Umbilical cord blood mononuclear cell separation method and application thereof

A separation method and nuclear cell technology, applied in the direction of cell dissociation method, blood/immune system cells, animal cells, etc., can solve the problems of high technical requirements and complicated operation, and achieve the reduction of technical requirements, stable separation effect, and time-consuming short effect

Inactive Publication Date: 2020-08-18
广州市天河诺亚生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to diluting the blood, this method must be carefully operated so that the blood and the layered liquid form a clear interface, s

Method used

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  • Umbilical cord blood mononuclear cell separation method and application thereof
  • Umbilical cord blood mononuclear cell separation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Add 20mL Ficoll to a 50mL centrifuge tube (recorded as tube ①).

[0028] 2. Add 20mL of umbilical cord blood into a 50mL centrifuge tube (denoted as tube ②), shake well, and perform cell counting. The mononuclear cells in the whole blood are 13.1×10 9 pc / L, whole red blood cell is 3.42×10 12 a / L.

[0029] 3. Use a pipette to directly inject the umbilical cord blood in tube ② into tube ① containing Ficoll, mix it upside down, and let it stand for processing.

[0030] 4. After standing still for 40 minutes, it can be seen that the tube is divided into 3 layers, the upper layer is plasma, the lower layer is red blood cells and granulocytes, and the middle layer is buffy coat mainly composed of mononuclear cells.

[0031] 5. Aspirate the buffy coat with a pipette, transfer the buffy coat into a 50mL centrifuge tube (denoted as tube ③), wash it twice with normal saline, and centrifuge at 800g for 8 minutes each time.

[0032] 6. Finally, resuspend the cells with 10ml o...

Embodiment 2

[0034] 1. Add 30mL Ficoll to a 50mL centrifuge tube (marked as tube ①).

[0035] 2. Add 20mL of umbilical cord blood into a 50mL centrifuge tube (denoted as tube ②), shake well, and perform cell counting. The mononuclear cells in the whole blood are 7.5×10 9 pc / L, whole blood red blood cell is 3.18×10 12 a / L.

[0036] 3. Use a pipette to directly inject the umbilical cord blood in tube ② into tube ① containing Ficoll, mix it upside down, and let it stand for processing.

[0037] 4. After standing still for 40 minutes, it can be seen that the tube is divided into 3 layers, the upper layer is plasma, the lower layer is red blood cells and granulocytes, and the middle layer is buffy coat mainly composed of mononuclear cells.

[0038] 5. Aspirate the buffy coat with a pipette, transfer the buffy coat into a 50mL centrifuge tube (denoted as tube ③), wash it twice with normal saline, and centrifuge at 800g for 8 minutes each time.

[0039] 6. Finally, resuspend the cells with 10m...

Embodiment 3

[0041] 1. Add 40mL Ficoll to a 250mL centrifuge tube (recorded as tube ①).

[0042] 2. Add 20mL of umbilical cord blood into a 250mL centrifuge tube (denoted as tube ②), shake well, and perform cell counting. The mononuclear cells in the whole blood are 9.8×10 9 pc / L, whole blood red blood cell is 3.16×10 12 a / L.

[0043] 3. Use a pipette to directly inject the umbilical cord blood in tube ② into tube ① containing Ficoll, mix it upside down, and let it stand for processing.

[0044] 4. After standing still for 40 minutes, it can be seen that the tube is divided into 3 layers, the upper layer is plasma, the lower layer is red blood cells and granulocytes, and the middle layer is buffy coat mainly composed of mononuclear cells.

[0045] 5. Aspirate the buffy coat with a pipette, transfer the buffy coat into a 50mL centrifuge tube (denoted as tube ③), wash it twice with normal saline, and centrifuge at 800g for 8 minutes each time.

[0046] 6. Finally, resuspend the cells with...

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Abstract

The invention discloses an umbilical cord blood mononuclear cell separation method and an application thereof, and belongs to the technical field of cell separation. According to the method, only umbilical cord blood and a Ficoll reagent need to be mixed and subjected to standing treatment, a layering liquid is obtained, then cells of a middle leukocyte layer are sucked and washed, and obtained sediment contains umbilical cord blood mononuclear cells. The method disclosed by the invention adopts a standing mode, is easy to operate and short in consumed time, avoids damage of centrifugation tocells, and ensures the survival rate of the cells. According to the method, blood does not need to be diluted, and consumed time is shorter. According to the method, the technical requirements on experimenters are reduced, and a more stable separation effect can be obtained.

Description

technical field [0001] The invention belongs to the technical field of cell separation, and in particular relates to a method for separating umbilical cord blood mononuclear cells and an application thereof. Background technique [0002] White blood cells (WBC) are a type of colorless, spherical, nucleated blood cells. The total number of normal adult white blood cells is (4.0~10.0)×10 9 / L can vary within a certain range due to different time of day and different functional states of the body. Leukocytes are not a homogeneous cell population, and can be divided into three categories according to their shape, function, and origin: granulocytes, monocytes, and lymphocytes, among which monocytes and lymphocytes belong to mononuclear cells (PBMC). [0003] The main separation method of mononuclear cells is Ficoll-hypaque density gradient centrifugation. Ficoll-diatrizoate is an ideal cell layering solution. Its main component polysucrose is a synthetic sucrose polymer with ...

Claims

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Application Information

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IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00
Inventor 嵐山芮魏伟徐佳
Owner 广州市天河诺亚生物工程有限公司
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