CN111500528A

CN111500528A - Method for elutriating and amplification culture of liver stem cells and application thereof

Info

Publication number: CN111500528A
Application number: CN201910098063.8A
Authority: CN
Inventors: 李尹雄; 伍飞马; 陈彦; 谭圣林; 潘廷才
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2020-08-07
Also published as: WO2020155909A1

Abstract

The invention provides a culture medium for elutriating and amplifying liver stem cells and application thereof. Based on the stem cell clone forming ability, the chemical micromolecules and the cytokines in the elutriation culture medium are optimally combined, the flow sorting of cell surface antigen markers is skipped, and the culture medium with clear components is developed. The optimized culture medium can be directly used for panning, separating and amplifying adult liver stem cells with liver/gallbladder bidirectional differentiation potential in adult livers, and has wide application prospect and huge market value.

Description

Method for elutriating and amplification culture of liver stem cells and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a culture medium for elutriating and amplifying liver stem cells and application thereof.

Background

China is a big country with liver diseases, and various acute and chronic liver diseases form great threats to the health of people in China. Different physical, chemical and biological factors including viral infection, alcoholic and non-alcoholic excessive diet, poisoning, liver injury and repeated stimulation of inflammation caused by autoimmunity and bile stasis and the like not only lead to death of parenchymal liver cells, but also induce fibrosis of liver interstitial tissues, and further develop into end-stage liver diseases such as liver cirrhosis, liver failure, even liver cancer and the like.

The only current option for effective treatment of end-stage liver disease is liver organ transplantation, which is limited by an extreme shortage of donor liver sources. Of hundreds of thousands of people waiting for liver transplantation, less than ten thousand people in a year have a fortunate chance to obtain liver sources. Proliferation can be achieved in the host after transplantation of mature hepatocytes. Thus, for the treatment of end-stage liver disease, especially acute liver failure, hepatocyte transplantation is considered as an alternative therapy to liver organ transplantation. However, the application of this method is also limited by the shortage of organ donations, high transplantation cost, difficulty in ex vivo expansion of obtained parenchymal hepatocytes, immune response after transplantation, and the like. Meanwhile, studies in animal models of liver injury have found that even though mature hepatocytes briefly expand and survive in the host liver, they still cause bile leakage and persistent liver tissue damage due to the inability to form neobile ducts.

At present, mesenchymal stem cells or hematopoietic stem cells are mostly used in the clinical treatment research of the end-stage liver diseases. Although these cells can improve part of the liver function of a patient in a short period of time, it remains a question of whether liver tissue can be substantially regenerated. Compared with liver organs or mature liver parenchymal cells, obtaining adult liver stem cells with bidirectional differentiation potential of liver parenchymal cells and bile duct epithelial cells becomes an internal hotspot and one of the hopes of cell transplantation treatment of end-stage liver diseases. Although not yet entered clinical studies, the potential of allograft liver stem cells for the treatment of advanced liver disease has been successfully validated several times in different laboratories, in different animal models of liver injury.

CN104818245A discloses a culture medium and a culture method of liver stem cells, the culture medium and the culture method adopt immunomagnetic beads to carry out flow sorting on liver stem cells and culture the liver stem cells in a liver stem cell culture medium containing a basal medium, HGF, SCF and L IF, according to the disclosed content, we cannot judge whether the method provided by the invention can amplify liver stem cells with liver/gallbladder bidirectional differentiation potential and capability of repairing damaged liver in vivo, CN104024401B discloses a culture medium and a method for amplifying and differentiating stem cell groups and obtaining organoids and application thereof in drug screening, toxicity testing and regenerative medicine, the culture medium comprises BMP inhibitors, Wnt agonists, receptor tyrosine kinase ligands, nicotinamide, p38 inhibitors, prostaglandin inhibitors, testosterone signal transduction activators and testosterone signal transduction activators, the culture medium and the activator of liver stem cells can be used for screening liver cells in vitro and forming liver diseases by a simple liver cell screening method based on liver cell surface antigen markers, and the research on liver stem cell differentiation potential and liver cancer, and liver cancer.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a culture medium for panning and amplifying liver stem cells and application thereof, the inventor develops a culture medium with definite components by culture optimization combination of chemical micromolecules and cytokines based on stem cell clone forming capability, skips flow sorting of cell surface antigen markers, and directly pans and amplifies adult liver stem cells with liver/gall bladder bidirectional differentiation potential in adult livers, thereby having wide application prospect and huge market value.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a medium for screening for expanded liver stem cells, the medium comprising a liquid basal medium, cytokines, an adenylate cyclase activator, a Wnt signaling pathway activator, a TGF β signaling pathway inhibitor and a ROCK1 inhibitor.

Preferably, the culture medium further comprises a cell culture supplement.

Aiming at the problems existing in the existing separation and amplification of adult liver stem cells, the inventor carries out design optimization based on stem cell clone forming capability, provides a culture medium with clear components and excellent effect by culture optimization combination of chemical micromolecules and cytokines, and the components are matched with each other to realize synergistic interaction and jointly promote the panning and amplification of the liver stem cells. Meanwhile, the invention can obtain a large amount of epithelioid liver stem cell clones which can be stably amplified in a short time without flow sorting based on cell surface antigen markers. The epithelial-like clone formed by single cell can be selected, cloned and purified, and still can retain the bidirectional differentiation potential of liver/gallbladder after being greatly expanded. More importantly, the expanded liver stem cells can repair damaged liver tissues in vivo.

Preferably, the liquid basal medium comprises any one of DMEM, DMEM/F12, RPMI1640 medium or Advance DMEM/F12 medium or a combination of at least two thereof.

Preferably, the cell culture supplement comprises any one of N2 cell culture supplement, B27 cell culture supplement, glutamine replacement, nicotinamide, or Insulin-Transferrin-sodium selenite mixture (ITS) ITS, or a combination of at least two thereof.

The glutamine substitutes, ITS, N2 and B27 are commercial products of Thermo Scientific.

The nicotinamide is a commercial product of Stem Cell company.

Preferably, the glutamine replacement concentration is 0.1-1%, for example may be 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.8% or 1%.

Preferably, the concentration of nicotinamide is 1-10mM, and may be, for example, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, or 10 mM.

Preferably, the concentration of the insulin-transferrin-sodium selenite mixture is 0.1-1%, for example it may be 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.8% or 1%.

Preferably, the concentration of the N2 cell culture supplement is 0.1-1%, and may be, for example, 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, or 1%.

Preferably, the concentration of the B27 cell culture supplement is 0.1-1%, and may be, for example, 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, or 1%.

