Method for separating and culturing primary chicken hepatocytes

Abstract

The invention discloses a method for separating and culturing primary chicken hepatocytes. According to the method, EGTA (Ethylene Glycol Tetraacetic Acid) with low hepatotoxicity is added into a perfusate A to loosen the junction between hepatocytes, so that the dispersion degree of the to-be-separated hepatocytes is increased; double resistance is added into the perfusate A, a perfusate B and D-Hanks flushing fluid, so that the pollution is further prevented; and circulating perfusion digestion is adopted during digestion perfusion, so that the perfusion cost is greatly reduced. A serum-free cultural method of the chicken hepatocytes is established; compared with a traditional serum cultural method, the method disclosed by the invention has the same effect on the aspects of cell quantity, activity, function and the like, so that the problems caused by the existence of serum are solved and a foundation is laid for the substance metabolism study and biological product preparation on the basis of the chicken hepatocyte culture.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating and culturing primary chicken hepatocytes. Background technique [0002] Compared with other in vitro and in vivo experimental methods, the use of primary hepatocytes for in vitro experiments has its own significant advantages. The primary cultured hepatocytes are not affected by the complex neuroendocrine system in the body, and can maintain the specific functions of the hepatocytes and maintain the responsiveness to some hormones. Therefore, the primary cultured hepatocytes are ideal for studying liver substance metabolism and An excellent model of its regulatory mechanism. [0003] The isolation of hepatocytes was initially performed by the isolated perfusion method. By 1975, Seglen successfully isolated rat hepatocytes with high activity rate by in situ two-step collagenase perfusion method. In 1992, Fraslin et al. improved Seglen's in situ two-step perfu...

AI Technical Summary

Problems solved by technology

However, the addition of serum to culture cells also has its disadvantages: First, the composition of serum is very complex and unclear. The use of serum in hepatocyte culture will affect the basic metabolism of hepatocytes and affect the research and production based on hepatocyte culture. Second, some biologically active substances in serum can poison liver cells and interfere with its biotransformation; third, serum may bring the danger of microbial contamination such as viruses, fungi and mycoplasma; fourth, certain proteins in serum will Interference with biological assays, inconvenient for analysis of experimental results

For the serum-free culture of chicken hepatocytes, there is no mature method with long culture time and high stability in China.

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