Preparation method and application of liver puncture tissue dissociation single-cell suspension

Abstract

The invention discloses a preparation method and application of a liver puncture tissue dissociation single-cell suspension. According to the invention, fresh trace liver puncture tissues are firstly cross-linked and fixed by using DTSSP, and then are stored and transported in a Meitenyi assembly protective solution, so that the problem of too low cell viability caused by excessive death of fragile parenchymal liver cells in the storage process is obviously improved; the tissue stability of the primary liver puncture tissue in the storage or transportation period is remarkably improved, the time required for dissociation is shortened, and the common defects of small cell number, low cell viability, high mitochondrial gene expression, cell type preference and the like in the traditional liver puncture tissue single cell sequencing are remarkably improved. The method can be applied to single cell sequencing, liver primary cell dissociation and the like. The method has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of cell molecular biology, and particularly relates to a method for preparing a single cell suspension from liver puncture tissue dissociation and an application thereof. Background Art

[0002] The liver is the largest digestive organ, and it also undertakes many important physiological functions such as immunity, metabolism, and synthesis. The hepatic lobule is the basic structural unit of the liver. In the hepatic lobule, the hepatic plates arranged by hepatocytes, the hepatic sinusoids between the hepatic plates, and the bile ducts interspersed between the adjacent surfaces of the hepatocytes form various independent but closely related responsible networks. The liver contains hepatocytes, bile duct cells, hepatic stellate cells, etc., of which hepatocytes account for 80%. The hepatic sinusoids contain blood cells, hepatic macrophages (Kupffer cells), and a large number of NK cells. These cells have different functions, participate in liver metabolism and synthesis through cell-to-cell interactions, and jointly maintain the immune tolerance state in the liver.

[0003] Compared with traditional bulk sequencing, single-cell sequencing can effectively solve the problem of inter-tissue heterogeneity and help discover new rare cell types. After rapid development in recent years, single-cell sequencing has gradually become a commonly used research method in medical and biological research, and has been widely used in the study of various diseases in different organs and tissues of humans and mice. The realization of high-throughput single-cell sequencing technology depends on the preparation of high-quality single-cell suspensions. The liver is a prone site for diseases such as human inflammation and cancer. It is also an important tissue site for multi-factor research such as immune microenvironment. It is a tissue type that is often concerned and studied in single-cell transcriptome sequencing and single-cell metabolomics of multiple species including humans and mice. Although there are currently public methods for dissociation of liver tissue and many single-cell sequencing articles related to liver tissue, most of the research focuses on liver tumor tissue and adjacent tissue, and there is no reliable dissociation method for liver puncture tissue for inflammatory diseases.

[0004] In actual dissociation of liver puncture tissue, there are many factors that limit the success rate of dissociation and data quality of such tissue. First, some cell types in liver tissue are relatively fragile (such as liver epithelial cells, B cells, neutrophils, etc.). If researchers are concerned about these cell types, they generally require the collected fresh tissue to be dissociated immediately. However, liver puncture tissue is limited by operation time and site, and often needs to be stored in tissue protection fluid for several hours or even longer after collection before dissociation, resulting in a more serious dissociation preference. For example, immune cells account for a large proportion of single-cell data, and T cells are the main immune cells. Secondly, the biopsy needle used for liver puncture tissue sampling in clinical practice is often 18G, the diameter of the tissue taken is 1.2mm, and the length is 1 to 2cm. The extremely small amount of tissue makes it impossible for conventional dissociation methods to obtain sufficient and high-viability single-cell suspensions. Furthermore, since liver cells have a relatively vigorous metabolism and are fragile, long-term storage in tissue protection solution (more than 2 hours) often leads to overexpression of mitochondrial genes in some cells and increased liver cell fragments, which seriously affects the quality of single-cell data and the reliability of subsequent data analysis. Finally, other tissue preservation methods, such as gradient freezing or resuscitation dissociation with cell freezing solution or extraction of cell nuclei after quick freezing in liquid nitrogen, are also not feasible due to the limitation of tissue quantity.

[0005] Fixing tissues with fixatives can maintain the inherent morphology and structure of tissues and cells, and maintain a state that is basically consistent with that before ex vivo. Commonly used fixatives include formalin, 4% paraformaldehyde, methanol, etc. Among them, formalin and paraformaldehyde are irreversible in the fixation and crosslinking of tissues, and will cause RNA degradation, so they cannot be used in the early tissue processing of single-cell transcriptome sequencing projects based on polyA capture of mRNA. Methanol also has a relatively good fixation effect on tissues and is widely used in immunohistochemistry or spatial transcriptome sequencing based on tissue sections. However, during the methanol fixation process, proteins and nucleic acids will crosslink with methanol and coagulate. Long-term fixation will destroy cell and chromatin structures, leading to tissue deformation and hardening, information offset or loss, etc. Therefore, the time for methanol fixation is generally controlled within 30 minutes, and it is not suitable for long-term storage or transportation of freshly collected animal tissues before single-cell dissociation. 3,3'-Dithiobis(sulfosuccinimidyl propionate) (DTSSP) is a water-soluble crosslinker that can cleave disulfide bonds. It has a reactive NHS ester end group and can react quickly with any primary amine-containing molecule. DTSSP does not enter the cell membrane, so it can be used for fixed crosslinking of membrane proteins. The disulfide bonds in the spacer arms formed by DTSSP crosslinked proteins can be easily cleaved by 10-50mM dithiothreitol (DTT), thereby releasing the crosslinking reaction without affecting cell activity. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for preparing a single cell suspension of liver puncture tissue dissociation and its application. First, DTSSP is used to fix and cross-link the proteins on the cell surface to improve the stability of fragile cell types such as hepatocytes, and then DTT is used to remove the cross-linking during dissociation, and a single cell suspension is prepared by two enzymatic hydrolysis. The first digestion uses collagenase to digest the intercellular matrix so that the cells in the tissue are detached from the fibrous skeleton. Trypsin is used to continue digestion to separate the clustered cells. In addition, on the basis of the existing ones, the dissociation experimental system (combination of enzymes) and process are optimized, the dissociation time is controlled within 2.5h, and a stable dissociation result with rich cell types can be obtained.