Preferably, the adenylate cyclase activator is Forskolin.

Preferably, the concentration of the adenylate cyclase activator is 1 to 50. mu.M, and may be, for example, 1. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M, 35. mu.M, 40. mu.M, 45. mu.M or 50. mu.M.

Preferably, the cytokine includes any one of Epidermal Growth Factor (EGF), Bone morphogenic protein 4 (BMP 4), or Fibroblast growth factor 10(Fibroblast growth factor 10, FGF10), or a combination of at least two thereof.

Preferably, the cytokine concentration is 3-150ng/m L, for example 3ng/m L, 5ng/m L0, 10ng/m L1, 15ng/m L2, 20ng/m L3, 25ng/m L4, 30ng/m L5, 35ng/m L6, 40ng/m L7, 45ng/m L8, 50ng/m L9, 60ng/m L, 70ng/m L0, 80ng/m L1, 90ng/m L, 100ng/m L, 110ng/m L, 120ng/m L, 130ng/m L, 140ng/m L or 150ng/m L.

Preferably, the epidermal growth factor concentration is 1-50ng/m L, such as 1ng/m L, 5ng/m L0, 10ng/m L1, 15ng/m L, 20ng/m L, 25ng/m L, 30ng/m L, 35ng/m L, 40ng/m L, 45ng/m L or 50ng/m L.

Preferably, the concentration of said bone morphogenic protein 4 is 1-50ng/m L, and may for example be 1ng/m L, 5ng/m L0, 10ng/m L1, 15ng/m L, 20ng/m L, 25ng/m L, 30ng/m L, 35ng/m L, 40ng/m L, 45ng/m L or 50ng/m L.

Preferably, the fibroblast growth factor 10 is present at a concentration of 1-50ng/m L, which may be, for example, 1ng/m L, 5ng/m L0, 10ng/m L1, 15ng/m L, 20ng/m L, 25ng/m L, 30ng/m L, 35ng/m L, 40ng/m L, 45ng/m L or 50ng/m L.

Preferably, the Wnt signaling pathway activator comprises glycogen synthase kinase 3 α/β inhibitor CHIR99021 and/or recombinant R-spondin protein.

Preferably, the molar concentration of the glycogen synthase kinase 3 α/β inhibitor CHIR99021 is 1-20. mu.M, which may be, for example, 1. mu.M, 5. mu.M, 10. mu.M, 15. mu.M or 20. mu.M.

Preferably, the mass concentration of the recombinant R-spondin protein is 1-100ng/m L, and can be, for example, 1, 10ng/m L, 20ng/m L0, 30ng/m L, 40ng/m L, 50ng/m L, 60ng/m L, 70ng/m L, 80ng/m L, 90ng/m L or 100ng/m L.

Preferably, the TGF β signalling pathway inhibitor comprises A83-01 and/or Repsox, preferably A83-01.

Preferably, the concentration of RepSOX is 1-5. mu.M, and may be, for example, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, or 5. mu.M. Preferably, the molar concentration of A83-01 is 0.5-10. mu.M, and may be, for example, 0.5. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M or 10. mu.M.

Preferably, the ROCK1 inhibitor comprises Y-27632 and/or Thiazovivin, preferably Y-27632.

Preferably, the concentration of Thiazovivin is 0.5-10. mu.M, and may be, for example, 0.5. mu.M, 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, or 10. mu.M.

Preferably, the molar concentration of Y-27632 is 1-10. mu.M, and may be, for example, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, or 10. mu.M.

Preferably, the medium components include:

the liquid basal medium is RPMI1640, 1% N2, 1% B27, 1% insulin-transferrin-sodium selenite (ITS) mixture, 1% glutaminase substitute, 10mM nicotinamide, 50ng/M L epidermal growth factor, 20ng/M L bone morphogenetic factor-4, 100ng/M L fibroblast growth factor-10, 100ng/M L RSPO1, 3 μ M CHIR99021, 5 μ M A83-1, 10 μ M Y-27632.

In addition, the invention provides a method for separating and stably amplifying liver stem cells of mammals including adult mice and human adults according to the culture medium, and prepares liver stem cells with liver/gallbladder bidirectional differentiation potential in vitro in large quantity.

In a second aspect, the present invention provides a method for panning and expanding liver stem cells, using the culture medium of the first aspect;

preferably, the method comprises the steps of:

(1) pretreating the liver to obtain cell suspension;

(2) removing the liver parenchymal cells from the cell suspension obtained in the step (1) to obtain non-liver parenchymal cells;

(3) resuspending and centrifuging the non-liver parenchymal cells obtained in the step (2) to obtain cell precipitates;

(4) suspending and inoculating the pellet of step (3) with a medium as described in the first aspect;

(5) and (4) selecting the liver stem cell clone selected in the step (4), inoculating the liver stem cell clone into the culture medium of the first aspect, amplifying, digesting and passaging.

Preferably, the pretreatment in the step (1) comprises perfusing the liver by a two-step method, and filtering by a sterilized gauze and a cell sieve;

preferably, the mesh number of the cell sieve comprises any one of 125 mesh, 250 mesh or 320 mesh or a combination of at least two;

preferably, the method for removing the parenchymal hepatocytes in the step (2) is centrifugation and cell sieve filtration;

preferably, the centrifugation in step (2) is performed 1 to 3 times.

Preferably, the centrifugation in the step (2) is carried out under the conditions of 40-60g, 2-6 ℃ and 1-3 min;

preferably, the mesh number of the cell sieve comprises any one of 400 mesh, 500 mesh, 600 mesh, 800 mesh or 1250 mesh or a combination of at least two thereof;

preferably, the resuspended medium in step (3) is high-sugar DMEM containing 1% penicillin-streptomycin;

preferably, the centrifugation in step (3) is performed 1 to 3 times.

Preferably, the centrifugation in the step (3) is performed under the condition of centrifugation at room temperature of 350-500g for 3-7 min;

the culture utensil for inoculation in the step (4) is a cell culture dish coated by 0.5-1% Matrigel;

the step (4) also comprises a step of removing nonadherent cells 18-24 hours after inoculation;

the culture time in the step (5) is 4-7 days.

