LU500561B1 - In vitro construction method and use of liver organoids - Google Patents

Abstract

An in vitro construction method and use of liver organoids. Mouse hepatocytes are extracted and induced to construct liver organoids, the morphology of the liver organoids is observed under a microscope, and the sternness and epithelial cell attributes of the liver organoids are identified by immunofluorescence and PCR. Acute liver failure (ALF) mouse models undergoing 70% liver resection are established and transplanted with the liver organoids, liver function is evaluated on day 1, day 4, and day 7 after the transplantation, and liver weight-to-body weight ratios are compared. The therapeutic effect of the liver organoids on ALF is verified by HE staining and immunohistochemistry. Inflammation and proliferation indexes such as HE staining and ki-67 staining show that the proliferation is enhanced after the liver organoids are transplanted. The liver organoids can alleviate the liver function failure and increase the liver weight-to-body weight ratio of ALF mice undergoing 70% liver resection.

Description

DESCRIPTION 7500561

IN VITRO CONSTRUCTION METHOD AND USE OF LIVER ORGANOIDS

TECHNICAL FIELD The present disclosure relates to the technical field of biomedicine, and in particular to an in vitro construction method and use of liver organoids.

BACKGROUND Acute liver failure (ALF) is a clinical syndrome of massive liver cell necrosis and severe liver injury caused by virus, drug, toxin, alcohol, or other factors in a short period of time, which often leads to jaundice, hepatic encephalopathy, multiple-organ failure (MOF), and other complications and has a high mortality rate. Currently, liver transplantation is the only effective treatment method. However, donor livers are in short supply, and the supply of donor livers is far from meeting clinical needs. Therefore, many patients die while waiting for a donor liver. In addition, medical treatment is merely a symptomatic and supportive treatment and cannot improve the prognosis for a patient. Therefore, there is an urgent need to develop a replacement therapy.

Stem cells have unique functions in self-renewal and differentiation potential, and stem cell transplantation is regarded as the most promising therapeutic method for tissue repair and regeneration. Compared with liver transplantation, stem cell transplantation has the advantages of low invasiveness and repeatable transplantation, but also faces problems such as low implantation efficiency and short survival time of implanted cells. Studies have shown that a three-dimensional (3D) structure is conducive to the proliferation and differentiation of cells. 3D cultivation can simulate an extracellular environment in vivo, and efficient energy transfer and various molecular signal exchanges between cells and an environment can be realized in long-term cultivation. Induced pluripotent stem cells (iPSCs) obtained from 3D cultivation have significantly improved urea secretion and albumin (ALB) synthesis abilities compared with iPSCs obtained from traditional two-dimensional (2D) cell cultivation. Organoids are cell clusters constructed in vitro based on a 3D cultivation system, which have the abilities of automatic renewing and assembly and show partial functions of target organs in vivo. Existing studies have proved that, after liver organoids derived from stem cells are implanted in pigs and mice, the liver function maintenance ability is significantly enhanced.

Huch, et al. isolated EPCAM” epithelial cells from a human liver sample and constructed liver -7500561 organoids in vitro from the epithelial cells. The chromosome structure and genetic constitution of the liver organoids remain stable after amplification. The liver organoids have the potential to differentiate into hepatocytes and cholangiocytes, which can successfully differentiate into functional hepatocytes after being implanted into nude mice. The construction of organoid transplants in vitro through a 3D cultivation model is a development direction of replacement therapy for ALF in the future.

Considerable progress has been made in cell transplantation-based treatment for liver diseases, involving embryonic stem cells (ESCs), iPSCs, mesenchymal stem cells (MSCs), and hepatic progenitor cells (HPCs). Hepatocyte-like stem cells can be induced from pluripotent stem cells (PSCs), but the clinical application of the hepatocyte-like stem cells is still limited due to cell differentiation efficiency, immunological rejection, ethical issues, teratogenic risks, and other problems. Although pure cell transplantation leads to a given curative effect, there is a lack of ideal cell sources, the curative effect is inexact and unstable, and the clinical application has the risk of infiltrating other organs, which limits the large-scale promotion. At present, the combination of cells with tissue engineering is a hot research topic. Organoids are cell clusters constructed in vitro based on a 3D cultivation system. Compared with a traditional cultivation mode, in 3D cultivation, signal effects among cells and between cells and an environment are further enhanced, and obtained organoids have the abilities of automatic renewing and assembly to realize partial functions of original organs in vivo. Current studies have proved that in chronic liver diseases, HPCs can mediate liver regeneration, and liver organoids have the functions of protecting hepatocytes and promoting liver regeneration. Whether exogenous implantation of liver organoids with properties similar to HPCs after acute liver injury such as partial hepatectomy can promote liver regeneration has not been reported yet.

SUMMARY The present disclosure is intended to provide an in vitro construction method and use of liver organoids in view of the problems in the prior art. Mouse hepatocytes are extracted and induced to construct liver organoids, the morphology of the liver organoids is observed under a microscope, and the stemness and epithelial cell attributes of the liver organoids are identified by immunofluorescence and PCR. ALF mouse models undergoing 70% liver HUS00561 resection are established and transplanted with the liver organoids, liver function is evaluated on day 1, day 4, and day 7 after the transplantation, and liver weight-to-body weight ratios are compared. The therapeutic effect of the liver organoids on ALF is verified by HE staining and immunohistochemistry.

