A method for preparing a liver tissue sample suitable for single cell sequencing

Abstract

The application discloses a liver tissue sample preparation method for single cell sequencing, comprising ex vivo liver tissue perfusion treatment and single cell suspension preparation method. The application also discloses a kit used in the foregoing method. The application makes up the shortage of too few or lost cells of some cell types caused by previous experimental methods. The application also discloses the application of the method in single cell sequencing, single cell suspension preparation and liver primary cell dissociation.

Description

Technical Field

[0001] This invention relates to the field of single-cell sequencing technology, and in particular to a technical method for single-cell sequencing suitable for the preservation of ex vivo liver tissue samples. Background Technology

[0002] Single-cell or single-nucleus sequencing technology is increasingly widely used in clinical and research fields, enabling studies at the single-cell level on growth and development, disease occurrence, tumor formation and development, tumor microenvironment, gene editing, or gene therapy. Single-cell sequencing first requires the preparation of high-quality single-cell suspensions. Common methods for preparing single-cell suspensions include mechanical methods, homogenization methods, and enzymatic digestion methods. Among these, enzymatic digestion methods are suitable for samples derived from most animal organs or tissues.

[0003] The liver is a vital organ for metabolism and immune function. Therefore, from both clinical and research perspectives, single-cell sequencing of human or animal liver or liver cancer tissue to obtain rich single-cell gene expression data is crucial for understanding the development of liver diseases or tumors and for developing related therapeutic or preventative drugs. However, the liver's basic structural unit (hepatic lobule) is complex. Hepatocytes, which make up about 70% of the total liver cells, are sandwiched between sinusoidal endothelial cells, and numerous tubular structures (blood vessels, bile ducts, etc.) run through them. Furthermore, the liver is a metabolically active organ; once isolated, hepatocytes, which perform liver function, can no longer perform aerobic glycolysis, and their activity rapidly declines. Therefore, single-cell sequencing data obtained from isolated liver tissue after mechanical or enzymatic digestion primarily consists of non-parenchymal cells (NPCs) and immune cells, with hepatocytes making up a small proportion, sometimes even too few for downstream data analysis. Furthermore, the rupture of dead liver parenchymal cells can lead to a high fragmentation rate in single-cell suspensions, which in turn affects the capture efficiency of other normal living cells and the final data quality.

[0004] Compared to single-cell sequencing, nuclear sequencing allows for the direct extraction of cell nuclei from liquid nitrogen-frozen tissues to prepare single-cell nuclear suspensions for subsequent sequencing and analysis. Single-cell nuclear sequencing of liver tissue can yield a large number of hepatocytes. However, because hepatocytes constitute a high percentage (approximately 70%) of liver tissue, the single-cell nuclear sequencing data from liver tissue shows an excessively high proportion of hepatocytes and a low proportion of immune cells and non-parenchymal cells. This poses a significant obstacle for researchers to conduct more in-depth studies (such as the immune microenvironment) and fails to meet their experimental and data needs.

[0005] Currently, the most widely used single-cell sequencing platforms are 10×Genomics and BD platforms, both of which require relatively expensive equipment and reagents, and demand extensive experimental experience from the researchers to ensure success. Therefore, experiments such as single-cell preparation, library construction, and high-throughput sequencing of single-cell sequencing samples still largely rely on technical service companies. However, from the time fresh samples are collected and placed in tissue preservation solution to their delivery to the service company under low-temperature (0-4℃) conditions, it typically takes 24–36 hours, sometimes even longer. For relatively fragile liver tissue (especially hepatocytes), excessively long in vitro transport time can lead to a significant decrease in cell viability and a large proportion of apoptosis or death of parenchymal cells.

[0006] Therefore, there is an urgent need for a method to prepare ex vivo liver tissue samples that is suitable for single-cell sequencing and can simultaneously obtain a richer and more comprehensive range of cell types. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a liver tissue processing and dissociation method that is relatively simple to operate and can obtain multiple cell types, including liver parenchymal cells, non-parenchymal cells, and immune cells, for single-cell sequencing in a single operation.

[0008] This invention provides a method for preparing liver tissue samples for single-cell sequencing, specifically including the following steps:

[0009] I. Perfusion treatment of isolated liver tissue

[0010] The liver tissue was cleaned and perfused with lavage solution I to obtain lavage material, which was then set aside. Then, lavage solution II was perfused into the liver tissue, and the tissue was allowed to stand in culture medium to obtain perfused liver tissue blocks.

[0011] In step (1), the irrigation solution I is an HBSS solution that does not contain calcium or magnesium ions.

[0012] In step (I), the specific steps for obtaining the lavage fluid by perfusing lavage fluid I are as follows: Using a sterile syringe, pre-cooled lavage fluid I at 4°C is evenly perfused to multiple different sites in order from the center of the liver tissue block to the periphery. The perfusion sites are the central vein and portal vein, or the surrounding blood vessels. During perfusion, the syringe should be perpendicular to the tissue surface. Each site should be perfused at least twice, with 200-500 μl of lavage fluid I each time. The visible red residual blood (approximately 1-4 ml depending on the tissue size) flowing out at the beginning of the perfusion should be discarded. Continue perfusing each site 2-3 times, and collect the lavage fluid that is no longer visible as red. Stop perfusion.

[0013] In this invention, 0.5mm is generally used. 3For tissue blocks of approximately 100 mg in size, the total volume of irrigation fluid I is 2.0-4.0 ml. This invention increases the volume of irrigation fluid I proportionally according to the size of the tissue block.

[0014] In step (i), the lavage fluid obtained by perfusion I refers to the lavage fluid that flows out of the liver tissue from the point where no red color is visible to the naked eye during collection until the end of perfusion with lavage fluid I.

[0015] In step (I), the components and contents of the irrigation solution II are: 0.5-2 ml irrigation solution I, 3-8% trehalose, and 5-10 mg / mL Matrigel matrix gel.

[0016] In step (I), the specific steps for perfusing irrigation fluid II are as follows: using a pre-cooled sterile syringe, the pre-cooled irrigation fluid II is perfused vertically and evenly into different parts of the liver tissue according to the same perfusion site and similar perfusion method as irrigation fluid I (central vein and portal vein or surrounding blood vessels), with each site perfused 1-2 times, 100 μl each time.

[0017] In this invention, 0.5mm is generally used. 3 For tissue blocks of approximately 100 mg in size, the volume of irrigation fluid II is 0.5-1.0 ml. This invention increases the volume of irrigation fluid II proportionally according to the size of the tissue block.

[0018] In this invention, the ratio of irrigation solution I to irrigation solution II is approximately 4:1.

[0019] In step (1), the settling temperature is 22-35℃; the settling time is 5-10 minutes.

