CN116355836A - Expansion or passage system of biliary tree stem cells and its application - Google Patents

Abstract

The invention provides an expansion or subculture system of biliary tree stem cells and its application. With the system of the present invention, there is no need to introduce exogenous genes into the biliary tree stem cells, and conventional culture can realize cell expansion, and the expansion efficiency is very high, and the obtained biliary tree stem cells can be passaged. The invention can maintain the stemness of tissue stem cells while ensuring a large amount of cell expansion.

Description

Expansion or passage system of biliary tree stem cells and its application

technical field

The invention belongs to the field of regenerative medicine; more specifically, the invention relates to the expansion or passage system of bile duct tree stem cells and its application.

Background technique

Human biliary tree stem cells (hBTSCs) exist in the peribiliary glands of the human biliary tree wall. Semeraro et al. initially determined that EpCAM ⁺ PDX1 ⁺ Sox17 ⁺ is a marker of BTSCs. hBTSCs have the potential to differentiate into hepatocytes, islet cells, and bile duct cells. hBTSCs are heterogeneous, hBTSCs in the common hepatopancreatic bile duct have a higher potential to differentiate into the liver and pancreas than hBTSCs in other parts of the biliary tree. However, hBTSCs isolated from extrahepatic bile ducts (excluding pancreatic duct) have stronger hepatic differentiation ability (expressed in higher expression of Sox17, low or no expression of PDX1), so it is more ideal for liver failure Rescue seeded cell types. Through previous studies, the inventors have proved that transplanted pig or human biliary tree stem cells can repair liver injury and diabetes model mice, and has clinical application prospects. However, the current research on biliary tree stem cells is mainly based on their primary isolation and culture, and the conditions for their expansion and passage have yet to be established.

To achieve in vitro expansion and passage of biliary tree stem cells to obtain a sufficient amount of cells is the basis for cell transplantation to treat ESLDs and diabetes. Establishing a good in vitro expansion system is the key to maintaining cell stemness and normal physiological functions of cells. At present, the culture and expansion of hBTSCs are mainly based on the in vitro expansion of other endoderm organ stem cells (such as gastrointestinal stem cells, liver stem cells, etc.) system. In view of the complicated operation of the co-culture system and the possible contamination of the feeder layer preparation process, the three-dimensional culture method based on hydrogel has gradually become the preferred method for the in vitro expansion of endoderm organ-derived epithelial stem cells.

The current common three-dimensional culture system is mainly based on the Matrigel-based gel embedding method. Matrigel is a matrix component extracted from mouse chondrosarcoma, and its main components include laminin, type IV collagen, nestin, heparan sulfate glycoprotein, growth factors, matrix metalloproteinase (MMP) and so on. Huch M et al. used the Matrigel base glue system to culture and clone hepatic stem cells into organoids in vitro for several months. However, Matrigel's animal origin and tumor stroma origin lead to its low application safety and cannot be used for the preparation of stem cells for clinical use.

In this field, there have been studies on the application of small molecular compounds or cytokines to some types of stem cell culture, but different types of stem cells have different cell properties and stemness regulation mechanisms, so the nutritional requirements and culture methods for culture maintenance are also very important. different. In particular, there are many types of small molecule compounds, the mechanism of action of small molecule compounds at different concentrations is also different, and there are many types of cytokines, and the use of different cytokines may have synergistic and antagonistic effects, whether it can be used for the cultivation of biliary tree stem cells , amplification and passaging, and how to achieve this goal with rational component and condition optimization, have not yet obtained answers in the art.

Contents of the invention

The purpose of the present invention is to provide an expansion or passage system of biliary tree stem cells and its application.

In the first aspect of the present invention, there is provided a method for culturing biliary tree stem cells (including: primary biliary tree stem cells) in vitro, comprising culturing biliary tree stem cells with a culture medium; wherein, the culture medium includes: basal medium, and small Molecular compounds and growth factors: RG108, A83-01, Forskolin, Bay K8644 and R-Spondin1.

In one or more embodiments, the small molecule compound and growth factor further include one or more selected from the group consisting of Bix01294, SB431542, EGF, Noggin, Wnt3a.

In one or more embodiments, the basal medium is a serum-free medium; preferably, the basal medium is selected from: Kubota's stem cell growth medium (KM medium), Advanced RPMI 1640 medium, AdvancedDMEM/F-12 medium, RPMI1640 medium, DMEM medium, MEM medium or Fischers medium; preferably Kubota's stem cell growth medium or Advanced RPMI 1640 medium; more preferably, the Advanced RPMI1640 medium , Advanced DMEM/F-12 medium, RPMI1640 medium, DMEM medium, MEM medium or Fischers medium were added albumin, nicotinamide, insulin, transferrin, selenic acid, zinc sulfate heptahydrate.

In one or more embodiments, the concentration of the small molecular compound and the growth factor in the basal medium is:

Preferably, it also includes:

In one or more embodiments, albumin 0.1% (w/v), nicotinamide 0.05% (w/v), insulin 5 μg/ml, transferrin 10 μg/ml, selenium Acid 3x10 ^-8 M, zinc sulfate heptahydrate 10 ^-10 M; preferably, the components can fluctuate by 60%, 50%, 40%, 30%, 20%, 10% or 5%.

In one or more embodiments, expansion culture or subculture is carried out by the method.

In one or more embodiments, the culturing is performed in a three-dimensional (3D) system to obtain organoids.

In one or more embodiments, the culturing is performed in a two-dimensional (2D) system to obtain expanded cells.

In one or more embodiments, the culture is carried out at a glycosaminoglycan (polysaccharide, "disaccharide unit" with a degree of polymerization of 10-10,000, such as 50, 100, 200, 500, 1000, 2000, 5000 , 8000) based on the formation of fluid state (glycosaminoglycan concentration 10 ~ 1000ng/ml, such as 100, 300, 500, 700, 900ng/ml) or colloidal state (glycosaminoglycan concentration 10 ~ 500mg/ml, such as 30, 50, 100, 200, 300mg/ml) hydrogel (Glycogel system).

In one or more embodiments, the skeleton matrix of the hydrogel includes (but is not limited to) selected from: Matrigel, hyaluronic acid (HA) and heparan sulfate (HS) hybrid hydrogel (HAHS), Collagen hydrogel, hyaluronic acid hydrogel, silk fibroin hydrogel.

In one or more embodiments, in the mixed hydrogel of hyaluronic acid and heparan sulfate, the final concentration of hyaluronic acid (HA) is 0.01-0.5% (w/v) (preferably 0.02-0.2%) ; more preferably 0.03～0.1%; such as 0.05%, 0.75%, 0.1%), heparan sulfate (HS) final concentration of 10～400ng/ml (preferably 10～100ng/ml; more preferably 30～100ng/ml ml, eg 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml).

In one or more embodiments, the biliary tree stem cells are human biliary tree stem cells and porcine biliary tree stem cells, including primary human biliary tree stem cells and porcine primary biliary tree stem cells.

