Expansion or passaging system of biliary tree stem cells and applications thereof

Abstract

The application provides an amplification or subculture system of biliary tree stem cells and application thereof. By using the system of the application, the amplification of the cells can be realized by conventional culture without introducing an exogenous gene into the biliary tree stem cells, the amplification efficiency is very high, and the obtained biliary tree stem cells can be subcultured. The application can maintain the stemness of the tissue stem cells while ensuring the large-scale amplification of the cells.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine; more specifically, this invention relates to the expansion or passage system of bile duct tree stem cells and its application. Background Technology

[0002] Human bile duct tree stem cells (hBTSCs) exist within the peribiliary glands of the bile duct tree wall. Semeraro et al. preliminarily identified EpCAM... + PDX1 + Sox17 + These are biomarkers for BTSCs. hBTSCs have the potential to differentiate into hepatocytes, pancreatic islet cells, and bile duct cells. hBTSCs exhibit heterogeneity; hBTSCs located in the common hepatopancreatic bile duct have a higher potential to differentiate into the liver and pancreas than hBTSCs from other parts of the bile duct tree. hBTSCs isolated from extrahepatic bile ducts (excluding the pancreatic duct) have an even stronger hepatic differentiation capacity (manifested by higher expression of Sox17 and lower or absent expression of PDX1), making them a more ideal seed cell type for the treatment of liver failure. Through previous studies, the inventors have demonstrated that transplantation of porcine or human bile duct tree stem cells has a repairing effect on liver injury and diabetic mouse models, and shows promise for clinical application. However, current research on bile duct tree stem cells is mainly based on their primary isolation and culture; conditions for their expansion and passage still need to be established.

[0003] Achieving in vitro expansion and passage of bile duct tree stem cells (hBTSCs) to obtain sufficient cell quantities is fundamental for their cell transplantation therapy in ESLDs and diabetes. Establishing a robust in vitro expansion system is crucial for maintaining cell stemness and normal physiological function. Currently, the culture and expansion of hBTSCs primarily rely on the in vitro expansion of other endoderm organ stem cells (such as gastrointestinal stem cells and liver stem cells), mainly through two systems: co-culture with feeder cells and Matrigel culture. Given the complexity of co-culture systems and the potential for contamination during feeder layer preparation, hydrogel-based three-dimensional culture methods have gradually become the preferred approach for in vitro expansion of endoderm organ-derived epithelial stem cells.

[0004] Currently, common three-dimensional culture systems are mainly based on the Matrigel embedding method. Matrigel is a matrix component extracted from mouse chondrosarcoma, and its main components include laminin, type IV collagen, nestin, heparin sulfate glycoprotein, growth factors, matrix metalloproteinases (MMPs), etc. Huch M et al. used the Matrigel system to culture liver stem cells in vitro for extended periods and clone them into organoids for several months. However, the animal-derived and tumor matrix-derived nature of Matrigel leads to low safety profiles, making it unsuitable for the preparation of stem cells for clinical use.

[0005] While there are existing studies in this field applying small molecule compounds or cytokines to the culture of certain types of stem cells, different types of stem cells have different cellular properties and stemness regulation mechanisms, resulting in vastly different nutritional requirements and culture methods for maintenance. In particular, there are numerous types of small molecule compounds, and different concentrations of these compounds have different mechanisms of action. Cytokines are also diverse, and different cytokines may exhibit synergistic or antagonistic effects. Whether they can be used for the culture, expansion, and passage of bile duct tree stem cells, and how to optimize the composition and conditions to achieve this goal, remains unanswered in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a system for the expansion or passage of bile duct tree stem cells and its application.

[0007] In a first aspect of the invention, a method for in vitro culture of bile tree stem cells (including: primary bile tree stem cells) is provided, comprising culturing bile tree stem cells in a culture medium; wherein the culture medium comprises: a basal culture medium, and small molecule compounds and growth factors: RG108, A83-01, Forskolin, Bay K8644 and R-Spondin1.

[0008] 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, and Wnt3a.

[0009] In one or more embodiments, the basal culture medium is a serum-free culture medium; preferably, the basal culture medium is selected from: Kubota's stem cell growth medium (KM medium), Advanced RPMI 1640 medium, Advanced DMEM / F-12 medium, RPMI 1640 medium, DMEM medium, MEM medium, or Fischers medium; preferably, it is Kubota's stem cell growth medium or Advanced RPMI 1640 medium; more preferably, albumin, nicotinamide, insulin, transferrin, selenic acid, and zinc sulfate heptahydrate are added to the Advanced RPMI 1640 medium, Advanced DMEM / F-12 medium, RPMI 1640 medium, DMEM medium, MEM medium, or Fischers medium.

[0010] In one or more embodiments, the concentrations of the small molecule compound and growth factor in the basal culture medium are:

[0011]

[0012]

[0013] Preferably, it also includes:

[0014]

[0015] In one or more embodiments, the culture medium contains 0.1% (w / v) albumin, 0.05% (w / v) nicotinamide, 5 μg / ml insulin, 10 μg / ml transferrin, and 3 x 10 μg selenate. -8 M, Zinc sulfate heptahydrate 10 -10 M; Preferably, the component may fluctuate by 60%, 50%, 40%, 30%, 20%, 10% or 5%.

[0016] In one or more embodiments, the method is used for amplification culture or subculture.

[0017] In one or more embodiments, the culture is carried out in a three-dimensional (3D) system to obtain organoids.

[0018] In one or more embodiments, the culture is carried out in a two-dimensional (2D) system to obtain expanded cells.

[0019] In one or more embodiments, the cultivation is carried out in a hydrogel (Glycogel system) based on glycosaminoglycans (polysaccharides, "disaccharide units" with a degree of polymerization of 10-10,000, such as 50, 100, 200, 500, 1000, 2000, 5000, 8000) in a fluid state (glycosaminoglycan concentration of 10-1000 ng / ml, such as 100, 300, 500, 700, 900 ng / ml) or a colloidal state (glycosaminoglycan concentration of 10-500 mg / ml, such as 30, 50, 100, 200, 300 mg / ml).

[0020] In one or more embodiments, the skeletal matrix of the hydrogel includes (but is not limited to) selected from: Matrigel, hyaluronic acid (HA) and heparan sulfate (HS) mixed hydrogel (HAHS), collagen hydrogel, hyaluronic acid hydrogel, and silk fibroin hydrogel.

[0021] In one or more embodiments, the final concentration of hyaluronic acid (HA) in the hyaluronic acid and heparan sulfate mixed hydrogel is 0.01-0.5% (w / v) (preferably 0.02-0.2%; more preferably 0.03-0.1%; for example 0.05%, 0.75%, 0.1%), and the final concentration of heparan sulfate (HS) is 10-400 ng / ml (preferably 10-100 ng / ml; more preferably 30-100 ng / ml, for example 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml).

