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Sub-totipotent stem cell of human placenta and stem cell bank construction method thereof

A technology of subpluripotent stem cells and human placenta is applied in the field of subpluripotent stem cells to induce differentiation. The field of preparation of subpluripotent stem cells can solve the problem that cells are difficult to meet the requirements of storing cell resources, and mesenchymal cells contaminate epithelial cells and mesenchymal cells. problems such as low cell stemness, to achieve the effect of easy acquisition, low cost, and simple cell acquisition

Active Publication Date: 2014-08-06
CHONGQING CELL BIOENG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional cell digestion methods are difficult to distinguish between the two layers of tissue, so it will cause mesenchymal cells to contaminate epithelial cells, and mesenchymal cells have low stemness, which greatly reduces the stemness of digested cells
In addition, the mesenchymal cells obtained from the digestion of the mesenchymal layer grow faster and will compete with epithelial cells for growth during the culture process, thereby inhibiting the growth of epithelial cells (i.e., placental subtotipotent cells)
Therefore, the cells obtained by conventional digestion methods are difficult to meet the requirements for storing cell resources

Method used

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  • Sub-totipotent stem cell of human placenta and stem cell bank construction method thereof
  • Sub-totipotent stem cell of human placenta and stem cell bank construction method thereof
  • Sub-totipotent stem cell of human placenta and stem cell bank construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Collection of placenta

[0061] ABO / Rh blood type testing, HLA (Human Leukocyte Antigen, human leukocyte antigen) typing testing, microbial immune testing, physical examination HIV (Human Immunodeficiency Virus, human immunodeficiency virus), HBV (Hepatitis B virus for short, B Hepatitis virus), HCV (Hepatitis C virus, hepatitis C virus); and then collect the placenta of the parturient in the operating room.

[0062] Before the placenta is collected, the puerpera fills in the informed consent form and personal basic information, and then the puerpera is identified. The collection process should strictly prevent the contamination of pathogenic microorganisms, and it is required to be carried out in a sterile place, and then the collected placenta is transported to the designated place. The temperature of the transportation environment should be within the range of 4-8oC; when the placenta is received, the receiving form should be filled out, and the placenta bo...

Embodiment 2

[0063] Example 2 Acquisition and isolation of human placental subtotipotent stem cells

[0064] The amniotic membrane was stripped from the placenta, washed repeatedly with D-Hank’s balanced salt solution with a pH value of 7.2-7.4 to remove residual blood, and the amniotic membrane was cut into pieces of 5×5 cm 2 small pieces. One of the amniotic membranes was taken to make a paraffin section and subjected to immunofluorescent staining (SSEA-4, see figure 1 ). figure 1 The results showed that SSEA-4 positive cells were mainly distributed in the outermost layer of the amniotic membrane, that is, the amniotic epithelial layer.

[0065] The amniotic membrane fragments were evenly divided into two groups, A and B, wherein group A was separated by the method described in the present invention, and group B was digested and separated by conventional methods (without separating the epithelial layer from the mesenchymal layer) as a comparison.

[0066] Drop 1mL of D-Hank's balanced...

Embodiment 3

[0076] Example 3 Culture of placental subtotipotent stem cells

[0077] Divide the obtained stem cells into 2×10 5 / cm 2 Inoculate in a culture bottle, add 10mL stem cell culture medium, place at 37oC, CO 2 Cultivate in an incubator with a content of 5%, replace the new stem cell culture medium after 4-5 days, discard the non-adherent cells, and replace the stem cell culture medium every 3-4 days thereafter, until the cells grow to 80% confluence, in the culture Add protease II solution to the bottle for digestion; put the culture bottle in a 37°C incubator for 5 minutes, observe the cells under an inverted microscope, and gently pat the culture bottle with the palm of your hand to ensure that the cells and tissue pieces are completely digested. Add 10mL of stem cell culture medium to the medium, blow and beat repeatedly to clean the cells in the culture flask, transfer the washed cell suspension into a 50mL centrifuge tube, and centrifuge at 1500 rpm for 5 minutes. After th...

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Abstract

The invention provides a sub-totipotent stem cell of a human placenta. The sub-totipotent stem cell of the human placenta is derived from stripped human placenta amnion, aiming at the characteristics of cells contained by an epithelial layer and a mesenchyme layer of the amnion, a step-by-step separation method is adopted, pollution of the sub-totipotent stem cell of the placenta by the mesenchyme layer of the amnion is reduced to the greatest extent, and the stemness of the sub-totipotent stem cell of the human placenta is improved effectively. The method has the characteristics of low cost, broad application prospect and capacity of supplying a large quantity of stem cell resources to clinic and research. The invention further provides a method for preparing the sub-totipotent stem cell of the human placenta from the human placenta amnion and constructing a stem cell bank.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a subtotipotent stem cell derived from amniotic membrane of human placenta. At the same time, the present invention also relates to a method for preparing the subtotipotent stem cells and a method for inducing differentiation. Background technique [0002] Stem cell research is a research hotspot that has emerged in recent years. Its rapid research progress and broad application prospects have attracted more and more attention to stem cell research. Stem cells are a type of cells with self-renewal and differentiation potential, which can be divided into embryonic stem cells and adult stem cells according to their source. Among them, adult stem cells are considered as potential seed cells for cell therapy because they are not ethically controversial and easy to obtain. [0003] Studies in recent years have shown that in adult placenta tissue, there is a kind of sub-pluripotent stem cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12Q1/06C12Q1/04A01N1/02
Inventor 朱德琳赵雅宁徐萌徐志国聂艳波
Owner CHONGQING CELL BIOENG TECH CO LTD
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