CN113025567A

CN113025567A - Separation method of intervertebral disc single cells

Info

Publication number: CN113025567A
Application number: CN202110345193.4A
Authority: CN
Inventors: 甘翼搏; 刘鹏; 朱军; 胡欧; 王钟
Original assignee: Chinese Peoples Liberation Army Army Specialized Medical Center
Current assignee: Chinese Peoples Liberation Army Army Specialized Medical Center
Priority date: 2021-03-31
Filing date: 2021-03-31
Publication date: 2021-06-25

Abstract

The invention discloses a method for separating single cells of intervertebral disc, which comprises the following steps: obtaining a living body intervertebral disc tissue sample, removing mixed tissues and blood on a biological clean workbench, and separating an end plate, an annulus fibrosus and a nucleus pulposus to obtain a single tissue sample; digesting the single tissue sample by trypsin, pronase and type II collagenase in sequence to obtain a tissue suspension; filtering the tissue suspension to obtain cell suspension, centrifuging, discarding supernatant, re-suspending with erythrocyte lysate, and sorting by magnetic bead sorting or flow cytometry to obtain single cell sample. The intervertebral disc single cell separation method provided by the invention adopts a high-specificity enzyme digestion and cell sorting strategy for the first time, fully digests extracellular matrix, efficiently removes residual digestive impurities and cell diploid, and obtains intervertebral disc single cell primary (P0) generation cells which represent the real expression and distribution in an intervertebral disc cell body, have high purity and high activity and meet the single cell sequencing and library building standard under the condition of not in-vitro amplification culture.

Description

Separation method of intervertebral disc single cells

The technical field is as follows:

the invention relates to the technical field of biology, in particular to a method for separating intervertebral disc single cells.

Background art:

an intervertebral disc is a cartilaginous junction between the vertebral bodies of two adjacent vertebrae, consisting of upper and lower cartilaginous endplates, a peripheral annulus fibrosus, and a central nucleus pulposus. Lumbago and leg pain is one of the diseases clinically with high incidence. Research reports that the pain in waist and lower extremities of most patients is caused by intervertebral disc degeneration, the incidence rate of people over 40 years old is up to 40-70%, and the relevant diagnosis and treatment cost causes huge economic burden to the society. The existing treatment strategies for intervertebral disc degeneration mainly comprise traditional means such as physical treatment, drug treatment, interventional therapy, surgical treatment and the like, and only can treat symptoms, but cannot improve the pathological process of intervertebral disc degeneration, so that the occurrence of intervertebral disc degeneration cannot be eradicated. Recent studies have found that biological treatment strategies are promising therapeutic approaches for degenerative disc diseases, and among them, cell transplantation techniques based on cell therapy and tissue engineering are currently the most interesting, and the treatment focus is on repairing or replacing the degenerated disc tissue to restore disc function. However, the current progress in disc biotherapy is critically limited by the lack of understanding of the physiological and pathological microenvironment of the disc, and in particular, the lack of intensive research into cellular heterogeneity within the disc.

Single cell RNA sequencing (scRNA-seq) is a high throughput sequencing technology that has been rapidly developed in recent years and is considered to be a powerful tool to address cell heterogeneity and resolve cellular components of complex cellular niches. The requirement of single cell RNA sequencing on the quality of a cell sample is extremely strict, and special requirements such as high activity (more than or equal to 80 percent), high purity (removal of tissue fragments, cell adhesion bodies and the like) and the like are required to ensure the accuracy and reliability of single cell sequencing information. In the method for separating primary intervertebral disc cells by using the traditional technology, the cell activity is low due to poor enzyme specificity, long digestion time and incomplete digestion, and the generated tissue fragments and cell adhesion bodies are more, so that the requirement of single cell RNA sequencing cannot be met. There are also methods to obtain P1 generation cells after in vitro culture to improve the survival rate and concentration of cells, but the phenotype of cells after in vitro culture often changes greatly, and cell subsets which are not adherent and have low proliferation activity are lost, and the cell subsets cannot represent the real expression and distribution condition of intervertebral disc cells in vivo, so the method is not suitable for single cell sequencing.