In another aspect of the present invention, there is provided a medium for culturing biliary tree stem cells in vitro, which includes: basal medium, and small molecular compounds and growth factors: RG108, A83-01, Forskolin, Bay K8644 and R- Spondin1; preferably, the small molecular compound and growth factor also include one or more selected from the group consisting of: Bix01294, SB431542, EGF, Noggin, Wnt3a. Preferably, each component is present in an effective amount.

In another aspect of the present invention, a culture system (Glycogel system) for biliary tree stem cells is provided, which includes: a hydrogel containing a skeleton matrix, and the culture medium.

In one or more embodiments, the matrix matrix includes (but is not limited to) selected from: Matrigel, hyaluronic acid (HA) and heparan sulfate (HS) hybrid hydrogel (HAHS), collagen hydrogel Glue, hyaluronic acid hydrogel, silk fibroin hydrogel.

In another aspect of the present invention, the use of the medium for culturing biliary tree stem cells is provided.

In one or more embodiments, the culturing includes: expanding biliary tree stem cells; subculture the biliary tree stem cells; prepare organoids; or subculture and stably culture the biliary tree stem cell organoids.

In another aspect of the present invention, a kit for culturing biliary tree stem cells in vitro is provided, including: the culture medium; or, the culture system (Glycogel system) for biliary tree stem cells.

In another aspect of the present invention, there is provided the biliary tree stem cell culture obtained by any one of the above methods or the biliary tree stem cell or organoid isolated and purified from the culture.

In one or more embodiments, the biliary tree stem cells or organoids express stemness markers EpCAM, Sox17, PDX1, CK19, Prom1, and ICAM1, but do not express the marker Albumin of mature hepatocytes, and do not express the markers of hepatic progenitor cells. Flag AFP.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. Flow chart of the construction of the Glycogel system and its application in the expansion of biliary tree stem cells and differentiation and maturation into the liver.

Figure 2. Identification of gene expression levels of primary BTSCs cultured in KM. (A) Morphology of primary BTSCs cultured in vitro. (B) Immunocytochemical staining results of primary BTSCs cultured in serum-free conditions. (C) Identification of gene expression levels of primary BTSCs cultured in KM. *<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 no statistical difference.

Figure 3. Gene expression and surface marker expression identification of BTSCs after primary expansion; (A) Schematic diagram of the components of the expansion culture system of BTSCs, including 4 growth factors and 6 small molecule compounds, abbreviated as 10PKM. (B) Morphology of primary BTSCs cultured on day 7 under serum-free KM and 10PKM conditions, respectively. (C) The number of primary BTSCs isolated and cultured from each C57 WT mouse under different conditions (serum-free KMday21, 10PKM day7). The number of cells obtained by 10PKM is about 20 times that of KM. *P<0.05 compared with other groups. (D) Identification of gene expression levels of primary BTSCs cultured at 10PKM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 not statistically different.

Figure 4. Characterization of the P1 generation of BTSCs two-dimensional amplification based on the Glycogel system (Matrigel-based). (A) Morphology of P1BTSCs cultured at 10PKM after plated with Matrigel. (B) The positive ratio of EpCAM+ sorting for BTSCs obtained under P0-KM and P0-10pKM conditions, respectively. (C) Identification of gene expression levels of P1 BTSCs cultured in 10PKM (Matrigel). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 not statistically different.

Figure 5. In vitro expansion of 10PKM P1 BTSCs under HAHS conditions. (A) Morphology of P1 BTSCs cultured at 10PKM in serum-free 0.05% hyaluronic acid (HA) and 0.05% hyaluronic acid (HA) mixed with heparan sulfate (HS) without using Matrigel. (B) Morphology of P1BTSCs cultured at 10PKM under the condition of 0.05% hyaluronic acid (HA) mixed with heparan sulfate (HS) under the microscope of 20X. (C) Statistics of cell number. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 not statistically different.

Figure 6. Three-dimensional expansion of BTSCs organoids based on Glycogel system; (A) Characterization of BTSCs organoids formed under 10PKM conditions after embedding with Matrigel. Scale bar is 100 μm. After mixing the cells with Matrigel at a ratio of 1:1, add 5000 BTSCs per 10 ul dropwise onto the culture plate and place it upside down after solidification, then add the overlay medium). (B) Immunocytochemical staining of organoids of P1 BTSCs under 10PKM conditions. (C) The viability and viability of organoids were judged by life-and-death staining. (D) Gene expression level identification of P1 BTSCs organoids under 10PKM condition. (E) Fold change graph of BTSCs cell number during passaging. (F) Identification of gene expression levels in BTSCs organoids between different passages. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 not statistically different.

Figure 7. The necessity of RG108, A83-01, Forskolin, and Bay K8644 for the in vitro expansion of BTSCs; (A) Mix the cells with Matrigel to form small droplets, which are dropped onto the culture plate in the form of 5000 BTSCs per 10ul Turn it upside down, put it upright after it solidifies, and then cover it with two culture media of 10PKM and 10PKM-A83-01-FOSK respectively. Characterization of BTSCs-org organoids at day2 and day5 under different culture conditions. (B) The number of organoids was counted on day 2 after inoculation. Number of formed BTSCs-org in each drop. (C) The BTSCs cultured in KM on the 14th day were digested, embedded and passaged, and the medium with different conditions was added for screening experiments. According to the pathway of action, growth factors and small molecule compounds were divided into 8 groups for screening. Representative morphology of P1BTSCs organoids under 10PKM and 10PKM-RG108 conditions on day 5. (D) The number of P1 BTSCs-org formed in each drop under 10PKM and different screening conditions were counted on day 5. The number of P1 BTSCs organoids was significantly reduced in the 10PKM-RG108 condition. (E) Identification of gene expression levels of P1 BTSCs organoids under 10PKM and other screening conditions. (F) Morphology of P1 BTSCs under 4PKM and 4PKM+WNT culture conditions after Matrigel plating. And carry out cell counting (choose RG108, A83-01, FOSK, BAY K8644 as 4PKM). (G) The number of formed BTSCs-org in each drop of P1 BTSCs under 4PKM and other conditions after embedding with Matrigel. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 not statistically different.

Figure 8. RSPO1 is an essential component of BTSCs in vitro expansion; (A) Organoid characterization of P1 BTSCs under 4PKM and other conditions after embedding with Matrigel , adding different conditions of media for screening experiments). (B) Identification of gene expression levels of P1 BTSCs organoids under 4PKM and other screening conditions. Through the expression of stemness gene and proliferation gene, 4PKM+RSPO1 was screened to form 5PKM. (C) Morphology of primary BTSCs cultured in 5PKM for different days after plated with Matrigel. (D) KI67 immunocytofluorescent staining of primary BTSCs under KM and 5PKM conditions, respectively. (E) Identification of gene expression levels of primary BTSCs and P1 organoid BTSCs under 5PKM conditions. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 not statistically different.