As a preferred technical scheme, a method for screening and amplifying liver stem cells, which adopts the culture medium of the first aspect, specifically comprises the following steps:

(1) perfusing the liver by a two-step method, filtering by sterile gauze, and filtering by 125-mesh, 250-mesh and 320-mesh cell sieves to obtain cell suspension;

(2) centrifuging the cell suspension obtained in step (1) for 1-3 times at 40-60g, 2-6 deg.C for 1-3min, and filtering with 400, 500, 600, 800 and 1250 mesh cell sieve to remove liver parenchymal cells to obtain non-liver parenchymal cells;

(3) resuspending the non-liver parenchymal cells in the step (2) by using high-glucose DMEM containing 1% penicillin-streptomycin, and centrifuging for 1-3 times at room temperature with the concentration of 350-500g for 3-7min each time to obtain cell precipitates;

(4) suspending and inoculating the precipitate of step (3) with the culture medium according to the first aspect, and removing non-adherent cells 18-24h after inoculation;

(5) and (4) selecting the liver stem cell clone which grows out after being cultured for 4-7 days after being inoculated in the step (4), inoculating the liver stem cell clone into the culture medium of the first aspect, amplifying, digesting and passaging.

The method comprises the following specific steps:

1) perfusing mouse liver/human liver tissue by two-step method to obtain cell suspension;

2) filtering the cell suspension with sterilized gauze to remove undigested tissue and connective tissue;

3) filtering the preliminarily filtered cell suspension through a 125-mesh, 250-mesh and 320-mesh cell sieve respectively;

4) the cell suspension obtained in the step 3 is subjected to low-speed centrifugation twice (50g, 4 ℃, 2min) to precipitate liver parenchymal cells, and the liver parenchymal cells are removed;

5) filtering the cell suspension obtained in the step 4 by using cell sieves of 400 meshes, 500 meshes, 600 meshes, 800 meshes and 1250 meshes in sequence to further remove the parenchymal hepatocytes;

6) resuspending the non-hepatic parenchymal cells obtained in step 5 with high-glucose DMEM containing 1% penicillin-streptomycin, and centrifuging at 450g for 5min at room temperature;

7) repeating the step 6;

8) directly suspending the cell sediment obtained in the step 7 by using a liver stem cell screening and amplification culture medium and inoculating the cell sediment into a cell culture dish coated by 1% Matrigel;

9) after the cells are inoculated for 24 hours, the culture dish is gently shaken, and the nonadherent cells and cell fragments are sucked and discarded, and then the culture dish is washed for 5 times by using sterile phosphate buffer solution;

10) continuously culturing the cells in a 5% carbon dioxide incubator for 4-7 days until liver stem cell clones grow out;

11) cloning the liver stem cells by selecting and inoculating the liver stem cells into a 48-well plate which is coated by Matrigel and contains a liver stem cell screening and amplification culture medium;

12) when cells in the well plates grew to more than 80% confluency, the cells were digested with Accutase and passaged.

The invention provides a method for rapidly and efficiently screening and massively amplifying liver stem cells with liver/gallbladder bidirectional differentiation potential in adult liver parenchymal cells based on stem cell in-vitro cloning forming capability without flow sorting.

In a third aspect, the present invention provides a liver stem cell obtained by screening and amplifying the method according to the first aspect or the culture medium according to the first aspect.

The liver stem cell has the following characteristics:

1) expressing liver stem cell markers at the gene level and protein level;

2) expressing tissue-specific stem cell genes, liver line genes and biliary line genes at the gene level and thus not expressing mature hepatocyte genes;

3) the liver stem cells after continuous passage still keep the clone forming ability and quickly proliferate in vitro;

4) the adult liver stem cells after being expanded and established have the function of being differentiated in vitro into liver-like cells and bile duct-like cells with physiological functions;

5) the liver stem cells after the line establishment is amplified can save the Fah homozygous mutant lethal phenotype and regenerate host liver tissues.

In a fourth aspect, the present invention provides a liver stem cell according to the third aspect for use in screening drugs, studying toxicology, discussing pathogenesis of hepatitis virus, cell therapy of end-stage liver disease, in vitro bioartificial liver support system and personalized therapy, i.e., an adult liver stem cell according to the third aspect is applied to drug screening, toxicology research, investigation of pathogenesis of hepatitis virus, treatment of end-stage liver disease or in vitro bioartificial liver support system before and after hepatic/biliary differentiation.

Compared with the prior art, the invention has the following beneficial effects:

(1) the culture medium provided by the invention can enable adult liver parenchymal cells to form typical epithelioid liver stem cell clone in vitro, meanwhile, the culture medium can also be used for the dryness maintenance and the in vitro large-scale stable amplification of adult liver stem cells, the adult liver stem cells cultured in vitro can differentiate liver-like cells and bile duct cells with physiological functions in vitro, and can regenerate Fah in vivo to knock out liver parenchymal cells of mice;

(2) the invention provides a new, rapid and efficient method for separating and amplifying adult liver stem cells, which provides reliable research materials for cell therapy and drug screening of clinical late-stage liver diseases; the method provided by the invention is based on the characteristic that the stem cells form clone in vitro, does not depend on expensive and precise instruments and antibodies, and directly elutriates adult liver stem cells in non-parenchymal liver cells efficiently and quickly, so that the method has potential of popularization and application; based on the method provided by the invention, an adult liver stem cell bank of different crowds can be established, and a basis is provided for drug screening and personalized treatment of end-stage liver diseases;

(3) the adult liver stem cells obtained by the method can be frozen and recovered repeatedly, and are favorable for storage and transportation of the cells.

Drawings

FIG. 1 is a schematic diagram of screening adult liver stem cells according to the present invention;

FIG. 2 is a diagram showing the result of RT-PCR detection of the liver stem cell-related gene of the present invention;

FIG. 3 is an immunofluorescence identification chart of the established line liver stem cell of the invention, wherein A is an expression chart of a liver stem cell marker Afp and a bile duct marker Krt19, B is an expression chart of Sox9 and EpCAM, C is an expression chart of L gr5, D is an expression chart of a cell fate regulatory factor Numb, and E is an expression chart of a liver nuclear transcription factor Hnf4 α and an epithelial cell marker E-Cadhrin;

FIG. 4(A) is a cell morphology diagram of the established line liver stem cell of the present invention after serial passages;

FIGS. 4(B) -4 (C) are graphs showing the division and proliferation patterns of the liver stem cells of the established line of the present invention;

FIGS. 5(A) -5 (B) are graphs of proliferation curves and doubling times of liver stem cells after serial passages according to the present invention;

FIG. 6(A) is the in vitro hepatic differentiation expression of the established liver stem cell line of the present invention to mature hepatocyte marker protein Alb

(Albumin) and A1AT (Alpha-1-Antitrypsin) results plots;