Therefore, it is urgently needed to develop a method for separating primary (P0) generation cells of intervertebral disc with high activity and high purity, which is suitable for the requirement of single-cell RNA sequencing samples, and the separated intervertebral disc cells can reflect the real expression and distribution condition in vivo, so as to provide a high-quality cell specimen for the subsequent research of the cell heterogeneity of intervertebral discs.

The invention content is as follows:

the invention aims to provide a method for separating a single-cell sample of an intervertebral disc, which comprises the following steps:

1) obtaining a living body intervertebral disc tissue sample, cleaning the tissue with PBS containing streptomycin on a biological clean workbench until no macroscopic blood contamination exists, then separating an end plate, a fiber ring and nucleus pulposus, and respectively storing to obtain a single tissue sample;

2) fully shearing single tissue samples respectively, re-suspending with PBS containing streptomycin, centrifuging, and discarding supernatant to obtain a first precipitate;

3) mixing the first precipitate with 0.25% trypsin solution, digesting at 37 ℃ for 30 minutes, centrifuging after digestion, and removing supernatant to obtain a second precipitate;

4) mixing the second precipitate with 0.2% streptokinase protease solution, digesting at 37 deg.C for 60 min, centrifuging, and removing supernatant to obtain third precipitate;

5) mixing the third precipitate with collagenase II solution, and digesting at 37 ℃ until the tissue is completely digested to obtain tissue suspension;

6) filtering the tissue suspension by a cell filter screen of 40 mu m to obtain a cell suspension, centrifuging the cell suspension, and removing supernatant to obtain a fourth precipitate;

7) resuspending the fourth precipitate with erythrocyte lysate, standing at room temperature for 5 minutes, adding 2mL of PBS containing streptomycin, centrifuging, and removing the supernatant to obtain a fifth precipitate;

8) and (3) obtaining cells from the fifth precipitate by a magnetic bead sorting method or a flow cytometry sorting method to obtain a single cell sample.

Compared with the traditional one-step digestion method (0.2% type II collagenase for 2-4h), the intervertebral disc single cell separation method provided by the invention can be used for fully digesting the extracellular matrix due to the high-specificity enzyme digestion and cell sorting strategy, efficiently removing the digestion residual impurities and the cell diploid, and obtaining the intervertebral disc single cell suspension (shown in figures 3A and 3C) which is high in purity and activity and meets the single cell sequencing and library building standard.

In one embodiment according to the invention, the streptomycin-containing PBS is a PBS containing 2% streptomycin.

In one embodiment according to the invention, the concentration of collagenase ii solution in step 5) is 0.1% if the single tissue sample is nucleus pulposus tissue; if the single tissue sample is an endplate or an annulus fibrosus, the collagenase type II solution concentration is 0.2%.

In one embodiment according to the invention, the centrifugation conditions in steps 2), 3), 4), 5), 6) or 7) are centrifugation at 300g for 5min at 4 ℃.

In one embodiment according to the invention, said sufficient mincing in step 2) is mincing said single tissue sample to 1mm by circular and ophthalmic scissors³Size.

In one embodiment according to the present invention,

in step 3), the ratio of the first precipitate to the trypsin solution in g: ml is 1: 5;

in step 4), the ratio of the second precipitate to the streptokinase protease solution is 1: 5;

in the step 5), the ratio of the third precipitate to the collagenase II solution in g: ml is 1: 5.

in one embodiment according to the present invention,

step 8) before sorting, sucking a proper amount of the resuspended cell suspension, and staining and counting the resuspended cell suspension by using an AO/PI solution;

preferably, the suspended cell suspension and the AO/PI solution are mixed evenly in a volume ratio of 1:1 and then counted immediately; preferably, the counting is accomplished using a Counter Star Counter.

The AO/PI cell fluorescence dead-live staining method can accurately judge the quality of a cell sample, does not produce nonspecific staining compared with the traditional trypan blue cell live rate staining, and can more accurately judge the live rate of primary (P0) generation cells of the human intervertebral disc by applying the AO/PI cell fluorescence dead-live staining function of a Countstar Rigel S1 cell counter.

