CN112481195B - Pulmonary artery tissue single cell suspension and preparation method and application thereof - Google Patents
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Abstract
The invention provides a pulmonary artery tissue single cell suspension for single cell sequencing and a preparation method and application thereof. The preparation method of the invention can obtain a large amount of separated cells from the denuded tissue of the pulmonary artery intima at one time while ensuring the cell quality, thus being conveniently used for single cell sequencing. The used reagent is simple, the operation is convenient, and the method has good practical application value.
Description
The application is a divisional application of an invention patent application with the application number of 2020105177713 and the title of the invention is 'a preparation method and application of a pulmonary artery tissue single cell suspension'.
Technical Field
The invention provides a pulmonary artery intimal denudation tissue single-cell suspension for single-cell sequencing and a preparation method and application thereof, belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of a pulmonary artery intimal denudation tissue single-cell suspension for single-cell sequencing.
Background
Chronic thromboembolic pulmonary hypertension (CTEPH) is a disease characterized by dyspnea, weakness and reduced endurance, and is a serious, progressive and fatal disease due to the increase of pulmonary vascular resistance, the progressive increase of pulmonary arterial pressure and the gradual increase of right heart load, and the final progression to right heart failure caused by the blockage of the proximal pulmonary artery by undissolved thromboemboli, accompanied by the remodeling of the distal pulmonary vessels. The CTEPH morbidity is on the rising trend year by year, and the CTEPH has become a global medical care problem seriously harming human life and health due to the hidden morbidity, rapid progress and high mortality. Clinical diagnosis and treatment of patients with CTEPH still faces many challenges, proximal organized thrombi and corresponding intimal thickening can be mostly solved by pulmonary artery thrombi intimal denudation surgery, while distal lesions are inoperable and residual pulmonary hypertension after surgery may require internal targeted drug therapy.
Numerous studies have shown that the occurrence and development of CTEPH are processes in which multiple genes, multiple cells, multiple factors and multiple signal transduction systems are involved together, but which factors and specific molecular mechanisms are not completely elucidated. At present, various cells play a role in the occurrence and development of diseases, the dysfunction of pulmonary artery endothelial cells, the abnormal proliferation of pulmonary artery smooth muscle cells, and various inflammatory cells cause inflammatory reaction, finally cause pulmonary vascular remodeling, and play an important role in the development of CTEPH, so that a comprehensive and complete cell map which is definitely involved in the occurrence and development process of CTEPH is very important.
In recent years, with the rapid development of technologies in the field of biological research, single cell sequencing technology has come to be developed, which is a new technology for performing high-throughput sequencing analysis on genomes, transcriptomes and epigenomes on the level of single cells. Single cell sequencing can reveal the gene structure and gene expression of single cell, and can reflect the heterogeneity of cells, and may be used in tumor, development biology, immunology, microbiology and other fields.
At present, single cell sequencing research related to diseases is increasing day by day, single cell sequencing can capture unique development change of each cell, simultaneously, a cell map of a complex organism is drawn at a single cell level, types, effects and development processes of organism cells are explored, a single cell level digital gene expression profile is obtained, thousands of single cell population analysis can be realized, the defects of the conventional method in the aspects of flux and data quantity are overcome, a new thought is developed for single cell research, the division of cell populations and the detection of gene expression difference among the cell populations are realized, the method is an important method in the fields of cell heterogeneity, cell development, cell population detection research and the like, and a new direction is provided for disease typing, disease treatment strategies, disease drug treatment efficacy prediction, new drug research and the like.
Single cell sequencing techniques have been successfully used to define complex subpopulations in normal tissues, which can study molecular differences at the single cell level and reconstruct lineage hierarchy, and therefore we can further map cells associated with CTEPH disease using single cell sequencing to define cell types and subtypes of pulmonary artery denuded tissues.