[0007] Specifically, a method for preparing a single cell suspension from liver puncture tissue dissociation of the present invention comprises:

[0008] The collected primary liver puncture tissue was washed with 1640 culture medium containing BSA, placed in a tissue fixative, fixed on ice, and then placed in a tissue preservation solution for low-temperature storage to obtain a primary liver puncture tissue with stable tissue;

[0009] Within 48 hours, the primary liver biopsy tissue stabilized by the above treatment is subjected to single cell dissociation treatment to form a liver biopsy tissue dissociated single cell suspension, specifically including:

[0010] The stabilized primary liver puncture tissue obtained through the above treatment is placed in a culture dish pre-cooled on ice, the tissue is washed several times with culture medium, and minced into minced pieces;

[0011] The liver tissue was initially digested using digestion solution I containing collagenase, deoxyribonucleotidase I, and dithiothreitol;

[0012] The liver tissue was digested again with digestion solution II containing trypsin, and the obtained cell suspension was sieved, and red blood cells were lysed and washed to obtain a liver puncture tissue dissociated single cell suspension.

[0013] Preferably, the tissue fixative is PBS containing 0.25-5 mM DTSSP.

[0014] Preferably, the fixation time on ice is 1.5-2.5 hours;

[0015] Preferably, the tissue preservation solution is Miltenyi tissue preservation solution; the low-temperature preservation refers to preservation at 2-8°C; and the preservation time is less than 48 hours.

[0016] Preferably, the digestion solution I comprises 1640 culture medium, 0.03%-0.05% BSA, 0.1%-0.3% M / V collagenase II, 0.1%-0.3% M / V collagenase IV, 0.05%-0.15 mg / mL DNase I, and 20-50 mM DTT.

[0017] Preferably, the digestive solution II is digestive solution I with trypsin added.

[0018] Preferably, the DTT in the digestion solution I reduces the disulfide bonds in the spacer arm to release the cross-linking effect of DTSSP on the protein; the reducing conditions are 37° C. and 20-30 min.

[0019] Preferably, the digestion conditions for the initial digestion and dissociation of liver tissue are 35° C.-39° C., 21 rpm-23 pm; and the digestion time is 20-25 min.

[0020] Preferably, the trypsin is used to continue digesting the liver suspension, so that the liver tissue is thoroughly digested and dissociated into single cells; the final concentration of trypsin is 0.23%-0.27% W / V, and the digestion time is 5-10 min.

[0021] Applications of the present invention include sequencing of single cell suspensions dissociated from liver puncture tissue, single cell flow analysis or sorting, or primary liver cell culture.

[0022] The present invention innovatively cross-links and fixes the freshly collected trace liver puncture tissue with DTSSP, and then stores and transports it in Miltenyi assembly protection solution, which can significantly improve the problem of low cell viability caused by excessive death of fragile liver parenchymal cells during storage; DTT is added to the enzymatic digestion solution to reduce the cross-linked protein, and collagenase is used to gradually digest and dissociate the tissue into a single cell suspension, and then trypsin is used for a short time to help fully release the single cells in the tissue, which significantly increases the cell yield of liver puncture trace tissue dissociation, and reduces the fragmentation rate and agglomeration rate of the single cell suspension through further optimization, and finally obtains high-quality single cell suspension for subsequent single cell sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the single cell dissociation microscopic examination results of liver puncture tissue obtained by the method of the present invention, wherein A is a liver puncture sampling specimen diagram; B is a microscopic examination diagram of a single cell suspension of liver puncture tissue;

[0024] Figure 2 The figure is a schematic diagram of the single-cell dissociation microscopic examination results of liver puncture tissue obtained using conventional methods, wherein A is a picture of the liver puncture sampling specimen; and B is a picture of the single-cell suspension microscopic examination of the liver puncture tissue.

[0025] Figure 3 It is a schematic diagram of the comparison results of the number of genes, number of reads and proportion of mitochondrial genes of liver puncture single cells obtained by using the method of the present invention and the method of preparing single cell suspension by conventional method; wherein A adopts the method of the present invention; B adopts the conventional method.

[0026] Figure 4 It is a schematic diagram of the comparison results of the proportion of each cell type in liver puncture single cells obtained by using the method of the present invention and the method for preparing single cell suspension by conventional methods; wherein A adopts the method of the present invention; and B adopts the conventional method.

[0027] Figure 5 The present invention is a schematic diagram of a human liver cancer tissue puncture tissue single-cell suspension and single-cell sequencing results prepared by the method of the present invention; wherein A is a liver puncture sampling specimen diagram; B is a microscopic examination diagram of a liver puncture tissue single-cell suspension; C is a violin diagram of the number of genes, number of reads and proportion of mitochondrial genes in single-cell sequencing data; and D is a umap clustering diagram of cell types in single-cell sequencing data. DETAILED DESCRIPTION

[0028] The present invention proposes a pretreatment method for liver puncture tissue and a two-step dissociation optimization system for single cell dissociation.

[0029] The tissue pretreatment method is as follows: the collected liver puncture tissue is washed twice with 1 mL PBS (pH 7.4) (Gibco, 10010031), placed in a pretreatment solution containing DTSSP, fixed and cross-linked on ice for 2 hours, and then stored in Miltenyi tissue protection solution (Miltenyi, 130-100-008), stored at 2-8°C and dissociated within 48 hours.

[0030] The pretreatment solution is a PBS solution with a pH value of 7.4 containing 0.5-2mM DTSSP (Thermo, #21578). PBS (pH7.4) is a solvent. DTSSP is a water-soluble cross-linking agent that chemically cross-links the surface proteins of tissue cells to slow down cell death during storage and transportation of liver tissue. Preferably, the concentration of DTSSP is 1mM.