In one embodiment according to the present invention, the magnetic bead sorting method comprises:

a) resuspending the cells with an appropriate amount of Dead Cell Removal Micro Beads, gently mixing well and incubating at room temperature for 15 minutes;

b) the MACS Separator was adsorbed to the MACS MultiStand scaffold and the MS column was adsorbed to the Separator and rinsed with 1 × Binding Buffer;

c) adding a proper amount of 1 × Binding Buffer into the incubated cells, gently mixing uniformly, transferring to an MS column, suspending and dripping, and allowing the living cells to pass through the column by gravity;

d) when the liquid in the MS column is about to drip, suspending and adding 1 × Binding Buffer to wash for several times, centrifuging, discarding the supernatant, and resuspending the cells with PBS + 0.04% BSA with proper volume.

The method has the advantages that the dead cells are removed by using the magnetic bead sorting method, so that the cell survival rate can be effectively improved, the cell impurities are reduced, and compared with the magnetic bead sorting method, the magnetic bead sorting method can effectively reduce the primary cell impurities in the human intervertebral disc, reduce the cell agglomeration rate, improve the cell survival rate, and enable the cell sample to better meet the requirement of single cell sequencing.

In one embodiment according to the present invention, the flow cytometric sorter method comprises:

i) resuspending the cells in PBS containing 3% BSA, staining with 7-AAD, centrifuging, discarding the supernatant, resuspending the cells in PBS, and then filtering with a flow filter;

II) starting the flow cytometry sorter, and sequentially carrying out the following operations in flow cytometry analysis software:

creating a dot pattern of FSC-A and SSC-A, and gating to remove impurities;

creating a dot diagram of FSC-H and FSC-W, and drawing a gate to remove the adhesion body;

creating a dot pattern of SSC-H and SSC-W, and then performing gate marking again to remove the adhesive;

creating a dot plot of SSC-H and PerCP (7-AAD), and gating off dead cells;

iii) connect to a flow cytometer and adapt the appropriate settings: FSC setting Voltage E-1.Ampgain7.21.Linear mode, SSC setting Voltage 271to 358.Ampgain 1.00, Linear mode; the main threshold parameters are: FSC, value 162;

IV) connecting the samples and operating at the slowest flow rate; preferably, the operating conditions are Sheet pressure 4.5PSIG, sample pressure 5.0PSIG,12 μ L/min.

The cell survival rate can be effectively improved and the cell impurities can be reduced by utilizing the sorting method of the flow cytometry sorter, and compared with the method for removing dead cells by using the flow cytometry sorter, the sorting method of the flow cytometry sorter can effectively reduce the primary (P0) generation cell impurities of the human intervertebral discs.

The invention also provides application of the intervertebral disc single cell sample prepared by the separation method in single cell RNA sequencing.

The invention has the beneficial effects that: compared with the traditional intervertebral disc primary cell separation method, the intervertebral disc single cell separation method disclosed by the invention adopts high-specificity enzyme digestion and cell sorting strategies, fully digests the extracellular matrix, efficiently removes the digestion residual impurities and the cell diploid, can obtain the high-quality intervertebral disc primary (P0) single cell suspension meeting the single cell sequencing and library building standard without in-vitro amplification culture, and the obtained cell population can represent the real expression and distribution condition of the intervertebral disc in a human body. The survival rate of the intervertebral disc P0 generation cells is more than or equal to 80%, the cell purity is high, the agglomeration rate is less than or equal to 10%, and the impurities are few, so that the method can be used for sequencing the high-quality intervertebral disc unicellular RNA.

Drawings

FIG. 1 is a schematic diagram of a sorting cross-gate strategy of a flow cytometer: A. removing impurities; B. removing the adhesive body; C. removing the adhesive body for the second time; D. removal of dead cells

FIG. 2 is a comparison graph of the viability of primary cells of nucleus pulposus of human intervertebral disc assessed by different staining methods. Wherein, A is trypan blue staining method; b is AO/PI fluorescent staining method; c is flow cytometric sorter analysis (7-AAD staining);

