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Method for separating bone marrow mononuclear cells

A nuclear cell and bone marrow technology, applied in the field of isolation of bone marrow mononuclear cells, can solve the problems of unfavorable MNCs collection and achieve the effect of simple and convenient operation

Inactive Publication Date: 2015-07-15
银广悦
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since some red blood cells are still suspended in Ficoll solution after centrifugation using this method, the collected MNCs may be mixed with red blood cells, which is not conducive to the collection of MNCs

Method used

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  • Method for separating bone marrow mononuclear cells
  • Method for separating bone marrow mononuclear cells
  • Method for separating bone marrow mononuclear cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Ficoll one-step separation of MNCs

[0028] Take 20 mL of bone marrow fluid and mix it with an equal amount of low-sugar DMEM medium (purchased from Invitrogen, USA). After mixing evenly, slowly add to the surface of an equal amount of Ficoll solution. Carry out centrifugation at room temperature (centrifugation operating parameters are 800g, 20min). After the centrifugation, the middle cloudy cell layer was carefully sucked, washed twice with PBS, and then centrifuged again (centrifugation operating parameters were 600g, 5min). The concentration was 1×10 in low-sugar DMEM medium containing 10% fetal bovine serum 6 The mmol / L suspension was used for future use, and then the MNCs were counted and their viability was detected by the trypan blue exclusion method.

Embodiment 2

[0030] Gelatin-Ficoll two-step separation of MNCs

[0031] Mix 20 mL of bone marrow fluid with an equivalent amount of 3% gelatin (purchased from SIGMA, USA) and settle naturally for 1 h, then take the gelatin suspension layer for centrifugation (centrifugation operation parameters are 350 g, 8 min), discard the supernatant, and add PRMI1640 culture medium (purchased from Hyclone, USA) was used to resuspend the cells, and then the MNCs were separated according to the Ficoll one-step method.

Embodiment 3

[0033] Detection of CD34 on the surface of MNCs by flow cytometry + and CD44 + / CD71 +

[0034] Take 2 mL of the MNCs suspension and wash it twice by centrifugation, and adjust the concentration to 5×10 with PBS. 6 cells / ml, take 100uL of the above cell suspension, add the corresponding antibodies respectively, and incubate at 4°C for 30min in the dark.

[0035] The antibody addition amount is as follows:

[0036] Table 1 Names and contents of antibodies added

[0037]

[0038] After the incubation, the cells were washed twice with PBS and then centrifuged (centrifugation operating parameters were 600 g, 5 min). The cells were resuspended in 400 μL PBS, filtered through a 400-mesh sieve, and flow cytometry was performed, and data were collected using CELLQUEST software.

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a method for separating bone marrow mononuclear cells. The separation of the mononuclear cells in bone marrow is carried out by using one point that a gelatin solution can destroy red blood cell surface charges, accelerates the deposition of red blood cells and has no influence on other principal components covering the mononuclear cells through density difference between the mononuclear cells in the bone marrow and the other components according to the principle that when a lymphocyte separation medium is used for density gradient centrifugation, various cellular components are redistributed and gathered by density gradient, thus obtaining the mononuclear cells. By using the method for separating the bone marrow mononuclear cells, the loss of the MNCs (mononuclear cells) is not increased, and bone marrow hematopoietic stem cells and mesenchymal stem cells can be reserved very well; and the method is simple and convenient to operate and can be popularized clinically.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for isolating bone marrow mononuclear cells. Background technique [0002] Mononuclear cells in the bone marrow include a mixture of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial cells (EPCs), and stromal cells. For now, hematopoietic stem cells in bone marrow mononuclear cells have been used in transplantation and widely used in clinical treatment of various blood diseases and achieved good results. In recent years, a large number of experimental studies have proved that bone marrow also contains a certain number of bone marrow mesenchymal stem cells (BMSCs). The pluripotent tissue stem cells differentiated from various cells such as cells have the following advantages: (1) easy to obtain in vitro, easy to isolate and culture, and fast to proliferate; (2) have unique immunological characteristics: they do not express MHC class II m...

Claims

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Application Information

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IPC IPC(8): C12N5/0789C12N5/0775C12N5/071
Inventor 银广悦杨洪涛丁俊丽刘继勇周志伟孙岩徐翠玲武彩虹汪方圆张龙
Owner 银广悦
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