Preparation method and application of high-quality liver non-parenchymal cell single-cell suspension

Abstract

The invention relates to the technical field of biology, and particularly discloses a preparation method and application of a high-quality liver non-parenchymal cell single-cell suspension. The problems of high cell agglomeration rate, many tissue fragments, heavy impurity background pollution and the like are effectively solved, and meanwhile, high cell activity is ensured. After fresh tissues are in vitro, if a single-cell suspension is prepared according to the method within 2 hours, the cell activity can be kept at 90% or above, and if the cell activity is prepared within 24 hours, the cell activity can reach 85% or above. The operation is simple and streamlined, the repeated operation effect stability is good, and partial operation is compatible with various kits or instrument consumables. Moreover, the method disclosed by the invention is suitable for related scientific experiment research in the single cell field, and solves the problem that various laboratories and various biological companies are difficult to prepare the liver non-parenchyma single cell suspension in liver disease scientific research work.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a method for preparing a high-quality single-cell suspension of liver non-parenchymal cells and its application. It can be widely applied in the fields of biomedical research, disease diagnosis and treatment, drug development, etc., providing strong technical support for in-depth understanding of the mechanisms of liver diseases, the interactions between liver non-parenchymal cells and their subsets, and the development of personalized treatment plans. Background Art

[0002] The main components of liver non-parenchymal cells (NPCs) include hepatic sinusoidal endothelial cells, Kupffer cells, and dendritic cells, and the minor components mainly consist of innate immune cells, some bile duct epithelial cells, and hepatic stellate cells. A variety of non-parenchymal cells play important roles in the induction of liver diseases, the maintenance of liver immune homeostasis, liver metabolism, tumor metastasis, etc. Therefore, in-depth understanding of the heterogeneity of NPCs is crucial for understanding the structure and function of the liver. At present, many researchers focus on purifying liver non-parenchymal cells, aiming to deeply study the biochemical characteristics of different types of non-parenchymal cells and their subsets in the liver through single-cell sequencing, flow cytometry, primary in situ culture, induction, etc., and conduct multi-omics, biochemical, morphological, etc. research.

[0003] At present, there are some common technical difficulties in the field of liver non-parenchymal cell purification and single cell suspension preparation: the liver is a complex functional organ, and its basic structural unit includes multiple components such as hepatocytes, vascular system and bile duct network. Although traditional separation methods (such as perfusion enzymatic method, mechanical-enzymatic combined dissociation method and commercial non-parenchymal cell dissociation kit) can achieve tissue dissociation, they all have the problem of residual hepatocyte fragments and serious red blood cell contamination. These impurities not only significantly increase the complexity of subsequent cell sorting, but are also likely to cause abnormal function of surviving cells by releasing intracellular substances, thereby affecting the reliability of downstream analysis data. It is worth noting that unscientific solutions (such as the combination of de-fragmentation reagents and red blood cell lysis buffer) have limitations: on the one hand, it is difficult to maintain the physiological activity of target cells, and on the other hand, it cannot solve the abnormal cell adhesion caused by the exposure of adhesion factors caused by cell membrane rupture and the release of extracellular DNA, which ultimately leads to an increase in cell aggregation rate. Affecting the quality of downstream analysis. How to solve the high aggregation rate with appropriate treatment methods and suitable working fluids while avoiding secondary adhesion before downstream experiments is also a technical problem that most researchers have not yet overcome. Chinese invention patent applications CN202010196718.8 and CN116875529A disclose methods for preparing active single-cell suspensions of liver tissue. The above methods use multiple enzymes for digestion on the basis of conventional tissue dissociation, which are cumbersome and time-consuming to operate, and cannot purify high-quality non-parenchymal cells. They cannot solve the high fragmentation and high agglomeration rate in the preparation process of non-parenchymal cells, and cannot ensure high cell activity while solving the above difficulties.

[0004] In summary, many research teams will face problems such as a lot of tissue fragments, heavy impurity background contamination, high cell agglomeration rate, low cell viability, loss of cell subpopulations, and poor signal-to-noise ratio after bioinformatics analysis during preparation, which ultimately leads to reduced accuracy and credibility of the research results and difficulty in repeating. How to solve the above problems through a complete dissociation scheme and a supporting buffer system is still a technical barrier that needs to be broken through. To this end, the present application provides an innovative method for preparing high-quality liver non-parenchymal cell single-cell suspensions, which aims to solve the technical difficulties such as a lot of tissue fragments, heavy impurity background contamination, high cell agglomeration rate, low cell viability, loss of cell subpopulations, and poor signal-to-noise ratio after bioinformatics analysis in the preparation process of non-parenchymal single-cell suspensions after traditional parenchymal cell fragmentation, and highly retain the expression of cell antigens and antibodies. This method ensures the integrity and activity of cells through a series of carefully designed steps, significantly improving the accuracy and reliability of single-cell experiments. Summary of the invention

[0005] The object of the present invention is to solve the technical problems raised in the above-mentioned background art, and to provide a method process and application for preparing a high-quality single-cell suspension of non-parenchymal liver cells. The method of the present invention is compatible with various tissue enzymatic digestion methods for extracting single cells. Finally, the obtained single-cell suspension has a high cell activity of more than 90%, almost no impurities, almost no cell clumping, a clean cell suspension background, good staining effect of single-cell antibody labels, and rich and complete non-parenchymal cell populations, especially immune cells.