FIG. 6(B) is a diagram showing the in vitro hepatic differentiation of the established liver stem cells of the present invention to express Alb and ASGR1 (Asiaoglycoprotein)

Receptor 1) result graph;

FIG. 7(A) is a graph showing ICG uptake by the cells after hepatic differentiation according to the present invention;

FIG. 7(B) is a graph of the stored neutral fat of the cells of the invention after hepatic differentiation;

FIG. 7(C) is a photograph of glycographic representation of the cells of the present invention after hepatic differentiation;

FIG. 7(D) is a graph of the hepatic differentiated cell transport ac-L D L of the present invention;

FIG. 8(A) is a graph showing the secretion of Alb by the hepatic differentiated cells of the present invention;

FIG. 8(B) is a graph of cellular metabolism of Midazolam (Midazolam) following hepatic differentiation in accordance with the present invention;

FIG. 8(C) is a graph of Diclofenac sodium (Diclofenac) metabolism by the cells after hepatic differentiation according to the present invention;

FIGS. 9(A) -9 (B) and 10(A) -10 (C) are graphs for identifying the in vitro biliary differentiation ability of the established liver stem cells of the present invention;

FIGS. 11(A) -11 (B) are graphs of the established line of liver stem cells of the present invention at Fah^-/-A liver proliferation condition graph of a chronic liver injury mouse;

FIG. 12 is a graph showing the efficiency of regenerating parenchymal hepatocytes in vivo by using the liver stem cells established by the present invention.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.

The experimental methods in the examples are all conventional and commonly used experimental methods unless otherwise specified.

In the examples, the relevant articles such as the experimental consumables and the reagents are all commercial products unless otherwise specified.

EXAMPLE 1 preparation of culture Medium

The culture medium comprises the following components:

the liquid basal medium is RPMI1640, 1% N2, 1% B27, 1% insulin-transferrin-sodium selenite (ITS) mixture, 1% glutaminase substitute, 10mM nicotinamide, 50ng/M L epidermal growth factor, 20ng/M L bone morphogenetic factor-4, 100ng/M L fibroblast growth factor-10, 100ng/M L RSPO1, 3 μ M CHIR99021, 5 μ M A83-01, 10 μ M Y-27632.

Example 2 screening of liver Stem cells

1.1 two-step perfusion of mouse liver

1) Pretreatment of the constant flow pump: the pipeline of the constant flow pump is perfused once by absolute ethyl alcohol, then washed once by high-pressure sterilization deionized water, and finally washed once by D-Hanks for standby;

2) dissecting experimental mice, namely, after anesthetizing the mice by intraperitoneal injection of 20 mu L avermectin per gram of body weight, fixing the mice on a dissecting plate, cutting the chest cavity of the mice, finding out the inferior vena cava, inserting a disposable indwelling needle into the inferior vena cava and fixing the indwelling needle by a surgical suture line, and injecting 3m L anticoagulant into the livers of the mice through the indwelling needle;

3) the mouse liver perfusion method comprises the steps of firstly adjusting the rotating speed of a constant flow pump to 5rpm/min, then starting perfusion with D-Hanks, cutting off a portal vein, ligating a superior vena cava, timing for 15min, adjusting the rotating speed of the constant flow pump to 20rpm/min, after D-Hanks perfusion, changing the perfusion liquid into collagenase perfusion liquid (50m L) preheated in water bath at 37 ℃, and adjusting the rotating speed of the constant flow pump to 10 rpm/min.

1.2 preparation of mouse liver nonparenchymal cells.

1) Preparation of mouse liver cell suspension: after collagenase perfusion is finished, the liver is carefully taken out and washed once by high-sugar DMEM containing 1% double antibody;

2) the liver is transferred to a 10cm cell culture plate containing 20m L precooled high-sugar DMEM and transferred to a superclean workbench, and the capsule on the surface of the liver is torn by forceps and continuously stirred until the tissue mass disappears;

3) respectively sieving the cell suspension obtained in the step 2) through a cell sieve of 120 meshes, 250 meshes and 320 meshes to remove undigested tissue blocks and connective tissues;

4) transferring the obtained cell suspension into a 50m L centrifuge tube, centrifuging for 2min at 50g and 4 ℃;

5) collecting the supernatant into a new 50m L centrifuge tube, resuspending the cell pellet with 20m L high-glucose DMEM, and repeating step (4) for 2 times;

6) respectively sieving the cell supernatant suspension obtained in the step 5) through 400 meshes, 500 meshes, 600 meshes, 800 meshes and 1250 meshes of cell sieves;

7) collecting the cell filtrate to a new sterile 50m L centrifuge tube, centrifuging 450g at 4 deg.C for 5 min;

8) the supernatant was aspirated, the cell pellet was resuspended in 20m L high-glucose DMEM and step 7) was repeated twice;

9) the supernatant was aspirated off, and the cell pellet was resuspended in an appropriate amount of high-glucose DMEM and used for subsequent screening of liver stem cells.

1.3 panning liver Stem cells from non-liver parenchymal cells

1) Dividing the obtained non-liver parenchymal cell suspension into a plurality of equal parts, 450g, and centrifuging for 5min at normal temperature;

2) the supernatant was aspirated and the cell pellet was resuspended in panning medium separately;

3) seeding the resuspended non-hepatocyte parenchymal cells in a 1% Matrigel coated well plate;

4) transfer plates to 5% CO₂Culturing in an incubator for 24 hours;

5) gently shaking the plate and discarding the non-adherent cells and cell debris;

6) washing the pore plate with PBS until all non-adherent cells and cell fragments disappear;

7) replacing the fresh elutriation culture medium, wherein the culture medium is replaced once a day;

8) the cells were further cultured in the incubator for 3-6 days.

A schematic of adult liver stem cell panning is shown in FIG. 1.

Example 3 establishment and expansion of liver Stem cells

1. Experimental methods

1.1 establishment of liver Stem cells

1) Culturing the non-parenchymal liver cells in a panning culture medium for 4-7 days;

2) gently scraping the epithelial clones with uniform cell morphology and cell number more than 50 by using a self-made elbow glass needle, and transferring the epithelial clones to a 48-hole cell culture pore plate coated by 1% Matrigel for culture for 3 days;

3) the fresh panning medium containing 1-fold double antibody was changed, after which the medium was changed every two days;

4) when cells grew to 80% confluence, they were passaged sequentially at a 1:2 ratio to 24-, 12-and 6-well plates.