[0022] In one or more embodiments, the bile duct tree stem cells are human bile duct tree stem cells or porcine bile duct tree stem cells, including primary human bile duct tree stem cells and primary porcine bile duct tree stem cells.

[0023] In another aspect of the invention, a culture medium for in vitro culture of bile duct tree stem cells is provided, comprising: a basal culture medium, and small molecule compounds and growth factors: RG108, A83-01, Forskolin, Bay K8644, and R-Spondin1; preferably, the small molecule compounds and growth factors further include one or more selected from the group consisting of: Bix01294, SB431542, EGF, Noggin, and Wnt3a. Preferably, each component is in an effective amount.

[0024] In another aspect of the present invention, a culture system for bile duct tree stem cells (Glycogel system) is provided, comprising: a hydrogel containing a scaffold matrix, and the culture medium.

[0025] In one or more embodiments, the scaffold matrix includes (but is not limited to) a selection from: Matrigel, hyaluronic acid (HA) and heparan sulfate (HS) mixed hydrogel (HAHS), collagen hydrogel, hyaluronic acid hydrogel, and silk fibroin hydrogel.

[0026] In another aspect of the invention, the use of the culture medium is provided for culturing bile duct tree stem cells.

[0027] In one or more embodiments, the culture includes: expanding bile duct tree stem cells; passage culture of bile duct tree stem cells; preparing organoids; or passage and stabilizing bile duct tree stem cell organoids.

[0028] In another aspect of the invention, a kit for in vitro culture of bile duct tree stem cells is provided, comprising: the culture medium; or, the culture system for bile duct tree stem cells (Glycogel system).

[0029] In another aspect of the invention, a bile duct tree stem cell culture obtained by any of the methods described above, or bile duct tree stem cells or organoids isolated and purified from said culture, is provided.

[0030] In one or more embodiments, the bile duct tree stem cells or organoids express stem cell markers EpCAM, Sox17, PDX1, CK19, Prom1, and ICAM1, but do not express the marker Albumin of mature hepatocytes or the marker AFP of hepatic progenitor cells.

[0031] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0032] Figure 1 A flowchart illustrating the construction of the Glycogel system and its application in the expansion and hepatic differentiation and maturation of bile duct tree stem cells.

[0033] Figure 2 Identification of gene expression levels in primary BTSCs cultured in KM. (A) Morphological image of primary BTSCs cultured in vitro. (B) Immunocytochemical staining results of primary BTSCs cultured under serum-free conditions. (C) Identification of gene expression levels in primary BTSCs cultured in KM. *<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 No statistically significant difference.

[0034] Figure 31. Identification of gene expression and surface marker expression in primary amplified BTSCs; (A) Schematic diagram of the components of the BTSC amplification culture system, including 4 growth factors and 6 small molecule compounds, abbreviated as 10 PKM. (B) Morphological images of primary BTSCs cultured on day 7 under serum-free KM and 10 PKM conditions, respectively. (C) Number of primary BTSCs isolated and cultured from each C57 WT mouse under different conditions (serum-free KM day 21 and 10 PKM day 7). The number of cells obtained under 10 PKM was approximately 20 times that under KM conditions. *Compared with other groups, P<0.05. (D) Identification of gene expression levels in primary BTSCs cultured under 10 PKM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 No statistical difference.

[0035] Figure 4 Characterization of P1 generation BTSCs amplified using the Glycogel system (Matrigel-based). (A) Morphological image of P1 BTSCs after Matrigel plating under 10 pKM culture conditions. (B) Positive proportions of BTSCs obtained under P0-KM and P0-10 pKM conditions by EpCAM+ sorting. (C) Identification of gene expression levels of P1 BTSCs cultured at 10 pKM (Matrigel). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05: No statistically significant difference.

[0036] Figure 5 In vitro expansion of P1 BTSCs at 10 pkM under HAHS conditions. (A) Morphological images of P1 BTSCs cultured at 10 pkM under serum-free conditions of 0.05% hyaluronic acid (HA) and 0.05% hyaluronic acid (HA) mixed with heparan sulfate (HS) without Matrigel. (B) Morphological images of P1 BTSCs cultured at 10 pkM under 0.05% hyaluronic acid (HA) mixed with heparan sulfate (HS) under 20x magnification. (C) Cell count statistics. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 No statistically significant difference.

[0037] Figure 61. Three-dimensional expansion of BTSC organoids based on the Glycogel system; (A) Characterization of BTSC organoids formed under 10 pkM conditions after embedding with Matrigel. Scale bar: 100 μm. Cells were mixed with Matrigel at a 1:1 ratio and added to a culture plate containing 5000 BTSCs per 10 μL. The plate was inverted and allowed to solidify before being placed upright and covered with covering medium. (B) Immunocytochemical staining of P1 BTSC organoids under 10 pkM conditions. (C) Determination of organoid viability and survival status using live / dead staining. (D) Identification of gene expression levels in P1 BTSC organoids under 10 pkM conditions. (E) Fold change in the number of BTSC cells during passage. (F) Identification of gene expression levels in BTSC organoids across different passages. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 No statistical difference.

[0038] Figure 7The necessity of RG108, A83-01, Forskolin, and Bay K8644 for in vitro expansion of BTSCs; (A) Cells were mixed with Matrigel to form small droplets, and added to a culture plate containing 5000 BTSCs per 10 μL. The plate was inverted and allowed to solidify before being placed upright. Then, two different culture media, 10 pKM and 10 pKM-A83-01-FOSK, were covered on top. BTSCs-org organoids were characterized on day 2 and day 5 under different culture conditions. (B) The number of organoids was counted on day 2 after inoculation. The number of BTSCs-org formed in each drop. (C) BTSCs cultured in KM for day 14 were digested, embedded, and passaged. Screening experiments were conducted by adding culture media with different conditions. Growth factors and small molecule compounds were divided into 8 groups according to their pathway of action for screening. Representative morphology of P1BTSCs organoids under 10 pKM and 10 pKM-RG108 conditions on day 5. (D) On day 5, the number of P1 BTSCs-org formed per drop under 10 PKM and different selection conditions was counted. The number of P1 BTSC organoids under the 10 PKM-RG108 condition was significantly reduced. (E) Identification of gene expression levels of P1 BTSC organoids under 10 PKM and other selection conditions. (F) Morphological images of P1 BTSCs cultured under 4 PKM and 4 PKM+WNT conditions after Matrigel plating. Cell counting was performed (RG108, A83-01, FOSK, and BAY K8644 were selected as 4 PKM). (G) The number of BTSCs-org formed per drop of P1 BTSCs under 4 PKM and other conditions after Matrigel embedding. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns p>0.05 showed no statistically significant difference.

