Method for inducing stem cells into liver cells and kit

A kit and stem cell technology, which can be used in cell culture active agents, embryonic cells, animal cells, etc., can solve problems such as high cost and complications

Active Publication Date: 2020-03-27
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, orthotopic liver transplantation is a complicated surgical operation, which will bring a high probability of complications, and the selection and matching of donor-recipient, surgery, and long-term immunosuppressive treatment will result in high costs

Method used

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  • Method for inducing stem cells into liver cells and kit
  • Method for inducing stem cells into liver cells and kit
  • Method for inducing stem cells into liver cells and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] This experiment takes clinical-grade human embryonic stem cells (CTS-hES) as the object (see Qi Gu, et al., Accreditation of Biosafe Clinical-Grade Human Embryonic Stem Cells According to Chinese Regulations (2017). stem cell reports, vol 9,1-15), evaluating the ability of the culture system of the present invention to induce the differentiation of embryonic stem cells into hepatocytes. Wherein, the differentiation degree and differentiation efficiency of CTS-hES during culture were detected by immunofluorescence and flow cytometry.

[0091] 1. Detection method

[0092] (1) Detection of protein expression by immunofluorescence staining

[0093] Specific operation steps: Put slides in a 24-well plate, dilute Matrigel matrix with CTS Knock-out DMEM culture medium (that is, add 50 μL Matrigel to 6 mL CTS Knock-out DMEM culture medium), spread it in a 24-well plate, 200 μL per well, put in CO 2 After standing in the incubator for 30 minutes, the cells were attached to th...

Embodiment 2

[0110] Example 2 Evaluation of the ability of the culture system described in this application to induce differentiation of human induced pluripotent stem cells (hiPSCs) into hepatocytes

[0111]This experiment takes clinical grade hiPSCs as the object (see Juan Wang, et al., Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions, Stem Cell Research & Therapy (2015), 6:223 for the cell acquisition method), Referring to the differentiation induction method in Example 1, the ability of the culture system described in this application to induce the differentiation of hiPSCs into hepatocytes was evaluated, and the results are shown in Figure 5 .

Embodiment 3

[0112] Example 3 In vitro functional testing of differentiated hepatocytes

[0113] Through the following experiments, the liver cells differentiated in Example 1 were tested for in vitro function, and the results are shown in Figure 6 .

[0114] 1. Oil red staining experiment

[0115] (1) Preparation of Oil Red O: Weigh 0.5g of oil red dry powder that has been ground and crushed in advance, dissolve it in 10mL of isopropanol, then add isopropanol to 100ml, seal the brown bottle (or wrap it in tin foil to avoid light) at 4°C Preservation, as a storage solution, can be preserved for a long time. When in use, take 6 mL of oil red solution and add 4 mL of three-distilled water, mix well, filter with qualitative filter paper, and use up within 3 hours after dilution.

[0116] (2) Cell fixation: Discard the culture supernatant, add 100 μL PBS to each well and wash once, taking a 24-well plate as an example, add 200 μL 4% PFA to each well and fix at room temperature (25°C) for...

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Abstract

The invention relates to a method for inducing stem cells into liver cells and a kit. Particularly, the invention relates to the kit which has clear chemical ingredients, and comprises a second culture medium and/or a third culture medium; and optionally, the kit further comprises a first culture medium. A culture system provided by the invention is simple in formula and clear in ingredients, doesnot contain animal derived ingredients and does not involve any ethical issue; and the induction efficiency can be improved, and the system has a very high clinical application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method and a kit for inducing stem cells into liver cells. Background technique [0002] Liver diseases are generally divided into the following four categories: viral hepatitis, cirrhosis, liver cancer, and metabolic liver diseases, including familial hypercholesterolemia, hyperlipidemia, and phenylketonuria. Hepatitis has a very high incidence worldwide, especially in East Asia and Southeast Asia. About 360 million people in the world suffer from hepatitis. In hepatitis, viral hepatitis B accounts for a large proportion, and China is a high-incidence area of ​​hepatitis B. At present, the conservative and relatively effective treatment method is antiviral treatment. Liver cirrhosis is mostly caused by the deterioration of liver fibrosis, liver injury and other conditions leading to extensive apoptosis, the liver loses the function of spontaneous reconstruc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0735C12N5/10
CPCC12N5/067C12N2500/38C12N2500/25C12N2501/15C12N2501/42C12N2501/237C12N2501/12C12N2501/39C12N2501/727C12N2500/32C12N2500/62C12N2506/02C12N2506/45C12N2510/00
Inventor 周琪郝捷李仲文吴骏李伟王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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