To achieve the above objective, the present disclosure provides the following technical solutions.

An in vitro construction method of liver organoids is provided, including the following steps: S1: preparation of a tissue suspension: aseptically collecting a liver from a normal mouse, and rinsing and chopping the liver to obtain the tissue suspension; S2: preparation of a cell suspension: adding a liver tissue digestion liquid to the tissue suspension prepared in S1, and transferring a resulting mixture to a water bath for static digestion; after the digestion, mechanically pipetting the mixture up and down, and allowing cell sedimentation; and collecting a resulting supernatant to obtain the cell suspension; S3: cell plating: subjecting the cell suspension obtained in S2 to filtration, centrifugation, and lysis to obtain a cell precipitate; resuspending the cell precipitate with matrigel, and inoculating a resulting cell suspension in central positions of a 24-well plate; and after the matrigel is fixed, adherently adding a liver organoid induction medium for cultivation to obtain initial organoids; and S4: passaging the initial organoids obtained in S3, and selecting organoids of p3 to p5 as final liver organoids.

In order to optimize the above technical solution, the present disclosure further includes: In S1, the liver may be first rinsed in 30 mL of a pre-cooled DMEM/F-12 medium to remove residual blood, and then chopped into pieces with a particle size of 1 mm in another mL of a DMEM/F-12 medium.

S2 may specifically include: transferring the tissue suspension with a pipette to a 50 mL centrifuge tube at room temperature, allowing the tissue suspension to stand for 1 min, and removing a resulting supernatant; adding 10 mL of the liver tissue digestion liquid, transferring the centrifuge tube to a 37°C water bath to allow static digestion for 20 min,

mechanically pipetting a resulting mixture up and down, and allowing the resulting mixture to -7500561 stand for 1 min; after cells settle at a bottom of the centrifuge tube, removing a resulting supernatant, and continuing to add 10 mL of the liver tissue digestion liquid; transferring the centrifuge tube to a 37°C water bath to allow static digestion for 20 min, mechanically pipetting a resulting mixture up and down, and allowing the resulting mixture to stand for 1 min; after cells settle at the bottom of the centrifuge tube, collecting a resulting supernatant in a pre-cooled 50 mL centrifuge tube; and repeating the above digestion and collection steps 4 times to collect 50 mL of a cell supernatant as the cell suspension.

The liver tissue digestion liquid may be prepared specifically as follows: adding 7.5 mL of a neutral protease and 7.5 mL of collagenase type IV to every 45 mL of a DMEM/F12 medium.

S3 may specifically include: S31: filtering the collected cell suspension with a 100 um cell strainer to obtain a filtrate in which cells have a diameter of less than 100 um; filtering the filtrate once again with a 30 pm cell strainer, and discarding a resulting filtrate; and placing the 30 um cell strainer upside down on a 50 mL centrifuge tube, and rinsing the cell strainer with a DMEM/F-12 medium to collect cells trapped on the 30 um cell strainer; S32: centrifuging a resulting cell suspension for 5 min at 300 g and 4°C, and discarding a resulting supernatant; S33: adding a red blood cell (RBC) lysing buffer at a volume 3 to 5 times of a volume of cells, gently pipetting a resulting mixture up and down, and conducting lysis for 4 min to 5 min; and centrifuging a resulting lysate at 4°C and 100 g for 5 min, and discarding a resulting supernatant to obtain a cell precipitate; S34: resuspending the cell precipitate with 240 pL of the matrigel, pipetting a resulting mixture up and down for thorough dispersion (where air bubbles are avoided), and inoculating a resulting cell suspension in central 8 wells of the 24-well plate; and S35: allowing the plate to stand in a 37°C cell incubator for 10 min; after the matrigel is fixed, adherently adding 750 pL of the liver organoid induction medium; and replacing the liver organoid induction medium every 2 d to 3 d according to cultivation conditions to obtain the initial organoids.

S4 may specifically include: -7500561 S41: removing the liver organoid induction medium, gently rinsing the initial organoids with PBS, and removing PBS; and adding the DMEM/12 medium to allow static digestion for 1 min, and mechanically pipetting a resulting mixture up and down; S42: centrifuging the resulting mixture for 5 min at 300 g and 4°C, and discarding a resulting supernatant; S43: using a pre-cooled pipette tip to resuspend a resulting cell precipitate with 240 pL of matrigel, pipetting a resulting mixture up and down for thorough dispersion (where air bubbles are avoided), and inoculating a resulting cell suspension in central 8 wells of a 24-well plate; and S44: allowing the plate to stand in a 37°C cell incubator for 10 min; and after the matrigel is fixed, adherently adding 750 pL of the liver organoid induction medium, where passage is conducted at a ratio of 1:4.

The liver organoid induction medium may be prepared specifically as follows: mixing a liver organoid basal medium with a liver organoid basal medium additive at a volume ratio of 10:1, adding an anti-mycoplasma reagent, thoroughly mixing and dispensing a resulting mixture, and storing a resulting medium at -20°C.

The present disclosure also claims use of liver organoids prepared by the method described above in the treatment of ALF.

