Stem cell detection and separation method based on flow cytometry

Abstract

The invention relates to a stem cell detection and separation method based on flow cytometry, relates to the technical field of cell detection, and aims to provide a novel, efficient and accurate technology for identifying and separating stem cells. According to the method, high-throughput analysis of a flow cytometry and a specific stem cell marker are combined, so that rapid and accurate identification and separation of the stem cells are realized. First, the method selects one or more novel stem cell markers, which have high selectivity and sensitivity to stem cells. The markers can clearly distinguish the stem cells from other cell types, and a foundation is laid for accurate identification of the stem cells. And secondly, by optimizing parameter setting of the flow cytometer, including laser power, a fluorescence detection channel and the like, efficient analysis of the stem cells is realized. Through multi-parameter analysis, fluorescence signals and other related parameters of the cells can be obtained, and the accuracy and sensitivity of stem cell detection are further improved. In addition, the method is also combined with a cell sorting function of a flow cytometer, and an efficient cell separation strategy is provided. According to the analysis result of the flow cytometry, the stem cells can be independently separated and collected, so that a stem cell subset with relatively high purity is obtained. Therefore, a reliable basis is provided for subsequent stem cell research and application.

Description

Technical Field

[0001] The invention belongs to the technical field of cell detection, and in particular relates to a stem cell detection and separation method based on flow cytometry. Background Art

[0002] Stem cells have broad application prospects in the fields of regenerative medicine and biological science research, and can be used in the fields of tissue engineering, disease treatment, drug screening, etc. However, the detection and separation of stem cells are key steps to realize their application. Traditional stem cell detection and separation methods have some limitations, such as complex operation, low efficiency, and low accuracy, which restrict the progress of stem cell research and application.

[0003] Flow cytometry is a commonly used cell analysis and classification technique that uses flow cytometers to perform high-throughput analysis and separation of cells by suspending them in liquid. Flow cytometry can accurately identify and analyze cells based on a variety of parameters, such as fluorescent markers, cell size, and morphology. However, there are still some challenges in applying flow cytometry to the detection and separation of stem cells.

[0004] First, there are limited specific markers between stem cells and other cell types, and conventional cell surface markers are not clearly expressed in stem cells or overlap, making it difficult to accurately identify and separate stem cells. Second, stem cells are usually few in number and unevenly distributed in mixed cell populations, requiring efficient separation methods to obtain stem cell subsets with higher purity. Summary of the invention

[0005] The purpose of the present invention is to solve the above technical problems and provide a stem cell detection and separation method based on flow cytometry.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: specifically comprising the following steps:

[0007] S1: Collect the cell sample to be tested from tissue or cell culture of stem cells from embryonic tissue, bone marrow or body fluid;

[0008] S2: Dispersing the collected tissue samples to obtain a single cell suspension, treating the tissue samples with a trypsin cell dispersion buffer having a concentration of 0.05% to 0.25% (mass / volume ratio) to enable the cells to be dissociated into single cells;

[0009] S3: Pass the dispersed cell suspension through a cell strainer to remove cell aggregates and large cell clumps. This can be done using a cell strainer or mesh to obtain a uniform cell suspension.

[0010] S4: Wash the cell suspension to remove the cell culture medium, enzymes and cell debris in the suspension. This can be achieved by adding PBS and centrifuging. Repeat the washing steps to ensure that the cell suspension is clean.

[0011] S5: Add cell preservation solution, fluorescent dye, etc. to maintain cell viability and stability and prepare for subsequent flow cytometry analysis;

[0012] S6: Select Oct-4, Nanog and Sox2 to stain or label the collected stem cell samples. These markers have high selectivity and sensitivity for stem cells and can accurately distinguish stem cells from other cell types;

[0013] S7: The labeled stem cell samples were analyzed by flow cytometry. For cells labeled with FITC (excitation wavelength is about 488 nm, emission wavelength is about 530 nm), a laser with an excitation wavelength of 488 nm should be selected, and the FL1 channel should be selected;

[0014] S8: Based on the results of flow cytometric analysis, the stem cells are separated and collected using the cell sorting function of the flow cytometer.

[0015] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0016] The present invention provides a stem cell detection and separation method based on flow cytometry, which can accurately and efficiently detect and separate stem cells. By selecting appropriate fluorescent markers and optimizing experimental conditions, stem cell populations can be clearly identified and separated, and high-purity stem cell separation can be achieved.