[0031] The two-step dissociation optimization system is: first, digestion is performed with digestion solution I containing dithiothreitol (DTT), collagenase and deoxyribonucleotidase (DNase I) to degrade protein cross-linking and separate the intercellular matrix from liver cells; then digestion is continued with digestion solution II containing trypsin. Then, cell loss is reduced by rinsing with a cell sieve, increasing the sieving system and washing multiple times, and the cell recovery rate is increased by increasing the washing volume and secondary centrifugation. Finally, the cell suspension is counted under a microscope and loaded on a machine.

[0032] The digestion solution I is a digestion solution containing DTT, collagenase, and DNase I, which includes but is not limited to RPMI 1640 culture medium (Gibco, 11875093) as a solvent, 0.02%-0.1% (W / V) BSA (MACS, 130-091-376), 20-50mM DTT (Solarbio, D8220), 0.05-0.2mg / ml DNase

[0033] I (Applichem, A3778.0050) and 0.2-0.3% (W / V) collagenase II (Gibco, 17101015) and 0.2-0.3% (W / V) collagenase IV (Gibco, 17104019). Preferably, the concentration of DTT is 25 mM, the concentration of BSA is 0.04%, the amount of DNase I is 0.1 mg / ml, and the amounts of collagenase I and collagenase IV are both 0.2%.

[0034] The function of DTT in the digestion solution I is to cut the disulfide bond formed by the cross-linking of DTSSP and proteins, thereby releasing the cross-linking of proteins on the cell membrane surface.

[0035] The collagenase II and collagenase IV in the digestive solution I are commonly used proteases for dissociating animal tissues into single cell suspensions, and can degrade triple-helical natural collagen fibers of connective tissues.

[0036] The function of DNase I in the digestion solution I is to degrade DNA released from dead and ruptured cells, reduce cell agglomeration, and improve the quality of cell suspension. It is a commonly used reagent in single cell dissociation.

[0037] The function of BSA in the digestive fluid I is to provide nutrition for tissue cells and ensure cell activity.

[0038] The digestion solution II is prepared by adding trypsin to the digestion solution I, which includes but is not limited to RPMI 1640 medium (Gibco, 11875093) as a solvent, 0.02%-0.1% (W / V) BSA (MACS, 130-091-376), collagenase contained in the digestion solution I, and 0.2%-0.3% trypsin (Gibco, 15090046). Preferably, the working concentration of trypsin is 0.25%.

[0039] The role of trypsin in the digestion solution II is to quickly dissociate small cell clusters into single cells, thereby increasing the single cell yield.

[0040] Specifically, the digestion conditions are 37° C. and 22 rpm, and the digestion time of the digestion solution I is 20-30 min. Preferably, the digestion is 20 min.

[0041] Specifically, the digestion conditions are 37° C. and 22 rpm, and the digestion time of the digestion solution II is 5-10 min. Preferably, the digestion is 5 min.

[0042] The optimized system refers to reducing the loss of total cell volume by optimizing details such as rinsing the cell sieve, enlarging the sieving system and washing multiple times, and improving the cell recovery rate by increasing the washing volume and secondary centrifugation.

[0043] Specifically, firstly, 2 mL of culture medium is used to fully rinse the 40 μm cell sieve, and 2-4 times the volume of culture medium is added to the digestion solution to terminate the digestion and sieve. Preferably, 3 times the volume of culture medium is added.

[0044] Specifically, the original tube is washed 1-3 times with 2-4 mL of culture medium and sieved, and the filtrates from the two sieves are combined. Preferably, 3 mL of culture medium is used for each wash. Preferably, the washing is performed twice.

[0045] Specifically, a two-stage centrifugation method is used: the first centrifugation condition is 4° C., 300G centrifugation for 5-10 minutes. Preferably, the centrifugation time is 5 minutes.

[0046] Specifically, the supernatant obtained from the first centrifugation is transferred to a new centrifuge tube for a second centrifugation at 4° C. and 400 G for 5-10 min. Preferably, the centrifugation time is 7 min.

[0047] Specifically, the cell pellet from the second centrifugation was resuspended in 500 μL of culture medium and then combined with the cell pellet from the first centrifugation.

[0048] The method proposed by the present invention fixes and crosslinks the tissue by DTSSP treatment, thereby reducing the problem of low cell viability of single cell suspension caused by apoptosis or death of fragile hepatic parenchymal cells during storage and transportation; by adding DTT to the digestion solution, the tissue is dissociated and digested while removing the protein crosslinking effect, thereby reducing the impact of debris and releases generated after excessive hepatic parenchymal cell death on living cells. Removing the crosslinking of proteins in the tissue is conducive to more thorough tissue digestion; by adding DNase I to digest the DNA released by the ruptured cells, the viscosity of the system is reduced to improve the cell agglomeration rate; by a relatively short two-step digestion, the cells in the liver puncture tissue are fully released more gently; by optimizing the screening, washing and centrifugation processes, the tissue fragments in the single cell suspension are reduced while the single cell recovery rate of the micro-puncture tissue is increased. And by optimizing the digestion system and experimental process, the dissociation of the tissue is achieved within about 2.5 hours. In the first dissociation, the combination of collagenase and DNase I first peels off the hepatic parenchymal cells and non-parenchymal cells from the connective tissue. In the continued dissociation, trypsin is used to further digest the clumped cells so that they are completely dissociated into a single cell suspension. Compared with previous methods, the method of the present invention can obtain more abundant cell types using milder experimental conditions within about 2.5 hours. Compared with the conventional method of only one or two collagenases, the addition of trypsin can hydrolyze elastin to destroy the tight connection of the tissue, which is more conducive to the release of cells such as epithelial and endothelial cells in the tissue, and fully dissociate small cell clusters into single cells, thereby increasing the yield of single cells. Through a variety of optimized combinations in the screening, washing and centrifugation processes, the loss of total cell volume is reduced while removing tissue fragments, and the recovery rate of single cells is increased. The single cell suspension after dissociation, screening and centrifugal enrichment is lysed, washed and counted under a microscope, and then put on the machine according to the research purpose.