The present disclosure has the following beneficial effects:

1. After the mouse liver organoids prepared by the method of the present disclosure are cultivated in vitro for 3 d, the liver organoids proliferate from a cystic structure with a diameter of 20 um to a cell mass with a diameter of about 100 um, liver stemness genes EPCAM, SOX9, and CK19 are significantly up-regulated, and EPCAM, SOX9, CK19, TBX3, AFP, SOX17, FOXA2, HNF4A, CEBPA, and CEBPB remain stable before and after passage.

2. In the present disclosure, immunofluorescence results show that CK8, Desmin, AFP, and PCNA are positive, the liver weight-to-body weight ratio increases significantly after the implantation of liver organoids, and the liver functions such as ALT, AST, ALB, and TG are recovered on day 4. Inflammation and proliferation indexes such as HE staining and ki-67 staining show that the proliferation is significantly enhanced after the liver organoids are transplanted. -7500561

3. The liver organoids of the present disclosure have strong stemness and proliferation function. The liver organoids can effectively alleviate the liver function failure and increase the liver weight-to-body weight ratio of ALF mice undergoing 70% liver resection, which promotes the liver regeneration and repair of ALF mice undergoing 70% liver resection by reducing liver inflammation infiltration.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the morphologies of organoids cultivated for 1 d, 2 d, and 3 d under an optical microscope.

FIG. 2 is a statistical diagram of diameters of the organoids shown in FIG. 1.

FIG. 3 is a comparison diagram of gene detection for P1 liver organoids, P3 liver organoids, and liver tissue.

FIG. 4 is a schematic diagram illustrating the proliferation ability of organoids.

FIG. 5 is a schematic diagram illustrating the liver weight-to-body weight ratios of the control group and the treatment group in Application Example 1.

FIG. 6 is a comparison diagram of alanine aminotransferase (ALT) change for the control group and the treatment group in Application Example 1.

FIG. 7 is a comparison diagram of aspartate aminotransferase (AST) change for the control group and the treatment group in Application Example 1.

FIG. 8 is a comparison diagram of liver ALB change for the control group and the treatment group in Application Example 1.

FIG. 9 shows the effects of transplanted liver organoids to alleviate ALF inflammatory responses and promote liver regeneration after 70% liver resection.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present disclosure is described in further detail below with reference to the accompanying drawings and examples. If specific conditions are not specified in an example, the example is conducted according to conventional conditions well-known in the art or the conditions recommended by a manufacturer. The instruments or reagents used are all conventional products that can be purchased commercially, and details can be seen in the following table.

In the examples, 6-week-old C57B/6 mice were selected as experimental animals, which -7500561 were raised under the following conditions: 12h/12h light/dark photoperiod (06:00 to 18:00), free eating and drinking, and temperature: 22°C. The mice were purchased from the Laboratory Animal Center of Nanjing Medical University by the agent of Laboratory Animal Center of Affiliated Drum Tower Hospital, Medical School of Nanjing University. À raising site was managed by the Laboratory Animal Center of Affiliated Drum Tower Hospital, Medical School of Nanjing University, and all animal experiments were also conducted in the Laboratory Animal Center of Affiliated Drum Tower Hospital, Medical School of Nanjing University. All animal experiments were approved by the Animal Experiment Ethics Committee of Affiliated Drum Tower Hospital, Medical School of Nanjing University (approval number: 2018010017).

Example 1 In vitro construction of organoids

1. Preparation of reagents 1) Organoid induction medium: HepatiCuLt™ OGM Mouse Basal Medium was selected as a liver organoid basal medium, HepatiCuLt™ OGM Mouse Supplement was selected as a liver organoid basal medium additive, and Plasmocin™ treatment was selected as an anti-mycoplasma reagent. The HepatiCuLt™ OGM Mouse Basal Medium and HepatiCuLt™ OGM Mouse Supplement were mixed at a volume ratio of 10:1, then 200 pL of the Plasmocin™ treatment was added, and a resulting mixture was well mixed, dispensed, and stored at -20°C.

2) Liver tissue digestion liquid: 45 mL of Advanced DMEM/F12 was taken and added with 7.5 mL of Dispase and 7.5 mL of Collagenase type IV, and a resulting mixture was well mixed, which should be immediately used.

2. Material collection In a clean bench, a 6 to 8 week-old C57BL/6 normal mouse was selected, subjected to inhalation anesthesia, and then sacrificed by cervical dislocation. Limbs of the mouse were fixed on an operating table, and the abdomen was disinfected with alcohol cotton balls. The liver was collected aseptically, rinsed in 30 mL of a pre-cooled DMEM/F-12 medium once to remove residual blood, and then chopped into pieces with a particle size of 1 mm in another mL of a DMEM/F-12 medium. HUS00561

3. Digestion A tissue suspension obtained was transferred with a pipette to a 50 mL centrifuge tube at room temperature and then stood for 1 min, and a resulting supernatant was removed; 10 mL of a liver tissue digestion liquid was added, a resulting mixture was transferred to a 37°C water bath to allow static digestion for 20 min, then mechanically pipetted up and down, and stood for 1 min; after cells settled at a bottom of the centrifuge tube, a resulting supernatant was removed, and 10 mL of the liver tissue digestion liquid was added; a resulting mixture was transferred to a 37°C water bath to allow static digestion for 20 min, mechanically pipetted up and down, and stood for 1 min; after cells settled at the bottom of the centrifuge tube, a resulting supernatant was collected in a pre-cooled 50 mL centrifuge tube; and the above digestion and collection steps were repeated 4 times to collect about 50 mL of a cell supernatant.

