Application of dihydroartemisinin in construction of liver organoid

Abstract

The invention discloses application of dihydroartemisinin in construction of liver organoid, which is characterized in that by adding dihydroartemisinin into a liver organoid culture medium, the maturity of the liver organoid can be obviously improved, the maturity period is shortened, and the liver function characteristics (such as metabolic function and gene expression maturity) of the organoid are enhanced. The defects of an existing liver organ model in the aspect of simulating primary liver functions are effectively overcome, a more accurate in-vitro model is provided for research of liver related diseases, and a new platform and possibility are provided for development of liver regeneration and treatment methods.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of dihydroartemisinin in the construction of liver organoids. Background Technology

[0002] For a long time, research on human organ development and disease mechanisms has mainly relied on animal models and two-dimensional (2D) cell culture models. Although animal models can simulate the dynamic cell-cell and cell-matrix interactions of complete organs, due to species differences, especially in pathogenicity and immune response, there are significant differences between animal and human models, which limits their application in human disease research [1]. Although two-dimensional cell culture helps to reveal cellular mechanisms, long-term culture can lead to changes in cell morphology, loss of hepatocyte polarity, and decreased metabolic function, which in turn affects its effectiveness in maintaining liver characteristics and function [2][3].

[0003] In order to overcome these limitations, new research models such as three-dimensional (3D) cell culture, liver organoids and liver organ microarrays have gradually emerged in recent years. Compared with traditional two-dimensional culture, three-dimensional cell culture can more realistically simulate the physiological state of the liver, maintain the cell polarity, morphology and function of the liver, and thus provide a more effective research tool for liver development, disease research and drug screening[3]. In addition, with the advancement of stem cell technology, the construction of liver organoids has become possible. Through organoids derived from pluripotent stem cells such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), key features of the human liver, such as cell composition and physiological function, can be simulated, which greatly promotes the research and application in the fields of disease model establishment, drug development and regenerative medicine[4].

[0004] However, organoids based on pluripotent stem cells still face some challenges. First, organoids derived from iPSCs are not mature enough, usually exhibiting fetal-like tissue characteristics and not yet fully mimicking the mature state of the adult liver. The limitations of in vitro culture conditions, especially the lack of specific in vivo environmental factors, inhibit further maturation of organoids[5]. In addition, the life cycle of organoids is often affected by nutrient diffusion limitations. Once a certain volume is reached, the cells cannot obtain sufficient nutrient support, thus affecting their long-term development and stability[6].

[0005] To address these issues, researchers have promoted organoid maturation by regulating culture conditions, introducing small molecule factors, non-solid cells (such as endothelial cells and mesenchymal stem cells), and acellular matrix (ECM) components, thereby enhancing their application value in drug screening and disease modeling.

[0006] Nevertheless, current organoids still lag behind adult livers in terms of maturity, structure, and function. Therefore, how to further promote the maturation of organoids, especially in terms of cell differentiation, metabolic function, and physiological function, remains the core challenge of current liver organoid research. Summary of the Invention

[0007] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide the use of dihydroartemisinin in the construction of liver organoids.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a liver organoid culture medium comprising: dihydroartemisinin and a cell growth medium.

[0010] Dihydroartemisinin (DHA) is a reduction product of artemisinin and possesses significant antimalarial activity. It is widely used in the treatment of malaria, especially against drug-resistant Plasmodium infections. Its molecular formula is C6H2O. 15 H 24 O5, chemical structural formula is

[0011] The molecular weight is 284.348.

[0012] In some embodiments, the cell growth medium includes Roswell Park Memorial Institute (RPMI) medium; Dulbecco modified Eagle medium (DMEM); derivatives of DMEM (e.g., Iscove modified Dulbecco medium (IMDM) or Advanced Dulbecco modified Eagle medium (ADMEM)); or combinations thereof.

[0013] The liver organoid culture medium provided in the first aspect of this invention is used for the passage culture of liver organoids. "Organoid passage" refers to the process of dissociating organoids cultured to a certain size and in good condition into small cell clusters or single-cell suspensions using physical mechanical dispersing or combined enzymatic digestion, followed by re-inoculation and continued organoid culture. During the passage of liver organoids, the addition of the small chemical molecule DHA to the culture medium can promote hepatic differentiation and functional maturation of the liver organoids.