FIG. 3 is a graph comparing the quality of intervertebral disc cells obtained by different cell separation methods: a, comparing the quality of cell samples processed by a traditional intervertebral disc cell separation method and an intervertebral disc single cell separation method (light microscope pictures and dead and live cell staining pictures); B. comparing the agglomeration rate of the cell samples processed by the traditional intervertebral disc cell separation method and the intervertebral disc single cell separation method; C. comparing the cell viability of the cell samples processed by the traditional intervertebral disc cell separation method and the intervertebral disc single cell separation method;

FIG. 4 is a comparison of the quality of a primary cell suspension sample of human intervertebral disc before and after magnetic bead sorting; a, comparing the quality of primary cells of human intervertebral discs before and after magnetic bead sorting treatment (dead and live cell staining by using a light microscope picture); B. comparing the cell clustering rate of the primary human intervertebral disc cells before and after the magnetic bead sorting treatment; C. comparing the cell viability of the primary human intervertebral disc cells before and after the magnetic bead sorting treatment;

FIG. 5 is a graph comparing the quality of a primary cell suspension sample of human intervertebral disc before and after flow cytometric sorting; a, light microscope photos and dead and live cell staining photos of a primary (P0) generation cell suspension sample of a human intervertebral disc before and after flow cytometry sorting treatment; B. comparing the cell clustering rate of the primary (P0) generation cell suspension sample of the human intervertebral disc before and after the flow cell sorting treatment; C. cell viability comparison before and after flow cytometric sorting of primary (P0) generation cell suspension samples from human intervertebral discs.

FIG. 6 shows that the heterogeneity of the disc cell subsets is reflected by the single cell sequencing of the human disc samples isolated by the method and the profile of the disc cells obtained by bioinformatics analysis.

The specific implementation mode is as follows:

the following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly define the scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Reagent, consumptive material and equipment:

(I) reagent

1. 0.25% Trypsin-EDTA digest (U.S. HyClone, J200033)

2. Collagenase type II (US, Sigma-Aldrich, C6885-5G)

3. Streptolase protease (U.S. Sigma-Aldrich,41844620)

4. Tissue preservation fluid (Germany, Miltenyi Biotec, MB17-R0061)

5. PBS phosphate buffer (China, Solarbio,1022Q024)

6. Penicillin streptomycin (U.S. Hyclone, J190014)

7. AO/PI (Chinese, Counter Star, A20011)

8. 7-AAD (US, Carlsbad,2115592)

9. Erythrocyte lysate (Germany, Miltenyi Biotec, MB17-R0335)

10. BSA (Germany, Miltenyi Biotec,5190527355)

11. 0.4% Trypan blue dye liquor (China, Solarbio, Cat # C0040)

12. DMEM/F12 medium (U.S. Gibco,8120305)

13. 20x Binding Buffer (Germany, Miltenyi Biotec,5191216516)

14. Dead Cell Removal MicroBeads (Miltenyi Biotec,5191216516, Germany)

(II) instruments and consumables:

1. surgical knife, surgical scissors and forceps X2

2. Several disposable filter screens (70 μm: Chinese, BIOFIL, CSS 013070; 40 μm: Chinese, BIOFIL, CSS013040)

3. Several 22 μm filter heads (China, Milllex GP, R9BA69583)

4. Several sterile 50mL,15mL centrifuge tubes (China, Nest)

5. 10cm sterile culture dish (China, Nest)

6. Magnetic force tube (Germany, Miltenyi Biotec,5190220010)

7. Flow tube (US, FALCON,352235)

8. EP tube (U.S. Thermo, UH2788641)

9. Pipettor (1000 ul: Germany, Eppendorf, K32968B; 10 ul: Germany, Eppendorf,4908112)

10. Pipette tip (1000 ul: USA, Bioderck, BK-1000-T; 10 ul: China, Beijing golden microphone, JX0102)

Laboratory apparatus

11. BCM-1300 biological clean bench (China, Suzhou Antai air technology Co., Ltd., A05020262)

12. Flow type cell sorter (BD, AriaII)

13. Magnetic force rack (Germany, Miltenyi Biotec)

14. Rotary mixer (Germany, Miltenyi Biotec, MACSmix)

15. Cell Counter (China, Counter Star, Rigel S1)

EXAMPLE 1 solution preparation

1.1 × erythrocyte lysate: by ddH₂Diluting 10 Xerythrocyte lysate with O, and storing at 4 deg.C

2. Pronase with 0.2% (w/v): 50mL of distilled water was added to 0.1g, and the solution was filtered through a 0.22 μm membrane filter and stored at 4 ℃.