[0006] The above object of the present invention is achieved as follows:

[0007] The solution of the present invention provides a method for preparing a high-quality single-cell suspension of non-parenchymal liver cells, comprising the following steps:

[0008] S1: Tissue sampling, rinsing and preservation: For mouse liver, it is recommended to directly and quickly remove the whole liver within 5 minutes without perfusion or take a part of the liver in vivo, and cut fresh liver tissue of 300 mg - 900 mg (preferably 500 - 800 mg, as too small or too large a volume will affect the enzymatic digestion effect, and it is easy to cause a decrease in cell activity or a high clumping rate due to excessive or insufficient enzymatic digestion). After sampling, quickly rinse the whole tissue with pre-cooled DPBS or pre-cooled PBS or pre-cooled DMEM to maintain tissue activity. After rinsing, it can be cut into tissue blocks of ≤1 cm * 1 cm and stored in pre-cooled tissue preservation solution (if the fresh tissue can be directly subjected to subsequent enzymatic digestion operations within 20 minutes, the tissue preservation solution can be not used). Tested by our research group, for the tissue stored in the tissue preservation solution and operated according to this whole process, if the single-cell suspension is prepared within 1 hour, the cell activity can be maintained above 90%, and if the single-cell suspension is prepared within 24 hours, the cell activity can be maintained at 85%). The subsequent processes of this experiment have been tested to be compatible with various enzymatic digestion methods while maintaining high activity, extremely few fragments and extremely few clumps. However, for non-parenchymal cell purification, after enzymatic digestion by perfusion method, low-speed centrifugation is required to separate liver parenchymal cells and non-parenchymal cells. Tested by our research group, about 5% of CD45 + cells will be mixed with parenchymal cells and lost or interfered. Therefore, non-perfusion mechanical dissociation is recommended.

[0009] S2: Tissue digestion: Taking the use of a non-parenchymal cell enzymatic digestion kit as an example, mechanical dissociation is carried out at 37°C. (If using the Miltenyi non-parenchymal cell dissociation kit, 1 cm tissue blocks can be directly mechanically dissociated; if using the Reword non-parenchymal cell dissociation kit, the liver tissue needs to be minced and then mechanically dissociated; it is recommended to sharply cut the tissue, and grinding and crushing the tissue is not recommended to prevent cell damage.)

[0010] S3: Terminate digestion and filter once to remove debris: After dissociation in step S2, filter the suspension directly through a pre-rinsed 70-100um cell mesh. After filtration, rinse the dissociation container with 4ml of pre-cooled DMEM to collect residual cells and filter. Then rinse the cell mesh with 1ml-2ml of pre-cooled DMEM to recover cells to the maximum extent.

[0011] S4: Washing and purifying cells: After washing and purifying cells by centrifugation at 4 degrees, wash the supernatant as much as possible (this research group has tested that centrifugation at 4 degrees throughout the process can help improve cell viability compared to centrifugation at room temperature).

[0012] S5: Secondary removal of debris by density gradient centrifugation: Use a debris removal kit density gradient centrifugation or percoll density gradient centrifugation to remove the debris. After centrifugation at 4 degrees, use a 1 ml or wider pipette tip to first aspirate the debris layer and then the supernatant (this research group tested that a smaller pipette tip could not completely aspirate the debris layer. Aspirating the debris layer first helps protect the cell layer).

[0013] S6: Washing purified cells: Wash the purified cells by centrifugation at 4 degrees and remove as much supernatant as possible.

[0014] S7: Lysis of red blood cells: After centrifugation in step S6, red blood cell sedimentation will be visible to the naked eye. Depending on the amount of red blood cells, use 2.5ml of pre-cooled mild tissue lysis solution to lyse for 2-3 minutes and then centrifuge and wash. Depending on the amount of red blood cells, use 2ml to lyse for 1-2 minutes and then centrifuge and wash. (According to the test of this research group, in the case of 500-900mg liver tissue volume, two-step short-time lysis does not affect cell activity. On the contrary, one-time lysis for too long is easy to affect cell activity because it is difficult to grasp the time).

[0015] S8: Washing purified cells: Wash the purified cells by centrifugation at 4 degrees and remove as much supernatant as possible.

[0016] S9: Disperse cell agglomerates First: Use DNase I to dissolve cell agglomerates caused by cell entanglement due to the release of DNA chains from hepatocytes. This research group tested that 15 minutes of incubation with DNase I at room temperature or 20 minutes on ice had the same effect on cell activity, but room temperature incubation was more ideal for removing agglomerates. After DNase I incubation, filter through a pre-rinsed 45um mesh.

