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Technical method for co-cultivating hepatocytes and hepatic sinusoidal endothelial cells and application of technical method

A technology of endothelial cells and co-cultivation, applied in the fields of medicine and biology, to achieve the effects of enhanced support, huge economic and social values, and broad application prospects

Inactive Publication Date: 2019-03-01
苏州瑞徕生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Journal of Southeast University (Medical Edition) 2018, 37(4): 566-572), but the function of the co-culture group was only 10-20% higher than that of the hepatocyte group alone, and the ability of the co-culture group to reduce bilirubin at 72h was still less than 50%. %, which needs to be improved for the application fields that require relatively high liver cell function

Method used

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  • Technical method for co-cultivating hepatocytes and hepatic sinusoidal endothelial cells and application of technical method
  • Technical method for co-cultivating hepatocytes and hepatic sinusoidal endothelial cells and application of technical method
  • Technical method for co-cultivating hepatocytes and hepatic sinusoidal endothelial cells and application of technical method

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Embodiment 1

[0027] Embodiment 1: co-culture method 1

[0028] Cultivate 2 bottles of human liver sinusoidal endothelial cell line LSEC under normal conditions (37°C incubator with 95% air and 5% carbon dioxide) to 60-80% confluence, then continue normal culture in 1 bottle, and put oxygen concentration in the other bottle. In a 3% three-gas incubator, close the door of the incubator, start timing when the oxygen concentration drops to 3%, take out the culture bottle after 24 hours of anoxic culture, and collect the normal cells respectively with 0.1% trypsin / 0.1% EDTA digestion method. cultured and hypoxic cultured LSECs, and the normal cultured human hepatocyte line IHH. Divide into three groups for inoculation, each group has 6 wells. Simple IHH group: take 1×10 4 Inoculate a 24-well plate at a cell density of 1 IHH / well; normal LSEC co-culture group: mix IHH and normal cultured LSEC at a ratio of 1:0.25, inoculate in a 24-well plate, and each well contains 1×10 4 IHH cells and 2.5×1...

Embodiment 2

[0033] Embodiment 2: co-culture method 2

[0034] Cultivate 2 bottles of human liver sinusoidal endothelial cell line LSEC under normal conditions (37°C incubator with 95% air and 5% carbon dioxide) to 60-80% confluence, then continue normal culture in 1 bottle, and put oxygen concentration in the other bottle. In a 2% three-gas incubator, close the door of the incubator, start timing when the oxygen concentration drops to 2%, take out the culture bottle after hypoxic culture for 12 hours, and collect normal cells by 0.1% trypsin / 0.1% EDTA digestion method. cultured and hypoxic cultured LSECs, and the normal cultured human hepatocyte line IHH. Divide into three groups for inoculation, each group has 6 wells. Simple IHH group: take 1×10 4 Inoculate a 12-well plate at a cell density of 1 IHH / well; normal LSEC co-culture group: mix IHH and normal cultured LSEC at a ratio of 1:4, inoculate in a 12-well plate, and each well contains 1×10 4 IHH cells and 4×10 4 LSEC; anoxic LSEC...

Embodiment 3

[0037] Embodiment 3: co-culture method 3

[0038] Cultivate 2 bottles of human liver sinusoidal endothelial cell line LSEC under normal conditions (37°C incubator with 95% air and 5% carbon dioxide) to 60-80% confluence, then continue normal culture in 1 bottle, and put oxygen concentration in the other bottle. In a 1% three-gas incubator, close the door of the incubator, start timing when the oxygen concentration drops to 1%, take out the culture bottle after 6 hours of anoxic culture, and collect the normal cells respectively with 0.1% trypsin / 0.1% EDTA digestion method. cultured and hypoxic cultured LSECs. In addition, on the day before harvesting LSEC, inoculated the normal cultured human hepatocyte line IHH into 24-well plate, 1×10 4 A total of 18 wells were inoculated with each IHH / well. After harvesting LSECs, they were divided into 5 x 10 3 The amount of 1 LSEC / well was added to the IHH culture wells, and 6 wells were each inoculated with LSECs from normal culture a...

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Abstract

The invention discloses a technical method for co-cultivating hepatocytes and hepatic sinusoidal endothelial cells and application of the technical method. The technical method is characterized in that a hepatic sinusoidal endothelial cell line is subjected to anaerobic cultivation for a certain period of time, and then the hepatic sinusoidal endothelial cell line is harvested and co-cultured witha hepatocyte line at a certain ratio. Co-culture with the hepatic sinusoidal endothelial cell line can partially simulate a liver environment in a human body, hepatocyte functions are facilitated, after the hepatic sinusoidal endothelial cells are activated by hypoxia, the ability of promoting the hepatocyte functions is enhanced, and no exogenous endothelial cell activator is needed, the introduction of exogenous materials is reduced, and subsequent cell application is facilitated. The invention further provides the application of the co-culture method in fields including drug screening, bioartificial liver preparation and the like.

Description

technical field [0001] The invention relates to the fields of medicine and biology, in particular to a co-cultivation method of hepatic cells and hepatic sinusoidal endothelial cells and the application of the co-cultured hepatic cells and hepatic sinusoidal endothelial cells in the preparation of bioartificial liver or drug screening. Background technique [0002] Hepatocytes cultured in vitro have application or potential application value in many aspects, such as: drug or toxic substance screening, hepatitis virus infection mechanism and treatment research, and the use of cultured hepatocytes to prepare bioartificial liver for the treatment of liver failure, etc. However, with the prolongation of culture time, hepatocytes easily lose their structural features and functions during monolayer culture in vitro (Elaut G, Henkens T, Papeleu P, et al. Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. CurrDrug Metab, 2006, 7...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02A61L27/38
CPCA61L27/3804A61L27/3839A61L27/3886A61L27/3895A61L2430/28C12N5/067C12N2502/14G01N33/5067G01N2500/10
Inventor 娄晋宁张文健
Owner 苏州瑞徕生物科技有限公司
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