[0049] The present invention places the freshly collected liver puncture tissue in a tissue protection solution for storage or transportation, and performs tissue dissociation within 48 hours. The "two-step dissociation optimization system" described in the present invention means that the liver puncture tissue is divided into two enzymatic digestion steps of collagenase digestion and trypsin digestion, and the detailed optimization of the steps of sieving, red lysis, centrifugation, and washing after enzymatic digestion is combined to improve the cell yield and cell viability. Specifically, the tissue pretreatment and two-step liver puncture tissue single cell suspension preparation method proposed in the present invention include but are not limited to the following steps: tissue collection, tissue fixation, tissue placement in tissue preservation solution, cross-linking removal and tissue dissociation, tissue continued digestion, sieving, washing, centrifugation, microscopic counting, final resuspension and cell loading.

[0050] In the method, freshly collected liver puncture tissue is placed in PBS containing DTSSP (Thermo, #21578) for fixation. The concentration of DTSSP ranges from 0.5 to 2 mM, preferably, 1 mM. The fixation temperature ranges from 0 to 4°C, preferably, 0°C. The fixation time is 1.5 to 2 hours, preferably, 2 hours.

[0051] In the method, the freshly collected liver puncture tissue is placed in Miltenyi tissue protection solution (Miltenyi, 130-100-008) for storage at a temperature range of 2-8°C, preferably 4°C.

[0052] In the method, both digestion solution I and digestion solution II used for dissociation include DTT (Solarbio, D8220) in a concentration range of 20-50 mM, preferably 25 mM.

[0053] In the method, the collagenase in the digestion solution I used for the initial dissociation includes collagenase II (Gibco, 17101015), collagenase IV (Gibco, 17104019), and DNase I (Applichem, A3778.0050). The hepatic parenchymal cells and non-parenchymal cells are peeled off from the tissue; preferably, the treatment time is 20 minutes. The content range (W / V) of collagenase II, collagenase IV, and DNase I is 0.2%-0.3%, 0.2%-0.3%, and the concentration is 0.05-0.2mg / ml. More preferably, the content ranges are 0.2%, 0.2%, and 0.1mg / ml, respectively.

[0054] In the method, the digestion solution II used for further dissociation is digestion solution I with trypsin (Gibco, 15090046) added thereto, with a content of trypsin (W / V) ranging from 0.2-0.3%, preferably 0.25%.

[0055] In the present invention, the "trypsin" is a broadly specific serine protease extracted and prepared from porcine pancreas, which mainly decomposes proteins by hydrolyzing peptide bonds. It can hydrolyze a variety of proteins, especially peptide bonds at tyrosine, phenylalanine and tryptophan residues. It can hydrolyze elastin and digest hemoglobin, casein and fibrin.

[0056] The "collagenase II" and "collagenase IV" described in the present invention are two isozyme forms of collagenase extracted and prepared from Clostridium histolyticum, and mainly hydrolyze collagen components in connective tissue.

[0057] In the method of the present invention, the dissociation of a tissue sample is controlled to be completed within ≤ 2.5 hours. Preferably, it is 2-2.5 hours. More preferably, it is 2 hours. In a specific embodiment, the present invention achieves a two-step gentle dissociation of liver puncture tissue within 2.5 hours by optimizing the type of digestive enzymes and the experimental process.

[0058] Compared with the previous methods, the treatment method of the present invention can obtain more abundant cell types in a shorter time using milder experimental conditions. The combination of trypsin and collagenase replaces the dissociation method of using only collagenase in previous reports. Compared with the conventional method of using only one or two collagenases, the combination of trypsin and collagenase shortens the dissociation time and is more conducive to the release of various cell types in the tissue. It is also conducive to the laboratory to establish a more stable and more universal single cell suspension preparation system for different species.

[0059] The cell viability of the single cell suspension obtained by the method of the present invention is >90%, preferably 90%-98%, and more preferably >95%.

[0060] The present invention also proposes the application of the method in aspects including but not limited to single-cell sequencing, single-cell suspension preparation, liver primary cell dissociation, single-cell flow cytometry analysis or sorting, and liver primary cell culture.

[0061] In a specific embodiment, the method of optimization of the present invention comprises the following specific steps:

[0062] (1) Collection of tissue samples: A piece of freshly collected liver puncture tissue (~25 mg) was placed in a culture dish pre-cooled on ice and washed thoroughly twice with ice-cold PBS (pH 7.4) (Gibco, 10010031), 1 ml each time.

[0063] (2) The tissue was transferred and immersed in 500 μl of PBS containing 1 mM DTSSP (Thermo, #21578) for 2 hours on ice.

[0064] (3) Tissue preservation and transfer: Transfer the tissue and immerse it in a centrifuge tube containing tissue protection solution and store or transport it at 4°C for no more than 48 hours.

[0065] (4) Initial dissociation of liver cells: Remove the liver tissue and place it in a small culture dish pre-cooled on ice. Wash it twice with 1 ml of pre-cooled culture medium (1640 + 0.04% BSA) on ice. Cut it into chyme with sterile scissors, transfer the tissue to a 5 ml centrifuge tube, and add 2.5 ml of digestion solution I. The formula of digestion solution I is: RPMI

[0066] 1640 medium (Gibco, 11875093) + 0.04% (W / V) BSA (MACS, 130-091-376)

[0067] +25mM DTT+0.2% collagenase II (Gibco, #17101015)+0.2% collagenase IV (Gibco, 17104019)+0.1mg / ml DNase I (Applichem, #A3778), digest for 20min at 37°C and 22rpm in a hybridization oven. Take out the centrifuge tube and gently pipette the suspension to help disperse and mix the tissue.

