Method for preparing exosome freeze-dried powder of human amniotic mesenchymal stem cells

A technology of mesenchymal stem cells and human amniotic membrane, applied in the field of stem cells, can solve the problems of unstable source of exosomes, stable and controllable product quality, etc., and achieve the effect of maintaining shape and activity and simple operation

Active Publication Date: 2015-04-08
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the existing technology, the source of exosomes is unstable, and most of them come from body fluids such as blood or cells from various sources, which cannot make the product quality stable and controllable
In addition, since exosomes need to be cryopreserved, it brings a lot of inconvenience in storage, transportation and application.

Method used

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  • Method for preparing exosome freeze-dried powder of human amniotic mesenchymal stem cells
  • Method for preparing exosome freeze-dried powder of human amniotic mesenchymal stem cells
  • Method for preparing exosome freeze-dried powder of human amniotic mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Culture of human amniotic mesenchymal stem cells and collection of culture supernatant

[0035] 1. Culture of human amniotic mesenchymal stem cells:

[0036] Well-growing human amniotic mesenchymal stem cells were taken, cultured in serum-free medium, digested and passaged with 0.25% trypsin, passed to the P2 generation, and when they grew to a confluence of 80%, the culture medium was removed, washed three times with PBS, and added 1640 basal medium, collect the culture supernatant every day, and replace it with fresh 1640 medium, collect and culture continuously for 3 to 5 days, then collect the culture supernatant for exosome extraction. Among them, the cell state used to collect the culture supernatant is as follows: figure 1 shown.

[0037] 2. Extraction of exosomes (unless otherwise specified, the whole extraction process is carried out at 4°C):

[0038] (1), centrifuge all collected culture supernatants at 300 g for 10 min, retain the supernatant, dis...

Embodiment 2

[0043] Example 2. Preparation of human amniotic mesenchymal stem cell exosome freeze-dried powder:

[0044] Take the exosomes purified in Example 1, add trehalose to a final concentration of 10%, mix well, pass through a 0.22 μm filter membrane, divide and store in an ultra-low temperature refrigerator (-80°C) for 12 hours, and then transfer the sample to Freeze-dried in a vacuum freeze-drying chamber, the vacuum degree of freeze-drying was kept at 10 Pa, and the temperature was frozen at -50°C for 12 to 48 hours to obtain the human amniotic mesenchymal stem cell exosome freeze-dried powder.

Embodiment 3

[0045] Example 3. Morphological detection of exosomes:

[0046] The first group was stored in an ultra-low temperature refrigerator (-80° C.); the second group (the human amniotic mesenchymal stem cell exosome freeze-dried powder prepared in Example 2) was stored at room temperature. One month later, the first group was thawed for detection, and the second group was reconstituted with PBS buffer solution for detection.

[0047] The morphology of exosomes was observed by atomic force microscopy. After the exosomes to be detected were diluted in a certain proportion with pure water, 10 μL of the diluted solution was added dropwise to the surface of the newly dissociated mica. After standing for about 30 minutes, rinse carefully with pure water and blow dry with nitrogen. AFM imaging was performed using the amplitude-modulated tapping mode (resonance frequency 56 kHz, elastic coefficient 0.24 N / m). see results figure 2 .

[0048] Depend on figure 2 The results show that t...

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Abstract

The invention relates to the field of stem cells, discloses a method for preparing exosome freeze-dried powder of stem cells, and particularly relates to a method for preparing exosome freeze-dried powder of human amniotic mesenchymal stem cells. The method for preparing the exosome freeze-dried powder of the human amniotic mesenchymal stem cells disclosed by the invention comprises the following steps: mixing trehalose with exosomes of the human amniotic mesenchymal stem cells as a cryoprotectant; filtering and removing bacteria by virtue of a filter membrane; and firstly carrying out ultralow temperature pre-freezing, and then carrying out low-temperature freezing at a certain vacuum degree. The preparation method disclosed by the invention is simple to operate, safe and effective. An experiment proves that compared with a conventional preparation method of the freeze-dried powder, the morphologies and the activity of the exosomes of the human amniotic mesenchymal stem cells can be well kept by the method for preparing the exosome freeze-dried powder of the human amniotic mesenchymal stem cells disclosed by the invention; and the method is suitable for long-term preservation and application of the exosomes of the human amniotic mesenchymal stem cells.

Description

technical field [0001] The present invention relates to the field of stem cells, in particular to a method for preparing freeze-dried powder of stem cell exosomes, in particular to a method for preparing freeze-dried powder of human amniotic mesenchymal stem cell exosomes. Background technique [0002] Exosomes were first discovered in the supernatant of sheep erythrocytes cultured in vitro. They are vesicle-like bodies actively secreted by cells with a uniform size, a diameter of 40-100 nm, and a density of 1.10-1.18 g / ml. The application of exosomes mainly focuses on clinical diagnosis and anti-tumor. [0003] Mesenchymal stem cells (MSCs) are cells with self-renewal and multi-directional differentiation capabilities, produce and release nutrients, promote cell repair and angiogenesis through paracrine pathways, and play an important role in the field of regenerative medicine. Lai et al. analyzed by mass spectrometry and found that exosomes of MSCs contained 857 proteins ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 陈海佳王一飞葛啸虎麦锦连马岩岩王小燕舒辉萍
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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