[0020] II. Methods for preparing single-cell suspensions

[0021] 1. Preparation of single-cell suspension from lavage fluid:

[0022] The cells were collected by centrifugation of the lavage material prepared in step (I) above; after resuspending in culture medium, red blood cell lysis buffer was added and the mixture was allowed to stand at room temperature; then the cells were collected by centrifugation a second time. After no red blood cells remained, the cells were resuspended in culture medium to obtain a single-cell suspension of the lavage material.

[0023] The conditions for the first centrifugation were: 4℃, 500g, centrifugation for 5-10 minutes;

[0024] The time for standing at room temperature is 3-5 minutes;

[0025] The conditions for the second centrifugation were: 4℃, 500g, centrifugation for 5-10 minutes.

[0026] 2. Preparation of single-cell suspension from liver tissue:

[0027] The liver tissue blocks perfused in step (I) were washed with DMEM culture medium, chopped, enzymatically digested, sieved, centrifuged for the first time, and then resuspended to obtain a single-cell suspension.

[0028] The enzymes used in the enzymatic hydrolysis refer to collagenase II, collagenase IV, and DNase I at a final concentration of 0.1-0.25% and 20-40 U / ml, respectively.

[0029] The enzymatic digestion time is 30-40 minutes.

[0030] The conditions for the first centrifugation were 4℃, 300-500g, for 5-10 minutes.

[0031] The present invention also includes a single-cell sequencing step: the prepared lavage single-cell suspension is combined with the single-cell suspension, fragments are removed, and then the mixture is centrifuged, washed, resuspended, and sequenced.

[0032] In one specific implementation, the method includes the following steps:

[0033] I. Perfusion treatment of isolated liver tissue

[0034] 1. Preparation of Matrigel: One day before use, take the pre-parsed Matrigel (Corning, CAT#354248, concentration 8-12 mg / mL) out from -20℃, place it on ice, and put it in the refrigerator (4℃) overnight to thaw.

[0035] 2. Freshly cut liver tissue was immediately placed in a sterile culture dish pre-cooled on ice and the surface blood was washed away with 1×PBS (pH=7.4) pre-cooled at 4°C.

[0036] 3. Using a sterile syringe, evenly infuse 4°C pre-cooled irrigation fluid I (HBSS solution free of calcium and magnesium ions) into multiple different sites, starting from the center of the liver tissue block and moving outwards. Infuse the central vein and portal vein, or areas surrounding these vessels. Hold the syringe perpendicular to the tissue surface during infusion. Infuse each site at least twice, using 200-500 μl each time, until the outflowing irrigation fluid shows no visible red color. Generally, 0.5 mm... 3 For tissue blocks of approximately 100 mg in size, the total volume of irrigation fluid I is 2.0-4.0 ml. Increase the volume of irrigation fluid I proportionally according to the size of the tissue block.

[0037] 4. After perfusion with lavage fluid I, the lavage material flowing out of the liver tissue (the lavage material refers to the lavage material flowing out of the liver tissue from the point when no visible red color is collected until the end of perfusion with lavage fluid I) is transferred to a new sterile centrifuge tube using a sterile pipette tip and stored at 2-8°C for later use.

[0038] 5. Prepare perfusion solution II on ice: First, add 250 μl of pre-prepared and filtered sterilized 20% trehalose to 800 μl of perfusion solution I, mix thoroughly by pipetting, and prepare perfusion solution I containing 5% trehalose. Pre-cool on ice. According to the expected usage, use a pre-cooled pipette tip to draw an appropriate amount of Matrigel matrix gel melted overnight at 4°C into the pre-cooled perfusion solution I containing 5% trehalose, mix thoroughly by pipetting, and place on ice for later use (perfusion solution II).

[0039] 6. Using a pre-cooled sterile syringe, vertically and evenly infuse the pre-cooled irrigation fluid II into different parts of the liver tissue, following the same infusion sites and similar infusion methods as irrigation fluid I (central veins, portal veins, and other vessels or perivascular areas). Infuse each site 1-2 times, 100 μl each time. Generally, 0.5 mm... 3 For tissue blocks of approximately 100 mg in size, use 0.5-1.0 ml of irrigation fluid II. Increase the volume of irrigation fluid II proportionally according to the size of the tissue block. The ratio of irrigation fluid I to irrigation fluid II is approximately 4:1.

[0040] 7. Transfer the liver tissue treated with irrigation solution II to a sterile environment at 22-35℃. Add 1 ml of calcium- and magnesium-free culture medium (such as DMEM) at 22-35℃ to the culture dish and let it stand for 5-10 minutes to allow the matrix gel to completely solidify in the liver tissue.

[0041] The matrix gel is characterized by melting at 4°C and solidification at 22-35°C. This invention utilizes the temperature-sensitive properties of the matrix gel, allowing it to solidify in perfused blood vessels and other sites while simultaneously providing support and nutrition to hepatocytes. This reduces damage to hepatocytes caused by hypoxia during prolonged immersion in tissue preservation fluid, and enhances the viability of hepatocytes during subsequent single-cell dissociation.

[0042] 8. The perfused liver tissue blocks can be immersed in cryovials containing Miltenyi tissue preservation solution (Cat#130-100-008) and sent to the company at low temperature (2-8℃) within 48 hours along with the perfusion material to complete the preparation of single-cell suspension.

[0043] II. Methods for preparing single-cell suspensions

[0044] 1. Preparation of single-cell suspension from lavage fluid:

[0045] (1) The irrigated material is centrifuged at 4℃ and 500g for 5-10 min (preferably, centrifuged at 4℃ and 500g for 10 min) to collect cells.

[0046] (2) Discard the supernatant, add 100-300 μl of culture medium to resuspend the cells according to the amount of cell pellet, then add 10 times the volume of Miltenyi erythrocyte lysis buffer (Cat#130-094-183), mix well by pipetting, and let stand at room temperature for 3-5 min (preferably 5 min).

[0047] (3) Add an equal volume of culture medium and mix well. Immediately centrifuge at 4℃ and 500g for 5-10 min (preferably, centrifuge at 4℃ and 500g for 10 min) to collect the cells.

[0048] (4) If there are residual red blood cells in the precipitate, repeat steps 2 and 3 once. After confirming that there are no residual red blood cells, add an appropriate amount of culture medium to resuspend the cells according to the amount of precipitate.

[0049] (5) Take 9-18 μl of cell suspension, add 1-2 μl of AO / PI fluorescent dye, mix well, and then perform fluorescence counting and quality inspection.

[0050] 2. Preparation of single-cell suspension from liver tissue:

[0051] (1) Transfer the liver tissue perfused with matrix gel from the tissue preservation solution to a sterile culture dish, place it on an ice plate, and wash it twice with DMEM medium (unless otherwise specified, all the mediums mentioned below are DMEM medium containing 0.04% BSA), and discard the washing solution.

