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Building method of pig-source 3D placental organ model

A method for establishing a pig placenta, which is applied in the fields of biomedicine and agricultural biology, can solve problems such as easy mutation, large differences in cell physiology, and inability to simulate three-dimensional structure, so as to achieve stable passage and ensure the effect of long-term culture.

Pending Publication Date: 2019-08-16
INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the two-dimensional (2D) cell line model has many inherent shortcomings: it grows in a single layer, which cannot simulate the three-dimensional structure in vivo; it only contains a single cell type, which is far from the multicellular composition of tissues and organs in vivo; immortalized or cancerous cells The system is prone to variation, and its physiology is quite different from that of in vivo cells; primary cells cannot be cultured in vitro for a long time
So far there is no research and report on porcine placental organoids

Method used

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  • Building method of pig-source 3D placental organ model
  • Building method of pig-source 3D placental organ model

Examples

Experimental program
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Effect test

Embodiment 1

[0028] A method for establishing a pig-derived 3D placental organoid model, the steps of which are as follows:

[0029] (1) Prepare two 3ml isotonic 30% and isotonic 60% Percoll respectively, and slowly add the two into a 15ml sterile centrifuge tube layer by layer according to the concentration from large to small, and at the same time take 5ml of lymphocyte separation solution and add into the 15ml sterile centrifuge tube.

[0030] (2) Put the pig placenta on the ultra-clean workbench and rinse it with sterile antibiotic saline repeatedly 8 times, then use ophthalmic scissors to cut off the pig placenta villi and shred it into multiple sections.

[0031] (3) Soak the shredded fluff in a 35°C incubator containing a mixture of trypsin and DNase, and the digestion time is 30 minutes;

[0032] (4) Add DMEM / F12 culture medium containing 10% FBS to the cell suspension obtained after digestion to stop the digestion, pipette into a cell suspension, filter the cell suspension throug...

Embodiment 2

[0037] A method for establishing a pig-derived 3D placental organoid model, the steps of which are as follows:

[0038] (1) Prepare two 3ml isotonic 30% and isotonic 60% Percoll respectively, and slowly add the two into a 15ml sterile centrifuge tube layer by layer according to the concentration from large to small, and at the same time take 5ml of lymphocyte separation solution and add into the 15ml sterile centrifuge tube.

[0039] (2) Put the pig placenta on the ultra-clean workbench and wash it with sterile antibiotic saline repeatedly 10 times, then use ophthalmic scissors to cut off the pig placenta villi and cut it into pieces.

[0040] (3) Soak the shredded fluff in a 40°C incubator containing a mixture of trypsin and DNase, and the digestion time is 40 minutes;

[0041](4) Add DMEM / F12 culture medium containing 10% FBS to the cell suspension obtained after digestion to stop the digestion, pipette into a cell suspension, filter the cell suspension through a 100-mesh c...

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PUM

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Abstract

The invention discloses a building method of a pig-source 3D placental organ model. The method includes steps: separating a placental stem cell, wrapping in matrigel, adding an organoid growth factor,and putting into a cell culture box for culture. The defects that cell line models and primary cell models are unitary in cell type and have immortalization or canceration features and primary cellsdo not support long-time culture and passage are overcome, 3D pig-source placental organs cultured by using the technical means support permanent passage and cryopreservation, and the 3D pig placentalorgans put forward by the invention for the first time are about to be of important value in fundamental researches and application-oriented researches.

Description

technical field [0001] The invention belongs to the fields of biomedicine and agricultural biotechnology, and in particular relates to an in vitro biological model, in particular to a method for establishing a pig-derived 3D placental organoid model. Background technique [0002] The placenta contains cells with different functions, and different cells work together to maintain the function of the placenta. In previous studies, cell lines or primary cells were mostly used in the study of placental function, and most of the cell lines were immortalized or cancer cells. Therefore, they were quite different from normal cells in the body and could not truly simulate the characteristics and functions of the placenta in vivo. . However, the placental trophoblast cell line has many disadvantages: such as single cell type, two-dimensional growth, and easy to mutate. Although primary placental cells are derived from in vivo placental tissue, their single-cell type characteristics, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0605C12N2533/54C12N2513/00
Inventor 吴信殷跃帮印遇龙
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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