Combined Reagents for Isolation of Primary Hepatocytes

Abstract

The invention provides a combined reagent for separating primary hepatocyte. The combined reagent comprises perfusate I, perfusate II and perfusate III, wherein each liter of perfusate I contains the following components by weight: 8.1-8.5g of sodium chloride, 0.5g of potassium chloride, 2.2-2.6g of HEPES, 3.5-4.5g of NAC, 0.9-1.1g of EGTA and the balance of deionized water; each liter of perfusate II contains the following components by weight: 8.1-8.5g of sodium chloride, 0.5g of potassium chloride, 2.2-2.6g of HEPES and the balance of deionized water; and each liter of perfusate III contains the following components by weight: 3.8-4g of sodium chloride, 0.5g of potassium chloride, 23-25g of HEPES, 0.07g of calcium chloride dehydrate, 3.5-4.5g of albumin, 162-260 Wunsch units of collagenase and the balance of deionized water. The invention further provides a preparation method of the combined reagent. The combined reagent provided by the invention is simple in formula, and convenient to prepare, and can be applied to primary hepatocyte separation; and hepatocyte damages are effectively prevented.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and in particular relates to a combination reagent for separating 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 hi...

AI Technical Summary

Problems solved by technology

On the basis of this method, many researchers have improved the hepatocyte separation method and the perfusate, 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 with four reagents, and the other is four-step perfusion with four reagents. Two reagents are used for two-step perfusion. When four-step perfusion is used, the viability of liver cells and the total number of liver cells are higher, which are 90-99% and 6.25×10 7 Liver tissue per gram, but the purity is low, only 90-95%, and there are many reagents for perfusion, and the cost is high; while the two-step perfusion uses few reagents, but the liver cell viability, total number of liver cells and purity are not high. Ideal, 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 with three reagents, but the total number of hepatocytes obtained, The activity rates were all low, being 1.12×10 7 Individual / gram liver tissue, 85.7%

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