Hepatocyte three-dimensional culture method

A three-dimensional culture and cell culture technology, applied in the field of cell culture, can solve the problems of reducing R&D efficiency and repetitive labor, and achieve the effect of reducing time, improving work efficiency and high culture activity.

Pending Publication Date: 2021-07-23
NAT INST FOR NUTRITION & HEALTH CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Three-dimensional cell culture technology is a technology between monolayer cell culture and animal experiments. It can not only simulate the microenvironment of cell growth, proliferation, migration, differentiation and apoptosis in the body to the greatest extent, but also easily form cells with reasonable morphology. It can also show the advantages of intuitive and controllable conditions of cell culture, so three-dimensional cell culture technology is often used in the research of drug carriers, drug toxicology, drug screening, tumor treatment, etc., but , the existing training methods usually require targeted experiments for different application requirements, which will cause a lot of repetitive labor and reduce the efficiency of research and development

Method used

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  • Hepatocyte three-dimensional culture method
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  • Hepatocyte three-dimensional culture method

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The cell culture method in this example adopts the method in the specific embodiment, the difference is that the hydrogel dilution ratio is 1:2, and the final cell concentration is 1×10 6 / ml, 5×10 5 / ml, 1×10 5 / ml, 5×10 4 / ml, 1×10 4 Cultivate at 0, 3, 6, 9, and 12 days, measure cell viability, measure the number of living cells: adopt the CCK-8 method to measure the number of living cells in three-dimensional cell culture, and CCK-8 is provided by Dongren Chemical Technology Co., Ltd. ; After the three-dimensional cell culture was cultured for a period of time, the growth medium was discarded, the three-dimensional cell culture was washed with PBS, and 100 μl of serum-free medium containing 10% CCK8 was added to each well to act for 2 hours, and the SpectraMax i3x microplate reader was used to measure the Absorbance.

[0029] The specific data results are shown in Table 1 below, and the corresponding curves are shown in figure 1 .

[0030] Table 1

[0031] ...

Embodiment 2

[0033] The cell culture method of this embodiment adopts the method in the specific embodiment, the difference is that the final concentration of cells is 1×10 5 / ml, hydrogel dilution 1:2, cultured for 14 days, cell viability was detected every day, and the number of living cells was measured: the number of living cells in three-dimensional cell culture was measured by the CCK-8 method, and CCK-8 was produced by Dongren Chemical Technology Co., Ltd. Provided by the company; Discard the growth medium after the three-dimensional cell culture has been cultured for a period of time, wash the three-dimensional cell culture with PBS, add 100 μl of serum-free medium containing 10% CCK8 to each well and act for 2 hours, use a SpectraMax i3x microplate reader to measure 450nm absorbance at .

[0034] The specific data results are shown in Table 2 below, and the corresponding curves are shown in figure 2 .

[0035] Table 2

[0036]

Embodiment 3

[0038] The cell culture method of this embodiment adopts the method in the specific embodiment, the difference is that the final concentration of cells is 1×10 5 / ml, the dilution of the hydrogel is 1:0, 1:1, 1:2, 1:3, 1:4 respectively, the cell viability was measured at 0, 3, 6, 9, and 12 days of culture, and the number of living cells was determined: The number of living cells in three-dimensional cell culture was determined by the CCK-8 method. CCK-8 was provided by Dongren Chemical Technology Co., Ltd.; the growth medium was discarded after the three-dimensional cell culture was cultivated for a period of time, and the three-dimensional cell culture was washed with PBS, and added to each well. After 100 μl of serum-free medium containing 10% CCK8 acted for 2 hours, the absorbance at 450 nm was measured using a SpectraMax i3x microplate reader.

[0039] The specific data results are shown in Table 3 below, and the corresponding curves are shown in image 3 .

[0040] tabl...

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Abstract

The invention relates to a hepatocyte three-dimensional culture method. The hepatocyte three-dimensional culture method comprises the following steps: step 1, putting hepatocytes into a culture dish containing a culture solution, putting the culture dish into an incubator containing CO2 with the volume content of 5% at the temperature of 37 DEG C for culture, and performing passage when the cell density in the culture dish reaches 80-90%, so as to obtain a cell suspension for three-dimensional cell culture; step 2, adding the cell suspension obtained in the step 1 and diluted hydrogel into a pore plate culture dish to be mixed, adjusting the final concentration of the cells to be 1*10<5> / ml to 5*10<5> / ml, performing standing at the room temperature till solidification so as to obtain a cell hydrogel mixture, wherein the volume ratio of the hydrogel to hydrogel diluent in the diluted hydrogel is (1: 2)-(1: 4); and step 3, adding a cell culture solution into the pore plate culture dish in the step 2, placing the pore plate culture dish in the incubator for culturing, replacing half of the cell culture solution every day, and performing culturing for a period of time to obtain a three-dimensional cell culture product. By implementing the method provided by the invention, a model for drug screening and poison pathway research can be obtained.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for three-dimensional culture of hepatocytes. Background technique [0002] The liver is an important vital organ of the human body. The construction of three-dimensional tissue and organ chips of human-derived hepatocytes in vitro can not only be used for the treatment of liver diseases such as liver necrosis, but also can be used as a model for liver development research and drug screening for liver diseases. It is useful for proposing health guidance values ​​for important pollutants and providing technical support for food-related policies. [0003] Three-dimensional cell culture (TDCC) refers to the co-cultivation of carriers with different three-dimensional structures and various types of cells in vitro, and the cells and the extracellular matrix (ECM) in the surrounding microenvironment , Various cytokines, chemical factors, physical and mechanical forces, a...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0671C12N2513/00
Inventor 王晶波陈晨马妍沈葹秦文杨倬王丽媛霍军生卓勤宫照龙
Owner NAT INST FOR NUTRITION & HEALTH CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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