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Preparation method of sub-totipotent mesenchymal stem cell secretin

A mesenchymal stem cell and secretin technology, which is applied in the field of preparation of subtotipotent mesenchymal stem cell secretin, can solve the problem that large-dose dressing application, industrial application, and large-scale and continuous in vitro culture cannot be achieved. and other issues, to achieve the effect of easy large-scale continuous production, high active ingredients, and increased value-added

Pending Publication Date: 2019-12-10
深圳刚华健医疗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many ways to culture mesenchymal stem cells in vitro, but usually only on a laboratory scale, which cannot achieve the purpose of large-scale and continuous in vitro culture, and thus cannot realize industrial applications, especially large-dose dressings. application

Method used

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  • Preparation method of sub-totipotent mesenchymal stem cell secretin
  • Preparation method of sub-totipotent mesenchymal stem cell secretin
  • Preparation method of sub-totipotent mesenchymal stem cell secretin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Pre-cultivation of mesenchymal stem cells

[0041] Take adipose-derived mesenchymal stem cells at 5000 cells / cm 2 Inoculated at a density of 10% fetal bovine serum (FBS) and modified Eagle medium (DMEM) supplemented with 1% antibiotics, cultured in a single layer for 2 generations, and obtained mesenchymal cells with a cell density of 100,000 / 100mL-10 million / 100mL Mesenchymal stem cell suspension, in this embodiment, the density of the mesenchymal stem cell suspension is preferably 1 million / 100ml.

[0042] 2. Large-scale culture of mesenchymal stem cells

[0043] Inoculate the mesenchymal stem cell suspension obtained in step 1 into a microcarrier base solution made of sterile biological microcarriers and cell culture medium at a ratio of 3g: 100mL, mix evenly, transfer to a bioreactor, and let stand for 2h. Stirring at 60rpm / min, and at 37°C, 5% CO 2 , 20%O 2 Under the condition of cultured for 7 days, the base fluid of mesenchymal stem cells was obtained; in ...

Embodiment 2

[0049] 1. Pre-cultivation of mesenchymal stem cells

[0050] Take adipose-derived mesenchymal stem cells at 5000 cells / cm 2Inoculated at a density of 10% fetal bovine serum (FBS) and modified Eagle medium (DMEM) supplemented with 1% antibiotics, cultured in a single layer for 2 generations, and obtained mesenchymal cells with a cell density of 100,000 / 100mL-10 million / 100mL Mesenchymal stem cell suspension, in this embodiment, the density of the mesenchymal stem cell suspension is preferably 1 million / 100ml.

[0051] 2. Large-scale culture of mesenchymal stem cells

[0052] Inoculate the mesenchymal stem cell suspension obtained in step 1 into a microcarrier base solution made of sterile biological microcarriers and cell culture medium at a ratio of 3g: 100mL, mix evenly, transfer to a bioreactor, and let stand for 2h. Stirring at a speed of 60 rpm / min, and culturing at 37° C., 5% CO2, 10% O2 for 10 days to obtain the base fluid of mesenchymal stem cells; in this embodiment,...

Embodiment 3

[0058] 1. Pre-cultivation of mesenchymal stem cells

[0059] Take adipose-derived mesenchymal stem cells at 5000 cells / cm 2 Inoculated at a density of 10% fetal bovine serum (FBS) and modified Eagle medium (DMEM) supplemented with 1% antibiotics, cultured in a single layer for 2 generations, and obtained mesenchymal cells with a cell density of 100,000 / 100mL-10 million / 100mL Mesenchymal stem cell suspension, in this embodiment, the density of the mesenchymal stem cell suspension is preferably 1 million / 100ml.

[0060] 2. Large-scale culture of mesenchymal stem cells

[0061] Inoculate the mesenchymal stem cell suspension obtained in step 1 into a microcarrier base solution made of sterile biological microcarriers and cell culture medium at a ratio of 3g: 100mL, mix evenly, transfer to a bioreactor, and let stand for 2h. Stirring at 60rpm / min, and at 37°C, 5% CO 2 , 5%O 2 Under the condition of culturing for 14 days, the base fluid of mesenchymal stem cells was obtained; in...

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Abstract

The invention provides a preparation method of sub-totipotent mesenchymal stem cell secretin. The preparation method comprises steps including preculture of mesenchymal stem cells, large-scale cultureof the mesenchymal stem cells, preparation of a mesenchymal stem cell secretin stock solution and preparation of mesenchymal stem cell secretin lyophilized powder. The preparation method of the sub-totipotent mesenchymal stem cell secretin is simple and easy to implement, rapid, convenient and favorable for large-scale continuous production. According to the method, rapid proliferation of the mesenchymal stem cells can be realized without adding biological factors, the mesenchymal stem cell secretin can be efficiently and continuously produced only through a cell culture solution replacementmanner, and the production cost is greatly reduced; and the mesenchymal stem cell secretin has higher content of active ingredients and has remarkably improved skin cell and bone cell proliferation capability.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing secretin from subtotipotent mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cell, MSC) are stem cells with strong proliferation ability and multi-lineage differentiation potential. They have all the common characteristics of stem cells, namely, self-renewal and multi-lineage differentiation capabilities. The combined application of mesenchymal stem cells and hematopoietic stem cells can improve the success rate of transplantation and accelerate hematopoietic reconstruction. After the patient receives high-dose chemotherapy, the infusion of mesenchymal stem cells and hematopoietic stem cells can significantly accelerate the recovery time of the patient's blood cells, and it is safe and has no adverse reactions. [0003] Mesenchymal stem cells exist not only in bone marrow, but also in skeletal muscle, periosteum an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/645C07K1/14C07K1/34C12N5/0775C12N5/073
CPCC07K14/645C12N5/0605C12N5/0663C12N5/0665C12N5/0667C12N5/0668C12N2531/00
Inventor 王彬林思恩李刚
Owner 深圳刚华健医疗有限公司
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