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Dual tangential flow filtration system for exosome extraction and preparation method and application of exosome

A technology of tangential flow filtration and exosomes, applied in the field of biomedicine, can solve the problems of small sample size, poor repeatability, and complicated operation

Pending Publication Date: 2021-06-04
瑞太生物科技(沈阳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For this reason, the present invention provides a dual tangential flow filtration system for exosome extraction and the preparation method and application of exosomes, so as to solve the complex operation, small sample size and poor repeatability in the prior art, which cannot be used for Issues with large-scale GMP-grade preparation of exosomes

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  • Dual tangential flow filtration system for exosome extraction and preparation method and application of exosome
  • Dual tangential flow filtration system for exosome extraction and preparation method and application of exosome
  • Dual tangential flow filtration system for exosome extraction and preparation method and application of exosome

Examples

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Embodiment 1

[0049] Example 1 The present invention provides a method for preparing exosomes using a dual tangential flow filtration system for exosome extraction

[0050] 1. Umbilical cord mesenchymal stem cell culture

[0051] 1. Extraction and preparation of umbilical cord mesenchymal stem cells

[0052] Collect the newborn umbilical cord from the operating room, rinse the umbilical cord with sterile PBS buffer, remove the blood vessels with scissors and tweezers, peel off the Wahrenheit tissue inside, and cut the tissue to 1mm 3 The size of the organization block. Spread the tissue pieces evenly in the culture dish, and cultivate them in the incubator for 1 h (the culture condition is 5% CO 2 , 37°C). When the tissue block is firmly attached to the wall, add an appropriate amount of stem cell culture medium and place it in an incubator for culture (the culture condition is 5% CO 2 , 37°C). After the cells adhere to the wall well, gently suck off the culture medium, and gently rins...

Embodiment 2

[0065] Example 2 A dual tangential flow filtration system for exosome extraction

[0066] The double tangential flow filtration system comprises a first storage tank 1, a second storage tank 4, a first filter device 3, a second filter device 5, a first peristaltic pump 2, and a second peristaltic pump 6;

[0067] Wherein, the outlet of the first storage tank is connected to the inlet of the first filter device through the first peristaltic pump, the outlet of the first filter device is connected to the inlet of the first storage tank, and the filtered liquid outlet is connected to the first filter of the second storage tank. Inlet connection; the outlet of the second storage tank is connected with the inlet of the second filter device through the second peristaltic pump, the outlet of the second filter device is connected with the second inlet of the second storage tank, and the liquid filtered by the second filter device is directly outflow system.

[0068] Wherein, the firs...

experiment example 1

[0073] Experimental Example 1 Comparison of Different Extraction Methods

[0074] 1. Electron microscope comparison of exosomes

[0075] The exosomes obtained in the examples and the comparative examples were respectively fixed on the sample-loading copper grid by using glutaraldehyde solution, and after reacting for 5 minutes, the exosomes were fixed with ddH 2 O wash the copper grid, then place the copper grid in uranyl oxalate solution and incubate for 5 minutes, and finally absorb the excess liquid with filter paper. After the copper grid is dry, put it in the sample box, and take electron microscope photos at 80kV. Such as Figure 10 As shown in the example, the exosomes extracted and purified by the electron microscope are shown in the figure, Figure 9 As shown, the results of exosomes extracted from the comparative example by electron microscopy. The results showed that the saucer-shaped double-layer vesicle structure was visible under the electron microscope, the p...

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Abstract

The invention discloses a dual tangential flow filtration system for exosome extraction and a preparation method and application of an exosome. The method comprises the following steps: putting a sample solution containing exosomes into a first storage tank, pumping the sample solution into a first filtering device, circularly concentrating and filtering, and collecting a filtrate into a second storage tank; when a volume of the liquid in the first storage tank is concentrated to the minimum operation volume, pumping the liquid in the second storage tank into a second filtering device, and carrying out circulating concentration and filtration; and collecting the liquid in the second storage tank to obtain the extracted and purified exosome solution. The exosome can be used for preparing exosome medical beauty products, exosome liquid dressings for promoting wound healing, exosome spray for relieving asthma and acute respiratory distress, and exosome related biological products. According to the invention, the exosome is extracted and purified through two tangential flow filtering membranes with different pore diameters, so that cell microvesicles and cell debris with larger particles in a sample can be removed, and the exosome can be extracted and concentrated to the greatest extent.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a double tangential flow filtration system for extracting exosomes and a preparation method and application of exosomes. Background technique [0002] Exosomes are small vesicles with a phospholipid bilayer structure secreted by living cells, with a diameter of 30-150nm and a density of 1.13-1.19g / ml, which can exist in various body fluids, such as serum, plasma, saliva, urine fluid, ascites, spinal fluid, milk, etc. Exosomes contain a variety of biomolecules, such as mRNA, miRNA, proteins, lipids, etc., which can be delivered to recipient cells, thereby changing the physiological or pathological functions of recipient cells. In recent years, exosomes have attracted widespread attention as intercellular information transfer tools and biomarkers of various diseases. Exosomes have great potential in the fields of biomedicine and disease diagnosis. [0003] Obtaining high-puri...

Claims

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Application Information

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IPC IPC(8): C12N5/071A61K8/98A61K8/99A61K9/12A61K35/28A61L26/00A61P11/00A61P11/06A61Q19/00
CPCC12N5/0602A61K8/99A61K8/982A61Q19/00A61L26/0057A61L26/0061A61K35/28A61K9/12A61P11/06A61P11/00C12N2509/10A61K2800/10
Inventor 张海心
Owner 瑞太生物科技(沈阳)有限公司
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