[0052] (2) Add 1 ml of culture medium to the culture dish and use sterile surgical scissors to cut the tissue block into a paste in the culture medium.

[0053] (3) Transfer the minced tissue to a 15ml centrifuge tube using a disposable dropper. Wash the culture dish 2-3 times with culture medium, transferring the washings to the 15ml centrifuge tube as well. Adjust the culture medium volume to 3-6ml based on the amount of tissue. Add collagenase II, collagenase IV, and DNase I to a final concentration of 0.1-0.25% and 20-40U / ml respectively (preferably, add collagenase II, collagenase IV, and DNase I to a final concentration of 0.2% and 30U / ml respectively). Place the tube in a hybridization oven at 37℃ and 20-25rpm for 30-40 minutes for enzymatic digestion.

[0054] (4) After digestion, the tissue is blown and aspirated 5-8 times with a dropper, transferred to a 40μm cell sieve and filtered into a 15ml centrifuge tube. The cell sieve is washed 3-4 times with culture medium and the washing solution is also transferred to a 15ml centrifuge tube.

[0055] (5) Centrifuge at 300-500g at 4℃ for 5-10 min (preferably, centrifuge at 500g at 4℃ for 8-10 min).

[0056] (6) Based on the amount of cell pellet, resuspend the cells with an appropriate amount of culture medium, stain with AO / PI fluorescent dye (9 μl of cell suspension mixed with 1 μl of dye), and then detect the cell number, cell size, cell viability and clumping rate on an automated fluorescence counter.

[0057] The single-cell suspension of the lavage fluid obtained by the method of the present invention consists of immune cells, etc., remaining in the blood vessels or microenvironment of the liver tissue block; the single-cell suspension of the liver tissue consists of liver parenchymal cells, non-parenchymal cells, and immune cells, etc., in the tissue microenvironment.

[0058] The immune cells mentioned include lymphocytes, dendritic cells, macrophages, etc.

[0059] The non-solid cells mentioned are endothelial cells, Kuffer cells, stellate cells, etc.

[0060] The following steps (7)-(10) are the single-cell sequencing preprocessing steps:

[0061] (7) Combine the single-cell suspension of the irrigation material prepared above with the single-cell suspension prepared in step (6) into the same 15ml centrifuge tube.

[0062] (8) De-debris removal: Use MACS debris removal solution (Meitianni, Cat#130-109-398) to perform de-debris removal according to the standard operating procedure in the product manual.

[0063] (9) Washing: Resuspend the cells in 1-2 ml of culture medium and centrifuge at 300 g for 5-8 min at 4℃. Repeat the washing once.

[0064] (10) The cell pellet is resuspended in an appropriate amount of 1×PBS (generally 100-1000μl) and the suspension concentration is adjusted to 700-1200 cells / μl. It is then placed on ice for later use.

[0065] 3.10×Genomics platform single-cell sequencing:

[0066] Using the 10×Genomics single-cell sequencing platform, the separately prepared liver tissue single-cell suspension and the irrigation fluid single-cell suspension were mixed with equal or specific cell numbers, and the sequencing, library preparation and next-generation sequencing were completed according to the 10×Genomics transcriptome sequencing protocol.

[0067] This invention also proposes applications of the method in single-cell sequencing, single-cell suspension preparation, and primary liver cell dissociation.

[0068] The key technical point of this invention is the ex vivo liver tissue perfusion treatment in the liver tissue sample preparation method for single-cell sequencing proposed in this invention. On the one hand, this invention utilizes the temperature-sensitive properties of matrix gel to support and protect hepatocytes that have been immersed in tissue preservation solution for a long time; on the other hand, the addition of 5% trehalose to perfusion solution II helps to maintain a high viability of hepatocytes during the subsequent single-cell dissociation process.

[0069] The present invention also provides a kit comprising perfusion solution I (HBSS solution free of calcium and magnesium ions), Matrigel matrix gel (8-20 mg / mL, stored at -20°C), and trehalose stock solution (20%, filtered for sterilization).

[0070] This invention also provides the application of the kit in single-cell sequencing, single-cell flow cytometry analysis or sorting, single-cell proteomics, etc.

[0071] The advantages of this invention are:

[0072] 1. In single-cell sequencing, existing methods for single-cell dissociation of ex vivo liver tissue samples yield only a few or no hepatocytes. This invention prepares single-cell suspensions separately from liver tissue lavage fluid (containing immune cells in the microenvironment) and liver tissue (hepatocytes and non-parenchymal cells). (The lavage fluid obtained using lavage fluid I in this invention mainly contains cells from the blood vessels and microvessels of the tissue block, including erythrocytes and immune cells from the tissue microenvironment. Without separate collection and preparation of single-cell suspensions, some immune cells from the tissue microenvironment would be lost. The purpose of lavage with lavage fluid I in this invention is to remove most of the blood cells from the blood vessels and microvessels, facilitating perfusion with lavage fluid II. A two-step perfusion method is commonly used to obtain hepatocytes: the first step is perfusion with culture medium.) After rinsing the blood out of the blood vessels, the second step is to in-situ dissociate the tissue by perfusing digestive enzyme solution, and then collect the dissociated hepatocytes. However, conventional methods, when dissociating liver tissue samples soaked in tissue preservation solution for 48 hours into a single-cell suspension, mainly yield immune cells and other non-parenchymal cells, making it difficult to obtain hepatocytes. Therefore, this invention innovatively proposes collecting the lavage fluid I separately and using lavage fluid II to increase the proportion of hepatocytes in the suspension during subsequent dissociation. Then, according to experimental needs, these are mixed in a certain proportion for sequencing, overcoming the shortcomings of currently used experimental methods that result in insufficient cell numbers or loss of certain cell types.

[0073] The purpose of this invention is to obtain a relatively rich variety of cell types, such as hepatocytes, non-parenchymal cells, and immune cells, from single-cell sequencing data, which is conducive to a more comprehensive mining of data information and biological significance.

[0074] 2. Matrices are used for organoid culture and in-situ perfusion at surgical sites to provide support or protection. This invention is the first to use Matrices for perfusion of isolated liver tissue. Utilizing the temperature-sensitive properties of Matrices (melting at 0-4℃ and rapid solidification at 22-37℃), it provides support and nutrition to isolated liver tissue, minimizing apoptosis and necrosis of hepatocytes (especially fragile hepatocytes) during long-distance transport.