[0014] In some embodiments, the concentration of dihydroartemisinin in the liver organoid culture medium is 1-100 μM. Specifically, the concentration of dihydroartemisinin can be 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, or a range of any two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0015] In some embodiments, the concentration of dihydroartemisinin in the liver organoid culture medium is 1-50 μM. Specifically, the concentration of dihydroartemisinin can be 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0016] In some embodiments, the concentration of dihydroartemisinin in the liver organoid culture medium is 5-15 μM. Specifically, the concentration of dihydroartemisinin can be 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0017] In some embodiments, the liver organoid culture medium further contains FBS, ITSE, NEAA, L-glutamine additive, nicotinamide, GSK-3 inhibitor, TGF-β signaling pathway inhibitor, forskolin, fibroblast growth factor, bone morphogenetic protein, and epidermal growth factor.

[0018] Insulin-Transferrin-Selenium-Ethanolamine (ITSE) is a mixture of insulin, transferrin, selenium, and ethanolamine. As a basal culture medium supplement, it enhances the performance of serum-free media and reduces the need for serum supplementation in conventional cell culture media.

[0019] Non-essential amino acid solution (NEAA) includes seven non-essential amino acids: L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine. It can effectively improve the cell culture medium ratio, reduce the side effects of cells producing non-essential amino acids themselves during cell culture, and promote cell proliferation and metabolism. It is one of the commonly used additives in cell culture.

[0020] L-glutamine additive is GlutaMAX supplement, an alternative to L-glutamine with better stability. It contains L-alanyl-L-glutamine dipeptide, which cells will cleave the dipeptide bond to release L-glutamine as needed.

[0021] In some embodiments, the GSK-3 inhibitor is selected from at least one of SB216763 (CAS No.:280744-09-4), TWS119 (CAS No.:601514-19-6), NP031112 (CAS No.:865854-05-3), CHIR-98014 (CAS No.:252935-94-7), AZD2858 (CAS No.:486424-20-8), AZD1080 (CAS No.:612487-72-6), SB415286 (CAS No.:264218-23-7), LY2090314 (CAS No.:603288-22-8), and CHIR99021 (CAS No.:252917-06-9).

[0022] In some embodiments, the GSK-3 inhibitor is CHIR99021.

[0023] In some embodiments, the TGF-β signaling pathway inhibitor is selected from LY2157299 (CAS No.:700874-72-2), EW-7197 (CAS No.:1352608-82-2), LY3200882 (CAS No.:1898283-02-7), SB-431542 (CAS No.:301836-41-9), LY2109761 (CAS No.:700874-71-1), TP-0427736 (CAS No.:864374-00-5), IN-1130 (CAS No.:868612-83-3), R-268712 (CAS No.:879487-87-3), A-83-01 (CAS No.:700874-82-2), EW-7197 (CAS No.:1352608-82-2), LY3200882 (CAS No.:1898283-02-7), SB-431542 (CAS No.:301836-41-9), LY2109761 (CAS No.:700874-71-1), TP-0427736 (CAS No.:864374-00-5), IN-1130 (CAS No.:868612-83-3), R-268712 (CAS No.:879487-87-3), A-83-01 (CAS No.:879487-87-3), and EW-7197 (CAS No.:1352608-82-2). At least one of the following: No.:909910-43-6), SB-525334 (CAS No.:356559-20-1), GW788388 (CAS No.:452342-67-5), RepSox (CAS No.:446859-33-2), A-77-01 (CAS No.:607737-87-1), SB-505124 (CAS No.:694433-59-5), SD-208 (CAS No.:627536-09-8), and LY364947 (CAS No.:396129-53-6).

[0024] In some embodiments, the TGF-β signaling pathway inhibitor is SB431542.

[0025] In some embodiments, the fibroblast growth factor is selected from at least one of FGF1, FGF2, FGB, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0026] In some embodiments, the fibroblast growth factor is FGF4.

[0027] In some embodiments, the bone morphogenetic protein is selected from at least one of BMP4 and BMP2.

[0028] In some embodiments, the bone morphogenetic protein is BMP4.

[0029] In some embodiments, the liver organoid culture medium further contains FBS, ITSE, NEAA, L-glutamine additive, nicotinamide, CHIR99021, SB431542, trichomoniasis, FGF4, BMP4, and epidermal growth factor.

[0030] In some embodiments, the liver organoid culture medium further contains 5%-15% FBS, 0.5%-2% ITSE, 0.5%-2% NEAA, 0.5%-2% L-glutamine additive, 5-15mM nicotinamide, 4-6μM CHIR99021, 5-15μM SB431542, 5-15μM trichomoniasis, 10-30ng / mL FGF4, 10-30ng / mL BMP4, and 10-30ng / mL epidermal growth factor.

