Decellularized extracellular matrix (DECM) template used for simulating liver cancer environment

A decellularization and template technology, applied in the direction of liver cells, tumors/cancer cells, cell culture supports/coatings, etc., can solve the problems of time-consuming and long-term cycle, materials are not easy to obtain, etc., achieve low price, promote proliferation and Adhesive, easy handling effect

Active Publication Date: 2020-06-30
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the existing bionic liver scaffolds are prepared by the perfusion method, which requires a whole piece of liver with well-preserved bronchi, and the materials are not easy to obtain.
[0003] On this basis, in order to find a more convenient and simple test method, the present invention has developed a low-cost, easy-to-make drug test template that is separated from living animals, and simulates the cell growth environment through the decellularized extracellular matrix of the tissue. At the same time, the same-layer contact mixed culture of normal cells and cancer cells is used to create a cancer-inducing effect, which realizes a test template used in vitro, which can be used to detect the effect of the currently prepared nano-synthetic materials on liver tumor cells. The effect of drug therapy, photothermal therapy and chemokinetic therapy, and solve the ethical and time-consuming and long-term problems caused by animal experiments

Method used

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  • Decellularized extracellular matrix (DECM) template used for simulating liver cancer environment
  • Decellularized extracellular matrix (DECM) template used for simulating liver cancer environment
  • Decellularized extracellular matrix (DECM) template used for simulating liver cancer environment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] (1) Combine DPPC, DPPE, DPPA, DSPE-PEG 2000 and Fe 3 o 4 Dissolve in a mixed solution of 10 mL methanol and 5 mL chloroform, and obtain solution S1 by ultrasonication, in which the concentration of each substance is 2 mg / mL DPPC, 5 mg / mL DPPE, 5 mg / mLDPPA, 5 mg / mL DSPE- PEG 2000 and 1.33 mg / mL Fe 3 o 4 ;

[0064] (2) After ultrasonication for 1 min, the solution S1 was rotary evaporated at 55 °C for 1 h to form a film to obtain P-FeNBs;

[0065] (3) Mix 500 µL glycerol, 5 mL PBS and 3 mg F-68 to obtain hydration solution S2;

[0066] (4) Add the hydration solution S2 into the P-FeNBs, and ultrasonically dissolve the liposome membrane for 5 min to obtain FeNBs;

[0067] (5) DOX was added to the obtained FeNBs solution in an amount of 3.64 mg / mL, and after 1 h in an oil bath at 75 °C, it was shaken at 500 rpm for 2 h in a perfluoropropane atmosphere, and centrifuged at 800 rpm for 10 min, centrifuged and washed 3 times with PBS at a centrifugation speed of 20,000 rp...

Embodiment 2

[0083] (1) Combine DPPC, DPPE, DPPA, DSPE-PEG 2000 and Fe 3 o 4 Dissolve in a mixed solution of 80 mL methanol and 1 mL chloroform, and obtain solution S1 by ultrasonication, in which the concentration of each substance is 5 mg / mL DPPC, 2 mg / mL DPPE, 4 mg / mLDPPA, 0.02 mg / mL DSPE- PEG 2000 and 0.01 mg / mL Fe 3 o 4 ;

[0084] (2) After ultrasonication for 5 min, the solution S1 was rotary evaporated at 20 °C for 10 min to form a film to obtain P-FeNBs;

[0085] (3) Mix 1 µL glycerol, 10 mL PBS and 15 mg F-68 to obtain hydration solution S2;

[0086] (4) Add the hydration solution S2 into the P-FeNBs, and ultrasonically dissolve the liposome membrane for 20 min to obtain FeNBs;

[0087] (5) DOX was added to the obtained FeNBs solution in an amount of 0.005 mg / mL. After 3 h in an oil bath at 100 °C, it was shaken at 300 rpm for 5 h in a perfluoropropane atmosphere, and centrifuged at 800 rpm for 5 h. min, washed once with PBS at a centrifugation speed of 3000 rpm for 1 min to...

Embodiment 3

[0094] (1) Combine DPPC, DPPE, DPPA, DSPE-PEG 2000 and Fe 3 o 4 Dissolve in a mixed solution of 40 mL methanol and 20 mL chloroform, and obtain solution S1 by ultrasound, in which the concentration of each substance is 5 mg / mL DPPC, 4.8 mg / mL DPPE, 2 mg / mLDPPA, 0.02 mg / mL DSPE- PEG 2000 and 0.005 mg / mL Fe 3 o 4 ;

[0095] (2) After ultrasonication for 5 min, the solution S1 was rotary evaporated at 20 °C for 10 min to form a film to obtain P-FeNBs;

[0096] (3) Mix 1 µL glycerol, 10 mL PBS and 15 mg F-68 to obtain hydration solution S2;

[0097] (4) Add the hydration solution S2 into the P-FeNBs, and ultrasonically dissolve the liposome membrane for 20 min to obtain FeNBs;

[0098] (5) DOX was added to the obtained FeNBs solution in an amount of 0.005 mg / mL, and after 3 h in an oil bath at 100 °C, it was shaken at 300 rpm for 5 h in a perfluoropropane atmosphere, and centrifuged at 4000 rpm for 40 min, washed once with PBS at a centrifugation speed of 3000 rpm for 1 min ...

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Abstract

The invention discloses a decellularized extracellular matrix (DECM) template used for simulating liver cancer environment. The DECM template used for simulating the liver cancer environment is prepared by the following steps: performing decellularization on a fresh animal liver section so as to maintain the original net-like structure with troponin; and then, co-implanting hepatocytes LO2 and hepatoma cells HepG2 on a hepatic DECM template so as to simulate actual internal environment of liver tumor lesions. The DECM template used for simulating the liver cancer environment prepared accordingto the invention is safe, non-toxic, good in biocompatibility, low in price, and convenient to operate. Being introduced with a magnetic targeting drug vector to be tested in the system, the DECM template is capable of accurately simulating treatment of liver cancer by a drug in vivo, thereby reducing overuse of laboratory animals and effectively decreasing cost of scientific researches.

Description

technical field [0001] The invention belongs to the field of biological material preparation and biomedical application, and in particular relates to a decellularized scaffold template for simulating the environment of liver cancer, which can be used in the research of drug-targeted therapy for liver cancer. Background technique [0002] Mature nano-drug delivery systems usually require animal experiments when testing their therapeutic effects (Li Y, J.Z. et al. ACS Applied Materials & Interfaces, 2017, 9(41): 35604-35612). Such experimental research will cause great harm to animals and deprive animals of their right to live. Therefore, the ethics and morals of scientific research involving animal experiments have been widely concerned by all walks of life. What's more, some radical animal rights defenders even demand a complete cessation of animal experiments, which will bring great inconvenience to scientific research activities in medicine, biology and other disciplines a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0671C12N5/0693C12N2533/90
Inventor 张进林哲郑辉东何晶晶刘宁李飞翰
Owner FUZHOU UNIV
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