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A method for establishing a liver cancer xenograft tumor model based on the hanging drop culture method of circulating tumor cells in liver cancer

A technology of biological gel and hanging drop culture, which is applied in the field of biomedicine, can solve the problems of high price and low success rate of modeling, and achieve the effect of low cost, simple preparation method and mature process

Active Publication Date: 2020-08-21
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, according to current reports, this method requires a large amount of peripheral blood for CTC isolation, and the success rate of modeling is not high, and it is expensive

Method used

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  • A method for establishing a liver cancer xenograft tumor model based on the hanging drop culture method of circulating tumor cells in liver cancer
  • A method for establishing a liver cancer xenograft tumor model based on the hanging drop culture method of circulating tumor cells in liver cancer
  • A method for establishing a liver cancer xenograft tumor model based on the hanging drop culture method of circulating tumor cells in liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preoperative CTC culture results of a patient with liver cancer

[0047] 1. Experimental method:

[0048] (1) Take 10 mL of preoperative peripheral blood from the patient, mix it with HBSS buffer 1:1, and place it in a 50 mL centrifuge tube. Add 10mL of Ficoll density gradient solution to another 50mL centrifuge tube, and gently add the diluted peripheral blood to the density gradient solution to form an obvious layer. Centrifuge at room temperature at 2000rpm for 20min. At this time, the liquid is divided into 4 layers. Use a 3mL Pasteur pipette to absorb the nucleated cell layer, add 10mL of HBSS buffer, and centrifuge at 1000rpm for 5min to collect the cells and fully remove the supernatant. Add 1mL of HBSS (containing 2% serum ) to resuspend the cells and transfer to a 5 mL sterile flow tube.

[0049] (2) Add 20 μL of StemCell CD45 negative selection magnetic beads, mix gently and incubate at room temperature for 15 minutes, put them in a magnetic field...

Embodiment 2

[0056] Example 2: Detection of Subcutaneous Tumor Formation Ability of Peripheral Blood MC and OC Type Clones in Nude Mice from 7 Cases of Liver Cancer Patients

[0057] 1. Experimental method:

[0058] (1) Take 10 mL of preoperative peripheral blood from the patient, mix it with HBSS buffer 1:1, and place it in a 50 mL centrifuge tube. Add 10mL of Ficoll density gradient solution to another 50mL centrifuge tube, and gently add the diluted peripheral blood to the density gradient solution to form an obvious layer. Centrifuge at room temperature at 2000rpm for 20min. At this time, the liquid is divided into 4 layers. Use a 3mL Pasteur pipette to absorb the nucleated cell layer, add 10mL of HBSS buffer, and centrifuge at 1000rpm for 5min to collect the cells and fully remove the supernatant. Add 1mL of HBSS (containing 2% serum ) to resuspend the cells and transfer to a 5 mL sterile flow tube.

[0059] (2) Add 20 μL of StemCell CD45 negative selection magnetic beads, mix gentl...

Embodiment 3

[0069] Example 3: Establishment of a PDX model of tumor formation under the liver capsule of CTC nude mice

[0070] 1. Experimental method:

[0071] (1) Pick cell clones from the CTC culture system and suspend them in 20 μL HBSS buffer for later use; after nude mice are anesthetized with isoflurane inhalation, a 0.4-0.5 cm incision is made on the upper abdomen to expose a small part of the liver, and 20 μL of the cells are injected into an insulin syringe. The suspension was injected under the liver capsule and sutured. Two weeks later, the tumor was observed by laparotomy.

[0072] (2) Embed the tumor tissue in wax block, and detect with HE staining: xylene (I) 5-10 min, xylene (II) 5-10 min, 95% ethanol (I) 1-3 min, 95% ethanol (II) 1 -3min, 80% ethanol for 1min, distilled water for 1min, hematoxylin semen staining for 5-15min, running water to wash away the hematoxylin semen for 1-3s, 1% hydrochloric acid ethanol for 1-3s, washing with water for 10-30s, and the blue-promo...

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Abstract

The invention relates to the technical field of biomedicines, and concretely relates to a method for establishing liver cancer xenograft tumors based on temperature-sensitive biological gel hanging-drop culture of a liver cancer circulating tumor system. The method comprises the following steps: carrying out in-vitro amplification on the circulating tumor cells of a liver cancer patient, forming a multicellular clone, picking the clone to inoculate naked mice to form tumors in subcutaneous tissues, the liver and the lung, and establishing the xenograft tumor model. The invention also provides a reagent used in the liver cancer circulating tumor cell biological gel hanging-drop culture technology. Experiments prove that the liver cancer circulating tumor cells undergo amplified growing in the temperature-sensitive biological gel system, and are used to inoculate the naked mice to form the tumors. The method provides a model animal for the individual treatment of the liver cancer for the establishment of the liver cancer xenograft tumor animal model.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for establishing a liver cancer xenograft tumor model based on a hanging drop culture method of liver cancer circulating tumor cells biogel. Background technique [0002] It has been revealed 150 years ago that tumor cells can fall from the lesion into the blood to form tumor cells, but there have been few major discoveries in this field in the past 100 years, because CTCs are very rare in the blood, how to enrich and Identification of tumor cells in peripheral blood was once a great technical bottleneck. For quite a long period of time, the research on CTC has stagnated in detecting the expression of specific tumor molecules in whole blood by PCR or flow cytometry, so as to reflect the number of circulating tumor cells in peripheral blood. However, the reliability of this method is extremely low. Taking liver cancer as an example, the detection of AFP mRNA expressi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09
CPCC12N5/067C12N5/0693C12N2500/32C12N2500/84C12N2501/105C12N2501/11C12N2501/113C12N2501/117C12N2523/00C12N2533/30
Inventor 张璐定郭猛刘芳滕飞张铭健展洋洋鲍蕾蕾施晓敏曹雪涛王全兴
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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