[0039] Figure 8RSPO1 is an essential component for the in vitro expansion of BTSCs. (A) Organoid characterization of P1 BTSCs under 4PKM and other conditions after embedding with Matrigel (BTSCs cultured for 7 days at 10KM were digested, embedded, and passaged, and screening experiments were conducted using culture media with different conditions). (B) Identification of gene expression levels of P1 BTSC organoids under 4PKM and other screening conditions. 4PKM+RSPO1 was obtained by screening for the expression of stemness genes and proliferation genes, constituting 5PKM. (C) Morphological images of primary BTSCs cultured for different days under 5PKM conditions after Matrigel plating. (D) Immunofluorescence staining of KI67 on primary BTSCs under KM and 5PKM conditions. (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 No statistical difference. Detailed Implementation

[0040] Through in-depth research, the inventors have for the first time disclosed a system for expanding or passageing bile duct tree stem cells (BTSCs). Using this system, there is no need to introduce exogenous genes into the BTSCs; conventional culture can achieve cell expansion with very high efficiency, and the obtained BTSCs are suitable for passage. This invention can maintain the stem cell nature of BTSC tissues while ensuring large-scale cell expansion. Furthermore, the method is simple to use, low in cost, and safe and stable.

[0041] As used in this invention, the terms “containing” or “comprising” include “comprising,” “mainly composed of,” “substantially composed of,” and “composed of.”

[0042] Unless otherwise stated, the culture medium in this invention is bile tree stem cells, which can be primary bile tree stem cells, and more particularly (primary) bile tree stem cells derived from humans, pigs and mice.

[0043] A first aspect of the present invention provides a method for expanding primary bile duct tree stem cells in vitro, comprising the following analysis and optimization processes:

[0044] (1) By adhering culture in serum-free medium (KM), the mixture of bile duct tree cells obtained from the gallbladder and bile duct was screened and purified to obtain a population of bile duct tree stem cells with clonal growth.

[0045] (2) Analysis of the bile tree stem cell population / gallbladder and bile duct tissue showed that the obtained bile tree stem cells expressed stem cell markers EpCAM, Sox17, PDX1, CK19, Prom1, and ICAM1; but did not express the marker Albumin of mature hepatocytes or the marker AFP of hepatic progenitor cells.

[0046] (3) The proportion of EpCAM+ in primary culture of bile tree stem cells obtained from gallbladder and bile duct tissue was higher than 85%.

[0047] (4) Establish a pKM component that can be used to expand primary bile 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, and Forskolin; named 10P(pKM).

[0048] (5) By using 10P (pKM), more than 50 times the number of cells in serum-free culture medium KM were obtained in 7 days of cell culture, resulting in gene-phenotype-stable and cell-morphotype-stable bile duct tree stem cells.

[0049] (6) By optimizing the pKM components, it was determined that 5P (RG108, A83-01, Forskolin (FOSK), BayK8644, RSPO1) can achieve the same amplification capability for primary BTSCs as 10P.

[0050] A second aspect of this invention provides a Glycogel system for the in vitro passage and expansion of bile duct tree stem cells, including the following analysis and optimization process:

[0051] (1) Establish a Glycogel system for in vitro expansion of bile duct tree stem cells based on Glycogel+pKM.

[0052] (2) By screening the components of Glycogel, it was determined that hyaluronic acid hydrogel with heparan sulfate (HAHS) and commercially available Matrigel can be used as Glycogel combined with pKM for passage culture of bile duct tree stem cells.

[0053] (3) In hyaluronic acid hydrogels with heparan sulfate (HAHS), the heparan sulfate is essential for supporting the cells in the hyaluronic acid hydrogel. Hyaluronic acid hydrogel alone is insufficient to support the in vitro expansion and maintenance of bile duct tree stem cells.

[0054] (4) The 3-O sulfuric acid modification of heparan sulfate is the main active structural component of heparan sulfate in Glycogel.

[0055] A third aspect of this invention establishes a method and expansion system for in vitro 2D adhesion or 3D organoid passage of bile duct tree stem cells, including the following analysis and optimization process:

[0056] (1) In a two-dimensional culture system based on Glycogel, the digested cells were mixed with the required culture medium and seeded onto pre-coated Matrigel plates at a ratio of 60,000 cells per 12-well plate. Bile duct tree stem cells were passaged at a 1:2 ratio on average every 4 days, and the cell doubling time was 3.5 days.

[0057] (2) In the three-dimensional culture conditions based on the Glycogel system, the digested cells were mixed with Matrigel at a 1:1 volume ratio for embedding, ensuring that each 10 μL contained 5000 BTSCs. Multiple 10 μL volumes were pipetted and added dropwise to the wells of the culture plate (with a certain distance between droplets). The plate was then inverted in an incubator, and after 2 hours of solidification, it was removed, covered with 10 pKM of medium, and Y-27632 Rock inhibitor was added. The plate was then placed upright in the incubator for further culture. Bile duct tree stem cells formed cystic organoids, and the organoid volume gradually increased with the number of culture days, with an average passage ratio of 1:3 every 4 days.

[0058] (3) During cell passage, evaluation is conducted on two aspects: firstly, by assessing the size and number of organoid clusters; and secondly, by collecting organoid samples and performing qPCR to evaluate gene expression levels. In a more specific embodiment, the method for digesting cells during cell passage includes:

[0059] (i) Primary bile duct tree stem cells cultured in KM: Y-27632 Rock inhibitor was added to the culture medium one day before digestion. The supernatant was removed the next day, and cell digestion solution TrypLe was added. Digestion was carried out for 15 minutes, during which 1 ml pipette was used to pipette every 3 minutes (until no adherent cells were visible on the culture plate). Digestion was stopped by adding culture medium, and single-cell pellets were obtained by centrifugation.

[0060] (ii) Primary bile tree stem cells cultured with pKM: Y-27632 Rock inhibitor was added to the culture medium one day before digestion. The supernatant was removed the next day, and cell digestion solution TrypLe was added. After digestion for about 5 minutes, the cells were mixed by pipetting with 1 ml pipette and observed under a microscope. After observing that the cytoplasm retracted and the intercellular spaces increased, culture medium was added to stop the digestion. Single-cell pellets were obtained by centrifugation.

[0061] (iii) pKM cultured bile tree stem cells (3D Matrigel): One day before digestion, Y-27632 Rock inhibitor was added to the culture medium. The next day, the supernatant was removed, and cell digestion solution TrypLe was added. The organoids were broken up by mechanical pipetting. After digestion for about 5 minutes, the mixture was mixed by pipetting with a 1 ml pipette and observed under a microscope. When a mixture of small cell clusters (composed of three to five cells) and single cells was seen, culture medium was added to stop the digestion. The cell pellet was collected by centrifugation.

