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Method for culturing bladder cancer organs in vitro

An in vitro culture, bladder cancer technology, applied in the field of in vitro culture of bladder cancer organoids, can solve the problems of unsuitable tumor immune drug screening, less successful bladder cancer organoid culture, lack of tumor microenvironment fidelity, etc. The effect of large cell loss, improved culture success rate, and simplified operation

Active Publication Date: 2020-05-26
上海嗣新医药科技有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been reported that the sources of successfully cultured tumor organoids include breast cancer, colon cancer, lung cancer, and esophageal cancer, among which the success rate of breast cancer and colon cancer organoids is as high as 80%, but the successful culture of bladder cancer organoids has been reported. Rarely, with a low success rate (less than 30%)
In addition, although the existing organoid culture technology can maintain the high heterogeneity of tumor samples, it still lacks the fidelity of the tumor microenvironment. For example, the existing tumor organoids do not contain the tumor microenvironment Immune cells, resulting in the cultured organoids not suitable for the screening of tumor immune drugs

Method used

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  • Method for culturing bladder cancer organs in vitro
  • Method for culturing bladder cancer organs in vitro
  • Method for culturing bladder cancer organs in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0032] 1. Processing of freshly isolated bladder cancer tissue:

[0033] The obtained fresh isolated bladder cancer tissue was trimmed to remove non-tumor tissue and obvious necrotic tissue, washed several times with pre-cooled PBS containing 2% double antibody, and then minced in a sterile environment to a diameter of less than 0.1 mm, that is, minced meat, and then filtered with a 100 μm cell sieve, discarding larger pieces of tissue, and the filtrate was centrifuged and washed twice with basal medium; the composition of the basal medium is: advanced DMEM / F12 medium , 500-fold dilution of Primocin primary cell antibiotics, 10 μM Y-27632 ROCK inhibitor (dihydrochloride) and 2.5% fetal bovine serum;

[0034] 2. Make a gas-liquid interactive culture system, namely:

[0035] 21. Preparation of Rat Tail Collagen Solution

[0036] Under an ice bath, take 1267 μL rat tail type I collagen (3.79 mg / mL) and place it in a 2 mL EP tube, then add 200 μL of 10X concentration Ham’s F-12 ...

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Abstract

The invention discloses a method for culturing bladder cancer organs in vitro. The method comprises the following steps: manufacturing a gas-liquid interaction culture system, namely manufacturing a uniformly paved rat tail collagen supporting layer on the surface of a porous culture membrane in a Transwell upper chamber; re-suspending fresh in-vitro bladder cancer tissues by using a rat tail collagen solution, uniformly mixing, adding the obtained mixture onto the rat tail collagen supporting layer, and then putting the rat tail collagen supporting layer into an incubator at 37 DEG C to solidify so as to obtain a rat tail collagen layer containing the bladder cancer tissues; adding an organoid culture medium into a Transwell lower chamber, and enabling the liquid level of the culture medium to be lower than the rat tail collagen layer containing bladder cancer tissues; and carrying out passage and cryopreservation. The culture success rate of the bladder cancer organoid is remarkablyincreased, the bladder cancer organoid with reserved immune cells can be obtained through culture, operation is easy, the utilization rate of tumor tissue is high, and great significance and value areachieved for bladder cancer drug screening research.

Description

technical field [0001] The invention relates to a method for culturing bladder cancer organoids in vitro, and belongs to the technical field of in vitro culture of organoids. Background technique [0002] Bladder cancer is the most common malignant tumor of the urinary system, and the incidence of bladder cancer in my country has been increasing year by year in recent years. Many patients with locally advanced or metastatic bladder cancer have unsatisfactory treatment outcomes, and some patients will change regimens or even stop medication because they cannot bear the side effects of chemotherapy. Targeted therapy, as a new treatment model with definite curative effect and good patient tolerance, has emerged in the treatment of solid malignant tumors. However, the preliminary screening of bladder cancer targeted drugs still lacks efficient and accurate in vitro models for drug screening. [0003] Tumor organoids are an in vitro three-dimensional tumor cell culture technolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N5/0684C12N2533/54C12N2509/00Y02A50/30
Inventor 王宗耀李宜璇
Owner 上海嗣新医药科技有限公司
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