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Human placenta mesenchymal stem cell, preparation method and application thereof

A technology for stem cells and human placenta, which is applied in the field of biomedicine, can solve the problems of lowering production cost and need to be further improved, and achieves the effects of easier control of process stability, good activity and shortened culture period.

Active Publication Date: 2019-08-02
山西省干细胞基因工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent CN102586184A discloses a method for establishing a placental mesenchymal dry cell bank, which uses tissue digestion enzyme solution (containing dispase, trypsin, deoxyribonuclease I, collagenase IV, hyaluronidase) to digest placental lobular tissue, Obtain placental mesenchymal stem cells by clonal picking and expansion. The above method needs to be further improved in terms of process simplification and production cost reduction

Method used

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  • Human placenta mesenchymal stem cell, preparation method and application thereof
  • Human placenta mesenchymal stem cell, preparation method and application thereof
  • Human placenta mesenchymal stem cell, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Collection, transport and receipt of human placenta

[0056] With the informed consent of the puerpera, the placenta of healthy fetuses delivered by cesarean section at term was collected. Before the placenta is collected, the puerpera needs to be tested for HIV, hepatitis B virus, hepatitis C virus, syphilis and other infectious infectious diseases, and all of them are qualified to ensure safety before collection.

[0057] Wash the collected placenta with normal saline to remove blood and meconium around the placenta to prevent contamination, and then put it into a sterile bag containing 40,000 IU / L gentamicin sulfate placental protection solution. Seal the sterile bag and put it into the collection box, put the collection box and 5ml of collected maternal blood into the constant temperature collection box, and placenta protection solution is the culture medium;

[0058] 4 ~ 8 ℃ cold chain transportation, use a constant temperature collection box to send the placenta,...

Embodiment 2

[0061] Isolation of human placental mesenchymal stem cells

[0062] First, wipe the collection container with 75% alcohol for disinfection, then put it into the ultra-clean workbench, open the placenta collection container in a sterile environment, and wipe the blood around the sterile collection bag with gauze soaked in 75% alcohol , open the sterile collection bag, and transfer the placenta from the collection bag to 400mm with hemostatic forceps 2 in a disc with the fetus facing up.

[0063] Wash the blood on the placenta with D-Hanks solution, gently peel off the amniotic membrane with sterilized tweezers to expose the chorion layer of the subamniotic layer, and use 18cm sterilized surgical scissors to cut along the section of the umbilical cord and cut 5cm around the umbilical cord 2 For the chorion layer of left and right size, cut along the chorion as much as possible during the cutting process, remove the villi and capillaries connected under the chorion, put the obta...

Embodiment 3

[0068] Subculture of human placental mesenchymal stem cells

[0069] Observe under a microscope that when the culture confluence of the isolated human placental mesenchymal stem cells reaches about 80%, they are digested with 0.25% trypsin (Gibco). Leave for 1 to 2 minutes, observe the cells under the microscope, gently tap the culture bottle with the palm of your hand to ensure that the cells are completely digested, then add 0.5ml fetal bovine serum to stop the digestion, add an appropriate amount of D-Hanks solution to the culture bottle, and dissolve the cell suspension Place in a 50ml sterile centrifuge tube, pipette and mix well, take an appropriate amount of cell suspension to detect cell viability, and perform cell counting, centrifuge the remaining cell suspension at 2000r / min for 10min, and then collect the cells.

[0070] According to 1×10 4 cells / cm 2 Density seeding in T75 culture flasks, and add 10mL complete medium for subculture.

[0071] Generally, the cell...

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Abstract

The invention provides a human placenta mesenchymal stem cell, a preparation method and an application thereof. The preparation method comprises the following steps: collecting, separating, culturing,cryopreserving, detecting, recovering, and the like. A high-purity high-activity mesenchymal stem cell can be acquired by only performing slide adherent culture after a human placenta chorion tissueblock is acquired in the separating process; the technological process is simplified; the cost of enzymic digestion is saved; the adherence and growth rate of primary cells are accelerated; the cell culture period is shortened; the introduction of more external interference factors is avoided, so that the process stability can be easily controlled. The multiplication capacity of the human placentamesenchymal stem cell acquired according to the invention is more stable than that of other mesenchymal stem cells; after the human placenta mesenchymal stem cell passes to P20 generation, the cell still can stably proliferate, and the cellular morphology, molecular surface antigen and adipogenesis osteogenesis differentiative potential thereof all meet the regulations for minimum standard of MSCidentification from international cell therapy association.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a human placenta mesenchymal stem cell and its preparation method and application. Background technique [0002] Mesenchymal Stem Cell (MSC) is a kind of stem cell. It is named for its ability to differentiate into mesenchymal tissue. It has subpotential differentiation potential and can be induced to differentiate into various tissues under specific in vivo and in vitro environments. Cells, for example, can be differentiated into nerve, heart, liver, bone, cartilage, tendon, fat, epithelial and other cells. Mesenchymal stem cells have the common characteristics of stem cells, that is, the ability of self-renewal, multidirectional differentiation and homing. Mesenchymal stem cells are widely distributed in bone marrow, periosteum, cancellous bone, fat, synovium, skeletal muscle, fetal liver, deciduous teeth, umbilical cord, and umbilical cord blood. Its content ...

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 任鹏陈淑倩魏瑞冬武晓东苏圆李建强张艳茹刘晓玲李林蔚李旭杰
Owner 山西省干细胞基因工程有限公司
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