Method for reversibly separating human adipose mesenchymal stem cells through monoclonal antibody L-NGFR immunomagnetic beads

A technology of immune magnetic beads and mesenchymal stem cells, which is applied in the direction of animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of poor activity, time-consuming and laborious, and low purity of mesenchymal stem cells, and achieve low cost and pain. Small, active effect

Active Publication Date: 2013-08-21
冯文峰
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Problems solved by technology

The traditional separation method of adipose-derived mesenchymal stem cells still adopts the adherent culture method, which is time-consuming and labor-intensive, and the isolated mesenchymal stem cells are not high in purity and activity, or can only be called stromal vascular cells

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  • Method for reversibly separating human adipose mesenchymal stem cells through monoclonal antibody L-NGFR immunomagnetic beads

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Embodiment 1

[0024] (1) Preparation of immunomagnetic beads: Co-precipitation method was used to prepare supercis Fe with a particle size of 20±5nm 3 o 4 magnetic nanoparticles.

[0025] Measure an equal volume of 1M FeSO 4 ·7H 2 O solution and 1.75M FeCl 3 solution, mixed rapidly, where [Fe 3+ ]: [Fe 2+ ]=1.75:1; dropwise add 5M NaOH solution to pH=7, at this time, brown particles are formed; then add dropwise 0.5M NaOH solution to pH=11, continue to stir, add absolute ethanol, after standing for 10min, the temperature Raise to 80°C for aging; stir, and add 100 μL, 0.001mol / L surfactant sodium dodecylbenzenesulfonate at the same time; after 30 minutes, use a strong magnet to settle, separate the supernatant, and use distilled water and anhydrous The precipitate was washed repeatedly with ethanol until the pH of the washing water was about 7; the precipitate was placed in a vacuum drying oven and dried at 75°C for 5 hours to obtain super-compliant Fe3O4 nano-magnetic particles.

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Abstract

The invention relates to a method for reversibly separating human adipose mesenchymal stem cells through monoclonal antibody L-NGFR immunomagnetic beads. The method comprises the following steps of: (1) preparing the immunomagnetic beads; (2) coupling the immunomagnetic beads with biotin analogues; (3) combining the biotin analogues with streptavidin; (4) coupling the streptavidin with monoclonal antibody L-NGFR; (5) separating the mesenchymal stem cells from adipose suspension digested by collagenase I; and (6) eluting the adipose mesenchymal stem cells. According to the method, the mesenchymal stem cells can be effectively separated from human adipose tissues through utilizing the specific monoclonal antibody L-NGFR of the adipose mesenchymal stem cells and adopting a reversible protein combination method with controllable pH and an immunomagnetic bead enrichment method; and the adipose mesenchymal stem cell separating method is simple, environment-friendly, and low in cost, ensures high activity of the stem cells and has better application prospect.

Description

technical field [0001] The invention relates to a method for sorting stem cells, in particular to a method for reversibly sorting human adipose-derived mesenchymal stem cells by monoclonal antibody L-NGFR immunomagnetic beads. Background technique [0002] Mesenchymal stem cells (MSCs) are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. Mesenchymal stem cells were originally discovered in the bone marrow, and have attracted increasing attention because of their multi-lineage differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication. Under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells. It has multi-directional differentiation potential and can be used as an ideal seed cell fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 冯文峰佘志勇方海庆陈建兴
Owner 冯文峰
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