[0017] S10: Washing purified cells: Centrifuge the cell suspension filtered in step S9 at 4 degrees to wash the purified cells and remove the supernatant as much as possible.

[0018] S11: Second dispersion of cell aggregates: Use a working solution of high concentration 5 mM / mL EDTA (0.5% BSA + 5 mM EDTA + DPBS). The research group of this study tested that after first preparing 300 mM EDTA with a pH ≈ 8 from DPBS and NaOH, diluting it to 5 mM with DPBS, and finally adding BSA to a final concentration of 0.5% BSA, the pH of the working solution can be maintained at ≈ 7.3 while solving the problem of easy precipitation of crystals in high-concentration EDTA. Using DNase I alone or high-concentration EDTA alone has a worse effect than using DNase I and EDTA in combination, and it does not affect cell viability. When using them in combination, DNase I needs to be incubated for a short time first because adding EDTA first easily causes DNase I inactivation.

[0019] S12: Wash and purify the cells: Centrifuge and wash the cells at 4°C, and then wash away the supernatant as thoroughly as possible.

[0020] S13: Replace the working solution and wash once: You can choose BD Stain Buffer or Miltenyi MACS or a solution of 5% BSA + 2 mM EDTA + DPBS for cleaning;

[0021] S14: Prepare for cell preservation and single-cell library construction: The research group of this study tested that the above working solutions have no difference in affecting cell viability). However, it is recommended to use 5% BSA + 2 mM EDTA + DPBS for cleaning and preservation, which helps to maintain a Ca-free environment and prevent cells from re-adhering. However, when using the working solution of 5% BSA + 2 mM EDTA, it is recommended to perform subsequent operations within 1 hour to avoid cell respiratory inhibition.

[0022] Furthermore, before rinsing the tissue specimen, an ice box filled with crushed ice needs to be prepared, and pre-cooled serum-free rinsing solution is added to the rinsing container to ensure that the liver tissue immediately enters the pre-cooled solution after being removed from the body. In the embodiment of the present invention, the ice box used is a foam ice box filled with crushed ice (such an ice box can slow down the melting speed of ice and is not likely to have ice water on the surface, which helps to avoid water pollution of reagents and cell rupture), the rinsing container is a culture dish, and the rinsing solution is 3 ml.

[0023] Furthermore, the rinsing solution is pre-cooled DPBS solution or serum-free medium DMEM.

[0024] Furthermore, the liver tissue includes normal mouse liver tissue or liver tissue of various disease models.

[0025] Furthermore, the specimen tissue is 300 - 900 mg of freshly cut tissue, or specimen tissue stored at 4°C in tissue preservation solution within 24 hours.

[0026] Furthermore, after taking out the tissue from the tissue preservation solution, it needs to be placed in a culture dish containing 3 ml of pre-cooled serum-free medium solution to wash away the preservation solution on the tissue surface.

[0027] Further, pre-digestion is also included in step S1. The specific steps are as follows: After soaking the rinsed tissue in a pre-warmed medium at 37°C containing digestive enzymes, it is mechanically dissociated in a dissociator at 37°C according to the preset program described in the corresponding mechanical dissociation equipment instructions or placed at a 45-degree angle on a 37°C shaker with a shaking speed of 200 RPM and shaken for 40 minutes.

[0028] Further, after enzymatic digestion of the tissue, it is filtered into a 15 ml centrifuge tube. The test of this research group shows that if it is filtered into a 50 ml centrifuge tube, due to a large amount of precipitation after the liver parenchymal cells are broken and a large number of cells, the precipitate after centrifugation is loose and easy to be contaminated or lose cells.

[0029] Further, the centrifugation conditions in step S4 are: 4°C, 300 g, 10 minutes, acceleration 5 and deceleration 5. The centrifugation conditions in step S6 are: 4°C, 300 g, 10 minutes, acceleration 5 and deceleration 5. In step S5, if a debris removal kit is selected, the recommended centrifugation conditions are 4°C, 3000 g, 10 minutes, acceleration 3 and deceleration 1 (the test of this research group shows that the debris removal effects are different according to the recommended centrifugation conditions of the debris removal kit by different brands of centrifuges, and even the debris stratification is not obvious. The main influencing factor is the difference in the control of acceleration and deceleration. Uniformly adopting acceleration 3 and deceleration 1 can adapt to centrifugation of different models). The centrifugation conditions in step S6 are: 4°C, 1000 g, 10 minutes, acceleration 9 and deceleration 9. The centrifugation conditions in steps S8, S10, and S12 are all: 4°C, 300 g, 5 minutes, acceleration 5 and deceleration 5.

[0030] Further, except for step S9, all operations are carried out on ice; except for step S2, the reagents are all pre-cooled at 4 degrees.

