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Method for non-enzymatic 3D culture and amplification of therapeutic mesenchymal stem cells

A mesenchymal stem cell and therapeutic technology, applied in the field of enzyme-free 3D culture and expansion of therapeutic mesenchymal stem cells, to achieve high yield and high space efficiency

Pending Publication Date: 2022-07-05
唐颐惠康干细胞产业平台(天津)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is clear that 2D culture systems are insufficient for future commercial viability; instead, s

Method used

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  • Method for non-enzymatic 3D culture and amplification of therapeutic mesenchymal stem cells
  • Method for non-enzymatic 3D culture and amplification of therapeutic mesenchymal stem cells
  • Method for non-enzymatic 3D culture and amplification of therapeutic mesenchymal stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation steps of PLGA porous microspheres are as follows:

[0039] (1) At room temperature, mix PLGA, cholesterol and NH 3 HCO 3 Dissolve into ether at a ratio of 10:1:1, add water (water: ether volume ratio is 3:1) to the mixture ether solution, and ultrasonicate for 30 minutes to form a W / O emulsion;

[0040] (2) Using a spray dryer, the above-mentioned emulsion is spray-dried to form PLGA porous microspheres.

Embodiment 2

[0042] The preparation steps of PLGA-PEG-PLGA thermosensitive coating microcarriers are as follows:

[0043] (1) Under the condition of 25°C, prepare a 10-15% PLGA-PEG-PLGA aqueous solution by mass fraction, add the dried PLGA porous microspheres to the PLGA-PEG-PLGA aqueous solution, and stir for 15-20 minutes;

[0044] (2) The PLGA porous microspheres were then centrifuged and stored at 37°C for subsequent use.

[0045] Scanning electron microscopy images of thermosensitive gel coating on the surface of microcarriers and rheological results of thermosensitive gel coating on the surface of microcarriers, see figure 2 , image 3 .

Embodiment 3

[0047] Culture expansion of mesenchymal stem cells:

[0048](1) The mesenchymal stem cells extracted from the bone marrow were mixed with thermosensitive coated PLGA porous microspheres in a non-heterologous medium and placed in a stirred tank bioreactor.

[0049] (2) Adjust the reaction temperature to 37°C, continuously supply oxygen, stir and mix for 1 to 3 days, and the cells are expanded on the microcarrier.

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Abstract

The invention discloses a method for non-enzymatic 3D culture and amplification of therapeutic mesenchymal stem cells. The method comprises the following steps: S1, preparing PLGA porous microspheres; s2, a PLGA-PEG-PLGA temperature-sensitive coating microcarrier is prepared; s3, culturing and amplifying the mesenchymal stem cells; and S4, non-enzymatic separation of the mesenchymal stem cells: reducing the culture temperature to be below the phase change critical temperature, centrifuging the culture solution, and collecting the stem cells. According to the method for non-enzymatic 3D culture and amplification of the therapeutic mesenchymal stem cells, PLGA porous microspheres are adopted as a cell culture microcarrier support, the surface of the PLGA porous microspheres is coated with temperature-sensitive hydrogel PLGA-PEG-PLGA, an additional enzymolysis process is not needed, and therefore the stem cells are efficiently amplified.

Description

technical field [0001] The invention relates to the technical field of mesenchymal stem cell culture, in particular to a method for culturing and expanding therapeutic mesenchymal stem cells in an enzyme-free 3D manner. Background technique [0002] Cell therapy, the use of human cells as drugs through complex mechanisms that cannot be achieved with a single compound, promises to transform the treatment of a wide variety of diseases, including cancer, neurodegenerative and autoimmune diseases. Cell therapy, on the other hand, may be the result of a "swarm effect," meaning that cells needed for regeneration or immune processes in the body need to be present in large numbers. Therefore, only by increasing the number of therapeutic cells in a pathological state can the balance of therapy be tilted toward repair. In these cases, expansion of sufficient numbers of cells is critical to provide an effective therapeutic dose. [0003] Currently, suspension culture and adhesion cul...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C08J9/28C08L67/04
CPCC12N5/0662C12N5/0663C08J9/28C12N2531/00C12N2539/10C12N2513/00C12N2533/40C08J2367/04A61K9/1647A61K9/1694C12N5/0075A61K9/1652
Inventor 郑斌曹毓琳彭文畅程世翔赵杰
Owner 唐颐惠康干细胞产业平台(天津)有限公司