BL/6 normal mice were selected, and they were sacrificed by cervical dislocation after inhalation anesthesia. Fix his limbs on the operating table, and disinfect the abdomen with alcohol cotton balls. Take the liver under aseptic conditions, rinse the liver in 30mL DMEM/F-12 medium, rinse once in pre-cooled 30mL DMEM/F-12 to remove residual blood, and then put it in a new 30mL DMEM/F-12 medium. Cut the F-12 medium to a size of 1mm.

3. Digestion

Use a pipette to transfer the tissue suspension to a 50 mL centrifuge tube at room temperature, let stand for 1 minute to remove the supernatant, add 10 mL of liver tissue digestion solution, and transfer to a 37°C water bath for 20 minutes for digestion. After mechanical pipetting, let it stand for 1 After the cells settle at the bottom and remove the supernatant, continue to add 10 mL of liver tissue digestion solution, transfer to a 37°C water bath and let stand for 20 minutes for digestion. After mechanical pipetting, let stand for 1 minute. After the cells settle on the bottom, collect the supernatant. In a 50mL pre-cooled centrifuge tube, continue to repeat the above digestion and collection steps 4 times to collect about 50mL of cell supernatant.

4. Seed plate (sterile operation)

1) The collected cell suspension is first filtered with a 100um cell mesh, leaving cells with a diameter of <100um mesh. Filter the filtrate again with a 30um cell sieve and discard the filtrate. Invert the 30um filter on a 50mL centrifuge tube, rinse with Advanced DMEM/F12, and collect the cells trapped on the 30um filter;

2) Centrifuge at 300g for 5 minutes at 4°C and remove the supernatant;

3) Add 3 to 5 times the cell volume of red blood cell lysate, gently pipette, lyse for 4-5 minutes, 4℃, 100g centrifugation for 5 minutes, discard the supernatant, and get the precipitate; remove the red blood cell lysate with a disposable syringe filter before use Bacteria, protect from light operation;

4) Use 240uL Matrigel to resuspend the cell pellet (operating on ice), pipetting up and down evenly, taking care to avoid air bubbles, planting in the center of the 24-well plate at the position of 8 holes;

5) Leave it in a 37°C cell incubator for 10 minutes. After the matrigel is fixed, add 750uL of the liver organoid induction medium to the wall, and replace the liver organoid induction medium for 2 to 3 days according to the culture condition.

5. Passing down

1) Remove the liver organoid induction medium, rinse gently with PBS, remove the PBS, then add Advanced DMEM/F12, stand for 1 minute for digestion, and mechanically pipette the organoids;

2) Centrifuge at 300g for 5 minutes at 4°C and remove the supernatant;

3) Pre-cool the pipette tip, use 240uL Matrigel to resuspend the cell pellet (operating on ice), pipe it up and down evenly, taking care to avoid air bubbles, plant it in the center of the 24-well plate at 8 holes;

4) Let it stand for 10 minutes in the cell culture incubator. After the matrigel is fixed, 750uL of the liver organoid induction medium is added to the wall and passaged at a ratio of 1:4, and 3 to 5 generations are selected as the liver organoids.

Refer to Figure 1-2. It can be seen from the figure that liver organoids are highly clonal. Under certain culture conditions, they can be rapidly expanded from a small number of starting cells. 3 days old, the diameter grows from 20um to more than 100um.

Example 2

Phenotypic identification of liver organoids

1. Gene identification:

1) Cell RNA extraction

a) Remove the culture medium from the organoids and rinse twice with PBS to ensure that the residual culture medium does not affect the purity of RNA. In a 24-well plate, add 500uL of Trizol to one well. In order to fully lyse, pipette repeatedly and let stand at room temperature for 10 minutes. , After fully separating the protein and nucleic acid complexes, transfer to an enzyme-free 1.5mL EP tube;

b) Add chloroform in a 5:1 ratio of Trizol to chloroform, shake vigorously up and down for 10 seconds, and let it stand for 5 minutes at room temperature to allow it to separate naturally.