In order to individually mark information in each cell for data analysis, the cells before single cell sequencing starts must be single suspended and complete living cells, so the single cell sequencing has high requirements on samples, including cell suspension state, concentration, living rate and the like. The current implementation method is to send single cell separation equipment to a hospital, and the fresh tissue obtained by operation is immediately digested and subsequently separated, so that the experiment is greatly limited in cost, time, place and the like. Moreover, a large amount of isolated cells with cell activity higher than 90% (primary cells with activity higher than 70%) are needed in single cell sequencing experiments, and most of the current cell methods for researching blood vessel sources, namely, isolating fresh tissues to obtain cells and obtaining primary culture cells, are not suitable for single cell sequencing. The pulmonary artery denudation tissue is not easy to obtain single cell suspension with high survival rate, and the single cell suspension is also not easy to transport because the cell survival rate can not be ensured in the transport process. Therefore, it is urgent to develop a simple, convenient and easy-to-operate method for separating denuded tissue of human pulmonary artery and obtaining a single cell suspension with high viability.
Disclosure of Invention
The preparation method comprises the steps of separating, cleaning, trimming, incubating and carrying out enzymolysis and digestion on a pulmonary artery intimal denudation tissue specimen obtained after an operation to obtain a single cell suspension. The single cell suspension of the pulmonary artery intima-denudation tissue prepared by the invention can ensure the cell quality and simultaneously can obtain a large amount of separated cells from the pulmonary artery intima-denudation tissue at one time. The method only needs simple reagent consumables, does not need highly specialized equipment, reagents or skills, is not limited by time and places, can obtain sufficient and complete cells from few stripped tissue tissues of the pulmonary artery intima for single cell sequencing, and is convenient for researchers to operate, so the method has good practical application value.
Specifically, the method of the present invention comprises the steps of:
separating, cleaning and repairing a pulmonary artery intimal denuded tissue specimen obtained by an operation, selecting an intimal faint yellow tissue component, and removing a blood thrombus component in the denuded tissue and a myogenic intimal tissue as far as possible, wherein the tissue of the part almost contains no cell component, so that the efficiency of subsequent single cell suspension preparation is influenced, and the preparation of the single cell suspension is not facilitated. And (3) putting the separated, cleaned and trimmed inner membrane faint yellow tissue components into a tissue preservation solution for preservation, and then carrying out subsequent single cell suspension preparation.
The tissue specimen is treated in the enzymolysis solution 1, and the treatment temperature is 30-40 ℃, and the optimal treatment temperature is 37 ℃. Adding DNAseI into the enzymolysis liquid, filtering the treated sample by a filter screen, resuspending cells, adding the enzymolysis liquid 2, and adding various collagenases in the preparation of the solution of the enzymolysis liquid 2, thereby being beneficial to cell dissociation, reducing the occurrence of cell agglomeration, improving the cell recovery rate and carrying out enzymolysis and digestion.
Taking the supernatant, filtering with a filter screen, adding a lysis red liquid after heavy suspension, and eliminating interference of red blood cells; after the completion of the red fissure, the mixture was collected by centrifugation.
And (4) removing dead cells, resuspending the cells by magnetic beads, incubating at room temperature, adding the cells into a rinsed magnetic bead column, collecting the solution, and centrifuging.
Resuspending the cells and diluting to different concentrations; the "AO/PI activity" assay was chosen and then tested by the instrument to observe the cell diameter distribution. AO/PI double fluorescence counting method is used for detecting cell concentration and survival rate.
The cell staining solution is prepared by mixing Acridine Orange (AO) (green fluorescent nucleic acid dye) and Propidium Iodide (PI) (red fluorescent nucleic acid dye). Propidium iodide is not membrane permeable and can only enter cells with damaged cell membranes, whereas acridine orange can pass through all cell populations. When both dyes are present in the nucleus, propidium iodide can cause a decrease in acridine orange fluorescence by fluorescence resonance energy transfer. Thus, nucleated cells with intact cell membranes fluoresce green and are counted as live cells, while nucleated cells with damaged cell membranes fluoresce only red and are counted as dead cells.
