Method for isolating and culturing human olfactory mucosa mesenchymal stem cells

A technique for isolating and culturing mesenchymal stem cells, which is applied in the field of isolating and culturing human olfactory mucosa mesenchymal stem cells, and can solve the problems of limited acquisition, decreased quantity and proliferation and differentiation potential, and high viral infection rate.

Inactive Publication Date: 2014-09-10
中国人民解放军联勤保障部队第九二一医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As for adult bone marrow-derived mesenchymal stem cells, their quantity and proliferation and differentiation potential decline with age, and the virus infection rate is high, and bone marrow collection from donors requires bone marrow aspiration, so their acquisition is subject to certain restrictions. limit

Method used

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  • Method for isolating and culturing human olfactory mucosa mesenchymal stem cells
  • Method for isolating and culturing human olfactory mucosa mesenchymal stem cells
  • Method for isolating and culturing human olfactory mucosa mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Two pieces of olfactory mucosal tissue (size 2×3mm) were obtained, and washed three times with 1% penicillin repeatedly under aseptic conditions to remove residual blood. Cut it to a length of approximately 1 mm with sterile ophthalmic scissors in complete medium 3 The tissue block was placed in a 15ml centrifuge tube, mixed with complete medium and shaken, evenly planted in a cell culture dish, and placed in a 37°C, 5% CO2 incubator. On the 7th day of culture, the cells climbed out and observed adherent spindle-shaped or polygonal cells under the microscope, and then the medium was replaced every 3 days. On the 15th day of culture, the cells were about 80% confluent, and the cells were passaged at a ratio of 1:3, and then every 1.5-2 days, and sufficient mesenchymal stem cells were finally obtained.

Embodiment 2

[0037] Three pieces of olfactory mucosa tissue (size 2×2mm) were obtained, and washed repeatedly with 1% penicillin under aseptic conditions to remove residual blood. Cut it to a length of approximately 1 mm with sterile ophthalmic scissors in complete medium 3 The tissue block was placed in a 15ml centrifuge tube, mixed with complete medium and shaken, evenly planted in a cell culture dish, and placed in a 37°C, 5% CO2 incubator. On the 8th day of culture, adherent spindle-shaped or polygonal cells were observed under the microscope, and the medium was replaced every 3 days thereafter. On the 14th day of culture, the cells were about 80% confluent, and they were subcultured at a ratio of 1:3, and thereafter every three days to obtain sufficient mesenchymal stem cells.

Embodiment 3

[0039] Two pieces of olfactory mucosal tissue (size 2×2mm) were obtained, and washed repeatedly with 1% penicillin under sterile conditions to remove residual blood. Cut it to a length of approximately 1 mm with sterile ophthalmic scissors in complete medium 3 The tissue block was placed in a 15ml centrifuge tube, mixed with complete medium and shaken, evenly planted in a cell culture dish, and placed in a 37°C, 5% CO2 incubator. On the 6th day of culture, adherent spindle-shaped or polygonal cells were observed under the microscope, and the medium was replaced every 3 days thereafter. On the 14th day of culture, the cells were about 80% confluent, and they were subcultured at a ratio of 1:3, and thereafter every three days to obtain sufficient mesenchymal stem cells.

[0040] The cultured olfactory mucosal mesenchymal stem cells were subjected to microscopic examination, flow cytometry to detect characteristic surface markers, osteogenesis and adipogenic induction and staini...

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Abstract

The invention provides a method for isolating and culturing human olfactory mucosa mesenchymal stem cells. The method comprises the following steps: 1, preparing a basic culture liquid by mixing DMEM and F12 in the ratio of 1:1; 2, immediately collecting AS of a patient; 3, adding the collected AS into the human olfactory mucosa mesenchymal stem cell culture medium, and adding 3 to 7 percent of CO2 at the temperature of 35 to 38 DEG C. By adopting the method, the high-purity olfactory mucosa mesnchymal stem cells can be efficiently and rapidly obtained, the cells have strong cloning forming capacity, self updating capacity and multi-direction differential potential. The olfactory mucosa cells acquired through the method are strong in grease forming capacity, bone forming capacity, neural stem cell forming capacity and nerve cell forming capacity, and a novel method is provided for obtaining a great amount of stem cells which are applied to the cell and tissue regeneration.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and relates to a method for separating and culturing human olfactory mucosa mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from the mesoderm and ectoderm in the early stages of development, with the ability of self-renewal and multidirectional differentiation. MSCs currently studied are mainly derived from adult bone marrow and umbilical cord blood. For adult bone marrow-derived mesenchymal stem cells, because the number and proliferation and differentiation potential of adult bone marrow decrease with age, and the virus infection rate is high, and bone marrow collection from donors requires bone marrow aspiration, so its acquisition is subject to certain restrictions. limit. [0003] Olfactory mucosa mesenchymal stem cells (OM-MSCs) are also called ectomesenchymal stem cells (OE-MSCs). Because they are loca...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 卢明葛丽特段答
Owner 中国人民解放军联勤保障部队第九二一医院
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