c) After centrifuging at 12000g for 15 minutes at 4°C, carefully save the upper aqueous phase and transfer it to another new 1.5mL EP tube;

d) Add an equal volume of isopropanol to the supernatant and mix, and place at -20°C for 1 hour to increase RNA precipitation;

e) Centrifuge at 12000g for 10 minutes at 4°C, discard the supernatant, and deposit the RNA on the bottom of the tube.

f) RNA precipitation is mixed with 1mL 75% ethanol (prepared with DEPC water), and the centrifuge tube is gently shaken to suspend the precipitation;

g) Centrifuge at 7500g for 5 minutes at 4°C, and carefully aspirate and discard the supernatant with a pipette.

h) Dry at room temperature for 5-10 minutes to let the last remaining small amount of ethanol evaporate.

i) Dissolve the RNA precipitate with 20uL DEPC water, and store the RNA solution at -80°C.

2) Determine RNA concentration

a) Zero the nucleic acid protein analyzer with ddH2O, draw 1uL RNA to determine the purity, OD260/OD280 is between 1.8~2.0, and the concentration is 500ng/uL;

b) Adjust the RNA concentration to 500ng/uL with DEPC water. After testing a specimen, wipe it with lens paper to avoid mutual influence between specimens.

3) Reverse transcription of RNA to cDNA

Using China's Shanghai Yisheng Technology Biological Company

Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR reverse transcription kit, the specific steps are operated in accordance with the instructions.

a) Removal of residual genomic DNA: Prepare the following mixture in a RNase free EP tube, and gently pipette to mix. Incubate at 42°C for 2 minutes. Prepare the following reaction system in the RNase free EP tube:

b) Reverse transcription reaction system preparation (20μL system): directly add 2×HifairTM II SuperMix plus to the reaction tube of step a), and gently pipette to mix. The dosage is as follows:

c) Reverse transcription program setting: reverse transcription of the above mixed solution according to the program of 25°C (5 minutes), 42°C (30 minutes), 85°C (5 minutes), and the reverse transcription product is stored at -20°C.

4) Real-time fluorescence quantitative PCR

Using China's Shanghai Yisheng Technology Biological Company

qPCR SYBR Green Master Mix (High Rox Plus) kit, follow the instructions.

a) Configure the following reaction system (operate on ice and avoid light)

Set up the PCR reaction according to the following conditions

b) Using β-actin as an internal reference, calculate 2 ^{(-△△CT) to} compare the relative expression of the target gene. The primer sequence of the target gene used is shown in the table below.

Refer to Figure 3, the first-generation liver organoids and the third-generation liver organoids were extracted from RNA, and compared with liver tissues at the genetic level. The expression of hepatic stemness genes EPCAM, SOX9 and CK19 were significantly up-regulated compared with liver tissues. Before and after passaging, EPCAM, SOX9, CK19, TBX3, AFP, SOX17, FOXA2, HNF4A, CEBPA and CEBPB did not change significantly, and hepatocytes were induced into dry liver organoids with strong proliferation ability.

2. Immunofluorescence

1) Dry frozen sections in a ventilated place for 5 minutes;

2) Pre-cooling 4% paraformaldehyde to fix at room temperature for 20 minutes;

3) Wash 3 times with 1X PBS, 5 minutes/time, discard PBS;

4) 1mL 0.5% TritonX-100 membrane rupture, 10 minutes at room temperature;

5) Wash 3 times with 1X PBS, discard PBS;

6) PBS containing 2% BSA was blocked for 1 hour at room temperature;

7) Use an immunofluorescent pen to delineate the area.