Detailed ways

After in-depth research, the present inventors revealed for the first time a system for the expansion or passage of Biliary Tree StemCells (BTSCs). With the system of the present invention, there is no need to introduce exogenous genes into the biliary tree stem cells, and conventional culture can realize cell expansion, and the expansion efficiency is very high, and the obtained biliary tree stem cells can be passaged. The invention can maintain the stemness of the bile duct tree stem cell tissue stem cell while ensuring a large amount of cell expansion. Moreover, the method has simple culture conditions, low cost, safety and stability.

As used herein, the term "comprising" or "comprising" includes "comprising", "consisting essentially of (made of)", "consisting essentially of" and "consisting of".

Unless otherwise specified, the culture object in the present invention is biliary tree stem cells, which may be primary biliary tree stem cells, more particularly (primary) biliary tree stem cells derived from humans, pigs and mice.

The first aspect of the specific embodiments of the present invention provides a method for expanding primary biliary tree stem cells in vitro, including the following analysis and optimization process:

(1) Through adherent culture in serum-free medium (KM), the mixture of biliary tree cells obtained from gallbladder bile ducts was screened and purified to obtain clonal-like growth of biliary tree stem cell populations.

(2) Analyze the biliary tree stem cell population/gallbladder bile duct tissue, showing that the obtained biliary tree stem cells express stemness markers EpCAM, Sox17, PDX1, CK19, Prom1, ICAM1; do not express the marker Albumin of mature hepatocytes and the markers of liver progenitor cells Flag AFP.

(3) The ratio of EpCAM+ in primary culture of biliary tree stem cells obtained from gallbladder bile duct tissue is higher than 85%.

(4) Establish pKM components that can be used for the expansion of primary biliary tree stem cells. pKM includes 4 growth factors and 6 small molecule compounds, including: EGF, R-Spindin1, Noggin, Wnt3a, Bix01294, Bay K8644, RG108, SB431542, A83-01, Forskolin; named 10P(pKM).

(5) Through 10P (pKM), biliary tree stem cells with a stable gene and phenotype and stable cell morphology and phenotype were obtained after cell culture for 7 days, which was more than 50 times the number of cells in serum-free medium KM.

(6) By optimizing the pKM components, it is determined that 5P (RG108, A83-01, Forskolin (FOSK), BayK8644, RSPO1) can achieve the same ability to expand primary BTSCs as 10P.

The second aspect of the specific embodiments of the present invention provides a Glycogel system for passage and expansion of biliary tree stem cells in vitro, including the following analysis and optimization process:

(1) Establish a Glycogel system for in vitro expansion of biliary tree stem cells based on Glycogel+pKM.

(2) By screening the components of Glycogel, it was determined that hyaluronic acid hydrogel with defined chemical composition plus heparan sulfate (HAHS) and commercial Matrigel can be used as Glycogel combined with pKM to subculture biliary tree stem cells.

(3) In Glycogel with hyaluronic acid hydrogel plus heparan sulfate (HAHS), heparan sulfate is essential for the cell support effect of this Glycogel. Hyaluronic acid hydrogel alone is insufficient to support the expansion and maintenance of biliary tree stem cells in vitro.

(4) The 3-O sulfate modification of heparan sulfate is the main active structural component of heparan sulfate in Glycogel.

In the third aspect of the specific embodiments of the present invention, a 2D adherent or 3D organoid passage method and expansion system for biliary tree stem cells in vitro are established, including the following analysis and optimization process:

(1) In the two-dimensional culture condition based on the Glycogel system, the digested cells were mixed with the required medium and seeded on the culture plate previously plated with Matrigel at a ratio of 60,000 cells per 12-well plate. Biliary tree stem cells were subcultured 1:2 for 4 days on average, and the cell doubling time was 3.5 days.

(2) In the three-dimensional culture condition based on the Glycogel system, mix and embed the digested cells with Matrigel at a volume of 1:1, and ensure that each 10ul contains 5000 BTSCs. In the orifice of the culture plate (with a certain distance between the droplets), place it upside down in the incubator, take out the culture plate after 2 hours of solidification, add 10PKM medium to cover it, and add Y-27632Rock inhibitor, and then culture The plate is being incubated in the incubator. Biliary tree stem cells will form cystic organoids, and the volume of organoids will gradually increase with the days of culture, with an average of 1:3 passage in 4 days.

(3) In the process of cell subculture, on the one hand, the size and quantity of the organoid clusters are evaluated, and on the other hand, the organoid samples are collected for QPCR to evaluate the gene expression level. In a more specific embodiment, the method for digesting cells in the cell passage includes:

(i) Primary biliary tree stem cells cultured in KM: the day before digestion, add Y-27632Rock inhibitor to the medium, remove the supernatant the next day, add cell digestion solution TrypLe, digest for 15 minutes, and use it every 3 minutes Pipette with a 1 ml pipette (until no adherent cells can be seen on the plate). Add culture medium to stop the digestion, and collect by centrifugation to obtain a single cell pellet.

(ii) Primary biliary tree stem cells cultured in pKM: the day before digestion, add Y-27632Rock inhibitor to the medium, remove the supernatant the next day, add cell digestion solution TrypLe, digest for about 5 minutes, use a 1ml pipette Pipette and mix well, place it under a microscope for observation, and find that the cytoplasm retracts and the intercellular space increases, then add medium to stop digestion, and collect by centrifugation to obtain a single cell pellet.

(iii) Biliary tree stem cells cultured in pKM (3D Matrigel): the day before digestion, add Y-27632Rock inhibitor to the medium, remove the supernatant the next day, add cell digestion solution TrypLe, mechanically pipette to make cystic cells The organ is broken and digested for about 5 minutes. Use a 1ml pipette to blow and mix well, and place it under a microscope for observation. When you see a mixture of small cell clusters (composed of three to five cells) and single cells, add medium to stop digestion , and the cell pellet was collected by centrifugation.

Based on the inventor's above-mentioned new discovery, the present invention discloses a method for expanding biliary tree stem cells in vitro: the biliary tree stem cells are cultured in the biliary tree stem cell medium of the present invention to obtain a large number of expanded cells. The cells express stemness markers EpCAM, Sox17, PDX1, CK19, Prom1, and ICAM1; they do not express the marker Albumin of mature hepatocytes and the marker AFP of hepatic progenitor cells. The method for expanding biliary tree stem cells in vitro includes: cultivating biliary tree stem cells with a basal medium added with small molecule compounds and growth factors; wherein, the small molecule compounds and growth factors include: RG108, A83-01, Forskolin, Bay K8644 and R-Spondin1. In some preferred modes, the small molecular compound and growth factor also include one or more selected from the following: Bix01294, SB431542, EGF, Noggin, Wnt3a.

In a preferred embodiment of the present invention, the concentrations of the small molecule compounds and growth factors are shown in Table 1.

