Tissue dissociation method and kit

Abstract

The embodiment of the invention provides an enzyme-based tissue dissociation method which comprises the following steps: sequentially carrying out first digestion, second digestion and third digestion on a tissue by using a first enzyme, a second enzyme and a third enzyme so as to dissociate the tissue, the second enzyme is a mixed enzyme and comprises a mixed enzyme of collagenase I and collagenase IV; the third enzyme comprises trypsin. According to the embodiment of the invention, the enzyme-based tissue dissociation method which is low in cost, high in dissociation efficiency, short in treatment time and few in generated cell debris impurities is successfully established for the first time, and the human scalp tissue single-cell suspension with complete cell types can be obtained; and good technical support is provided for single-cell-based researches such as single-cell sequencing, single-cell apparent group research and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a tissue dissociation method, a tissue dissociation kit and a single cell analysis method. BACKGROUND

[0002] Hair loss is a common disease, which is affected by environmental and genetic factors. In recent years, the patients with hair loss are gradually becoming younger, and the proportion of the population with hair loss is increasing year by year. Therefore, the demand for effective hair loss treatment methods is also increasing. Hair follicle is an important factor directly affecting the quality and quantity of hair growth, which is composed of more than ten cell subtypes of different layers of different types and functions. The skin tissue cells around the hair follicle are crucial for maintaining the microenvironment of the hair follicle and regulating the function of the hair follicle.

[0003] However, compared with other skin tissues of the human body, the scalp tissue has significantly increased thickness and obvious tissue heterogeneity, and the composition of the hair follicle cells and the surrounding microenvironment thereof are relatively complex. Therefore, the research on the scalp tissue has certain difficulties, that is, on the one hand, traditional transcriptome analysis will ignore and lose cell heterogeneity; on the other hand, the new generation of single cell RNA sequencing technology (scRNA-seq) requires a better method to fully dissociate the tissue to better obtain all types of cells.

[0004] Therefore, there is an urgent need for an efficient tissue dissociation method for single cell sequencing. SUMMARY

[0005] The present application aims to at least solve one of the technical problems in the related art to some extent.

[0006] To this end, the first aspect of the embodiments of the present application proposes an enzyme-based tissue dissociation method, comprising: using a first enzyme, a second enzyme and a third enzyme to sequentially perform first digestion, second digestion and third digestion on the tissue to dissociate the tissue, wherein the first enzyme comprises neutral protease II; the second enzyme is a mixed enzyme comprising a mixture of collagenase I and collagenase IV; and the third enzyme comprises trypsin. The enzyme-based tissue dissociation method is successfully established for the first time, which has low cost, high dissociation efficiency, short processing time and few cell debris impurities, can obtain a single cell suspension of human scalp tissue with complete cell types, and provides good technical support for single cell sequencing, single cell epigenome research and other single cell-based research.

[0007] In some embodiments, the first enzyme, the second enzyme and the third enzyme are provided in the form of a solution.

[0008] In some embodiments, in the first digestion, the final concentration of the first enzyme in the digestion system is 3.5-4.5 mg / mL.

[0009] In some embodiments, the first digestion is performed at 25-37 °C. In some embodiments, the first digestion is performed at 37 °C. In some embodiments, the first digestion lasts for 10-100 min. In some embodiments, the first digestion lasts for 10-80 min. In some embodiments, the first digestion lasts for 45-55 min.

[0010] In some embodiments, the first enzyme is neutral protease II.

[0011] In some embodiments, based on the first enzyme being provided in solution form, the concentration of the first enzyme is 3.5-4.5 mg / mL. In some embodiments, the concentration of the first enzyme is 4 mg / mL.

[0012] In some embodiments, the first enzyme solution further comprises DMEM / F-12.

[0013] In some embodiments, in the second digestion: the second enzyme comprises a 2-1 enzyme and a 2-2 enzyme, wherein the final concentrations of the 2-1 enzyme and the 2-2 enzyme in the digestion system are 0.5-1.0 mg / mL and 0.75-1.25 mg / mL, respectively.

[0014] In some embodiments, the 2-1 enzyme is collagenase I and the 2-2 enzyme is collagenase IV.

[0015] In some embodiments, based on the second enzyme being provided in solution form, the concentration of the collagenase I is 1.0-2.0 mg / mL and the concentration of the collagenase IV is 1.5-2.5 mg / mL.

[0016] In some embodiments, the concentration of the collagenase I is 1.6 mg / mL and the concentration of the collagenase IV is 2.0 mg / mL.

[0017] In some embodiments, the second enzyme solution further comprises CaCl2.