8) Add the primary antibody with appropriate dilution ratio and incubate (CK8, AFP, Desmin, PCNA), add the primary antibody dilution dropwise to the negative control, put it in a humid box, overnight at 4°C;

9) Recover the primary antibody, wash 3 times with 1X PBST, 5 minutes/time, discard PBST;

10) Incubate the fluorescent secondary antibodies (goat anti-mouse IgG H&L and goat anti-rabbit IgG H&L) in the dark for 30 minutes;

11) Wash 3 times with 1X PBST, 5 minutes/time, discard PBST;

12) Hoest nuclear staining, incubate for 5 minutes in the dark;

13) Wash 3 times with 1X PBST, 5 minutes/time, discard PBST;

14) Add glycerin dropwise, lightly cover the cover glass, fix the cover glass, and observe under a fluorescence microscope (Leica, USA).

Refer to Figure 4, which shows the positive expression of Desmin, AFP, PCNA, and CK8, indicating that hepatocytes are induced into liver organoids with strong proliferation ability and dryness.

Application example 1

Liver organ transplantation improves liver function in 70% of acute liver failure after hepatectomy

Animal model construction: 6-8 weeks old, C57BL/6 mice, randomly divided into 3 groups, 3 mice in each group, one group does not do any treatment, namely the blank group, the remaining two groups are constructed for 70% acute liver failure after hepatectomy Model: a group of postoperative liver capsules injected with 200uL PBS as the control group (ie control group), a group of postoperative liver capsules transplanted with 200uL organoid suspension (1×10 ⁶ cells) as the treatment group (ie treatment group) . The control group and the treatment group were sacrificed on days 1, 4, and 7 to collect blood from the orbit of the mice. The liver was weighed and fixed with 10% neutral formaldehyde.

The specific steps for constructing a mouse model of acute liver failure after 70% hepatectomy are as follows: prohibit eating and drinking for 1 day before the operation, set the anesthesia parameters of the small animal anesthesia machine according to the weight of the mouse, put the mouse in the anesthesia box, and turn on the anesthesia aisle. After the mouse is anesthetized, the distal ends of the limbs are fixed with tape, the back is against the mouse board, and the breathing mask is put on. Use iodophor to disinfect the surgical site (range up to the level of the axillary line, and down to the line of the upper edge of the groin). The surgical incision is made at the junction of the lower edge of the costal arch and the white line of the abdomen: first cut a small incision of about 0.5 cm, clamp the arteries in the abdominal wall on both sides with hemostatic forceps, and continue to extend the white line up and down to an appropriate length. With the aid of a wet cotton swab, cut the mesangium and hepatogastric ligament between the tail lobe of the liver, the stomach and the diaphragm and the left lobe of the liver. Make each liver lobe completely free, pre-moisten the 3-0 surgical thread, and cooperate with the cotton swab to ligate the root of the left lobe until the color becomes darker, then cut off the lobe to remove the remaining blood. Continue to ligate the middle liver lobe with a 3-0 surgical suture and wet cotton swabs. After the color becomes darker, cut off the lobe to remove residual blood. Observe the vital signs of the mice, whether there is a lot of blood oozing.

The control group was injected with 200uL PBS under the liver capsule and compressed with dry cotton balls to stop bleeding. In the treatment group, 200 uL of liver organ suspension was injected under the liver capsule (slow bolus injection), and light pressure was used to stop bleeding with dry cotton balls. Remove the residual blood in the abdominal cavity, start to suture the abdominal muscle layer and fur layer layer by layer and disinfect the wound. After the operation, the mice were resuscitated in an incubator, and the activities of the mice were observed. It is necessary to extend the resuscitation time.

Liver function test: An automated biochemical analyzer (Mindray, China) was used to determine the synthesis level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and liver albumin (ALBumin, ALB).

Referring to Figure 5, the difference in liver-to-body ratio was counted, and it was found that mice in the treatment group increased significantly on the 1st and 7th days after 70% hepatectomy.

Refer to Figure 6-8. On the fourth day, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the treatment group were lower than those in the control group, and the liver albumin (ALBumin, ALB) synthesis was also significantly increased. It shows that liver function is restored after organ transplantation.

Application example 2

Transplantation of liver organoids reduces 70% of the inflammatory reaction of acute liver failure after hepatectomy and promotes liver regeneration.