Table 1

Dosage better amount example RG108 0.01～0.2μM 0.02～0.1μM 0.03, 0.04, 0.05, 0.06, 0.08μM A83-01 0.2～4μM 0.5～3μM 0.6, 0.8, 1, 1.5, 2, 2.5, 3.5μM Forskolin 2～40μM 5～30μM 6, 8, 10, 12, 15, 20, 25μM Bay K8644 0.4～8μM 1～6μM 1.2, 1.5, 2, 2.5, 3, 4, 5μM R-Spondin1 20～400ng/ml 50～300ng/ml 60, 80, 100, 150, 200, 250ng/ml Bix01294 0.1～2μM 0.2～1.5μM 0.3, 0.5, 0.6, 0.8, 1, 1.2μM SB431542 0.4～8μM 1～6μM 1.5, 2, 2.5, 3, 4, 5μM EGF 5～100ng/ml 12～70ng/ml 15, 18, 20, 25, 30, 50, 60, 70ng/ml Noggin 20～400ng/ml 50～300ng/ml 60, 80, 100, 150, 200, 250ng/ml Wnt3a 10～200ng/ml 25～150ng/ml 30, 35, 40, 50, 60, 80, 100, 120ng/ml

Using the culture method and culture medium of the present invention, it can be cultivated/passed by two-dimensional or three-dimensional culture system. As a preferred mode of the present invention, the culture/passaging is culture under three-dimensional conditions, so that organoids can be formed.

The biliary tree stem cells obtained by the method of the present invention can be cryopreserved, revived, subcultured and maintained for a long time. In addition, it should also be understood that the biliary tree stem cells (primary biliary tree stem cells) used as the starting strain in the present invention may be the established primary biliary tree stem cells, or may be isolated from organisms.

It should be understood that in addition to the specific small molecule compounds and growth factors listed in the examples of the present invention, other small molecule compounds or growth factors with the same function should also be included in the present invention.

Likewise, analogs, proteins with the same function (such as those of growth factors) or compounds, equivalent compounds that induce the same target, analogs, derivatives and/or their salts, hydrates or Precursors can also be used to replace the above-mentioned specific components to achieve the same technical effect. These analogs, functional proteins or compounds should also be included in the present invention. Analogs of compounds include, but are not limited to: isomers and racemates of compounds. Compounds possess one or more asymmetric centers. These compounds may thus exist as racemic mixtures, individual enantiomers, individual diastereoisomers, diastereomeric mixtures, cis or trans isomers. The "salts" include but are not limited to: (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, butanediol acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine, etc. Other salts include those formed with alkali metals or alkaline earth metals such as sodium, potassium, calcium or magnesium, and the like. The "precursor of the compound" refers to a compound that can be converted into a compound of any of the above-mentioned compounds by the precursor of the compound in the culture medium after being administered or treated by an appropriate method, or a compound of any of the above-mentioned compounds composed of salts or solutions.

As a preferred mode of the present invention, components for preventing bacterial contamination of cell culture, especially Gram-positive and negative bacterial contamination, such as but not limited to penicillin and streptomycin, are added to the medium.

The basal cell culture medium can include: Kubota's stem cell growth medium (KM medium), Advanced RPMI 1640 medium, Advanced DMEM/F-12 medium, RPMI1640 medium, DMEM medium, MEM medium, Neuronal basal medium or Fischers medium, etc. It should be understood that those skilled in the art are familiar with the preparation or purchase methods of the basal cell culture medium, therefore, the basal cell culture medium may not be limited to those exemplified in the present invention.

In a preferred mode of the present invention, a system and method for in vitro expansion of biliary tree stem cells with defined components and non-animal-derived Glycogel polysaccharide glue are also provided. The Glycogel system includes basal medium, growth factor combination, small molecule compound combination, glycosaminoglycan, or consists of these substances. The biliary tree stem cell expansion method provided by the present invention can not only provide a stable 3D microenvironment for the biliary tree stem cells, but also increase the space for cell expansion, reduce the adverse effects of external stimuli on stem cells, and ensure a large number of cell expansion. Maintain the totipotency of pluripotent stem cells/stemness of other stem cells. The invention solves the problems of safety, high cost and difficulty in large-scale expansion in the in vitro expansion of biliary tree stem cells, so as to provide a large number of seed cells to realize the clinical application of endoderm stem/progenitor cells. At the same time, it also provides a reference expansion method for other difficult-to-culture stem cells.

In the present invention, by substituting growth factors with small molecule compounds, the safety in the process of stem cell expansion is greatly increased, and the cost required for stem cell expansion is effectively reduced at the same time.

In the present invention, culture conditions including activation/inhibition of Wnt signaling pathway and TGFβ signaling pathway are used to expand biliary tree stem cells in the form of organoids. Methods of expanding primary biliary tree stem cells, populations of expanded biliary tree stem cells, and medical applications of the expanded biliary tree stem cells are provided.

The present invention also provides a kit containing the medium described in the present invention. In some preferred embodiments, the kit also includes instructions for use, so as to facilitate the research or clinical application by those skilled in the art.

Based on the new discovery of the present invention, a biliary tree stem cell culture obtained by the method of the present invention or a biliary tree stem cell or organoid isolated and purified from the biliary tree stem cell culture is also provided. The biliary tree stem cells or organoids express stemness markers EpCAM, Sox17, PDX1, CK19, Prom1, and ICAM1 but do not express mature hepatocyte marker Albumin and liver progenitor cell marker AFP.

Methods for enriching or separating and purifying cells from cell cultures are also well known to those skilled in the art, for example, enrichment can be performed based on the morphological characteristics of biliary tree stem cells; or based on special proteins expressed by biliary tree stem cells (such as EpCAM, etc.) or Molecular markers for selective collection (eg, using specific antibodies or ligands). As an optional embodiment, flow cytometry technology can be used to separate and purify the cells through the molecular markers on the surface of the biliary tree stem cells.

The inventor's analysis has shown that the biliary tree stem cells cultured in the system have multi-directional differentiation potential to differentiate into mature hepatocytes, biliary epithelial cells, pancreatic endocrine cells and other types of cells. Therefore, the biliary tree stem cells cultured in the present invention have various uses.

The biliary tree stem cells or organoids cultured in the present invention can be used to prepare compositions (pharmaceutical compositions) for promoting regeneration of the liver/biliary duct/pancreas; Obesity, acute liver failure, hepatitis, liver fibrosis, liver cancer, liver metabolic disease or liver damage caused by liver failure) composition (pharmaceutical composition); for the preparation of a composition for treating bile duct or pancreas damage; for use as In vitro models for liver/biliary/pancreas-related diseases or drug efficacy studies, such as for the study of drug transport, drug metabolism, liver formation, liver regeneration, for liver/biliary/pancreatic toxicity testing, screening of biliary tree stem cytotoxic compounds, Screening compounds that regulate the function of biliary tree stem cells, etc.