[0075] The application of matrix gel for perfusion of ex vivo liver tissue in this invention is not a routine or easily conceived procedure. The technical difficulties this invention needs to overcome include: ① The application of matrix gel is not widespread, and its properties are not universally understood. Currently, it is mainly used in organoid culture and organ remodeling. Organoid culture requires obtaining relatively pure cells of a specific functional type (such as hepatocytes), and avoids mixing multiple cell types. Single-cell sequencing, on the other hand, aims to obtain a richer variety of cell types for data mining. Therefore, there has been little overlap between organoid and single-cell sequencing technologies. Only recently, with technological advancements, have a few organoid researchers begun to attempt single-cell sequencing studies on organoids. ② The currently mature method for preparing hepatocytes is the two-step perfusion method. However, this method requires in situ perfusion of liver tissue in vivo, or perfusion of fresh ex vivo tissue within a very short time (generally within a few hours), which is unsuitable for samples that need to be preserved in tissue preservation solution for a longer period (24-48 hours) for single-cell sequencing. ③ Due to the high cost and technical requirements of single-cell sequencing experiments, most single-cell sequencing projects are currently completed by service companies. However, the dissociation results of some special samples (such as liver tissue) are greatly affected by various factors, including sample collection, storage conditions, storage time, and transportation conditions, from the time researchers collect samples to when they are sent to the company for dissociation. Analyzing the reasons for unsatisfactory dissociation results requires extensive experience in single-cell sequencing projects and strong comprehensive analytical skills. It also requires targeted experimental design and verification for different samples. Those without sufficient project experience and analytical skills will find it difficult to identify the causes and devise solutions. ④ Matte gel is a basement membrane matrix extracted from EHS mouse tumors rich in extracellular matrix proteins. If large quantities are used, the cost is relatively high. However, the tissue sample volume used for single-cell sequencing is small (generally in the milligram range), requiring very little Matte gel (less than 1 ml), making the cost controllable. The current two-step perfusion method for primary hepatocyte dissociation requires a continuous flow of perfusion fluid through the tissue, and the amount of perfusion fluid required is large (generally more than 10 ml). Therefore, given the high cost, it is impractical to obtain liver tissue samples by using matrix gel for perfusion.

[0076] 3. Previous studies have shown that trehalose has a protective effect against liver ischemia-reperfusion injury under both in vivo and in vitro conditions, and can reduce cell apoptosis. This invention discovers that adding an appropriate amount of trehalose to the matrix gel (irrigation solution II) can play a certain protective role in reducing hepatocyte damage during long-term preservation of isolated liver tissue (within 48 hours after in vitro) and during single-cell dissociation. Attached Figure Description

[0077] Figure 1 A is a single-cell suspension of isolated mouse liver tissue prepared using the method of this invention.

[0078] Figure 1 B is a single-cell suspension of isolated mouse liver tissue prepared using the control method.

[0079] Figure 1 C represents the cell atlas and corresponding cell types of mouse isolated liver tissue samples prepared using the method of this invention.

[0080] Figure 1 D represents the cellular atlas and corresponding cell types of mouse ex vivo liver tissue samples prepared using a comparative method. Detailed Implementation

[0081] The invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the invention are all common knowledge and general knowledge in the art, and the invention does not have any particular limitations.

[0082] Example 1: A method for preparing a single-cell suspension from isolated mouse liver tissue samples for single-cell sequencing. Details are as follows:

[0083] (1) The complete liver was removed from the freshly killed mouse and placed in a culture dish on ice. After washing the surface of the liver tissue with PBS pre-cooled at 4°C, the liver tissue was placed in a new culture dish pre-cooled on ice. Using a 5ml syringe, 5ml of pre-cooled irrigation fluid I was slowly injected into the liver through the portal vein. A large amount of blood was observed flowing out, and the color of the liver tissue block in the injection area gradually changed from dark red. Then, 2ml of pre-cooled irrigation fluid I was drawn and injected into the vascular area around the liver tissue block. The color of the tissue in the injection area gradually changed from dark red to light yellow.

[0084] (2) Transfer the rinsing material from the petri dish to a 15ml sterile centrifuge tube using a pipette and store at 4℃ for later use.

[0085] (3) Using a 1ml pre-cooled syringe, draw up the pre-cooled perfusion fluid II from ice and inject it at each injection site as in (1). Inject 600μl into the central vein and 200μl into each of the surrounding sites. Then add 1ml of DMEM medium at 35℃ to the culture dish. Cover the culture dish and immediately transfer the tissue to a 35℃ incubator and let it stand for 5min.

[0086] (4) Place the culture dish containing the perfused liver tissue back on ice, cut it into small pieces the size of soybeans with a sterile blade, immerse it in cryovials containing Miltenyi tissue preservation solution (Cat#130-100-008), and send it to the company at low temperature (4°C) within 48 hours along with the perfused material.

[0087] (5) The lavage fluid was centrifuged at 500g for 10 min at 4℃ to collect cells. 3 ml of Miltenyi erythrocyte lysis buffer (Cat#130-094-183) was added, and the mixture was aspirated and incubated at room temperature for 5 min. The cells were then centrifuged at 300g for 10 min at 4℃ to collect cells. The lysis process was repeated once until no visible red color was visible in the cell pellet. The cells were then resuspended in 500 μl of culture medium. 9 μl of the cell suspension was taken and 1 μl of AO / PI fluorescent dye was added. After mixing, fluorescence counting and quality control were performed.

[0088] (6) Remove the perfused liver tissue from the tissue preservation solution and place it in a pre-chilled sterile culture dish on ice. Wash twice with DMEM medium containing 0.04% BSA, and discard the washing solution. Use sterile surgical scissors to cut the tissue block into a paste in 1 ml of medium, and transfer it to a 15 ml centrifuge tube. Wash the culture dish three times with 5 ml of medium, and transfer the mixture to the centrifuge tube. Add collagenase II (0.2%), collagenase IV (0.2%), and DNase I (30 U / ml) respectively. Incubate at 37°C and 22 rpm for 40 min for enzymatic digestion.

[0089] (7) After digestion, the tissue was blown 8 times and filtered through a 40μm cell sieve into a new centrifuge tube. The cell sieve was washed with an equal volume of culture medium, and the washing solution was also transferred to the centrifuge tube. Centrifuged at 500g for 5 min at 4℃.

[0090] (8) Resuspend the cells in 2.5 ml of culture medium, stain with AO / PI fluorescent dye (9 μl of cell suspension mixed with 1 μl of dye), and then detect the cell number, cell size, cell viability and clumping rate on an automated fluorescence counter.

[0091] (9) Combine the single-cell suspension of the lavage material prepared in (5) with the single-cell suspension in (8) into the same 15ml centrifuge tube.

[0092] (10) De-debris removal: Use MACS debris removal solution (Meitianni, Cat#130-109-398) to perform de-debris removal according to the standard operating procedure in the product manual.

[0093] (11) Washing: Resuspend the cells in 1 ml of culture medium and centrifuge at 300 g for 5 min at 4 °C. Repeat the washing once.

[0094] (12) The cell pellet was resuspended in 200 μl PBS + 0.04% BSA, and the suspension concentration was adjusted to about 1140 cells / μl. It was then placed on ice for later use.

