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Placenta sub-totipotential stem cell active factor for beauty and skin care and preparation and application thereof

A sub-totipotent stem cell and active factor technology is applied in the field of placental sub-totipotent stem cell active factor and its preparation and application, which can solve the problems of unsatisfactory effect and large side effects, and achieve improvement of skin physiological state, simple ingredients, easy quantitative and qualitative Effect

Inactive Publication Date: 2018-01-05
奥尔文(深圳)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the beauty and skin care products on the market are fine chemical products, the effect of effectively improving the physiological state of the skin is not ideal, and the side effects of long-term use are large

Method used

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  • Placenta sub-totipotential stem cell active factor for beauty and skin care and preparation and application thereof
  • Placenta sub-totipotential stem cell active factor for beauty and skin care and preparation and application thereof
  • Placenta sub-totipotential stem cell active factor for beauty and skin care and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Isolation and culture of placental subtotipotent stem cells

[0055] Take the placenta and cord blood, mechanically separate the amniotic membrane tissue on the surface of the placenta, and rinse with normal saline to remove blood cells. The specific steps are as follows:

[0056] (1) Place the amnion in a petri dish containing 10 ml (1% penicillin and 1% streptomycin) PBS containing antibiotics;

[0057] (2) Hold the amniotic membrane with surgical tweezers for rinsing, repeat rinsing 3 times, and try to remove clean blood cells;

[0058] (3) Cut the amnion tissue into small pieces and transfer the amnion tissue to a 50mL centrifuge tube;

[0059] (4) Add 0.25% trypsin to the centrifuge tube, then place it in a shaker at 37°C and 250rpm / min for oscillating digestion for 15min; wherein, the volume ratio of amnion tissue to trypsin is: amnion tissue:trypsin=1: 2,

[0060] (5) After the shaking digestion is completed, remove the digestion supernatant, and re...

Embodiment 2

[0069] The P3 placental subtotipotent stem cells were taken, and the immunophenotype of the cells was detected by indirect immunofluorescence method; for the surface antigen of the placental subtotipotent stem cells, the cells were digested with trypsin, washed with washing solution (PBS containing 0.5% BSA), and added a Incubate at 4°C for 30 min and wash twice with washing solution. The primary antibody is mouse anti-human monoclonal antibody CD31, CD34, CD45, HLA-DR, Flk1. To detect intracellular antigens, cells were fixed with 2% paraformaldehyde at 4°C for 15 min before incubation with primary antibodies, and permeabilized with 0.1% saponin for 1 h at room temperature.

[0070] In addition, choose the same isotype non-related IgG antibody as the negative control. After washing with washing solution, add FITC-labeled goat anti-mouse secondary antibody and incubate at 4°C for 30 minutes, wash the cells three times, suspend them in 300ml of PBS, and place them on ice until ...

Embodiment 3

[0073] 1. Adipose-induced differentiation

[0074] Get the P3 placental subtotipotent stem cells obtained in Example 1, with 2x10 4 The density per well was inoculated in a 6-well plate. After 12 hours, the culture medium was replaced with adipose-inducing culture medium, and the medium was changed in half every 3 days. On the 21st day, oil red O staining was used for identification. The normally cultured P3 placental subtotipotent stem cells without adipogenic induction medium were used as negative control group. The induction medium comprises 10% FCS, 10 -6 M Dexamethasone, 100 μg / ml 1-methyl-3-isobutyl-xanthine and 50 μg / ml ascorbic acid.

[0075] The results of the experimental group and the comparison group are as follows: Figure 9-10 As shown, it can be seen that the placental subtotipotent stem cells of the present invention have the ability of adipogenic differentiation.

[0076] 2. Osteogenic differentiation

[0077] Get the P3 placental subtotipotent stem cells...

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Abstract

The invention relates to a preparation method of a placenta sub-totipotential stem cell active factor for beauty and skin care. The preparation method comprises the steps of separating and culturing placenta sub-totipotential stem cells and preparing the active factor. The preparation of the active factor comprises the steps of taking P3 generation placenta sub-totipotential stem cells, carrying out inoculation and digestion, carrying out ice ultrasonic cell breakage, and collecting supernatant; then, dropping in trichloroacetic acid, performing an ice bath, carrying out low-temperature centrifugation, and abandoning the supernatant; adding precooling acetone to wash sediment, conducting low-temperature centrifugation, abandoning the acetone, resuspending the sediment, and conducting filtering the product with a filter film of which the thickness is 0.1 micrometer, wherein the concentrations of the obtained SCF, a FGF, VEGF, EGF, and KGF are all larger than 20000 pg / ml. The preparationmethod of the placenta sub-totipotential stem cell active factor for beauty and skin care adopts a culture system free of heterologous animal serum, and reduces the risks of introducing heterologousanimal viruses; at the same time, there is no hormone at all in the culture system, so that possible skin allergic reaction caused by the hormones is avoided; the active factor is prepared by the adoption of the low-temperature precipitation method, so that the activity of the active factor is guaranteed.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a placental subtotipotent stem cell active factor for beauty and skin care and its preparation and application. Background technique [0002] Stem cells, known as pluripotent cells because they have cells that can differentiate into other tissue types, have become a research hotspot in the field of regenerative medicine. Studies in recent years have suggested that there is a primitive stem cell population in adult tissues that is different from hematopoietic stem cells, neural stem cells, and other pluripotent stem cells that can only differentiate into specific germ layers. This type of primitive stem cell population can differentiate into tissue cells of different germ layers. In addition, they are also different from embryonic stem cells, which gradually lose part of their differentiation potential during growth and development, and exhibit some special phenotypes or molecular mar...

Claims

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Application Information

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IPC IPC(8): C12N5/073C07K14/475C07K14/485C07K14/50C07K14/515A61K8/64A61K8/99A61Q19/00
Inventor 李陶张晓文
Owner 奥尔文(深圳)生物科技有限公司
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