[0031] Further, after incubation of DNase I at room temperature or on ice, it is first passed through a pre-rinsed cell sieve to remove the debris after the DNA strand entanglement is released to the greatest extent.

[0032] Further, high-concentration EDTA configured with 0.5% BSA + 5 mM EDTA + DPBS can effectively solve cell aggregation caused by calcium ion adhesion on the basis of ensuring cell viability. And it can solve the problem that high-concentration EDTA is prone to precipitate crystals while satisfying PH≈7.3.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The present invention solves the technical problems existing in the preparation of non-parenchymal single-cell suspensions during the preparation of non-parenchymal single-cell suspensions after traditional parenchymal cell disruption, such as a large number of tissue fragments, heavy impurity background pollution, high cell aggregation rate, low cell viability, and loss of cell subsets, and highly retains the expression of cell antigens and antibodies. Moreover, through a series of carefully designed method processes, the method of the present invention can ensure the integrity and activity of cells, facilitating a significant improvement in the accuracy and reliability of single-cell experiments.

[0035] 2. Single cells obtained by the method for preparing active non-parenchymal cells of liver tissue of the present invention have relatively high cell viability. If the single-cell suspension is prepared within 1 hour, the cell viability can be maintained above 90%. If the single-cell suspension is prepared within 24 hours, the cell viability can be maintained at 85%.

[0036] 3. The method of the present invention is relatively simple to operate, has good stability in repeated operations, and some operations are compatible with a variety of reagent kits or instrument consumables. It is applicable to relevant scientific experimental research in the field of single cells, solving the problems encountered in the preparation of non-parenchymal single cells of the liver in the scientific research work of liver diseases in each laboratory and biological company. Description of the Drawings

[0037] Figure 1 is the bright-field image of non-parenchymal cells of liver tissue in the single-cell suspension in the embodiment of the present invention;

[0038] Figure 2 is the cell viability staining image of non-parenchymal cells of liver tissue in the single-cell suspension in the embodiment of the present invention;

[0039] Figure 3 is the detection of the cell viability of non-parenchymal cells of liver tissue in the single-cell suspension (BD Rhapsody platform; the cell viabilities are: 93.38%; 91.21%; 94.74%) when simultaneously processing 3 portions of non-parenchymal cells of liver tissue in the embodiment of the present invention;

[0040] Figure 4 is the detection of single-cell capture of non-parenchymal cells of liver tissue in the embodiment of the present invention (BD Rhapsody platform);

[0041] Figure 5 is the TSNE map of single-cell sequencing and the overall non-parenchymal cell clustering heat map of non-parenchymal cells of liver tissue in the embodiment of the present invention (it can be seen that the embodiment retains the non-parenchymal cell population mainly composed of immune cells and endothelial cells; there are very few residual parenchymal cells; the cell heat map has clear clustering, facilitating subsequent fine analysis of cell subsets);

[0042] Figure 6 is the bright-field image of non-parenchymal cells of mouse liver tissue in the single-cell suspension in the comparison between the embodiment of the present invention and Comparative Example 1 (the left is the embodiment; the right is the comparative example);

[0043] Figure 7 Figure 1 shows a bright-field image of a single-cell suspension of non-parenchymal cells in mouse liver tissue, comparing the example with Comparative Example 2 (the example on the left; the comparative example on the right).

[0044] Figure 8 Figure 2 shows a bright-field image of a single-cell suspension of non-parenchymal cells in mouse liver tissue using only DNase I to solve cell clumping (the quality is inferior to the DNase I combined with 5 mM EDTA protocol used in the example).

[0045] Figure 9 Figure 3 shows a bright-field image of a single-cell suspension of non-parenchymal cells in mouse liver tissue using only 5 mM EDTA to solve cell clumping (the quality is inferior to the DNase I combined with 5 mM EDTA protocol used in the example). Detailed implementation mode

[0046] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0048] Refer to the attached Figures 1-9 As shown, it is a preferred embodiment provided by the present invention.

[0049] Example: The solution of the embodiment of the present invention provides a method for preparing a high-quality single-cell suspension of non-parenchymal liver cells, and the method includes the following implementation steps:

[0050] S1: Item preparation: a foam ice box filled with crushed ice (outer dimensions: 34 cm * 22 cm * 14 cm), two 35 cm culture dishes; one cryogenic tube (1.5 ml), pre-cooled DPBS solution, dissection instruments, disinfected alcohol, tissue preservation solution (Miltenyi TissueStorage Solution). Operation: After anesthetizing or decapitating the mouse, immediately dissect the liver, take 500 mg - 900 mg of liver tissue within 5 minutes and place it in a 35 cm culture dish containing 3 ml of pre-cooled DPBS solution for rinsing. Gently pick up the liver and rinse it once in another 35 cm culture dish in the same way. Cut the tissue into tissue blocks of about 1 cm * 1 cm in DPBS solution and store it in pre-cooled tissue preservation solution for enzymatic digestion preparation.