Specifically, the preparation method of the pulmonary artery tissue single cell suspension comprises the following steps:
selecting a faint yellow tissue component of the pulmonary artery intima as a tissue specimen; wherein the faint yellow tissue component of the pulmonary artery intima is obtained by operation, and is separated, cleaned and trimmed to remove the bloody thrombus component in the denuded tissue and the myogenic intima tissue as far as possible;
placing the selected tissue specimen into a tissue preservation solution for preservation;
treating the tissue specimen obtained in the step 2) with enzymolysis liquid 1, wherein the enzymolysis liquid 1 is prepared by adding collagenase I and CaCl into a 1640 culture medium2The treatment temperature is 30-40 ℃; the final concentration of the collagenase I is 2.5mg/mL, and the treatment temperature is preferably 37 ℃;
adding DNAseI into the enzymolysis solution 1, and fully digesting the tissues; the final concentration of DNAseI is preferably 50U/mL;
filtering the processed sample, collecting cells, adding an enzymolysis solution 2 for further enzymolysis, wherein the enzymolysis solution 2 comprises collagenase I, collagenase II, collagenase XI, elastase and DNAseI; the enzymolysis liquid 2 is prepared by adding 1mg/ml collagenase I, 1mg/ml collagenase II, 0.2mg/ml collagenase XI, 1.8U/ml elastase, 50U/ml DNAseI and 2.5mMCaCl into 1640 culture medium2Preparing the product;
filtering the sample obtained in the step 5), adding a lysis red liquid after resuspension, and eliminating interference of red blood cells; after the red crack is finally stopped, centrifugally collecting;
the filtration step is performed through a sieve.
Resuspending cells by using dead cell magnetic beads, incubating at room temperature, adding into a rinsed magnetic bead column, collecting a filtered solution, and centrifuging;
resuspending the cells and diluting to different concentrations;
optionally, an "AO/PI viability" assay is selected to detect cell concentration and viability.
The steps of the preparation method are carried out under the aseptic condition, so that the influence on the subsequent single cell sequencing caused by bacterial pollution is avoided. The single cell suspension of the stripped tissue of the pulmonary artery intima can be prepared by the preparation method. The pulmonary artery intima denudation tissue single cell suspension obtained by the preparation method has high cell activity, good quality, higher cell separation degree and low agglomeration rate, and is beneficial to subsequent single cell sequencing.
The invention is based on a 10x Genomics platform, can separate and mark 5000 single cells at one time, and performs gene expression detection at the single cell level, thereby providing a more extensive platform for single cell research. Encapsulating gel beads and single cells with barcodes and primers in oil droplets; next within each oil droplet, the gel beads are lysed and the cells lysed to release the mRNA, which by reverse transcription generates barcoded cDNA for sequencing. And after the liquid oil layer is damaged, subsequently constructing a cDNA library, carrying out sequencing detection and quality control on the library by using an Illumina sequencing platform, acquiring original data, analyzing the original data, carrying out normalization processing on the original data, carrying out t-SNE (t-distribution random neighborhood embedding) dimension reduction analysis for data visualization, clustering cells based on gene expression level for deeply analyzing heterogeneity of a sample, wherein the cell pairs close to each other have more similar gene expression profiles, and finally obtaining a cell clustering result of the pulmonary artery intima-denudation tissue applied to the single cell sequencing technology. The single cell suspension prepared by the invention has high quality, high activity and high application value. The single cell suspension prepared by the method can be conveniently, quickly and efficiently applied to single cell sequencing detection.
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FIG. 1 is an example of tissue selection and treatment of a stripped tissue specimen of a pulmonary artery intima, wherein FIG. 1A is a schematic overview of the stripped tissue specimen of the pulmonary artery intima; FIG. 1B is a schematic view of the yellowish tissue of the inner membrane preserved in the tissue preservation solution; FIG. 1C is a schematic representation of the enzymatic hydrolysate treated inner membrane buffy coat tissue components for single cell suspension preparation.