1.2 identification of division mode of established liver Stem cells (PHK26 staining)

1) Cells were digested into single cells with accutase (sigma);

2) resuspending the fine cells in appropriate medium, counting on a hemocytometer, and collecting 5 × 10⁵The cells are cultured in a medium such as water,

300g, centrifuging for 5min at normal temperature, and removing supernatant as much as possible;

3) cells 2 × cell suspension (25. mu. L) was prepared using Diluent C in the kit;

4)2 × staining solution was prepared with Diluent C (0.2. mu. L dye was added to 25. mu. L Diluent C);

5) quickly adding 2 × cell suspension into 2 × staining solution, gently blowing and beating uniformly, and incubating at room temperature for 5min

6) Adding 50 mu L1% BSA, mixing well, and incubating for 1min at room temperature to stop the reaction;

7)300g, centrifuging for 5min at room temperature;

8) the cells were resuspended in 1m L medium, 300g, centrifuged at room temperature for 3 min;

9) repeat step 8) twice, cells were resuspended with 2m L medium and seeded in 1% Matrigel coated well plates;

10) after 24h, 48h and 72h of cell inoculation, the distribution of the PHK26 dye in the clones was observed under a fluorescence microscope.

1.3 drawing of proliferation Curve of liver Stem cells of established line

1) Selecting a single liver stem cell line from non-hepatocyte sources, and continuously expanding and culturing the liver stem cell line in vitro to P10, P20 and P30 (the clone grows to more than 80% and is subjected to passage 1:4 when fused);

2) the cells of different generations were seeded in the wells of a six-well plate (1 × 10)⁵Per well) for six days;

3) taking cells in three multiple wells every 24h, digesting the cells into single cells by using Accutase, and counting the single cells by using a cell counter;

4) cell doubling time was calculated using the web page calculation tool http:// www.doubling-time. com/computer. php. proliferation curves were plotted using GraphPad Prism 5.0.

Example 4 Gene expression of established lines of liver Stem cells and detection of markers of liver Stem cells

1. Experimental methods

1.1RT-PCR detection of the gene expression of the established liver stem cells, the result is shown in figure 2;

1) extracting total RNA of cells, namely fusing 80% of liver stem cells of a construction line in a six-hole plate, cracking the cells for 5min at room temperature by using 1m L Trizol, adding 0.2m L chloroform into 1m L Trizol cell lysate, violently shaking for 15s, standing for 3min at room temperature, centrifuging for 15min at 12000rpm at 4 ℃, taking an upper aqueous phase solution into a new centrifuge tube, adding 0.5m L isopropanol, reversing, uniformly mixing, standing for 10min at room temperature, centrifuging for 15min at 12000g at 4 ℃, discarding supernatant, re-suspending precipitates by using 75% precooled ethanol, centrifuging for 5min at 4 ℃, discarding supernatant, drying the precipitates for 3-5min at room temperature, dissolving the precipitates by using RNA-free water, and transferring the precipitates to an ultra-low temperature refrigerator at-80 ℃ for later use.

2) Synthesis of cDNA by reverse transcription (25. mu. L system, 2. mu.g RNA):

RNA was diluted to 500 ng/. mu. L, and a reaction solution (M1) was prepared as shown in Table 1 below;

TABLE 1

Mixing the reaction solution M1, centrifuging slightly, denaturing in 80 deg.C water bath for 5min, and then freezing for 2 min; the reaction mixture (M2) was prepared as in Table 2 below;

TABLE 2

Mixing M1 reaction solution and M2 reaction solution, centrifuging slightly at low speed, transferring to 42 deg.C water bath, incubating for 1h, and then incubating at 99 deg.C for 5 min; cooling on ice, and storing the product at-20 ℃ for later use.

3) PCR detection of gene expression of the established liver stem cells:

preparing PCR reaction solution according to the following table 3;

TABLE 3

TABLE 4 RT-PCR primer Table

The reaction solution is placed in a PCR instrument, reaction conditions are set, wherein the reaction conditions comprise 95 ℃ for 3min, 94 ℃ for 30s, 58-60 ℃ for 30s (the annealing temperature is set according to a primer Tm value), 72 ℃ for 40s, the steps 2-4 are repeated for 30 cycles, the temperature is kept at 72 ℃ for 10min and 16 ℃, 10 mu L PCR products are taken and mixed with 2 mu L ×L applying buffer (containing 1 SYBR green) and are spotted in a hole of 2% agarose gel (prepared by 1 × TAE buffer), 110V electrophoresis is carried out for 30min, the gel is photographed in a micro gel imager, and the result is shown in figure 2, and the result shows that the established liver stem cell line expresses liver stem cell genes Afp, L gr5, Sox9 and Ephnf 1 α, Hnf4 α, Krt 6338, Krt18 and tTr at the mRNA level, biliary gene Hnf6, Krt 2 and Cyt 19, mature liver gene CAM, liver gene gp 638, liver gene 638 and Taa 638.

1.2 immunofluorescence detection of liver stem cell markers.

1) The liver stem cells of the 10 th generation are inoculated in an eight-hole cell culture dish (Thermo) according to 8000 cells/hole;

2) cells were fixed with 4% paraformaldehyde for 15min at room temperature, followed by cell temperature block-through in block-through fluid (PBS with 10% FBS and 0.2% Triton X-100) for 1 h;

3) washing the cells with PBS for 3 times, 15min each time;

4) the antibody was diluted to appropriate concentration with blocking solution (10% FBS in PBS) and subsequently incubated with the cells overnight at 4 ℃;

5) the primary antibody was discarded by aspiration, and the cells were washed with PBS 3 times, 15min each time;

6) the secondary antibody was added with blocking solution in a ratio of 1: 500, diluting, then adding the diluted solution into cells, and transferring the pore plate into an aluminum lunch box to incubate for 1h in a dark place;

7) the secondary antibody was aspirated, and the cells were washed three times with PBS, 20min each time;

8) nuclei were stained with DAPI for 3min, followed by washing 3 times with PBS for 5min each;

9) the results of the images taken by the microscope and the confocal microscope show in FIG. 3 show that the established liver stem cell lines express the liver stem cell marker Afp (FIG. 3A), Sox9 (FIG. 3B), EpCAM (FIG. 3B) and L gr5 (FIG. 3C), the cell fate regulator Numb (FIG. 3D), the bile duct marker Krt19 (FIG. 3A), the hepatonuclear transcription factor Hnf4 α (FIG. 3E) and the epithelial cell marker E-Cadherin (FIG. 3E).

