Preparation method of placental-chorionic-plate-tissue-derived mesenchymal stem cells
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A technology for stem cells and placental tissue, applied in the field of cell biology, can solve problems such as difficulty in ensuring cell separation efficiency, cell viability, cell inconvenience, and impact on cell use effects.
Pending Publication Date: 2020-01-14
GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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[0006] However, the placental chorionic tissue is located at the junction of the umbilical cord and the placenta, and is a thin and transparent tissue. In the process of tissue separation, it is easy to be mixed with epithelial tissue components, and then a large number of epithelial cells are mixed in the cell culture process. Affects the purity of chorionic MSCs
It will cause inconvenience to the later use of cells and even affect the use effect of cells
In addition, the separation, preparation and cryopreservation methods of placental chorionic MSCs are not fixed at present, and it is difficult to guarantee the separation efficiency, cell survival rate and cell activity after recovery of different batches of cells.
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Embodiment 1
[0036] A method for preparing mesenchymal stem cells derived from placental chorionic plate tissue, comprising the following steps:
[0037] 1. Organization collection
[0038] The full-term fetal placental tissue is collected in the hospital, and the customer's informed consent is signed. At the same time, the placental blood sample is tested for virus factors and microorganisms, and the tissue that meets the testing standards will proceed to the next step.
[0039] Placental tissue that passed the test was placed in a special tissue collection bag, added with tissue protection solution, and transported to the laboratory at 4°C in a refrigerated environment.
[0040] The tissue protection solution added in it is to protect the chorionic tissue and maintain its biological activity during the delivery to the laboratory. Its main components are: normal saline + 25 μg / ml gentamicin sulfate and 5 μg / ml amphoteric Formulated with Mycin B to ensure no bacterial and fungal contamina...
Embodiment 2
[0062] A preparation method of mesenchymal stem cells derived from placental chorionic plate tissue, which is basically the same as the method in Example 1, the only difference being that the cryopreservation solution used in this example is: serum-free medium + non-essential amino acids (2%)+trehalose (0.2mol / L)+dimethyl sulfoxide (10%)+human albumin (5%).
[0063] In this example, the preparation method of the cells is roughly the same as in Example 1, except that the components of the cryopreservation solution are adjusted on the original basis, and no dextran-based sucrose is added in this example. Through the above operations, mesenchymal stem cells were isolated and prepared. However, for frozen cells, there are some aggregations of cell clumps.
Embodiment 3
[0065] A method for preparing mesenchymal stem cells derived from placental chorionic plate tissue is basically the same as the method in Example 1, the only difference being that the cryopreservation solution used in this example is: serum-free medium + dextran-based sucrose (5%) + non-essential amino acid concentration is 2% + trehalose (0.2mol / L) + dimethyl sulfoxide (10%).
[0066] In this example, the preparation method of the cells is roughly the same as in Example 1, and the components of the freezing solution are adjusted on the basis of the original experiment. In this example, human serum albumin is not added, and the cells after freezing are prone to cell death Phenomenon.
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Abstract
The invention relates to a preparation method of placental-chorionic-plate-tissue-derived mesenchymal stem cells, and belongs to the technical field of cell biology. The preparation method of the placental-chorionic-plate-tissue-derived mesenchymal stem cells comprises the following steps: carrying out tissue separation, namely to obtain a placenta tissue sample, separate out chorion tissue, and carry out washing with a tissue cleaning solution until the chorion tissue is semi-translucent; carrying out tissue cryopreservation, namely to put the washed chorion tissue into a cryopreservation solution, and carry out cooling and cryopreservation according to predetermined procedures; carrying out tissue treatment, namely to take out the chorion tissue after the cryopreservation, soak the chorion tissue with ethanol, remove impurities, and perform shearing so as to obtain tissue masses; and then, carrying out cell culture, namely to put the tissue masses into a culture flask, add a mesenchymal stem cell selection medium so as to obtain a mixture, put the mixture into a CO(2) incubator so as to carry out incubation until cell fusion degree reaches 80+/-10%, carry out cell passage, and continue amplification culture and/or cryopreservation of the passage cells. The preparation method of the placental-chorionic-plate-tissue-derived mesenchymal stem cells enables long-term storage of placental chorionic plate tissue and mesenchymal stem cells separated from the placental-chorionic plate tissue in liquid nitrogen; and moreover, the placental chorionic plate tissue and the mesenchymalstem cells still have maintained cell activity after reconstruction.
Description
technical field [0001] The invention relates to the technical field of cell biology, in particular to a method for preparing mesenchymal stem cells derived from placental chorionic plate tissue. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from mesoderm with high self-renewal and multi-lineage differentiation capabilities. They are widely found in various tissues throughout the body and can be cultured and expanded under in vitro conditions. Under specific conditions, differentiate into bone cells, chondrocytes, fat cells, muscle cells, etc. As a representative of pluripotent stem cells, MSC has the ability of "horizontal differentiation" or "cross-lineage differentiation", and can differentiate into various tissue cells under different induction conditions. MSCs have broad clinical application prospects, are the preferred seed cells for cell replacement therapy and tissue engineering, and are research hotspots in ...
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