Preparation method and application of respiratory tract sample single-cell suspension

Abstract

The invention relates to a preparation method and application of a respiratory tract sample single-cell suspension. The method comprises the following steps: putting a fresh respiratory tract sample into a sterile collection bottle; adding a Dulbecco improved Eagle culture medium until the volume is 5-30ml, then adding a liquefying agent for liquefying, and liquefying in a water bath at the temperature of 30-40 DEG C for 10-30 minutes; centrifuging, and absorbing and discarding the supernatant; adding a mild cell digestive enzyme to resuspend the cells, and incubating to obtain a cell suspension; filtering is performed; centrifuging, and absorbing and discarding the supernatant; dMEM and 9-11% of fetal calf serum are added to resuspend the cells, and a single-cell suspension is obtained.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to a method for preparing a single cell suspension of a respiratory sample and an application thereof. Background Art

[0002] Respiratory samples, such as airway aspirates, sputum, bronchoalveolar lavage fluid (BALF), pharyngeal swabs and nasopharyngeal swabs, are the main specimens used in the study of the pathogenic mechanism of respiratory infections. They are mainly composed of mucus and inflammatory exudates, including mucin and other proteins, enzymes, foreign matter, pathogenic microorganisms, various inflammatory cells and necrotic and detached mucosal epithelial cells. They are characterized by high viscosity, high protein content and complex composition. It is difficult to directly use respiratory samples to prepare single-cell suspensions with high cell viability, and pre-treatment is required before subsequent operations can be performed. However, there are currently very few literatures that study how to efficiently obtain fresh single-cell suspensions from respiratory samples. Most of them focus on sputum liquefaction (Efthimiadis A, Spanevello A, Hamid Q, et al. Methods of sputum processing for cell counts, immunocytochemistry and insitu hybridisation. Eur Respir J Suppl. 2002; 37: 19s-23s) or filtration (Ren X, Wen W, Fan X, et al. COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas [published correction appears in Cell. 2021 Nov 11; 184 (23): 5838]. Cell. 2021; 184 (7): 1895-1913. e19). The number of live cells obtained is low, which brings great technical difficulties to subsequent single-cell research.

[0003] Therefore, it is of great significance to develop a single-cell suspension of respiratory samples that can efficiently obtain high cell viability. Summary of the invention

[0004] In a first aspect, the present invention relates to a method for preparing a single cell suspension of a respiratory sample, wherein the method comprises the following steps:

[0005] S1 Take a fresh respiratory sample and place it in a sterile collection bottle;

[0006] S2 was supplemented with Dulbecco's modified Eagle medium (DMEM) to 5-30 ml, and then liquefied with a liquefier and liquefied in a 30-40°C water bath for 10-30 minutes;

[0007] S3 centrifuge, discard the supernatant;

[0008] S4: add mild cell digestion enzyme to resuspend the cells and incubate to obtain a cell suspension;

[0009] S5 filtering;

[0010] Centrifuge at S6 and discard the supernatant;

[0011] S7 added with DMEM + 9-11% fetal bovine serum (FBS)

[0012] Resuspend cells to obtain a single cell suspension.

[0013] In a second aspect, the present invention also relates to an application of the preparation method according to the present invention in single-cell sequencing, primary cell culture or flow cytometry detection.

[0014] Compared with the prior art, the advantages and beneficial effects of the present invention are particularly:

[0015] 1. The method of the present invention can be used to obtain a single cell suspension of respiratory samples with high cell viability, which is difficult to obtain with the prior art.

[0016] 2. The method of the present invention can separate as many viscous, flaky or clumpy parts of respiratory samples as possible into single cells.

[0017] 3. The single cell suspension of respiratory samples obtained using the method of the present invention contains less cell debris, debris, and residual nuclei, and the number of single cells obtained is large and the activity is strong. DETAILED DESCRIPTION

[0018] General Definition

[0019] The term "fresh respiratory sample" used in this article refers to a respiratory sample collected on the same day and stored at 0℃-8℃ within 4 hours.

[0020] The term "respiratory sample" as used herein includes airway aspirates, sputum, bronchoalveolar lavage fluid, throat swabs, and nasopharyngeal swabs, etc.

[0021] The term "airway aspirate" as used herein refers to a respiratory sample collected from a patient with an artificial airway, including airway aspirates from the upper respiratory tract, airway aspirates from the lower respiratory tract, and airway aspirates from both the upper and lower respiratory tracts.

