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Method for separating, amplifying, cryopreserving and reviving placenta sub-totipotent stem cell

A subpluripotent stem cell and stem cell technology, applied in cell dissociation methods, embryonic cells, animal cells, etc., can solve the problems of decreased cell viability, weak proliferation ability, and cells are susceptible to bacterial infection, and can improve the activity rate, improve quality, The effect of improving purity

Active Publication Date: 2018-07-03
TANGYI HLDG(SHENZHEN) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The placenta of a full-term fetus is disc-shaped, with a diameter of about 15-20cm, a thickness of about 2.5-3.0cm, and a weight of about 450-500g. The placental stem cells discovered by science include: placental mesenchymal stem cells, placental subtotipotent stem cells, placental hematopoiesis Stem cells, sub-totipotent stem cells refer to a type of stem cell subgroup with the potential to differentiate into three germ layers. Under certain circumstances, they can theoretically be induced to differentiate into any tissue cells in the human body, mainly derived from mesenchymal cells such as bone marrow, fat, umbilical cord and placenta. Due to the strong differentiation ability, low immunogenicity and immunoregulatory function of subtotipotent stem cells derived from umbilical cord and placenta, its clinical application value has attracted more and more attention. Placental tissue contains a variety of subtotipotent stem cells Stem cells have many collection sites, such as amniotic membrane, decidua, placental lobules, etc. There are certain differences in the growth of subtotipotent stem cells from different parts, and when different parts are mixed together to isolate placental subtotipotent stem cells, the isolated The differences in the morphology and survival rate of the cells are relatively large. Studies have found that the fetal face chorion is an enriched area for placental subtotipotent stem cells, but the fetal face chorion also contains a large number of mesenchymal stem cells. If the cells are not removed , will affect the adherent growth of subtotipotent stem cells in the subsequent culture process, placental subtotipotent stem cells also have problems such as easy aging of cells after cryopreservation and recovery
[0003] Chinese invention patent CN200810240040.8 discloses a subtotipotent stem cell, its preparation method and its use. The placental subtotipotent stem cell prepared by this method is derived from a cut human umbilical cord or placenta, which grows adherently in a culture container and can In vivo, mesodermal, and ectodermal tissue differentiation, the placental subtotipotent stem cells provided by this method need to be expanded to more than four generations to obtain enough cells for the establishment of subtotipotent stem cell seed banks. The proliferation ability is weak and the proliferation speed is slow. As the number of cell passages increases, the viability of the cells decreases, and after the obtained cells are frozen and re-cultivated after treatment, the cells are prone to bacterial infection

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  • Method for separating, amplifying, cryopreserving and reviving placenta sub-totipotent stem cell
  • Method for separating, amplifying, cryopreserving and reviving placenta sub-totipotent stem cell
  • Method for separating, amplifying, cryopreserving and reviving placenta sub-totipotent stem cell

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Effect test

Embodiment 1

[0034] A method for isolating, expanding, freezing, and resuscitating stem cells from placental sub-pluripotent stem cells, which is characterized in that the method comprises the following steps:

[0035] 1) Collect samples: Take healthy placenta, remove amniotic membrane and congestion, sterilize the placenta with normal saline, reduce fetal chorionic villi in a glass dish, and cut to 0.5mm 3 Place the small pieces in the collection bottle, add 200IU / mL streptomycin sulfate to the collection bottle, and separate within 24h;

[0036] 2) Isolation of placental sub-pluripotent stem cells: perform cell separation on the sample collected in step 1) to obtain placental sub-pluripotent stem cells;

[0037] 3) Primary culture of placental sub-pluripotent stem cells: primary culture of the placental sub-pluripotent stem cells obtained in step 2) to obtain primary placental sub-pluripotent stem cells;

[0038] 4) Passage expansion and culture of placental sub-pluripotent stem cells: add the pr...

Embodiment 2

[0043] A method for isolating, expanding, freezing, and resuscitating stem cells from placental sub-pluripotent stem cells, which is characterized in that the method comprises the following steps:

[0044] 1) Collect samples: take a healthy placenta, remove amniotic membrane and congestion, disinfect the placenta with normal saline, reduce fetal chorionic villi in a glass dish, and cut to 0.5mm 3 Place the small pieces in the collection bottle, add 300IU / mL streptomycin sulfate to the collection bottle, and separate within 24h;

[0045] 2) Isolation of placental sub-pluripotent stem cells: perform cell separation on the sample collected in step 1) to obtain placental sub-pluripotent stem cells;

[0046] 3) Primary culture of placental sub-pluripotent stem cells: primary culture of the placental sub-pluripotent stem cells obtained in step 2) to obtain primary placental sub-pluripotent stem cells;

[0047] 4) Passage expansion culture of placental sub-pluripotent stem cells: add the prim...

Embodiment 3

[0055] A method for isolating, expanding, freezing, and resuscitating stem cells from placental subtotipotent stem cells, which is characterized in that the method comprises the following steps:

[0056] 1) Collect samples: take healthy placenta, remove amniotic membrane and congestion, disinfect the placenta with normal saline, reduce fetal chorionic membrane in a glass dish, and cut it to 0.5mm 3 Place the small pieces in the collection bottle, add 400IU / mL streptomycin sulfate to the collection bottle, and separate within 24h;

[0057] 2) Isolation of placental sub-pluripotent stem cells: the samples collected in step 1) are subjected to cell separation to obtain placental sub-pluripotent stem cells;

[0058] 3) Primary culture of placental sub-pluripotent stem cells: primary culture of the placental sub-pluripotent stem cells obtained in step 2) to obtain primary placental sub-pluripotent stem cells;

[0059] 4) Passage expansion and culture of placental sub-pluripotent stem cells:...

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Abstract

The invention relates to a method for separating, amplifying, cryopreserving and reviving a placenta sub-totipotent stem cell. The method comprises the following steps: (1) collecting a sample; (2) separating the placenta sub-totipotent stem cell; (3) performing primary culture on the placenta sub-totipotent stem cell; (4) performing secondary multiplication culture on the placenta sub-totipotentstem cell; (5) cryopreserving; and (6) reviving. The placenta sub-totipotent stem cell separated by adopting the method has high purity, the primary cultured cell has high survival rate, the cryopreserved cell has low pollution rate, and the amplified cell quality of the revived placenta sub-totipotent stem cell can be remarkably improved.

Description

Technical field [0001] The present invention relates to the field of biomedicine, in particular to a method for separating, expanding, freezing and resuscitating stem cells from placental subtotipotent stem cells. Background technique [0002] The placenta of a full-term fetus is disc-shaped, with a diameter of about 15-20cm, a thickness of about 2.5-3.0cm, and a weight of about 450-500g. The placental stem cells found in current science contain: placental mesenchymal stem cells, placental subtotipotent stem cells, and placental hematopoietic cells Stem cells, sub-pluripotent stem cells refer to a type of stem cell subgroup with the potential for differentiation of the three germ layers. Under certain circumstances, they can theoretically be induced to differentiate into any tissue cell of the human body, mainly derived from bone marrow, fat, umbilical cord and placenta. Because of the strong differentiation ability, low immunogenicity and immunomodulatory function of subtotal st...

Claims

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Application Information

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IPC IPC(8): C12N5/071A01N1/02
CPCA01N1/0284C12N5/0605C12N2501/91C12N2509/10
Inventor 曹毓琳刘俊江时兆田王沾白志惠
Owner TANGYI HLDG(SHENZHEN) LTD
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