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Construction method of mesenchymal stem cell bank

A technology of quality stem cells and cells, which is applied in the field of cell bank construction, can solve the problems of low cell purity, increased tissue exposure time, contamination risk, and numerous preparation steps, so as to achieve good purity, improve the purity of primary cells, and improve preparation efficiency Effect

Inactive Publication Date: 2017-11-24
章毅 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing hpMSCs library construction methods still have many shortcomings, such as: numerous preparation steps, increased tissue exposure time and contamination risk; low cell purity, and the introduction of exogenous animal protein, etc.

Method used

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  • Construction method of mesenchymal stem cell bank
  • Construction method of mesenchymal stem cell bank
  • Construction method of mesenchymal stem cell bank

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 Preparation of placental mesenchymal stem cells

[0050] Neonatal placenta authorized with maternal consent was used as a source of mesenchymal stem cells.

[0051] One postpartum fresh placenta was taken under aseptic conditions, and the placenta was preserved and transported using placenta preservation solution (see Chinese invention patent No. ZL201410028687.X) and placenta collection and storage device (see Chinese utility model patent No. ZL2014203904164). Before receiving the placenta, the preparation personnel must inspect and ensure that the collection bag (box) is undamaged, the maintenance solution has no leakage, and the placenta is in complete shape without severe hemolysis. The placental fetal surface villi plate and villi leaflet tissue were peeled off from the placenta tissue by mechanical method, and the connective tissue adhering to the chorion was removed as far as possible, soaked and rinsed with PBS for 5 times, placed in a 50mL centrifu...

Embodiment 2

[0052] Subculture and Morphological Observation of Embodiment 2 Placental Mesenchymal Stem Cells

[0053] Primary hpMSCs in 5×10 4 / mL for subculture, when the adherent growth reaches 80-90% confluence, discard the original culture medium, add 5mL PBS buffer solution to gently wash the cells, discard the washing solution, add 1mL 0.25% trypsin solution, infiltrate At the bottom of the dish, observe under an inverted microscope. When the intercellular binding protein is digested and the cells begin to take on the shape of short shuttle balls, add 3mL DMEM complete medium to stop the trypsinization, gently blow down the cells with a sterile dropper, and place them in a 15mL centrifuge tube , centrifuge at 1,500rpm for 5min at room temperature, discard the supernatant, add 5mL serum-free MSC medium to resuspend the cells, and measure 5×10 live cells 4 / mL density passage, 37°C, 5% CO 2 cultured in a cell culture incubator with saturated humidity, and counted as the first genera...

Embodiment 3

[0054] Example 3 Frozen Storage of Placental Mesenchymal Stem Cells

[0055] The cells were digested according to the method in Example 2 (generally, the P3-P6 generation cells can not only meet the number requirements of cryopreserved cells, but also have the best cell state). per 1×10 7Add 1mL of serum-free freezing solution to each cell, pipette gun or a sterile dropper to mix the cells gently, divide the cells of each sample into 5 tubes, and each tube contains 1×10 7 cells; place the cryopreservation tube in a -80°C refrigerator overnight, transfer it to a gas-phase liquid nitrogen tank according to the ABO / Rh typing and HLA typing of the sample, and store it in a deep low temperature of -196°C for long-term storage. Placental information (maternal information, fetal information, delivery hospital, placental transfer personnel), cell preparation information (preparation number, preparation experimental conditions, number of primary cells, preparation personnel and time),...

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Abstract

The invention relates to a construction method of a mesenchymal stem cell bank. The construction method comprises the following steps of first, adding 0.2w / v% collagenase II of 1-3 times of volume into tissue fragments containing mesenchymal stem cells for digestion; after digestion is finished, adding an eluent, mixing uniformly and centrifuging, and taking supernate to obtain MSCs; adding the eluent into the supernate again, mixing uniformly and centrifuging, then discarding the supernate, and acquiring white MSCs precipitate; next, adding a red cell lysis buffer into the acquired MSCs precipitate for lysis; after lysis, adding the eluent, centrifuging and discarding supernate, and performing cell counting and inoculation culture on the MSCs; and then cultivating and propagating the MSCs till the quantity of the cells reaches a storage standard, and digesting and freezing to store. The method comprises the easy operation steps and is completely suitable for clinical tests, clinical application and scientific research; the cell purity is high, and the stored seed cells contain no exogenous serum.

Description

technical field [0001] The present invention relates to a method for constructing a cell bank, in particular to a method for constructing a mesenchymal stem cell bank, through preparation, purification, expansion and cryopreservation, so as to make the mesenchymal Stem cells, especially neonatal placental mesenchymal stem cells (Human Placenta Mesenchymal Stem Cells, hpMSCs) are used to construct solid stem cell banks. Background technique [0002] Mesenchymal Stem Cells (MSCs) are a type of stem cells derived from mesoderm with self-renewal and multilineage differentiation potential. People first discovered CD34 mesenchymal stem cells from human bone marrow in 1999, but MSCs in bone marrow only accounted for 0.001-0.010% of the total number of nuclear cells, and decreased with age. Later, a small amount of MSCs were found in periosteum, muscle connective tissue, fat, synovial fluid, fetal liver, dermis and umbilical cord blood. In contrast, the placental chorionic tissue ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N5/0775C12N5/073A01N1/02
CPCA01N1/0221C12N5/0605C12N5/0665C12N2509/00C40B50/06
Inventor 章毅伍婷陈侃俊陈亮李萍李冉金魏名王磊
Owner 章毅
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