[0062] Based on the aforementioned new discoveries of the inventors, this invention discloses a method for in vitro expansion of bile duct tree stem cells: bile duct tree stem cells are cultured in the bile duct tree stem cell culture medium of this invention to obtain a large number of proliferated cells. The cells express stem cell markers EpCAM, Sox17, PDX1, CK19, Prom1, and ICAM1; they do not express the marker for mature hepatocytes, Albumin, or the marker for hepatic progenitor cells, AFP. The method for in vitro expansion of bile duct tree stem cells includes: culturing bile duct tree stem cells in a basal culture medium supplemented 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 embodiments, the small molecule compounds and growth factors further include one or more selected from the following: Bix01294, SB431542, EGF, Noggin, and Wnt3a.

[0063] In a preferred embodiment of the present invention, the concentrations of the small molecule compounds and growth factors are shown in Table 1.

[0064] Table 1

[0065] Dosage Optimal amount For 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, 5 μM 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

[0066] Using the culture method and culture medium of this invention, organoids can be cultured / passaged in two-dimensional or three-dimensional culture systems. As a preferred embodiment of this invention, the culture / passaging is performed under three-dimensional conditions, thereby enabling the formation of organoids.

[0067] The bile duct tree stem cells obtained by the method of this invention can be cryopreserved, thawed, passaged, and maintained in culture for a long time. Furthermore, it should be understood that the bile duct tree stem cells (primary bile duct tree stem cells) used as the starting line in this invention can be primary bile duct tree stem cells from established lines, or they can be isolated from organisms.

[0068] It should be understood that other small molecule compounds or growth factors with the same function, besides those specifically listed in the embodiments of the present invention, should also be included in the present invention.

[0069] Similarly, analogues, homofunctional proteins (such as homofunctional proteins of growth factors), or compounds of the components specifically listed above, equivalent compounds inducing the same target, analogues, derivatives, and / or their salts, hydrates, or precursors may also be used to replace the components specifically listed above to achieve the same technical effect. These analogues, homofunctional proteins, or compounds should also be included in this invention. Analogues of compounds include, but are not limited to, isomers of compounds and racemic mixtures. Compounds have one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis or trans isomers. The term "salt" includes, but is not limited to, salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.; and (2) salts formed with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine, etc. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium). The term "precursor of the compound" refers to a compound, or a salt or solution of any of the aforementioned compounds, which, when applied or treated by appropriate methods, can be converted into a compound in a culture medium.

[0070] As a preferred embodiment of the present invention, the culture medium is further provided with components for preventing bacterial contamination of cell culture, particularly Gram-positive and Gram-negative bacterial contamination, such as, but not limited to, penicillin, streptomycin, etc.

[0071] The basal cell culture medium may include, but is not limited to, Kubota's stem cell growth medium (KM medium), Advanced RPMI 1640 medium, Advanced DMEM / F-12 medium, RPMI 1640 medium, DMEM medium, MEM medium, Neuronal basal medium, or Fischers medium. It should be understood that those skilled in the art are familiar with the preparation or purchase of the basal cell culture medium, and therefore, the basal cell culture medium is not limited to those exemplified in this invention.

[0072] In a preferred embodiment of the present invention, a bile duct tree stem cell in vitro expansion system and method with a defined composition and containing non-animal-derived Glycogel polysaccharide gel are also provided. The Glycogel system comprises a basal culture medium, a combination of growth factors, a combination of small molecule compounds, glycosaminoglycans, or a combination of these substances. The bile duct tree stem cell expansion method provided by the present invention not only provides a stable 3D microenvironment for bile duct tree stem cells but also increases the cell expansion space, reduces the adverse effects of external stimuli on stem cells, and maintains the pluripotency of pluripotent stem cells / the stemness of other stem cells while ensuring large-scale cell expansion. The present invention solves the problems of safety, high cost, and difficulty in large-scale expansion of bile duct tree stem cells in in vitro expansion, so as to provide a large number of seed cells for the clinical application of endoderm stem / progenitor cells. It also provides a reference expansion method for other difficult-to-culture stem cells.

[0073] In this invention, by using small molecule compounds to replace growth factors, the safety of stem cell expansion is greatly increased, while the cost of stem cell expansion is also effectively reduced.

[0074] This invention utilizes culture conditions involving activation / inhibition of the Wnt and TGFβ signaling pathways to expand organoid-like bile duct tree stem cells. It provides a method for expanding primary bile duct tree stem cells, expanded bile duct tree stem cell populations, and the medical applications of these expanded stem cells.

[0075] The present invention also provides a kit containing the culture medium described herein. In some preferred embodiments, the kit further includes instructions for use, thereby facilitating application by those skilled in the art in research or clinical practice.

[0076] Based on the new findings of this invention, a bile duct tree stem cell culture obtained by the method described in this invention, or bile duct tree stem cells or organoids isolated and purified from such bile duct tree stem cell culture, is also provided. The bile duct tree stem cells or organoids express stem cell markers EpCAM, Sox17, PDX1, CK19, Prom1, and ICAM1, but do not express the marker of mature hepatocytes, Albumin, or the marker of hepatic progenitor cells, AFP.

[0077] Methods for enriching or purifying cells from cell cultures are well known to those skilled in the art. For example, enrichment can be based on the morphological characteristics of bile duct tree stem cells; or selective collection can be based on specific proteins (such as EpCAM) or molecular markers expressed by bile duct tree stem cells (e.g., using specific antibodies or ligands). As an alternative implementation, flow cytometry can be used to separate and purify cells by means of molecular markers on the surface of bile duct tree stem cells.

[0078] The inventors' analysis has shown that the bile duct tree stem cells cultured in the described system possess multi-directional differentiation potential, capable of differentiating into various cell types such as mature hepatocytes, bile duct epithelial cells, and pancreatic endocrine cells. Therefore, the bile duct tree stem cells cultured in this invention have multiple applications.

[0079] The bile duct tree stem cells or organoids cultured in this invention can be used to prepare compositions (pharmaceutical compositions) that promote liver / bile duct / pancreas regeneration; compositions (pharmaceutical compositions) for treating liver injury (e.g., end-stage liver disease, cirrhosis, alcoholic liver disease, diabetes, obesity, acute liver failure, hepatitis, liver fibrosis, liver cancer, liver metabolic diseases, or liver failure); compositions for treating bile duct or pancreatic injury; and as in vitro models for studying liver / bile duct / pancreas-related diseases or drug efficacy, such as for studying drug transport, drug metabolism, liver formation, liver regeneration, liver / bile duct / pancreas toxicity testing, screening for toxic compounds of bile duct tree stem cells, and screening for compounds that regulate the function of bile duct tree stem cells.

[0080] The bile duct tree stem cells or organoids cultured in this invention can be used for liver / bile duct / pancreas toxicology research, and can also be applied to cell transplantation therapy for liver diseases, construction of bioartificial liver / bile duct / pancreas, detection of new drug toxicity (such as hepatotoxicity), efficacy evaluation, and drug target identification. They can provide bile duct tree stem cell sources or bile duct tree stem cell models for basic research and clinical applications in biology, medicine, and pharmacy. Their induction differentiation process can also provide a research platform for the development and differentiation process of human liver / bile duct / pancreas cells, with broad application prospects.