[0018] In some embodiments, the second digestion is performed at 25-37 °C. In some embodiments, the second digestion is performed at 37 °C.

[0019] In some embodiments, the second digestion lasts for 10-100 min. In some embodiments, the second digestion lasts for 10-80 min. In some embodiments, the second digestion lasts for 25-35 min.

[0020] In some embodiments, based on the second enzyme being provided in solution form, the second enzyme solution further comprises DMEM / F-12.

[0021] In some embodiments, in the third digestion: the third enzyme is provided in a solution at a concentration of 1.25-2.5 mg / mL.

[0022] In some embodiments, the third enzyme is trypsin.

[0023] In some embodiments, based on the third enzyme being provided in a solution, the concentration of the trypsin is 1.25-2.5 mg / mL.

[0024] In some embodiments, the third enzyme solution further comprises EDTA.

[0025] In some embodiments, the third digestion is performed at 25-37 °C. In some embodiments, the third digestion is performed at 37 °C.

[0026] In some embodiments, the third digestion lasts for 10-100 min. In some embodiments, the third digestion lasts for 10-80 min. In some embodiments, the third digestion lasts for 15-20 min.

[0027] In some embodiments, the method further comprises: after the second digestion and before the third digestion, performing a first filtration on the second-digested first digestion solution to capture first undigested tissue; and performing the third digestion on the first undigested tissue.

[0028] In some embodiments, the first filtration is performed at a filter pore size of 70-100 pm.

[0029] In some embodiments, the first filtration is performed at a filter pore size of 70 pm.

[0030] In some embodiments, after the second digestion and before the first filtration, the method further comprises: adding a first termination solution to terminate the second digestion.

[0031] In some embodiments, the first termination solution is a DMEM / F-12 solution.

[0032] In some embodiments, the method further comprises: after the third digestion, performing a second filtration on the third-digested second digestion solution.

[0033] In some embodiments, the second filtration is performed at a filter pore size of 30-40 pm.

[0034] In some embodiments, the second filtration is performed at a filter pore size of 30 pm.

[0035] In some embodiments, after the third digestion, the method further comprises: adding a second termination solution to terminate the third digestion.

[0036] In some embodiments, the second stop solution is a DMEM / F-12 solution containing FBS.

[0037] In some embodiments, before performing the first digestion, the method further comprises: isolating the tissue.

[0038] In some embodiments, the isolating comprises a physical separation method and a chemical separation method, wherein the physical separation method comprises mechanical separation, and the chemical separation method comprises a lysate treatment.

[0039] In some embodiments, the isolating results in an average size of the tissue of 1-3 mm 3 .

[0040] In some embodiments, the isolating results in an average size of the tissue of 1 mm 3 .

[0041] In some embodiments, the isolating is performed in the first enzyme solution.

[0042] In some embodiments, the tissue is derived from a mammal or a non-mammal. In some embodiments, the tissue is selected from one or more of: epithelial tissue, connective tissue, muscle tissue, and neural tissue.

[0043] In some embodiments, the tissue is skin, thymus, spleen, lymph, bone marrow, artery, vein, oral cavity, nasopharynx, esophagus, colon, rectum, large intestine, thyroid, heart, lung, liver, gallbladder, head and neck, pancreas, stomach, breast, ovary, prostate, uterus, bladder, and / or brain taken from a mammal. In some embodiments, the tissue is taken from skin. In some embodiments, the tissue is taken from scalp.

[0044] A second aspect of embodiments of the present application provides a tissue dissociation kit comprising a first enzyme, a second enzyme, and a third enzyme, and optionally a cell strainer, a first stop solution, and a second stop solution, wherein the first enzyme comprises neutral protease II; the second enzyme is a mixed enzyme comprising a mixture of collagenase I and collagenase IV; and the third enzyme comprises trypsin.

[0045] In some embodiments, the first enzyme, the second enzyme, and the third enzyme are provided in solution.

[0046] A third aspect of embodiments of the present application provides a method of single cell analysis, the method comprising:

[0047] The method of enzyme-based tissue dissociation according to any of the embodiments of the first aspect of the present application dissociates the tissue to obtain a single cell suspension; and analyzes the single cells in the single cell suspension.

[0048] In some embodiments, the analysis comprises: sequencing analysis and / or morphological observation, wherein the sequencing analysis optionally comprises one or more of: single-cell epigenomics analysis, single-cell transcriptomics analysis, single-cell genomics analysis, single-cell metabolomics analysis, single-cell proteomics analysis, single-cell immunomics analysis, and single-cell multi-omics combined analysis of any combination thereof.

