Three-dimensional culture of primary cancer cells using tumor tissue

A tumor tissue, three-dimensional culture technology, applied in the direction of tumor/cancer cells, tissue culture, cell culture support/coating, etc., can solve practical problems, cancer cell proliferation ability, ease of processing, high throughput , There are problems in generality, and it is impossible to confirm the obvious proliferation of cell blocks, etc., to achieve the effect of high proliferation ability and high throughput

Pending Publication Date: 2019-11-19
MITSUBISHI CHEM MEDIENCE
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, only human lung cancer tumors and breast cancer xenograft tumors (Non-Patent Document 8: Sakamoto, Ruriko, et al."Time-lapse imaging assay using the BioStation CT: A sensitive drug-screening method for three-dimensional cellculture."Cancer Science 106.6(2015):757-765.), especially in breast cancer xenograft tumors, it is impossible to confirm the obvious proliferation of cell masses, and the verification of universality is not sufficient, and there are doubts about its practicality
[0008] As shown by these methods, the culture of primary cancer cells using tumor tissue increases the reliability of culturability by the three-dimensional culture method. There are problems in any aspect of performance, versatility, etc.

Method used

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  • Three-dimensional culture of primary cancer cells using tumor tissue
  • Three-dimensional culture of primary cancer cells using tumor tissue
  • Three-dimensional culture of primary cancer cells using tumor tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086]

[0087] According to the conventional method, in the safety cabinet, the patient-derived human cancer patient was aseptically extracted from the subcutaneous tissue of immunodeficient mice [Super SCID mice (system name: C3H / HeJ / NOs-scid; LPS-nonresponder)]. - Derived Xenograft (hereinafter referred to as "PDX") tumor, the necrotic area of ​​the tumor was removed with surgical scissors. The tumor was quickly immersed in the Japanese Pharmacopoeia saline solution and stored on ice. Next, the Japanese Pharmacopoeia saline solution was removed from the tumor, and the tumor was repeatedly washed three times with a specimen treatment solution (provided with the cancer organoid culture kit, ORGANOGENIX).

[0088] As a preparatory step for three-dimensional culture, the dispersion treatment of cancer cells was performed as follows. The washed tumor was collected in a 10 cm petri dish on ice, cut into about 1 mm square with surgical scissors, and recovered in a 50 mL tube. ...

Embodiment 2

[0089]

[0090] First, using the Cancer Organoid Culture Kit (ORGANOGENIX), which was first described as capable of three-dimensional culture of primary cancer cells, it was investigated whether three-dimensional culture using PDX tumors was possible. The cancer organoid culture kit is a method of culturing in a low-adhesion well plate having a concave-convex scaffold structure and a medium containing 1% by volume or more of serum in order to form a cell mass of cancer cells.

[0091] According to Example 1, primary cancer cells were prepared using pancreatic cancer (1) PDX tumors (obtained from the Basic Medicine Research Institute). Aliquot the required amount of cells counted after the dispersion treatment, put them in a 15mL tube, and centrifuge at 300×g for 5min. ) so that the number of cells is 1×10 5 cells / mL to prepare cell suspension. Add 150 μL of NanoCulture Medium P type to the NanoCulturePlate (cancer organoid culture kit accessory, ORGANOGENIX company) which ...

Embodiment 3

[0095]

[0096]According to Example 1, primary cancer cells were prepared using pancreatic cancer (1) PDX tumors (obtained from the Basic Medicine Research Institute). The required amount of cells counted after dispersion treatment was collected and placed in a 15 mL tube, centrifuged at 300 × g for 5 min, and the supernatant was removed, and used in StemPro hESC SFM (Thermo Fisher Scientific Company) according to the final concentration of 0 , 0.5, 1, 2, 5, 10, 20 or 50v / v% of the medium after adding Corning Matrigel GFR (Corning Company), according to the number of cells is 5 × 10 4 cells / mL to prepare cell suspension. Inoculate 200 μL of it on PrimeSurface (SumitomoBakelite Company), set at 37°C, 5% CO 2 CO 2 Start static culture in the incubator. The number of seeded cells was 1×10 4 cells / 200μL / well, set the inoculation day as Day 0. No medium change was performed and on Day 14 the possibility of performing a half medium change was assessed. show the result in f...

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Abstract

The present invention addresses the problem of providing a method for forming a cell mass by three-dimensional culture of primary cancer cells using a tumor tissue as a starting material while inhibiting the proliferation of cells (e.g., fibroblasts) other than cancer cells, said method ensuring a high proliferation ability of the cell mass which contains primary cancer cells as the main component, being easy to handle and having a versatility and high throughput properties. To solve this problem, provided is a method for forming a cell mass by three-dimensional culture of primary cancer cellsusing a tumor tissue, said method comprising a three-dimensional culture step for culturing cells, which are obtained from the tumor tissue, in a medium containing not more than 5 v / v% of an extracellular matrix on a cell culture base material substantially having a low adhesiveness.

Description

technical field [0001] The present invention relates to a method for producing a cell block, a cell block, a screening method, a determination method, and a kit based on a three-dimensional culture method of primary cancer cells using tumor tissue. Background technique [0002] Strained cancer cells (cancer cell lines) have conventionally been used as methods for evaluating tumors in vitro. However, since cancer cell lines are approximately homogeneous cell populations adapted to the in vitro environment, accumulation of gene mutations is confirmed through long-term maintenance, etc., it is pointed out that most of the basic tumor properties are lost. In addition, it has also been pointed out that tumors are obviously composed of cancer cells with various genetic backgrounds, and from the viewpoint of this heterogeneity, pathological conditions cannot be fully explained by a limited number of cancer cell lines. Therefore, in order to understand tumors more accurately, cultu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/02G01N33/15G01N33/50
CPCG01N33/15G01N33/5011G01N33/5082C12N5/0693C12N2513/00C12N2533/90C12N2533/10C12N2533/52C12N2533/54
Inventor 柿沼秀明嶋田有纪子井上裕章森川崇
Owner MITSUBISHI CHEM MEDIENCE
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