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Preparation of mesenchymal stem cells (MSCs) derived exosomes and application of the same in acute lung injury

A technology of stem cells and exosomes, applied in the field of stem cell research, can solve the problems of inability to distinguish large protein aggregates, shorten the preparation time, and incapable of large-scale preparations, achieve high uniformity, ensure extensiveness, and be widely used in clinical practice. The effect of applying the foreground

Inactive Publication Date: 2015-06-10
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the preparation technology of Exosomes mainly relies on the traditional ultra-high-speed density gradient centrifugation method, which requires special materials such as large-scale ultra-high-speed centrifuges and heavy water, but it cannot distinguish Exosomes from other small vesicle structures or large protein aggregates , and due to the limitation of the special rotor used in the centrifuge and the volume of the centrifuge tube, it cannot be prepared in large quantities, which greatly limits its application
Although the biofilm separation technology has simple requirements for equipment and materials, simplified steps, and greatly shortened the preparation time, it still has not solved the key problem

Method used

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  • Preparation of mesenchymal stem cells (MSCs) derived exosomes and application of the same in acute lung injury
  • Preparation of mesenchymal stem cells (MSCs) derived exosomes and application of the same in acute lung injury
  • Preparation of mesenchymal stem cells (MSCs) derived exosomes and application of the same in acute lung injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Isolation and cultivation of mesenchymal stem cells

[0037] 1.1 Isolation and culture of human umbilical cord blood mesenchymal stem cells

[0038] With the consent of the newborn's parents, 50-80ml of umbilical cord blood was collected from healthy full-term cesarean section fetuses, and the collected samples were processed within 4 hours. Dilute the anticoagulated umbilical cord blood (20U / ml heparin anticoagulated) to one time with the same volume of PBS, mix well, and then mix it with 3% gelatin pre-warmed at 37°C in equal proportions, and let it stand at room temperature until the interface between plasma and red blood cells is formed. , absorb the plasma layer, centrifuge at 2000 r / min for 5 minutes, and collect the cells; resuspend the obtained cells with 5ml DMEM / F12, add to the surface of human lymphocyte separation medium (1.077g / ml) at a volume ratio of 2:1, and collect the cells at 2000 r / min min Centrifuged for 20 min, buffy coat layer was draw...

Embodiment 2

[0043] Example 2: Isolation of Exosomes from Mesenchymal Stem Cells

[0044] Well-grown mesenchymal stem cells of passage 3-50 were taken. When the cell confluence reached 70%-80%, the supernatant was discarded, washed with PBS, and replaced with serum-free medium (adding parathyroid hormone 1ng / ml) for culture. After culturing for 48 hours, the culture supernatant was collected, centrifuged at 3000 rpm at low temperature (4°C) for 10 minutes, and filtered through a 0.2 μm sterile filter membrane to obtain crudely extracted Exosomes.

Embodiment 3

[0045] Example 3: Extraction and purification of Exosomes

[0046] 3.1 Discontinuous Ficoll density gradient method

[0047] 3.1.1 Preparation of Ficoll-glycerol separation solution

[0048] ①Dissolve 2g of Ficoll dry powder in 5ml of distilled water, and then fully mix with 40ml of 60% glycerin. Take 22ml of the mixed solution and distilled water to finally adjust the volume to 32ml. After mixing, when the temperature is cooled to 4°C, use a hydrometer to measure the density of 1.125g / ml; % glycerin and mix well, when the temperature is cooled to 4°C, the density is measured as 1.206g / ml with a hydrometer.

[0049] 3.1.2 Discontinuous Ficoll density gradient centrifugation

[0050] Put the Ficoll-glycerol separation solution in the same centrifuge tube (50ml) in order of density from low to high (that is, the bottom layer is 1.206g / ml Ficoll-glycerol separation solution 5ml, and the upper layer is 1.125g / ml Ficoll-glycerol separation solution 5ml), let it stand until it i...

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Abstract

The invention relates to preparation and a purification production process of mesenchymal stem cells (MSCs) derived exosomes, and application of the exosomes as a non-cell therapy in the treatment of acute lung injury. The MSCs deriving the exosomes belong to the wall adhesion type of stem cells, and the preparation and purification process have the characteristics that (1) parathyroid hormone is added specially for the preparation of MSCs condition medium, and (2) discontinuous Ficoll density gradient method and immunomagnetic beads separation and purification method are combined for separation and purification so as to obtain the exosomes. Rat acute lung injury (ALI) model experiments confirm that the exosomes can be used for treatment of ALI, so as to lay the foundation for the realization of non-stem cell therapies to substitute stem cells therapies.

Description

[0001] technical field [0002] The invention relates to stem cell research, which belongs to the fields of epigenetics, cell biology and molecular biology, and specifically relates to an improved preparation process of exosomes (Exosomes) secreted and released by mesenchymal stem cells (MSCs). Its homing mechanism, to study its mechanism of action in the treatment of acute lung injury (ALI). [0003] Background technique [0004] ALI is the main cause of acute respiratory failure in clinically critically ill patients. Its pathogenesis is complex and has not been fully elucidated. The condition is prone to sudden changes, and the mortality rate is still higher than 40%. MSCs are a kind of pluripotent cells with self-replication ability. Under certain conditions, they can differentiate into a variety of functional cells. They are an important basis for organisms to maintain normal shape and function. Diseases are closely related. Studies have confirmed that MSCs implanted ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/28A61P11/00
Inventor 田新瑞周楠牛勃张霆陈彦王建华徐文婧季成杨利军
Owner SHANXI MEDICAL UNIV
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