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Method for separating and culturing primary hepatocyte

A technology of primary hepatocytes and culture methods, applied in the biological field, can solve the problems of difficult acquisition and in vitro perfusion, and achieve the effect of less cell damage, less time and cost, and better activity

Active Publication Date: 2018-07-20
SHANGHAI CELLIVER BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the invention patent with the publication number CN102061284A discloses a method of separating and culturing human primary hepatocytes from human liver tissue obtained by surgical resection by using a multi-point puncture collagenase perfusion method. Disadvantages of extracorporeal perfusion
However, this method requires the sample to have a certain volume and weight, and has a relatively complete envelope, which is difficult to obtain clinically, thus limiting the application of this method in general laboratories and primary human hepatocytes in medicine

Method used

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  • Method for separating and culturing primary hepatocyte
  • Method for separating and culturing primary hepatocyte
  • Method for separating and culturing primary hepatocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] 1. Isolation and culture of primary human hepatocytes from liver biopsy samples

[0100] 1. Obtaining Liver Tissue Samples

[0101] Use a surgical puncture needle to take a normal biopsy tissue from a patient with liver disease, wherein the type of surgical needle used is 18Gx15cm to obtain a liver tissue sample; the diameter of the liver tissue sample is 1-1.2mm, the length is 1.5cm, and the weight is 0.02-0.05 g. The liver tissue samples were transported to the laboratory at low temperature in HNAKS solution containing antibiotics and sodium heparin. The liver tissue samples were obtained from the Department of Tumor Intervention, Renji Hospital, Shanghai Jiaotong University after informed consent.

[0102] Take out the liver tissue sample with sterile surgical forceps and place it in a 10cm 2 The culture dish was infiltrated with HANKS solution to keep the liver tissue sample in a moist state. Cut the liver tissue sample into a volume of 1 mm with a very thin blade...

Embodiment 2

[0126] 1. Isolation and culture of primary human hepatocytes from liver biopsy samples

[0127] 1. Obtaining Liver Tissue Samples

[0128] The acquisition of liver tissue samples was the same as in Example 1.

[0129] 2. Isolation of Primary Hepatocytes

[0130] Move the liver tissue fragments into a centrifuge tube, which contains 10 mL of HANKS solution containing 0.5 mg / mL type IV collagenase, and the preparation method is: add 0.05 g of type IV collagen to 100 mL of HANKS solution Enzyme; place the centrifuge tube on a shaker in an incubator at 37°C, shake it for 30 minutes at a shaking frequency of 10rmp / min, then filter it with a 70μm filter to remove the solution, and then use HANKS solution to crush the tissue The blocks were washed twice to obtain the initially digested tissue fragments. Transfer the initially digested tissue fragments into a centrifuge tube containing TrypLE digestion solution, put it back on a shaker in a 37°C incubator, shake for 10min at a shak...

Embodiment 3

[0147] 1. Isolation and culture of primary human hepatocytes from liver biopsy samples

[0148] 1. Obtaining Liver Tissue Samples

[0149] The acquisition of liver tissue samples was the same as in Example 1.

[0150] 2. Isolation of Primary Hepatocytes

[0151] The tissue fragments were transferred into a centrifuge tube, which was filled with 10 mL of HANKS solution containing 0.5 mg / mL type IV collagenase and 5 mg / mL fetal bovine serum albumin preheated at 37 °C, and its preparation method For: add 0.05g type IV collagenase and 0.5g fetal bovine serum albumin to 100mL HANKS solution. The centrifuge tube was placed on a shaker in a 37°C incubator, shaken for 30min at a shaking frequency of 10rmp / min, then filtered through a 70μm filter, and then washed twice with HANKS solution to obtain Preliminary digested tissue fragments; transfer the primary digested tissue fragments into a centrifuge tube containing TrypLE digestion solution, put them back on the shaker in a 37°C in...

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Abstract

The invention provides a method for separating and culturing primary hepatocyte. The method comprises the following steps: S1, performing aspiration biopsy on liver tissue so as to obtain a liver tissue sample, and crushing the liver tissue sample so as to obtain tissue fragments; S2, digesting the tissue fragments, and filtering so as to obtain a second tissue fragment; and S3, inoculating the second tissue fragment into a culture device, and performing multiplication culture. The method is low in liver tissue source requirement, wide in tissue source, small in operation risk, simple and feasible in step, high in operability, short in time and low in cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating and culturing primary hepatocytes. Background technique [0002] The liver is an important organ in the human body, which has the functions of participating in the metabolism of substances, the synthesis of secreted proteins, and the biotransformation of toxins. As the main body of the liver, hepatocytes are widely used in the field of life medicine. For example, they have high application requirements in hepatocyte transplantation, bioartificial liver, gene therapy and drug research and development. However, under general culture conditions, primary hepatocytes are cultured for a short time in vitro, the differentiation function is maintained for a short time, and the specific function is rapidly reduced, which limits the application of hepatocytes. [0003] At present, the main primary hepatocyte isolation method is the two-step in situ perfusio...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2509/00C12N2533/50C12N2533/54
Inventor 鄢和新周徐翟博黄伟健吴红平唐为忠
Owner SHANGHAI CELLIVER BIOTECHNOLOGY CO LTD
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