[0095] (13) Complete the sequencing and library construction according to the 10×Genomics single-cell sequencing protocol.

[0096] Comparative Example 1:

[0097] (1) The complete liver was removed from the freshly killed mouse and placed in a culture dish on ice. The surface of the liver tissue was washed with PBS pre-cooled at 4°C. The liver tissue was then cut into small pieces the size of soybeans with a sterile blade and immersed in cryovials containing Miltenyi tissue preservation solution (Cat#130-100-008). The liver tissue was then sent to the company at low temperature (4°C) within 48 hours.

[0098] (2) Liver tissue from the control group was removed from the tissue preservation solution and placed in a pre-chilled sterile culture dish on ice. The tissue was washed twice with DMEM medium containing 0.04% BSA, and the washings were discarded. The tissue block was cut into a paste in 1 ml of medium using sterile surgical scissors and transferred to a 15 ml centrifuge tube. The culture dish was washed three times with 5 ml of medium and transferred to the centrifuge tube. Collagenase II (0.2%), collagenase IV (0.2%), and DNase I (30 U / ml) were added to each tube. The mixture was incubated at 37°C and 22 rpm for 40 min.

[0099] (3) After digestion, the tissue was blown 8 times and filtered through a 40μm cell sieve into a new centrifuge tube. The cell sieve was washed with an equal volume of culture medium, and the washing solution was also transferred to the centrifuge tube. Centrifuged at 500g for 5 min at 4℃.

[0100] (4) Add 3 ml of Miltenyi erythrocyte lysis buffer (Cat#130-094-183), mix well, and incubate at room temperature for 5 min. Centrifuge at 300 g for 10 min at 4℃ to collect cells. Repeat the lysis process once more until no visible red color remains in the cell pellet. Resuspend the cells in 1 ml of culture medium. Take 9 μl of the cell suspension, add 1 μl of AO / PI fluorescent dye, mix well, and then perform fluorescence counting and quality control.

[0101] (5) De-debris removal: Use MACS debris removal solution (Meitianni, Cat#130-109-398) to perform de-debris removal according to the standard operating procedure in the product manual.

[0102] (6) Washing: Resuspend the cells in 1 ml of culture medium and centrifuge at 300 g for 5 min at 4 °C. Repeat the washing once.

[0103] (7) The cell pellet was resuspended in 200 μl PBS + 0.04% BSA, and the suspension concentration was adjusted to 1040 cells / μl. It was then placed on ice for later use.

[0104] (8) Complete the sequencing and library construction according to the 10×Genomics single-cell sequencing protocol.

[0105] Experimental results of Example 1 and Comparative Example 1:

[0106] (1) Quality control results of single-cell suspensions prepared from mouse liver tissue in Example 1 and samples from Comparative Example 1 before being processed (see Appendix) Figure 1 A, 1B):

[0107] Table 1

[0108]

[0109] (2) Comparison of the proportion of each cell type in the single-cell data analysis of mouse liver tissue prepared in Example 1 of the present invention and the sample prepared in Comparative Example 1 (see Appendix) Figure 1 C, 1D):

[0110] Table 2

[0111]

[0112] As can be seen from the comparative data in Table 2, the proportions of immune cells (T cells, B cells, centrifugal cells, etc.) and non-hepatic parenchymal cells (astrocytes, endothelial cells, macrophages, etc.) captured by the conventional method described in Comparative Example 1 (single-cell sequencing of a single-cell suspension prepared from dissociated fresh liver tissue using tissue preservation solution) in the total cells are sufficient for subsequent analysis. However, the proportion of captured mouse hepatic parenchymal cells is very low (0.6%), meaning that only 60 hepatic parenchymal cells were detected out of 10,000 captured cells, which is unsuitable for subsequent bioinformatics analysis. In contrast, Example 1, based on the method described in this invention, not only were appropriate proportions of immune cells and non-hepatic parenchymal cells captured, but also a higher proportion (14.3%) of hepatic parenchymal cells (1430 hepatic parenchymal cells out of 10,000 cells) were captured, which can be used for subsequent bioinformatics analysis and mining of related biological data. These experimental results demonstrate that the implementation method of this invention is significantly effective in mouse liver tissue samples.

[0113] Example 2: A method for preparing a single-cell suspension from a human liver cancer tissue sample for single-cell sequencing.

[0114] Specifically as follows:

[0115] (1) Liver cancer samples and adjacent normal control samples (260 mg and 100 mg, respectively) collected during human liver cancer surgery were immediately placed in culture dishes on ice. After washing the surface of the liver tissue with PBS pre-cooled at 4°C, the liver tissue was placed in a new culture dish pre-cooled on ice. Using a 5 ml syringe, 5 ml of pre-cooled irrigation fluid I was slowly injected into the liver from the visible blood vessels until the liver tissue block in the injection area changed from dark red to light yellow. Then, injections were made into the blood vessel areas around the liver tissue block until the liver tissue block turned light yellow.

[0116] (2) Using a 1ml pre-cooled syringe, draw up the pre-cooled perfusion fluid II from ice and inject 200μl into each injection site as in (1). Then, add 1ml of DMEM medium at 35℃ to the culture dish. Cover the culture dish and immediately transfer the tissue to a 35℃ incubator and let it stand for 5min.

[0117] (3) Immerse the perfused liver tissue in cryopreservation tubes containing Mitenni tissue preservation solution (Cat#130-100-008) and send them to the company at low temperature (4°C) within 48 hours.

[0118] (4) Remove the perfused liver tissue from the tissue preservation solution and place it in a pre-chilled sterile culture dish on ice. Wash twice with DMEM medium containing 0.04% BSA, and discard the washing solution. Use sterile surgical scissors to cut the tissue block into a paste in 1 ml of medium, and transfer it to a 15 ml centrifuge tube. Wash the culture dish 2-3 times with 5 ml of medium, and transfer it to the centrifuge tube. Add collagenase II (0.2%), collagenase IV (0.2%), and DNase I (30 U / ml) respectively. Incubate at 37°C and 22 rpm for 40 min for enzymatic digestion.

[0119] (5) After digestion, the tissue was blown 8 times and filtered through a 40μm cell sieve into a new centrifuge tube. The cell sieve was washed with an equal volume of culture medium, and the washing solution was also transferred to the centrifuge tube. Centrifuged at 500g for 5 min at 4℃.

[0120] (6) Resuspend the cells in 2.5 ml of culture medium, stain with AO / PI fluorescent dye (9 μl of cell suspension mixed with 1 μl of dye), and then detect the cell number, cell size, cell viability and clumping rate on an automated fluorescence counter.

[0121] (7) De-debris removal: Use MACS debris removal solution (Meitianni, Cat#130-109-398) to perform de-debris removal according to the standard operating procedure in the product manual.

