Method for in-vitro amplification of tumor stem cells

A technology for tumor stem cells and tumor cells, which is applied in the field of preparation of alginate gel scaffolds, can solve the problems of expensive growth factors and unfavorable expansion of culture systems, avoid expensive materials and processes, low cost, and facilitate large-scale expansion Effect

Active Publication Date: 2014-02-12
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, the medium and growth factors are very expensive...

Method used

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  • Method for in-vitro amplification of tumor stem cells
  • Method for in-vitro amplification of tumor stem cells
  • Method for in-vitro amplification of tumor stem cells

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0031] Example 1: Expansion of tongue squamous cell carcinoma stem cells

[0032] 1) Confirm that the matrix stiffness of tongue squamous cell carcinoma stem cells in the human body is 8-17kPa (Engler, A.J., et al., Matrix Elasticity Directs Stem Cell Lineage Specification. Cell, 2006.126(4): p.677-689. );

[0033] 2) Mix tongue squamous cell carcinoma cells with different concentrations of 1.5%, 2.5%, 3% (g / 100ml) sodium alginate (molecular weight 200kDa, GM ratio 0.4) solution, adjust the cell density to 10 6 cells mL -1 , using the electrostatic droplet method to drop 100mmol·L -1 CaCl 2 In the solution, calcify at room temperature for 30 minutes to form a calcium alginate gel with a particle size of 400-500um. The matrix stiffness of the gel support is 4kPa, 10kPa, 20kPa;

[0034] 3) Afterwards, wash with DMEM medium for 3 times, then add DMEM medium containing 10% fetal bovine serum, and store at 37°C, 5% CO 2 , Cultivate under saturated humidity conditions, and rep...

Embodiment 2

[0036] Example 2: Expansion of liver cancer stem cells

[0037] 1) Confirm that the matrix stiffness of liver cancer stem cells in the human body is 0.6-3kPa (YEH, W.-C., Elastic modulus measurements of human liver and correlation with pathology. Ultrasound in Med. & Biol, 2002.28 (4): p .467-474.);

[0038] 2) Mix liver cancer cells with 0.5% (g / 100ml) sodium alginate (molecular weight 200kDa, GM ratio 0.4) solution, adjust the cell density to 10 6cells mL -1 , using the electrostatic droplet method to drop 100mmol·L -1 CaCl 2 In the solution, calcify at room temperature for 30 minutes to form a calcium alginate gel with a particle size of 300-400um. The matrix stiffness of its gel scaffold is 1kPa.

[0039] 3) Afterwards, wash with culture medium for 3 times, then add DMEM culture medium containing 10% fetal bovine serum, at 37°C, 5% CO 2 , Cultivate under saturated humidity conditions, and replace the culture medium every 2 days.

[0040] After culturing for 15 days ...

Embodiment 3

[0041] Example 3: Changing the Elastic Modulus of the Gel Scaffold by Changing the Calcium Ion Concentration

[0042] Mix liver cancer cells with 0.5% (g / 100ml) sodium alginate (molecular weight 430kDa, GM ratio 0.4) solution, adjust the cell density to 10 6 cells mL -1 , using the electrostatic droplet method to drop 25mmol·L -1 , 50mmol·L -1 , 100mmol·L -1 , 200mmol·L -1 CaCl 2 In the solution, calcify at room temperature for 30 minutes to form a calcium alginate gel with a particle size of 300-400um. Afterwards, wash with culture medium three times, then add DMEM culture medium containing 10% fetal bovine serum, and incubate at 37°C, 5% CO 2 , Cultivate under saturated humidity conditions, and replace the culture medium every 2 days.

[0043] After culturing for a period of time, use 55mmol·L -1 Sodium citrate was used to dissolve the calcium alginate gel, and the internal tissue-like cell mass was harvested, RNA was extracted, and the expression of related genes wa...

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Abstract

The invention discloses a method for in-vitro amplification of tumor stem cells. The method comprises the following steps of carrying out in-vitro three-dimensional culture on tumor cells by an alginate gel support, and adjusting and controlling the rigidity of the alginate gel support by a change of support preparation parameters such as alginate molecular weight, a ratio (GM) of guluronic acid content to mannuronic acid content, a calcium ion concentration and calcification time, so that hardness of an in-vitro matrix in different tumor tissue microenvironments is simulated and in-vitro amplification of tumor stem cells in different tissue is realized. The method can be operated simply, has a low cost and controllable performances, can be industrialized and is a good method for in-vitro amplification on tumor stem cells having different sources.

Description

technical field [0001] The invention relates to providing a method for expanding tumor stem cells in vitro, in particular to the preparation of an alginate gel scaffold with controllable matrix stiffness. Background technique [0002] Cancer stem cells have been proven to be the root cause of metastasis and recurrence of malignant tumors, and their research is of great significance for elucidating the mechanism and biological behavior of tumors and finding a radical treatment plan targeting tumor stem cells in the future. However, the content of tumor stem cells in tumor tissues and tumor cell lines is very low, and it is difficult to obtain a sufficient amount of high-purity tumor stem cells for research, which is one of the obstacles faced by current cancer stem cell research. [0003] The cellular microenvironment can be divided into biochemical factors and biophysical factors according to their properties. As one of the biophysical factors, substrate stiffness has been ...

Claims

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Application Information

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IPC IPC(8): C12N5/095C12N11/10C12N11/04
Inventor 马小军刘畅孙广炜徐小溪刘洋于炜婷
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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