Exosome freeze-drying protective agent, and freeze-dried powder resuscitation fluid and application thereof

A lyophilized protective agent, exosome technology, applied in the application, preservation of human or animal body, biochemical equipment and methods, etc., can solve the problems of loss of activity, difficulty in efficient and convenient application in experiments and clinical

Inactive Publication Date: 2021-01-12
广州佳为生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Exosomes can be stored in a frozen state for a short time, but as the storage time prolongs, the bimolecular membrane of exosomes gradually

Method used

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  • Exosome freeze-drying protective agent, and freeze-dried powder resuscitation fluid and application thereof
  • Exosome freeze-drying protective agent, and freeze-dried powder resuscitation fluid and application thereof
  • Exosome freeze-drying protective agent, and freeze-dried powder resuscitation fluid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of exosomes from human adipose-derived mesenchymal stem cells

[0031] Absorb human fat to prepare adipose-derived mesenchymal stem cells, and culture the adipose-derived mesenchymal stem cells. When the cell confluence reaches 75%-85%, wash the cells with normal saline, and then transfer the cells to a serum-free medium After culturing for 20-30 hours, collect the cell culture supernatant and replace it with a fresh medium. After continuous collection and culture for 3-5 days, collect the culture supernatant for exosome extraction. figure 1 Shown is a diagram of the cell state in the culture supernatant, where figure 1 The magnification of 1 is 40 times, figure 1 The magnification of 2 is 100x.

[0032] Centrifuge the supernatant at 4°C, 400×g for 10 minutes, transfer the supernatant to remove cell debris; centrifuge at 4°C, 2000×g for 10 minutes, transfer the supernatant to remove dead cells; filter with a 0.22 μm sterile filter membrane Remov...

Embodiment 2

[0033] Example 2 Freeze-drying of exosomes

[0034] Experiment 1. Take 10ml of the exosomes prepared in Example 1, add 0.25g of sucrose (2.5%), 100μl (0.11g) of Tween 80, and incubate the mixture at 4°C for 1h, every 20 minutes Vibrate once. Finally, the above-incubated mixed solution was placed in a 37°C incubator and equilibrated for 0.5h to obtain the exosome freeze-dried solution. The freeze-dried solution was placed at -80°C, pre-frozen for 12 hours, and then freeze-dried at -50°C and 10 Pa for 24 hours to obtain the freeze-dried powder of exosomes.

[0035] Experiment 2. Take 10ml of the exosomes prepared in Example 1, add 0.5g of trehalose (5%), 50μl (0.055g) of DMSO, and incubate the mixture at 4°C for 1h, shaking every 20 minutes once. Finally, the above-incubated mixed solution was placed in a 37°C incubator and equilibrated for 0.5h to obtain the exosome freeze-dried solution. The freeze-dried solution was placed at -80°C, pre-frozen for 12 hours, and then freez...

Embodiment 3

[0040] Example 3 Preparation of exosome freeze-dried powder resuscitation solution

[0041] The preparation process of the resuscitation solution includes: adding hydroxyethyl starch and liposomes to physiological saline at 4°C, stirring slowly until completely mixed, and storing in a refrigerator at 4°C after preparation. All lipids The body was purchased from Shanghai Jiake Biotechnology Co., Ltd.

[0042] Experiment 1. Take 50ml of normal saline, add 0.25g of hydroxyethyl starch (0.5%), and 10g of DOPC liposomes (20%) with a diameter of 25nm, dissolve and stir evenly to obtain the exosome freeze-dried powder resuscitation solution.

[0043] Experiment 2. Take 50ml of normal saline, add 1.25g of hydroxyethyl starch (2.5%), and 5g of DLPC liposomes (10%) with a diameter of 1000nm, dissolve and stir evenly to obtain the exosome freeze-dried powder resuscitation solution.

[0044] Experiment 3. Take 50ml of normal saline, add 0.75g of hydroxyethyl starch (1.5%), and 0.5g of DM...

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Abstract

The invention discloses an exosome freeze-drying protective agent, freeze-dried powder resuscitation fluid and application thereof, the freeze-drying protective agent is composed of sugar and a surfactant, the resuscitation fluid comprises normal saline, hydroxyethyl starch and lipidosome, the hydroxyethyl starch accounts for 0.5-2.5% of the resuscitation fluid, and the lipidosome accounts for 1-20% of the resuscitation fluid. Liposome with a structure similar to that of a vesicle membrane is added into the resuscitation fluid and is used for repairing damaged vesicles. The process follows thecharacteristic of biological membrane fluidity, and effectively repairs the exosome vesicle membrane structure, thereby being beneficial to keeping the activity of the exosome.

Description

technical field [0001] The invention relates to an exosome freeze-drying protective agent, a freeze-dried powder resuscitation solution and applications thereof. Background technique [0002] Exosomes are small vesicles (30-150nm in diameter) that contain complex RNA and proteins, and a variety of cells can secrete exosomes under normal and pathological conditions. It is mainly derived from the multivesicular body formed by the invagination of lysosomal particles in the cell, and is released into the extracellular matrix after the fusion of the outer membrane of the multivesicle and the cell membrane. [0003] All cultured cell types secrete exosomes, and exosomes are naturally present in bodily fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk. The function of exosomes depends on the cell type from which they originate, which can participate in the body's immune response, antigen presentation, cell migration, cell differentiation, tumor invasion,...

Claims

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Application Information

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IPC IPC(8): A01N1/02C12N5/0775
CPCC12N5/0667A01N1/0221A01N1/0226C12N2509/00C12N2500/12C12N2500/34C12N2500/36
Inventor 王治元方燕海
Owner 广州佳为生物科技有限公司
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