[0122] (8) Washing: Resuspend the cells in 2 ml of culture medium and centrifuge at 300 g for 5 min at 4 °C. Repeat the washing once.

[0123] (9) The cell pellet was resuspended in 300 μl PBS + 0.04% BSA, and the suspension concentration was adjusted to 1060 cells / μl for cancer samples and 960 cells / μl for adjacent normal samples. The pellet was then placed on ice for later use.

[0124] (10) Complete the sequencing and library construction according to the 10×Genomics single-cell sequencing protocol.

[0125] Example 2 Experimental Results:

[0126] (1) Quality control results of single-cell suspensions prepared from human liver cancer samples and adjacent normal samples before processing:

[0127] Table 3

[0128]

[0129] (2) In the single-cell data analysis of human liver cancer samples and adjacent normal samples, the proportion of each cell type was as follows:

[0130] Table 4

[0131]

[0132] According to the data in Table 4, Example 2 of this invention describes the application of the method of this invention to human liver cancer and adjacent normal tissue samples. In Example 2, a total of 8558 cells were captured from the liver cancer tissue sample using single-cell sequencing technology, of which 607 were cancer cells (7.1%) and 370 were liver parenchymal cells (4.3%). A total of 10140 cells were captured from the paired adjacent normal tissue sample, of which 1642 were liver parenchymal cells (16.2%). These experimental results demonstrate that the method of this invention can be applied to single-cell sequencing and analysis of human liver cancer tissue and its paired adjacent normal tissue.

[0133] Example 3: A method for preparing a single-cell suspension from a porcine liver tissue sample for single-cell sequencing.

[0134] The specific implementation examples for the experimental group are as follows:

[0135] (1) Weigh approximately 300 mg of freshly removed pig liver tissue and place it in a culture dish on ice. After washing the surface of the liver tissue with PBS pre-cooled to 4°C, place the liver tissue into a new culture dish pre-cooled on ice. Using a 5 ml syringe, draw 5 ml of pre-cooled irrigation fluid I and slowly inject it into the liver from the visible blood vessels. Blood can be seen flowing out, and the color of the liver tissue block in the injection area gradually changes from dark red. Then, draw 5 ml of pre-cooled irrigation fluid I and inject it into the blood vessel areas around the liver tissue block. The color of the tissue in the injection area can be seen to gradually change from dark red to light yellow.

[0136] (2) Transfer the rinsing material from the petri dish to a 15ml sterile centrifuge tube using a pipette and store at 4℃ for later use.

[0137] (3) Using a 1ml pre-cooled syringe, draw up the pre-cooled perfusion fluid II from ice and inject it at each injection site as in (1). Inject 600μl into the central vein and 200μl into each of the surrounding sites. Then add 1ml of DMEM medium at 35℃ to the culture dish. Cover the culture dish and immediately transfer the tissue to a 35℃ incubator and let it stand for 5min.

[0138] (4) Place the culture dish containing the perfused liver tissue back on ice, cut it into small pieces the size of soybeans with a sterile blade, immerse it in cryovials containing Miltenyi tissue preservation solution (Cat#130-100-008), and send it to the company at low temperature (4°C) within 48 hours along with the perfused material.

[0139] (5) The lavage fluid was centrifuged at 500g for 10 min at 4℃ to collect cells. 3 ml of Miltenyi erythrocyte lysis buffer (Cat#130-094-183) was added, and the mixture was aspirated and incubated at room temperature for 5 min. The cells were then centrifuged at 300g for 10 min at 4℃ to collect cells. The lysis process was repeated once until no visible red color was visible in the cell pellet. The cells were then resuspended in 500 μl of culture medium. 9 μl of the cell suspension was taken and 1 μl of AO / PI fluorescent dye was added. After mixing, fluorescence counting and quality control were performed.

[0140] (6) Remove the perfused liver tissue from the tissue preservation solution and place it in a pre-chilled sterile culture dish on ice. Wash twice with DMEM medium containing 0.04% BSA, and discard the washing solution. Use sterile surgical scissors to cut the tissue block into a paste in 1 ml of medium, and transfer it to a 15 ml centrifuge tube. Wash the culture dish three times with 5 ml of medium, and transfer the mixture to the centrifuge tube. Add collagenase II (0.2%), collagenase IV (0.2%), and DNase I (30 U / ml) respectively. Incubate at 37°C and 22 rpm for 40 min for enzymatic digestion.

[0141] (7) After digestion, the tissue was blown 8 times and filtered through a 40μm cell sieve into a new centrifuge tube. The cell sieve was washed with an equal volume of culture medium, and the washing solution was also transferred to the centrifuge tube. Centrifuged at 500g for 5 min at 4℃.

[0142] (8) Resuspend the cells in 2.5 ml of culture medium, stain with AO / PI fluorescent dye (9 μl of cell suspension mixed with 1 μl of dye), and then detect the cell number, cell size, cell viability and clumping rate on an automated fluorescence counter.

[0143] (9) Combine the single-cell suspension of the lavage material prepared in (5) with the single-cell suspension in (8) into the same 15ml centrifuge tube.

[0144] (10) De-debris removal: Use MACS debris removal solution (Meitianni, Cat#130-109-398) to perform de-debris removal according to the standard operating procedure in the product manual.

[0145] (11) Washing: Resuspend the cells in 2 ml of culture medium and centrifuge at 300 g for 5 min at 4 °C. Repeat the washing once.

[0146] (12) The cell pellet was resuspended in 500 μl PBS + 0.04% BSA, and the suspension concentration was adjusted to 1020 cells / μl. It was then placed on ice for later use.

[0147] (13) Complete the sequencing and library construction according to the 10×Genomics single-cell sequencing protocol.

[0148] Comparative Example 2:

[0149] (1) Weigh approximately 300 mg of freshly excised pig liver tissue and place it in a petri dish placed on ice. After washing the surface of the liver tissue with PBS pre-cooled at 4°C, place the liver tissue in a new petri dish pre-cooled on ice and cut it into small pieces the size of soybeans with a sterile blade. Immerse the pieces in cryovials containing Miltenyi tissue preservation solution (Cat#130-100-008) and send them to the company at low temperature (4°C) within 48 hours.

[0150] (2) Liver tissue from the control group was removed from the tissue preservation solution and placed in a pre-chilled sterile culture dish on ice. The tissue was washed twice with DMEM medium containing 0.04% BSA, and the washings were discarded. The tissue block was cut into a paste in 1 ml of medium using sterile surgical scissors and transferred to a 15 ml centrifuge tube. The culture dish was washed three times with 5 ml of medium and transferred to the centrifuge tube. Collagenase II (0.2%), collagenase IV (0.2%), and DNase I (30 U / ml) were added to each tube. The mixture was incubated at 37°C and 22 rpm for 40 min.

