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Separation method of human placenta sub-totipotent stem cells

A sub-totipotent stem cell and separation method technology, applied in the direction of cell dissociation methods, embryonic cells, animal cells, etc., can solve the problems of unclean collection, affecting the effect of enzymatic hydrolysis, and many miscellaneous cells, so as to reduce the separation time and improve the separation Efficiency, the effect of ensuring the effect of enzymatic hydrolysis

Inactive Publication Date: 2019-08-27
内蒙古银宏干细胞生命科技投资有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] In the existing method for separating placental subtotipotent stem cells, the amniotic membrane is partially lifted with tweezers, and then cut off with scissors, all of which are irregular fragments, and the amniotic membrane is extremely thin and easy to peel off. On the one hand, it is impossible to ensure that the amniotic membrane is completely removed. Result in a lower yield of human placental subtotipotent stem cells;
[0010] On the other hand, in the existing method for isolating placental sub-totipotent stem cells, the fragments obtained by peeling off the amniotic membrane with tweezers are irregular, and it is not convenient to use a cell scraper to scrape off coagulation and jelly, which affects the effect of enzymatic hydrolysis. There are many miscellaneous cells in totipotent stem cells. In order to obtain human placental subtotipotent stem cells with higher purity, it is necessary to add Ficoll lymphocyte separation medium when resuspending. Smaller miscellaneous cells (such as monocytes) float on the liquid surface of the layered liquid, and the human placental subtotipotent stem cells are sandwiched between the two layers of liquid. Since the amount of human placental subtotipotent stem cells obtained by the traditional method is small, take out the operation Time-consuming and labor-intensive; and when human placental subtotipotent stem cells are taken out from between the two layers of liquid, it is easy to get dirty or take out together with other miscellaneous cells, which will affect the yield and purity of cells

Method used

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  • Separation method of human placenta sub-totipotent stem cells
  • Separation method of human placenta sub-totipotent stem cells
  • Separation method of human placenta sub-totipotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The junction of the umbilical cord and the placenta is used as a dot, and the line connecting the apex and the edge of the placenta is used as the axis circle, and the amniotic membrane is removed to obtain a circular amniotic membrane sample with a width of 3-5cm.

[0051] Rinse the removed circular placental amniotic membrane three times with phosphate buffer solution with a pH value of 7.3, then put it into a kidney-shaped dish, and use tweezers and a cell scraper to clean the clots and gelatinous substances on the membrane to obtain amniotic membrane Sample, to prevent affecting the digestion efficiency of the enzyme; the clot on the placenta is a black-red block that adheres to the membrane, and the jelly is a transparent viscous substance that is attached to the surface of the amniotic membrane. It is used in a kidney-shaped disc. Use tweezers to remove large pieces of coagulated blood, and then use a cell scraper to gently and slowly scrape off the remaining coagu...

Embodiment 2

[0058] The amniotic membrane was randomly stripped from the placenta, washed repeatedly with D-Hank’s balanced salt solution with pH=7.3 to remove residual blood, and the amniotic membrane was cut into 5×5cm pieces 2 Small piece;

[0059] Drop 1mL of D-Hank's balanced salt solution into a 6-cm petri dish, wet the bottom of the petri dish, place the epithelial layer of the amniotic membrane down in the petri dish, use slides to separate the gelatinous substance of the mesenchymal layer scrape off;

[0060] Put the scraped amniotic membrane into 10 mL of D-Hank's balanced salt solution containing 10 (w / v)% N-acetyl-L-cysteine, and incubate at room temperature for 15 minutes; remove the residual mucus on the amniotic membrane for easy digestion completely;

[0061] Add 20mL of protease solution to the amnion, digest it in an air bath at 37°C for 30min, collect the digestion solution and add 5mL of fetal bovine serum; wherein the protease solution is 0.15 (w / v)% collagenase II-E...

Embodiment 3

[0069] The cell suspensions of the three control groups were all grown in vitro, and the cell growth was observed with a microscope after three days and five days. The growth of the first control group was as follows: Figure 1-Figure 4 shown;

[0070] Because the shape of placental subtotipotent stem cells is round, Figure 1-3 The shape of the cells is basically round, and the Figure 4 There are spindle cells in it, it can be seen that the cells separated by the patent method are more pure than the human placental subtotipotent stem cells separated by the traditional method.

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Abstract

The invention discloses a separation method of human placenta sub-totipotent stem cells. The separation method comprises the following steps: S1, preparing an amniotic membrane sample; S2, preparing an amniotic membrane specimen; S3, performing enzymolysis; a scheme is short in technological process and simple in workstage, a partition amniotic membrane stripping way is adopted in the step of preparing the amniotic membrane specimen, and the amniotic membrane is not easy to break; on the one hand, the regular amniotic membrane is convenient for subsequent flushing, blood coagulation and jellyremoval, and primary enzymolysis and secondary enzymolysis work; on the other hand, the partition amniotic membrane stripping way can ensure that the amniotic membrane is totally taken out, and ensures the yield of the human placenta sub-totipotent stem cells. According to the method, the amniotic membrane which is stripped in partition is regular and tidy and is convenient to clean, the blood coagulation and jelly on the amniotic membrane are cleaned out, and the enzymolysis effects are ensured; the cleaned amniotic membrane specimen is subjected to the enzymolysis twice, parenchyma cells onthe amniotic membrane are removed in the primary enzymolysis, so that a Ficoll lymphocyte separating medium is not needed for separation, and the human placenta sub-totipotent stem cells with relatively high purity can also be obtained.

Description

[0001] Technical field: [0002] The invention relates to a method for separating human placental subtotipotent stem cells, belonging to the technical field of biomedicine. [0003] Background technique: [0004] The placenta is a temporary organ, which is the direct connection hub between the mother and the fetus. It is very important to the development and growth of the fetus throughout the gestation period. It has important physiological functions and can carry out nutrients, gases and metabolism between the fetus and the mother. The exchange of waste; not only that, the placenta is also involved in the immune protection of the fetus during pregnancy, so the placenta is a multifunctional organ. [0005] Studies have shown that after the baby is born, the placenta still retains rich life resources, and a large number of placental sub-totipotent stem cells can be isolated. Their developmental stage is close to that of embryonic stem cells, and they have the biological characte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0605C12N2509/00
Inventor 宋丽霞胡燕张潇李彬刘伟
Owner 内蒙古银宏干细胞生命科技投资有限公司
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