[0081] When needed, the bile duct tree stem cells or organoids cultured by this invention can be further applied to genetic engineering recombination to form recombinant cells. For example, in order to endow cells with further functions or characteristics, exogenous gene expression cassettes can be introduced into the cells, or gene knockout or gene editing can be performed on the cell genome.

[0082] The present invention also provides a composition (pharmaceutical) containing: an effective amount of the aforementioned bile duct tree stem cells (e.g., 1 × 10⁻⁶). 4 -1×10 12 One; the better 1×10 5 -1×10 10 The composition contains an effective amount of the described bile duct tree stem cells and a pharmaceutically acceptable carrier. The composition has no visible toxicity or side effects in animals.

[0083] The term "effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc. The carriers may also contain cell transfection reagents.

[0084] The present invention also provides a method for promoting liver / bile duct / pancreas regeneration or repair, the method comprising: administering an effective amount of bile duct tree stem cells cultured according to the present invention to a subject requiring treatment. The composition, when used for administration, is typically 1 × 10⁻⁶. 2 -1×10 10 Cells / kg body weight, optimal 1×10 3 -1×10 8 One cell / kg body weight is appropriate, but this also depends on the clinician's diagnosis and the severity of the patient's symptoms.

[0085] The present invention also provides a kit containing bile duct tree stem cells cultured according to the present invention or a composition containing such bile duct tree stem cells. Preferably, the kit also includes instructions for use, thereby facilitating use by those skilled in the art in research or clinical application.

[0086] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0087] The in vitro expansion of bile duct tree stem cells specifically includes: 1. Isolation, culture, and expansion of extrahepatic bile duct tree stem cells; 2. Screening of growth factor combinations and small molecule compound combinations to construct expansion culture media (the matrix includes: A. Matrigel 3D, B. hyaluronic acid and heparan sulfate); 3. Passaging of bile duct tree stem cells (primary cells are cultured in expansion culture medium, EpCAM sorting is performed during passaging, and Matrigel hydrogel is currently used for passaging).

[0088] Various aspects and embodiments of the invention are described in more detail below.

[0089] Materials, Methods and Terminology Explanation

[0090] Glycogel: A hydrogel solution that transitions from a fluid to a colloidal state based on glycosaminoglycans (polysaccharides). Small molecule compounds and growth factors are added according to the needs of cell expansion or differentiation and maturation to form the Glycogel system. It is used for the two-dimensional or three-dimensional expansion of bile duct tree stem cells and plays a supporting and protective role in the induction of bile duct tree stem cells into hepatocytes before and after expansion.

[0091] KM: Serum-free culture medium Kubota's Medium (Kubota's Stem Cell Growth Medium).

[0092] PKM: A bile duct tree stem cell expansion medium supplemented with serum-free medium and a combination of small molecule compounds and growth factors to promote the expansion of primary BTSCs. The small molecule compounds and growth factors included in PKM are:

[0093] Growth factors: 50 ng / ml EGF, 100 ng / ml R-Spondin1 (RSPO1), 100 ng / ml Noggin, 50 ng / ml Wnt3a;

[0094] Small molecule compounds: 0.5 μM Bix01294, 2 μM Bay K8644, 0.04 μM RG108, 2 μM MSB431542, 10 μM Forskolin, 1 μM A83-01;

[0095] 10P: PKM (10KPM) containing 10 small molecule compounds or growth factor components.

[0096] 8P: PKM containing 8 small molecule compounds or growth factor components.

[0097] Glycogel system: The amplification medium may contain or consist of the following:

[0098] (a) Matrix matrix (A, Matrigel hydrogel / solution; B, HAHS hydrogel / solution of 0.05% hyaluronic acid (HA) and 100 ng / ml heparan sulfate (HS);

[0099] (b) Growth factors: 50 ng / ml EGF, 100 ng / ml R-Spondin1, 100 ng / ml Noggin, 50 ng / ml Wnt3a;

[0100] (c) Small molecule compounds: 0.5 μM Bix01294, 2 μM Bay K8644, 0.04 μM RG108, 2 μM MSB431542, 10 μM Forskolin, 1 μM A83-01;

[0101] (d) KM medium (where the basal medium RPMI1640 is replaced by advanced DMEM / F12).

[0102] Two-dimensional (2D) culture: The system and method for the proliferation and passage of BTSCs in epidermal clones in a Glycogel system.

[0103] Three-dimensional (3D) culture: a system and method for organoid proliferation and passage of BTSCs embedded in Glycogel.

[0104] Cell characteristic identification: Two-dimensional cultured BTSCs are identified by gene identification, cell morphology, and surface markers; three-dimensional organoids are identified by organoid size, number, surface markers, and gene phenotype.

[0105] Hepatic differentiation and maturation of cells: Systems and methods for inducing differentiation and maturation of hepatocytes from two-dimensional or three-dimensional BTSCs based on the Glycogel system.

[0106] HMM: Hepatocyte maturation medium.

[0107] Cell wash (20ml) medium: RPMI 1640 medium supplemented with BSA, selenium, and double antibiotics (0.5g BSA, 30nM selenium, and 1× double antibiotics per 500ml RPMI 1640).

[0108] 4PKM: RG108 concentration 0.04μM, A83-01 concentration 1μM, FOSK concentration 10μM, BAY K8644 concentration 2μM.

[0109] In 4PKM+Wnt3a, the concentration of Wnt3a was 50 ng / ml.

[0110] In 4PKM+RSPO1, the concentration of RSPO1 was 100 ng / ml.

[0111] In 4PKM+EGF, the EGF concentration was 50 ng / ml.

[0112] In 4PKM+TGFβ, TGFβ refers to "the combination of TGFβ signaling pathway-related factors (composed of three small molecule compounds: A83-01 (1μM), SB431542 (2μM), and Noggin (100ng / ml)").

[0113] In 5PKM (4PKM + RSPO1), the concentration of RSPO1 was 100 ng / ml.

[0114] Example 1: Isolation, culture and identification of extrahepatic primary bile duct tree stem cells

[0115] Gallbladder and extrahepatic bile duct tissues from C57 WT mice were surgically obtained and placed in 20 ml of cold (4°C) cell wash medium supplemented with two antibiotics. The tissues were separated into small fragments with scissors and collected in 15 ml centrifuge tubes. After centrifugation at 1000 rpm for 2 minutes, the tissues were resuspended and digested with digestive enzymes (type IV collagenase / DNase) for 15 minutes (vibrating the centrifuge tube vigorously every 3 minutes). The tissues were then centrifuged at 800 rpm for 5 minutes and resuspended in 20 ml of cold (4°C) cell wash medium supplemented with two antibiotics. This step was repeated once. Finally, the tissues were resuspended in pKM amplification medium, filtered through a 40 μm filter, and the cells were counted and seeded into standard two-dimensional cell culture plates.

