Rapid induced differentiation method of mesenchymal stem cells, kit and application of rapid induced differentiation method

A mesenchymal stem cell and induced differentiation technology, applied in the field of cell engineering, can solve the problems of restricting the large-scale cultivation of autologous mesenchymal stem cells, high cost, restricting industrialization and marketization, etc., and achieve strong proliferation and immune regulation ability, good damage Repair and anti-aging, strong cell growth effect

Inactive Publication Date: 2021-10-01
CHENGNUO REGENERATIVE MEDICINE TECH (ZHUHAI HENGQIN NEW AREA) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The disadvantage of the embryoid body differentiation method is that the differentiation efficiency is low, the differentiation process is complicated and time-consuming, and more types of cytokines need to be added, which is costly
Therefore, the existing technology of iPSC differentiation into MSC has problems such as long time-consuming, low yield, high technical requirements, and limited cell purity, which restricts the large-scale cultivation of autologous mesenchymal stem cells and limits its industrialization and marketization.

Method used

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  • Rapid induced differentiation method of mesenchymal stem cells, kit and application of rapid induced differentiation method
  • Rapid induced differentiation method of mesenchymal stem cells, kit and application of rapid induced differentiation method
  • Rapid induced differentiation method of mesenchymal stem cells, kit and application of rapid induced differentiation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1 Human IPSC induced differentiation to obtain autologous MSC cells

[0111] 1, experimental materials

[0112] The main reagent information used in the embodiments of the present invention is shown in Table 1.

[0113] Table 1 Main reagent information used in the embodiments of the present invention

[0114]

[0115]

[0116] 2, induce the preparation of the former cell

[0117] (1) Experimental method

[0118] 1 Treatment of the plate: The culture plate was incubated for more than 30 minutes by Matrigel room temperature;

[0119] 2 Cell digestion and inoculation: IPSC (Beijing Mi Nuo Medical Biotechnology Co., Ltd.) cultured in T25 bottles, DPBS washed twice, 1 min 1 min; add 0.5mm EDTA digestive liquid 2ml; 37 ° C for 5 minutes, abandon EDTA, add E8 fully medium to terminate digestion;

[0120] Cell suspension, cell count; depending on cell density and different amounts of cell suspension, 1000 rock / separation center for 5 min; use the E8 to add Rock Inhibi...

Embodiment 2

[0150] Example 2 Flow cytometry detects IPSC differentiated MSC cell surface markers

[0151] 1, experimental method

[0152] (1) Cell digestion

[0153] The cell culture medium obtained by the IPSC differentiated MSC cell obtained in Example 1 was removed with a pipette, and the calcium magnesium PBS was added, and the culture flask was gently shaken, discarded the calcium magnesium PBS, and repeated the above process once; 4 times diluted Accutase, placement of cells at 37 ° C, observe the digestion of cells under microscope; when the cells are mostly delamed, tap petri dishes, collect cell suspensions to 50 ml of centrifuge tube;

[0154] (2) Single-cell suspension preparation

[0155] 30 μm aperture screen filtrates the cell suspension in the above 50 ml of centrifuge tube to a new 50 ml of centrifuge tube, 300 g × 5min collects cells, abandon the supernatant;

[0156] (3) Cell fixation

[0157] The collected cells were resuspended with 10 ml of curprownable magnesium PBS, and...

Embodiment 3

[0164] Example 3 Detection of MSC cells from IPSC differentiation

[0165] In this example, the MSC cells obtained by divided by IPSC in Example 1 conducted an osteo-inducible differentiation experiment, such as a cartilage inducing differentiation experiment, and an induced differentiation experiment to verify the differentiation of IPSC in the present invention. MSC cells obtained. Polygeneity.

[0166] 1, ingredient induced differentiation experiment

[0167] (1) The MSC of the IPSC source was cultured in MSC maturation medium;

[0168] (2) Before the MSC is differentiated into an osteogenic cell, it ensures that the cells reach a 100% conversion, and an osteoblastified medium is used;

[0169] (3) Cultivate cells for 2-3 weeks, change the medium every 3-4 days;

[0170] (4) After the PBS washing, 4% polymethylene formaldehyde solution was used for half an hour;

[0171] (5) Suffering the fixing liquid, wash the cells with distilled water;

[0172] (6) At room temperature, wate...

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Abstract

The invention discloses a rapid induced differentiation method of mesenchymal stem cells, a kit and application thereof. The rapid induced differentiation method of mesenchymal stem cells provided by the invention can be used for preparing enough number of mesenchymal stem cells capable of meeting clinical requirements in a short time at high yield, because there is no step of forming an embryoid body, the preparation process is simple, and the method has the advantages of high differentiation efficiency, convenience in purification and separation and the like. In addition, animal serum and other non-human additives are not involved in the differentiation process, the mesenchymal stem cells are more suitable for clinical requirements, and the mesenchymal stem cells obtained through differentiation are high in multiplication capacity and vigorous in cell activity.

Description

Technical field [0001] The present invention belongs to the field of cell engineering, and in particular, the present invention relates to a rapid inducing differentiation method, kit, and its application thereof in mesenchymal stem cells. Background technique [0002] Human Induced PLURPOTENT STEM CELLS, IPSC) is a pluripotent stem cell that is in vitro self-renewal, and maintains normal nuclear and developmental cells. Personality. IPSC cells are in terms of morphology, proliferation differentiation, cell surface antigen, gene expression model, etc., have great similarity with embryonic Stem Cell, ES, and because IPSC can come from supplier somatic cells, Therefore, in clinical application, it will not cause immunoassay effects, and can avoid the medical ethics of embryonic stem cells, so it has played an important role in all aspects of disease modeling, drug discovery, and cell therapy. Development of Cell Biology and Regenerative Medicine. [0003] MSenchymal Stem Cell, MSC)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P37/02A61P25/00A61P7/00A61P9/00A61P3/10A61P19/08A61P19/00A61P13/12A61P1/16A61P35/00
CPCC12N5/0662A61K35/28A61P37/02A61P25/00A61P7/00A61P9/00A61P3/10A61P19/08A61P19/00A61P13/12A61P1/16A61P35/00C12N2506/45C12N2500/38C12N2501/155C12N2501/15
Inventor 吴理达顾雨春
Owner CHENGNUO REGENERATIVE MEDICINE TECH (ZHUHAI HENGQIN NEW AREA) CO LTD
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