[0051] S2: Item preparation: DMEM pre-warmed to 37°C, pre-cooled DMEM, Liver Non-Parenchymal Cell Dissociation Kit (Miltenyi Biotec Liver Dissociation Kit, mouse #130 - 105 - 807, or Reword DHGT - 5004) or perfusion dissociation solution, mechanical dissociator and tissue processing tube or 37°C shaker, dissociator or 37°C shaker, 35 cm culture dish. Operation: Take out the tissue from the tissue preservation solution and place it in a 35 cm culture dish containing 3 ml of pre-cooled DMEM solution to wash off the preservation solution on the tissue surface. Taking Reword DHGT - 5004 as an example. Add 4.84 ml of pre-warmed DMEM to the tissue dissociation tube and add 110 uL of Enzyme A and 50 uL of Enzyme B to prepare the dissociation mixture. After cutting the liver tissue into pieces in the rinsing solution, place the liver tissue into the dissociation solution and dissociate it according to the preset program M_Liver_Heater_1 for 36 minutes or place it on a mechanical shaker at 37°C at a 45-degree angle, with a shaking speed of 200 RPM and shake for 40 minutes.

[0052] S3: Item preparation: Pre-cooled DMEM, pre-rinsed 70 um sieve, 15 m centrifuge tube, debris ice box that can be placed in a test tube rack or ice water ice box. After dissociation in step S2, directly filter the enzymatically digested cell suspension through a pre-rinsed 70 um cell sieve. After filtration, rinse the dissociation container with 4 ml of pre-cooled DMEM to collect the residual cells and filter them into a 15 ml centrifuge tube. Then rinse the cell sieve with 1 ml - 2 ml of pre-cooled DMEM to recover cells to the maximum extent.

[0053] S4: Item preparation: 4°C centrifuge (swing bucket centrifuge, horizontal rotor). Operation: Centrifuge at 4°C, 300 g, for 10 minutes, with an acceleration of 5 and a deceleration of 5. After centrifugation, aspirate as much supernatant as possible.

[0054] S5: Item preparation: Percoll density gradient centrifugation solution or pre-cooled debris removal kit (Miltenyi Biotec DebrisRemoval Solution), pre-cooled DPBS, 15 ml centrifuge tube. Operation: After centrifugation in step S4, pour out the supernatant. Referring to the working instructions of the debris removal reagent, add 6200 ul of pre-cooled DPBS and gently resuspend 10 times into a new 15 ml centrifuge tube, then add 1800 ul of pre-cooled debris removal reagent and gently resuspend 7 times. After resuspension, tilt the centrifuge tube at a 45-degree angle of the tube mouth, and gently drip 4 ml of pre-cooled DPBS onto the liquid surface with a 1 ml pipette tip close to the tube wall (do not mix with the lower layer during the operation process). Perform density gradient centrifugation at 4°C, 3000 g, for 10 minutes, with an acceleration of 3 and a deceleration of 1. After centrifugation is completed, gently pick up the centrifuge tube, first aspirate the middle debris layer with a 1 ml pipette, and then aspirate the top layer, always paying attention to keeping the liquid level at the 6 ml - 8 ml level. After aspirating the upper two layers, gently resuspend the cells 7 times with a 1 ml pipette tip. After resuspension, add pre-cooled DPBS to 15 ml, and gently invert the centrifuge tube 3 times.

[0055] S6: Item preparation: 4°C centrifuge (swing-bucket centrifuge, horizontal rotor). Operation: 4°C, 1000 g, 10 minutes, acceleration 9, deceleration 9. After centrifugation, aspirate as much supernatant as possible.

[0056] S7: Item preparation: Pre-cooled gentle red blood cell lysis buffer (no manufacturer requirement) to lyse red blood cells, pre-cooled DMEM. After centrifugation according to the steps of S6, a red blood cell pellet can be seen with the naked eye. Use 2.5 ml of pre-cooled gentle tissue red blood cell lysis buffer to gently resuspend the cell pellet, let it stand on ice for 2 - 3 minutes for lysis, then add pre-cooled DMEM to make the volume up to 11 ml, and centrifuge at 4°C, 300 g, 5 minutes, acceleration 5, deceleration 5 for washing. After centrifugation, gently resuspend with 2 ml of lysis buffer according to the amount of red blood cells, let it stand on ice for 1 - 2 minutes for lysis, and then add pre-cooled DMEM to make the volume up to 11 ml.

[0057] S8: Item preparation: 4°C centrifuge (swing-bucket centrifuge, horizontal rotor). Operation: 4°C, 300 g, 5 minutes, acceleration 5, deceleration 5 for centrifugation. After centrifugation, aspirate as much supernatant as possible.