The biliary tree stem cells or organoids cultured in the present invention can be used for liver/biliary/pancreas toxicology research, and can also be applied to cell transplantation to treat liver diseases, bioartificial liver/biliary/pancreas construction, new drug toxicity (such as liver toxicity) detection, drug efficacy Evaluation and drug target identification; can provide bile duct tree stem cell source or bile duct tree stem cell model for basic research and clinical application of biology, medicine and pharmacy; its induction and differentiation process can also provide a research platform for the development and differentiation process of human liver/biliary duct/pancreatic cells , with broad application prospects.

If necessary, the biliary tree stem cells or organoids cultured in the present invention can be further applied to genetic engineering recombination to form recombined cells, for example, in order to endow the cells with further functions or characteristics, exogenous genes are transferred into the cells Expression cassettes, or gene knockout or gene editing of the cell's genome, etc.

The present invention also provides a composition (medicine), which contains: an effective amount of the biliary tree stem cells (such as 1×10 ⁴ -1×10 ¹² ; preferably 1×10 ⁵ -1 ×10 ¹⁰ ); and a pharmaceutically acceptable carrier. It contains an effective amount of the biliary tree stem cells and a pharmaceutically acceptable carrier. The composition has no visible toxicity and side effects on animals.

The "effective amount" refers to the amount that can produce functions or activities on humans and/or animals and can be accepted by humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for the administration of therapeutic agents, including various excipients and diluents. The term refers to pharmaceutical carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in compositions may contain liquids, such as water, saline, buffers. In addition, there may also be auxiliary substances in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and the like. The carrier may also contain cell transfection reagents.

The present invention also provides a method for promoting regeneration or repair of the liver/biliary duct/pancreas, the method comprising: administering an effective amount of the cultured biliary tree stem cells of the present invention to a subject in need of treatment. When the composition is used for administration, usually 1×10 ² -1×10 ¹⁰ cells/kg body weight, preferably 1×10 ³ -1×10 ⁸ cells/kg body weight is suitable, which is also It depends on the clinician's diagnosis and the severity of the patient's symptoms.

The present invention also provides a kit containing the cultured biliary tree stem cells of the present invention or a composition containing the biliary tree stem cells. Preferably, the kit also contains instructions for use, so as to facilitate the research or clinical application by those skilled in the art.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, generally follow the conditions described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, or according to the manufacturer's suggestion conditions of.

In vitro expansion of biliary tree stem cells, the specific research contents include: 1. Realize the isolation, culture and expansion of extrahepatic biliary tree stem cells; 2. Screen growth factor combinations and small molecular compound combinations to construct expansion medium. (skeleton matrix includes: A. matrigel 3D B. hyaluronic acid and heparan sulfate); 3 realize the subculture of biliary tree stem cells (cultivate with expansion medium after primary isolation, EpCAM sorting during subculture, and temporarily use water for current subculture) Gel Matrigel).

Aspects and various embodiments of the invention are described in more detail below.

Materials, Methods and Explanation of Terms

Glycogel: a hydrogel solution based on glycosaminoglycans (polysaccharides) from a fluid state to a colloidal state. According to the needs of cell expansion or differentiation and maturation, a combination of small molecule compounds and growth factors are added to form a Glycogel system. It is used in the two-dimensional or three-dimensional expansion of biliary tree stem cells, and it has a supporting and protective role in the induction of differentiation of biliary tree stem cells into liver cells before and after expansion.

KM: serum-free medium Kubota's Medium (Kubota's Stem Cell Growth Medium).

PKM: On the basis of serum-free medium, a biliary tree stem cell expansion medium is added with a combination of small molecule compounds and growth factors that promote the expansion of primary BTSCs. Small molecule compounds and growth factors contained in PKM:

Growth factors: 50ng/ml EGF, 100ng/ml R-Spondin1 (RSPO1), 100ng/ml Noggin, 50ng/ml Wnt3a;

Small molecule compounds: 0.5μM Bix01294, 2μM Bay K8644, 0.04μM RG108, 2μM MSB431542, 10μM Forskolin, 1μM A83-01;

10P: PKM (10KPM) containing 10 small molecular compounds or growth factor components.

8P: PKM containing 8 small molecule compounds or growth factor components.

Glycogel System: Expansion medium may contain or consist of:

(a) skeleton matrix (A, Matrigel hydrogel/solution; B, hyaluronic acid (HA) 0.05% and heparan sulfate (HS) 100ng/ml mixed HAHS hydrogel/solution;

(b) Growth factors: 50ng/ml EGF, 100ng/ml R-Spondin1, 100ng/ml Noggin, 50ng/ml Wnt3a;

(c) Small molecule compounds: 0.5 μM Bix01294, 2 μM Bay K8644, 0.04 μM RG108, 2 μM SB431542, 10 μM Forskolin, 1 μM A83-01;

(d) KM medium (in which the basal medium RPMI1640 is replaced by advanced DMEM/F12).

Two-dimensional (2D) culture: a system and method for the proliferation and passage of BTSCs in epithelial-like clones under the Glycogel system.

Three-dimensional (3D) culture: systems and methods for BTSCs embedded in Glycogel to proliferate and pass in organoids.

Identification of cell characteristics: two-dimensionally cultured BTSCs are identified by gene identification, cell morphology, and surface markers; three-dimensional organoids are identified by organoid size, quantity, surface markers, and gene phenotype.

Hepatic differentiation and maturation of cells: a system and method for inducing differentiation and maturation of two-dimensional or three-dimensional BTSCs into hepatocytes based on the Glycogel system.

HMM: hepatocyte maturation medium hepatocyte maturation induction medium.

cell wash (20ml) medium: on the basis of RPMI 1640 medium, add BSA, selenium, and double antibiotics (add 0.5g of BSA, Selenium 30nM and 1× double antibiotics per 500ml RPMI 1640).

4PKM: RG108 concentration 0.04 μM, A83-01 concentration 1 μM, FOSK concentration 10 μM, BAY K8644 concentration 2 μM.

In 4PKM+Wnt3a, the concentration of Wnt3a is 50ng/ml.

In 4PKM+RSPO1, the concentration of RSPO1 is 100ng/ml.

In 4PKM+EGF, the EGF concentration is 50ng/ml.

In 4PKM+TGFβ, TGFβ refers to "the combination of TGFβ signaling pathway-related factors (composed of three small molecule compounds, A83-01 (1 μM), SB431542 (2 μM), and Noggin (100ng/ml))".

In 5PKM (4PKM+RSPO1), the concentration of RSPO1 is 100 ng/ml.

Example 1. Isolation, culture and identification of extrahepatic primary biliary tree stem cells

The gallbladder and extrahepatic bile duct tissues of C57 WT mice were obtained by surgery, placed in cold (4°C) cell wash (20ml) medium supplemented with double antibiotics, separated into small pieces with scissors, collected in a 15ml centrifuge In the tube, after centrifugation at 1000rpm for 2 minutes, use digestive enzymes (type IV collagenase/DNase) to resuspend the digestion treatment for 15 minutes (violently shake the centrifuge tube every 3 minutes), centrifuge at 800rpm for 5 minutes, add double antibiotics in cold (4°C) Cell wash (20ml) culture medium resuspended, this step was repeated once. Finally, resuspend with the expansion medium pKM, filter with a 40 μm filter, count the cells, and inoculate in a common two-dimensional cell culture well plate.