[0068] (5) Continue cell dissociation: Add trypsin with a final concentration of 0.25% (W / V) to the initially dissociated cell suspension (the digestion solution at this time is named digestion solution II, with a total volume of about 3 mL), and place in a hybridization oven at 37°C and 22 rpm to continue digestion for 5 min.

[0069] (6) Rinse the 40 μm cell sieve thoroughly with 2 mL of culture medium (1640 + 0.04% BSA), add 6 mL (2 times the volume) of culture medium to the cell suspension obtained in (5) to terminate digestion, pass through a 40 μm cell sieve, wash the original centrifuge tube twice with culture medium, 3 mL each time, pass the washing solution through a sieve and combine it with the previously sieved cell suspension into one tube, and centrifuge at 4°C, 300 g for 5 min.

[0070] (7) The supernatant was transferred to a new centrifuge tube and centrifuged a second time at 400G for 7 min at 4°C. The precipitate was resuspended in 200 μL of culture medium and transferred to the cell precipitate obtained in (6).

[0071] (8) Add 2 mL of erythrocyte lysis buffer (Miltenyi Biotec, lot #130-094-183) to the cell pellet and resuspend it. Place it on ice for 3 min and add 10 mL of culture medium to terminate erythrocyte lysis. Centrifuge at 400 g for 7 min at 4°C. Resuspend the cell pellet in 3 mL of culture medium and wash it.

[0072] (9) Centrifuge at 4°C and 300 g for 7 min. Resuspend the cell pellet in 100 μL 1640 + 0.04% BSA and place on ice.

[0073] (10) Microscopic counting: The obtained cells were stained with acridine orange / propidium iodide (AO / PI) and then counted under a microscope and the cell viability was detected using a fluorescent cell counter.

[0074] (11) Finally resuspend the cells and load them onto the machine: Use 1640 + 0.04% BSA to adjust the cell concentration to 700-1200

[0075] cells / μL and immediately used for 10x single cell loading.

[0076] Compared with the prior art, the advantages and beneficial effects of the present invention include:

[0077] The present invention fixes a trace amount (minimum about 25 mg) of liver puncture tissue with DTSSP, then stores and transports it in a tissue protection solution and dissociates it within 48 hours, and adds DTT to the digestion solution to perform enzymatic hydrolysis on the tissue while degrading protein cross-links in the tissue to release single cells, thereby overcoming the shortcomings of low viability, high agglomeration and fragmentation of single cell suspensions caused by the death of liver parenchymal cells during storage in the tissue protection solution.

[0078] The present invention adopts a two-step method for dissociation and tries to control the experimental time of each step: the time of the two dissociations is relatively short, the first dissociation is about 20 minutes, the second dissociation is only 5 minutes, and the red lysis time is controlled at 3 minutes. Generally, the dissociation of a tissue sample is controlled to be completed in a relatively short time (≤2.5 hours), which ensures that the obtained single cell suspension can maintain a high cell viability (>90%) and makes up for the shortcomings of too few cells or loss of specific cell types caused by the previous single enzymatic hydrolysis system. The processing method of the present invention can obtain more abundant cell types (such as B cells, etc.) in a shorter time using milder experimental conditions.

[0079] In the dissociation, the present invention uses a sequential combination of collagenase and trypsin instead of the dissociation method of using only collagenase in previous reports. Compared with collagenase which only acts on the hydrolysis of collagen, trypsin is more conducive to the release of cells such as endothelial cells and epithelial cells in the tissue. Therefore, the combination of trypsin and collagenase is more conducive to the release of various types of cells in the tissue, and is also conducive to the laboratory to establish a more stable and more universal liver tissue single cell suspension preparation system suitable for different species.

[0080] The present invention reduces the loss of the total amount of cells by optimizing methods such as increasing the volume of cell digestion solution during sieving, combining two washes and sieving, and performing secondary centrifugation on the supernatant after sieving.

[0081] The present invention provides a method for first fixing the tissue and then placing it in a tissue protection solution for storage and transportation, as well as a two-step method for preparing a single-cell suspension of liver puncture tissue, that is, an optimized two-step method for preparing a single-cell suspension of liver puncture tissue. By optimizing the digestive enzyme type and the experimental process, the two-step mild dissociation of the liver puncture tissue is completed within 2.5 hours, and high-quality single-cell suspensions are obtained respectively, which makes up for the single-cell suspension quality problems such as few cells, low viability, high clumping and fragmentation caused by directly placing the liver puncture tissue in a tissue protection solution and then dissociating it with a single digestive enzyme in the past, as well as the shortcomings of cell type preference or overly complicated two-step experimental process.

[0082] The method provided by the present invention can be applied to aspects including but not limited to single-cell sequencing, single-cell suspension preparation, liver primary cell dissociation, single-cell flow cytometry analysis or sorting, and liver primary cell culture.

[0083] Example 1 Human hepatitis puncture tissue is subjected to single cell dissociation and sequencing using the method of the present invention. The tissue processing and experimental steps include:

[0084] (1) Collection of tissue samples: A freshly collected human hepatitis puncture tissue was placed in a pre-cooled culture dish on ice.

[0085] After removing the stain, wash thoroughly twice with ice-cold PBS (pH 7.4) (Gibco, 10010031), 1 ml each time.

[0086] (2) Transfer the tissue and immerse it in 500 μl of PBS containing 1 mM DTSSP for 2 hours on ice.

[0087] (3) Tissue preservation and transfer: Transfer the tissue and immerse it in a centrifuge tube containing tissue protection solution and store or transport it at 4°C for no more than 48 hours.