3. Configuration of 0.2% (w/v) collagenase II: 0.1g of collagenase II was weighed, added to 50mL of serum-free DMEM/F12, and the solution was filtered through a 0.22 μm membrane filter, ready to use, for digestion of the annulus fibrosus and endplates.

4. Configuration of 0.1% (w/v) collagenase II: 0.05g of collagenase II was weighed, 50mL of serum-free DMEM/F12 was added, and the solution was filtered through a 0.22 μm membrane filter and ready to use for digesting the nucleus pulposus.

5. Preparation of 2% streptomycin PBS: pipette 1mL of streptomycin and 49mL of PBS, mix well, and store at 4 ℃.

6. PBS containing 3% BSA was prepared by adding 1.5mL of BSA to 48.5mL of PBS, mixing, and storing at 4 ℃.

Example 2 disc cell digestion

1. Sample acquisition:

in disc surgery, electro-coagulation of disc tissue is avoided to prevent tissue necrosis. Taking out intervertebral disc tissues as much as possible by using nucleus pulposus pincers and an end plate scraper, distinguishing nucleus pulposus, annulus fibrosus and end plate tissues, and then placing the tissues in centrifuge tubes (with sterile tissue preservation solution arranged inside) with corresponding marks;

2. sample separation:

disc tissue was rinsed with PBS (2% streptomycin added) at a biological clean bench until the tissue surface was red-free, then the tissue was transferred to a 10cm dish and the disc tissue was again isolated under a stereomicroscope: the end plate is a transparent cartilage tissue with the thickness of about 1 mm; the annulus fibrosus is a cricoid cartilage tissue, and adjacent layers have opposite slopes and cross each other; the nucleus pulposus is milky semitransparent colloid and is elastic.

3. Tissue cutting:

the circular scissors and the ophthalmic scissors can sufficiently cut the tissue to 1mm³The cells were transferred to a 15mL centrifuge tube (not more than 5mL per tube), resuspended in 2% streptomycin PBS, centrifuged at 300g/5min/4 ℃ and the supernatant discarded and weighed.

4. And (3) trypsinization:

0.25% trypsin (10 mL per 2g tissue) was added and the mixture was vortexed at 37 ℃ for 30 minutes, centrifuged at 300g/5min/4 ℃ and the supernatant discarded.

5. Streptoenzyme protease digestion:

adding 0.2% streptokinase protease solution (10 mL per 2g tissue), rotating the mixer at 37 deg.C to digest tissue for 1 hr, centrifuging at 300g/5min/4 deg.C, and discarding the supernatant.

6. Type II collagenase digestion:

nucleus pulposus tissue:

adding 0.1% type II collagenase solution (10 mL solution per 2g tissue) into nucleus pulposus, rotating and digesting for 2 hours on a rotary mixer at 37 ℃, observing digestion results every half hour in the digestion process, and stopping digestion when the tissue is basically completely digested;

fiber ring organization:

adding 0.2% type II collagenase solution (10 mL per 2g tissue) and rotating and digesting for 2 hours on a rotary mixer at 37 ℃, observing digestion results every half hour in the digestion process, and stopping digestion when the tissue is basically completely digested;

③ end plate tissue:

7. and (3) filtering the cells:

transferring the collagenase II digestive juice to a 50mL centrifuge tube through a 40-micron cell filter screen to obtain cell suspension, centrifuging at 300g/5min/4 ℃, and discarding the supernatant.

8. And (3) cracking red blood cells:

2mL of erythrocyte lysate was added, the cells were resuspended, and the mixture was allowed to stand at room temperature for 5 minutes, then 2mL of PBS containing 2% streptomycin was added, and the mixture was centrifuged at 300g/5min/4 ℃ to discard the supernatant.

