Culture method for long-term maintenance of functional state of human primary hepatocytes

A technology of primary hepatocytes and functional status, which is applied in the field of medicine and can solve problems such as complex operation techniques of culture equipment

Pending Publication Date: 2020-05-29
SHANGHAI CELLIVER BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these culture methods require special culture equipment and complex operating techniques, which to some extent limit the selection of PHH's analytical methods for evaluating drug effectiveness and their applicability in high-throughput sequencing

Method used

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  • Culture method for long-term maintenance of functional state of human primary hepatocytes
  • Culture method for long-term maintenance of functional state of human primary hepatocytes

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Experimental program
Comparison scheme
Effect test

Embodiment

[0024] 1. Experimental materials

[0025] The recombinant protein HGF used in this example was purchased from Peprotech; the remaining small molecule compounds such as Y27632, A83-01 and CHIR99021 were purchased from TargetMol; the primary cells used were purchased from BioreclamationIVT, and specific cell culture basal (i.e. hepatocyte expansion medium) culture; penicillin and streptomycin double antibiotics and the basal medium used for subsequent primary hepatocyte maintenance experiments were purchased from Yuanpei Biotechnology Company; fetal bovine serum (FBS) was purchased from in Gibco; Matrigel used in cultured primary hepatocytes was purchased from BD.

[0026] 2. Experimental method

[0027] 1. Cell recovery and inoculation

[0028] After the frozen cells were taken out of the liquid nitrogen tank, they were quickly shaken in a water bath at 37°C until they were completely melted, centrifuged at 700rpm at 4°C for 3 minutes, the supernatant was discarded, and 1ml o...

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Abstract

The invention relates to the technical field of medicines, in particular to a culture method for long-term maintenance of functional state of human primary hepatocytes. The method is of culturing thehuman primary hepatocytes in a hepatocyte amplification medium with the inoculum density of 3 x 104 / cm2. According to the method in the invention, the function of the primary hepatocyte (PHH) can bemaintained within at least 30 days through high-density culture in the hepatocyte amplification culture medium, the long-term in vitro culture and function maintenance of the primary human hepatocytecan be realized, and a basic guarantee is provided for drug metabolism evaluation and hepatocyte treatment.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a culture method for maintaining the functional state of human primary hepatocytes for a long time. Background technique [0002] Primary human hepatocytes (PHH) are considered to be the most ideal cells for hepatocyte therapy, and even the gold standard cells for drug metabolism evaluation and modeling research. However, during the in vitro culture of PHH, the extremely low proliferation rate and the loss of metabolic function hinder the application of PHH. The important reason is that the stable function of terminally differentiated cells in vivo is highly dependent on the precise spatiotemporal regulation of microenvironmental signals, while isolated primary cells usually lose their core gene expression characteristics and specific functions. In order to overcome this problem, researchers have proposed many methods for long-term maintenance of liver cell function in vitro, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/067G01N33/5067C12N2500/25C12N2501/11C12N2501/2306C12N2501/12C12N2501/115
Inventor 鄢和新陈依
Owner SHANGHAI CELLIVER BIOTECHNOLOGY CO LTD
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