Method for preparing high-activity exosome through heterologous serum-free 3D culture of MSC stem cells

A stem cell and exosome technology, applied in the field of natural biological nanomaterials, can solve the problems of increasing the production of MSC exosomes, and achieve the effect of enhancing the production of exosomes, enhancing the effect, and having high anti-cancer activity.

Active Publication Date: 2021-05-07
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the 3D culture environment also has the potential to increase the exosome production of MSCs

Method used

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  • Method for preparing high-activity exosome through heterologous serum-free 3D culture of MSC stem cells
  • Method for preparing high-activity exosome through heterologous serum-free 3D culture of MSC stem cells
  • Method for preparing high-activity exosome through heterologous serum-free 3D culture of MSC stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The preparation of embodiment 1 human source platelet lysate (HPL)

[0048] (1) Obtain human platelets from the blood product center, and perform freezing / thawing treatment: freeze at -80°C for 48 hours, thaw in a 37°C water bath for 2 hours, repeat the freezing / thawing operation once; then centrifuge at 1500g for 10 minutes, and collect the supernatant Filter through a 0.22 μm filter membrane to obtain sterile human-derived platelet lysate, and store it at -20°C; or directly purchase human-derived platelet lysate for the following steps;

[0049] (2) Thaw the human platelet lysate at 37°C before use, then centrifuge at 10,000 g for 10 min at 4°C, and collect the supernatant; filter the supernatant with a 0.22 μm filter membrane to obtain human platelet lysate (HPL) ;

[0050] (3) The human platelet lysate (HPL) prepared in step (2) was assayed using an ELISA kit for assaying human fibrinogen (Molecular Innovations), wherein the content of fibrinogen was 2.0 mg / mL.

Embodiment 2

[0051] Example 2 Preparation and Characterization of TRAIL Genetically Engineered MSC Stem Cells

[0052] (1) Preparation of TRAIL genetically engineered MSC stem cells

[0053] ① Preparation of lentivirus expressing TRAIL:

[0054] The TRAIL expression plasmid pCCL-CMV-flT (for the construction method of this plasmid, refer to the literature Yuan et al., 2015. Cytotherapy; 17:885-896) and the lentiviral packaging plasmid pCMV-dR8.74 and pMD2.G (given from London Dr. Thrasher, University College, or purchased from Addgene) co-transfected 293T cells according to conventional methods; 48 hours after transfection, the cell culture supernatant (containing the TRAIL-expressing lentivirus released by cell synthesis) was collected and subjected to ultracentrifugation (using Beckman The company’s Optima LE80K centrifuge, SW28 rotor, 4°C, 17,000rpm centrifuge for 2h) to precipitate the virus, and finally resuspended the virus pellet and dissolved it in PBS solution, and stored it at -...

Embodiment 3

[0061] Example 3 No heterologous serum 3D culture MSCflT cells

[0062] In this example, the DF-12 medium (Gibco, Life Technologies, Gaithersburg, MD, USA) containing 10% FBS was used as a control, and the DF-12 medium containing 5% of the HPL prepared in Example 1 was used to compare the exocrine The output of the body, the specific method is as follows:

[0063] (1) MSCflT cells (prepared in Example 2) resuscitated in the DF-12 medium of 10% FBS were cultured to 80% density, then replaced with DF-12 medium without any serum and cultivated for 24 hours; wait for the cells to consume FBS-derived protein, then trypsinize and dissociate the cells, and wash twice with PBS;

[0064] (2) The human-derived platelet lysate (HPL) prepared in Example 1 is mixed with DF-12 medium to obtain an HPL medium for culturing MSC stem cells, wherein the HPL content is 5%; after washing step (1) The cells were resuspended with HPL medium for culturing MSC stem cells at 11400 / cm 2 The cell dens...

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Abstract

The invention belongs to the field of natural biological nano materials, and particularly relates to an HPL culture medium for culturing MSC stem cells and a method for preparing a high-activity exosome by heterologous serum-free 3D culture of the MSC stem cells. According to the HPL culture medium for culturing the MSC stem cells, provided by the invention, human-derived platelet lysate is used for replacing animal serum FBS for cell culture. The method for preparing the high-activity exosome through heterologous serum-free 3D culture of the MSC stem cells is simple in preparation process, efficient and low in cost. The yield of the exosome is much higher than that of a conventional 2D cell culture method, the prepared exosome is higher in anti-cancer activity and safer to use, and a good foundation is laid for potential clinical application of MSC stem cell-derived biological nano-drug carrier treatment on cancers and the like.

Description

technical field [0001] The invention belongs to the field of natural biological nanomaterials, and in particular relates to an HPL medium for cultivating MSC stem cells and a method for preparing highly active exosomes by culturing MSC stem cells in 3D without heterologous serum. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells that can be isolated and cultured from adult bone marrow and adipose tissue or from neonatal accessory tissues such as umbilical cord and placenta. MSC cells are easy to expand in vitro, naturally have tumor tropism, and have the characteristics of inhibiting tumor growth, anti-inflammation, and protecting the body from damage. They are a good resource for cell therapy. It has been found that the main mechanism for MSCs to exhibit biological activity is to release a class of small extracellular vesicles (EVs) to the extracellular environment through the paracrine pathway, with a diameter of 50-130 nm, which ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/12C12N15/867C12N7/01A61K35/28A61K38/17A61P35/00
CPCC07K14/70575C12N5/0665C12N5/0663C12N5/0667C12N5/0664C12N5/0668C12N5/0605C12N15/86C12N7/00A61K35/28A61K38/177A61P35/00C12N2500/90C12N2500/84C12N2510/00C12N2513/00C12N2740/15043C12N2740/15021Y02A50/30
Inventor 袁正强苏奎候欢袁倩黎淑怡孙健武黄超鸿柯长洪
Owner GUANGDONG UNIV OF TECH
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