[0088] (4) Initial dissociation of liver cells: Remove the liver tissue and place it in a small culture dish pre-cooled on ice. Wash it twice with 1 ml of pre-cooled culture medium (1640 + 0.04% BSA) on ice. Cut it into chyme with sterile scissors, transfer the tissue to a 5 ml centrifuge tube, and add 2.5 ml of digestion solution I. The formula of digestion solution I is: RPMI

[0089] 1640 medium (Gibco, 11875093) + 0.04% (W / V) BSA (MACS, 130-091-376)

[0090] +25 mM DTT + 0.2% collagenase II (Gibco, #17101015) + 0.2% collagenase IV (Gibco,

[0091] 17104019) + 0.1 mg / ml DNase I (Applichem, #A3778), and digest for 20 min at 37°C and 22 rpm in a hybridization oven. Take out the centrifuge tube and gently pipette the suspension with a wide-mouth pipette to help the tissue disperse and mix.

[0092] (5) Continue cell dissociation: Add trypsin with a final concentration of 0.25% (W / V) to the initially dissociated cell suspension (the digestion solution at this time is named digestion solution II, with a total volume of about 3 mL), and place in a hybridization oven at 37°C and 22 rpm to continue digestion for 5 min.

[0093] (6) Rinse the 40 μm cell sieve thoroughly with 2 mL of culture medium (1640 + 0.04% BSA), add 6 mL (2 times the volume) of culture medium to the cell suspension obtained in (5) to terminate digestion, pass through a 40 μm cell sieve, wash the original centrifuge tube twice with culture medium, 3 mL each time, pass the washing solution through a sieve and combine it with the previously sieved cell suspension into one tube, and centrifuge at 4°C, 300 g for 5 min.

[0094] (7) The supernatant was transferred to a new centrifuge tube and centrifuged a second time at 400G for 7 min at 4°C. The precipitate was resuspended in 200 μL of culture medium and transferred to the cell precipitate obtained in (6).

[0095] (8) Add 2 mL of erythrocyte lysis buffer (Miltenyi Biotec, lot #130-094-183) to the cell pellet and resuspend it. Place it on ice for 3 min and add 10 mL of culture medium to terminate erythrocyte lysis. Centrifuge at 400 g for 7 min at 4°C. Resuspend the cell pellet in 3 mL of culture medium and wash it.

[0096] (9) Centrifuge at 4°C and 300 g for 7 min. Resuspend the cell pellet in 100 μL 1640 + 0.04% BSA and place on ice.

[0097] (10) Microscopic counting: The obtained cells were stained with acridine orange / propidium iodide (AO / PI) and then counted under a microscope and the cell viability was detected using a fluorescent cell counter.

[0098] (11) Final resuspending of cells for loading onto the microarray: Use 1640 + 0.04% BSA to adjust the cell concentration to 700-1200 cells / μL and immediately use for 10x single cell loading onto the microarray.

[0099] Example 1 The method of the present invention was used to dissociate a trace amount (about 25 mg) of liver puncture tissue, and the final single cell count obtained was 60,000, the cell viability was 91%, the agglomeration rate was 8%, and the fragmentation rate was less than 20%.

[0100] at the same time Figure 1 This is a diagram showing the dissociation effect of the present invention. Figure 1 A is a picture of the collected liver puncture sample. Figure 1 B is a microscopic image of a single cell suspension of liver puncture tissue prepared by the method of the present invention. It can be seen that the single cell suspension dissociated by the method of the present invention has a high cell viability, a clean background, and relatively low cell agglomeration and fragmentation rates. The quality of the single cell suspension can meet the requirements for single cell sequencing.

[0101] Comparative Example 1 Human hepatitis puncture tissue was subjected to single cell dissociation tissue processing using conventional methods and the experimental steps included:

[0102] (1) Collection of tissue samples: A piece of freshly collected human inflamed liver puncture tissue was placed in a culture dish pre-cooled on ice. After removing stains, it was thoroughly washed twice with ice-cold PBS (pH 7.4) (Gibco, 10010031), 1 ml each time.

[0103] (2) Tissue preservation and transfer: Transfer the tissue and immerse it in a centrifuge tube containing tissue protection solution and store or transport it at 4°C for no more than 48 hours.

[0104] (3) Initial dissociation of liver cells: Remove the liver tissue and place it in a small culture dish pre-cooled on ice. Wash it twice with 1 ml of pre-cooled culture medium (1640 + 0.04% BSA) on ice. Use sterile scissors to cut it into chyme, transfer the tissue to a 15 ml centrifuge tube, and add 3 ml of digestion solution. The formula of the digestion solution is: 1640 culture medium + 0.04% BSA + 0.2% collagenase II (Gibco, #17101015) + 0.25% trypsin and 0.1 mg / ml DNase I (Applichem, #A3778). Digest in a hybridization oven at 37°C and 22 rpm for 30 minutes. Take out the centrifuge tube and use a wide-mouthed pipette to gently pipette the suspension to help the tissue disperse and mix. (4) Rinse the cell sieve with culture medium (1640 + 0.04% BSA), pass the cell suspension after digestion through a 40 μm cell sieve, wash the original centrifuge tube twice with 2 ml of culture medium, pass the washing solution through a sieve and combine it with the previously sieved cell suspension into one tube, and centrifuge at 4°C and 400 g for 10 min.

[0105] (5) Add 2 mL of erythrocyte lysis buffer (Miltenyi Biotec, lot #130-094-183) to the cell pellet and resuspend it. Place it on ice for 3 min, and add 10 ml of culture medium to stop erythrocyte lysis. Centrifuge at 4°C and 500 g for 5 min. Resuspend the cell pellet with 1 mL of 1640 + 0.04% BSA and place it on ice.

[0106] (6) Microscopic counting: The obtained cells were stained with acridine orange (AO) / propidium iodide (PI) and then counted under a microscope and tested for cell viability using a fluorescent cell counter.

