Isolation and preparation method of primary hepatocytes

Abstract

The invention discloses a method for separating and preparing primary hepatocytes, which comprises the following steps: a. Extracorporeal perfusion of the liver: take the isolated animal liver, and first perfuse it with perfusate I at a rate of 300-1000mL / min, The perfusion time is 10-12 minutes; then perfusate with perfusate II, the perfusion rate is 300-1000mL / min, and the perfusion time is 2-3 minutes; finally perfusate with perfusate III, the perfusion rate is 400-1200mL / min, the perfusion time 25‑40 minutes; b. Separation of liver cells: wash the liver treated in step a, collect the liver cell suspension, filter, centrifuge, and collect the precipitate. The method for separating and preparing primary hepatocytes of the present invention can isolate and obtain hepatocytes with high quantity, viability and purity, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of medical biotechnology and relates to a method for separating and preparing primary hepatocytes. Background technique [0002] As an important metabolic organ of the body, the liver plays an important role in various physiological and pathological processes. Hepatocytes perform the main functions of the liver, such as removing toxins, participating in biosynthesis and biotransformation, metabolizing various nutrients, storing glucose, and secreting active substances that promote the growth of liver cells. Primary hepatocytes isolated from the body are used in in vitro studies such as pharmacology and toxicology, immunology, and cell biology. With the application of bioartificial liver in the field of liver failure, a large number of highly active primary hepatocytes are also required as biological material. Therefore, how to separate and prepare primary hepatocytes with high yield, high purity and high survival ra...

AI Technical Summary

Problems solved by technology

On the basis of this method, many researchers have improved the hepatocyte separation method, which has increased the total amount and viability of hepatocytes to a certain extent, but it still cannot meet the needs of practical applications.

[0004] For example: the publication number is CN1632107A, and the patent titled "Preparation Method of Pig and Human Hepatocytes for Bioartificial Liver" reports two methods for separating hepatocytes, one is four-step perfusion and the other is two-step perfusion , when four-step perfusion was used, the viability and total number of hepatocytes obtained were higher, which were 90-99% and 6.25×10 7 Liver tissue per gram, but the purity is low, only 90-95%, and there are too many perfusion steps, and the cost is high; while two-step perfusion has fewer steps, but the liver cell viability, total number of liver cells and purity are not ideal. Respectively only 80-90%, 1.25×10 7 pc / g liver tissue and 80-90%

[0005] Zhang ZG et al., An updated method for the isolation and culture of primary calf hepatocytes, Vet J, 2012, 191: 323-326 discloses a method for separating hepatocytes by three-step perfusion, but the total number and viability of the obtained hepatocytes are relatively low low, respectively 1.12×10 7 Individual / gram liver tissue, 85.7%

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