Compared with the traditional one-step digestion method (0.2% type II collagenase 2-4h), the single-cell separation method for the intervertebral disc can improve the cell separation survival rate and purity, fully digest extracellular matrix and efficiently remove digestion residual impurities due to the adoption of high-specificity enzyme digestion, improve the purity and survival rate of cells (A and C in figure 3), and reduce impurities and cell adhesion bodies (A and B in figure 3)

Example 3 cell quality testing

AO/PI staining: 1mL of 2% streptomycin solution PBS is added to resuspend the cells, 10. mu.l of the cell suspension is sucked and mixed with 10. mu.l of AO/PI solution in a ratio of 1:1, and the mixture is immediately detected.

2. Cell counting: 20 μ l of the stained cell suspension was aspirated and counted using a Counter Star Counter.

The cell sample quality can be accurately judged by using an AO/PI cell fluorescence dead-live staining method, compared with the traditional trypan blue cell live rate staining method, the cell sample quality can not be subjected to non-specific staining, and the cell live rate of primary (P0) generation cells of the human intervertebral disc can be more accurately judged by using the AO/PI cell fluorescence dead-live staining function of a Countstar Rigel S1 cell counter. As shown in FIG. 2, primary (P0) generation cells isolated from a patient' S degenerated disc nucleus pulposus were found to have a viability of 32.0% by trypan blue staining (A in FIG. 2), and 85.2% by AO/PI cytofluorimetric cell viability staining using a Countstar Rigel S1 cytometer (B in FIG. 2), which was found to have a viability of 87.7% by flow sorting (C in FIG. 2), which is closer to the results of AO/PI cytofluorimetric cell viability staining.

Example 4 removal of dead cells

Scheme A: magnetic bead sorting

1. Preparation reagent

Preparing 1 XPBS containing 0.04% BSA, and storing at 4 ℃;

preparing 1 XBinding Buffer by ddH₂And (O) preparation.

2. Removal of dead cells

1) Selecting consumable materials: miltenyi Biotec MS columns require a total cell count of 2X 10⁸Within, and the number of dead cells is 1X 10⁷The content of the compound is less than the content of the compound; miltenyi Biotec LS columns require a total cell count of 2X 10⁹Within, and dead cells are at 1X 10⁸Within.

2) Taking no more than 1 × 10⁷Dead cells and 2X 10⁸Within the total cell count, the cells were collected by centrifugation at 300g/5min/4 ℃ and the supernatant was discarded.

3) Cells were resuspended in 100. mu.l of Dead Cell Removal Micro Beads, gently mixed and incubated for 15 minutes at room temperature.

4) The MACS Separator was adsorbed to the MACS MultiStand holder, a column of the appropriate type was selected and adsorbed to the Separator, and a 15mL centrifuge tube was placed under the MS column and rinsed with 500. mu.l of 1 × Binding Buffer.

5) Add 500ul 1 × Binding Buffer to the incubated cells, mix gently, transfer to MS column, suspend and drop, live cells pass through the column by gravity.

6) After the liquid block was dropped, 500. mu.l of 1 XBinding Buffer was added to the block for washing, 4 times in total, centrifuged at 300g/5min/4 ℃, the supernatant was discarded, and the cells were resuspended in PBS + 0.04% BSA of appropriate volume.

7) And (4) detecting the cell quality, wherein the cell survival rate is more than or equal to 80 percent, and the single cell RNA can be used for sequencing.

According to the invention, the dead cells are removed by using the magnetic bead sorting method, so that the cell survival rate can be effectively improved, the cell impurities are reduced, and compared with the method before the magnetic bead sorting, the magnetic bead sorting method can effectively reduce the primary (P0) generation cell impurities (A in figure 4), reduce the cell agglomeration rate (B in figure 4), improve the cell survival rate (C in figure 4) and enable the cell sample to better meet the requirement of single cell sequencing.

The method B comprises the following steps: sorting with flow cytometer

1) After the primary (P0) generation cells were extracted for the first quality inspection, the cell suspension was centrifuged at 300g/5min/4 ℃, the supernatant was discarded, and 1mL of PBS containing 3% BSA was added for resuspension.

2) The cell suspension was centrifuged at 300g/5min/4 ℃, the supernatant was discarded, and the suspension was resuspended by adding 0.5mL of PBS containing 3% BSA.

