Human primary hepatocyte separation and culture method

Abstract

The invention provides a human primary hepatocyte separation and culture method. The method comprises the following steps of (1) cutting a fresh human hepatocyte under an aseptic condition, sealing at4 DEG C and then carrying back to a lab; (2) placing the tissue into a precooled sterile PBS buffer liquid containing double antibodies, and washing for multiple times to remove blood and connectivetissues; (3) irrigating through a preheated PBS buffer liquid through a needle-punching method; (4) irrigating through a preheated HBSS buffer liquid through a needle-punching method; (5) irrigating through a preheated GBSS mixed enzyme liquid through a needle-punching method; (6) cutting up the tissue, and using the GBSS mixed enzyme liquid for digesting, centrifuging, and removing a supernatant;(7) using trypan-blue for dyeing a cell filter liquor to detect cell activity; (8) after carrying out cell counting, resuspending a complete medium, inoculating into a coated culture flask, and culturing at 37 DEG C in an environment with 5 percent of CO2; (9) identifying cell morphology; (10) detecting through an ELISA (Enzyme-linked Immuno Sorbent Assay) method; (11) detecting through RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The invention provides the human primary hepatocyte separation and culture method which is simple to operate, the number of the obtained cells is large, the survival rate is high, the method is the ideal human primary hepatocyte separation and culture method, and a reliable cell resource is provided for experiments.

Description

technical field [0001] The invention belongs to the technical field of cell culture of modern biotechnology, and specifically relates to a method for separating and culturing primary human hepatocytes. Background technique [0002] Hepatocytes are used as seed cells for cell transplantation to treat chronic liver failure and as target cells for drug screening. The preparation and cultivation of large-scale, highly viable hepatocytes is the most basic link in these studies. Therefore, the technology of separation and culture of hepatocytes is attracting increasing attention and has become a hot spot in current research. [0003] At present, the separation methods of hepatocytes include mechanical separation, chelation and enzymatic digestion. The mechanical method is simple to operate, but the number of isolated hepatocytes is small and their activity is poor. Howard created the collagenase perfusion method, and Seglen further developed this method into a two-step perfusion...

AI Technical Summary

Problems solved by technology

However, this method requires a large volume of liver tissue, and is not suitable for surgically resected irregular small pieces of human liver tissue, and cannot meet the needs of human hepatocytes in basic research.

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