[0031] Specifically, the volume concentration of FBS can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or a range of any two of the above values, not limited to the listed values; other unlisted values ​​within this range also apply. The volume concentration of ITSE can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values, not limited to the listed values; other unlisted values ​​within this range also apply. The volume concentration of the NEAA can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values, not limited to the listed values; other unlisted values ​​within this range also apply. The volume concentration of the L-glutamine additive can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values, not limited to the listed values; other unlisted values ​​within this range also apply. The concentration of nicotinamide can be 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, or a range of any two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable. The concentration of CHIR99021 can be 4 μM, 4.1 μM, 4.2 μM, 4.3 μM, 4.4 μM, 4.5 μM, 4.6 μM, 4.7 μM, 4.8 μM, 4.9 μM, 5 μM, 5.1 μM, 5.2 μM, 5.3 μM, 5.4 μM, 5.5 μM, 5.6 μM, 5.7 μM, 5.8 μM, 5.9 μM, 6 μM, or a range of any two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable. The concentration of SB431542 can be 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 11μM, 12μM, 13μM, 14μM, 15μM, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable.The concentration of the laryngin can be 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, or any range of two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable. The concentration of the FGF4 can be 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, or any range of two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable. The concentration of BMP4 can be 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within the range are also applicable. The concentration of the epidermal growth factor can be 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within the range are also applicable.

[0032] In some embodiments, the liver organoid culture medium further contains 10% FBS, 1% ITSE, 1% NEAA, 1% L-glutamine additive, 10 mM nicotinamide, 5 μM CHIR99021, 10 μM SB431542, 10 μM trichomoniasis, 20 ng / mL FGF4, 20 ng / mL BMP4, and 20 ng / mL epidermal growth factor.

[0033] A second aspect of the present invention provides a culture medium combination, comprising the liver organoid culture medium and the hepatocyte maturation culture medium of the first aspect.

[0034] In some embodiments, the hepatocyte maturation culture medium comprises hepatocyte culture medium, hepatocyte growth factor (HGF), tumor suppressor factor (oncostatin M), glucocorticoid receptor agonist, fibroblast growth factor, and B27 additive.

[0035] Hepatocyte culture medium refers to a culture medium suitable for culturing hepatocytes. Any existing hepatocyte culture medium can be used, such as the Hepatocyte Culture Medium Bullet Kit. TM Any one of Lonza (HCM), HepatoZYME-SFM (ThermoFisher Scientific, HepatoZYME), or Cellartis Power Primary HEP Medium (Cellartis, Primary HEP). In some embodiments, the hepatocyte culture medium is a Hepatocyte Culture Medium Bullet Kit. TM (Lonza, HCM). The B27 additive is a well-defined serum-free additive containing antioxidants, proteins, vitamins, and fatty acids. Original formulations of the serum-free neural cell culture additive can be found in Brewer et al., J Neuroscience Res 35:567-576, 1993 and Brewer and Cotman, Brain Res 494:65-74, 1989.

[0036] In some embodiments, the glucocorticoid receptor agonist is dexamethasone.

[0037] In some embodiments, the fibroblast growth factor is selected from at least one of FGF1, FGF2, FGB, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0038] In some embodiments, the fibroblast growth factor is FGF4.

[0039] In some embodiments, the hepatocyte maturation culture medium contains HGF, tumor suppressor factor, dexamethasone, FGF4, and B27 additives.

[0040] In some embodiments, the hepatocyte maturation culture medium contains 10-30 ng / mL HGF, 40-60 ng / mL tumor suppressor, 50-150 nM dexamethasone, 10-30 ng / mL FGF4, and 0.5%-2% B27 additive by volume.

[0041] Specifically, the concentration of HGF can be 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable. The concentration of the tumor suppressor factor may be 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, 60 ng / mL, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within the range are also applicable. The concentration of dexamethasone can be 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within the range are also applicable. The concentration of FGF4 can be 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within the range are also applicable. The volume concentration of the B27 additive can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values, and is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0042] In some embodiments, the hepatocyte maturation culture medium contains 20 ng / mL HGF, 50 ng / mL tumor suppressor, 100 nM dexamethasone, 20 ng / mL FGF4, and 1% B27 additive (v / v).

[0043] A third aspect of the present invention provides a method for constructing liver organoids, comprising: performing a first-stage culture using the liver organoid culture medium of the first aspect during the passage of liver organoids.

[0044] In some embodiments, the construction method includes: dissociating liver organoids into small cell clusters or single-cell suspensions by physical mechanical blowing or combined enzymatic digestion, and then re-inoculating them into the liver organoid culture medium of the first aspect for organoid culture.