4. Cell plating (aseptic operation) 1) The collected cell suspension was first filtered with a 100 um cell strainer to obtain a filtrate in which cells had a diameter of < 100 um. The filtrate was filtered with a 30 pm cell strainer once again, and a resulting filtrate was discarded. The 30 um cell strainer was placed upside down on a 50 mL centrifuge tube, and then rinsed with Advanced DMEM/F12 to collect cells trapped on the 30 pm cell strainer.

2) A resulting cell suspension was centrifuged for 5 min at 300 g and 4°C, and a resulting supernatant was discarded.

3) An RBC lysing buffer was added at a volume 3 to 5 times of a volume of cells, a resulting mixture was gently pipetted up and down, subjected to lysis for 4 min to 5 min, and centrifuged at 4°C and 100 g for 5 min, and a resulting supernatant was discarded to obtain a cell precipitate, where the RBC lysing buffer was sterilized with a disposable syringe filter in the dark before use.

4) The cell precipitate was resuspended with 240 uL of matrigel (operating on ice), and a resulting mixture was pipetted up and down for thorough dispersion (where air bubbles were avoided), and then inoculated in central 8 wells of a 24-well plate.

5) The plate stood in a 37°C cell incubator for 10 min; after the matrigel was fixed, 750 pL of the liver organoid induction medium was adherently added; and the liver organoid -7500561 induction medium was replaced every 2 d to 3 d according to cultivation conditions.

5. Passage 1) The liver organoid induction medium was removed, the organoids were gently rinsed with PBS, and PBS was removed: and Advanced DMEM/F12 was added to allow static digestion for 1 min, and a resulting mixture was mechanically pipetted up and down.

2) A resulting cell suspension was centrifuged for 5 min at 300 g and 4°C, and a resulting supernatant was discarded.

3) A pre-cooled pipette tip was used to resuspend a resulting cell precipitate with 240 pL. of matrigel (operating on ice), and a resulting mixture was pipetted up and down for thorough dispersion (where air bubbles were avoided), and then inoculated in central 8 wells of a 24-well plate.

4): The plate stood in a cell incubator for 10 min; and after the matrigel was fixed, 750 pL of the liver organoid induction medium was adherently added; and liver organoids of p3 to p5 were selected as final liver organoids, where passage was conducted at a ratio of 1:4.

It can be seen from FIG. 1 and FIG. 2 that liver organoids had high clonality, and could rapidly expand from a small number of initiating cells under specified cultivation conditions; and the organoids expanded from a cystic structure with a diameter of 20 um on day 1 to a spherical structure with a diameter of more than 100 um on day 3.

Example 2 Phenotypic identification of the liver organoids

1. Gene identification: 1) Cell RNA extraction a) The medium was removed from an organoid suspension, and obtained organoids were rinsed twice with PBS to ensure that the residual medium would not affect the RNA purity, and then added to a 24-well plate. 500 pL of Trizol was added to each well of the 24-well plate. In order to achieve thorough lysis, a resulting mixture was pipetted up and down repeatedly, and then stood at room temperature for 10 min. A protein-nucleic acid complex, after being completely separated, was transferred to a 1.5 mL enzyme-free EP tube.

b) Chloroform was added at a Trizol-chloroform ratio of 5:1, and a resulting mixture was vigorously shaken up and down for 10 s, and then stood for 5 min at room temperature to -7500561 allow natural phase separation.

c) Then the mixture was centrifuged at 4°C and 12,000 g for 15 min, and an upper aqueous phase was carefully pipetted to another 1.5 mL EP tube.

d) The aqueous phase was added with an equal volume of isopropanol, and a resulting mixture was thoroughly mixed and placed at -20°C for 1 h to increase RNA precipitation.

e) The mixture was centrifuged at 4°C and 12,000 g for 10 min, and a resulting supernatant was discarded to obtain an RNA precipitate at a bottom of the tube.

f) 1 mL of 75% ethanol (prepared with DEPC water) was added to the RNA precipitate, and a resulting mixture was well mixed; and the tube was gently shaken to make the precipitate suspended.

g) À resulting suspension was centrifuged for 5 min at 7,500 g and 4°C, and a resulting supernatant was carefully removed with a pipette.

h) A resulting precipitate was dried at room temperature for 5 min to 10 min to allow a small amount of residual ethanol to volatilize.

i) The RNA precipitate was dissolved with 20 uL of DEPC water, and a resulting RNA solution was stored at -80°C.

2) Determination of RNA concentration a) Zero adjustment was conducted for a nucleic acid and protein analyzer with ddH2O, and 1 pL of RNA was pipetted to determine a purity. OD260/OD280 was of 1.8 to 2.0, and a concentration of 500 ng/uL was preferred.

b) The RNA concentration was adjusted with DEPC water to 500 ng/uL. After each sample was tested, the analyzer was wiped with lens paper to avoid mutual influence of samples.

3) Reverse transcription of RNA to cDNA The Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR reverse transcription kit from Shanghai Yeasen Biotech Co., Ltd. in China was used, and specific operations were conducted according to the instructions.

a) Removal of residual genomic DNA (gDNA): The following mixture was prepared in an RNase free EP tube, gently pipetted up and down for thorough mixing, and incubated at

42°C for 2 min.

