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Functional hepatocyte induction method and special three-dimensional induction culture medium and application thereof
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A technology of induction medium and culture medium, applied in the field of bioengineering, can solve the problem of hepatocytes expressing immature functional marker alpha-fetoprotein
Active Publication Date: 2020-04-14
ACADEMY OF MILITARY MEDICAL SCI
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For example, in 2014, the Hans.Clever team isolated cells from surgically resected primary liver tissue and screened the cells through the surface marker EPCAM, using extracellular matrix gel Matrigel or BME, combined with cytokines and small molecular compounds for three-dimensional organoid culture. A large number of hepatic stem cells with bidirectional differentiation potential were expanded in vitro (Huch M, Gehart H, van Boxtel R, et al. Long-Term Culture of Genome-Stable Bipotent StemCells from Adult Human Liver[J]. Cell, 2015, 160: 299-312.), this liver organoid culture method has laid a good foundation for the large-scale expansion of liver stem cells and functional liver cells in vitro. Clinical or pharmaceutical research has certain limitations
The inventors have also used human embryonic stem cells (commercially sourced hESCs) as a source to obtain liver organoids with the potential to bidirectionally differentiate into functional hepatocytes and cholangiocytes ( Wang Xuan, Research on the Construction of Human Pluripotent Stem Cell-derived Liver Organoids with Bidirectional Differentiation Potential, Hebei Medical University, Master Thesis, 2018), using this liver organoids to obtain a sufficient amount of functional hepatocytes through three-dimensional induced culture , but the hepatocytes obtained by this method express a large amount of immature functional marker alpha-fetoprotein (AFP), which has many limitations in its application
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[0065] In this embodiment, pluripotent stem cells (cell line H9, human embryonic stem cells (human EmbryonicStem Cells), purchased from Wicell Research Center, USA) are used as starting cells, such as figure 1 As shown, through the qualitative endoderm stage, the liver stem / progenitor cell stage and the liver organoid stage in sequence, hepatocytes are then induced. Specifically include the following steps:
[0066] 1.1. Culture of cell line H9 cells
[0067] 1) Take Matrigel (Corning) out of the -80°C refrigerator one day in advance, and thaw it in a 4°C refrigerator. After it melts, dilute Matrigel at a ratio of 1:100 with pre-cooled DMEM / F12 medium (Gibco).
[0068] 2) Evenly spread the diluted Matrigel on a six-well plate, 1 mL per well, and incubate for 1 hour in a 37°C incubator for later use.
[0069] 3) Take out the H9 cells to be subcultured from the incubator at 37°C, suck off the waste liquid, and wash once with...
[0197] 2.1, RNA extraction, reverse transcription and real-time quantitative PCR
[0198] In this step, the RNA extraction kit (Qiagen) was used for RNA extraction, the reverse transcription kit (TOYOBO) was used for reverse transcription, and the real-time quantitative PCR instrument (Bio-Rad) was used for real-time quantitative PCR.
[0199] 1) Collect the hepatocytes (three-dimensional cell spheres) induced in the above-mentioned Example 1, and add an appropriate amount of lysis solution Buffer RLT (Qiagen) to the collected cells to fully lyse the cells.
[0200] 2) Add an equal volume of 70% ethanol to the above lysate, mix with a pipette gun, and transfer to spincolume, room temperature 12000rpm, centrifuge for 30s.
[0201] 3) Discard the waste liquid, add 700 μL of Buffer RW1 to the spin column, centrifuge at 12000 rpm at room temperature for 30 s.
[0202] 4) Discard the waste liquid, add 500 μL o...
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Abstract
The invention discloses a functional hepatocyteinduction method as well as a special three-dimensional induction culture medium and an application thereof, which belongs to the technical field of bioengineering. The functional hepatocyte three-dimensional induction culture medium provided by the invention contains a plurality of small molecules and cell factors, can be effectively applied to themethod for obtaining functional hepatocytes through induction of liver organs with stem / progenitorcell characteristics. The functional hepatocytes obtained by induction culture can be used for artificial liver production and in-vitro liver functionsimulation, and can also be used as seed cells for establishing a liver disease treatment drug screening model and an in-vitro liver diseasesimulation model.
Description
technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for inducing functional hepatocytes and its special three-dimensional induction medium and its application, in particular to a three-dimensional method for inducing functional hepatocytes using liver organoids with stem / progenitorcell characteristics. The medium and the application of the induced functional hepatocytes in the preparation of medicines for treating liver diseases. Background technique [0002] The liver is the largest digestive organ in the human body. It regulates many important physiological functions of the body, including the metabolism of nutrients, the synthesis of urea, the synthesis of various important proteins, the secretion of bile, and the detoxification of heterologous substances. Liver damage or diseases caused by various reasons usually cause serious consequences, such as viral hepatitis, liver fibrosis, genetic metabolic liver disease...
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