[0058] S9: Item preparation: Thawed DNase I with a concentration of 1 mg / mL (Solarbio D8071 or STEAMCELL07900), pre-cooled DMEM, pre-wetted 45-μm cell strainer, 15-ml centrifuge tube. After step S8, resuspend the cell pellet with 1 - 2 ml of pre-cooled DMEM, add 100 - 200 μL of DNase I with a concentration of 1 mg / mL to the suspension. Let it stand at room temperature for 15 minutes (or on ice for 20 minutes). After standing, gently tap the tube wall, gently resuspend 2 times with a 1-ml pipette, and transfer the resuspended solution through a pre-wetted 35-μm cell strainer into a new 15-ml centrifuge tube for centrifugation, and rinse the strainer with 1 ml of pre-cooled DMEM to solve the problem of cell clumping caused by DNA strand release from hepatocyte breakage resulting in cell entanglement.

[0059] S10: Item preparation: 4°C centrifuge (swing-bucket centrifuge, horizontal rotor). Operation: 4°C, 300 g, 5 minutes, acceleration 5, deceleration 5 for centrifugation. After centrifugation, aspirate as much supernatant as possible.

[0060] S11: Item preparation: Prepare 5 mM EDTA working solution according to the aforementioned preparation method, pre-cooled DMEM. After the centrifugation in step S10 is completed, pour out the supernatant, aspirate the supernatant with a 20-μl pipette, resuspend the cells with 2 ml of pre-cooled 5 mM EDTA working solution, and let it stand on ice for 3 min.

[0061] S12: Item preparation: 4°C centrifuge (swing-bucket centrifuge, horizontal rotor). Operation: 4°C, 300 g, 5 minutes, acceleration 5, deceleration 5 for centrifugation. After centrifugation, aspirate as much supernatant as possible.

[0062] S13: After the centrifugation in step S12 is completed, the cell pellet can be gently resuspended with 2 ml of BD Stain Buffer or Miltenyi MACS or 5% BSA + 2 mM EDTA + DPBS solution, and then centrifuged again at 4°C, 300 g for 5 minutes with an acceleration of 5 and a deceleration of 5. After centrifugation, aspirate as much supernatant as possible.

[0063] S14: Preparation of items: A pre-rinsed 45-μm cell strainer. After washing according to S13, the cell pellet can be gently resuspended with 2 ml of BD Stain Buffer or Miltenyi MACS or 5% BSA + 2 mM EDTA + DPBS solution, and then passed through a pre-rinsed 30-40-μm cell strainer. The preparation of a high-quality single-cell suspension of liver non-parenchymal cells is completed.

[0064] After operating according to the above steps S1-S14, downstream library construction can be directly carried out. This specification recommends that cell counting and viability detection can be performed before the centrifugation in step S13, and antibody tagging staining can be directly performed before single-cell library construction. For adult mice, after operating according to this specification, the cell viability of the final single-cell sample can be over 90%. There are very few double-cell or multi-cell aggregates caused by DNA enzyme chain entanglement or calcium ion adhesion, and almost no cell debris caused by hepatocyte fragmentation or cell necrosis.

[0065] Comparative Example 1: Comparing the method of dissociating liver non-parenchymal cell tissue of Miltenyi Biotec GmbH alone (refer to the digestion enzyme instruction manual). The cell debris dissociated by the method of Comparative Example 1 is numerous and visible throughout the field of view. The cell aggregation rate is very high, >30%. The red blood cell precipitation is obvious. The overall cell viability is <70% (considered related to debris, cell aggregation, and red blood cell interference). The cell suspension far fails to meet the requirements for single-cell research and library construction on the machine.

[0066] Comparative Example 2: Comparing the method of dissociating liver non-parenchymal cell tissue of Miltenyi Biotec GmbH in combination with a debris removal kit and red blood cell lysate. Although the cells dissociated by the method of Comparative Example 2 can solve the problems of debris and red blood cells to a certain extent, the cell aggregation rate is still very high, >30%. Especially as the number of washes before library construction increases, the hepatocyte debris that is not sufficiently enzymolyzed will gradually be released and increase, and the cell aggregation will become higher and higher. Eventually, the quality of the cell suspension deteriorates as the number of washes increases, far failing to meet the requirements for single-cell research and library construction on the machine.

[0067] Comparative Example 3: The comparative document (CN116875529A) discloses a method for preparing a single-cell suspension for single-cell sequencing of all types of liver cells. The following defects exist when this prior art is compared with the present invention: 1. The full-process operation is only applicable to perfusion dissociation, and it cannot be used by researchers without perfusion conditions. However, this solution can be adapted to various enzymatic digestion schemes, and it is universal for lysing red blood cells, removing debris, and solving cell clumping in liver tissue of corresponding volume. 2. Even if the enzymatic digestion method is optimized, the cell viability is usually <90%, and the operation is cumbersome, making it difficult to accurately control. 3. Debris and multi-cell clumps can still be seen under bright field, affecting cell capture and sequencing accuracy. 4. There is a large difference between the theoretical sequencing capture and the reagent sequencing capture. 5. The obtained cells include parenchymal cells and non-parenchymal cells. CN116875529A focuses on parenchymal cells, and it is not applicable to the sequencing of non-parenchymal cells that require in-depth subdivision of subpopulations.