[0022] The criteria for determining whether the method described in this article uses red blood cell lysis buffer are as follows:

[0023] 1. Do not use: The collected cell pellet has no visible blood color;

[0024] 2. Use: The collected cell pellet contains blood visible to the naked eye.

[0025] As used herein, the terms "comprising" and "including" mean "including but not limited to", and are not intended to exclude, for example, other steps or sample types.

[0026] In a first aspect, the present invention relates to a method for preparing a single cell suspension of a respiratory sample, wherein the method comprises the following steps:

[0027] S1 Take a fresh respiratory sample and place it in a sterile collection bottle;

[0028] S2 is supplemented with DMEM to 5-30 ml, preferably 6-20 ml, more preferably 8-15 ml, and particularly preferably 9-12 ml, and then a liquefier is added to liquefy it, and liquefied in a 30-40°C water bath for 10-30 minutes;

[0029] S3 centrifuge, discard the supernatant;

[0030] S4: add mild cell digestion enzyme to resuspend the cells and incubate to obtain a cell suspension;

[0031] S5 filtering;

[0032] Centrifuge at S6 and discard the supernatant;

[0033] S7 added DMEM + 9-11% FBS to resuspend the cells to obtain a single cell suspension.

[0034] Optionally, a step is added between step S6 and step S7: adding red blood cell lysing solution to lyse red blood cells, centrifuging, and discarding the supernatant. The method of adding red blood cell lysing solution can be operated according to any conventional method known to those skilled in the art.

[0035] Preferably, the fresh respiratory sample refers to a respiratory sample collected on the same day and stored at 0°C-8°C for 4 hours, preferably within 2 hours.

[0036] Preferably, the fresh respiratory samples include airway aspirates, sputum, bronchoalveolar lavage fluid, throat swabs and nasopharyngeal swabs.

[0037] In a preferred embodiment of the present invention, the sterile collection bottle containing the fresh respiratory sample in S1 is immediately placed on ice for subsequent operations.

[0038] In a preferred embodiment of the present invention, DMEM pre-cooled to 2-8°C is added to S2 to make up to 5-30 ml, preferably 6-20 ml, more preferably 8-15 ml, and particularly preferably 9-12 ml, and then a liquefier is added to liquefy it, and it is liquefied in a 30-40°C water bath for 10-30 minutes.

[0039] In a preferred embodiment of the present invention, the liquefier is a dithiothreitol (DTT) aqueous solution, wherein the aqueous solution is a sterile aqueous solution; the liquefier may also be a commonly used clinical liquefier whose main component includes DTT.

[0040] Preferably, the amount of the liquefier added is 20 μl-10 ml, preferably 30 μl-8 ml, more preferably 40 μl-6 ml.

[0041] Preferably, the concentration of the dithiothreitol aqueous solution is 0.5-100 mmol / l, preferably 1-50 mmol / l, more preferably 5-20 mmol / l.

[0042] In a preferred embodiment of the present invention, the centrifugation method in S3 is: centrifugation at 350-500g for 3-15 minutes at 2-8°C, preferably centrifugation at 380-480g for 4-12 minutes at 3-5°C, such as 4°C.

[0043] Preferably, the centrifugation in S3 uses a horizontal centrifuge, more preferably a horizontal refrigerated centrifuge.

[0044] Preferably, after centrifugation in S3, the liquid level of the sample mixture is kept horizontal, and the supernatant is slowly aspirated and discarded.

[0045] When the precipitate obtained in S3 is resuspended in cells and directly subjected to cell counting, the cell viability in the resuspended cell solution is low.

[0046] In a preferred embodiment of the present invention, the mild cell digestion enzyme in S4 is Accutase cell digestion enzyme, dispase II or other mild cell digestion enzyme, preferably Accutase cell digestion enzyme. The activity of the Accutase cell digestion enzyme is 50-1500U / ml, preferably 100-1000U / ml, more preferably 200-900U / ml, and particularly preferably 400-800U / ml. When the Accutase cell digestion enzyme is replaced with trypsin, the cell viability in the obtained single cell suspension is low and the markers on the cell surface are destroyed.

[0047] Preferably, the incubation method in S4 is: incubating at 30-40°C for 5-40 minutes, preferably incubating at 30-40°C for 10-35 minutes, more preferably incubating at 30-40°C, such as 37°C, at 200-500 rpm, such as 300-400 rpm, for 15-30 minutes.

