Tumor stem cell suspension culture method

A cancer stem cell and suspension culture technology, which is applied in the direction of tumor/cancer cells, tissue culture, animal cells, etc., can solve the problems of different types and doses of growth factors, no company production, and inconsistent culture methods, etc., to achieve simple and feasible operation, Improve the success rate and reduce the cost

Inactive Publication Date: 2013-10-16
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the reagents currently used for the suspension culture of tumor stem cells are not produced by specific companies, and the culture methods proposed in the literature are not uniform. There are also differences in the types and doses of basal media and growth factors used for tumor cells from different tissues. different

Method used

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  • Tumor stem cell suspension culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Culture of liver cancer cell line Huh7 stem cells

[0046] The liver cancer cell line Huh7 was purchased from Shanghai Chinese Academy of Sciences.

[0047] 1. Glue compounding: Dissolve 0.12g of PHEMA in 20ml of 95% alcohol to a final concentration of 12mg / ml, and gently shake at a low speed at 65°C for 8 hours.

[0048] 2. Spread the plate: Use a pipette to spread 1ml of the prepared glue to the bottom of the 6-well plate in the ultra-clean bench, cover evenly, avoid repeated operations, and complete it at one time. Absorb the excess glue, blow air for 10 hours in the ultra-clean table, irradiate with ultraviolet light for 1 hour, and store in a dry and ventilated place away from light for 1-2 months.

[0049] 3. Culture medium preparation: add IGF1ug, bFGF50ng, EGF1ug to 49.9ml DMEM / F12 by pipetting and mixing, store at 4°C for 1-2 months.

[0050] 4. Huh7 was stably cultured and subcultured in DMEM medium containing 10% FBS, and subcultured when the adh...

Embodiment 2

[0056] Example 2: Glioma cell line U251 stem cell culture

[0057] Glioma cell line U251 was purchased from Shanghai Academy of Sciences.

[0058] 1. Glue compounding: Dissolve 0.12g of PHEMA in 20ml of 95% alcohol to a final concentration of 12mg / ml, and gently shake at a low speed at 65°C for 8 hours.

[0059] 2. Spread the plate: Use a pipette to spread 1ml of the prepared glue to the bottom of the 6-well plate in the ultra-clean bench, cover evenly, avoid repeated operations, and complete it at one time. Absorb the excess glue, blow air for 10 hours in the ultra-clean table, irradiate with ultraviolet light for 1 hour, and store in a dry and ventilated place away from light for 1-2 months.

[0060] 3. Culture medium preparation: add IGF1.25ug, bFGF75ng, EGF1ug to 49.9ml DMEM / F12, pipette and mix well, store at 4°C for 1-2 months.

[0061] 4. U251 was stably cultured and subcultured in DMEM medium containing 10% FBS, and subcultured when the adherent growth reached 90%. ...

Embodiment 3

[0067] Example 3: Stem cell culture after primary culture of liver cancer tissue

[0068] Liver cancer tissue was derived from clinical liver cancer resection.

[0069] 1. Glue compounding: Dissolve 0.12g of PHEMA in 20ml of 95% alcohol to a final concentration of 12mg / ml, and gently shake at a low speed at 65°C for 8 hours.

[0070] 2. Spread the plate: Use a pipette to spread 1ml of the prepared glue to the bottom of the 6-well plate in the ultra-clean bench, cover evenly, avoid repeated operations, and complete it at one time. Absorb the excess glue, blow air for 10 hours in the ultra-clean table, irradiate with ultraviolet light for 1 hour, and store in a dry and ventilated place away from light for 1-2 months.

[0071]3. Culture medium preparation: add IGF1.5ug, bFGF75ng, EGF1.5ug to 49.9ml DMEM / F12, mix by pipetting, store at 4°C for 1-2 months.

[0072] 4. The primary cultured cells of liver cancer tissue were stably cultured and passaged in DMEM medium containing 10%...

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Abstract

The invention belongs to the technical field of modern biotechnology cell culture. A purpose of the invention is to provide a tumor stem cell suspension culture method, which can be used for culture of tumor cells with different tissue sources, and tumor stem cells obtained through the culture method have characteristics of self-renewal capacity and enrichment. The tumor stem cell suspension culture method comprises: carrying out digestion centrifugation collection on tumor cells requiring culture, adopting PBS to carry out resuspending centrifugation, adding to a prepared suspension culture medium of the invention, carrying out beating-up into a single cell suspension, inoculating into a paved ultra-low absorption culture dish, and carrying out cell culture. The tumor stem cell suspension culture method has the following characteristics that: ball forming rates of various in vitro cultured tumor cells are high, multi-function factors are expressed, the OCT4 proportion and the NANOG proportion are increased, rich and comprehensive experimental materials are provided for tumor stem cell researches, and broad application prospects are provided in basic researches and clinical applications.

Description

technical field [0001] The invention belongs to the technical field of cell culture of modern biotechnology, and in particular relates to a suspension culture method of tumor stem cells. Background technique [0002] Tumors are composed of heterogeneous cells with different proliferation and invasion abilities. Studies have shown that tumors also follow the architecture of normal tissue cells, in which there is a very small group of cells with long-term self-renewal and differentiation capabilities, leading to the continuous occurrence and progression of tumors. Because of the similar function to normal tissue stem cells, this subset is called cancer stem cells (cancer stem cells), or tumor initiating cells (tumor initiating cells). It participates in the growth and metastasis of primary tumors, has the potential of high self-renewal and multi-directional differentiation similar to stem cells, and is the source of differentiation and proliferation of different tumor cells. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/095C12N5/02
Inventor 刘善荣肖潇胡晶晶
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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