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Extracting and multiplication culture method of urine-derived mesenchymal stem cells (USCs)

A technology of mesenchymal stem cells and culture methods, which is applied in the field of extraction and in vitro expansion and culture of urine-derived mesenchymal stem cells, can solve the problems of high cost, ethical obstacles to the application of mesenchymal stem cells, and inconvenient material collection, so as to achieve low cost and avoid Risk of disease transmission, no ethically controversial effects

Inactive Publication Date: 2019-04-05
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional mesenchymal stem cell extraction has always had the disadvantages of inconvenient, invasive, and high cost, and ethical issues are also one of the important reasons hindering the application of mesenchymal stem cells

Method used

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  • Extracting and multiplication culture method of urine-derived mesenchymal stem cells (USCs)
  • Extracting and multiplication culture method of urine-derived mesenchymal stem cells (USCs)
  • Extracting and multiplication culture method of urine-derived mesenchymal stem cells (USCs)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Isolation and Extraction of Urine-derived Mesenchymal Stem Cells from Clean Urine

[0034] Under sterile conditions, take 200ml of non-morning urine from volunteers' clean midsection, and immediately separate and extract. Aliquot the clean urine into 50ml centrifuge tubes, centrifuge at 400g for 10min at 4°C, and discard the supernatant. Add basal medium containing antibiotics, centrifuge at 300g for 5min at 4°C and discard the supernatant. Cells were resuspended in 1ml of complete medium, inoculated into 24-well plates, and cultured in an incubator at 37°C with 5% CO saturated humidity.

[0035] After culturing for 3 days, observe under a microscope, discard if bacterial contamination is observed, change the medium if there is no contamination, and continue culturing. After 5-7 days, a single cell can be seen to adhere to the wall, mark the position of a single cell in the 24-well plate, change the medium every 2 days, and observe the formation of single c...

Embodiment 2

[0036] Example 2: Differentiation of Urine-derived Mesenchymal Stem Cells into Osteocytes

[0037] (1) The third generation of urine-derived mesenchymal stem cells obtained in Example 1 was used for in vitro differentiation function identification

[0038] (2) Osteoblast induction and differentiation medium containing 50 μg / ml vitamin C, 10 mM β-glycerophosphate sodium, and 0.1 μM dexamethasone was added to urine-derived mesenchymal stem cells for induction culture, and the medium was changed twice a week.

[0039] (3) Alizarin red S staining was performed after 21 days of differentiation induction, and the formation of calcium nodules could be observed ( Figure 8 ), indicating that urine-derived mesenchymal stem cells have the ability to differentiate into osteoblasts.

Embodiment 3

[0040] Example 3: Differentiation of Urine-derived Mesenchymal Stem Cells into Adipocytes

[0041] (1) Take the third-generation culture of urine-derived mesenchymal stem cells obtained in Example 1

[0042] (2) Induce the adipogenic differentiation medium containing 0.5 μM dexamethasone, 50 μM 3-isobutyl-1-methylxanthine, 50 μM indomethacin, and 10 μg / ml insulin in urine-derived mesenchymal stem cells to cultivate.

[0043] (3) After induction of differentiation for 14 days, oil red O staining was performed. After oil red O staining, a large number of red oil droplet-like particles can be seen ( Figure 9), indicating that urine-derived mesenchymal stem cells have the ability to differentiate into adipocytes.

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Abstract

The invention discloses an extracting and in-vitro multiplication culture technical method of urine-derived mesenchymal stem cells (USCs). In-vitro multiplication is mainly conducted in the mode thatthe mesenchymal stem cells (MSCs) are separated from sterile urine, and a culture medium easy to prepare is adopted; the extracted USCs subjected to multiplication culture can be used for therapy of clinical immunology related diseases and damage repair of various tissue and organs; the extracting and in-vitro multiplication culture technical method has the advantages that the separating and extracting processes of the USCs are completely free of wounds, special instruments are not needed, the cost is low, and the extracting process is easy to accept by patients; the USCs have other advantagesof being capable of achieving autotransplantation, free of immune rejection response and ethical arguments, and capable of avoiding the pathophoresis risk; and the USCs show the good multiplication ability, and the number of the cells needed during cell therapy can be met.

Description

technical field [0001] The invention relates to a method for extracting and in vitro amplifying and culturing urine-derived mesenchymal stem cells, belonging to the fields of cell biology, regenerative medicine and tissue engineering. Background technique [0002] Mesenchymal stem cells (MSCs) are a kind of multipotent adult stem cells derived from mesoderm, which have low immunogenicity, multipotential differentiation potential and immunomodulatory effects. At present, mesenchymal stem cells are increasingly used in the treatment of clinically refractory immune diseases, providing new ideas and directions for the treatment of immune diseases. At the same time, MSC, as an ideal seed cell, combined with regenerative medicine and tissue engineering technology, provides a new means for tissue regeneration and repair. [0003] Mesenchymal stem cells were first isolated from bone marrow. With further research, researchers are now able to isolate and extract mesenchymal stem cel...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/077
CPCC12N5/0653C12N5/0654C12N5/0668C12N2500/12C12N2500/32C12N2500/38C12N2500/40C12N2506/1392C12N2501/33
Inventor 刘毅杨嫄罗玉斌赵毅
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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