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Establishment and applications of three-dimensional liver model based on qualitative filtration paper

A qualitative filter paper and model technology, applied in the field of tissue engineering and drug research, to achieve reasonable design, high albumin expression level, and maintain the effect of liver cell structure and function

Inactive Publication Date: 2018-06-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of filter paper materials to culture hepatocytes, and to construct a three-dimensional liver tissue model that can be cultured in vitro for a long time

Method used

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  • Establishment and applications of three-dimensional liver model based on qualitative filtration paper
  • Establishment and applications of three-dimensional liver model based on qualitative filtration paper
  • Establishment and applications of three-dimensional liver model based on qualitative filtration paper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Three-dimensional liver models were constructed using qualitative filter paper.

[0040] as follows figure 1 and figure 2 As indicated, the filter paper was made into a 24-well or 96-well disc with a hole puncher, and after autoclaving, it was coated with 100ug / ml type I rat tail collagen overnight, and then washed with PBS to remove the residual Collagen, press HUVEC cells in 0.4X10 6 Cells per ml were seeded in a 24-well plate, and 500ul of cell suspension was added to each well. After culturing on paper for two days, the original culture medium was sucked off, and hiHep cells were inoculated according to the ratio of HUVEC / hiHep=1:1~2. , change the hiHep medium and continue the culture, so as to obtain a three-dimensional liver model with better hepatocyte function. Afterwards, the medium was changed every other day and the cell culture medium was collected for subsequent detection of albumin and urea.

[0041] The model is designed by figure 2 The three-dimensi...

Embodiment 2

[0045] Use this 3D liver model to study hepatocyte-specific functions.

[0046] The three-dimensional model was established according to the above-mentioned method, and the medium was changed every other day during the culture period. From the hepatocyte inoculation incubation until the 14th day, the hepatocyte function reached a good state. Monolayer hepatocyte culture was used as a control, and the lipid and glycogen synthesis of hepatocytes and the expression level of albumin were detected. The three-dimensional liver model cultured for 14 days was washed once with warm PBS, fixed with 4% PFA at room temperature for 15 minutes, washed three times with PBS, and oil red O working solution (oil red: deionized water = 3:2) was added. Stain at 37 degrees for about 20 minutes, decolorize, rinse with 75% alcohol or 60% isopropanol to remove excess dye, then rinse with PBS, and observe the lipid synthesis under a microscope. Use the same fixation method as above to fix and culture...

Embodiment 3

[0049] The three-dimensional liver model was used to study its cell phenotype and liver function during long-term culture in vitro.

[0050] The three-dimensional liver model was also established according to the aforementioned method, and the medium was changed every other day during the culture period, and the liver tissue morphology in the three-dimensional model was observed on the 7th day, the 31st day, the 60th day, and the 103rd day respectively. Since the liver cells themselves emit green fluorescence, the result ( Figure 6 ) showed that the cells had formed dispersed small cell aggregates on the 7th day, and large microstructures had formed on the 31st day, presenting a three-dimensional shape. The cultured three-dimensional liver model is more stable than the three-dimensional liver model alone, and the cell shape is better maintained.

[0051] The three-dimensional liver model was also established according to the above method. During the culture period, the mediu...

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Abstract

The present invention provides establishment and applications of a three-dimensional liver model based on qualitative filtration paper. The three-dimensional liver model is mainly based on the normalphysiological structure characteristics of liver, wherein liver cells hiHep obtained through transdifferentiation are directly induced with fibroblasts, a traditional primary liver cell or liver cellline monolayer culture model is improved, and qualitative filtration paper as a scaffold material for cell culture is placed in a 24-well plate, and is suspended in a culture solution, such that the complete material transfer and nutrient component exchange can be performed so as to achieve the three-dimensional growth of cells on the filtration paper. According to the present invention, the co-culture of hiHep cells and umbilical vein endothelial cells (HUVECs) is performed on the qualitative filtration paper to obtain the liver model based on the filtration paper, such that the three-dimensional liver tissue capable of being subjected to long-term in vitro culture and maintaining the specific functions of liver is achieved; with the obtained three-dimensional liver tissue, the in vitro test and evaluation can be performed on a series of hepatotoxicity drugs; and the obtained three-dimensional liver tissue is mainly used for the in vitro research of the long-term toxicity of drug molecules to liver tissues and the liver function injury process.

Description

technical field [0001] The invention belongs to the field of tissue engineering and drug research, in particular to the establishment and application of a three-dimensional liver model based on qualitative filter paper. Background technique [0002] Among the many organs in the human body, the liver is the largest gland in the human body. It has a very complex microstructure and has various functions such as substance metabolism, blood detoxification and plasma protein secretion, especially the drug and toxin metabolism function, which is very important for maintaining the stability of the body. and metabolism play an important role. However, at present, various liver diseases seriously threaten human health, and most of the research on liver diseases, drug screening, and toxicity detection still rely on animal experiments and clinical studies. Due to the differences between species, it is difficult to fully translate the results of animal experiments into the clinic, and d...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02G01N33/68G01N33/62
CPCC12N5/0671C12N2502/28C12N2503/02C12N2533/54G01N33/5014G01N33/5067G01N33/62G01N33/6827G01N2333/765
Inventor 秦建华王亚清王丽
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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