[0017] The present invention introduces a variety of stem cell markers, such as Oct-4 and Nanog, which are of great significance in the identification and separation of stem cells. By utilizing the specific fluorescent signals of these markers, stem cells can be accurately identified and separated, thereby improving the efficiency and reliability of stem cell research and application.

[0018] By using the method of the present invention, a high-purity stem cell subset can be obtained, reducing contamination by other cell types, which is of great significance for further stem cell research and application, and can ensure the accuracy and repeatability of experimental results. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example:

[0021] A method for detecting and separating stem cells based on flow cytometry, the method for detecting and separating stem cells using flow cytometry comprising the following steps:

[0022] S1: Collect cell samples to be tested from hESCs;

[0023] S2: Dispersing the collected tissue samples to obtain a single cell suspension, treating the tissue samples with a trypsin cell dispersion buffer having a concentration of 0.05% to 0.25% (mass / volume ratio) to enable the cells to be dissociated into single cells;

[0024] S3: Pass the dispersed cell suspension through a cell strainer to remove cell aggregates and large cell clumps. This can be done using a cell strainer or mesh to obtain a uniform cell suspension.

[0025] S4: Wash the cell suspension to remove the cell culture medium, enzymes and cell debris in the suspension. This can be achieved by adding PBS and centrifuging. Repeat the washing steps to ensure that the cell suspension is clean.

[0026] S5: Add cell preservation solution, fluorescent dye, etc. to maintain cell viability and stability and prepare for subsequent flow cytometry analysis;

[0027] S6: Oct-4 and Nanog were used as specific markers for stem cells. Through fluorescence staining and flow cytometry analysis, high expression of Oct-4 and Nanog on the surface of hESC was observed. The fluorescent stain used was DAPI or Hoechst33342, which can distinguish the cell nucleus from the cytoplasm, further assisting in the identification of stem cells;

[0028] S7: The labeled stem cell samples were analyzed by flow cytometry, using a 532nm laser to excite the fluorescence signals of Oct-4 and Nanog, and a 488nm laser to excite the fluorescence signal of DAPI. By setting appropriate fluorescence detection channels, accurate fluorescence signals can be obtained and fluorescence cross-interference can be eliminated;

[0029] S8: Using the cell sorting function of the flow cytometer, Oct-4 and Nanog double-positive hESCs were separated and collected based on fluorescence positivity. This can obtain a high-purity hESC subset and reduce contamination by other cell types.

[0030] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting and isolating stem cells based on flow cytometry, characterized in that: The stem cell detection and separation method using flow cytometry comprises the following steps: S1: Collect the cell sample to be tested from tissue or cell culture of stem cells from embryonic tissue, bone marrow or body fluid; S2: Dispersing the collected tissue samples to obtain a single cell suspension, treating the tissue samples with a trypsin cell dispersion buffer having a concentration of 0.05% to 0.25% (mass / volume ratio) to enable the cells to be dissociated into single cells; S3: Pass the dispersed cell suspension through a cell strainer to remove cell aggregates and large cell clumps. This can be done using a cell strainer or mesh to obtain a uniform cell suspension. S4: Wash the cell suspension to remove the cell culture medium, enzymes and cell debris in the suspension. This can be achieved by adding PBS and centrifuging. Repeat the washing steps to ensure that the washing is clean; S5: Add cell preservation solution, fluorescent dye, etc. to maintain cell viability and stability and prepare for subsequent flow cytometry analysis; S6: Select Oct-4, Nanog and Sox2 to stain or label the collected stem cell samples. These markers have high selectivity and sensitivity for stem cells and can accurately distinguish stem cells from other cell types. S7: The labeled stem cell samples were analyzed by flow cytometry. For cells labeled with FITC (excitation wavelength is about 488 nm, emission wavelength is about 530 nm), a laser with an excitation wavelength of 488 nm should be selected, and the FL1 channel should be selected; S8: Based on the results of flow cytometric analysis, the stem cells are separated and collected using the cell sorting function of the flow cytometer.

2. The method for detecting and isolating stem cells based on flow cytometry according to claim 1, characterized in that: The fluorescent stain used in step S5 is DAPI or Hoechst33342.