After 11 days of culture in serum-free KM medium, biliary tree stem cells grew as adherent clones (Fig. 2A). The cells are densely arranged and have a high nuclear-cytoplasmic ratio (scale bar 100 μm).

EpCAM, CK19, ALB, HNF4α, CPS-1 antibodies were used to identify BTSCs clones grown in KM culture conditions for 21 days. After immunohistochemical staining, fluorescence microscope showed that the edge and inner cells of biliary tree stem cell clones on day 21 expressed EpCAM and CK19 (green fluorescence), and the edge signal was stronger than that in the center, and neither expressed ALB, HNF4α, CPS- 1 (red fluorescence); DAPI (blue) was used as a contrasting stain in the staining, indicating cell nuclei (Fig. 2B).

BTSCs grown on day 21 were collected for QPCR identification. From the perspective of gene expression level, the biliary tree stem cells on the 21st day expressed EpCAM, SOX17, PDX1, CK19, Prom1 (markers of endoderm stem cells, wherein EpCAM, SOX17, PDX1 surface markers of biliary tree stem cells), ICAM1, (stem cell markers); ALB (mature hepatocyte marker), AFP was expressed, and primary hepatocytes were used as a control (Fig. 2C).

Example 2. Construction of the Glycogel system: Screening growth factor combinations and small molecule compound combinations to construct expansion media

Expansion media may include basal media Advanced DMEM/F-12. Basal medium can be supplemented with medium supplements and/or one or more additional components, including selenic acid (3×10 ⁻⁸ M), bovine serum albumin (0.1%), nicotinamide (0.05%), Zinc sulfate heptahydrate (10 ^-10 M), hydrocortisone, transferrin (10 μg/ml), insulin (5 μg/ml), high-density lipoprotein, and free fatty acid mixture.

In the determination of the pKM components of the Glycogel system, the inventors studied the gene expression profile of BTSC, combined with experimental verification, and after a large number of analyzes and screenings, determined 4 kinds of growth factors and 6 kinds of small molecule compounds, including: EGF, R -Spindin1, Noggin, Wnt3a, Bix01294, Bay K8644, RG108, SB431542, A83-01, Forskolin. The above-mentioned growth factors and small molecular compounds were added to KM to constitute the expansion medium pKM, and BTSCs were cultured (Fig. 3A).

For the hydrogel composition of the Glycogel system, the choice of matrix matrices includes chemically defined types, such as silk fibroin, collagen, or hyaluronic acid hydrogels, as well as non-chemically defined composite protein hydrogels, including Matrigel et al. Suitable composite protein hydrogels may comprise extracellular matrix components such as laminin, collagen IV, fibronectin and hyaluronic acid, heparan sulfate proteoglycans. Two are used here: one is a plateable, embedded A-skeleton matrix: a hydrogel of extracellular matrix proteins from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, which are available from commercial sources and include Matrigel (Corning Life Sciences). The other is the B-skeleton matrix added to the culture medium, a HAHS hydrogel that is mixed with hyaluronic acid (HA) and heparan sulfate (HS) at an appropriate ratio.

Embodiment 3, in vitro culture of primary BTSCs in pKM

Matrigel is not required for primary culture, and a skeleton matrix is needed to assist growth due to poor adherence of biliary tree stem cells during subculture. Since BTSCs directly grow slowly in KM medium (cell count on day 14, each C57 WT mouse can obtain 2×10 ⁴ cells), consider adding expansion factors on the basis of KM to construct an improved medium pKM to achieve primary generation Expansion of cells.

In the same method as in Example 1, after the gallbladder and extrahepatic bile duct tissues were obtained by surgery, separated and digested with enzymes, they were resuspended in a serum-free modified medium pKM, and seeded in ordinary two-dimensional cell culture well plates. After 7 days of culture in the modified serum-free medium pKM, BTSCs grew as adherent clones (Fig. 3B). Cells are densely arranged, and most cells are in the state of nuclear division (scale bar 100 μm).

The cell number of BTSCs was significantly increased in pKM medium. On day 7 of cell counting, 4.2×10 ⁵ cells could be obtained from each C57 WT mouse. 20 times more than the cells collected on the 14th day, and if the multiple relationship of the time difference is calculated, the number of cells is 50 times more than that of the KM maintenance medium (Fig. 3C).

Primary BTSCs cultured in pKM on day 7 were collected for qPCR identification, and primary hepatocytes were used as controls for gene expression detection. From the perspective of gene expression level, compared with the BTSCs cultured in KM on the 7th day, the expression level of EpCAM in the former group was lower than that in the KM group (p<0.001), but the expression level of SOX17 was significantly higher than that in the KM group (p<0.001). 0.001), there was no significant difference in the expression changes of PDX1, AFP and albumin.

This result indicated that 10pKM, while promoting cell proliferation, also increased the stemness of BTSCs' ability to differentiate into hepatocytes (Fig. 3D). The decrease of EpCAM expression and the increase of SOX17 expression indicated that the stemness of liver differentiation ability increased.

Example 4. Two-dimensional (2D) passage of biliary tree stem cells based on the Glycogel system

When subcultured, remove the medium of primary BTSCs, use TrypLE (GIBCO, 12604021) to digest the cells at 37°C, and pipette with a pipette every 3 minutes (add 1ml TrypLE to each well of a 6-well plate). The cells were digested and collected in a centrifuge tube, centrifuged at 200g for 5min, resuspended in pKM, and filtered through a 40μm filter. Primary BTSCs were collected for EpCAM ⁺ flow sorting to obtain EpCAM ⁺ target cells to remove mesenchymal stem cells that may be present during the separation process. After the primary BTSCs cultured in KM or pKM were sorted by EpCAM ⁺ , the positive ratio was above 84%, which was consistent with the previous experimental results (Fig. 4A-B). Sorted cells were collected in medium containing 10% serum. After centrifugation at 200g for 5 minutes, resuspend in expansion medium.

Due to the problem that the primary biliary tree stem cells hardly adhere to the wall after digestion, pKM needs to be combined with Glycogel to realize the two-dimensional subculture of BTSCs during the passage process. In the present invention, the two-dimensional passage expansion of BTSCs based on Matrigel with uncertain composition and Glycogel with HAHS with chemical composition is realized respectively:

1. 2D passage amplification based on Matrigel hydrogel/solution

After the primary BTSCs were digested with enzymes, EpCAM ⁺ BTSCs were obtained by flow sorting for passage. After counting, the biliary tree stem cells were adhered to culture plates coated with Matrigel, and when the cells reached 80%-90% confluence, they were passaged at a ratio of 1:2 (the passage ratio was determined according to the actual number of cells), Medium was changed every 48 hours and cultured for 4-7 days for subsequent digestion.

