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Culture medium and culture method for hepatocytes and application of hepatic cells

A culture method and technology for hepatocytes, applied in the field of in vitro culture of primary cells, can solve problems such as differences, incomplete differentiation of hepatocytes, and tumorigenic risks, and achieve the effect of small differences in liver function and reducing the risk of cancer.

Inactive Publication Date: 2020-06-23
SHANGHAI TECH UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2019, the laboratory of Professor Deng Hongkui of Peking University reported a technique for stably culturing primary hepatocytes in vitro for a long time (greater than 4 weeks) by adding five small molecular compounds. However, this technique can only be used to maintain performance of hepatocytes, but not for in vitro expansion of hepatocytes
The currently established in vitro culture system of hepatocytes cannot realize real in vitro expansion and culture of hepatocytes, but can only be carried out in the form of first dedifferentiation and passage and then inducing their differentiation into target cells
Although induced mature hepatocytes have certain liver functions, they still have significant differences compared with primary hepatocytes
Simultaneously induced hepatocytes have the possibility of incomplete differentiation and therefore have potential tumorigenic risks

Method used

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  • Culture medium and culture method for hepatocytes and application of hepatic cells
  • Culture medium and culture method for hepatocytes and application of hepatic cells
  • Culture medium and culture method for hepatocytes and application of hepatic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] The preparation of embodiment 1 proliferation medium and mature medium

[0097] Proliferation medium (YACD medium): 500ml DMEM / F12 containing 2g / L galactose, 1% insulin-transferrin-sodium selenite [(ITS)-X], 0.3g / L proline, 0.61 g / L Nicotinamide, 0.1g / L Ornithine, 40ng / mL Transforming Growth Factor α (TGFa), 40ng / mL Epidermal Growth Factor (EGF), 10μM Dexamethasone (Dex), 10μM Y-27632, 0.5μM Mixture of A-83-01, 3 μM CHIR99021 and penicillin and streptomycin (Solarbio, P1400).

[0098] Maturation medium (ODF medium): 500ml DMEM / F12 containing 2g / L galactose (manufacturer BBI, product number GB0215) 5ml insulin-transferrin-sodium selenite [(ITS)-X] (manufacturer Original Culture , product number S450), 0.3g / L proline (manufacturer Alfa Aesar, product number A10199.22), 0.61g / L nicotinamide (manufacturer Sigma, product number N7004), 0.1g / L ornithine (manufacturer Sigma, product number O2375 ), 40ng / ml TGFα (manufacturer Peprotech, product number AF-100-16A), 40ng / ml EGF...

Embodiment 2

[0099] Example 2 In vitro expansion and culture of mouse hepatocytes

[0100]Take 50 μg of collagen (manufacturer Sigma, product number C7661) and prepare it with water to make a coating solution with a concentration of 20 μg / mL, and coat a 6 cm cell culture plate with the prepared coating solution. Liver perfusion was used to obtain fresh primary mouse hepatic parenchymal cells, which were divided into 2x10 4 / cm 2 Density seeding on collagen-coated cell culture plate, cultured with DMEM medium containing 10% fetal bovine serum (FBS), to make it adhere to the wall.

[0101] After 6 hours, the unattached cells were removed, and the YACD medium prepared in Example 1 was added to the original medium for culture, and the medium was changed every two days. Observe every day, if the cells proliferate densely, passage them in time. At the same time, a control group was set up, and the adherent cells were cultured in the traditional hepatocyte HMM medium in the control group.

[...

Embodiment 3

[0104] Example 3 Real-time PCR verification of liver function gene expression in primary mouse hepatocytes cultured in YACD medium

[0105] The cells cultured to the 5th and 11th passages were washed with PBS, and fully lysed with an appropriate amount of Trizol. Add 20% chloroform, centrifuge at 4°C for 20 minutes, take the upper aqueous phase, add an equal volume of isopropanol and mix well, let stand at room temperature for 15 minutes, centrifuge at 4°C for 15 minutes, wash the precipitate with 75% ethanol, dry at room temperature, add water Dissolve the extracted RNA.

[0106] Using the reverse transcription kit (manufacturer Takara, Cat. No. RR047B), first take 500ng RNA and add 2.0 μl of 5x gDNA Eraser Buffer, 1.0 μl of gDNA Eraser, add RNase Free dH 2 O Prepare the system to 10 μl, mix well and incubate at 42°C for 2 minutes to remove genomic DNA contamination. Then add PrimeScript RTEnzyme Mix I 1.0μl, RT Primer Mix 1.0μl, 5x PrimeScript Buffer 2 (for Real Time) 4.0μ...

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Abstract

The invention relates to the field of culture of primary cells in vitro, in particular to a culture medium and culture method for hepatocytes and application of the hepatocytes. The culture medium forthe hepatocytes comprises a proliferation culture medium comprising the following components: a liquid basic culture medium, galactose, insulin-transferrin-sodium selenite, proline, niacinamide, ornithine, a transforming growth factor alpha, an epidermal growth factor, dexamethasone, Y-27632, A-83-01 and CHIR99021. A long-term in-vitro amplification system for primary hepatocytes is established;and the hepatocytes are completely differentiated without cell dedifferentiation or transdifferentiation during passage amplification culture, the carcinogenic risk is reduced, and the difference between hepatic functions of hepatocytes matured by induction and primary hepatocytes is small.

Description

technical field [0001] The invention relates to the field of in vitro culture of primary cells, in particular to a hepatocyte culture medium, a culture method and an application thereof. Background technique [0002] The in vitro culture of hepatocytes is of great significance for the advancement of liver cell therapy, the mechanism of liver cell death and liver regeneration, and the research of drugs for liver diseases. [0003] Several methods for culturing hepatocytes in vitro have been reported. For example, Takeshi Katsuda’s research group reported in 2016 that by adding small chemical molecules Y-27632, A-83-01, and CHIR99021 to the culture medium, rats can achieve And the in vitro expansion of mouse hepatocytes, the cultured cells dedifferentiate into bipotential hepatic progenitor cells, which can be further induced to differentiate into hepatocytes or cholangiocytes. In 2018, Hui Lijian's research group at the Shanghai Institute of Biochemical Cells, Chinese Academ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2500/34C12N2500/25C12N2500/32C12N2501/148C12N2501/11
Inventor 黄鹏羽毛一雷庄绪冉刘俊来彭文博
Owner SHANGHAI TECH UNIV
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