[0068] Comparative Example 4: The comparative document (CN202010196718.8) discloses a method for preparing a single-cell suspension of human liver tissue. The following disadvantages exist when its preparation method is compared with that of the present invention: 1. The cost is relatively high, and pre-digestion is required first; using a variety of commercial enzymes for long-term digestion is likely to cause over-digestion. In Comparative Example 1, the Miltenyi dead cell removal reagent (#130-090-101) was finally introduced for dead cell removal treatment, indicating that there are many dead cells in this solution, and the final cell viability is still the lowest. 2. The tissue treatment is not good. Even though the hepatic parenchymal cells are concerned, the parenchymal cells are not well protected. 3. It is not applicable to the research that requires in-depth subdivision of subpopulations and in-depth exploration of the interaction of non-parenchymal cells.

[0069] Through the above embodiments of the present invention, the method of the present invention has the following advantageous features: Technical features: 1. The method of the present invention adopts an efficient and high-quality cell separation technology: simplifies the cell separation process, and the processes of removing debris, removing red blood cells, and eliminating clumps are universal for various enzymatic digestion methods, which is convenient for application; precisely separates non-parenchymal liver cells to ensure cell purity and activity; 2. The present invention optimizes the crushing and cleaning processes: effectively reduces cell debris and impurities by optimizing the steps of removing debris and cleaning, and improves the purity of the cell suspension; 3. The present invention solves the problem of cell clumping: innovatively uses a specific method to solve cell clumping caused by various reasons without affecting cell viability, greatly improves the single-cell rate, and is compatible with various single-cell separation methods; 4. The method of the present invention realizes a live cell protection mechanism: ensures that the cell quality and activity are not affected during the preparation and subsequent processing processes by optimizing the working fluid; 5. The method of the present invention has compatibility and universality: this method is applicable to a variety of single-cell sequencing and flow cytometry analysis platforms, and has good compatibility and universality.

[0070] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a high-quality single-cell suspension of liver non-parenchymal cells, characterized in that, The following steps are involved: S1. Tissue sampling, rinsing and storage: No in vivo or in vitro liver perfusion is performed. After the mouse is euthanized, the whole liver or part of the liver is quickly removed within 5 minutes and 300-900 mg of fresh liver tissue is cut; after sampling, the whole tissue is quickly rinsed in pre-cooled DPBS or pre-cooled DMEM to maintain tissue activity and wash away surface blood stains; after rinsing, step S2 is performed directly within 20 minutes. Before implementing step S2, the tissue needs to be continuously immersed in pre-cooled DPBS or pre-cooled DMEM; if step S2 cannot be performed immediately, it can be cut into smaller tissue blocks and stored in pre-cooled tissue preservation solution at 4°C; S2. Tissue digestion: Use any liver non-parenchymal cell enzymatic dissociation kit to mechanically dissociate the tissue sampled in step S1 at 37°C; S3, terminate digestion and filter once to remove debris: after the dissociation in step S2 is completed, filter the suspension directly through a pre-rinsed 70-100um cell mesh, rinse the mechanical dissociation container with pre-cooled DMEM after filtration to collect residual cells and filter through the cell mesh again, rinse the cell mesh with pre-cooled DMEM after filtration to maximize cell recovery, and finally fix the volume of the cell suspension to 5-10 ml; S4, washing and purifying cells: centrifuge at 4 degrees, centrifugation parameters: 300g, 10min, acceleration 5, deceleration 5. After washing and purifying cells, aspirate the supernatant; S5. Secondary removal of debris: Use a debris removal kit or percoll density gradient centrifugation to remove debris for a second time. After centrifugation, use a 1ml or wide pipette tip to first aspirate the debris layer, then aspirate the supernatant; and complete the debris removal operation according to the debris removal kit or percoll density gradient centrifugation process; S6. Wash the purified cells again: centrifuge at 300g for 5 min at 4°C, speed up 5, speed down 5, wash the purified cells again, and aspirate the supernatant; S7, lysing red blood cells: After centrifugation in step S6, red blood cell sediments are generally visible to the naked eye. Depending on the amount of red blood cells, 2.5 ml of pre-cooled mild tissue lysing solution is used for lysis in an ice bath for 3 minutes, followed by centrifugation at 4 degrees at 300 g for 5 minutes, with a speed of 5 and a speed of 5 deceleration for centrifugation and washing. Depending on the amount of red blood cells after washing and purification, 2 ml of pre-cooled solution is used for lysis for 1-2 minutes, followed by centrifugation and washing; S8. Wash and purify cells: centrifuge at 300g for 5 min at 4°C, speed up 5, speed down 5, wash and purify cells, pour out the supernatant and aspirate the supernatant; S9. Disperse cell agglomerates for the first time: Resuspend the cells with 1mg / mL DNase I solution and let stand for 15-20min at room temperature. During this period, gently tap the tube wall several times to remove cell agglomerates caused by cell entanglement due to the release of DNA chains from liver cell fragmentation. After standing, filter through a pre-rinsed 45um cell mesh. S10, washing purified cells: centrifuging the cell suspension filtered in step S9 at 300 g for 5 min at 4 degrees, with a speed of 5 and a speed of 5, washing purified cells, and aspirating the supernatant; S11. Disperse cell aggregates for the second time: resuspend the cells with a high concentration of 5 mM / mEDTA working solution prepared with 0.5% BSA + 5 mM EDTA + DPBS solution and place on ice for 10-15 min to disperse the cell aggregates caused by calcium adhesion; S12, washing and purifying cells: centrifuging at 300g for 5 min at 4 degrees, speed up 5 and decelerate 5, washing and purifying cells, and aspirating the supernatant; S13, replace the working solution and wash once: use BD Stain Buffer or Miltenyi MACS or 5% BSA + 2mM EDTA + DPBS solution for washing to complete the preparation of high-quality liver non-parenchymal cell single cell suspension; S14. Short-term storage of cells: Use BD Stain Buffer or Miltenyi MACS or 5% BSA + 2mM EDTA + DPBS solution for washing and storage; and perform subsequent operations according to experimental requirements within 1 hour.

