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Method for simply preparing cytokines from placenta mesenchymal stem cells

A technology of stem cells and cytokines, which is applied to the isolation of exosomes from human placental mesenchymal stem cells, and the application in cell repair. In the field of membrane bubbles, it can solve the problems of complex process and low output.

Active Publication Date: 2019-12-06
BOYALIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] However, in the prior art, when culturing mesenchymal stem cells from placenta, umbilical cord blood, bone marrow, and fat, the medium supernatant of these MSCs is often used to extract exosomes. These methods all have the disadvantages of low yield and complicated process.

Method used

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  • Method for simply preparing cytokines from placenta mesenchymal stem cells
  • Method for simply preparing cytokines from placenta mesenchymal stem cells
  • Method for simply preparing cytokines from placenta mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1: Preparation of placental mesenchymal stem cells

[0155] (1) Processing placental tissue

[0156] Remove the amniotic membrane from a fresh human placenta, cut the membranous tissue on the surface of the placenta leaflet, and wash it with normal saline;

[0157] Shred the membranous tissue on the surface of the placental lobules into tissue fragments with a volume of about 0.2 cm3;

[0158] Put the tissue fragments into a centrifuge tube, add an appropriate amount of 0.9% normal saline (100ml), filter through a 300-mesh filter, and wash twice with an appropriate amount of 0.9% normal saline (100ml) until the filtrate is clear;

[0159] Add the cleaned tissue to the HBSS digestion solution (100ml) containing 0.005% Liberase MNP-S enzyme and 0.05% DNA type I enzyme, mix thoroughly, and shake and digest in a shaker for 30min (37°C, 100rpm) ;

[0160] (2) Obtaining placental blast cells

[0161] After the digestion, add fetal bovine serum (2ml) to the centr...

Embodiment 2

[0174] Example 2: Isolation and extraction of exosomes from placental mesenchymal stem cells

[0175] 1) Provide human placental mesenchymal stem cells of the 2nd to 5th passages in the logarithmic growth phase (this example takes the 3rd passage obtained in Example 1 as an example), at a rate of 5,000 to 15,000 / cm 2 The density inoculation (inoculation density of this embodiment: 10000 / cm2) is cultured in the DMEM / F12 medium that contains the L-glutamine of 2mM, after cultivating 3-4 days (this embodiment cultivates 4 days), collects Clear, remove cells and debris with 20000g centrifugal force for 20min, then filter through a 0.22 μm filter membrane, add mannose phosphate sodium to dissolve it, and obtain the culture supernatant; [in this embodiment, the amount of mannose phosphate sodium added accounts for The mass / volume percent concentration of filtrate is 0.08%]

[0176] 2) Drain the Pudex G-25 dextran gel filtration medium with a glass rod into the chromatographic col...

Embodiment 3

[0194] Example 3: Isolation and extraction of exosomes from placental mesenchymal stem cells

[0195] Carry out with reference to Example 2.

[0196] 1) Provide the second generation human placental mesenchymal stem cells in the logarithmic growth phase at 5000 / cm 2 The density was inoculated into DMEM / F12 medium containing 2mM L-glutamine for culture. After 3 days of culture, the supernatant was collected, centrifuged at 20000g for 20min to remove cells and debris, and then filtered through a 0.22μm filter membrane, and added 0.1% sodium mannose phosphate was dissolved to obtain the culture supernatant;

[0197] 2) Drain the Pudex G-25 dextran gel filtration medium with a glass rod into the chromatography column (Sephadex G-25 column), and stir with a glass rod while loading;

[0198] 3) After installing the column, use a pipette gun to suck out the water above the column, and then load the culture supernatant collected in step 1) into the chromatography column;

[0199]...

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Abstract

The invention relates to a method for simply preparing cytokines from placenta mesenchymal stem cells. On the one hand, the invention relates to a method for separating and extracting exosome and cytokines from placenta mesenchymal stem cells. The method comprises the following steps: culturing the placenta mesenchymal stem cells to obtain a culture supernatant; carrying out sephadex column chromatography on the cultured supernatant to obtain a chromatographic solution; concentrating the chromatographic solution by using a tangential flow ultrafiltration system to obtain an exosome concentrate, acquiring an ultrafiltration filtrate at the same time, concentrating the chromatographic solution again by using the tangential flow ultrafiltration system, and filtering and concentrating the ultrafiltration filtrate to obtain a cytokine concentrate; and carrying out cryopreservation or freeze drying on the exosome concentrate or the cytokine concentrate. The invention also relates to a methodfor preparing the cytokine concentrate. The method of the present invention exhibits excellent properties as described in the specification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for obtaining membrane vesicles involved in cell biological activities, in particular to a method for separating and obtaining exosomes (Exosomes, or Exosomes) from human placental mesenchymal stem cells (MSCs). method, and its application in cell repair. In particular, the present invention relates to a method for isolating exosomes from human placental mesenchymal stem cells. In addition, the present invention also relates to a method for simply preparing cytokines of placental mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a kind of non-hematopoietic pluripotent stem cells derived from mesoderm. It has been confirmed that MSCs not only have the potential of self-renewal and multilineage differentiation, but also have strong anti-inflammatory and inhibitory effects. The ability of a variety of immune cells and the ability to induce ...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/0775C07K14/52C07K1/14C07K1/34
CPCC07K14/52C12N5/0605C12N5/0668C12N2509/00
Inventor 刘冰肖海蓉李诣书
Owner BOYALIFE
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