FIG. 2 shows a distribution diagram of cell diameters measured by a fluorometer, and the distribution of cell diameters is observed.
FIG. 3 shows the measurement of cell concentration and viability by a fluorometer instrument using the double fluorometric method (AO/PI). FIG. 3A-1: live cell fluorescence intensity profile, FIG. 3A-2: dead cell fluorescence intensity profile. FIG. 3B: bright field and fluorescence images of cell suspensions.
FIG. 4 shows the results of the Illumina sequencing platform sequencing quality control data and cell number.
FIG. 5 shows a two-dimensional display of cell populations.
Detailed Description
The invention is further illustrated by the following examples.
Obtaining and processing a tissue specimen:
a pulmonary endarterium denuded tissue specimen was obtained intraoperatively (fig. 1A). Selecting a pulmonary artery intima denuded tissue specimen obtained by an operation for separation, cleaning and block repair (figure 1B), selecting an intima faint yellow tissue component for single cell suspension preparation, and removing a blood thrombus component in the denuded tissue and a myogenic intima tissue as far as possible, wherein the tissue does not contain a cell component, so that the subsequent single cell suspension preparation efficiency is influenced, and the single cell suspension preparation is not facilitated. Endomal light yellow tissue fraction treated with enzymatic hydrolysate for single cell suspension preparation (FIG. 1C).
Preparing single cell suspension:
the preparation and counting of the single cell suspension of the stripped tissue of the pulmonary artery specifically comprises the following steps:
(1) 1mL of enzymolysis solution 1 is prepared by using a 2.0mL centrifuge tube, and the specific method comprises the following steps: add collagenase I and CaCl 2.5mM to 1640 medium at final concentration of 2.5mg/mL2；
(2) Thoroughly washing the tissue with PBS to remove debris, and transferring the tissue to an enzymatic solution (FIG. 1C); shearing the tissue with scissors, placing on a rotary shaker in an incubator at 30-40 deg.C, preferably 37 deg.C, digesting for 30-60min, preferably 40min at about 20rpm, which is favorable for obtaining tissue source single cell with high activity and subsequent single cell sequencing treatment; meanwhile, the method is also beneficial to improving the recovery rate of the cells;
(3) taking out the centrifuge tube, standing at room temperature for 30s, transferring the supernatant to a new centrifuge tube, storing in ice bath, adding 1mL of enzymolysis solution 1 into the incompletely digested tissue precipitate again, and performing rotary digestion for 20min to fully digest the tissue;
(4) adding DNAseI with the final concentration of 50U/mL into the enzymolysis liquid, performing rotary digestion for 10min, and fully digesting the tissues;
(5) combining the supernatant collected in step 4 with the solution in step 5, filtering with 30 μm (in combination with the distribution of the obtained cell diameter, see fig. 2, preferably using a filter screen of this type), washing the filter screen with 10mL 1640 culture medium, 400g, centrifuging at 4 deg.C for 5 min;
(6) removing supernatant, resuspending cells in 1mL 1640 culture medium, reducing the vibration to the cells in the process of resuspension, being beneficial to improving the activity of the cells, and counting (the number of the cells is too low and does not support the subsequent experiment);
(7) washing the residual tissue with 1640 culture medium, adding enzymolysis solution 2, adding 1mg/ml collagenase I, 1mg/ml collagenase II, 0.2mg/ml collagenase XI, 1.8U/ml elastase, 50U/ml DNAseI and 2.5mM CaCl to 1640 culture medium2Blowing, beating, mixing, placing into 37 deg.C incubator, and rotating shaking table for 20prm digestion for 40 min;
(8) sucking supernatant, filtering with 30 μm filter screen, washing filter screen with 10ml 1640 culture medium, centrifuging at 300g and 4 deg.C for 5 min;
(9) removing supernatant, resuspending with 1ml PBS, adding 3ml crackle solution, blowing, mixing, standing for 8min, stopping crackle with 4ml PBS, centrifuging at 300g and 4 deg.C for 5 min;
(10) removing supernatant, taking 100 mu L of dead cell-removing magnetic beads to resuspend cells, incubating at room temperature for 15min, adding into a rinsed magnetic bead column, collecting the filtered solution, and centrifuging at 200g and 4 ℃ for 5 min;
(11) resuspend cells in 200 μ L PBS and count;
(12) diluting the sample with PBS to 5 different concentrations;
(13) add 12. mu.L of LAO/PI staining solution to 12. mu.L of sample and mix gently with a pipette;
(14) 20 μ L of the mixture was pipetted into a cell counting plate;
(15) allowing the cells to stand on a counting plate for about 1min, and putting the counting plate into a counting instrument for counting;
(16) the "AO/PI Activity Rate" assay was chosen and then tested by an instrument to observe the cell diameter distribution (FIG. 2). AO/PI dual fluorescence counting was used to measure cell concentration and viability (FIG. 3).