[0045] In some implementations, the first-stage culture period is 5-7 days. Specifically, the first-stage culture period can be 5 days, 6 days, 7 days, or a range of any two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0046] In some implementations, a matrix gel is also added to the culture system during the first stage of culture. The matrix gel is added to the culture system after cell seeding to provide three-dimensional culture substrate support for the cells.

[0047] In some embodiments, the matrix adhesive is selected from at least one of Laminin-521, Vitronectin, and Matrigel.

[0048] In some embodiments, the matrix adhesive is Matrigel.

[0049] In some embodiments, the volume concentration of the matrix adhesive is 4%-6%. Specifically, the volume concentration of the matrix adhesive can be 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, or a range of any two of the above values. It is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0050] In some embodiments, the construction method further includes a second-stage culture using the hepatocyte maturation medium from the culture medium combination of the second aspect.

[0051] In some embodiments, the construction method further includes collecting cells from the culture after the first stage of culture and adding hepatocyte maturation culture medium for a second stage of culture.

[0052] In some implementations, the second-stage culture period is 7-9 days. Specifically, the first-stage culture period can be 7 days, 8 days, 9 days, or any range of two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0053] In some implementations, a matrix gel is also added to the culture system during the second stage of culture. The matrix gel is added to the culture system after cell seeding to provide three-dimensional culture substrate support for the cells.

[0054] In some embodiments, the matrix adhesive is selected from at least one of Laminin-521, Vitronectin, and Matrigel.

[0055] In some embodiments, the matrix adhesive is Matrigel.

[0056] In some embodiments, the volume concentration of the matrix adhesive is 4%-6%. Specifically, the volume concentration of the matrix adhesive can be 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, or a range of any two of the above values. It is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0057] In some embodiments, the liver organoids are liver organoids derived from human pluripotent stem cells. Pluripotent stem cells are stem cells that possess the pluripotency to differentiate into all cells present in an organism, namely the three germ layers (endoderm, mesoderm, and ectoderm), and also have proliferative capacity. There are no particular limitations on the aforementioned pluripotent stem cells; examples include embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained through nuclear transfer (ntES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells. The human embryonic stem cells described in this invention are not human embryonic stem cells isolated or obtained from human embryos that have undergone in vivo development, nor are they stem cells isolated or obtained from human embryos that have not undergone in vivo development and have been fertilized for more than 14 days.

[0058] In some embodiments, the pluripotent stem cells are selected from at least one of ES cells, ntES cells, and iPS cells.

[0059] In some embodiments, the pluripotent stem cells are iPS cells or ES cells.

[0060] The fourth aspect of this invention provides the use of dihydroartemisinin in any of the following aspects:

[0061] Preparation of liver organoid culture medium;

[0062] Construct liver organoids.

[0063] The fifth aspect of the present invention provides a liver organoid obtained according to the construction method described in the third aspect.

[0064] The beneficial effects of this invention are as follows: This invention provides the use of dihydroartemisinin in the construction of liver organoids. By adding dihydroartemisinin to the liver organoid culture medium, the maturity of liver organoids can be significantly improved, the maturation cycle shortened, and the liver function characteristics of the organoids (such as metabolic function and gene expression maturity) enhanced. This effectively overcomes the shortcomings of existing liver organoid models in simulating primary liver function, providing not only a more accurate in vitro model for the study of liver-related diseases, but also a new platform and possibilities for the development of liver regeneration and treatment methods. Attached Figure Description

[0065] Figure 1 This is a schematic diagram of organoid differentiation.

[0066] Figure 2 This study demonstrated the expression levels of genes related to DHA's promotion of hepatocyte maturation.

[0067] Figure 3 This study demonstrated that DHA increases the gene expression levels of drug-metabolizing enzymes in hepatocytes.

[0068] Figure 4 This study demonstrated that DHA enhances the expression of genes related to ammonia metabolism in hepatocytes.

[0069] Figure 5 This study demonstrated that DHA promotes albumin secretion in hepatocytes. Detailed Implementation

[0070] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0071] The experimental materials used in some embodiments of this invention are as follows:

[0072] DHA (Dihydroartemisinin, CAS No.: 81496-82-4) (MCE, Catalog#: HY-N0176) was dissolved in DMSO to prepare a 10 mM stock solution, which was stored at -20°C in the dark.

