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Method for separating different kinds of single cells in ovary

An oocyte and cell technology, applied in the field of cell biology, can solve the problems of cell damage, cells that cannot obtain activity, and influence

Pending Publication Date: 2016-11-09
SDIVF R&D CENT LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of the existing follicle isolation method is that the separation process often needs to go through the step of centrifugation and resuspension. The disadvantage is that it will reduce the recovery efficiency of cells, and due to improper control of the action of digestive enzymes, it is easy to cause certain damage to the cells.
LCM technology is used to separate single cells, although it is very accurate, but the tissue must be fixed and sectioned first, and active cells cannot be obtained
In addition, the cell structure after slice processing is often incomplete, so it will have a certain impact on subsequent research and analysis

Method used

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  • Method for separating different kinds of single cells in ovary
  • Method for separating different kinds of single cells in ovary
  • Method for separating different kinds of single cells in ovary

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 prepares test material and prepares animal

[0034] Prepare the following test materials:

[0035] Dissecting fluid: Leibowitz L15 medium: containing 4% fetal bovine serum (FBS) and penicillin-streptomycin double antibody 100ug / ml.

[0036] Digestion solution 1: αMEM medium: containing 0.05mg / ml Liberase TM TL Research Grade (Sigma, product number 05401020001, purchase information http: / / www.sigmaaldrich.com / catalog / product / roche / 05401020001?lang=zh®ion=CN) and 0.2mg / ml collagenase IV (ThermoFisher, 17104-019 ) and 0.1% DNase.

[0037] Digestive juice two: Cell Dissociation Reagent (ThermoFisher, product number A1110501, purchase information https: / / www.thermofisher.com / order / catalog / product / A1110501). Accutase is a widely used digestive enzyme preparation in recent years, which has protease and collagenase activity and does not contain any animal or bacterial source components. Often used as a substitute for pancreatic enzymes, it has a milder dig...

Embodiment 2

[0042] Example 2 Isolation of single oocytes of mouse primordial follicles and primary follicles and single granulosa cells of the oocytes

[0043] Experimental steps:

[0044] (1) The mouse ovary was taken out and dissociated, transferred to a petri dish containing dissection solution and washed 3 times. Then transfer to a Petri dish containing 2ml of digestion solution.

[0045] The ovarian tissue was mechanically dissected with a sterile needle, and the tissue was divided under a dissecting microscope into approximately 1×10 6 -4×10 6 μm 3 of pieces.

[0046] (2) Place the culture dish in a sterile culture cell incubator for digestion at 37°C for 30 minutes, and pipette the digestion solution every 10 minutes during the digestion period. During the operation, the separation of follicles was observed in real time through a microscope, and the next step was performed when most of the follicles were in a free state and no large pieces of tissue were observed.

[0047] (3...

Embodiment 3

[0061] Example 3 Identification of single oocytes of mouse primordial follicles and primary follicles and single granulosa cells of the oocytes

[0062] According to the cell morphology and size of the single cells obtained in Example 2, it was judged that the single oocyte of the mouse primordial follicle and the primary follicle and the single granulosa cell of the oocyte were obtained.

[0063] figure 1 Separation of oocytes and granulosa cells in a single early follicle of a mouse observed under a microscope. figure 1A is the free early follicle observed under the microscope; figure 1 B is the separated oocytes and granulosa cells observed under the microscope.

[0064] Such as figure 2 Single oocytes from mouse primordial and primary follicles were obtained as shown. figure 2 A is the free single oocyte of the primordial follicle isolated according to the method of embodiment 2 observed under a microscope; figure 2 B is a free single oocyte of the primary follicle...

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Abstract

The invention provides a method for obtaining single oocyte and single granulosa cells thereof by separating early follicles of mammals. By means of the method, the early follicles of the mammals can be separated to obtain single oocyte with activity and the corresponding granulosa cells thereof. The invention further provides a kit for obtaining the single oocyte and the single granulosa cells thereof of the early follicles of the mammals.

Description

technical field [0001] The present invention relates to the field of cell biology. In particular, the present invention relates to methods of processing mammalian early follicles to obtain single oocytes and their granulosa cells. Background technique [0002] Cells are the basic unit of life. Since the theory of cells was proposed, human beings have been trying to find a way to study cells. In recent years, the major breakthroughs in nucleic acid amplification and sequencing technology have great significance for the physiological and biochemical research of single cells, making the technical methods of analyzing single cells a hot research direction. Therefore, it is particularly important to accurately capture a single specific type of cell for research and analysis. [0003] Follicle is the basic structural and functional unit of female mammalian ovary, which is mainly composed of oocyte in the middle and surrounded by one or several layers of granulosa cells. Follicl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075C12N5/071
CPCC12N5/0608C12N5/0609C12N2509/00C12N2501/31C12N2501/998C12N2500/25C12N5/0642C12N2501/734C12N2509/10
Inventor 陈子江马金龙吕跃赵涵梁子君王成栋
Owner SDIVF R&D CENT LTD
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