HUS00561 Reaction system 10 pL RNase free ddH,O to 10 pL x gDNA digester 2 uL gDNA digester 1 pL Template RNA Total RNA: 1 ng to 5 pg, or mRNA: 1 ng to 500 ng b) Preparation of the following reverse transcription reaction system (20 pL system): 2 x HifairTM II SuperMix plus was directly added to the reaction tube in step a), and a resulting mixture was gently pipetted up and down for thorough mixing.

Reaction system 20 uL Reaction solution in step a) 10 uL 2 x Hifair®IIuperMix plus 10 pL c) Setting of a reverse transcription program: The above mixture was subjected to reverse transcription according to the program of 25°C (5 min), 42°C (30 min), and 85°C (5 min), and a reverse transcription product was stored at -20°C. 4) Real-time fluorescent quantitative PCR The Hieff® qPCR SYBR Green Master Mix (High Rox Plus) kit from Shanghai Yeasen Biotech Co., Ltd. in China was used, and operations were conducted according to the instructions.

a) Preparation of the following reaction system (operating on ice in the dark) Reaction system 10 uL Hieff® qPCR SYBR Green Master Mix 5 uL (High Rox Plus) Forward Primer (10 pM) 0.2 uL Reverse Primer (10 pM) 0.2 uL Template DNA 1 uL Sterile ultrapure water (UPW) 3.6 uL PCR was conducted according to the following conditions. Stage 2 Cyclic reaction Reps: 40 Stage 3 Melting curve Reps: 1 “we | we b) With B-actin as an internal reference, 2 AAC’ was calculated to compare the relative expression levels of target genes. Primer sequences used for the target genes were shown in the table below.

GAPDH : SOX17 : ; FOXA2 ra

; i

CEBPA : a

CEBPB 1 z 7

EPCAM 5 x 5 7 As shown in FIG. 3, RNA was extracted from P1 liver organoids and P3 liver organoids and compared with that of liver tissue at the genetic level, where the expression levels of liver stemness genes EPCAM, SOX9, and CK19 were significantly up-regulated compared with that in liver tissue, and EPCAM, SOX9, CK19, TBX3, AFP, SOX17, FOXA2, HNF4A, CEBPA, and CEBPB did not change significantly before and after passage, indicating that hepatocytes were induced into liver organoids with strong proliferation ability and stemness.

2. Immunofluorescence HUS00561 1) A sample was subjected to frozen section and air-dried for 5 min at a ventilated place.

2) Then the sample was fixed for 20 min with pre-cooled 4% paraformaldehyde.

3) Washing was conducted 3 times with 1 X PBS, with 5 min for each time; and then PBS was discarded.

4) 1 mL of 0.5% TritonX-100 was used to break the cell membrane at room temperature for 10 min.

5) Washing was conducted 3 times with 1X PBS, and then PBS was discarded.

6) Blocking was conducted for 1 h at room temperature with 2% BSA-containing PBS.

7) An immunofluorescent pen was used to circle regions.

8) A primary antibody at an appropriate dilution ratio was added for incubation (CK8, AFP, Desmin, PCNA), and a primary antibody diluent was added dropwise to a negative control. A resulting mixture was incubated overnight at 4°C in a humidified box.

9) The primary antibody was recovered, and washing was conducted 3 times with 1X PBST, with 5 min for each time; and then PBST was discarded.

10) Fluorescent secondary antibodies (goat anti-mouse IgG H&L and goat anti-rabbit IgG H&L) were added, and a resulting mixture was incubated for 30 min in the dark.

11) Washing was conducted 3 times with 1X PBST, with 5 min for each time; and then PBST was discarded.

12) A Hoest nuclear stain was added, and a resulting mixture was incubated for 5 min in the dark.

13) Washing was conducted 3 times with 1X PBST, with 5 min for each time; and then PBST was discarded.

14) Glycerin was added dropwise, a cover glass was lightly covered and fixed, and then the sample was observed under a fluorescence microscope (Leica, United States).

As shown in FIG. 4, the Desmin, AFP, PCNA, and CK8 were positive, indicating that hepatocytes were induced into liver organoids with strong proliferation ability and stemness.

Application Example 1 Improvement of liver organoid transplantation on the liver functions of ALF mice undergoing 70% liver resection

Construction of animal models: 6 to 8 week-old C57BL/6 mice were selected and HUS00561 randomly divided into 3 groups, with 3 mice in each group. One group was subjected to no treatment, which was a blank group. The remaining two groups were used construct ALF models undergoing 70% liver resection: one group was postoperatively injected with 200 pL of PBS through the Glisson's capsule, which was a control group (i.e., control group); and the other group was postoperatively transplanted with 200 uL of an organoid suspension (1 x 10° cells) through the Glisson's capsule, which was a treatment group (i.e., treatment group). The mice in the control group and the treatment group were sacrificed on day 1, day 4, and day 7, blood was collected at the orbite, and the liver was collected, weighed, and fixed with 10% neutral formaldehyde.