HE staining, the steps are as follows:

1) Material: After inhalation anesthesia, the mouse is fixed to the operating table, the abdominal cavity is opened, and the heart is cut to death. The mesangium between the tail lobe of the liver, the stomach and the diaphragm and the left outer lobe of the liver, the hepatogastric ligament, the middle lobe of the liver and the diaphragm are put to death. Cut open the sickle ligament, peel off the intact liver tissue, and put it in 10% neutral formaldehyde after rinsing with PBS to fix the morphology and structure of the cells;

2) Washing: After fixing, _{rinse with ddH 2} O for several hours;

3) Dehydration: Dehydrate the tissue 3 times with 70%, 80%, and 90% ethanol, each for 30 minutes, and then use 95% and 100% ethanol for 2 times, each for 20 minutes;

4) Transparent: prepare a mixture of xylene and absolute alcohol (in a ratio of 1:1), put the tissue in it for 15 minutes, and then put it in xylene I and xylene II for 15 minutes each, until the tissue is transparent;

5) Wax penetration: prepare a mixture of paraffin wax and xylene (1:1 ratio), place the tissue in it for 15 minutes, and then use paraffin wax I and paraffin wax II to penetrate the wax for 1 hour respectively;

6) Embedding: Preheat the paraffin mold with an alcohol lamp, place it on a horizontal table, take out the wax cup from the incubator, pour in an appropriate amount of paraffin, and place the tissue in the wax mold with the cut surface facing down with heated tweezers , Arranged neatly, put the embedding box, pour the molten wax;

7) Slicing: Take out the wax block, install it on the inserting table of the microtome according to the instructions, install the blade with the knife edge upward, and start to push the spiral cutting wax block. The wax block is close to the knife edge, but not more than the knife edge. Adjust the two Adjust the thickness adjuster to a suitable gear for the angle and position between the two. Place the wax block on a horizontal table with a writing brush, and check that the section meets the standard;

8) Exhibit and patch: Adjust the temperature of the water bath to 40℃, take a clean glass slide, place the tissue section in the middle of the glass slide, mark it with a marker, and place it on the slice rack;

9) Dewaxing and rehydration: Adjust the water temperature of the water bath to 60°C, put the slices in a dry dyeing tank together with the rack, place in the water bath, and cover for 30 minutes until the wax melts. Then, paraffin sections were deparaffinized with xylene I and II for 15 minutes each, and then put into 100%, 95%, 90%, 80%, 70% alcohol solution for 5 minutes each time, and then ddH _{Rinse in 2} O for 3 minutes;

10) Staining: the sections are stained in hematoxylin staining solution for 5 minutes;

11) Washing with water: _{Rinse with ddH 2} O for 15 minutes, so that the color of the slices turns blue;

12) Differentiation: Put the slices in 1% hydrochloric acid ethanol, the slices will fade and then turn red;

13) Anti-blue: When the slice is placed in ddH ₂ O, it returns to blue;

14) Dehydration: the slices were placed in ethanol for 3 times, the concentration was 50%, 70%, 80%, 5 minutes each;

15) Counterstaining: stain with eosin ethanol solution (0.5%) for 2 minutes;

16) Dehydration: Put the slices into ethanol with a concentration gradient of 50%, 70%, 80%, 90%, 95%, and 100% in sequence for 5 minutes;

17) Transparent: Put the slices in xylene Ⅰ and Ⅱ for 5 minutes each;

18) Mounting: After sealing the film with neutral gum, place it in an oven at 37°C and bake the film overnight. The stained liver tissue sections were observed, analyzed and photographed under an inverted microscope (Leica, USA).

