Method and culture medium for in-vitro isolated culture of glioma stem cells

A technology of glioma stem cells and culture medium, which is applied in the field of glioma stem cell in vitro culture method and culture medium, can solve the problems of insufficient purity of glioma, and achieve the effect of avoiding the interference of mixed cells

Inactive Publication Date: 2020-11-24
SUZHOU HAIMIAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For example, Chinese Patent Application No. 201210299549.6 proposes a human glioma cell line, which reveals the cultivation process of glioma, but the purity of the glioma obtained by the cultivation is not enough

Method used

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  • Method and culture medium for in-vitro isolated culture of glioma stem cells
  • Method and culture medium for in-vitro isolated culture of glioma stem cells
  • Method and culture medium for in-vitro isolated culture of glioma stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Glioma stem cell specimens excised from a malignant glioblastoma (female, less than 12 years old).

[0050] The steps of its isolation culture include primary culture and subculture, wherein the steps of primary culture include:

[0051] Step 1: Place the fresh glioma tissue in PBS, and cut it into a minced shape under a sterile environment;

[0052] Specifically: the surgically excised fresh tumor tissue was put into a centrifuge tube filled with 5 mL of PBS, and placed on ice. The tumor tissue was taken out in a sterile ultra-clean bench, placed in a plate containing PBS buffer, and blood clots and necrotic tissue in the tumor tissue were carefully removed with tweezers. Cut the tumor tissue into mince within 5 minutes, add PBS to suspend it, and collect the suspension in a 15mL centrifuge tube for work. The working condition is 800rpm, 3min, then discard the supernatant.

[0053] Step 2: Add Accutase solution to dissolve the tissue block to form a cell suspension;...

Embodiment 2

[0098] Glioma stem cell specimens excised from a malignant glioblastoma (female, less than 12 years old).

[0099] The steps of its isolation culture include primary culture and subculture, wherein the steps of primary culture include:

[0100] Step 1: Place the fresh glioma tissue in PBS, and cut it into a minced shape under a sterile environment;

[0101] Specifically: the surgically excised fresh tumor tissue was put into a centrifuge tube filled with 20 mL of PBS, and placed on ice. The tumor tissue was taken out in a sterile ultra-clean bench, placed in a plate containing PBS buffer, and blood clots and necrotic tissue in the tumor tissue were carefully removed with tweezers. Cut the tumor tissue into mince within 5 minutes, add PBS to suspend it, and collect the suspension in a 15mL centrifuge tube for work. The working condition is 800rpm, 3min, then discard the supernatant.

[0102] Step 2: Add Accutase solution to dissolve the tissue block to form a cell suspension...

Embodiment 3

[0142] Glioma stem cell specimens excised from a malignant glioblastoma (female, less than 12 years old).

[0143] The steps of its isolation culture include primary culture and subculture, wherein the steps of primary culture include:

[0144] Step 1: Place the fresh glioma tissue in PBS, and cut it into a minced shape under a sterile environment;

[0145] Specifically: the surgically excised fresh tumor tissue was put into a centrifuge tube filled with 20 mL of PBS, and placed on ice. The tumor tissue was taken out in a sterile ultra-clean bench, placed in a plate containing PBS buffer, and blood clots and necrotic tissue in the tumor tissue were carefully removed with tweezers. Cut the tumor tissue into mince within 5 minutes, add PBS to suspend it, and collect the suspension in a 15mL centrifuge tube for work. The working condition is 800rpm, 3min, then discard the supernatant.

[0146] Step 2: Add Accutase solution to dissolve the tissue block to form a cell suspension...

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Abstract

The invention provides a method and culture medium for in-vitro isolated culture of glioma stem cells. The method comprises the following steps of 1, adding cell tumor tissues into a PBS buffer solution, removing blood clots and necrotic cells, and cutting the treated cell tumor tissues into paste; 2, adding an Accutase solution to digest tissue blocks into a cell suspension; 3, adding a GC gliomacell culture medium, performing uniform mixing, stopping digestion, and filtering the cell suspension through a filter screen (BD) to obtain a single-cell suspension; 4, adding PBS, performing re-suspending precipitation, washing the cells, and removing an enzyme solution; 5, adding an erythrocyte lysis solution to lyse erythrocytes, discarding a supernatant, and adding PBS to wash cell precipitates in order to remove the erythrocyte lysis solution; and 6, adding an NXFS glioma stem cell culture medium to a Laminin-coated 6-hole cell plate, and carrying out primary culture on the glioma stemcells. The glioma stem cells with the purity of about 80% can be obtained.

Description

technical field [0001] The invention relates to the technical field of stem cell cultivation, in particular to a method for culturing glioma stem cells in vitro and a culture medium. Background technique [0002] Malignant glioma is the most common intracranial malignant tumor, accounting for about 15% of the total incidence population. It can be transformed from healthy cells in the brain and primary astroglioma. In most cases, the cause is unknown and there are many types According to pathology, it can be divided into astrocytoma, medulloblastoma, glioblastoma multiforme, ependymoma, and oligodendroglioma. There is no way to prevent this type of tumor, and the general survival rate after diagnosis is 12 to 15 months. At present, the more effective treatment method is surgical resection combined with radiotherapy and chemotherapy. , the postoperative recurrence rate is extremely high, and gliomas are highly heterogeneous among individuals. [0003] The current difficultie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/095
CPCC12N5/0695C12N2509/00C12N2501/11C12N2501/115C12N2501/33C12N2501/91C12N2500/30
Inventor 王文元谭淼罗芳
Owner SUZHOU HAIMIAO BIOTECH CO LTD
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