[0073] Hepatic progenitor cell organoid expansion medium (HB expansion medium): composed of IMDM medium (Gibco, Catalog#: C12440500BT), 10% FBS (Excell bio, Catalog#: FND500), 1×ITSE (Biogems, Catalog#: 00-101-10), 1×NEAA (Gibco, Catalog#: 11140050), 1×GlutaMAX (Thermo, Catalog#: 35050061), 10mM nicotinamide (sigma, Catalog#: 72340), 5μM CHIR99021 (MCE, Catalog#: HY-10182), 10μM SB431542 (MCE, Catalog#: HY-10431), 10μM Forskolin (MCE, Catalog#: HY-15371), 20ng / mL Composed of FGF-4 (Peprotech, Catalog#: 100-31), 20ng / mL BMP-4 (Peprotech, Catalog#: 120-05), 20ng / mL EGF (Peprotech, Catalog#: AF-100-15).

[0074] Hepatocyte maturation medium consisted of Hepatocyte Culture Medium (Lonza, Catalog#:CC-3198), 20 ng / mL HGF (Peprotech, Catalog#:100-39), 50 ng / mL Oncostatin M (Peprotech, Catalog#:300-10), 100 nM dexamethasone (Sigma, Catalog#:D4902), 20 ng / mL FGF-4 (Peprotech, Catalog#:100-31), and 1×B27 (Gibco, Catalog#:17504044).

[0075] Human embryonic stem cell-derived liver progenitor cell organoids: These were constructed according to the method described in CN115851578A (Invention Title: A kit for 3D suspension-induced continuous expansion of liver progenitor cell organoids and / or hepatocyte organoids and its application). The specific steps are as follows:

[0076] 1. Dissociate hESCs cultured in the feeder layer into single cells using GCDR (StemCell, 100-0485), centrifuge to discard the supernatant, and then rehydrate at 2×10⁻⁶ cells / cells. 5Cells were seeded at a density of 100 cells per milliliter, with 3 ml of cell suspension per well in 6-well ultra-low adhesion plates, and cultured for 1 day in mTeSR1 medium containing 10 μM Y-27632. The mTeSR1 medium was then changed daily thereafter. The culture was continued for 48-96 hours until the cells reached a diameter of 100-200 μm.

[0077] 2. Subsequently, prepare endoderm cells: Replace the cells with: 1640 medium (Gibco, 61870036) + 80 ng / ml Activin A (Peprotech, 120-14) + 3 μM CHIR99021 (Selleck, CT99021). On the second day, aspirate the cell supernatant and add: 1640 medium + 80 ng / ml Activin A + 0.8% KSR (Thermo, 10828028). On the third day, replace the medium with: 1640 medium + 80 ng / ml Activin A + 8% KSR.

[0078] 3. Preparation of liver progenitor cells: Transfer DE stage cell spheres to the following culture medium and culture for 6 days. HB medium: IMD Mmedia (Gibco, 31980030) + 20% FBS (fetal bovine serum, VISTECH, SE100-B7953) + 1% w / w GlutaMAX (Gibco, 35050061) + 0.126 U / mL human insulin (recombinant human insulin, Sigma, 91077C-1) + 0.3 mM 1-thioglycerol (1-thioglycerol, Sigma, M6145) + 20 ng / mL FGF-4 (Peprotech, 100-18B) + 20 ng / mL HGF (Peprotech, 100-39) + 10 ng / mL BMP2 (Peprotech, 120-02) + 10 ng / mL BMP4 (Peprotech, 120-05) + 0.5% DMSO (dimethyl sulfoxide, MPBiomedical, 196055) + 100 nM dexamethasone (Sigma, D4902).

[0079] 4. Preparation of expandable hepatic progenitor cell organoids: Differentiated HB cell spheres were dissociated using TrypLE to prepare single-cell suspensions, and then the single cells were cultured at 2×10⁻⁶ cells / cells. 5Cells were seeded at a density of 100 cells / mL in low-adhesion six-well plates, with 2 mL of cell suspension per well. The culture medium was then replaced with hepatic progenitor cell expansion medium, and HB cells re-aggregated into cell spheroids within 24 hours. The next day, 5% (v / v) growth factor-reduced Matrigel was added to the culture medium, and the medium was changed daily for the next 5 days. Typical hepatic progenitor cell organoid morphology (HB-orgs) appeared within 5 days. At this stage, HB-orgs had continuous expansion capacity. During passage, HB-orgs were dissociated using TrypLE to prepare single-cell suspensions, and passaged every 5-6 days at a ratio of 1:4-6. 5% growth factor-reduced Matrigel was added to the cell seeding at each passage. For cryopreservation of HB-orgs, cells were dissociated using TrypLE and gently digested into small cell clusters, then cryopreserved using serum-free cryopreservation medium (NewCeMed, C40100). The culture medium for expanding hepatic progenitor cell (HB) organoids was HB medium + 5% Matrigel (Corning, 354230). The culture medium for expanding HB organoids was: IMDM + 10% w / w FBS + 1% w / w ITSE (Biogems, 00-101) + 1% w / w NEAA (Gibco, 11140050) + 1% w / w GlutaMAX (Gibco, 35050061) + 10 mM nicotinamide (Sigma, N0636) + 5 μM CHIR99021 + 10 μM SB431542 (Selleckchem, S1067) + 10 μM MSK (MCE, HY-15371) + 20 ng / mL FGF-4 + 20 ng / mL BMP4 + 20 ng / mL EGF (Peprotech, AF-100-15). Subsequent experiments were conducted using resuscitated and passaged HB progenitor cell organoids.

