Extraction method of specific mesenchymal stem cell exosome

Abstract

The invention discloses an extraction method of a specific mesenchymal stem cell exosome. The method comprises the following steps: collecting supernatant of serum-free mesenchymal stem cells: firstly, culturing mesenchymal stem cells, changing a culture medium for culturing, centrifuging, and removing cell debrises to obtain initial supernatant; taking the initial supernatant for carrying out further gradient centrifugation, transferring the centrifuged supernatant into a sterile centrifugal tube, centrifuging and collecting supernatant containing micro vesicles; centrifuging the supernatantcontaining the micro vesicles to obtain precipitate containing an exosome, washing and then centrifuging to obtain exosome precipitate; adding the obtained exosome precipitate into sterile PBS and filtering to obtain the specific mesenchymal stem cell exosome. The exosome collected in the extraction method disclosed by the invention adopts a filter membrane for filtration, so the extracted exosomeis enabled to be nanoscale; meanwhile, the rotational speed is reduced and optimized, high-speed mechanical damage is reduced, separation efficiency is high and the cost is low.

Description

technical field [0001] The invention belongs to the field of cell technology, and in particular relates to a specific method for extracting exosomes from mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (Mesenchyaml stem cells, MSCs) are a type of pluripotent stem cells, which originate from the mesoderm and ectoderm in the early stages of development, and can be isolated from various tissues such as bone marrow, fat, synovium, bone, muscle, and umbilical cord. After induction, it can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve liver, cardiac muscle and endothelium. In the past, it was often believed that mesenchymal stem cells could nest in the damaged site and then directly differentiate into target cells to play a role. However, studies have shown that the proportion of mesenchymal stem cells nesting in the injury site and the number of differentiated target cells are very limited, and ...

AI Technical Summary

Problems solved by technology

There are many problems in using PEG to precipitate exosomes: such as low purity and recovery, more impurities (false positives), uneven particle size, generation of difficult-to-remove polymers, mechanical force or chemical additives such as Tween-20 Will destroy exosomes, etc., so it is easy to be questioned when publishing an article

[0018] (1) Although there are many exosomes collected by ultracentrifugation, since there is no very uniform identification standard for microvesicles and exosomes, the purity of the collected exosomes is not good, so exosomes were found to aggregate during electron microscope identification Clumped, poor quality;

[0019] (2) The existing exosome extraction methods are not cell-specific, such as ultracentrifugation, cells of different sizes and types are all under the same ultracentrifugation conditions, that is, 300g, 2000g, 10000g, 100000g centrifugation, such ultracentrifugation method Of course, it is difficult to meet the centrifugation conditions of different cell types, so it is necessary to optimize the centrifugation conditions of exosomes of different cell types;

[0020] (3) Although the ultrafiltration centrifugation method is simple, the ultrafiltration tube centrifugation operation only takes 1 hour, which is close to 2.5 hours compared with the ultracentrifuge tube, which can save 1.5 hours, but the entire centrifugation process needs to replace the filter membrane at least twice, which is expensive

Moreover, the concentration of exosomes collected by the simple ultrafiltration method cannot reach a high level, which is also a limitation for applications such as detection and treatment that require higher concentrations of exosomes.

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