[0116] After 11 days of culture in serum-free KM medium, bile duct tree stem cells exhibited adherent clonal growth. Figure 2 A). The cells are densely packed and have a high nucleoplasmic ratio (scale bar 100 μm).

[0117] BTSC clones grown under KM culture conditions for 21 days were identified using antibodies against EpCAM, CK19, ALB, HNF4α, and CPS-1. After immunohistochemical staining, fluorescence microscopy revealed that cells at both the edge and interior of the 21-day-old BTSC clones expressed EpCAM and CK19 (green fluorescence), with stronger signal at the edge than at the center. Neither cell expressed ALB, HNF4α, nor CPS-1 (red fluorescence). DAPI (blue) was used as a backing stain to represent the cell nucleus. Figure 2 B).

[0118] Bile duct tree stem cells (BTSCs) were collected on day 21 for qPCR identification. Gene expression levels showed that day 21 BTSCs expressed EpCAM, SOX17, PDX1, CK19, Prom1 (endoderm stem cell markers, with EpCAM, SOX17, and PDX1 being surface markers of BTSCs), and ICAM1 (stem cell marker); they did not express ALB (mature hepatocyte marker) or AFP. Primary hepatocytes were used as a control. Figure 2 C).

[0119] Example 2: Construction of Glycogel system: Screening of growth factor combinations and small molecule compound combinations to construct amplification culture medium.

[0120] The amplification medium may include the basal medium Advanced DMEM / F-12. The basal medium may be supplemented with medium supplements and / or one or more additional components, including selenic acid (3 x 10⁻⁶). -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 a mixture of free fatty acids.

[0121] In determining the components of pKM in the Glycogel system, the inventors studied the gene expression profile of BTSCs and, combined with experimental verification, identified four growth factors and six small molecule compounds through extensive analysis and screening. These included: EGF, R-Spindin1, Noggin, Wnt3a, Bix01294, Bay K8644, RG108, SB431542, A83-01, and Forskolin. These growth factors and small molecule compounds were added to KM to construct the amplification medium pKM, and BTSCs were cultured. Figure 3 A).

[0122] For the determination of hydrogel composition in the Glycogel system, the selected scaffold matrix includes chemically limited components, such as silk fibroin, collagen, or hyaluronic acid hydrogels, as well as non-chemically defined complex protein hydrogels, including Matrigel. Suitable complex protein hydrogels may contain extracellular matrix components such as laminin, collagen IV, fibronectin, and hyaluronic acid, heparin sulfate proteoglycans. Two types are used here: one is a plate-layout, embedding-compatible A-scaffold matrix: a hydrogel of extracellular matrix proteins derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, which is commercially available and includes Matrigel (Corning Life Sciences). The other is a B-scaffold matrix added to the culture medium, an HAHS hydrogel using an appropriate ratio of hyaluronic acid (HA) and heparin sulfate (HS).

[0123] Example 3: In vitro culture of primary BTSCs in pKM

[0124] Primary culture does not require matrix gel; however, during passage, scaffold matrix is ​​added to assist growth due to poor adhesion of bile duct tree stem cells. BTSCs grow slowly directly in KM medium (cell count at day 14: 2 × 10⁶ cells per C57 WT mouse). 4 (Cells), considering adding amplification factors to KM to construct a modified culture medium pKM to achieve primary cell amplification.

[0125] Following the same method as in Example 1, after surgically obtaining gallbladder and extrahepatic bile duct tissues, which were then separated, enzyme-digested, and resuspended in serum-free modified pKM medium, they were seeded into ordinary two-dimensional cell culture plates. After 7 days of culture in serum-free modified pKM medium, BTSCs exhibited adherent clonal growth. Figure 3 B). Cells are densely packed, with most cells undergoing nuclear division (scale bar 100 μm).

[0126] The number of BTSCs cultured in pKM medium increased significantly. On day 7, cell counts showed 4.2 × 10⁶ cells per C57 WT mouse. 5 Cells. 20 times more cells than collected on day 14, and if the time difference is taken into account, 50 times more cells than in KM maintenance medium. Figure 3 C).

[0127] Primary BTSCs cultured in pKM on day 7 were collected for qPCR identification, with primary hepatocytes used as controls for gene expression detection. Regarding gene expression levels, compared to BTSCs cultured in KM, BTSCs cultured in pKM on day 7 showed decreased EpCAM expression (p<0.001), but significantly increased SOX17 expression (p<0.001). There were no significant differences in PDX1, AFP, and albumin expression.

[0128] This result indicates that 10 pKM, while promoting cell proliferation, also increased the stemness of BTSCs' ability to differentiate into hepatocytes. Figure 3 D). The decrease in EpCAM expression and the increase in SOX17 expression indicate an increase in the liver's ability to differentiate into stem cells.

[0129] Example 4: Two-dimensional (2D) passage of bile duct tree stem cells based on the Glycogel system

[0130] At passage, the culture medium of primary BTSCs was removed, and cells were digested using TrypLE (GIBCO, 12604021) at 37°C, with pipetting every 3 minutes (1 ml TrypLE per well in a 6-well plate). Digested cells were collected in centrifuge tubes, centrifuged at 200g for 5 minutes, resuspended in pKM, and filtered through a 40 μm filter. Primary BTSCs were then collected for EpCAM. + EpCAM obtained by flow cytometry + The target cells are used to remove any mesenchymal stem cells that may have been present during the isolation process. Primary BTSCs cultured using either KM or pKM methods are used for EpCAM. + After sorting, the positive rate was above 84%, which is consistent with the previous experimental results. Figure 4 (AB). Sorted cells were collected in a culture medium containing 10% serum. After centrifugation at 200g for 5 min, the cells were resuspended in amplification medium.

[0131] Due to the problem that primary bile duct tree stem cells (BTSCs) hardly adhere to the culture vessel after digestion, pKM needs to be combined with Glycogel to achieve two-dimensional passaging of BTSCs during passage. In this invention, two-dimensional passaging expansion of BTSCs based on Matrigel (of uncertain composition) and Glycogel based on HAHS (of defined chemical composition) were achieved respectively:

[0132] 1. 2D passage amplification based on Matrigel hydrogel / solution

[0133] Primary BTSCs were enzymatically digested and then flow-cytosored to obtain EpCAM. + BTSCs were passaged. After counting, the BTSCs were cultured in Matrigel-coated culture plates. When the cells reached 80%-90% confluence, they were passaged at a ratio of 1:2 (the passage ratio was determined based on the actual number of cells). The culture medium was changed every 48 hours and cultured for 4-7 days for subsequent digestion.

