Separation method for mantle epithelial cells in vitro of pinctada martensii

A Hepu pearl oyster, separation method technology, applied in the field of separation of a single type of cells, can solve the problems of less research on separation and purification, weak technology, unfavorable analysis of the properties of a single cell group in cell separation and culture, and achieve vitality and low cost , easy-to-operate effects

Inactive Publication Date: 2009-12-23
TSINGHUA UNIV
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Problems solved by technology

[0003] Due to the large number of cells migrated out during the culture of Hepu pinnacle primary cells, it is not conducive to the isolation and culture of cells and the analysis of th...

Method used

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Examples

Experimental program
Comparison scheme
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Embodiment

[0017] Example: Cell Isolation of Outer Mantle Epithelial Cells

[0018] (1) Preparation of Percoll gradient solution: In a centrifuge tube, mix 9 parts of Percoll stock solution with 1 part of D-MBSS to obtain 100% Percoll solution. 100% Percoll stock solution and D-MBSS solution were mixed in different proportions to prepare 15%, 30% and 36% Percoll gradient solutions.

[0019] (2) Preparation of gradient layer: take a centrifuge tube, soak the tube wall with 1% BSA solution for a period of time in advance, pour off the BSA solution, and add the above-mentioned 36%, 30%, and 15% Percoll gradient solution from bottom to top with a syringe to prepare gradient tubes .

[0020] (3) Adding cell suspension: Use a capillary to add 2ml of the cell suspension to be separated (containing about 5×10 7 cells) were gently spread on the 15% Percoll solution in the gradient tube, centrifuged at 1000 rpm / min for 5 min at room temperature, and continued to centrifuge at 2000 rpm / min for 15...

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Abstract

The invention relates to a separation method for mantle epithelial cells in vitro of pinctada martensii, belonging to the technical field of separation for histiocyte culture. The method mainly comprises steps of: (1) preparing Percoll gradient solution as follows: mixing nine parts of Percoll stock solution with one part of D-MBSS to obtain 100% of Percoll solution; and mixing the 100% Percoll stock solution with D-MBSS solution in different proportion to obtain the Percoll gradient solution; (2) preparing gradient layer as follows: previously using BSA solution to soak the tube wall of a centrifuge tube and preparing a gradient tube; (3) adding cell suspension as follows: adding cell suspension to be separated into the gradient tube; and (4) cell separation: after centrifugating, taking out cells, washing and heavy suspending. The invention can separate out single type of cell group from cells emigrated from primarily cultured tissue blocks, has low damage to cells, and can maintain activity of cells to the maximum extent. The cells after being separated still can be cultured. The operation is simple.

Description

technical field [0001] The invention relates to a tissue cell culture and separation technology, in particular to a technology for separating a single type of cell from a primary cultured tissue block of a pearl oyster, Hepu pinnacle oyster. Background technique [0002] Pinctada fucata Martenssi, also known as Pinctada fucata Martenssi, belongs to the phylum Mollusca, the class Bivalvia, the order Pterioida, the family Pteriidae, and the genus Pinctada ( Pinctada), mainly distributed in the coastal areas of my country, is currently the main shellfish used in the production of seawater pearls in the world. In recent years, due to the impact of global climate change and the economic purpose of cultivating shellfish, the quality of pearl oysters and the quality of pearls have declined. The culture and isolation of Hepu pearl oyster tissue cells are of great significance to the quality of pearl oyster and pearl. [0003] Because many cells migrated out during the culture proce...

Claims

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Application Information

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IPC IPC(8): C12N5/06
Inventor 张荣庆谢莉萍上官俊龙李琪刘晓军周玉娟
Owner TSINGHUA UNIV
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