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Method for culturing tumor organoid

A technology of organoids and tumors, applied in the field of biomedicine, can solve the problems of unsuitable tumor organoids, loss of three-dimensional scaffold function, high price, etc., achieve good water permeability, promote cell growth, and reduce culture costs

Pending Publication Date: 2022-04-12
SUZHOU GENOARRAY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Matrigel has successfully cultured a variety of tumor organoids, it is not suitable as a matrix gel for large-scale culture of tumor organoids due to its high price; secondly, Matrigel gels at 22-37°C and needs to be Operating in a low temperature environment may easily lead to improper gelation of Matrigel, resulting in inoculation failure; and, after mixing Matrigel with tumor cells, it needs to be left in a 37°C cell culture incubator for 30 minutes to completely gel, during which a large number of tumor cells The three-dimensional scaffolding effect of Matrigel is lost due to gravity sinking to the bottom of the culture dish; finally, Matrigel is currently only suitable for culturing organoids of malignant tumors derived from epithelial tissue, and there is no report that it can be used to culture organoids of malignant tumors derived from mesenchymal tissue

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  • Method for culturing tumor organoid
  • Method for culturing tumor organoid
  • Method for culturing tumor organoid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, the optimization of hydrogel preparation method

[0061] Different concentrations of calcium chloride and glutaraldehyde were added to sheep plasma (Zhengzhou Jiulong Biological Products Co., Ltd.). Human liver cancer HepG2 cells (ATCC, USA) were two-dimensionally cultured to the logarithmic growth phase, and then digested with 0.25% (% expressed in g / 100mL) trypsin (Sigma) to obtain a single cell suspension with a final concentration of 5 ×10 3 / mL mixed with the above groups (sheep plasma with different concentrations of calcium chloride and glutaraldehyde added) and spread on the bottom of the cell culture plate wells to observe the coagulation time of each group. DMEM medium (Thermo Scientific) containing 10% (v / v) fetal calf serum (Thermo Scientific) and 1% (m / v) penicillin / streptomycin was added on top of the gel with a solidification time≤30 minutes. At 37°C, 5% CO 2 Observe the state of cells in each group after culturing for 3 days. It was fou...

Embodiment 2

[0062] Embodiment 2, the preparation method of hydrogel

[0063] 1. Prepare 100× calcium chloride storage solution

[0064] Weigh 3.3294-6.6588 g of anhydrous calcium chloride and dissolve in 100 ml of deionized water, the concentration of calcium chloride is 300-600 mmol / L, and filter to sterilize with a filter membrane with a pore size of 0.22 μm.

[0065] 2. Prepare 100× glutaraldehyde storage solution

[0066] Measure 10-50 mL of glutaraldehyde, add it to 100 mL with deionized water, the concentration of glutaraldehyde is 10-50% (v / v), filter and sterilize with a filter membrane with a pore size of 0.22 μm.

[0067] 3. Sheep plasma: product of Zhengzhou Jiulong Biological Products Co., Ltd.

[0068] 4. Preparation of three-dimensional cell culture scaffolds

[0069] Mix 980 μL of sheep plasma with 10 μL of 100× calcium chloride stock solution (450 mmol / L) and 10 μL of 100× glutaraldehyde stock solution (25%, % indicates volume percentage) and add them to the wells of th...

Embodiment 3

[0071] Example 3, the cultivation of tumor organoids

[0072] This embodiment detects malignant tumors derived from multiple epithelial tissues (lung cancer, breast cancer, gastric cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, ovarian cancer and pancreatic cancer) and malignant tumors derived from mesenchymal tissues (chondrosarcoma and mesenchymal cancer) Organoid culture effect of tumor).

[0073] 1. Human tumor tissue samples were washed three times with pre-cooled Hank's balanced salt solution (containing 200 U / mL penicillin, 200 mg / mL streptomycin and 0.5 mg / mL amphotericin B, all purchased from Sigma).

[0074] 2. Cut the tumor tissue to about 0.5mm with sterile scissors 3 of small pieces. Transfer the shredded tissue to a 15mL centrifuge tube, and add about 10mL of pre-cooled Hank's balanced salt solution. Pipette up and down several times with a 10mL pipette.

[0075] 3. Let the centrifuge tube stand for 2-3 minutes to allow the tissue pie...

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Abstract

The invention discloses a method for culturing tumor organoids. The invention provides hydrogel for culturing tumor organs. The hydrogel is formed by taking isolated sheep plasma or other mammal plasma or human plasma as a matrix and adding a coagulant and a protein cross-linking agent. The hydrogel has the advantages of being low in cost, simple and convenient to prepare, loose in structure, moderate in strength, good in water permeability, capable of being completely gelatinized in a short time, good in cell biocompatibility and suitable for culture of malignant tumor organoid from both epithelial tissue sources and mesenchymal tissue sources.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for culturing tumor organoids. Background technique [0002] Tumor organoids are three-dimensional cell complexes in which primary tumor cells are cultured in vitro to form a certain spatial structure. They have structural and functional properties similar to those of the source tumor tissue, and can be stably expanded in an in vitro three-dimensional culture system. Because tumor organoids maintain the phenotype and genotype of the source tumor tissue, they provide an ideal platform for preclinical drug screening, drug safety evaluation, personalized therapy, and basic tumor research. [0003] The current method of culturing tumor organoids is to use Matrigel as a scaffold for three-dimensional culture in vitro. Matrigel is a soluble extracellular matrix extracted from mouse sarcoma, and its main components are laminin, type IV collagen, Entactin protein and heparan sulfate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09B01J13/00
Inventor 张函槊刘勇
Owner SUZHOU GENOARRAY