Tumor tissue implant liquid as well as preparation method and application thereof

A tumor tissue and tissue technology, applied in biochemical equipment and methods, tissue culture, tumor/cancer cells, etc., can solve the problem that the molecular composition cannot accurately simulate the human TME, affect the research on the invasion process of human cancer, and affect the success rate of pharmaceutical research and development and other problems, to achieve the effect of increasing the success rate of tumor formation, low cost and easy operation

Active Publication Date: 2020-11-03
江苏莱森生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the molecular composition of EHS cannot accurately simulate human TME, it will cause differences in the establishment of human tumor xenograft models, which will affect the research on the invasion process of human cancer in experiments.
Currently commercial EHS tumor derivative products such as Matrigel®, ECM Gel (Sigma), Cultrex® BME (Amsbio), Geltrex® (Gibco) and ECMatrix™ (Millipore) all use mouse tumor tissue homogenate as the main component, and are very It is difficult to completely simulate the natural microenvironment of human tumor growth, therefore, a mismatch between preclinical and clinical data often occurs, affecting the success rate of drug development

Method used

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  • Tumor tissue implant liquid as well as preparation method and application thereof
  • Tumor tissue implant liquid as well as preparation method and application thereof
  • Tumor tissue implant liquid as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Preparation of Human Ewing Sarcoma Cell Line RD-ES Cell Homogenate

[0019] Human Ewing sarcoma RD-ES cells were incubated at 37°C in 5% CO 2 Cultured in a humidified environment, trypsin (EDTA) was routinely passaged, and the medium (85% RPMI-1640+15% FBS) was replaced every 2-3 days. Inject 2×10 subcutaneously in nude mice at 4-6 weeks 6 RD-ES cells, a personal Ewing sarcoma cell line, when the sarcoma volume in mice reaches 250 mm 3 At the same time, the mice were sacrificed, the sarcoma tissue was taken out, and frozen with liquid nitrogen; the frozen sarcoma tissue was ground into a tissue powder with a freeze grinder, and 10 g of the tissue powder was resuspended in 20 ml of pre-cooled 3.4 M NaCl buffer (pH 7.4 ), after homogenization and centrifugation, resuspend in 20 ml of the above-mentioned NaCl buffer, use the DC protein assay (Bio-Rad) to measure the protein concentration, and dilute different batches of tissue fluid with cell culture medium to...

Embodiment 2

[0020] Example 2: Configuration of Tumor Tissue Implantation Solution

[0021] Take the human Ewing sarcoma cell line RD-ES cell homogenate prepared in Example 1, add different concentrations (5, 10, 20, 40 ng / ml) of EGF factors to prepare tumor implants containing different concentrations of EGF factors liquid. The cell adhesion test was performed on the obtained tumor implant fluid. In a 96-well plate, 100 μl PBS, 10 μg / ml BSA (Sigma-Aldrich), and the above-mentioned tumor implantation solutions containing different concentrations of EGF factors were added to each well, and each concentration was added to six wells, and coated for 24 hours. The implant solution was diluted to 1:10 in PBS, and the excess liquid was removed the next day, and the culture plate was incubated with 100 μl / well 0.1% BSA for 2 hours, and washed with PBS. Add 6000 A549 cells in 100 μl serum-free medium to each well, and culture them in an incubator for 2 h. Non-adherent cells were washed away with...

Embodiment 3

[0025] Example 3: Configuration of Tumor Tissue Implantation Solution

[0026] Take the human Ewing sarcoma cell line RD-ES cell homogenate prepared in Example 1, add different concentrations (5, 10, 20, 40 ng / ml) of hFGF to prepare tumor implantation solutions containing different concentrations of EGF factors . The same method as in Example 2 was used to conduct a cell adhesion test on the obtained tumor implantation fluid. Table 2 is a comparison table of the effects of tumor implant fluids containing different concentrations of hFGF factors on cell adhesion.

[0027] Table 2. Comparison table of the effect of tumor implantation fluid containing different concentrations of hFGF on cell adhesion

[0028]

[0029] It can be seen from Table 2 that the tumor implantation solution prepared after adding hFGF factor at a concentration of 10-40 ng / ml all had high absorbance, and the absorbance measured when the concentration of hFGF factor was 20 ng / ml was the highest.

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Abstract

The invention belongs to the technical field of biology, and particularly relates to tumor tissue implant liquid as well as a preparation method and application thereof. According to the tumor implantliquid provided by the invention, tissue homogenate after tumor formation of a human Ewing sarcoma cell line RD-ES is used as a tumor microenvironment simulation matrix, and EGF, hFGF, ITS, non-essential amino acids and L-glutamine are added as other components. The adhesive property of tumor tissues can be well improved, the tumor formation success rate of xenograft of human tumor cell strains can be greatly increased through verification of various tumor cells, and good effect on various tumor models is achieved. Besides, the preparation method provided by the invention is simple, low in cost, easy to operate and low in requirements for operators.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a tumor tissue implantation liquid and its preparation method and application. Background technique [0002] PDX (Patient-Derived tumor Xenograft) human-derived tumor xenograft model refers to a tumor model established by transplanting the tumor tissue of a tumor patient into an immunodeficient mouse and relying on the microenvironment provided by the mouse for growth. The simulated stromal composition of the tumor microenvironment (TME) is a very important factor in the construction of PDX models. Mouse EHS (Engelbreth-Holm Swarm) sarcoma-derived products are the most commonly used tumor microenvironment simulating substrates in experimental research. However, because the molecular composition of EHS cannot accurately simulate human TME, it will cause differences in the establishment of human tumor xenograft models, which will affect the research on the invasion process...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/84C12N2533/90
Inventor 刘晗青屠志刚
Owner 江苏莱森生物科技研究院有限公司
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