Separation liquid for removing dead liver cells and method for removing dead liver cells using same

A technology for hepatocytes and separation liquid, applied in the field of separation liquid for removing dead hepatocytes, can solve the problems of low cell viability and low efficiency of removing dead hepatocytes, and achieve high cell viability, good maintenance of liver cell shape, and efficient removal Effect

Active Publication Date: 2021-06-22
立沃生物科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For samples with large cell volume and low cell viability, the method is relatively inefficient in removing dead hepatocytes

Method used

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  • Separation liquid for removing dead liver cells and method for removing dead liver cells using same
  • Separation liquid for removing dead liver cells and method for removing dead liver cells using same
  • Separation liquid for removing dead liver cells and method for removing dead liver cells using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] The separation solution for removing dead liver cells in this embodiment is to add the following components to William's E medium: 0.1% v / v ITS general-purpose culture supplement, 2.5 mmol / L L-glutamine, 1 μg / L Epidermal growth factor, hydrocortisone of 20mg / L, dexamethasone of 35μg / L, penicillin streptomycin of 1% v / v and Nycodenz of 0.25g / mL, in the penicin streptomycin, contain 100U / mL of penicillin and 100 mg / mL of streptomycin.

[0094] The method for removing dead hepatocytes using the above-mentioned separation liquid for removing dead hepatocytes in this embodiment includes the following steps:

[0095] Step 1: Two-step collagenase perfusion method to isolate primary hepatocytes

[0096] Take fresh tree shrew liver tissue, perfuse it with perfusion solution I for 10 minutes until the blood in the liver tissue is washed away, and then perfuse it with perfusion solution II preheated at 37°C for 15 minutes until the liver tissue loses its elasticity, and the dige...

Embodiment 2

[0107] The separation solution for removing dead liver cells in this embodiment is to add the following components to William's E medium: 1% v / v ITS general-purpose culture supplement, 2 mmol / l L-glutamine, 10 μg / l epidermis Growth factors, hydrocortisone of 18 mg / l, dexamethasone of 40 μg / l, penicillin streptomycin of 1% v / v, fetal bovine serum of 5% v / v and Nycodenz of 0.3 g / mL, said Penicillin and streptomycin contain 100 U / mL of penicillin and 100 mg / mL of streptomycin.

[0108] The method for removing dead hepatocytes using the above-mentioned separation liquid for removing dead hepatocytes in this embodiment includes the following steps:

[0109] Step 1: Two-step collagenase perfusion method to isolate primary hepatocytes

[0110] Take fresh rat liver tissue and perfuse it with perfusion solution I for 20 minutes until the blood in the liver tissue is washed away, and then perfuse it with perfusion solution II preheated at 37°C for 22 minutes until the liver tissue lose...

Embodiment 3

[0121] The separation solution for removing dead liver cells in this embodiment is to add the following components to William's E medium: 1.5% v / v ITS general-purpose culture additive, 1.5 mmol / l L-glutamine, 20 μg / l Epidermal growth factor, 15 mg / l hydrocortisone, 45 μg / l dexamethasone, 0.5% v / v penicillin streptomycin, 10% v / v fetal bovine serum and 0.5 g / mL Nycodenz, all Among the above-mentioned penicillin and streptomycin, it contains 100U / mL penicillin and 100mg / mL streptomycin.

[0122] The method for removing dead hepatocytes using the above-mentioned separation liquid for removing dead hepatocytes in this embodiment includes the following steps:

[0123] Step 1: Two-step collagenase perfusion method to isolate primary hepatocytes

[0124] Take fresh chicken liver tissue and perfuse it with perfusion solution I for 30 minutes until the blood in the liver tissue is washed away, and then perfuse it with perfusion solution II preheated at 37°C for 30 minutes until the li...

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Abstract

The invention discloses a separation liquid for removing dead liver cells and a method for removing dead liver cells by using the same, belonging to the technical field of separation and purification of liver cells. The separation solution is added the following components in William'sE medium: 0.1% v / v-1.5% v / v ITS general-purpose culture supplement, 1.5mmol / L-2.5mmol / L L-glutamine, 1μg / L-20μg / L epidermal growth factor, 15mg / L-20mg / L hydrocortisone, 35μg / L-45μg / L dexamethasone, 0.5% v / v-1% v / v blue chain Mycin, 1% v / v-10% v / v of fetal bovine serum and 0.25g / mL-0.5g / mL of Nycodenz. The invention has the advantages of maintaining normal osmotic pressure of liver cells, repairing part of damaged liver cells and providing nutritional requirements for liver cells.

Description

technical field [0001] The invention relates to a separation liquid for removing dead liver cells and a method for removing dead liver cells by using the same, belonging to the technical field of separation and purification of liver cells. Background technique [0002] Primary hepatocytes refer to hepatocytes cultured immediately after removal from the liver tissue, which can be widely used in basic research and drug development, including liver research, hepatitis virus research, drug metabolism and toxicity research. However, when isolating primary hepatocytes, the cell viability is affected by many conditions, including the freshness of liver tissue, perfusion time, and composition of perfusion solution, etc., and there are large batch differences. The cell viability of the primary hepatocytes isolated in the prior art is 45%-95%, which seriously affects the experimental research and drug development based on the primary hepatocytes. [0003] Currently, there are two rou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2500/25C12N2500/30C12N2500/32C12N2501/11C12N2501/39C12N2509/00
Inventor 周明黄梓刚刘丹
Owner 立沃生物科技(深圳)有限公司
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