[0151] (3) After digestion, the tissue was blown 8 times and filtered through a 40μm cell sieve into a new centrifuge tube. The cell sieve was washed with an equal volume of culture medium, and the washing solution was also transferred to the centrifuge tube. Centrifuged at 500g for 5 min at 4℃.

[0152] (4) Add 3 ml of Miltenyi erythrocyte lysis buffer (Cat#130-094-183), mix well, and incubate at room temperature for 5 min. Centrifuge at 300 g for 10 min at 4℃ to collect cells. Repeat the lysis process once more until no visible red color remains in the cell pellet. Resuspend the cells in 1 ml of culture medium. Take 9 μl of the cell suspension, add 1 μl of AO / PI fluorescent dye, mix well, and then perform fluorescence counting and quality control.

[0153] (5) De-debris removal: Use MACS debris removal solution (Meitianni, Cat#130-109-398) to perform de-debris removal according to the standard operating procedure in the product manual.

[0154] (6) Washing: Resuspend the cells in 2 ml of culture medium and centrifuge at 300 g for 5 min at 4 °C. Repeat the washing once.

[0155] (7) The cell pellet was resuspended in 500 μl PBS + 0.04% BSA, and the suspension concentration was adjusted to 970 cells / μl. It was then placed on ice for later use.

[0156] (8) Complete the sequencing and library construction according to the 10×Genomics single-cell sequencing protocol.

[0157] Experimental results of Example 3 and Comparative Example 2:

[0158] (1) Quality control results of single-cell suspensions prepared from pig liver tissue in Example 3 and Comparative Example 2 before being processed:

[0159] Table 5

[0160]

[0161] (2) Comparison of the proportion of each cell type in the single-cell data analysis of pig liver (experimental group) in Example 3 of the present invention and Comparative Example 2:

[0162] Table 6

[0163]

[0164] As can be seen from the comparative data in Table 6, in the Comparative Example 2 sample sent using conventional methods, mainly immune cells and non-parenchymal cells were captured, while hepatic parenchymal cells accounted for only 1.5%. However, in Example 3 according to the method described in this invention, not only were appropriate proportions of immune cells and non-parenchymal cells captured, but also a higher proportion (15.9%) of hepatic parenchymal cells were captured, which can be used for subsequent bioinformatics analysis and mining of related biological data. These experimental results demonstrate that the implementation method of this invention is significantly effective in porcine liver tissue samples.

[0165] Example 4: A method for preparing single-cell suspensions from rat liver tissue samples for single-cell sequencing.

[0166] The specific implementation examples for the experimental group are as follows:

[0167] (1) Freshly excised rat liver tissue was placed in a culture dish on ice. After washing the surface of the liver tissue with PBS pre-cooled at 4°C, the liver tissue was placed in a new culture dish pre-cooled on ice. Using a 10ml syringe, 8ml of pre-cooled irrigation fluid I was slowly injected into the liver through visible blood vessels. Blood could be seen flowing out, and the color of the liver tissue block in the injection area gradually changed from dark red. Then, 5ml of pre-cooled irrigation fluid I was injected into the blood vessel areas around the liver tissue block. The color of the tissue in the injection area gradually changed from dark red to light yellow.

[0168] (2) Transfer the rinsing material from the petri dish to a 15ml sterile centrifuge tube using a pipette and store at 4℃ for later use.

[0169] (3) Using a 5ml pre-cooled syringe, draw up the pre-cooled perfusion fluid II from ice and inject it into each injection site as in (1). Inject 800μl into the central vein and 300μl into each of the surrounding sites. Then add 2ml of DMEM medium at 35℃ to the culture dish. Cover the culture dish and immediately transfer the tissue to a 35℃ incubator and let it stand for 8min.

[0170] (4) Place the culture dish containing the perfused liver tissue back on ice, cut it into small pieces the size of soybeans with a sterile blade, immerse it in cryovials containing Miltenyi tissue preservation solution (Cat#130-100-008), and send it to the company at low temperature (4°C) within 48 hours along with the perfused material.

[0171] (5) The lavage fluid was centrifuged at 500g for 10 min at 4℃ to collect cells. 3 ml of Miltenyi erythrocyte lysis buffer (Cat#130-094-183) was added, and the mixture was aspirated and incubated at room temperature for 5 min. The cells were then centrifuged at 300g for 10 min at 4℃ to collect cells. The lysis process was repeated once until no visible red color was visible in the cell pellet. The cells were then resuspended in 500 μl of culture medium. 9 μl of the cell suspension was taken and 1 μl of AO / PI fluorescent dye was added. After mixing, fluorescence counting and quality control were performed.

[0172] (6) Remove the perfused liver tissue from the tissue preservation solution and place it in a pre-chilled sterile culture dish on ice. Wash twice with DMEM medium containing 0.04% BSA, and discard the washing solution. Use sterile surgical scissors to cut the tissue block into a paste in 1 ml of medium, and transfer it to a 15 ml centrifuge tube. Wash the culture dish three times with 5 ml of medium, and transfer the mixture to the centrifuge tube. Add collagenase II (0.2%), collagenase IV (0.2%), and DNase I (30 U / ml) respectively. Incubate at 37°C and 22 rpm for 40 min for enzymatic digestion.

[0173] (7) After digestion, the tissue was blown 8 times and filtered through a 40μm cell sieve into a new centrifuge tube. The cell sieve was washed with an equal volume of culture medium, and the washing solution was also transferred to the centrifuge tube. Centrifuged at 500g for 5 min at 4℃.

[0174] (8) Resuspend the cells in 2.5 ml of culture medium, stain with AO / PI fluorescent dye (9 μl of cell suspension mixed with 1 μl of dye), and then detect the cell number, cell size, cell viability and clumping rate on an automated fluorescence counter.

[0175] (9) Combine the single-cell suspension of the lavage material prepared in (5) with the single-cell suspension in (8) into the same 15ml centrifuge tube.

[0176] (10) De-debris removal: Use MACS debris removal solution (Meitianni, Cat#130-109-398) to perform de-debris removal according to the standard operating procedure in the product manual.

[0177] (11) Washing: Resuspend the cells in 2 ml of culture medium and centrifuge at 300 g for 5 min at 4 °C. Repeat the washing once.

[0178] (12) The cell pellet was resuspended in 1000 μl PBS + 0.04% BSA, and the suspension concentration was adjusted to 1150 cells / μl. It was then placed on ice for later use.

[0179] (13) Complete the sequencing and library construction according to the 10×Genomics single-cell sequencing protocol.