FIG. 4 is the cell morphology and division proliferation mode identification of the established line liver stem cells after continuous passage; the results showed that the established liver stem cells still have clonogenic capacity after serial passage in vitro (FIG. 4(A)) and proliferate in a symmetrically dividing manner (FIG. 4(B) and FIG. 4 (C)). Meanwhile, the proliferation curve of the liver stem cells after serial passage still shows the typical 'S' -type stem cell proliferation curve (fig. 5(a)) and the doubling time of the cells also remains stable (fig. 5 (B)).

Example 5 identification of in vitro hepatic/biliary bidirectional differentiation Capacity of established line hepatic Stem cells

1. Experimental methods

1.1 identification of hepatic differentiation ability of established line liver stem cells.

1) Hepatic differentiation: cells were grown to 80% confluency, cells were washed twice with PBS, expansion medium was replaced with Hepatic Differentiation Medium (HDM) (HCM + 1% ITS +20ng/M L OSM +20ng/M L HGF +10ng/M L EGF +10ng/M L bFGF + 1. mu.M Dexamethasone + 0.5. mu. M A83.01) for 7 days, and hepatic differentiation was initiated on day eight with 20% Matrigel added to the hepatic differentiation medium and continued for 3 days.

2) Detecting mature hepatocyte gene expression by immunofluorescence: the experimental method is shown in immunofluorescence detection of liver stem cell markers.

3) Indolizine green (ICG) absorption experiment, wherein after the liver is differentiated for 10 days, Matrigel covered on cells is sucked and discarded, and the cells are washed twice by HDM;

the ICG was aspirated and the cells were washed 3 times with HDM; and (5) performing microscopic examination and photographing.

4) Dyeing with oil red O: after 10 days of hepatic differentiation, Matrigel coated on cells was aspirated away and washed twice with PBS; fixing the cells with 4% paraformaldehyde at room temperature for 15 min; the cells were washed twice with PBS for 5min each time; pretreating cells with 60% isopropanol for 2 min; incubating the cells with 0.2% oil red O (in 60% isopropanol) for 10-60min at room temperature, with occasional observation to terminate the reaction when lipid droplets are clearly visible; cells were nucleated with hematoxylin for 1min, followed by 3min bluing in PBS; washing the cells with distilled water until no background color is formed; and (5) performing microscopic examination and photographing.

5) Periodic acid-Schiff's Staining (Polyscience) (glycogen Staining): after 10 days of hepatic differentiation, Matrigel coated on cells was aspirated away and washed twice with PBS; fixing the cells with 4% paraformaldehyde at room temperature for 15min, and washing with deionized water for 3 times; balancing each component of the kit to room temperature; incubating the cells with a Periodic Acid on a horizontal shaker at room temperature for 5 min; washing cells with deionized water for 3 times; cells were incubated with Schiff's reagent on a horizontal shaker for 15min at room temperature; the cells were incubated with the potassiumtetablistate for one minute at room temperature and this step was repeated once; cells were washed slowly on a water tap for 10min to facilitate color generation.

6)Alexa

488-ac-L D L (Invitrogen) transport experiment (acetylation L D L transport) hepatic differentiation of liver stem cells was performed for 10 days, then Matrigel coated on the cells was aspirated and washed twice with HDM, Alexa

488-ac-L D L was diluted to 10. mu.g/m L with HDM and then incubated with cells in an incubator for 4h, cells were washed twice with PBS, cells were fixed with 4% paraformaldehyde at room temperature for 15min, cells were washed twice with PBS for 5min each, nuclei were stained with DAPI for 3min and PBS for 5min each, and cells were photographed under an inverted fluorescence microscope.

7) Extraction of total cell protein, namely, after hepatic differentiation of liver stem cells for 10 days, sucking and discarding Matrigel covered on the cells, washing the cells twice by PBS, adding a proper amount of RIPA lysate (containing 1 × Cocktail and 1 × PSFM) into the cells, cracking the cells on ice for 30min, gently scraping the cells by a cell scraper, transferring the cells into a 1.5m L centrifuge tube, centrifuging the cells at 4 ℃ and 12000rpm for 15min, collecting supernatant, and storing the supernatant at-80 ℃ for later use.

8) Quantification of total cellular protein (Thermo):

taking a proper amount of enzyme connecting strips, and adding related reagents according to the following table 5;

TABLE 5

Numbering	1	2	3	4	5	6	7	8
									Standard substance (mu L)	0	1	2	4	8	12	16	20
ddH₂O(μL)	20	19	18	16	12	8	4	0
									Protein content	0	1	2	4	8	12	16	20

ddH for sample to be tested₂Diluting by 20 times, adding 20 mu L into a hole of an enzyme-linked strip, preparing a proper amount of BCA working solution according to the proportion of 50:1 of solution A and solution B in the BCA kit, fully and uniformly mixing, adding 200 mu L BCA working solution into each hole, oscillating and uniformly mixing for 30s, incubating for 30min at 37 ℃, detecting the absorbance at 562nm wavelength by using a microplate reader with the hole No. 1 of a standard product as a reference, drawing a standard curve, and obtaining the protein concentration according to the absorbance and dilution times of a sample to be detected.

9) The E L ISA kit (Abcam) detects the albumin content in the medium:

after liver stem cell liver differentiating for 10 days, gently absorbing Matrigel covered on the liver stem cell liver, washing twice by using HDM, adding fresh HDM into the cell and incubating for 24h in a cell incubator, collecting cell supernatant, 3000g, centrifuging at normal temperature for 10min to remove cell debris, simultaneously extracting total protein from the cell and quantifying, preparing all reagents, a sample to be tested and a standard according to the kit specification, adding 50 mu L sample to be tested or standard into each enzyme-linked well, incubating for 2h at room temperature, washing the plate for 5 times by using Wash buffer, adding 50 mu L biotin coupling antibody into each well, incubating for 1h at room temperature, washing the plate for 5 times by using Wash buffer, adding 50 mu L Streptavidin-Peroxidase Conjugate into each well, incubating for 30min at room temperature, washing the plate for 5 times by using Wash buffer, adding 50 mu L chromosome Conjugate into each well, incubating for 15min at room temperature, adding 50 mu L p into each well, reading the absorbance curve of the albumin in an enzyme-analyzer according to the log curve of the absorbance curve defined by the standard curve of albumin/log of the absorbance curve of the sample to be tested and the log/log of albumin.

10) Testing the drug metabolism capability of the liver-like cells:

culturing liver stem cells in HDM for 10 days, adding Midazolam and Diclofenac into the cells respectively, incubating for 24h, collecting cell supernatant, and detecting metabolites α -hydroxymetazazolam and Diclofenac metabolite 4' -Hydroxydiclofenac of Midazolam respectively by mass spectrometry.

