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A kind of preparation method of clinical grade placental mesenchymal stem cells

A technology for stem cells and placenta, applied in the field of biomedicine, can solve the problems of unclear use for clinical-grade cell expansion and culture, complicated operation, and influence on cell adherent growth.

Active Publication Date: 2019-02-12
深圳华大基因细胞科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzymatic digestion method has many enzymes to choose from, the digestion steps are cumbersome, and enzymatic digestion has a great impact on cell proliferation and cell activity.
In addition, the digested tissue generally has to be filtered through a filter to obtain a single-cell suspension, and a large number of red blood cells are mixed in it, which affects the adherent growth of cells to a certain extent and prolongs the cycle of primary culture.
[0008] In addition, for the in vitro expansion of placental mesenchymal stem cells, most of them still use low-concentration human or other animal-derived serum culture systems, and the complexity and ambiguity of serum components limit their use in clinical-grade cell expansion and culture.
[0009] Some people also tried to add various cytokines such as bFGF to replace serum components in the stage of cell passage and expansion, and treated the cell culture dishes with various matrix protein colloids, such as Matrigel, gelatin, etc., for serum-free expansion culture. Although it can grow, the cell state is not uniform, the operation is complicated, the cost of the coating process is high, and the batch stability of the culture and expansion effect is difficult to control, so it is not suitable for large-scale preparation and production

Method used

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  • A kind of preparation method of clinical grade placental mesenchymal stem cells
  • A kind of preparation method of clinical grade placental mesenchymal stem cells
  • A kind of preparation method of clinical grade placental mesenchymal stem cells

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preparation example Construction

[0089] The invention provides a method for preparing clinical-grade placental mesenchymal stem cells, including the steps of cell separation, primary and passage serum-free expansion, and the like.

[0090] In the present invention, suitable materials include placental tissue materials, usually human placental amnion or chorion tissue.

[0091] In the method of the present invention, there is no particular limitation on the volume of the placental tissue material used as a raw material. Typically, the volume is 0.1-5ml, preferably 0.2-2ml, or by weight, 0.1-5g, preferably 0.2-2g.

[0092] The volume ratio of placental amnion or chorion tissue of the present invention to the combined digestive fluid of the present invention is not particularly limited, usually 1g:2-4ml (weight / volume ratio).

[0093] In the present invention, the digestion conditions with combined enzymes include: digestion at 37±2°C; and / or digestion for 6-24 hours, preferably 8-20 hours, more preferably 12-1...

Embodiment 1

[0138] Preparation of primary placental mesenchymal stem cells

[0139] 1. The placenta samples are qualified after strict infectious virus testing (hepatitis B virus, hepatitis C virus, HIV, syphilis and cytomegalovirus) and the results are negative, and enter the cell separation process.

[0140] 2. In a class 100 clean environment, such as an ultra-clean workbench, cut the placental amniotic membrane or chorionic tissue into 1% penicillin / streptomycin PBS solution, remove the remaining blood stains, cut it into pieces mechanically with scissors, and collect In a 15ml centrifuge tube, add 1:3-4ml volume ratio (80U / ml, 0.008wt% working concentration of collagenase type IV, trypsin) combined digestion solution; after digestion at 37°C for 12-18h, preferably 16h, fully blown Mix well, transfer the supernatant suspension to a new 15ml centrifuge tube, centrifuge at 300-500g for 5min, and harvest the cell pellet.

[0141] 3. Add 1-2ml red blood cell lysate to the cells harvested...

Embodiment 2

[0145] Serum-free subculture, harvest, and passaging of primary cells

[0146] Add the primary placental mesenchymal stem cells obtained in Example 1 to resuspend in serum-free medium, and press 1:3-6 (cell number dilution ratio) or 3000-6000 cells / cm 2 Density passage expansion.

[0147] After 1-2 amplification passages, low-passage, 10 7 Cells of the above order of magnitude were stored at -196°C, and the cells were analyzed by flow cytometry for surface markers.

[0148] Results of cell morphology identification

[0149] Cell morphology was observed on the amplified cells, and the results were as follows:

[0150] Placental mesenchymal stem cells obtained by the separation and serum-free expansion method. The morphology under the inverted microscope is as attached figure 1 As shown, the adherent cells of the P5 generation were spindle-shaped and polygonal, with uniform cell shape and size and strong cell proliferation ability.

[0151] Flow Cytometry Surface Marke...

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Abstract

The invention provides a clinical-grade placenta mesenchymal stem cell preparation method. Specifically, the method includes: using a combined digestive enzyme to digest a placenta tissue material after being cut up to obtain a digested tissue mixture, wherein the combined digestive enzyme comprises collagenase IV and trypsase; adding a red blood cell lysis buffer to obtain a mixture after going through red blood cell lysis treatment; centrifuging to obtain precipitate, namely primary-generation mesenchymal stem cells; performing serum-free passage amplification on the primary-generation mesenchymal stem cells obtained in the previous step to obtain clinical-grade placenta mesenchymal stem cells after going through passage. The cells prepared by the method are high in purity and proliferation capability and can guarantee needs on clinical use of the cells.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular, the invention provides a preparation method of clinical-grade placental mesenchymal stem cells. Background technique [0002] Human mesenchymal stem cell (MSC) is a type of tissue with a wide range of sources, including bone marrow, adipose tissue, deciduous tissue, peripheral blood, etc., as well as neonatal umbilical cord blood, umbilical cord, placental tissue, etc. Renewable adult tissue stem cells can differentiate into osteoblasts, chondrocytes, adipocytes, muscle cells, tenocytes, liver cells or glial cells in vitro and in vivo, secrete pluripotent cytokines and improve the microenvironment and lower Immunogenicity; based on the above characteristics, it quickly became a practical "seed cell" in the field of cell therapy and regenerative medicine after its discovery. [0003] Placental tissue-derived stem cells have typical characteristics of mesenchymal stem cells, inc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/0735
Inventor 王全磊商周春葛玉萍刘聪吴亮董国艺刘传宇郑俊强
Owner 深圳华大基因细胞科技有限责任公司
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