Separation and culture method for dog hepatocytes

Abstract

The invention belongs to the field of separation and culture of cells and in particular relates to a separation and culture method for dog hepatocytes. The separation and culture method comprises the following steps: S1, taking liver from a living body; S2, perfusing; S3, separating the hepatocytes; S4, culturing the hepatocytes. The perfusion is realized by the following steps: firstly, perfusing perfusate A preheated at the temperature of 37DEG C into a flushed liver at the flow speed of 50ml/min for 15 to 20 minutes; secondly, continuously perfusing perfusate B preheated at the temperature of 37DEG C at the flow speed of 50ml/min for 3 to 5 minutes; thirdly, perfusing perfusate C preheated at the temperature of 37DEG C at the flow speed of 20ml/min for 4 to 6 minutes; finally, pouring a basal culture medium which contains three antibodies and is precooled at the temperature of 4DEG C on the surface of the liver, wherein the volumetric ratio of the three antibodies to the basal culture medium is 1 to 99. The hepatocytes obtained by separating through the method are nearly pure parenchymal hepatic cells; various functions of the hepatocytes are retained; the hepatocytes have the advantages of high quantity, morphological integrity, good adherence and high activity.

Description

technical field [0001] The invention belongs to the field of cell separation and culture, in particular to a method for separation and culture of canine hepatocytes. Background technique [0002] A lot of research has been done on the separation method and culture conditions of hepatocytes, but it is still difficult to obtain hepatocytes with high viability and good function, especially canine primary hepatocytes. [0003] The early methods of separating hepatocytes were mainly non-enzymatic separation of hepatocytes, including mechanical methods (such as homogenization) and chelation methods. However, the mechanical method has great damage to the liver cells, and the viability of the isolated liver cells is low; the chelation method uses Ca 2+ , Mg 2+ The chelating agent (such as citrate or EDTA) is used to separate hepatocytes, but the effect of chelating agent alone is not good, and it is often used in combination with enzymatic method. In the later period, it was grad...

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Problems solved by technology

According to the results, the hepatocytes obtained by one-step perfusion combined with tissue block digestion method had poor morphological integrity, most of the cells were not att

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