Fig. 6(a) -fig. 6(B), fig. 7(a) -fig. 7(D), fig. 8(a) -fig. 8(C) are the identification of the in vitro hepatic differentiation capacity of the established line liver stem cells, experimental results show that the established line liver stem cells express mature hepatocyte marker proteins Alb (albumin), A1AT (Alpha-1-Antitrypsin) (fig. 6(a)) and ASGR1 (asialoglycoprotien receptor 1) (fig. 6(B)) after hepatic differentiation, physiological function tests show that the cells after hepatic differentiation can take up ICG (indocyninegren, ICG, indocyanine green) (fig. 7(a)), store neutral fat (fig. 7(B)), store glycogen (fig. 7(C)) and transport-L D L (fig. 7(D)), finally we also show that the cells after hepatic differentiation can secrete Alb (fig. 8(a)), metabolize reach (milnaclam) (fig. 8 (Diclofenac) and zolasepam (Diclofenac) into the culture medium.

1.2 the identification of the biliary differentiation ability of the established line liver stem cells.

1) Biliary differentiation of liver stem cells:

matrigel (bd bioscience) was thawed on ice, diluted one-fold with pre-cooled bile duct direction differentiation medium (CDM) (DMEM/F121:1(Hyclone) + 10% fbs (invitrogen)) + 1% its (thermo) +20ng/m L hgf (Peprotech) +50ng/m L TNF α (Peprotech)), and then the bottom of the culture plate was covered with it, the well plate was placed in a cell culture chamber for 30min to form a gel, more than 80% of the fused cells were digested into single cells with Accutase, 300g, centrifuged at room temperature for 5min to remove supernatant, the cells were resuspended with DMEM/F121:1, 300g, centrifuged at room temperature for 3min, the supernatant was removed, the cells were resuspended with an appropriate amount, and then seeded on gel plated with 50% Matrigel for 7 days, and the medium was replaced once a day.

2) Performing immunofluorescence detection on expression of bile duct markers:

fixing choledocholistic cells for seven days with 0.25% paraformaldehyde at room temperature for 10 min; the immunofluorescence experiment step is shown in the immunofluorescence detection of the liver stem cell marker.

3) Rhodamine123 uptake assay:

after the liver stem cells are induced to differentiate in the bile duct direction by CDM for 7 days, the differentiation medium is changed into serum-free DMEM/F12 to be continuously cultured for 24 hours; cells were incubated with 100. mu.M Rhodamine123 in DMEM/F12 for 5 min; the cells were washed three times with serum-free DMEM/F12; and (5) performing microscopic examination and photographing.

4) Rhodamine123 secretion inhibition assay:

after the liver stem cells are induced to differentiate in the bile duct direction by CDM for 7 days, the differentiation medium is changed into serum-free DMEM/F12 to be continuously cultured for 24 hours; cells were treated with DMEM/F12 containing 10. mu.M Verapamul (Selleck) for 30 min; the cells were washed three times with serum-free DMEM/F12; cells were incubated with 100. mu.M Rhodamine123 in DMEM/F12 for 5 min; cells were washed three times with serum-free DMEM/F12, examined microscopically and photographed.

FIGS. 9(A) -9 (B) and 10(A) -10 (C) are graphs showing the in vitro biliary differentiation ability of the established liver stem cells. The experimental results showed that liver stem cells can form a loop bile duct structure in 3D medium (fig. 10 (a)). Immunofluorescent staining revealed that bundled F-actin fibers surrounded the lumen of the induced bile duct structure, which was in the same location as it was in the bile duct in vivo (fig. 9(a) and (B), left column). Further validation found that cells in these bile duct structures all expressed the bile duct marker proteins Krt7 (fig. 9(a), middle panel) and Krt19 (fig. 9(B), middle panel). Secretory function tests showed that cholangiocytes in the bile duct structure could absorb Rhodamine123 and secrete it into the lumen of the bile duct (fig. 10(B)) and that this secretion could be inhibited by Verapamil (10 μ M) (fig. 10 (C)).

Example 6 identification of the ability of liver Stem cells to regenerate damaged liver

1. Experimental methods

1.1 transplantation of liver Stem cells

When the liver stem cells grow to 80% fusion, the cells are digested to single cells by Accuvatase, centrifuged, resuspended by PBS, counted by a hemocytometer, and 10 parts of the cells are counted⁶(100 ten thousand) cells/50. mu. L were dispensed and placed on ice for use, Fumarylacetoacetate hydrolase (Fah) knockout mice were anesthetized by intraperitoneal injection of avermectin (1.25g tribromoethanol +2.5m L t-butanol +97.5m L deionized water), 10 cells were placed in a cell transplantation needle⁶Injecting liver stem cells into the spleen, and suturing the wound; the NTBC in the drinking water of the mice was removed to the end of the experimental period.

1.2 identification of liver stem cell embedded host liver.

1) Immunohistochemical detection of liver tissue:

fresh liver tissue was fixed with 4% PFA at 4 ℃ overnight, embedded in paraffin, and sliced to 3-5 μm thick onto a glass slide; slicing, and sequentially dewaxing in 70% ethanol for 3min, 95% ethanol for 3min, anhydrous ethanol for 2min, xylene for 2min, and xylene for 5 min; antigen retrieval: soaking paraffin section in citric acid buffer solution (0.01M), boiling with high fire in microwave oven for 3min, and decocting with low fire for 15 min; soaking the slices in a buffer solution and naturally cooling to room temperature; the sections were washed 3 times with PBS, 5min each time; and (3) sealing: draining, slicing, adding goat serum blocking solution dropwise onto the tissue surface, sealing for 1h, washing with PBS for 2 times, each time for 5 min; the slice is dripped with primary antibody diluted by confining liquid and is kept overnight at 4 ℃ in a wet box; PBST washing 3 times, 10min each time, PBS washing 2 times, 5min each time; dripping HRP coupled secondary antibody into the slices, and incubating for 2h at room temperature; washing the slices with PBST for 3 times (10 min each time) and PBS for 2 times (5 min each time); DAB dyeing is carried out in a dark place, the dyeing condition can be observed in real time under a microscope in the dyeing process, and the dyeing can be stopped when the color of the tissue is changed; PBST washing 3 times, 10min each time, PBS washing 2 times, 5min each time; counterstaining with hematoxylin for 3 min; PBST washing 3 times, 10min each time, PBS washing 2 times, 5min each time; dropping sealing liquid into the tissue, covering a cover glass, preventing bubbles from generating, and finally sealing and fixing by using neutral quick-drying glue; and (6) microscopic examination and photographing.