[0180] Comparative Example 3:

[0181] (1) Freshly excised rat liver tissue was placed in a culture dish on ice. After washing the surface of the liver tissue with PBS pre-cooled at 4°C, the liver tissue was placed in a new culture dish pre-cooled on ice. It was cut into small pieces the size of soybeans with a sterile blade and immersed in cryovials containing Miltenyi tissue preservation solution (Cat#130-100-008). The tissue was then sent to the company at low temperature (4°C) within 48 hours.

[0182] (2) Liver tissue from the control group was removed from the tissue preservation solution and placed in a pre-chilled sterile culture dish on ice. The tissue was washed twice with DMEM medium containing 0.04% BSA, and the washings were discarded. The tissue block was cut into a paste in 1 ml of medium using sterile surgical scissors and transferred to a 15 ml centrifuge tube. The culture dish was washed three times with 5 ml of medium and transferred to the centrifuge tube. Collagenase II (0.2%), collagenase IV (0.2%), and DNase I (30 U / ml) were added to each tube. The mixture was incubated at 37°C and 22 rpm for 40 min.

[0183] (3) After digestion, the tissue was blown 8 times and filtered through a 40μm cell sieve into a new centrifuge tube. The cell sieve was washed with an equal volume of culture medium, and the washing solution was also transferred to the centrifuge tube. Centrifuged at 500g for 5 min at 4℃.

[0184] (4) Add 3 ml of Miltenyi erythrocyte lysis buffer (Cat#130-094-183), mix well, and incubate at room temperature for 5 min. Centrifuge at 300 g for 10 min at 4℃ to collect cells. Repeat the lysis process once more until no visible red color remains in the cell pellet. Resuspend the cells in 1 ml of culture medium. Take 9 μl of the cell suspension, add 1 μl of AO / PI fluorescent dye, mix well, and then perform fluorescence counting and quality control.

[0185] (5) De-debris removal: Use MACS debris removal solution (Meitianni, Cat#130-109-398) to perform de-debris removal according to the standard operating procedure in the product manual.

[0186] (6) Washing: Resuspend the cells in 2 ml of culture medium and centrifuge at 300 g for 5 min at 4 °C. Repeat the washing once.

[0187] (7) The cell pellet was resuspended in 1000 μl PBS + 0.04% BSA, and the suspension concentration was adjusted to 1179 cells / μl. It was then placed on ice for later use.

[0188] (8) Complete the sequencing and library construction according to the 10×Genomics single-cell sequencing protocol.

[0189] Experimental results of Example 4 and Comparative Example 3:

[0190] (1) Quality control results of rat liver tissue samples from Example 4 and Comparative Example 3 samples before single-cell suspension preparation:

[0191] Table 7

[0192]

[0193] (2) Comparison of the proportion of each cell type in the single-cell data analysis of the rat liver experimental group in Example 4 of the present invention and Comparative Example 3:

[0194] Table 8

[0195]

[0196] As can be seen from the comparative data in Table 8, in the comparative example 3 sample sent using conventional methods, mainly immune cells and non-parenchymal cells were captured, while hepatic parenchymal cells accounted for only 2.8%. However, in Example 4 according to the method described in this invention, not only were appropriate proportions of immune cells and non-parenchymal cells captured, but also a higher proportion (26.7%) of hepatic parenchymal cells were captured, which can be used for subsequent bioinformatics analysis and mining of related biological data. These experimental results demonstrate that the implementation method of this invention is also effective in rat liver tissue samples.

[0197] This invention is not based on simple condition matching on the basis of existing technology, nor is it based on the expectation of experimental results by arbitrarily changing the conditions of existing technology. Rather, it is based on the innovative application of special materials (matrix gel) in the preparation of single-cell sequencing samples.

Claims

1. A method for single cell sequencing of a liver tissue sample, characterized in that, Specifically, the following steps are included: (a) Perfusion treatment of isolated liver tissue The liver tissue was cleaned and perfused with lavage solution I to obtain lavage material, which was then set aside. Then, lavage solution II was perfused into the liver tissue, and the tissue was allowed to stand in culture medium to obtain perfused liver tissue blocks. The irrigation solution I is an HBSS solution that does not contain calcium or magnesium ions; The components and contents of the irrigation solution II are as follows: 0.5-2 ml of the irrigation solution I, 3-8% trehalose, and 5-10 mg / mL Matrigel. Prepare perfusion solution II on ice; The liver tissue treated with irrigation fluid II was transferred to 22-35°C to allow the matrix gel to completely solidify in the liver tissue; The temperature for static osmosis is 22-35℃; (II) Methods for preparing single-cell suspensions Preparation of single-cell suspension of lavage fluid: The cells were collected by centrifugation of the lavage material prepared in step (I) above; after resuspending in culture medium, red blood cell lysis buffer was added and the mixture was allowed to stand at room temperature; then the cells were collected by centrifugation a second time. After no red blood cells remained, the cells were resuspended in culture medium to obtain a single-cell suspension of the lavage material. Preparation of single-cell suspension from liver tissue: The liver tissue blocks perfused in step (I) above were washed with DMEM culture medium, cut into small pieces, digested with enzymes, sieved, centrifuged for the first time, and then resuspended to obtain a single-cell suspension of liver tissue. (iii) The single-cell suspension of the lavage material prepared in step (ii) is mixed with the single-cell suspension of liver tissue.

2. The method of claim 1, wherein, In step (1), the settling time is 5-10 minutes.

3. The method of claim 1, wherein, In step (ii), during the preparation of the single-cell suspension of the irrigation material, the conditions for the first centrifugation are: 4℃, 500g, centrifugation for 5-10 min; the time for standing at room temperature is 3-5 min; and the conditions for the second centrifugation are: 4℃, 500g, centrifugation for 5-10 min.

4. The method as described in claim 1, characterized in that, In step (ii), the enzymes used in the preparation of the liver tissue single-cell suspension are collagenase II, collagenase IV and DNase I with a final concentration of 0.1-0.25% and 20-40 U / ml, respectively; the digestion time is 30-40 min.

5. The method as described in claim 1, characterized in that, In step (ii), the conditions for the first centrifugation in the preparation of the liver tissue single-cell suspension are 4℃, 300-500g, centrifugation for 5-10min.

6. The method as described in claim 1, characterized in that, The lavage fluid single-cell suspension and the liver tissue single-cell suspension are mixed at an equal cell number or a specific cell number ratio; and / or The single-cell suspension of the lavage fluid obtained by the method consists of immune cells remaining in the blood vessels or microenvironment of the liver tissue block; and / or, the single-cell suspension of the liver tissue consists of liver parenchymal cells, non-parenchymal cells, and immune cells in the tissue microenvironment.

7. The method as described in claim 6, characterized in that, The immune cells include lymphocytes, dendritic cells, and macrophages; the non-parenchymal cells are endothelial cells, Kuffer cells, and stellate cells.

8. The application of the method as described in claim 1 in single-cell sequencing, single-cell suspension preparation, and primary liver cell dissociation.

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