Ki67 immunohistochemistry

The tissue acquisition and the preparation of paraffin sections are the same as above, and the specific steps of histochemistry are as follows:

1) Dewaxing: Use xylene Ⅰ and Ⅱ to dewax the paraffin sections for 15 minutes each, and then place them in an alcohol solution with a concentration gradient of 100%, 95%, 90%, 80%, and 70%. , 5 minutes each time, and finally rinse with distilled water for 3 minutes;

2) Inactivation: Treat the slices with 3% hydrogen peroxide for 10 minutes at room temperature;

3) Antigen retrieval: Place the slices in sodium citrate buffer (0.01M), heat to 100°C, cool for 5 minutes, and repeat twice;

4) Washing: After the section is cooled to room temperature, rinse with PBS 3 times, 5 minutes each time;

5) Blocking: Use BSA (3%) to block the section at 37°C for 1 hour;

6) Primary antibody binding: Combine the pre-diluted Ki67 primary antibody at an appropriate concentration, overnight at 4°C;

7) Rewarming: Place the slices in an oven at 37°C for rewarming for 1 hour;

8) Washing: Wash the sections with PBS 10 times, 2 minutes each time;

9) Secondary antibody binding: Combine the pre-diluted secondary antibody of the appropriate concentration and corresponding species at 37°C for 1 hour;

10) Washing: Wash the sections with PBS 10 times, 2 minutes each time;

11) Add SABC: Drop SABC on the slices, 37°C, 30 minutes;

12) Washing: Wash the slices 5 times with PBS, 2 minutes each time;

13) Color development: drop the configured DAB color development solution on the slice, place it at room temperature, observe the reaction time under the microscope (usually 1 to 5 minutes), when the best effect is reached, rinse with PBS to stop the color development;

15) Staining: the sections are counter-stained with hematoxylin staining solution for 1 minute, and rinsed thoroughly with PBS;

16) Dehydration: Place the slices in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 1 minute in sequence;

17) Transparent: Put the slices in xylene Ⅰ and Ⅱ for 5 minutes each;

18) Mounting the slides: After sealing the slides with neutral gum, bake the slides overnight in an oven at 37°C, and the stained liver tissue sections are observed and analyzed under an inverted microscope and photographed.

Refer to Figure 9, Figure A shows that the liver cells in the normal group are in regular strips, with clear hepatic sinusoidal spaces and no infiltration of inflammatory cells; the liver cells are normal in shape and size, without fat or balloon-like changes. In the treatment group and the control group, mice in the treatment group and the control group disappeared on the first day after the operation, the cell arrangement was disordered, the size was different, the inflammatory cells were infiltrated, the shape was not clear, there were a lot of fatty vacuoles, and the hepatic fatty vacuoles of the treatment group More serious; on the 4th day after surgery, the nucleolar staining of liver cells in the control group increased, the space between the liver sinusoids was enlarged, there were still inflammatory cell infiltrations, and there were areas of hepatocyte necrosis in some areas. The morphology and size of hepatocytes in the treatment group returned to normal, with clear morphology, no inflammatory cell infiltration, no fat or ballooning changes; from Figure B, 70% of the mice after hepatectomy, on the first day, the control group The proliferation was significantly stronger than that of the treatment group. On the 4th day, both the control group and the treatment group showed a proliferation state, while the treatment group's regeneration was significantly higher than that of the control group, showing vigorous proliferation.

In summary, it shows that organ transplantation can reduce liver inflammation and promote liver regeneration to treat acute liver failure.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, a number of modifications and improvements can be made, all of which belong to the present invention. The scope of protection of the invention.

Claims (8)

1. An in vitro construction method of liver organoids, which is characterized in that it comprises the following steps:

   S1: Prepare a tissue suspension, take the liver of a normal mouse under aseptic conditions, rinse the liver and cut it into pieces to obtain the tissue suspension;

   S2: prepare a cell suspension, add liver tissue digestion to the tissue suspension prepared in step S1, then transfer to a water bath for static digestion, perform mechanical pipetting after digestion, and collect the supernatant as the cell suspension after the cells settle;

   S3: Seed the plate, filter, centrifuge, and lyse the cell suspension obtained in step S2 to obtain the cell pellet. Use Matrigel to resuspend the cell pellet and plant it in the center of the 24-well plate. After the matrigel is fixed, add it to the liver. The organoid induction medium is cultivated to obtain the initial organoid;