2) Calculating the liver regeneration efficiency of the primary liver parenchymal cells/liver stem cells:

three sections of 2 parts of liver are respectively taken to carry out Fah immunohistochemical staining, the specific experimental steps are shown in the partial content of immunohistochemical identification of liver tissues, staining information of the sections is captured by a Miaodi digital section system L ite 1.0, and the ratio of optical density of Fah positive cells to the optical density of the whole section is used

And (6) analyzing software.

FIGS. 11(A), 11(B) and 12 show the proliferation of liver and the regeneration efficiency of parenchymal hepatocytes in Fah-/-chronic liver injury mice. Immunohistochemistry and immunofluorescence experiments found that liver stem cells could embed into damaged liver and differentiate into cells with binuclear mature liver parenchyma (fig. 11(B), arrows indicate cells), and the efficiency of liver stem cells in regenerating liver parenchyma cells was comparable to primary liver parenchyma cells (fig. 12).

Statistical analysis all statistical results in the present invention were obtained from the SPSS17.0 software (SPSS Inc., Chicago, I L.) the statistical differences between the two sets of data were obtained from the t-test (unpaired two-labeled Student's t-test). statistically significant differences were labeled as P.ltoreq.0.05,. ltoreq.0.01,. gtP.ltoreq.0.001.

In conclusion, the invention provides a culture medium for screening and amplifying liver stem cells and application thereof, which is based on stem cell clone forming capability, develops a culture medium with definite components by culture optimization combination of chemical molecules and cytokines, skips flow sorting of cell surface antigen markers, directly separates adult liver stem cells from non-liver parenchymal cells, is used for screening and amplifying the liver stem cells, and has wide application prospect and great market value.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangzhou biomedical and health research institute of Chinese academy of sciences

<120> culture medium for elutriating and amplifying liver stem cells and application thereof

<130>2019

<160>34

<170>PatentIn version 3.3

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Claims

1. A culture medium for panning and expanding liver stem cells, the culture medium comprising a liquid basal medium, cytokines, an adenylate cyclase activator, a Wnt signaling pathway activator, a TGF β signaling pathway inhibitor and a ROCK1 inhibitor.

2. The culture medium of claim 1, further comprising a cell culture supplement.

3. The culture medium according to claim 1 or 2, wherein the liquid basal medium comprises any one of DMEM, DMEM/F12, RPMI1640 medium or advanced DMEM/F12 medium or a combination of at least two thereof;

preferably, the cell culture supplement comprises any one of N2 cell culture supplement, B27 cell culture supplement, glutamine substitutes, nicotinamide, or insulin-transferrin-sodium selenite mixture, or a combination of at least two thereof;

preferably, the concentration of said glutamine substitutes is 0.1-1%;

preferably, the concentration of the nicotinamide is 1-10 mM;

preferably, the concentration of the insulin-transferrin-sodium selenite mixture is 0.1-1%;

preferably, the concentration of the N2 cell culture supplement is 0.1-1%;

preferably, the concentration of the B27 cell culture supplement is 0.1-1%.

4. The culture medium according to any one of claims 1 to 3, wherein the adenylate cyclase activator is Forskolin;

preferably, the concentration of the adenylate cyclase activator is 1-50 μ M;

preferably, the cytokine comprises any one of epidermal growth factor, bone morphogenetic protein 4, or fibroblast growth factor 10, or a combination of at least two thereof;

preferably, the concentration of the cytokine is 3-150ng/m L.

5. The culture medium of any one of claims 1-4, wherein the Wnt signaling pathway activator comprises glycogen synthase kinase 3 α/β inhibitor CHIR99021 and/or recombinant R-spondin protein;

preferably, the glycogen synthase kinase 3 α/β inhibitor CHIR99021 is present at a molar concentration of 1-20. mu.M;

preferably, the mass concentration of the recombinant R-spondin protein is 1-100ng/m L;

preferably, the TGF β signalling pathway inhibitor comprises a83-01 and/or RepSOX, preferably a 83-01;

preferably, the concentration of RepSOX is 1-5 μ M;

preferably, the molar concentration of the A83-01 is 0.5-10 mu M;

preferably, the ROCK1 inhibitor comprises Y-27632 and/or Thiazovivin, preferably Y-27632;

preferably, the concentration of Thiazovivin is 0.5-10 μ M;

preferably, the molar concentration of Y-27632 is 1-10. mu.M.

6. A method for panning and expanding liver stem cells, comprising the steps of using the culture medium according to any one of claims 1 to 5;

preferably, the method comprises the steps of:

(1) pretreating the liver to obtain cell suspension;

(4) suspending and inoculating the pellet of step (3) with the medium of any one of claims 1-5;

(5) picking the liver stem cell clone selected in the step (4), inoculating the liver stem cell clone into the culture medium of any one of claims 1-5, amplifying, digesting and passaging.

7. The method according to claim 6, wherein the pretreatment of step (1) comprises perfusing the liver with a two-step process, followed by filtration through sterilized gauze and cell sieve;

preferably, the centrifugation of step (2) is performed 1-3 times;

preferably, the centrifugation of step (3) is performed 1-3 times;

preferably, the culture device inoculated in the step (4) is a cell culture dish coated by 0.5-1% Matrigel;

preferably, step (4) further comprises the step of removing non-adherent cells 18-24h after seeding;

preferably, the culturing period in step (5) is 4 to 7 days.

8. The method according to claim 6 or 7, characterized in that it comprises in particular the steps of:

(2) centrifuging the cell suspension obtained in the step (1) for 1-3 times under the conditions of 40-60g, 2-6 ℃ and 1-3min, and filtering with 400-mesh, 500-mesh, 600-mesh, 800-mesh and 1250-mesh cell sieves to remove parenchymal hepatocytes to obtain non-parenchymal hepatocytes;

(4) suspending and inoculating the pellet of step (3) with a medium according to any one of claims 1-5, and removing non-adherent cells 18-24h after inoculation;

(5) picking the liver stem cell clone which grows out after culturing for 4-7 days after inoculation in the step (4), inoculating into the culture medium of any one of claims 1-5, amplifying, digesting and passaging.

9. A liver stem cell obtained by screening and expanding the method according to any one of claims 6 to 8 or the medium according to any one of claims 1 to 5.

10. A liver stem cell according to claim 9 for use in screening drugs, studying toxicology, investigating the pathogenesis of hepatitis virus, cell therapy of end-stage liver disease, in vitro bioartificial liver support system and personalized therapy.