The steps of the preparation method are carried out under the aseptic condition, so that the influence on the subsequent single cell sequencing caused by bacterial pollution is avoided. The single cell suspension of the stripped tissue of the pulmonary artery intima prepared by the preparation method has high cell activity, good quality, higher cell separation degree and low agglomeration rate, and is beneficial to subsequent single cell sequencing.
The invention is based on a 10x Genomics platform, can separate and mark 5000 single cells at one time, and performs gene expression detection at the single cell level, thereby providing a more extensive platform for single cell research. According to the process of marking a single cell library by 10x Genomics, a V3 single cell reagent kit is adopted, the prepared single cell suspension is combined with a mixture of Gel beads containing Barcode information and enzyme, and then the single cell suspension is wrapped by oil drops positioned In microfluid 'double cross' connection, so that GEMs (Gel Bead-In-EMulsions) are formed, cell lysis and reverse transcription reactions are carried out In the GEMs, In effective GEMs, 10x Barcode is connected with cDNA products, the GEMs are crushed and oil drops are broken, PCR amplification is carried out by taking the cDNA as a template, and after the cDNA amplification is finished, quality inspection is carried out on the amplified products. And after the quality of the amplification product is qualified, constructing a sequencing library. And performing library detection after the library is finished, sequencing by using an Illumina sequencing platform after the library detection is qualified, and obtaining sequencing sequence data, namely obtaining a large amount of gene expression data of single cells at one time, thereby realizing the purpose of performing expression sequencing at the single cell level. The original sequencing data is in a fastq format, data quality evaluation is carried out by adopting FastQC software, cell-barcode (sequence name of a single GEM droplet) and UMI (Unique Molecular Identifier) are extracted, an RNA sequence is compared with a reference genome, and UMI is corrected based on the Unique comparison result of the RNA sequence and the UMI sequence, so that PCR repetition appearing in sequencing is removed. After effective cells are identified, a Gene-barcode matrix is generated, and the expression values of genes of different cells are recorded. Cell ranger software groups cells based on gene expression levels. And (3) normalizing the expression data UMI, performing t-SNE (t distribution random neighborhood embedding) dimension reduction analysis for visualization, wherein the cell pairs close to each other have more similar gene expression profiles, and finally obtaining a cell grouping result. The results of sequencing quality control data and cell number are shown in figure 4, in order to deeply analyze the heterogeneity of samples, the cells are grouped based on gene expression level, the results are shown in figure 5, and the single cell suspension prepared by the method can be conveniently, quickly and efficiently applied to single cell sequencing detection. The results show that the single cell suspension prepared by the invention has high quality, high activity and high application value.
