Separating mediumon solution for removing dead hepatocytes and method for removing dead hepatocytes by utilizing separating mediumon solution

A liver cell and separation solution technology, applied in the field of separation solution for removing dead liver cells, can solve the problems of low cell viability and low efficiency of removing dead liver cells, and achieve the effect of improving recovery efficiency and maintaining osmotic pressure

Active Publication Date: 2019-09-13
立沃生物科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For samples with large cell volume and low cell viability, the

Method used

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  • Separating mediumon solution for removing dead hepatocytes and method for removing dead hepatocytes by utilizing separating mediumon solution
  • Separating mediumon solution for removing dead hepatocytes and method for removing dead hepatocytes by utilizing separating mediumon solution
  • Separating mediumon solution for removing dead hepatocytes and method for removing dead hepatocytes by utilizing separating mediumon solution

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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 separating mediumon solution for removing dead hepatocytes and a method for removing dead hepatocytes by utilizing the separating mediumon solution and belongs to the technical field of hepatocyte isolationliver cell separation and purification. The separating mediumon solution is prepared by adding the following components in a William's E culture medium: 0.1-1.5 percentv/v of ITS universal culture additives, 1.5-2.5 mmol/L of L-glutamine, 1-20 [mu]gmu g/L of an epidermal growth factor, 15-20mg/L of hydrocortisone, 35-45 [mu]gmu g/L of dexamethasone, 0.5-1 percent v/v of penicillin streptomycin, 1-10 percent v/v fetal calf serum and 0.25-0.5g/mL of Nycodenz. The separating medium tion solution has the advantages of maintaining normal osmotic pressure of hepatocytes, repairing partial damaged hepatocytes and providing nutritional requirement for hepatocytes.

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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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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