2. The method for preparing a high-quality single-cell suspension of non-parenchymal liver cells according to claim 1, wherein In step S1, before rinsing the tissue specimen, an ice box filled with crushed ice is prepared, and pre-cooled serum-free rinsing solution is added to the rinsing container to ensure that the liver tissue enters the pre-cooling solution immediately after being removed from the body.

3. A method for preparing a high-quality single-cell suspension of non-parenchymal liver cells according to claim 1, characterized in that, The rinse solution is pre-cooled DPBS solution or serum-free culture medium DMEM.

4. A method for preparing a high-quality single-cell suspension of non-parenchymal liver cells according to claim 1, characterized in that, The liver tissue includes normal liver tissue of mice or liver tissue of various disease models.

5. A method for preparing a high-quality single-cell suspension of non-parenchymal liver cells according to claim 1, characterized in that, The specimen tissue is 300-900 mg of fresh tissue, or tissue preserved in tissue preservation solution within 24 hours.

6. A method for preparing a high-quality single-cell suspension of liver non-parenchymal cells according to claim 1, characterized in that, After the tissue is removed from the tissue preservation solution within 24 hours, it needs to be placed in pre-cooled DPBS solution or serum-free culture medium DMEM to wash away the preservation solution on the surface of the tissue.

7. A method for preparing a high-quality single-cell suspension of non-parenchymal liver cells according to claim 1, characterized in that, Step S1 also includes pre-digestion, which specifically includes soaking the rinsed tissue in a 37°C pre-warmed culture medium with digestive enzymes added thereto, and then mechanically dissociating the tissue in a 37°C dissociation machine according to the preset program in the corresponding mechanical dissociation equipment instructions, or placing the tissue in a 37°C shaker at a 45-degree angle, shaking at a speed of 200 RPM, and shaking for 40 minutes instead of mechanical dissociation.

8. A method for preparing a high-quality single-cell suspension of liver non-parenchymal cells according to claim 7, characterized in that, After tissue enzymatic hydrolysis, filter into a 15ml or 50ml centrifuge tube.

9. A method for preparing a high-quality single-cell suspension of non-parenchymal liver cells according to claim 1, characterized in that, The centrifugation conditions in step S4 are: 4°C, 300g, 10 minutes, acceleration 5 and deceleration speed 5; the centrifugation conditions in step S6 are: 4°C, 300g, 10 minutes, acceleration 5 and deceleration speed 5; in step S5, if the fragmentation removal kit is selected, the first centrifugation condition is recommended to be 4°C, 3000g, 10 minutes, acceleration 3 and deceleration speed 1; the centrifugation conditions in step S6 are: 4°C, 1000g, 10 minutes, acceleration 9 and deceleration speed 9; the centrifugation conditions in steps S8, S10, S12 and S13 are all: 4°C, 300g, 5 minutes, acceleration 5 and deceleration speed 5.

10. A method for preparing a high-quality single-cell suspension of non-parenchymal liver cells according to claim 1, characterized in that, Except for step S9, all operations were performed on ice; except for step S2, all reagents were pre-cooled at 4 degrees.

11. A method for preparing a high-quality single-cell suspension of non-parenchymal liver cells according to claim 1, characterized in that, After the removal of fragments and lysis of red blood cells, 1 mg / mL DNase I solution and high concentration 5 mM / mL EDTA working solution were used in turn to remove cell entanglement caused by the release of DNA chains from broken cells and cell agglomeration caused by calcium adhesion.

Images