[0080] Example 1: Organoid Culture and DHA Treatment

[0081] Hepatic progenitor cell organoids derived from human embryonic stem cells were used with TrypLE TM Digested into a single-cell suspension, centrifuged, and resuspended in 2 ml of HB amplification medium. The cells were then seeded into 6-well plates (5 × 10⁻⁶ cells / well). 5Cells / well were added to the culture medium with 5% Growth-factor reduced Matrigel (Corning, Catalog #: 354230). Simultaneously, 2 μL of 10 mM DHA (Dihydroartemisinin, CAS No.: 81496-82-4) was dissolved in DMSO to prepare a 10 mM stock solution, bringing the final DHA concentration to 10 μM. A control group was prepared with 2 μL of DMSO. 2 mL of fresh HB amplification medium was added every 48 hours, and the cells were cultured at 37°C with 5% CO2 for 6 days until the cell diameter reached 100-200 μm.

[0082] like Figure 1 As shown in Figure A, after DHA treatment, the resulting liver organoids exhibited characteristics of increased vacuolation. Following DHA treatment, the liver progenitor cell organoids maintained a proliferative state and still exhibited a vacuolar morphology (HB-org Day 6). After further induction and differentiation into mature liver organoids, the organoids retained an independent spherical structure and exhibited a columnar epithelial morphology. Figure 1 (B)

[0083] Example 2: Hepatocyte maturation induction

[0084] Organoids from DHA-treated and control hepatocyte progenitor cells cultured for 6 days were collected separately, centrifuged, and the supernatant was removed. Then, 2 mL of pre-prepared hepatocyte maturation medium was added to the cell pellet, and the cells were transferred to new low-adhesion 6-well plates. Next, 5% Growth-factor reduced Matrigel (Corning, Catalog#:354230) was added to each well to provide three-dimensional culture medium support and promote hepatocyte maturation induction. Subsequent culture was carried out in a cell culture incubator at 37°C and 5% CO2, with 3 mL of hepatocyte maturation medium added every other day. Organoids were collected on day 6 for further analysis. Cell images are shown below. Figure 1 In the middle B group, the organoids still maintain an independent spherical structure and exhibit columnar epithelial morphology (P-Hep-org Day 6).

[0085] Example 3: DHA promotes the expression of marker genes related to hepatocyte maturation

[0086] The expression of hepatocyte maturation-related marker genes in the cell spheres collected in Example 2 was detected.

[0087] After cell culture, the cell pellet and supernatant were collected by centrifugation. 1 mL of Trizol was added to the pellet for lysis at room temperature for 5 minutes, followed by 200 μL of RNA Extraction Buffer. The mixture was thoroughly mixed and incubated at room temperature for 5 minutes. Then, it was centrifuged at 12000 g at 4°C for 15 minutes. After centrifugation, clear stratification occurred. The supernatant was transferred to a new centrifuge container, and an equal volume of isopropanol was added. The solution was thoroughly mixed by inverting and incubated at room temperature for 10 minutes to promote RNA precipitation. Subsequently, the mixture was centrifuged at 12000 g at 4°C for 10 minutes. The supernatant was carefully removed, and 1 mL of 75% ethanol was added to the centrifuge tube containing the RNA pellet for washing. The tube was then centrifuged again at 7500 g at 4°C for 5 minutes. After completely discarding the supernatant, the tube was left uncapped at room temperature to allow the pellet to dry for 10 minutes. Finally, RNase-free ddH2O was added to dissolve the pellet, and the concentration and purity of the RNA were determined using a Nanodrop instrument.

[0088] (2) RNA is reverse transcribed into cDNA

[0089] The genome removal system is prepared according to Table 1:

[0090] Table 1 Genome Removal System

[0091]

[0092] After gently mixing with a pipette, place the mixture into a PCR instrument and perform the genome removal reaction at 42°C for 2 minutes.

