Placental villus plate mesenchymal stem cells and extraction method thereof

A technique for stem cells and an extraction method, applied in the field of placental villus plate mesenchymal stem cells and their extraction, can solve the problems of requiring a large amount of cleaning solution and cleaning process, increasing the difficulty of the operation process, etc., and achieve the effects of high yield and high purity

Inactive Publication Date: 2016-11-09
四川华皓生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The placental tissue obtained by this method includes villi plate, villi layer, and maternal blood cell components, and the obtained chorionic mesenchymal stem cells are actually mixed components of villi plate mesenchymal stem cells, villous mesenchymal stem cells, and maternal blood cells, so It cannot be simply considered as chorionic mesenchymal stem cells, and the introduction of chorionic placenta tissue requires a large amount of cleaning solution and cleaning process for red blood cells during tissue separation, which increases the difficulty of the operation process

Method used

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  • Placental villus plate mesenchymal stem cells and extraction method thereof
  • Placental villus plate mesenchymal stem cells and extraction method thereof
  • Placental villus plate mesenchymal stem cells and extraction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Isolation of Placental Villi Plate Mesenchymal Stem Cells

[0038] (1) Sign an informed consent form with the donor, collect the full-term placenta, and process it as follows within 72 hours: Use sterile hemostatic forceps to peel off the amniotic membrane covering the outside of the villi plate; use sterile surgical scissors to cut a sufficient amount of the placenta villi plate organize;

[0039] (2) Use sterile tissue forceps to remove blood vessels and villi attached to the placenta villi;

[0040] (3) Wash the placental villi plate obtained in step (2) with sterile physiological saline until there is no blood color;

[0041] (4) Use sterile surgical scissors to cut the cleaned villi plate tissue into 2mm 2 The tissue block was washed 2-5 times with sterile saline, avoiding the use of antibiotics during the cleaning process;

[0042] (5) Collect the tissue blocks in step (4), divide them into 6 parts, 2g each, add 5 parts of them to the following tissue...

Embodiment 2

[0052] Example 2 Subculture and cryopreservation of placental villi plate mesenchymal stem cells

[0053] After the isolated placental villi plate mesenchymal stem cells were cultured until the cell confluence reached 70%-90%, the cells were digested with 0.25% (v / v) trypsin (Hyclone) containing EDTA, and centrifuged after digestion. Centrifuge at 2000r / min for 3min, collect the cells, detect the cell viability, and count the cells according to 1×10 4 cells / cm 2 Density inoculation in T175 culture flask, then add 20mL complete medium for subculture, and then subculture the cells at a density of 2×10 6 The cells / mL were resuspended in the cryopreservation solution, which consisted of complete medium containing 10% DMSO, and the cryopreservation volume was 1mL, and then placed in a liquid nitrogen tank for long-term storage after being cooled to -80°C by a program.

Embodiment 3

[0054] Example 3 Identification of Biological Characteristics of Placental Villi Plate Mesenchymal Stem Cells

[0055] 1. Identification of multi-lineage differentiation potential

[0056] The mesenchymal stem cells from P3 generation placental villi plate were inoculated in a six-well plate, and each well was inoculated with 1×10 5 Add 2 mL of complete medium to each well, set up 3 replicates for each sample, replace the medium every two days, and add osteogenic, adipogenic, and chondrogenic induction medium (Cyagen ) each 2mL, with the addition of complete medium as a blank control, the induction medium was replaced every 2-3 days, after continuous culture for 2-3 weeks, the induction medium was removed and washed once with PBS, and 2mL of paraformaldehyde solution (4% , v / v) fixed for 30min, then removed the paraformaldehyde solution and washed twice with PBS, stained with staining solution, the staining time for osteogenic induction and chondrogenic induction was 2-3min; ...

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Abstract

The invention discloses placental villus plate mesenchymal stem cells. An extraction method of the placental villus plate mesenchymal stem cells includes the following steps that 1, a placental villus plate is obtained; 2, blood vessels and villus tissue are removed; 3, the villus plate is cleaned till the blood color disappears; 4, the villus plate is sheared into 0.5-5 mm<2> tissue microblocks, and the tissue microblocks are cleaned; 5, tissue digestive juice containing pancreatin, collagenase II, collagenase IV and hyaluronidase is added for digestion; 6, filtration and centrifugation are carried out; 7, culture is carried out; 8, cell digestion is carried out with 0.25% pancreatin when the cell confluence reaches 70-90%; 9, subculturing is carried out; 10, detection is carried out; 11, cryopreservation is carried out; 12, database building is carried out. According to the method, the steps are simple, and a large number of mesenchymal stem cells can be rapidly obtained from the placental tissue; the separation process does not need too much cleaning operation; the use of antibiotics is omitted, so that the purity of the placental villus plate mesenchymal stem cells obtained after separation is high.

Description

technical field [0001] The invention relates to the technical field of preparation of mesenchymal stem cells, in particular to placental villi plate mesenchymal stem cells and an extraction method thereof. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells with multi-directional differentiation potential, which can differentiate into cells of various tissue origins under specific induction conditions, such as adipocytes, osteoblasts, and chondrocytes. and neuroblasts, etc. Mesenchymal stem cells not only have multidirectional differentiation potential, but also have various biological characteristics such as self-renewal, homing, immune regulation, hematopoietic support, secretion of various cytokines, and promotion of stem cell transplantation. They are involved in tissue damage repair and tissue regeneration. important cellular components. Currently, mesenchymal stem cells can be extracted from various adult tissues (such as bone m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/00
Inventor 黄海森
Owner 四川华皓生物科技有限公司
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