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Collagen material for promoting stem cell expansion and stemness maintenance and preparation method thereof

A technology of collagen materials and stem cells, applied in biochemical equipment and methods, cell culture supports/coatings, animal cells, etc., can solve problems such as the reduction of cell stemness, improve the microenvironment, promote in vitro proliferation, and promote tissue regeneration. The effect of damage repair

Inactive Publication Date: 2019-08-20
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the in vitro expansion and culture of stem cells, the stemness of the cells will gradually decrease as the cell proliferation and division time prolongs.

Method used

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  • Collagen material for promoting stem cell expansion and stemness maintenance and preparation method thereof
  • Collagen material for promoting stem cell expansion and stemness maintenance and preparation method thereof
  • Collagen material for promoting stem cell expansion and stemness maintenance and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the preparation of collagen material

[0053] 1. Take bovine fascia tissue and remove excess muscle and fat on the surface.

[0054] 2. Take the tissue obtained in step 1, wash it thoroughly with deionized water, and then place it in a liquid containing tributyl phosphate and soak it for 48 hours at 4°C (change the liquid every 12 hours, that is, replace with a new liquid containing tributyl phosphate ).

[0055] The liquid containing tributyl phosphate is obtained by mixing tributyl phosphate and water. In this embodiment, the concentration of tributyl phosphate is 1 g / 100 ml. In practical application, the concentration of tributyl phosphate is 0.5g / 100ml-1.5g / 100ml.

[0056] In practical application, 2°C-16°C is acceptable.

[0057] In practical applications, the soaking time can be 18 hours to 72 hours.

[0058] 3. Take the tissue that has completed step 2 and soak it in deionized water at 4°C (change the deionized water every 10 minutes, a total o...

Embodiment 2

[0106] Example 2, preparation of bone marrow mesenchymal stem cells

[0107] 1. SD rats were sacrificed by cervical dislocation, and bilateral femurs and tibias were taken. The bone marrow cavity was washed with serum-free DMEM culture medium and the liquid phase was collected, which was the bone marrow fluid.

[0108] 2. Slowly add 1 volume part of bone marrow fluid to 1 volume part of lymphocyte separation solution, centrifuge at room temperature and 2000rpm for 20min (the liquid in the centrifuge tube is divided into 3 layers, and monocytes are enriched in the middle layer), take the middle layer, and use 5ml PBS buffer was resuspended, and then centrifuged at 1200rpm for 6min to collect the cell pellet.

[0109] 3. Take the cells collected in step 2 and suspend them with stem cell culture medium to obtain a cell suspension.

[0110] 4. Inoculate the cell suspension obtained in step 3 into T25 cell culture flasks (approximately inoculate 6 × 10 7 cells) at 37°C, 5% CO 2 ...

Embodiment 3

[0115] Embodiment 3, collagen material can promote bone marrow mesenchymal stem cell proliferation

[0116] 1. Collect the third-generation bone marrow mesenchymal stem cells obtained in Example 2, and suspend them with stem cell culture medium to obtain a cell suspension.

[0117] 2. Group processing

[0118] Test group: 250 μl of the cell suspension prepared in step 1 (containing 5×10 3 bone marrow mesenchymal stem cells) mixed with 500 μl of the gelatinous collagen material prepared in Example 1, then added to a 35 mm cell culture dish, at 37° C., 5% CO 2 cultured in a humidified incubator.

[0119] Control group: 250 μl of the cell suspension prepared in step 1 (containing 5×10 3 Bone marrow mesenchymal stem cells) were added to a 35mm cell culture dish at 37°C, 5% CO 2 cultured in a humidified incubator.

[0120] Three replicates were performed and the results were averaged.

[0121] The number of cells was detected at different culture time points (1 day, 3 days, 5...

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Abstract

The invention discloses a collagen material for promoting stem cell expansion and stemness maintenance and a preparation method thereof. Firstly, the invention provides the preparation method of the collagen material, including the following steps: (1) taking cattle fascia tissues, removing muscle and fat on the surfaces of the fascia tissues, and cleaning the fascia tissues with water; (2) placing the fascia tissues in liquid containing tributyl phosphate to be soaked; (3) placing the fascia tissues in water to be soaked and cleaned; (4) placing the fascia tissues in a NaCl solution to be soaked; (5) placing the fascia tissues in water to be soaked and cleaned; (6) placing the fascia tissues in a surfactant solution to be soaked; (7) placing the fascia tissues in water to be soaked and cleaned; (8) placing the fascia tissues in a NaOH solution to be soaked; (9) placing the fascia tissues in water to be soaked and cleaned; (10) carrying out freeze-drying; (11) dissolving the freeze-dried tissues in an acetic acid solution; (12) carrying out dialysis; and (13) carrying out freeze-drying to obtain the collagen material. The invention also provides an application of the collagen material in the culture of stem cells. The collagen material provided by the invention has a great application and popularization value for the culture of stem cells.

Description

technical field [0001] The invention relates to a collagen material for promoting stem cell expansion and stemness maintenance and a preparation method thereof. Background technique [0002] Exogenous stem cell transplantation is an important means of repairing tissue damage. Stem cells have the ability to self-renew and differentiate, and can differentiate into specific types of tissue cells to promote tissue regeneration. At the same time, stem cells can also improve the damaged microenvironment through paracrine effects and promote damage repair. However, during the in vitro expansion and culture of stem cells, the stemness of the cells will gradually decrease with the extension of the cell proliferation and division time. Therefore, how to establish a culture method that can promote the expansion of stem cells and maintain the characteristics of the expanded stem cells is very important for the repair of tissue damage. Contents of the invention [0003] The object of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2533/54
Inventor 戴建武赵燕南陈冰
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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