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Stem cell culture medium and application thereof and stem cell cultivation method

A stem cell and culture medium technology, applied in the field of cell culture, can solve the problems of reduction, slow growth of stem cells, and increase in the number of cell potential passages, and achieve the effect of extending the number of passages and maintaining the potential.

Active Publication Date: 2015-01-21
苏州博棠再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a stem cell culture medium and its application and stem cell culture method, aiming to solve the problem of slow growth and cell potentiality in the culture of stem cells in the existing culture medium supplemented with bovine serum. The problem of greatly reducing the number of times

Method used

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  • Stem cell culture medium and application thereof and stem cell cultivation method
  • Stem cell culture medium and application thereof and stem cell cultivation method
  • Stem cell culture medium and application thereof and stem cell cultivation method

Examples

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Embodiment 1

[0058] Example 1. Acceleration of stem cell proliferation

[0059] The conventional medium formula for culturing stem cells is: DMEM / F12 basic medium, 10% fetal bovine serum (FBS). In order to compare the culture medium described in the present invention and to examine the influence of the culture medium described in the present invention on the expansion speed of human stem cell culture, human stem cells from three kinds of tissue and organ sources: fat, bone marrow and umbilical cord were selected in this example. (ADSC, BMSC, UMBSC) for in vitro cell culture experiments. The cell inoculation density is controlled between 10-20%. Before inoculation, the culture dish is treated with cell adhesion material at room temperature for 2 hours or incubated overnight at 4°C. After inoculation, culture medium is added (37°C, 5% CO 2 ). Cell density was measured every 12 hours by MTT assay. The test results are shown in Figure 1~ image 3 As shown, the stem cells (solid column) cul...

Embodiment 2

[0060] Example 2. Potential Analysis of Proliferating Mesenchymal Stem Cells Divided into Chondrogenic Cells in Vitro

[0061] In order to examine the influence of stem cells on their differentiation potential after being amplified by the medium described in the present invention for 10 generations, human stem cells derived from fat, bone marrow and umbilical cord were measured in this example according to the method described in the literature ( Chondrogenic differentiation assay after ADSC, BMSC, UMBSC) expansion (Estes BT et al. Nat Protoc2010; 5:1294–1311; Awad HA et al., Biomaterials.2004Jul; 25(16):3211-22; Cheng NC et al., Tissue Eng Part A. 2009 Feb;15(2):231-41). When the stem cells grow to a suitable density (cell density 20-80%), add chondrogenic induction medium (DMEM / F12 medium with 1% FBS, TGF-β1, 50nM vitamin C, 6.25mg / L insulin, 1% double antibody), the solution was changed once a day, and the total induction was 2-3 weeks. Alcian blue staining was performed...

Embodiment 3

[0062] Example 3. Potential analysis of adipogenic cells

[0063] In order to further examine the influence of stem cells on their differentiation potential after being amplified by the medium described in the present invention for 10 generations, in this embodiment, we followed the adipogenic induction and oil red O staining methods of adipose-derived stem cells (ADSC) described in the literature. The differentiation experiment of adipocytes after the expansion of human stem cells (ADSC, BMSC, UMBSC) derived from fat, bone marrow and umbilical cord three kinds of tissues and organs was determined according to the method described in the literature (Yu G et al., Methods in Molecular Biology, 2011, Volume702, Part3, 193-200; Bunnell BA et al., Methods. Volume45, Issue2, June2008, Pages115-120; Gimble JM&Guilak F. Cytotherapy.2003, Vol.5, No.5, Pages362-369) . The specific method is to replace the stem cell density with about 80% adipogenic medium for induction. The formulation...

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Abstract

Provided are a stem cell culture medium and use thereof and a stem cell culture method. The stem cell culture medium does not contain serum, and comprises amino acid, vitamin, salts, lipids, cytokine and polypeptide proteins. The stem cell culture medium is suitable for rapidly culturing stem cells derived from human and mammal tissues, including but not limited to adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and umbilical cord blood stem cells. Compared with an ordinary culture medium, the adoption of the stem cell culture medium can improve the amplification speed of cells by 3-5 times, and prolong the passage numbers, and can well maintain the potency of stem cells.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a stem cell culture medium, its application and a stem cell culture method. Background technique [0002] Many studies have shown that stem cells can not only self-renew, but also differentiate into other functional cells under suitable conditions. Therefore, stem cells are expected to become an effective means of treating difficult human diseases. However, the content of stem cells in normal adult tissues is very small. How to rapidly expand and cultivate stem cells in vitro is an important technology for studying the mechanism of stem cells and exploring their therapeutic methods in the treatment of human diseases. [0003] The content of stem cells is as small as possible, but they are widely distributed in various tissues and organs of mammals, including but not limited to bone marrow, umbilical cord, adipose tissue, brain tissue, retina, heart, liver, lung and skin. A large numb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/0775C12N5/0789
CPCC12N2500/33C12N5/0037C12N2501/115C12N2501/235C12N2501/165C12N2501/39C12N2501/105C12N2501/135C12N2501/91C12N2501/125C12N2501/113C12N2500/32C12N2501/11C12N2500/38C12N2500/36C12N5/0663C12N5/0665C12N5/0667
Inventor 刘小青应明陈光风郑龙坡
Owner 苏州博棠再生医学科技有限公司
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