[0049] The advantages and technical effects brought by the independent claims according to the embodiments of the present application are as follows:

[0050] (1) The tissue dissociation method according to the embodiments of the present application has high dissociation efficiency. Compared with the long time-consuming in the related art for processing scalp tissue, only 3 hours for enzymatic digestion, all dissociation steps of the tissue dissociation method according to the embodiments of the present application can be completed within 2 hours, greatly reducing the time for preparing single-cell suspension, and significantly increasing the dissociation efficiency.

[0051] (2) The tissue dissociation method according to the embodiments of the present application has low cost. Since the reagents involved in the tissue dissociation method according to the embodiments of the present application are all conventional reagents, without the need to match specific brand dissociation instruments for mechanical dissociation, the cost of a single sample is low, and therefore the cost of processing scalp tissue by using the tissue dissociation method according to the embodiments of the present application is low.

[0052] (3) The tissue dissociation method according to the embodiments of the present application produces less cell debris impurities, and can obtain single-cell suspension of human scalp tissue with complete cell types. The mechanical crushing process in the tissue dissociation method according to the embodiments of the present application is less, and the enzymatic treatment process is mild, so that when it is applied to dissociate scalp tissue, the generated cell debris is reduced, the background pollution is reduced, and therefore single cells of human scalp tissue with high activity, low pollution, low clumping rate and complete cell types can be obtained, which provides good technical support for single-cell based researches such as single-cell sequencing, single-cell epigenomics research, etc. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is a method flow diagram of enzyme-based tissue dissociation according to the embodiments of the present application.

[0054] Figure 2 is a human scalp tissue sample image diagram for enzyme-based tissue dissociation method according to the embodiments of the present application.

[0055] Figure 3 is a human scalp tissue single-cell precipitation result diagram obtained by enzyme-based tissue dissociation method according to the embodiments of the present application.

[0056] Figure 4 is a cell suspension fluorescence staining photo diagram obtained by enzyme-based tissue dissociation method according to the embodiments of the present application.

[0057] Figure 5 is a cell cDNA fragment distribution map obtained according to the enzyme-based tissue dissociation method of an embodiment of the present application.

[0058] Figure 6 is a cell subpopulation and annotation result schematic diagram obtained according to the enzyme-based tissue dissociation method of an embodiment of the present application. DETAILED DESCRIPTION

[0059] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below by reference to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0060] The present application is based on the following recognition of the inventor:

[0061] Hair loss is a common disease, which is affected by many factors such as environment and heredity. Hair follicle is an important factor that directly affects the quality and quantity of hair growth, and the scalp tissue as the skin tissue around the hair follicle is crucial for the maintenance of the hair follicle microenvironment and the regulation of the hair follicle function. However, compared with other skin tissues of the human body, the scalp tissue has significantly increased thickness and obvious tissue heterogeneity, and the composition of the hair follicle cells and the surrounding microenvironment thereof are relatively complex. Therefore, the research on the scalp tissue is difficult.

[0062] Specifically, due to the complexity of the composition of the hair follicle cells and the surrounding microenvironment thereof, the traditional transcriptome analysis, such as bulk cell RNA sequencing technology (Bulk RNA-seq), will ignore and lose the cell heterogeneity of different cells in the tissue, and cannot obtain the functional characteristics of different cell subtypes or single cells, and it is difficult to analyze the key problems such as the differentiation and development trajectory of the cells. Therefore, compared with the traditional bulk cell RNA sequencing technology, the new generation of single cell RNA sequencing technology (scRNA-seq) is particularly suitable for in-depth exploration of the tissue, especially the scalp tissue, because it can capture the transcriptome information at the single cell level, and can be used for cell differentiation and development trajectory analysis, intercellular interaction analysis and other benefits.

[0063] However, an important technical prerequisite for scRNA-seq is to obtain a high-quality single-cell suspension that meets the conditions for single-cell capture and tracking, which requires a better method to fully dissociate the tissue to better obtain all types of cells. Currently, there is no method for dissociating human scalp tissue and preparing a single-cell suspension for single-cell sequencing. Only the company Magenta Biosciences can provide a human whole skin dissociation kit product (Whole Skin Dissociation Kit, human; 130-101-540), but it is not specifically for scalp tissue and needs to be used with the same brand of tissue dissociation instrument. In addition to the cost of purchasing the dissociation instrument, the cost of the kit for a single sample is about 85 yuan, which is relatively high, but the efficiency is general, and only the enzyme digestion part takes 3 hours.