The first-generation BTSCs cultured under these conditions were collected for QPCR identification, and primary hepatocytes were used as controls. From the perspective of gene expression level, the expression levels of EpCAM, SOX17, PDX1, and ICAM1 in the first-generation BTSCs cultured in pKM (Matrigel) were similar to those in the KM group compared with the BTSCs cultured in KM, and AFP and ALB were still not expressed (Figure 4C ).

P0 generation BTSCs cells have hepatic differentiation potential (expression of SOX17) and pancreatic differentiation potential (expression of PDX1). Increase).

The above results indicated that the BTSCs amplified by the Glycogel system had better proliferation ability and hepatic differentiation ability.

2. Improved 2D passage expansion of BTSCs based on HAHS hydrogel/solution

In order to avoid the possible risk of tumor formation of Matrigel, a HAHS framework matrix containing hyaluronic acid and heparan sulfate was constructed as an in vitro expansion system for BTSCs. At this point the expansion medium was supplemented with mixed sulfate modified heparan sulfate and 0.05% HA. After the primary BTSCs were digested with enzymes, EpCAM ⁺ BTSCs were obtained by flow sorting for passage. After counting, the biliary tree stem cells were adhered to the culture plate, and when the cells reached 80%-90% confluence, they were passaged at a ratio of 1:2 (the passage ratio was determined according to the actual number of cells), and replaced every 48 hours Culture medium, cultivated for 4-7 days, for subsequent digestion. The cells were effectively amplified, and the expansion factor was about 3 times per generation ( FIG. 5 ).

Example 5, three-dimensional (3D) passage of biliary tree stem cells based on Glycogel

In order to obtain biliary tree stem cell organoids, P0 generation BTSCs cells were mixed with Matrigel at a ratio of 1:1, and added dropwise to the culture plate in the form of 5000 BTSCs per 10ul. After it was solidified, the culture plate was placed upright and added Cover with 10PKM medium. After 1 day, organoid formation was detectable under the microscope. After 3 days, portions reached a size visible to the naked eye. BTSCs organoids grow cystically, consisting of a single layer of cuboidal epithelium and inner cell mass. After 5 days, the maximum diameter of BTSCs organoids can reach 1000 μm, and the 1:3 passage is carried out every 3-5 days, and the shape of organoids can still be stably maintained when passaged to P6. Compared with P1 BTSCs, the number of P5 is expected to increase by 80 times (Fig. 6A, E).

EpCAM, CK19, and ki67 antibodies were used to identify P1 BTSCs organoids on day 3 of KM culture conditions. After fluorescent staining of immune cells, the P1BTSCs organoids on day 3 expressed EPCAM, CK19, and ki67 (green fluorescence) under a fluorescent microscope, and DAPI (blue) was used as a counterstain in the staining to indicate cell nuclei. The viability of BTSCs organoids can be judged by life-and-death staining. Organoids formed from first passage (P1) BTSCs were stained with a live/dead assay kit. Live cells (with esterase activity) are stained green and dead cells (damaged plasma membrane) are stained red. Magnified by 10 times, it shows that most of the cells in the organoid are stained green and are living cells, and a small amount of cells stained red are dead cells (Fig. 6B-C).

P1 BTSCs organoids (P1 BTSC organoids) on day 5 were collected for qPCR identification. From the perspective of gene expression level, compared with the primary BTSCs cultured in pKM (P0 BTSCs) on day 5, the expression levels of SOX17, ICAM1, EpCAM and CK19 were slightly decreased. increased, but the difference was not statistically significant. Among them, the expression level of PDX1 increased (p<0.05), and AFP and ALB were still not expressed. Primary hepatocytes were used as a control (Fig. 6D).

BTSCs organoids between different passages were identified by qPCR, and primary hepatocytes were used as controls. It can be seen from the results that the expression of EpCAM, SOX17, ICAM1, and CK19 surface gene markers of BTSCs organoids between different generations is stable, which proves that this method of 3D Matrigel 10PKM system can carry out stable passage expansion. The expression level of PDX1 was up-regulated during the passage process, indicating that the 3D-expanded BTSCs maintained the ability to differentiate into the liver and at the same time enhanced the ability to differentiate into pancreatic cells (Fig. 6F).

Example 6, RG108, A83-01, Forskolin, Bay K8644 (RAFB) are essential growth factors for the expansion of biliary tree stem cells

In this example, the necessary growth factors for the expansion of biliary tree stem cells were verified, except for the difference in components, other conditions were the same as in Example 5.

For the BTSCs digested and embedded on the 14th day of KM culture, the P1BTSCs organoids obtained under the conditions of 10PKM and 10PKM-A83-01-FOSK (minus A83-01 and Forskolin, the same below) were larger in size and more in number ( 7A-B ), illustrating that A83-01 and FOSK are necessary for the expansion of biliary tree stem cells.

The inventors optimized on the basis of 10PKM, and divided growth factors and small molecular compounds into 8 groups according to the pathway of action for group screening, and compared the formation ability and gene expression level of P1 BTSCs organoids. They are: 10PKM-EGF, 10PKM-TGFβ (A83-01, Noggin, SB431542), 10PKM-WNT (RSPO1, WNT3a), 10PKM-cAMP, 10PKM-bix01294, 10PKM-Bay K8644, 10PKM-RG108, and 10PKM as controls.

A83-01 is an inhibitor of TGFβ kinase/activin receptor-like kinase (ALK 5) (IC50=12nM), prevents phosphorylation of Smad2/3 and inhibits TGFβ-induced growth. A 83-01 blocks the phosphorylation of Smad2 and inhibits TGF-β-induced epithelial-mesenchymal transition. In addition, A83-01 can inhibit transcriptional activity induced by TGFβ type I receptor ALK-5, activin type IB receptor ALK-4, and nodular type I receptor ALK-7. It is confirmed in the present invention that A83-01 is necessary for the expansion of BTSCs in the Glycogel system.

Forskolin, a diterpene naturally occurring in the Indian plant Coleus forskolini, activates adenylyl cyclase and therefore increases intracellular cAMP concentrations. The second messenger cAMP activates cAMP-dependent protein kinase (PKA or cAPK), regulates multiple cellular mechanisms such as gene transcription, ion transport and protein phosphorylation, and is an essential component for the expansion of BTSCs in the Glycogel system.

Then in the 8 groups of screening experiments, the number of 10PKM-RG108 cells decreased significantly (Fig. 7C-D), indicating that RG108 is necessary for the expansion of biliary tree stem cells.

As a DNA methyltransferase inhibitor, RG108 can inhibit DNA methylation and activate the expression of cell proliferation-related genes, thereby achieving the proliferation of BTSCs.