[0093] The reverse transcription system is prepared according to the table below:

[0094] Table 2 Reverse Transcription System

[0095]

[0096] After gently mixing with a pipette, place the mixture into the PCR instrument and perform the reaction according to the following procedure (Table 3).

[0097] Table 3 PCR reaction procedure

[0098]

[0099] The obtained cDNA was stored at -80℃ or diluted 5-10 times before being directly used for qRT-PCR experiments.

[0100] (3) qRT-PCR detection

[0101] The qRT-PCR reaction system (10 μL system) is prepared as shown in Table 4 below:

[0102] Table 4 qRT-PCR reaction system

[0103]

[0104] The reaction procedure is shown in Table 5 below:

[0105] Table 5 PCR reaction procedure

[0106]

[0107] All tests were performed in three technical replicates. By analyzing the difference between the internal reference gene and the target gene, the data were finally analyzed using the relative quantification method 2-ΔΔCt to obtain the relative fold change of the target gene between different treated samples.

[0108] The primers for detecting marker genes related to hepatocyte maturation are shown in Table 6 below:

[0109] Table 6 Primers for detecting marker genes related to hepatocyte maturation

[0110]

[0111]

[0112] Test results as follows Figure 2 As shown, the addition of DHA significantly promoted the expression of hepatocyte maturation marker genes, including ALB (albumin), A1AT (α1-antitrypsin), AFP (α-fetoprotein), and HNF4A (hepatocyte nuclear factor 4α). The upregulation of these genes indicates that DHA can promote the maturation of liver organoids and enhance their liver function-related biological characteristics.

[0113] Example 4: DHA promotes drug metabolism in hepatocytes

[0114] To assess the impact of DHA on hepatocyte drug metabolism, this experiment used qPCR (specific steps as in Example 3) to detect changes in the expression of CYP (cytochrome P450) family genes related to drug metabolism in cell spheroids collected in Example 2. The primers used to detect drug metabolism genes are shown in Table 7 below:

[0115] Table 7 Primers for detecting drug metabolism genes

[0116]

[0117] Detection such as Figure 3As shown, the addition of DHA significantly upregulated the expression levels of several CYP genes closely related to drug metabolism in liver organoids, including CYP3A4, CYP2C19, CYP1A1, and CYP1A2. These genes are all important enzymes in liver drug metabolism, and their upregulated expression indicates that DHA can promote the drug metabolism capacity of liver organoids, thereby potentially enhancing their metabolism and clearance of exogenous drugs.

[0118] Specifically, CYP3A4 and CYP2C19 are two important hepatic drug-metabolizing enzymes involved in the metabolism of various drugs; while CYP1A1 and CYP1A2 are mainly involved in the metabolism of aromatic hydrocarbons and certain drugs. Therefore, upregulation of DHA expression in these genes may enhance the drug-metabolizing function of hepatic organoids, thereby increasing their role in drug metabolism and toxin clearance.

[0119] Example 5: DHA promotes hepatocyte ammonia metabolism.

[0120] Ammonia metabolism plays a vital physiological role in the liver, primarily through a series of metabolic pathways that convert ammonia into urea or eliminate it through other pathways. To investigate the effects of DHA on ammonia metabolism in hepatocytes, this experiment used qPCR technology (the steps are the same as in Example 3) to detect changes in the expression of key genes related to ammonia metabolism in the cell spheroids collected in Example 2.

[0121] Key genes involved in ammonia metabolism include CPS1 (carbonic anhydrase synthase 1), ASL (ornithine transaminase), OTC (ornithine aminotransferase), and ASS1 (ornithine synthase 1). These genes play crucial roles in the urea cycle and ammonia metabolism, participating in the conversion and elimination of ammonia.

[0122] The primers for detecting drug metabolism genes are shown in Table 8 below:

[0123] Table 8 Primers for detecting key genes related to ammonia metabolism

[0124]

[0125] Test results as follows Figure 4 As shown, DHA treatment significantly increased the expression levels of the aforementioned genes. Specifically, the expression of CPS1, ASL, OTC, and ASS1 genes was significantly upregulated in the DHA-treated group, indicating that DHA can promote the synthesis of ammonia metabolism-related enzymes in hepatocytes, thereby potentially enhancing the liver's ability to metabolize ammonia and optimizing the ammonia clearance process.

