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Method for enriching MNCs (mononuclear cells) in human blood

A technology for enriching human and blood, which is applied in the field of enriching human blood MNCs, can solve the problems of high cost, time-consuming and labor-intensive preparation of blood MNCs in batches, so as to reduce the preparation cost, reduce the amount of pipe laying and pipetting operations, The effect of reducing the amount of separation liquid used

Active Publication Date: 2018-06-01
北广再生医学科技(广东)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, preparing blood MNCs in batches is time-consuming, labor-intensive, and costly.

Method used

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  • Method for enriching MNCs (mononuclear cells) in human blood
  • Method for enriching MNCs (mononuclear cells) in human blood
  • Method for enriching MNCs (mononuclear cells) in human blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Collection of umbilical cord blood or peripheral blood samples

[0028] Collect 30 samples of umbilical cord blood or peripheral blood, blood volume 75-135mL, no hepatitis B, hepatitis C, syphilis, HIV-I / II virus infection, no bacteria, mycoplasma infection. The 30 samples were randomly divided into two groups, 15 samples in each group. CD34 phenotype detection and lymphocyte count test were performed. Among them, one group was the control group, and MNCs were isolated according to the "Instructions for Human Peripheral Blood Lymphocyte Separation Solution"; the other group For the experimental group, MNCs were separated by density gradient centrifugation after MNCs enrichment.

Embodiment 2

[0029] Example 2: Enrichment of cord blood MNCs

[0030] Transfer 15 parts of the experimental group to 50mL centrifuge tubes (model 373687, manufacturer NUNC), adjust the speed of the centrifuge to 0, and centrifuge horizontally at 200g for 10 minutes. The centrifuge tube is divided into two layers from top to bottom, and the lower layer is the cells. Layer 2, the upper layer is the plasma layer 1, and the cell layer and plasma layer between about 1 cm away from the plasma layer and about 0.5 cm away from the cell layer is the enriched MNCs layer 3 (see figure 1 ), carefully aspirate the enriched MNCs with a sterile Pasteur pipette, and combine the cell suspension.

Embodiment 3

[0031] Example 3: Separation of MNCs by Density Gradient Centrifugation

[0032] Add 20mL of lymphocyte separation solution into a 50mL centrifuge tube, dilute 15 cases of umbilical cord blood or peripheral blood samples with normal saline at a ratio of 1:1, dilute the enriched MNCs of 15 cases of experimental group to 25mL with normal saline, and spread carefully Centrifuge horizontally at 400g for 20 minutes, carefully aspirate the ring-shaped buffy coat layer in the middle, wash twice with normal saline, and collect the precipitated cells.

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Abstract

The invention discloses a method for enriching MNCs (mononuclear cells) in human blood. The method comprises steps as follows: S1, a blood sample is acquired; S2, enriched MNCs are sucked after the blood sample is centrifuged; S3, the enriched MNCs are separated with a density gradient centrifugation method, and the MNCs are obtained. The method has the benefits as follows: pipe pavement and pipetting operation can be reduced, the use quantity of a lymphocyte separation liquid can be reduced, and finally, the labor intensity and the preparation cost are effectively reduced.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for enriching human blood MNCs. Background technique [0002] Mononuclear cells (Mononuclear cells, hereinafter referred to as MNCs) are non-lobular monocytes, generally referring to lymphocytes and mononuclear macrophages in umbilical cord blood, placental blood, bone marrow, and peripheral blood. In fact, the composition of MNCs in blood is very complex, and MNCs from different tissue sources contain different proportions of hematopoietic stem / progenitor cells, mesenchymal stem cells, neural precursor cells, endothelial precursor cells, endothelial cells, pluripotent stem cells, small embryonic like cells, etc. Therefore, human blood MNCs have broad applications in the fields of regenerative medicine and tissue engineering. With the development of umbilical cord blood transplantation and immune cell therapy technology, the isolation method of MNCs from umbilical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00
Inventor 张洪钿苑春慧
Owner 北广再生医学科技(广东)有限公司