3) Filtering by a flow filter tube.

4) Adding 10. mu.l of 7-AAD, and dyeing at 4 ℃ for 5 minutes

5) The flow cytometer was turned on and normal fluid management settings were performed according to the manufacturer's instructions.

6) The CellQuest Pro (or other FCM accessory software) is started and a new file (or previously saved formatted file, and then all acquisition maps are switched for analysis) is opened.

7) A dot plot of FSC-A and SSC-A was created and gated to remove impurities (A in FIG. 1).

8) A dot plot of FSC-H versus FSC-W was created, and the gates were scored to remove the adhesions (B in FIG. 1).

9) A dot plot of SSC-H and SSC-W is created, and the gate is again scribed to remove the adhesive (C in FIG. 1).

10) Dot plots of SSC-H and PERCP (7-AAD) were created and the dead cells were removed by cross-hatching (D in FIG. 1).

11. Connect to flow cytometric sorter and adapt appropriate settings: FSC (Voltage E-1.Ampgain7.21.Linear mode), SSC (Voltage 271to 358.Ampgain 1.00, Linear mode) main threshold parameters: FSC, value 162, auxiliary threshold parameter: none.

12. Samples were attached and run at the slowest flow rate (4.5 PSIG for Sheet pressure, 5.0PSIG for sample pressure, 12 μ L/min).

13. All samples were sorted using the same setup.

14. After all samples were analyzed and data storage was complete, fluid management and cleaning was performed as instructed by the manufacturer, followed by data analysis.

15. And finally, the cells obtained by sorting are subjected to cell quality inspection, and the cell sample with the cell survival rate of more than or equal to 80 percent and the cell agglomeration rate of less than or equal to 10 percent can be used for single cell RNA sequencing.

Compared with the flow sorting method for removing dead cells, the flow sorting method can effectively reduce the primary (P0) generation cell impurities (A in figure 5), remove the diploid, reduce the cell agglomeration rate (B in figure 5) and improve the cell survival rate (C in figure 5) of the human intervertebral discs, so that the cell sample meets the requirement of single cell sequencing. Single cell sequencing was successfully performed using this sample, and bioinformatics analysis showed the profile of human disc cell subsets, reflecting disc cell heterogeneity (FIG. 6).

The above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements may be made by those skilled in the art within the spirit and principles of the invention. The scope of the invention is to be determined by the appended claims.

Claims

1.A method for separating a single-cell sample from an intervertebral disc, comprising:

2. The method for isolating a single cell sample from an intervertebral disc of claim 1, wherein the streptomycin-containing PBS is Ca-free with 2% streptomycin²⁺Mg²⁺PBS。

3. The method for isolating a single disc cell sample according to claim 1, wherein in step 5), if the single tissue sample is nucleus pulposus tissue, the concentration of the collagenase type ii solution is 0.1%; if the single tissue sample is an endplate or an annulus fibrosus, the collagenase type II solution concentration is 0.2%.

4. The method for isolating a sample of human intervertebral disc cells according to claim 1, wherein the centrifugation conditions in steps 2), 3), 4), 5), 6) and 7) are 300g at 4 ℃ for 5 min.

5. The method for separating a discogenic single cell sample as claimed in claim 1, wherein said sufficient cutting in step 2) is performed by cutting said single tissue sample to 1mm by using circular and ophthalmic scissors³Size.

6. The method for isolating a single-cell sample from an intervertebral disc according to claim 1,

7. the method for isolating a single-cell sample from an intervertebral disc according to claim 1,

8. The method of claim 1, wherein the magnetic bead sorting method comprises:

9. The method for isolating a disc unicell sample of claim 1, wherein the flow cytometric method comprises:

i) resuspending the cells in PBS containing 3% BSA, centrifuging, discarding the supernatant, resuspending the cells in PBS, then filtering with a flow filter tube, and performing 7-AAD staining;

creating a dot pattern of FSC-A and SSC-A, and gating to remove impurities;

creating a dot plot of SSC-H and PerCP (7-AAD), and gating off dead cells;

10. Use of a disc unicell sample prepared by the isolation method according to any one of claims 1to 9 for single cell RNA sequencing.