Specific steps for constructing ALF mouse models undergoing 70% liver resection were as follows: A mouse was prevented from eating and drinking 1 d before the operation, anesthesia parameters of a small animal anesthesia machine were set according to a body weight of the mouse, then the mouse was put in an anesthesia box, and an anesthesia channel was turned on. After the mouse was anesthetized, distal ends of limbs were fixed with adhesive tape, the back was against a mouse board, and a breathing mask was put on for the mouse. A surgical site (from a horizontal line connecting axillary fossas to a horizontal line connecting upper edges of groins) was disinfected with iodophor. A surgical incision was made at a junction between a line connecting lower edges of costal arches and a linea alba: a small incision of about 0.5 cm was made first, the epigastric arteries on both sides were clamped with hemostatic forceps, and then the small incision was extended up and down along the linea alba to an appropriate length. With the aid of a wet cotton swab, the mesangium and hepatogastric ligament between any one of the tail liver lobe, the stomach, and the diaphragm and the left liver lobe were cut off. After each liver lobe was completely free, a 3-0 surgical suture was pre-wetted and used to ligate a root of the left lobe with the aid of a cotton swab until it darkened, then the left lobe was cut off, and residual blood was cleared. The 3-0 surgical suture was used to further ligate the middle liver lobe with the aid of a cotton swab until it darkened, then the middle liver lobe was cut off, and residual blood was cleared. The vital signs of the mouse were observed, and whether there is heavy blood oozing was also observed.

The control group was injected with 200 pL of PBS through the Glisson's capsule, and an -7500561 injection site was compressed with a dry cotton ball to stop bleeding. The treatment group was injected with 200 pL of a liver organoid suspension through the Glisson's capsule (slow injection), and an injection site was slightly pressed with a dry cotton ball to stop bleeding. Residual blood in the abdominal cavity was cleared, then the abdominal muscle layer and the skin layer were sutured layer by layer, and the wound was disinfected. After the operation, the mice were resuscitated in an incubator, and activities of the mice were observed. À resuscitation time can be extended if necessary.

Liver function test: An automated biochemical analyzer (Mindray, China) was used to determine the synthesis levels of ALT, AST, and liver ALB.

As shown in FIG. 5, the difference in the liver weight-to-body weight ratio was counted, and it was found that the liver weight-to-body weight ratios of the mice in the treatment group increased significantly on day 1 and day 7 after 70% liver resection.

As shown in FIG. 6 to FIG. 8, on day 4, the synthesis levels of ALT and AST in the treatment group were lower than those in the control group, and the synthesis level of liver ALB also significantly increased, indicating that liver functions were restored after organoid transplantation.

Application Example 2 The liver organoid transplantation can alleviate ALF inflammatory responses and promote liver regeneration after 70% liver resection.

HE staining was conducted through the following steps: 1) Material: A mouse was subjected to inhalation anesthesia and then fixed on an operating table, then the abdominal cavity was opened, and the mouse was sacrificed by cutting the heart. The mesangium and hepatogastric ligament between any one of the tail liver lobe, the stomach, and the diaphragm and the left outer liver lobe and the falciform ligament between the middle liver lobe and the diaphragm were cut off to peel off the intact liver tissue, and then the liver tissue was rinsed with PBS and put in 10% neutral formaldehyde to fix the morphological structure of cells.

2) Washing: After the fixing, the liver tissue was rinsed with ddH2O for several hours.

3) Dehydration: The liver tissue was dehydrated 3 times successively with 70% ethanol,

80% ethanol, and 90% ethanol, with 30 min for each time. Then the liver tissue was 7500561 dehydrated 2 times successively with 95% ethanol and 100% ethanol, with 20 min for each time.

4) Transparency: A mixed solution of xylene and absolute alcohol (at a ratio of 1:1) was prepared, and the tissue was soaked in the mixed solution for 15 min and then in xylene I and xylene II successively (with 15 min in each) until the tissue was transparent.

5) Wax penetration: A mixed solution of paraffin wax and xylene (at a ratio of 1:1) was prepared, and the tissue was soaked in the mixed solution for 15 min, and then in paraffin wax I and paraffin wax II successively (with 1 h in each).

6) Embedding: A paraffin wax mold was pretreated with an alcohol lamp and placed on a horizontal tabletop; a wax cup was taken out from an incubator and poured with an appropriate amount of paraffin wax; then the tissue was put in the paraffin wax mold using heated tweezers with a section facing downward, and arranged neatly; and an embedding cassette was put, and molten wax was poured.

7) Sectioning: A wax block was taken out and placed on a clamping platform of a section cutter according to the instructions, a blade was assembled with a blade edge facing upward, and then the blade was pushed to spirally cut the wax block, where the wax block was close to the knife edge, but not crossed the knife edge, the angle between and the positions of the two were adjusted, and a thickness adjuster was adjusted to a suitable gear. The wax block was placed on a horizontal tabletop with a pen brush to check whether sections meet standards.

8) Stretching and attachment of sections: A water temperature in a water bath was adjusted to 40°C. A clean glass slide was taken, a tissue section was placed in the middle of the glass slide, and then the glass slide was marked with a marker and placed on a section rack.

9) Dewaxing and rehydration: A water temperature of the water bath was adjusted to 60°C. The section rack with the tissue section was placed in a dry staining jar, and then the dry staining jar was placed in the water bath for 30 min with the water bath being covered until the wax melted. Then, the paraffin section was dewaxed in xylene I and xylene II successively (with 15 min in each) and then in 100%, 95%, 90%, 80%, and 70% alcohol solutions successively (with 5 min in each), and then rinsed with ddHzO for 3 min.