[0107] (7) Final re-suspending of cells for loading: Centrifuge the cells obtained in (5) at 4°C, 400 g for 5 min. Resuspend the cell pellet with 200 μL 1640 + 0.04% BSA, count under a microscope, and adjust the concentration to the concentration required for loading on the 10xGenomics single cell machine (700-1200 cells / μL) for loading on the 10x single cell machine.

[0108] Comparative Example 1 used conventional methods to dissociate a small amount (about 25 mg) of liver puncture tissue, and the final single cell count obtained was 11,000, the cell viability was 77.5%, the agglomeration rate was 10%, and the fragmentation rate was 35%. Figure 2 This is a diagram showing the dissociation effect of conventional methods. Figure 2 A is a picture of the collected liver puncture sample. Figure 2 B is a microscopic examination of a single-cell suspension of liver biopsy tissue prepared using conventional methods.

[0109] It can be seen that compared with the method described in the present invention, the single cell suspension dissociated by conventional methods has a small number of cells, a relatively low cell viability, a relatively high cell aggregation and fragmentation rate, and the quality of the cell suspension cannot meet the requirements of single cell sequencing. Figure 3 and Figure 4 The single-cell sequencing quality control results, i.e., cell type identification results, obtained by the method of the present invention and the conventional method are shown respectively. Figure 3 A and Figure 4 A is the method of the present invention, Figure 3 B and Figure 4 B is a conventional method. It can be seen that in the sequencing data obtained by the conventional method, the proportion of mitochondrial genes is very high (>20%), the number of genes detected in each cell is relatively small (<1500), the number of cell types is small, and multiple cell types are lost. In the sequencing data obtained by the method described in the present invention, the proportion of mitochondrial genes is low (<5%), the number of genes detected in each cell is relatively small (>1500), and the cell types are rich. Not only can relatively fragile cell types such as parenchymal cells, bile duct cells, and endothelial cells be detected, but the proportion of B cells detected is significantly higher.

[0110] Example 2 Human liver cancer puncture tissue was subjected to single cell dissociation and sequencing using the method of the present invention. The tissue processing and experimental steps included:

[0111] (1) Collection of tissue samples: Two freshly collected human liver cancer puncture tissues (about 50 mg) were placed in a pre-cooled culture dish on ice. After removing stains, the culture dish was incubated with pre-cooled culture medium PBS (pH 7.4) (Gibco, 10010031).

[0112] Wash thoroughly twice, 1 ml each time.

[0113] (2) The tissue was transferred and immersed in 500 μl of PBS containing 1 mM DTSSP (Thermo, #21578) for 2 hours on ice.

[0114] (3) Tissue preservation and transfer: Transfer the tissue and immerse it in a centrifuge tube containing tissue protection solution and store or transport it at 4°C for no more than 48 hours.

[0115] (4) Initial dissociation of liver cells: Remove the liver tissue and place it in a small culture dish pre-cooled on ice. Wash it twice with 1 ml of pre-cooled culture medium (1640 + 0.04% BSA) on ice. Cut it into chyme with sterile scissors, transfer the tissue to a 5 ml centrifuge tube, and add 2.5 ml of digestion solution I. The formula of digestion solution I is: RPMI

[0116] 1640 medium (Gibco, 11875093) + 0.04% (W / V) BSA (MACS, 130-091-376)

[0117] +25mM DTT+0.2% collagenase II (Gibco, #17101015)+0.2% collagenase IV (Gibco, 17104019)+0.1mg / ml DNase I (Applichem, #A3778), digest for 20min at 37°C and 22rpm in a hybridization oven. Take out the centrifuge tube and gently pipette the suspension to help disperse and mix the tissue.

[0118] (5) Continue cell dissociation: Add trypsin with a final concentration of 0.25% (W / V) to the initially dissociated cell suspension (the digestion solution at this time is named digestion solution II, with a total volume of about 3 mL), and place in a hybridization oven at 37°C and 22 rpm to continue digestion for 5 min.

[0119] (6) Rinse the 40 μm cell sieve thoroughly with 2 mL of culture medium (1640 + 0.04% BSA), add 6 mL (2 times the volume) of culture medium to the cell suspension obtained in (5) to terminate digestion, pass through a 40 μm cell sieve, wash the original centrifuge tube twice with culture medium, 3 mL each time, pass the washing solution through a sieve and combine it with the previously sieved cell suspension into one tube, and centrifuge at 4°C, 300 g for 5 min.

[0120] (7) The supernatant was transferred to a new centrifuge tube and centrifuged a second time at 400G for 7 min at 4°C. The precipitate was resuspended in 200 μL of culture medium and transferred to the cell precipitate obtained in (6).

[0121] (8) Add 2 mL of erythrocyte lysis buffer (Miltenyi Biotec, lot #130-094-183) to the cell pellet and resuspend it. Place it on ice for 3 min and add 10 mL of culture medium to terminate erythrocyte lysis. Centrifuge at 400 g for 7 min at 4°C. Resuspend the cell pellet in 3 mL of culture medium and wash it.

[0122] (9) Centrifuge at 4°C and 300 g for 7 min. Resuspend the cell pellet in 100 μL 1640 + 0.04% BSA and place on ice.

[0123] (10) Microscopic counting: The obtained cells were stained with acridine orange / propidium iodide (AO / PI) and then counted under a microscope and the cell viability was detected using a fluorescent cell counter.

[0124] (11) Final resuspending of cells for loading onto the microarray: Use 1640 + 0.04% BSA to adjust the cell concentration to 700-1200 cells / μL and immediately use for 10x single cell loading onto the microarray.