[0126] CPS1 is a key enzyme in the urea cycle, responsible for converting ammonia to glutamate, the first step in ammonia metabolism. ASL and OTC play important roles in the intermediate stages of the urea cycle, helping to convert ammonia into urea. ASS1 plays a crucial role in the synthesis of ornithine, a process essential for maintaining the liver's ammonia clearance function. Therefore, DHA, by upregulating the expression of these genes, may enhance the function of liver organoids in ammonia metabolism, thereby improving the liver's detoxification capacity and ammonia metabolism levels.

[0127] Example 6: DHA promotes albumin secretion in hepatocytes

[0128] The supernatant from liver organoid culture (the supernatant collected in Example 3) was collected, and the albumin secretion level in the supernatant was detected by ELISA. The detection results are as follows: Figure 5 As shown, DHA significantly promoted albumin secretion in liver organoids. This is consistent with the upregulation of albumin-related gene expression levels in Example 3, indicating that DHA can significantly improve the maturity of liver organoids.

[0129] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0130] References:

[0131] [1]Starkel P,Leclercq I A.Animal models for the study of hepaticfibrosis[J].Best Pract Res Clin Gastroenterol,2011,25(2):319-33.

[0132] [2]Schyschka L, Sanchez JJ, wanj Z, et al.Hepatic 3D cultures but not2D cultures preserve specific transporter activity for acetaminophen-inducedhepatotoxicity[J].Arch Toxicol,2013,87(8):1581-93.

[0133] [3]Schutte M,Fox B,Baradez M O,et al.Rat primary hepatocytes showenhanced performance and sensitivity to acetaminophen during three-dimensional culture on a polystyrene scaffold designed for routine use[J].Assay Drug Dev Technol,2011,9(5):475-86.

[0134] [4]Rossi G,Manfrin A,Lutolf M P.Progress and potential in organoidresearch[J].Nat Rev Genet,2018,19(11):671-87.

[0135] [5]Grubb,M.S.&Thompson,I.D.The influence of early experience on thedevelopment of sensory systems.Curr.Opin.Neurobiol.14,503–512(2004).

[0136] [6]Qian,X.et al.Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure.Cell 165,1238–1254(2016).

Claims

1. Liver organoid culture medium, which contains: dihydroartemisinin and cell growth medium.

2. The liver organoid culture medium according to claim 1, characterized in that, The cell growth medium includes RPMI medium, DMEM, DMEM derivatives, or combinations thereof.

3. The liver organoid culture medium according to claim 1, characterized in that, The concentration of dihydroartemisinin in the liver organoid culture medium is 1-100 μM.

4. The liver organoid culture medium according to claim 1, characterized in that, The liver organoid culture medium also contains FBS, ITSE, NEAA, L-glutamine additive, nicotinamide, GSK-3 inhibitor, TGF-β signaling pathway inhibitor, trichodin, fibroblast growth factor, bone morphogenetic protein, and epidermal growth factor.

5. The liver organoid culture medium according to claim 3, characterized in that, The GSK-3 inhibitor is selected from at least one of TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR99021; Preferably, the TGF-β signaling pathway inhibitor is selected from at least one of LY2157299, EW-7197, LY3200882, SB-431542, LY2109761, TP-0427736, IN-1130, R-268712, A-83-01, SB-525334, GW788388, RepSox, A-77-01, SB-505124, SD-208, and LY364947; Preferably, the fibroblast growth factor is selected from at least one of FGF1, FGF2, FGB, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23; Preferably, the bone morphogenetic protein is selected from at least one of BMP4 and BMP2.

6. A culture medium composition comprising the liver organoid culture medium and the hepatocyte maturation culture medium according to any one of claims 1-5; Preferably, the hepatocyte maturation culture medium comprises hepatocyte culture medium, hepatocyte growth factor, tumor suppressor factor, glucocorticoid receptor agonist, fibroblast growth factor and B27 additive; Preferably, the glucocorticoid receptor agonist is dexamethasone; Preferably, the fibroblast growth factor is selected from at least one of FGF1, FGF2, FGB, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

7. A method for constructing liver organoids, comprising: During the passage of liver organoids, the liver organoid culture medium according to any one of claims 1-5 is used for the first stage of culture; Preferably, the first stage of cultivation lasts for 5-7 days; Preferably, the liver organoid is a liver organoid derived from human pluripotent stem cells.

8. The construction method according to claim 7, characterized in that, The construction method further includes a second-stage culture using the hepatocyte maturation medium in the culture medium combination of claim 6; Preferably, the second stage of cultivation lasts for 7-9 days.

9. Uses of dihydroartemisinin in any of the following aspects: Preparation of liver organoid culture medium; Construct liver organoids.

10. Liver organoids obtained by the construction method according to claim 7 or 8.

Images