[0134] First-generation BTSCs cultured under these conditions were collected for qPCR identification, with primary hepatocytes used as a control. In terms of gene expression levels, compared to BTSCs cultured in Matrigel (pKM), the expression levels of EpCAM, SOX17, PDX1, and ICAM1 in the first-generation BTSCs cultured in pKM were similar to those in the KM group, but AFP and ALB were still not expressed. Figure 4 C).

[0135] P0 generation BTSCs possess hepatic differentiation potential (expressing SOX17) and pancreatic differentiation potential (expressing PDX1). BTSCs amplified by pKM exhibit better proliferative capacity and increased hepatic differentiation potential (manifested as increased expression of SOX17 and ICAM1).

[0136] The above results indicate that BTSCs expanded using the Glycogel system have better proliferative capacity and hepatic differentiation capacity.

[0137] 2. Modified 2D passage amplification of BTSCs based on HAHS hydrogel / solution

[0138] To avoid the potential tumorigenic risk associated with Matrigel, a HAHS (hyaluronic acid and heparan sulfate) backbone matrix was constructed as the in vitro amplification system for BTSCs. The amplification medium was supplemented with a mixture of sulfuric acid-modified heparan sulfate and 0.05% HA. Primary BTSCs were enzymatically digested and then flow-cytosorized to obtain EpCAM. + BTSCs were passaged. After counting, the BTSCs were cultured in adherent culture plates. When the cells reached 80%-90% confluence, they were passaged at a ratio of 1:2 (the passage ratio was determined based on the actual number of cells). The culture medium was changed every 48 hours, and the cells were cultured for 4-7 days for subsequent digestion. The cells were effectively expanded, with an expansion rate of approximately 3 times per passage. Figure 5 ).

[0139] Example 5: Three-dimensional (3D) passage of Glycogel bile duct tree stem cells

[0140] To obtain bile duct tree stem cell organoids, P0 generation BTSCs were mixed with Matrigel at a 1:1 ratio and added dropwise to a culture plate containing 5000 BTSCs per 10 μL. The plate was inverted and allowed to solidify before being placed upright and covered with 10 pKM of medium. Organoid formation was detectable under a microscope after one day. After three days, some reached a visible size. BTSC organoids exhibited cystic growth, composed of a single layer of cuboidal epithelium and an inner cell mass. After five days, the maximum diameter of BTSC organoids reached 1000 μm. Passage was performed at a 1:3 ratio every 3-5 days. The organoid morphology was stably maintained even at P6 level. Compared to P1 BTSCs, the number of P5 BTSCs was expected to increase 80-fold. Figure 6 A, E).

[0141] Organoids of P1 BTSCs cultured under KM conditions on day 3 were identified using antibodies against EpCAM, CK19, and Ki67. After immunofluorescence staining, P1 BTSCs organoids on day 3 showed expression of EPCAM, CK19, and Ki67 (green fluorescence) under a fluorescence microscope. DAPI (blue) was used as a backing to represent the cell nucleus. The viability of BTSC organoids was determined using live / dead staining. Organoids formed from first-generation (P1) BTSCs were stained using a live / dead detection kit. Live cells (with esterase activity) stained green, and dead cells (with damaged plasma membranes) stained red. At 10x magnification, the vast majority of cells in the organoids were stained green (live cells), while a small number stained red (dead cells). Figure 6 BC).

[0142] P1 BTSC organoids were collected on day 5 for qPCR identification. In terms of gene expression levels, compared with primary BTSCs (P0 BTSCs) cultured in pKM, the expression levels of SOX17 and ICAM1 in day 5 P1 BTSC organoids were slightly decreased, and EpCAM and CK19 were slightly increased, but the differences were not statistically significant. PDX1 expression was increased (p<0.05), but AFP and ALB were still not expressed. Primary hepatocytes were used as a control. Figure 6 D).

[0143] qPCR was used to identify BTSC organoids from different passages, with primary hepatocytes as a control. The results showed that the expression of surface gene markers EpCAM, SOX17, ICAM1, and CK19 in BTSC organoids remained stable across different passages, demonstrating that this method can stably amplify BTSCs using the 3D Matrigel 10PKM system. The expression level of PDX1 was upregulated during passage, indicating that 3D-amplified BTSCs maintained their ability to differentiate into hepatocytes while also enhancing their ability to differentiate into pancreatic cells. Figure 6 F).

[0144] Example 6: RG108, A83-01, Forskolin, and Bay K8644 (RAFB) are essential growth factors for the expansion of bile duct tree stem cells.

[0145] In this embodiment, the necessary growth factors for the expansion of bile duct tree stem cells were verified. Except for the different components, the other conditions were the same as in Example 5.

[0146] BTSCs cultured in KM for 14 days were digested, embedded, and passaged. P1BTSC organoids obtained under 10PKM conditions were larger and more numerous than those obtained under 10PKM-A83-01-FOSK conditions (minus A83-01 and Forskolin, hereinafter the same). Figure 7 (AB) indicates that A83-01 and FOSK are necessary for the expansion of bile duct tree stem cells.

[0147] The inventors optimized the 10PKM compound, dividing the growth factor and small molecule compound into eight groups according to their pathways of action for screening. The groupings were compared based on the organoid formation ability and gene expression levels of P1 BTSCs. The groups included: 10PKM-EGF, 10PKM-TGFβ (A83-01, Noggin, SB431542), 10PKM-WNT (RSPO1, WNT3a), 10PKM-cAMP, 10PKM-bix01294, 10PKM-Bay K8644, 10PKM-RG108, with 10PKM serving as a control.

[0148] A83-01 is an inhibitor of TGFβ kinase / activin receptor-like kinase (ALK 5) (IC50 = 12 nM) that prevents Smad2 / 3 phosphorylation and inhibits TGFβ-induced growth. A83-01 blocks Smad2 phosphorylation and inhibits TGF-β-induced epithelial-mesenchymal transition. Furthermore, A83-01 inhibits transcriptional activity induced by TGFβ type I receptor ALK-5, activin type IB receptor ALK-4, and nodular type I receptor ALK-7. This invention demonstrates that A83-01 is essential for the amplification of BTSCs in the Glycogel system.

[0149] Forskolin is a naturally occurring diterpenoid found in the Indian plant *Forsythia suspensa* that activates adenylate cyclase, thus increasing intracellular cAMP concentration. The second messenger cAMP activates cAMP-dependent protein kinases (PKA or cAPK), regulating multiple cellular mechanisms such as gene transcription, ion transport, and protein phosphorylation, and is an essential component for the amplification of BTSCs in the Glycogel system.

[0150] Then, in the screening experiments of 8 groups, the number of 10PKM-RG108 cells decreased significantly. Figure 7 CD), indicating that RG108 is necessary for the expansion of bile duct tree stem cells.

[0151] RG108, as a DNA methyltransferase inhibitor, inhibits DNA methylation and activates the expression of cell proliferation-related genes, thereby promoting the proliferation of BTSCs.

