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Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same

A technology of complete culture medium and mesenchymal stem cells, which is applied to the complete culture medium for culturing mesenchymal stem cells and the field of using the culture medium to cultivate mesenchymal stem cells, can solve the problems that there is no report on the complete culture medium of mesenchymal stem cells, etc. Shorten cell operation time, simple and feasible operation, and accurate volume

Active Publication Date: 2009-09-09
INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there has been no report on a new complete medium for mesenchymal stem cells

Method used

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  • Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same
  • Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same
  • Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] (1) Isolation, culture and expansion of human bone marrow mesenchymal stem cells

[0068] Aseptically extract about 1ml of femoral bone marrow from the fetus (3-4 months old fetus induced by water bag, provided with the informed consent of the puerpera), transfer it into a sterile centrifuge tube containing 4ml of DMEM / F12 medium (product of GIBCO company), centrifuge to remove fat and supernatant. Add about 5ml of phosphate buffer to the cell mass to resuspend the cells, slowly add to an equal volume of Ficoll, and centrifuge at 900g for 15-18 minutes. Take the cloudy cell layer at the interface and add it to the complete medium, blow it into a single cell suspension, count the nucleated cells, and divide the cells in 10 5 / ml inoculated at 25cm 2 in a culture bottle. At 37°C, 5% CO 2 , saturated humidity CO 2 Cultivate in an incubator, remove non-adherent cells by changing the medium after 2-3 days, and change the medium every 3-4 days thereafter. When the cells...

Embodiment 2

[0088] (1) Isolation, culture and expansion of human umbilical cord mesenchymal stem cells

[0089] Rinse the aseptically collected human umbilical cord with phosphate buffered saline to remove residual blood, and cut it into about 1-1.5 mm with a sterile knife 3 of small pieces. Add 1 to 1.5 times the volume of the shredded tissue in phosphate buffer, add 1 / 2 to 1 / 3 of the total volume of type II collagenase, stir and digest at 37°C for 40-70 minutes, and use the mixture with 100-200 Filter through a mesh filter to separate the filtered cell suspension from the undigested tissue. Add phosphate buffer and trypsin to the undigested tissue block to continue digestion, stir and digest at 37°C for 20-30 minutes, stop the action of trypsin in serum, filter the cell suspension and undigested tissue with a 100-200 mesh filter Complete tissue block separation. Add an equal amount of phosphate buffer to all collected cell suspensions, mix well, slowly add to Ficoll separation medium...

Embodiment 3

[0105] (1) Isolation, culture and expansion of mesenchymal stem cells derived from human lung tissue

[0106] Take the lung tissue of the 3-4-month-old fetus induced by the water bag (provided with the informed consent of the puerpera), wash with phosphate buffer solution containing 100 U / ml penicillin / streptomycin and 250 μg / ml amphotericin B repeatedly to remove residual blood, and use Cut it into 1-2mm with sterile instruments 3 of small pieces. Add phosphate buffer with a final concentration of 0.1% type II collagen to 50ml, mix well, and digest at 37°C for 40-60 minutes under continuous magnetic stirring. After the mixture is filtered through a 100-200 mesh filter, the filtered cell suspension is separated from the undigested complete tissue. Use 40ml of phosphate buffer to wash the undigested tissue pieces off the filter, then add 20ml of phosphate buffer containing 12.5mg / ml trypsin, mix well and digest at 37°C for 20-30 minutes under continuous magnetic stirring , a...

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Abstract

The invention discloses a complete medium with low serum concentration for cultivating mesenchymal stem cells and a method for cultivating the mesenchymal stem cells using same. The complete medium comprises a cell basic medium, fetal calf serum with final concentration of 1-100 mul / ml, an epidermal growth factor with final concentration of 1-100 ng / ml, and a basic fibroblast growth factor with final concentration of 1-100 ng / ml. The complete medium with low serum concentration successfully reaches equal or even better function of promoting cell proliferation than a culture reagent with high serum concentration. The cultured cells have the typical biological characteristics of mesenchymal stem cells, and can also express an omnipotent mark of the embryonic stem cell and high express the idiosyncratic mark of the neuron under the condition of in vitro inducement. And the difference between the cell batches is little, the cost is low and the security is good. Compared with the prior cultivating method, the method has advantages of simple operation, low probability of pollution and high success ratio of cultivating cells.

Description

technical field [0001] The present invention relates to a culture medium, in particular to a complete culture medium for culturing mesenchymal stem cells and a method for cultivating mesenchymal stem cells using the culture medium. Background technique [0002] Stem cells have become the focus of research in the fields of regenerative medicine, tissue engineering, cell therapy and transplantation, and gene therapy. An ideal stem cell therapy strategy requires stem cells to have both totipotent differentiation potential and self-renewal ability. At present, mesenchymal stem cells are the most researched. The cells have the characteristics of self-renewal, multi-directional differentiation, homing to diseased tissues and immune regulation, making them promising seed cells in cell therapy and tissue engineering. In 2006, the International Cell Therapy Association clearly proposed the definition of mesenchymal stem cells: (1) adherent growth; (2) express CD105, CD13 and CD90, n...

Claims

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Application Information

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IPC IPC(8): C12N5/08
Inventor 韩忠朝郑翠玲杨少光卢士红韩之波
Owner INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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