   S4: Passage the initial organoids obtained in step S3, and select 3 to 5 generations as the final prepared liver organoids.
2. The method for constructing liver organoids in vitro according to claim 1, characterized in that, in the step S1, the liver is first rinsed in a pre-chilled 30mL DMEM/F-12 medium to remove residual blood, and then Cut into a new 30mL DMEM/F-12 medium to 1mm.
3. The method for constructing liver organoids in vitro according to claim 1, wherein the specific steps of step S2 include: transferring the tissue suspension to a 50 mL centrifuge tube at room temperature with a pipette, and letting it stand for 1 minute Remove the supernatant, add 10mL liver tissue digestion solution, transfer to 37℃ water bath and let stand for 20 minutes for digestion. After mechanical pipetting, let it stand for 1 minute. After the cells settle on the bottom, remove the supernatant, continue to add 10mL liver tissue digestion solution, and transfer Place it in a 37℃ water bath for 20 minutes for digestion. After mechanical pipetting, let it stand for 1 minute. After the cells settle at the bottom, collect the supernatant in a 50mL pre-cooled centrifuge tube. Repeat the above digestion and collection steps 4 times to collect 50mL. The cell supernatant was used as the cell suspension.
4. The method for in vitro construction of liver organoids according to claim 1 or 3, characterized in that: the specific preparation of the liver tissue digestive juice is: adding 7.5 mL of neutral to every 45 mL of DMEM/F12 medium Protease and 7.5 mL of type IV collagenase.
5. The method for constructing liver organoids in vitro according to claim 1, wherein said step S3 specifically comprises:

   S31: The collected cell suspension is first filtered with a 100um cell sieve, leaving cells with a diameter less than 100um, and then the filtrate is filtered again with a 30um cell sieve, the filtrate is discarded, and the 30um filter is poured into 50mL On the centrifuge tube, rinse with DMEM/F-12 medium, and collect the cells trapped on the 30um filter;

   S32: Centrifuge at 300g for 5 minutes at 4°C and remove the supernatant;

   S33: Add 3 to 5 times the cell volume of red blood cell lysate, gently pipette, lyse for 4-5 minutes, centrifuge at 4°C, 100g for 5 minutes, discard the supernatant, and obtain a cell pellet;

   S34: Use 240uL Matrigel to resuspend the cell pellet, pipe it up and down evenly to avoid air bubbles, plant it in the center of the 24-well plate at the position of the 8 holes;

   S35: Let the cell incubator stand at 37°C for 10 minutes. After the matrigel is fixed, 750uL liver organoid induction medium is adhered to the wall, and the liver organoid induction medium is replaced every 2 to 3 days according to the culture condition to obtain the initial organoids.
6. The method for constructing liver organoids in vitro according to claim 1, wherein said step S4 specifically comprises:

   S41: Remove liver organoid induction medium, rinse gently with PBS, remove PBS, add DMEM/12 medium, stand for 1 minute for digestion, mechanically pipette the initial organoids;

   S42: Centrifuge at 300g for 5 minutes at 4°C and remove the supernatant;

   S43: The pipette tip is pre-cooled, and 240uL Matrigel is used to resuspend the cell pellet, pipetting up and down evenly to avoid air bubbles, planted in the center of the 24-well plate at the position of 8 holes;

   S44: Let the cell culture incubator stand at 37°C for 10 minutes. After the matrigel is fixed, add 750uL liver organoid induction medium to the wall, and pass the culture at a ratio of 1:4.
7. The method for in vitro construction of liver organoids according to any one of claims 1 or 5 or 6, wherein the specific preparation of the liver organoid induction medium is: combining the liver organoid basal medium and The liver organoid basal medium additives are mixed in a volume ratio of 10:1, and an anti-mycoplasma reagent is added. After mixing, they are divided into aliquots and stored at a temperature of -20°C.
8. An application of the liver organoid prepared by the method of claim 1 in the preparation of a medicine for treating acute liver failure.

Images