[0125] Example 2 The method of the present invention was used to dissociate a small amount (about 50 mg) of liver puncture tissue, and the final single cell count obtained was 100,000, with a cell viability of 96.5%, a clumping rate of 7%, and a fragmentation rate of 19%. Figure 5 The dissociation effect diagram and sequencing data results are displayed. Figure 5 A is a picture of liver puncture sampling specimen; Figure 5 B is a microscopic examination of a single cell suspension of liver puncture tissue; Figure 5 C is a violin plot of the number of genes, number of reads, and proportion of mitochondrial genes in single-cell sequencing data; Figure 5 D is the umap cluster diagram of the cell type of the single cell sequencing data. It can be seen that the high-quality cell suspension was obtained by adopting the method of the present invention, and the proportion of mitochondrial genes in the sequencing data was low, the number of genes detected was high, the cell types were rich, including fragile cell types such as parenchymal cells, and a higher proportion of B cells were detected.

[0126] In summary, a method for preparing a single cell suspension from liver puncture tissue dissociation of the present invention comprises:

[0127] The collected primary liver puncture tissue was washed with 1640 culture medium containing BSA, placed in a tissue fixative, fixed on ice, and then placed in a tissue preservation solution for low-temperature storage to obtain a primary liver puncture tissue with stable tissue;

[0128] Within 48 hours, the primary liver biopsy tissue stabilized by the above treatment is subjected to single cell dissociation treatment to form a liver biopsy tissue dissociated single cell suspension, specifically including:

[0129] The stabilized primary liver puncture tissue obtained through the above treatment is placed in a culture dish pre-cooled on ice, the tissue is washed several times with culture medium, and minced into minced pieces;

[0130] The liver tissue was initially digested using digestion solution I containing collagenase, deoxyribonucleotidase I, and dithiothreitol;

[0131] The liver tissue was digested again with digestion solution II, and the obtained cell suspension was sieved, and red blood cells were lysed and washed to obtain a liver puncture tissue dissociated single cell suspension.

[0132] The tissue fixative is PBS containing 0.25-5 mM DTSSP, and the fixation time on ice is 1.5-2.5 hours.

[0133] Wherein, the tissue preservation solution is Miltenyi tissue preservation solution; the low-temperature preservation refers to preservation at 2-8°C; and the preservation time is less than 48 hours.

[0134] The digestion solution I includes 1640 culture medium, 0.03%-0.05% BSA, 0.1%-0.3% M / V collagenase II, 0.1%-0.3% M / V collagenase IV, 0.05%-0.15 mg / mL DNase I, and 20-50 mM DTT.

[0135] Among them, digestion solution II is digestion solution I with trypsin added.

[0136] Among them, DTT in digestion solution I reduces the disulfide bonds in the spacer arm to release the cross-linking effect of DTSSP on the protein; the reducing conditions are 37°C and 20-30min.

[0137] The digestion conditions for the initial digestion and dissociation of liver tissue are 35°C-39°C, 21rpm-23pm; and the digestion time is 20-25min.

[0138] Wherein, trypsin is used to continue digesting the liver suspension, so that the liver tissue is thoroughly digested and dissociated into single cells; the final concentration of trypsin is 0.23%-0.27% W / V, and the digestion time is 5-10 minutes.

[0139] Although the present invention is described in detail above, the present invention is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made based on the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. A method for preparing a single cell suspension from liver puncture tissue dissociation, comprising: The collected primary liver puncture tissue is washed with PBS or 1640 medium containing BSA, placed in a tissue fixative, fixed on ice, and then placed in a tissue preservation solution for low-temperature storage to obtain a primary liver puncture tissue with stable tissue; Within 48 hours, the primary liver biopsy tissue stabilized by the above treatment is subjected to single cell dissociation treatment to form a liver biopsy tissue dissociated single cell suspension, specifically including: The stabilized primary liver puncture tissue obtained through the above treatment is placed in a culture dish pre-cooled on ice, the tissue is washed several times with culture medium, and minced into minced pieces; The liver tissue was initially digested using digestion solution I containing collagenase, deoxyribonucleotidase I, and dithiothreitol; The liver tissue was digested again with digestion solution II, and the obtained cell suspension was sieved, and red blood cells were lysed and washed to obtain a liver puncture tissue dissociated single cell suspension.

2. The preparation method according to claim 1, wherein the tissue fixative is PBS containing 0.25-5 mM DTSSP.

3. The method for preparing a single-cell suspension from liver puncture tissue dissociation according to claim 2, wherein the fixation time on ice is 1.5-2.5 hours.

4. The preparation method according to claim 1, wherein the tissue preservation solution is Miltenyi tissue preservation solution; the low temperature preservation refers to preservation at 2-8°C; and the preservation time is less than 48 hours.

5. The method for preparing a single-cell suspension of liver puncture tissue dissociation according to claim 1, wherein the digestion solution I comprises 1640 culture medium, 0.03%-0.05% BSA, 0.1%-0.3% M / V collagenase II, 0.1%-0.3% M / V collagenase IV, 0.05%-0.15 mg / mL DNase I, and 20-50 mM DTT.

6. The preparation method according to claim 5, wherein the digestive solution II is obtained by adding trypsin to the digestive solution I.

7. The method for preparing a single-cell suspension from liver puncture tissue dissociation according to claim 5, wherein the DTT in the digestive solution I reduces the disulfide bonds in the spacer arm to release the cross-linking effect of DTSSP on the protein; the reducing conditions are 37°C and 20-30min.

8. The method for preparing a single-cell suspension from liver puncture tissue dissociation according to claim 5, wherein the digestion conditions for the initial digestion and dissociation of the liver tissue are 35°C-39°C, 21rpm-23pm; and the digestion time is 20-25min.

9. The method for preparing a single-cell suspension from liver puncture tissue dissociation according to claim 5, wherein the trypsin is used to continue digesting the liver suspension so that the liver tissue is completely digested and dissociated into single cells; the final concentration of trypsin is 0.23%-0.27% W / V, and the digestion time is 5-10 min.

10. A method for single-cell sequencing of liver biopsy tissue, comprising: Using the preparation method according to any one of claims 1 to 9 to prepare a liver puncture tissue dissociated single cell suspension; The liver puncture tissue dissociated single cell suspension is sequenced using a sequencing device.

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