The inventors collected P1 BTSCs from each group on day 5 for QPCR identification, and found that the stemness gene PROM1 and proliferation gene KI67 of BTSCs under 10PKM-Bay K8644 conditions were significantly decreased, but EPCAM was slightly increased, and PDX1 and SOX17 were almost unchanged. It can be concluded that Bay K8644 is essential for the expansion of biliary tree stem cells (Fig. 7E).

Bay K8644, as a classical calcium ion channel inhibitor, is essential to prevent the differentiation of BTSCs during expansion.

Example 7, RSPO1 can replace various components to expand biliary tree stem cells

The screened RG108, A83-01, FOSK, and BAY K8644 were used as 4PKM to carry out the experiment, except that the components were changed, and other conditions were the same as the above-mentioned Example 5 (3D) or Example 1 (2D). KM medium cannot achieve 2D passage expansion of BTSCs, nor can it support BTSCs to form organoids in 3D. Compared with KM medium, 4PKM can maintain the passage and expansion of BTSC organoids in 3D, and it needs to be optimized to maintain the growth of BTSC clones in 2D.

Next, the inventors further optimized the proliferation effect of 4PKM in 2D and 3D. After repeated screening and component adjustment, it was found that the addition of growth factors of the WNT signaling pathway resulted in significant cell proliferation (Fig. 7F-G, Fig. 8A). Therefore it is beneficial to optimize 4PKM by adding additional growth factors.

By grouping: 4PKM+Wnt3a, 4PKM+RSPO1, 4PKM+EGF, 4PKM+TGFβ, and then collecting the P1 BTSCs of each group on the fifth day for QPCR identification, it was found that the stemness genes SOX17, PROM1, The expression level of the proliferation gene KI67 was higher than that of other groups, thus confirming that RSPO1 is a surrogate factor for the expansion of biliary tree stem cells. In the 4PKM+EGF group, although the expression level of KI67 was the highest, the level of PROM1 decreased, so it was not suitable for the expansion and maintenance of BTSCs (Fig. 8B).

Under the condition of 5PKM (4PKM+RSPO1), the primary biliary tree stem cells grow as adherent clones. The cells were densely arranged (Fig. 8C), and most cells were in the state of nuclear division (scale bar 100 μm). Next, the inventors used the ki67 antibody to perform immunocytofluorescent staining of P1 BTSCs organoids on day 7 under 5PKM culture conditions. Under the fluorescent microscope, it was shown that the primary BTSCs on day 7 expressed ki67 (green fluorescence), and DAPI (blue) was used as a contrast stain in the staining to indicate the nucleus (Fig. 8D). BTSCs cultured in 5PKM on day 7 were collected for QPCR identification. From the perspective of gene expression level, compared with BTSCs cultured at 10PKM on day 7, the expression levels of EpCAM, PDX1, SOX17, PROM1, and KI67 in BTSCs cultured at 5PKM were stable or slightly still did not express ALB. Gene expression was performed using primary hepatocytes as a control (Fig. 8E).

Therefore, 5PKM after adding R-spondin1 can realize the growth and passage of BTSC clones in 2D, and can also significantly improve the 3D amplification effect of 4PKM.

Further, the inventor replaced the KM basal medium with Advanced RPMI 1640 and added 10 factors (RG108, A83-01, FOSK, BAY K8644, RSPO1, Bix01294, SB431542, EGF, Noggin, Wnt3a) or 5 factors (RG108, A83- 01, FOSK, BAY K8644, RSPO1), and can also realize the expansion and passage of BTSCs.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims. Also, all documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference.

Claims (10)

1. A method for cultivating biliary tree stem cells in vitro, comprising cultivating biliary tree stem cells with culture medium; wherein, said culture medium includes: basal medium, and small molecule compounds and growth factors: RG108, A83-01, Forskolin, BayK8644 and R-Spondin1. 2. The method according to claim 1, wherein the small molecular compound and growth factor further comprise one or more selected from the group consisting of Bix01294, SB431542, EGF, Noggin, Wnt3a. 3. The method according to claim 1 or 2, wherein the basal medium is a serum-free medium; preferably, the basal medium is selected from: Kubota's stem cell growth medium, Advanced RPMI 1640 culture Base, Advanced DMEM/F-12 medium, RPMI1640 medium, DMEM medium, MEM medium or Fischers medium; preferably Kubota's stem cell growth medium or Advanced RPMI 1640 medium; more preferably, the Advanced RPMI 1640 medium, Advanced DMEM/F-12 medium, RPMI1640 medium, DMEM medium, MEM medium or Fischers medium supplemented with albumin, nicotinamide, insulin, transferrin, selenic acid, zinc sulfate heptahydrate ;or The concentration of the small molecular compound and the growth factor in the basal medium is:

Preferably, it also includes:

4. The method according to claim 1 or 2, characterized in that, the expansion culture or subculture is carried out by the method; Preferably, the culture is carried out in a three-dimensional system to obtain organoids, or the culture is carried out in a two-dimensional system to obtain expanded cells; Preferably, the culturing is carried out in a fluid or colloidal hydrogel based on glycosaminoglycans; Preferably, the skeleton matrix of the hydrogel is selected from: Matrigel, hyaluronic acid and heparan sulfate mixed hydrogel, collagen hydrogel, hyaluronic acid hydrogel, silk fibroin hydrogel. 5. A medium for culturing biliary tree stem cells in vitro, comprising: basal medium, and small molecule compounds and growth factors: RG108, A83-01, Forskolin, Bay K8644 and R-Spondin1; preferably, the The small molecular compound and growth factor also include one or more selected from the following group: Bix01294, SB431542, EGF, Noggin, Wnt3a. 6. culture medium as claimed in claim 5, is characterized in that, the concentration of described small molecule compound and growth factor in basal medium is:

Preferably, it also includes:

7. A culture system for biliary tree stem cells, comprising: a hydrogel containing a skeleton matrix, and the culture medium according to claim 5 or 6; Preferably, the skeleton matrix is selected from: Matrigel, hyaluronic acid and heparan sulfate mixed hydrogel, collagen hydrogel, hyaluronic acid hydrogel, silk fibroin hydrogel. 8. The use of the medium according to claim 5 or 6, for culturing biliary tree stem cells; preferably, the culturing comprises: Expansion of biliary tree stem cells; Subculture the biliary tree stem cells; prepare organoids; or Passaging and stable culture of biliary tree stem cell organoids. 9. A kit for culturing biliary tree stem cells in vitro, comprising: The medium described in claim 5 or 6; or The culture system of biliary tree stem cells according to claim 7. 10. The biliary tree stem cell culture obtained by the method of any one of claims 1-4 or the biliary tree stem cell or organoid isolated and purified from the culture; preferably, the biliary tree stem cell or organoid Expresses stemness markers EpCAM, Sox17, PDX1, CK19, Prom1, ICAM1, does not express mature hepatocyte marker Albumin, and does not express hepatic progenitor cell marker AFP.

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