[0152] The inventors collected P1 BTSCs from each group on day 5 for qPCR identification and found that under the 10 pkM-Bay K8644 condition, the stemness gene PROM1 and the proliferation gene KI67 of BTSCs were significantly decreased, but EPCAM was slightly increased, and PDX1 and SOX17 were almost unchanged. This suggests that Bay K8644 is necessary for the expansion of bile duct tree stem cells. Figure 7 E).

[0153] Bay K8644, a classic calcium channel inhibitor, is crucial for preventing the differentiation of BTSCs during amplification.

[0154] Example 7: RSPO1 can replace multiple components to expand bile duct tree stem cells

[0155] The selected RG108, A83-01, FOSK, and BAY K8644 were used as 4PKM medium for experiments. Except for changes in components, other conditions were the same as in Example 5 (3D) or Example 1 (2D). KM medium could not achieve 2D passage amplification of BTSCs, nor could it support the formation of organoids in 3D. Compared with KM medium, 4PKM could maintain the passage and amplification of BTSC organoids in 3D conditions, but further optimization is needed to maintain BTSC clonal growth in 2D conditions.

[0156] Next, the inventors further optimized the proliferation effect of 4PKM in 2D and 3D cells. Through repeated screening and component adjustments, they found that the addition of growth factors from the WNT signaling pathway significantly increased cell proliferation. Figure 7 FG, Figure 8 A). Therefore, it is beneficial to optimize 4PKM by adding additional growth factors.

[0157] By dividing the cells into groups: 4PKM+Wnt3a, 4PKM+RSPO1, 4PKM+EGF, and 4PKM+TGFβ, P1 BTSCs from each group were collected on day 5 for qPCR identification. The results showed that BTSCs under the 4PKM+RSPO1 condition had higher expression levels of stemness genes SOX17, PROM1, and proliferation gene KI67 than those in other groups, thus confirming that RSPO1 is a viable alternative factor for biliary tree stem cell expansion. While the 4PKM+EGF group showed the highest KI67 expression level, its PROM1 level decreased, making it unsuitable for BTSC expansion and maintenance. Figure 8 B).

[0158] Under 5 PKM (4 PKM + RSPO1) conditions, primary bile duct tree stem cells exhibited adherent clonal growth. Cells were densely packed. Figure 8C), most cells were in the process of nuclear division (scale bar 100 μm). Next, the inventors used the Ki67 antibody to perform immunofluorescence staining on P1 BTSC organoids cultured for 7 days under 5 pkM conditions. Fluorescence microscopy showed that the primary BTSCs on day 7 expressed Ki67 (green fluorescence), with DAPI (blue) used as a backing to represent the cell nucleus (…). Figure 8 D). BTSCs cultured at 5 pkM on day 7 were collected for qPCR identification. In terms of gene expression levels, compared with BTSCs cultured at 10 pkM, BTSCs cultured at 5 pkM on day 7 showed stable or slightly elevated levels of EpCAM, PDX1, SOX17, PROM1, and KI67, but still did not express ALB. Primary hepatocytes were used as a control for gene expression. Figure 8 E).

[0159] Therefore, 5PKM with added R-spondin1 can achieve 2D BTSC clonal growth and passage, and can also significantly improve the 3D amplification effect of 4PKM.

[0160] Furthermore, the inventors 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) to achieve the amplification and passage of BTSCs.

[0161] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for in vitro culture of bile duct tree stem cells, comprising culturing bile duct tree stem cells in a culture medium; wherein, The culture medium includes: a basal medium, and small molecule compounds and growth factors: RG108, A83-01, Forskolin, BayK8644, and R-Spondin1, at concentrations of which in the basal medium are: The basal culture medium is a serum-free culture medium.

2. The method as described in claim 1, characterized in that, The small molecule compounds and growth factors also include: Bix01294, SB431542, EGF, Noggin, and Wnt3a, with the following concentrations in the basal medium: 。 3. The method as described in claim 2, characterized in that, The basal culture medium is selected from: Kubota's stem cell growth medium, Advanced RPMI 1640 medium, Advanced DMEM / F-12 medium, RPMI 1640 medium, DMEM medium, MEM medium or Fischers medium.

4. The method as described in claim 3, characterized in that, The basal culture medium is Kubota's stem cell growth medium or Advanced RPMI 1640 medium.

5. The method as described in claim 1, characterized in that, The concentrations of the small molecule compounds and growth factors in the basal culture medium are as follows: 。 6. The method as described in claim 2, characterized in that, The concentrations of the small molecule compounds and growth factors in the basal culture medium are as follows: 。 7. The method as described in claim 1 or 2, characterized in that, Amplification or subculture is performed using the method described above.

8. The method as described in claim 7, characterized in that, 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.

9. The method as described in claim 7, characterized in that, The culture is carried out in a fluid or colloidal hydrogel based on glycosaminoglycans.

10. The method as described in claim 9, characterized in that, The skeletal matrix of the hydrogel includes those selected from: Matrigel, a mixed hydrogel of hyaluronic acid and heparin sulfate, collagen hydrogel, hyaluronic acid hydrogel, and silk fibroin hydrogel.

11. A culture medium for in vitro culture of bile duct tree stem cells, comprising: The basal culture medium, along with small molecule compounds and growth factors: RG108, A83-01, Forskolin, Bay K8644, and R-Spondin1, were present at the following concentrations in the basal culture medium: The basal culture medium is a serum-free culture medium.

12. The culture medium as described in claim 11, characterized in that, The small molecule compounds and growth factors also include: Bix01294, SB431542, EGF, Noggin, and Wnt3a, with the following concentrations in the basal medium: 。 13. The culture medium as described in claim 11, characterized in that, The concentrations of the small molecule compounds and growth factors in the basal culture medium are as follows: 。 14. The culture medium as described in claim 12, characterized in that, The concentrations of the small molecule compounds and growth factors in the basal culture medium are as follows: 。 15. A culture system for bile duct tree stem cells, comprising: A hydrogel containing a scaffold matrix, and a culture medium according to any one of claims 11-14.

16. The culture system as described in claim 15, characterized in that, The scaffold matrix includes materials selected from: Matrigel, a mixed hydrogel of hyaluronic acid and heparin sulfate, collagen hydrogel, hyaluronic acid hydrogel, and silk fibroin hydrogel.

17. Use of the culture medium according to any one of claims 11-14 for culturing bile duct tree stem cells.

18. The use as described in claim 17, characterized in that, The cultivation includes: Expanding bile duct tree stem cells; Passage culture of bile duct tree stem cells; Preparation of organoids; or Passage and stable culture of bile duct tree stem cell organoids.

19. A kit for in vitro culture of bile duct tree stem cells, comprising: The culture medium according to any one of claims 11-14; or The culture system for bile duct tree stem cells as described in claim 15 or 16.

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