[0048] In a preferred embodiment of the present invention, the filtration in S5 is performed once with a 30-100 μm cell strainer, preferably once with a 35-70 μm cell strainer, and more preferably once with a 40-50 μm cell strainer.

[0049] Preferably, before filtering the cell suspension, use 1-5 ml, preferably 2-4 ml DMEM + 9-11% FBS to wet the cell strainer; preferably, before filtering the cell suspension, use 1-5 ml, preferably 2-4 ml DMEM + 9-11% FBS to wet the cell strainer, and after filtering the cell suspension, use 0.5-3 ml, preferably 0.8-2 ml DMEM + 9-11% FBS to rinse the sample tube and filter; more preferably, before filtering the cell suspension, use 1-5 ml, preferably 2-4 ml DMEM + 9-11% FBS to wet the cell strainer, and after filtering the cell suspension, use 0.5-3 ml, preferably 0.8-2 ml DMEM + 9-11% FBS to rinse the sample tube and filter, and after filtration, rinse the strainer with another 0.5-5 ml, preferably 1-4 ml DMEM + 9-11% FBS.

[0050] In a preferred embodiment of the present invention, the centrifugation method in S6 is: centrifugation at 350-500g for 3-15 minutes at 2-8°C, preferably centrifugation at 380-480g for 4-12 minutes at 3-5°C, such as 4°C.

[0051] Preferably, the centrifugation in S6 uses a horizontal centrifuge, more preferably a horizontal refrigerated centrifuge.

[0052] Preferably, after centrifugation in S6, the liquid level of the sample mixture is kept horizontal, and the supernatant is slowly aspirated and discarded.

[0053] In a preferred embodiment of the present invention, DMEM + 9-11% FBS or other suitable culture medium can be added to S7 to resuspend the cells according to conventional methods known to those skilled in the art so that the number of cells in the single-cell suspension is suitable for subsequent experimental operations, such as single-cell counting, single-cell sequencing, primary cell culture and flow cytometry.

[0054] In a second aspect, the present invention also relates to an application of the preparation method according to the present invention in single-cell sequencing, primary cell culture or flow cytometry detection.

[0055] The present invention will be described and illustrated in more detail below in conjunction with specific embodiments. It will be understood by those skilled in the art that the purpose of providing these embodiments is only for illustrative purposes and does not constitute any limitation to the scope of the present invention.

[0056] Example

[0057] General Description

[0058] In the following examples, all samples used were obtained from clinical collection, and all raw materials used were commercially purchased, wherein the purity of DTT was ≥98%, Accutase cell digestion enzyme was purchased from ThermoFisher (activity was 500-720U / ml), and cell counting was performed using a Countstar intelligent cell analyzer (model: Countstar Rigel S3) for fully automatic testing.

[0059] Cell counting test method: Take a single cell suspension, add AO / PI (Acridine Orange / Propidiumiodide) staining solution, transfer it to a cell counting plate, insert the cell counting plate into the Countstar intelligent cell analyzer to automatically calculate the number of cells and cell viability, where the calculation formula for cell viability is as follows:

[0060]

[0061] Example 1

[0062] The following experiments were performed on fresh BALF of 6 people: 5 ml of sample was placed in a sterile collection bottle and immediately placed on ice. Pre-cooled DMEM was added to make up to 10 ml, and then 5 ml of 10 mM DTT solution was added and liquefied in a 37°C water bath for 15 minutes. The sample mixture was centrifuged at 450g for 5 minutes at 4°C, and the supernatant was slowly aspirated. 1 ml of Accutase was added to resuspend the cells, and incubated at 350 rpm at 37°C for 20 minutes to obtain a cell suspension. The cell suspension was directly passed through a 40 μm cell strainer wetted with 4 ml of DMEM + 10% FBS, and then the sample tube was rinsed and filtered with 1.5 ml of DMEM + 10% FBS, and the strainer was rinsed with another 3 ml of DMEM + 10% FBS. The resulting mixture was centrifuged at 450g for 5 minutes at 4°C, and the supernatant was slowly aspirated. 200 μl of DMEM + 10% FBS was added to resuspend the cells to obtain a single cell suspension. After the cell counting test, the average cell number was 4.76×10 5 The average cell viability was 93.8%.