10) Staining: The section was stained in a hematoxylin stain for 5 min. -7500561 11) Water washing: The section was rinsed with ddH,O for 15 min until the section turned blue.

12) Differentiation: The section was placed in 1% hydrochloric acid-ethanol, where the section first faded and then turned red.

13) Recovery to blue: The section was placed in ddH2O, such that the section returned to blue.

14) Dehydration: The section was soaked 3 times in 50% ethanol, 70% ethanol, and 80% ethanol, respectively, where the soaking was conducted for 5 min each time.

15) Counterstaining: The section was stained with an eosin-ethanol solution (0.5%) for 2 min.

16) Dehydration: The section was soaked in 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol successively, with 5 min in each ethanol solution.

17) Transparency: The section was soaked in xylene I and xylene II successively (with 5 min in each).

18) Mounting: The section was mounted with neutral gum, and then baked overnight in an oven at 37°C. Stained liver tissue sections were observed, analyzed, and photographed under an inverted microscope (Leica, United States).

Ki67 immunohistochemistry The collection of tissues and the preparation of paraffin sections were the same as above, and specific immunohistochemical steps were as follows: 1) Dewaxing: The paraffin section was dewaxed in xylene I and xylene II successively (with 15 min in each) and then in 100%, 95%, 90%, 80%, and 70% alcohol solutions successively (with 5 min in each), and then rinsed with distilled water for 3 min.

2) Inactivation: The section was treated with 3% hydrogen peroxide for 10 min at room temperature.

3) Antigen retrieval: The section was placed in a sodium citrate buffer (0.01 M), a resulting mixture was heated to 100°C and then cooled for 5 min, and the process was repeated twice.

4) Washing: After being cooled to room temperature, the section was rinsed 3 times with -7500561 PBS, with 5 min for each time.

5) Blocking: The section was blocked with BSA (3%) for 1 h at 37°C.

6) Primary antibody binding: The section was added with pre-diluted Ki67 primary antibody at an appropriate concentration, and incubated overnight at 4°C.

7) Rewarming: The section was rewarmed for 1 h in an oven at 37°C.

8) Washing: The section was washed 10 times with PBS, with 2 min for each time.

9) Secondary antibody binding: The section was added with pre-diluted corresponding secondary antibody at an appropriate concentration, and incubated for 1 h at 37°C.

10) Washing: The section was washed 10 times with PBS, with 2 min for each time.

11) SABC addition: SABC was added dropwise on the section, and then the section was incubated at 37°C for 30 min.

12) Washing: The section was washed 5 times with PBS, with 2 min for each time.

13) Color development: A prepared DAB chromogenic solution was added dropwise on the section, then the section was placed at room temperature, and a reaction time (usually 1 min to 5 min) was observed under a microscope. When the optimal effect was achieved, the section was rinsed with PBS to stop the color development.

15) Staining: The section was counter-stained in a hematoxylin stain for 1 min, and then thoroughly rinsed with PBS.

16) Dehydration: The section was soaked in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, absolute ethanol I, and absolute ethanol II successively, with 1 min in each.

17) Transparency: The section was soaked in xylene I and xylene II successively, with 5 min in each.

18) Mounting: The section was mounted with neutral gum, and then baked overnight in an oven at 37°C. Stained liver tissue sections were observed, analyzed, and photographed under an inverted microscope.

As shown in FIG. 9, it can be seen from A that hepatocytes of mice in the normal group were in regular strips, with clear hepatic sinusoids and no inflammatory cell infiltration, and the hepatocytes were normal in size and morphology, without fatty or ballooning degeneration; on day 1 after the operation, for mice in the treatment group and the control group, the hepatic cord structure disappeared, hepatocytes were arranged disorderly and had different sizes and -7500561 unclear morphologies, and there was inflammatory cell infiltration and a lot of fatty vacuoles, where there were more hepatic fatty vacuoles in the treatment group; and on day 4 after the operation, for mice in the control group, the nuclear staining of hepatocytes increased, hepatic sinusoids were enlarged, there was still inflammatory cell infiltration, and there was hepatocyte necrosis in some areas; for mice in the treatment group, the morphology and size of hepatocytes basically returned to normal, and there was clear morphology, no inflammatory cell infiltration, and no fatty or ballooning degeneration. It can be seen from B that, after 70% liver resection, on day 1, the proliferation of mice in the control group was significantly stronger than that in the treatment group; and on day 4, both the control group and the treatment group showed a proliferation state, but the regeneration in the treatment group was significantly higher than that in the control group, indicating vigorous proliferation.

In summary, it indicates that organoid transplantation can treat ALF by reducing liver inflammatory responses and promoting liver regeneration.

The above are merely preferred implementations of the present disclosure. It should be noted that various modifications and variations can be made by those of ordinary skill in the art without departing from the conception of the present disclosure and the modifications and variations are within the protection scope of the present disclosure.

