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Novel method capable of enriching and amplifying suspended blood cancer stem cells, novel culture medium and application thereof to drug screening

A technology of tumor stem cells and culture medium, which can be used in tumor/cancer cells, animal cells, vertebrate cells, etc., and can solve the problem that the properties of tumor stem cells cannot be fully reflected

Inactive Publication Date: 2012-04-11
殷勤伟 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the side group cells are only relatively enriched in tumor stem cells, the study of the properties of tumor side group cells cannot fully reflect the properties of tumor stem cells

Method used

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  • Novel method capable of enriching and amplifying suspended blood cancer stem cells, novel culture medium and application thereof to drug screening
  • Novel method capable of enriching and amplifying suspended blood cancer stem cells, novel culture medium and application thereof to drug screening
  • Novel method capable of enriching and amplifying suspended blood cancer stem cells, novel culture medium and application thereof to drug screening

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1. Tumor stem / progenitor cell colony culture and expansion of T-lymphoma

[0022] Human T-lymphocyte tumor line Hut-102 was purchased from the American cell bank ATCC (TIB-162), and the cell culture used DMEM medium containing 10% fetal bovine serum (PAA, FCS500) (Beijing Niuyinhuaxin Technology Co., Ltd., DM10140021) , cultured in T-25 cell culture flasks produced by Coring Company, the culture conditions were 37 ° C, 5% CO 2 . Cells were passaged every two days. Colony culture of hematological tumor cells was carried out in Corning 35mm culture dishes. A layer of DMEM semi-solid medium containing 1% agarose was spread on the lower layer of the culture dish. Take another new blood tumor stem cell culture medium (recipe in Table 1) to make 1 ml of 0.3% agarose medium, inoculate 1000 Hut-102 cells in it, mix well and spread gently on 1% agarose medium above. The formulation of the suspension tumor stem cell culture medium disclosed in the present invention i...

Embodiment 2

[0027] Example 2. Cell stem / progenitor cell colony culture and expansion of promyelocytic leukemia K562 cell line

[0028] The human promyelocytic leukemia K562 cell line was purchased from the American cell bank ATCC (?), and the DMEM medium containing 10% fetal calf serum (PAA, FCS500) was used for cell culture (Beijing Niuyin Huaxin Technology Co., Ltd., DM10140021), and the cell culture was carried out at Coring Company. Produced T-25 cells were cultured in culture flasks at 37°C, 5% CO 2. Cells were passaged every 3 days. Colony culture of hematological tumor cells was carried out in Corning 35mm culture dishes. A layer of DMEM semi-solid medium containing 1% agarose was spread on the lower layer of the culture dish. Another new blood tumor stem cell culture medium (recipe in Table 1) was prepared into 1 ml of 0.3% agarose medium, and 1000 K562 cells were inoculated in it, mixed evenly and spread gently on top of 1% agarose. Cells at 37°C, 5% CO 2 cultured in a cell ...

Embodiment 3

[0029] Example 3. Stem Cell Gene Expression of Lymphoma Stem Cells

[0030] In order to verify that the screened and cultured new subtype of Hut-102 (Hut-102S) is a tumor stem cell, the changes in the expression of several stem cell characteristic genes of the cell subtype were determined. The four genes selected here are Nanog, Oct4 and Sox2, which are commonly used in embryonic stem cells. The PCR primers used are shown in Table 2. The PCR experiment was carried out using the RT-PCR kit (KR114) from Tiangen Biotechnology Company, and was carried out according to the kit operation instructions. Figure 4 Gel photographs of PCR products at Hut-102 and Hut-102S are given. The figure shows that the expression of the internal reference gene β-actin is the same in Hut-102 and Hut-102S, the stem cell marker gene NANOG is not expressed in Hut-102, and the expression in Hut-102S is significantly increased, while the expression of SOX2 gene in Hut-102S The expression of the stem cel...

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Abstract

The invention provides a novel method capable of largely and fast enriching and culturing stem / progenitor cell subsets in blood suspended cancer cells by an embryonic stem cell culture medium, and a culture medium composition. The embryonic stem cell culture medium is adopted, stem / progenitor cell cloning of blood cancer cells can be selected in a semisolid culture medium through adding other factors and inhibitor micromolecule compounds inhibiting glycogen synthase kinase (GSK) signal channels and tyrosine kinase, and the cloning can be massively cultured in a suspended way and amplified in the blood cancer stem cell culture medium provided by the invention. The blood cancer stem cells obtained by the method have special marks in aspects of cell morphology, stem cell gene expression, stem cell surface characteristic antigen expression and the like, the stem cell characteristic of sieved suspended stem cells is closer to that of embryonic stem cells, and the obvious drug resistance on chemotherapeutic drug is realized. The novel method and the culture medium composition have good application prospects in the aspects of blood suspended cancer stem cell enrichment and cloning, cancer stem cell special drum screening and identification and the like.

Description

technical field [0001] The invention belongs to the production of cell culture medium in the technical field of bioengineering, and more specifically, relates to a technology and method for screening, cultivating and expanding corresponding tumor stem cell subgroups from tumor cells in the blood system, as a method for screening and treating leukemia drugs. Biotherapy, screening of gene therapy, in vitro evaluation model methods and experimental materials. Background technique [0002] Tumor is an important disease that threatens human health and is the second killer of human diseases. Leukemia is a tumor of the human circulatory system. In recent years, the incidence of this disease has shown a significant growth trend, and it is showing a trend of younger age. People treat leukemia mainly through chemotherapy. Although bone marrow suppression is considered to be an effective means of treating leukemia and lymphoma, the wide application of bone marrow transplantation is s...

Claims

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Application Information

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IPC IPC(8): C12N5/095C12Q1/02
Inventor 殷勤伟张洪杰黄兵王珊珊
Owner 殷勤伟
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