Preparation method and detection method of human parathyroid gland single-cell suspension

A single-cell suspension, parathyroid technology, applied in the biological field, can solve the problem of not providing a specific preparation method of parathyroid single-cell suspension, and achieve the effect of improving cell viability and reducing damage

Pending Publication Date: 2020-08-11
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] Purpose of the invention: for the deficiencies in the prior art, the purpose of the present invention is to provide a preparation method of human parathyroid single cell suspension, to solve the problem that the prior art does not provide a specific preparation method of parathyroid single cell suspension It can stably and reliably prepare parathyroid single cell suspension, and then provide a research basis for comprehensive analysis of cell subpopulations in parathyroid single cell suspension

Method used

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  • Preparation method and detection method of human parathyroid gland single-cell suspension
  • Preparation method and detection method of human parathyroid gland single-cell suspension
  • Preparation method and detection method of human parathyroid gland single-cell suspension

Examples

Experimental program
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Effect test

Embodiment 1

[0044] The preparation method of the single cell suspension of human parathyroid gland specifically comprises the following processes:

[0045] (1) Take about 50 mg of hyperplastic parathyroid tissue from a human patient, place the tissue in a sterile petri dish, add a small amount of DMEM medium without serum and antibiotics to wash, and remove visible blood and adipose tissue on the surface of the tissue ; Transfer the tissue to a sterile culture dish, add a small amount of DMEM medium (just enough to cover the tissue; too much will easily cause cell loss; too little will affect cell viability), and use sterile blades or tissue scissors to process it into For fragments of about 1mm3, transfer all the mixed solution in the culture dish to a 25cm2 cell culture flask.

[0046] (2) Add 10ml type IV collagenase (preheated at 37°C) to the cell culture flask, mix well and put it into a constant temperature culture shaker, set the temperature and rotation speed to 37°C and 40rpm res...

Embodiment 2

[0053] Detect the human parathyroid single cell suspension obtained by the preparation method of Example 1, the specific method is as follows:

[0054] S1. Take the human parathyroid single cell suspension obtained above, add RPMI-1640 medium containing 10% fetal bovine serum, and culture at 37°C and 5% CO2. Observe the growth status of the primary and second generation cells under the microscope every day: the cell inoculation diary is the first day, such as image 3 As shown, under the light microscope, the cells can be seen scattered, round, homogeneous and transparent, with complete morphology and structure. The cell types are relatively single. Primary cells began to adhere to the wall on the 3rd day of culture, such as Figure 4 As shown, under the light microscope, a large number of spindle-shaped adherent cells can be seen, the cells are evenly distributed, and the shape is complete; it was passaged on the 5th day, as shown in Figure 5 As shown, the cells after pas...

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Abstract

The invention discloses a preparation method of a human parathyroid gland single-cell suspension. The preparation method comprises the following steps of taking parathyroid gland tissues, washing, andcutting into pieces; adding IV-type collagenase, uniformly mixing and digesting; stopping digestion, filtering, centrifuging, removing supernatant, and adding DPBS for suspension; performing filtering, centrifuging, removing supernatant, and adding DPBS for suspension; adding erythrocyte lysate to remove erythrocytes, centrifuging, removing supernatant, adding DPBS to clean the cells twice, removing the supernatant, and then adding DPBS to suspend to obtain the parathyroid gland single-cell suspension. The invention also discloses a detection method of the human parathyroid gland single-cellsuspension. The detection method comprises the following steps of carrying out culturing on the human parathyroid gland single-cell suspension; observing the growth state of primary and passage first-generation cells every day; and extracting RNA of the passage first-generation cells, and detecting RNA of a parathyroid gland cell specific marker by using a PCR method. The preparation method of thehuman parathyroid gland single-cell suspension can stably and reliably digest cells, and the obtained cells are high in content and activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of human parathyroid single cell suspension. Background technique [0002] Traditional transcriptomics and genomics sequencing obtains the overall state of biological tissues / organs, and uniformly analyzes the different cells that make up biological tissues / organs, while there is heterogeneity among cells in the same tissue. It is even worse in different tissues / organs, so the results of ordinary sequencing will affect the accuracy of cognition of cell gene function. Single cell RNA / DNA seq is the sequencing of the genome of a single cell, which can better help us understand the heterogeneity between cells, redefine cell subtypes, understand cell evolution and its role in disease occurrence Functional changes during development are of great significance, and preparing a high-quality single-cell suspension is a prerequisite for ensuring the quality of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/686C12N15/11
CPCC12N5/0617C12Q1/686C12N2509/00
Inventor 叶文欣倪侃姚敏刘莹卢红
Owner NANTONG UNIVERSITY
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