Method for separating and extracting cells from bronchoalveolar lavage fluid

A technology of alveolar lavage fluid and bronchi, applied in cell dissociation methods, blood/immune system cells, animal cells, etc., can solve problems such as inability to culture and affect cell separation

Active Publication Date: 2021-06-04
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT) +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the prior art, the isolated alveolar lavage fluid cells are directly separated with isotonic buffer solution combined with low-speed centrifugation, usually PBS or normal saline centrifugation, but there will be many impurities, including hyphae, sputum, etc. The cells prepared by the method are only used for analysis and cannot be cultured
[0004] However, the composition of the lavage fluid isolated from patients with pulmonary infection diseases and diffuse alveolar deposition diseases such as alveolar proteinosis and early pneumoconiosis is very complicated, because in addition to cellular components and cell debris, it also includes dust deposited in the alveoli, Drugs, surface active substances, secretions and necrosis, etc. These impurities directly cover or adhere to the cells, affecting the separation of cells

Method used

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  • Method for separating and extracting cells from bronchoalveolar lavage fluid
  • Method for separating and extracting cells from bronchoalveolar lavage fluid
  • Method for separating and extracting cells from bronchoalveolar lavage fluid

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Effect test

Embodiment 1

[0057] After the alveolar lavage fluid was taken from the patient with alveolar proteinosis, it was quickly transferred to a 4°C environment for storage, and then transferred to the laboratory as soon as possible. Filter the lavage solution with 4 layers of sterile gauze to remove impurities such as sputum and large sediment in the airway. The filtered lavage fluid was centrifuged at 500×g for 5 min. The resulting pellet was resuspended in PBS buffer containing 2% FBS. The suspension was centrifuged with a Percoll density gradient (composed of equal volumes of 30% and 60% Percoll layering solution), and the centrifugation conditions were 1500×g, 15min, increasing speed 6×g, decreasing speed 2×g. After successful centrifugation, the uppermost layer is the precipitation of surface active substances, and the cell layer is in the middle of the Percoll layer. The cells were washed with 5 times the volume of PBS buffer with a pH value of 7.4 and then centrifuged, and the erythrocy...

Embodiment 2

[0060] After the alveolar lavage fluid was taken from the patient with alveolar proteinosis, it was quickly transferred to a 4°C environment for storage, and then transferred to the laboratory as soon as possible. Filter the lavage solution with 5 layers of sterile gauze to remove impurities such as sputum and large sediment in the airway. The filtered lavage fluid was centrifuged at 500×g for 5 min. The resulting pellet was resuspended in PBS buffer containing 2% FBS. With a Percoll density gradient (consisting of equal volumes of 30% and 60% Percoll layers), the suspension was centrifuged under the conditions of 1400×g, 18min, increasing speed 4×g, and decreasing speed 1×g. After successful centrifugation, the uppermost layer is the precipitation of surface active substances, and the cell layer is in the middle of the Percoll layer. The cells were washed with 5 times the volume of PBS buffer with a pH value of 7.4 and then centrifuged, and the erythrocytes were lysed 1 to ...

Embodiment 3

[0062] After the alveolar lavage fluid was taken from the patient with alveolar proteinosis, it was quickly transferred to a 4°C environment for storage, and then transferred to the laboratory as soon as possible. Filter the lavage solution with 5 layers of sterile gauze to remove impurities such as sputum and large sediment in the airway. The filtered lavage fluid was centrifuged at 500×g for 5 min. The resulting pellet was resuspended in PBS buffer containing 2% FBS. The suspension was centrifuged with a Percoll density gradient (composed of equal volumes of 40% and 80% Percoll layering solution), and the centrifugation conditions were 1600×g, 12min, increasing speed 5×g, decreasing speed 2×g. After successful centrifugation, the uppermost layer is the precipitation of surface active substances, and the cell layer is in the middle of the Percoll layer. The cells were washed with 5 times the volume of PBS buffer solution with a pH value of 7.4, and then centrifuged, and the...

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Abstract

The invention provides a method for separating and extracting cells from bronchoalveolar lavage fluid, and belongs to the technical field of cell extraction. According to the method for separating and extracting cells from the bronchoalveolar lavage fluid, after the bronchoalveolar lavage fluid is subjected to impurity removal, solid-liquid separation is carried out, precipitates is collected to carry out resuspension, Percoll discontinuous density gradient centrifugation is carried out on cell suspension, and a cell layer between Percoll stratified liquid is collected. According to the method provided by the invention, cell components, cell debris components and impurities such as dust, drugs, surface active substances, secreta and the like deposited on an alveolar part in the alveolar lavage fluid can be efficiently removed, and the cell components with diagnostic significance are obtained through separation to a great extent and are used for clinical diagnosis and fundamental research of diseases.

Description

technical field [0001] The invention belongs to the technical field of cell extraction, and in particular relates to a method for separating and extracting cells from bronchoalveolar lavage fluid. Background technique [0002] The alveolar microenvironment is an important part that reflects the functional state of the alveoli. The epithelial cells that make up the alveolar structure and the immune cells in the alveolar cavity together form a natural immune barrier, accepting the impact of harmful substances in the inhaled gas, and protecting the stability of the internal environment of the body . Bronchoalveolar lavage is to infuse a specific amount of normal saline into a certain lobe / segment of lung through a bronchofiberscope, and recover the liquid through negative pressure or gravity, which contains cells in the alveolar cavity, and through the analysis of the shape, function or phenotype of the cells Carry out evaluation and complete the interpretation of cytology, so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0787C12N5/0786C12N5/0783C12N5/0781C12N5/071
CPCC12N5/0645C12N5/0635C12N5/0636C12N5/0642C12N5/0625C12N2509/10
Inventor 李时悦宋新宇朱易平罗钰龙蔡嘉慧郭文亮
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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