Library construction was performed based on the 10x Genomics platform. The invention is based on a 10x Genomics platform, can separate and mark 5000 single cells at one time, and performs gene expression detection at the single cell level, thereby providing a more extensive platform for single cell research. Encapsulating gel beads and single cells with barcodes and primers in oil droplets; next within each oil droplet, the gel beads are lysed and the cells lysed to release the mRNA, which by reverse transcription generates barcoded cDNA for sequencing. After the liquid oil layer is destroyed, the cDNA is subsequently subjected to library construction. And then, carrying out sequencing detection on the library by using an Illumina sequencing platform, wherein the results of sequencing quality control data and cell number are shown in figure 4, and the results are shown in figure 5, wherein the cells are grouped based on gene expression level in order to deeply analyze the heterogeneity of the sample, and the results show that the single cell suspension prepared by the invention has high quality and high activity.
Claims (10)
1. A single cell suspension of pulmonary artery tissue, prepared and optionally detected according to the following method, characterized in that it comprises the following steps:
1) selecting a faint yellow tissue component of the pulmonary artery intima as a tissue specimen;
2) placing the selected tissue specimen into a tissue preservation solution for preservation;
3) treating the tissue specimen obtained in the step 2) with enzymolysis liquid 1, wherein the enzymolysis liquid 1 is prepared by adding collagenase I and CaCl into a 1640 culture medium2The treatment temperature is 30-40 ℃;
4) adding DNAseI into the enzymolysis solution 1, and fully digesting the tissues;
5) filtering the processed sample, collecting cells, adding an enzymolysis solution 2 for further enzymolysis, wherein the enzymolysis solution 2 comprises collagenase I, collagenase II, collagenase XI, elastase and DNAseI;
6) filtering the sample obtained in the step 5), adding a lysis red liquid after resuspension, and eliminating interference of red blood cells; after the red crack is finally stopped, centrifugally collecting;
7) resuspending cells by using dead cell magnetic beads, incubating at room temperature, adding into a rinsed magnetic bead column, collecting a filtered solution, and centrifuging;
8) resuspending the cells and diluting to different concentrations;
9) optionally, an "AO/PI viability" assay is selected to detect cell concentration and viability.
2. The single cell suspension of pulmonary artery tissue according to claim 1, wherein the final concentration of collagenase I in step 3) is 2.5 mg/mL.
3. The single cell suspension of pulmonary artery tissue as claimed in claim 1, wherein the processing temperature in step 3) is 37 ℃.
4. The single cell suspension of pulmonary artery tissue of claim 1, wherein the final concentration of DNAaseI in step 4) is 50U/mL.
5. The single cell suspension of pulmonary artery tissue of claim 1, wherein the enzymolysis solution 2 of step 5) is prepared by adding collagenase I, collagenase II, 0.2mg/ml collagenase XI, elastase, 50U/ml DNAseI, and 2.5mM CaCl to 1640 medium to obtain the final concentration2And (3) preparing.
6. The single cell suspension of pulmonary artery tissue as claimed in claim 1, wherein the yellowish tissue component of the pulmonary artery intima is obtained by surgery and is isolated, washed, and trimmed to remove as much as possible of the thrombi component in the denuded tissue and the muscularized intima tissue.
7. The single cell suspension of pulmonary artery tissue as claimed in claim 1, wherein the filtration step is performed through a sieve.
8. The single cell suspension of pulmonary artery tissue according to any one of claims 1 to 7, wherein each step is performed under sterile conditions.
9. Use of a single cell suspension of pulmonary artery tissue according to any one of claims 1 to 8 for single cell sequencing.
10. The application of the pulmonary artery tissue single cell suspension as claimed in claim 9, wherein the prepared single cell suspension is applied to a 10x Genomics platform for library construction, and sequencing quality control data, sequence data and single cell gene expression data are obtained, so as to complete single cell level gene expression detection.
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| CN102168062A (en) * | 2010-12-24 | 2011-08-31 | 首都医科大学附属北京朝阳医院 | Human pulmonary artery smooth muscle cell separation and culturing method and application of same |
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