[0063] Example 2

[0064] Fresh airway aspirates from 6 people were subjected to the following experiments: Placed in a sterile collection bottle and immediately placed on ice. Add pre-cooled DMEM to make up to 10 ml, then add 50 μl of 15 mmol / l DTT solution, and liquefy in a 37°C water bath for 15 minutes. The liquefied mixture was centrifuged at 400g for 10 minutes at 4°C, and the supernatant was slowly aspirated. Add 1 ml of Accutase to resuspend the cells, and incubate at 300 rpm at 37°C for 20 minutes to obtain a cell suspension. The cell suspension was directly passed through a 40 μm cell strainer wetted with 3 ml of DMEM + 10% FBS, then rinse the sample tube with 1 ml of DMEM + 10% FBS and filter, and rinse the strainer with another 2 ml of DMEM + 10% FBS. The resulting mixture was centrifuged at 400g for 10 minutes at 4°C, and the supernatant was slowly aspirated. Add 200 μl of DMEM + 10% FBS to resuspend the cells to obtain a single cell suspension. After the cell counting test, the average cell number was 2.12×10 6 The average cell viability was 85.7%.

[0065] Example 3

[0066] The following experiments were performed on fresh sputum from 6 people: Place the sputum in a sterile collection bottle and immediately place it on ice. Add pre-cooled DMEM to make up to 11 ml, then add 80 μl of 10 mmol / l DTT solution, and liquefy it in a 37°C water bath for 20 minutes. Centrifuge the liquefied mixture at 400g for 10 minutes at 4°C, and slowly aspirate the supernatant. Add 1 ml of Accutase to resuspend the cells, and incubate at 300 rpm at 37°C for 20 minutes to obtain a cell suspension. Pass the cell suspension directly through a 40 μm cell strainer moistened with 3 ml of DMEM + 10% FBS, then rinse the sample tube with 1 ml of DMEM + 10% FBS and filter, and rinse the strainer with another 2 ml of DMEM + 10% FBS. Centrifuge the resulting mixture at 400g for 10 minutes at 4°C, and slowly aspirate the supernatant. Add 200 μl of DMEM + 10% FBS to resuspend the cells to obtain a single cell suspension. After the cell counting test, the average cell number was 2.00×10 6 The average cell viability was 76.2%.

Claims

1. A method for preparing a single cell suspension of a respiratory sample, wherein the method comprises the following steps: S1 Take a fresh respiratory sample and place it in a sterile collection bottle; S2 is supplemented with DMEM to 5-30 ml, preferably 6-20 ml, more preferably 8-15 ml, and particularly preferably 9-12 ml, and then a liquefier is added to liquefy it, and liquefied in a 30-40°C water bath for 10-30 minutes; S3 centrifuge, discard the supernatant; S4: add mild cell digestion enzyme to resuspend the cells and incubate to obtain a cell suspension; S5 filtering; Centrifuge at S6 and discard the supernatant; S7 added DMEM + 9-11% FBS to resuspend the cells to obtain a single cell suspension.

2. The method according to claim 1, wherein: Add a step between steps S6 and S7: add red blood cell lysis buffer to lyse red blood cells, centrifuge, and discard the supernatant.

3. The method according to claim 1, wherein: The fresh respiratory sample refers to a respiratory sample collected on the same day and stored at 0°C-8°C for 4 hours, preferably within 2 hours.

4. The method according to claim 1, wherein: The fresh respiratory samples include airway aspirates, sputum, bronchoalveolar lavage fluid, throat swabs and nasopharyngeal swabs.

5. The method according to any one of claims 1 to 4, wherein: Place the sterile collection bottles containing fresh respiratory samples in S1 on ice immediately for subsequent manipulations.

6. The method according to any one of claims 1 to 4, wherein: The DMEM in S2 is pre-cooled to 2-8°C.

7. The method according to any one of claims 1 to 4, wherein: The liquefier in S2 is a dithiothreitol aqueous solution, wherein the aqueous solution is a sterile aqueous solution.

8. The method according to any one of claims 1 to 4, wherein: The mild cell digestion enzyme in S4 is selected from Accutase cell digestion enzyme or Dispase II, preferably Accutase cell digestion enzyme.

9. The method according to any one of claims 1 to 4, wherein: In S5, the filtration is performed once using a 30-80 μm cell strainer, preferably once using a 35-70 μm cell strainer, and more preferably once using a 40-50 μm cell strainer.

10. Use of the preparation method according to any one of claims 1 to 9 in single-cell sequencing, primary cell culture or flow cytometry.

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