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Preparation method of subtotipotent stem cells

A subpluripotent stem cell and cell technology, applied in the field of stem cell engineering, can solve problems such as complex methods, achieve the effect of good differentiation ability and avoid pollution

Active Publication Date: 2017-12-29
钜威细胞(厦门)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

The method is complex and uses reagents such as bovine serum

Method used

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  • Preparation method of subtotipotent stem cells
  • Preparation method of subtotipotent stem cells
  • Preparation method of subtotipotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Tissue acquisition: Place the umbilical cord of the newborn under sterile conditions and separate it within 12 hours. The placenta is taken from a full-term healthy pregnant woman. The pregnant woman’s HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-Treponema pallidum antibody, ALT, and mycoplasma All other test items were negative. After the placenta was removed from the operating table, it was immersed in 0.9% physiological saline containing antibiotics and kept at 4°C for storage;

[0035] Disinfection treatment: After ligation at both ends of the umbilical cord, soak in 75% alcohol for 1-2 minutes, wash with 0.9% normal saline for 2-3 times, and remove residual blood on the surface of the tissue; Wipe clockwise with 75% alcohol cotton and repeat; the environment may not be able to achieve complete aseptic conditions and still ensure the safety of sub-totipotent stem cell extraction. The amniotic surface, chorion, and decidua surface of the umbilical cord and ...

Embodiment 2

[0042] Example 2 prepares 0.1% collagenase, other steps are the same as Example 1.

Embodiment 3

[0043] Example 3 prepares 2% collagenase, other steps are the same as Example 1.

[0044] Result analysis

[0045] Subtotipotent stem cell surface markers

[0046] Culture the collected stem cells for 2 to 3 generations, digest and centrifuge, wash the cell pellet twice with DPBS, and take about 0.5‐1X10 6 100ul of cell suspension was placed in a flow loading tube, 15ul of primary antibody was added, after incubation at 4°C for 30min, 400ul of DPBS was added, centrifuged at 1500rpm for 6min, the supernatant was discarded, 10ul of secondary antibody was added, and after 30min of incubation at 4°C, added Centrifuge 400ul DPBS at 1500rpm for 6min, discard the supernatant, add 500ul DPBS, mix well, and test on the machine. Positive indicators: Flk1, CD73, CD90, CD105, negative indicators: CD34, HLA-DR, positive rate ≥ 95%, negative rate ≤ 2%. It shows that what we have isolated are umbilical cord and placental subtotipotent stem cells rather than other cells, see Table 2 and Fi...

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Abstract

The invention discloses a method for preparing subtotipotent stem cells, which comprises the following steps: obtaining tissues, disinfecting, separating, washing and shredding tissues, digesting with collagenase, and obtaining subtotipotent stem cells. The invention discloses a method for isolating subtotipotent stem cells from umbilical cord, placental amniotic membrane, chorion or basal decidua tissue, and the obtained subtotipotent stem cells are uniform, stable and have a short growth cycle.

Description

technical field [0001] The invention relates to the technical field of stem cell engineering, in particular to a method for preparing subtotipotent stem cells. Background technique [0002] Stem cells are a kind of primitive cells with self-renewal ability and multi-lineage differentiation potential under suitable microenvironment. Stem cells can be divided into embryonic stem cells, neonatal stem cells, and adult stem cells according to the order in which they appear during individual development, and can be divided into totipotent stem cells, subtotipotent stem cells, pluripotent stem cells, and unipotent stem cells according to their differentiation potential. At present, stem cells have been used to treat myocardial infarction, lupus erythematosus, rheumatoid arthritis, nerve damage and muscle atrophy. [0003] Subtotipotent stem cells refer to a class of stem cell subsets with the potential to differentiate into three germ layers. Under specific circumstances, they can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 王军霞黄福来
Owner 钜威细胞(厦门)医学科技有限公司
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