Claims (8)

CLAIMS LU500561

1. An in vitro construction method of liver organoids, comprising the following steps: sl: preparation of a tissue suspension: aseptically collecting a liver from a normal mouse, and rinsing and chopping the liver to obtain the tissue suspension; s2: preparation of a cell suspension: adding a liver tissue digestion liquid to the tissue suspension prepared in sl, and transferring a resulting mixture to a water bath for static digestion; after the digestion, mechanically pipetting the mixture up and down, and allowing cell sedimentation; and collecting a resulting supernatant to obtain the cell suspension; s3: cell plating: subjecting the cell suspension obtained in s2 to filtration, centrifugation, and lysis to obtain a cell precipitate; resuspending the cell precipitate with matrigel, and inoculating a resulting cell suspension in central positions of a 24-well plate; and after the matrigel is fixed, adherently adding a liver organoid induction medium for cultivation to obtain initial organoids; and s4: passaging the initial organoids obtained in s3, and selecting organoids of p3 to p5 as final liver organoids.

2. The in vitro construction method of liver organoids according to claim 1, wherein in sl, the liver is first rinsed in 30 mL of a pre-cooled DMEM/F-12 medium to remove residual blood, and then chopped into pieces with a particle size of 1 mm in another 30 mL of a DMEM/F-12 medium.

3. The in vitro construction method of liver organoids according to claim 1, wherein s2 specifically comprises: transferring the tissue suspension with a pipette to a 50 mL centrifuge tube at room temperature, allowing the tissue suspension to stand for 1 min, and removing a resulting supernatant; adding 10 mL of the liver tissue digestion liquid, transferring the centrifuge tube to a 37°C water bath to allow static digestion for 20 min, mechanically pipetting a resulting mixture up and down, and allowing the resulting mixture to stand for 1 min; after cells settle at a bottom of the centrifuge tube, removing a resulting supernatant, and continuing to add 10 mL of the liver tissue digestion liquid; transferring the centrifuge tube to a 37°C water bath to allow static digestion for 20 min, mechanically pipetting a resulting mixture up and down, and allowing the resulting mixture to stand for 1 min; after cells settle at the bottom of the centrifuge tube, collecting a resulting supernatant in a pre-cooled 50 mL centrifuge tube; and repeating the above digestion and collection steps 4 times to collect 50 -7500561 mL of a cell supernatant as the cell suspension.

4. The in vitro construction method of liver organoids according to claim 1 or 3, wherein the liver tissue digestion liquid is prepared specifically as follows: adding 7.5 mL of a neutral protease and 7.5 mL of collagenase type IV to every 45 mL of a DMEM/F12 medium.

5. The in vitro construction method of liver organoids according to claim 1, wherein s3 specifically comprises: s31: filtering the collected cell suspension with a 100 um cell strainer to obtain a filtrate in which cells have a diameter of less than 100 um; filtering the filtrate once again with a 30 pm cell strainer, and discarding a resulting filtrate; and placing the 30 um cell strainer upside down on a 50 mL centrifuge tube, and rinsing the cell strainer with a DMEM/F-12 medium to collect cells trapped on the 30 um cell strainer; s32: centrifuging a resulting cell suspension for 5 min at 300 g and 4°C, and discarding a resulting supernatant; $33: adding a red blood cell (RBC) lysing buffer at a volume 3 to 5 times of a volume of cells, gently pipetting a resulting mixture up and down, and conducting lysis for 4 min to 5 min; and centrifuging a resulting lysate at 4°C and 100 g for 5 min, and discarding a resulting supernatant to obtain a cell precipitate; s34: resuspending the cell precipitate with 240 uL of the matrigel, pipetting a resulting mixture up and down for thorough dispersion (wherein air bubbles are avoided), and inoculating a resulting cell suspension in central 8 wells of the 24-well plate; and s35: allowing the plate to stand in a 37°C cell incubator for 10 min; after the matrigel is fixed, adherently adding 750 pL of the liver organoid induction medium; and replacing the liver organoid induction medium every 2 d to 3 d according to cultivation conditions to obtain the initial organoids.

6. The in vitro construction method of liver organoids according to claim 1, wherein s4 specifically comprises: s41: removing the liver organoid induction medium, gently rinsing the initial organoids with PBS, and removing PBS; and adding the DMEM/12 medium to allow static digestion for 1 min, and mechanically pipetting a resulting mixture up and down;

s42: centrifuging the resulting mixture for 5 min at 300 g and 4°C, and discarding a -7500561 resulting supernatant; s43: using a pre-cooled pipette tip to resuspend a resulting cell precipitate with 240 pL of matrigel, pipetting a resulting mixture up and down for thorough dispersion (wherein air bubbles are avoided), and inoculating a resulting cell suspension in central 8 wells of a 24-well plate; and s44: allowing the plate to stand in a 37°C cell incubator for 10 min; and after the matrigel is fixed, adherently adding 750 pL of the liver organoid induction medium, wherein passage is conducted at a ratio of 1:4.

7. The in vitro construction method of liver organoids according to any one of claims 1, 5, and 6, wherein the liver organoid induction medium is prepared specifically as follows: mixing a liver organoid basal medium with a liver organoid basal medium additive at a volume ratio of 10:1, adding an anti-mycoplasma reagent, thoroughly mixing and dispensing a resulting mixture, and storing a resulting medium at -20°C.

8. Use of liver organoids prepared by the method according to claim 1 in the preparation of a drug for treating acute liver failure (ALF).