[0064] Therefore, the first aspect of the embodiments of the present application proposes an enzyme-based tissue dissociation method, comprising: sequentially using a first enzyme, a second enzyme and a third enzyme to perform first digestion, second digestion and third digestion on the tissue to dissociate the tissue, wherein the first enzyme comprises neutral protease II; the second enzyme is a mixed enzyme comprising a mixture of collagenase I and collagenase IV; and the third enzyme comprises trypsin. The enzyme-based tissue dissociation method is successfully established for the first time, which has low cost, high dissociation efficiency, short processing time and few cell fragment impurities, can obtain a human scalp tissue single-cell suspension with complete cell types, and provides good technical support for single-cell-based research such as single-cell sequencing and single-cell epigenomics research.

[0065] In some embodiments, the first enzyme, the second enzyme and the third enzyme are provided in the form of a solution.

[0066] Tissue dissociation is a process dependent on cell types. Different cell types have different tolerances to stress. The enzyme concentration and enzyme treatment time used in single-cell tissue dissociation are not widely applicable parameters. Therefore, it is understandable to make appropriate adjustments based on the embodiments of the present application. For example, for a relatively short time, a higher concentration of enzyme should be used; and for a longer time, a lower concentration of enzyme should be used. In addition, for different tissue types, the first enzyme, the second enzyme and the third enzyme can be appropriately adjusted.

[0067] In addition, based on the fact that the first enzyme, the second enzyme and the third enzyme are provided in the form of a solution, the preparation system of the enzyme solution can be adjusted according to the tissue type.

[0068] In some embodiments, in the first digestion: the final concentration of the first enzyme in the digestion system is 3.5-4.5 mg / mL.

[0069] In some embodiments, the first digestion is performed at 25-37 °C. In some embodiments, the first digestion is performed at 37 °C. In some embodiments, the first digestion lasts for 10-100 min. In some embodiments, the first digestion lasts for 10-80 min. In some embodiments, the first digestion lasts for 45-55 min.

[0070] In some embodiments, the first enzyme is neutral protease II.

[0071] In some embodiments, based on the first enzyme being provided in solution form, the concentration of the first enzyme is 3.5-4.5 mg / mL. In some embodiments, the concentration of the first enzyme is 4 mg / mL.

[0072] In some embodiments, the first enzyme solution further comprises DMEM / F-12.

[0073] In some embodiments, in the second digestion: the second enzyme comprises a 2-1 enzyme and a 2-2 enzyme, wherein the final concentrations of the 2-1 enzyme and the 2-2 enzyme in the digestion system are 0.5-1.0 mg / mL and 0.75-1.25 mg / mL, respectively.

[0074] In some embodiments, the 2-1 enzyme is collagenase I and the 2-2 enzyme is collagenase IV.

[0075] In some embodiments, based on the second enzyme being provided in solution form, the concentration of the collagenase II is 1.0-2.0 mg / mL and the concentration of the collagenase IV is 1.5-2.5 mg / mL.

[0076] In some embodiments, the concentration of the collagenase I is 1.6 mg / mL and the concentration of the collagenase IV is 2.0 mg / mL.

[0077] In some embodiments, the second enzyme solution further comprises CaCl2.

[0078] In some embodiments, the second digestion is performed at 25-37 °C. In some embodiments, the second digestion is performed at 37 °C.

[0079] In some embodiments, the second digestion lasts for 10-100 min. In some embodiments, the second digestion lasts for 10-80 min. In some embodiments, the second digestion lasts for 25-35 min.

[0080] In some embodiments, based on the second enzyme being provided in solution form, the second enzyme solution further comprises DMEM / F-12.

[0081] In some embodiments, in the third digestion: the third enzyme is provided in a solution at a concentration of 1.25-2.5 mg / mL.

[0082] In some embodiments, the third enzyme is trypsin.

[0083] In some embodiments, based on the third enzyme being provided in a solution, the concentration of the trypsin is 1.25-2.5 mg / mL.

[0084] In some embodiments, the third enzyme solution further comprises EDTA.

[0085] In some embodiments, the third digestion is performed at 25-37°C. In some embodiments, the third digestion is performed at 37°C.

[0086] In some embodiments, the third digestion lasts for 10-100 min. In some embodiments, the third digestion lasts for 10-80 min. In some embodiments, the third digestion lasts for 15-20 min.

[0087] In some embodiments, the method further comprises: after the second digestion and before the third digestion, performing a first filtration on the second-digested first digestion solution to capture first undigested tissue; and performing the third digestion on the first undigested tissue.

[0088] In some embodiments, the first filtration is performed at a filter pore size of 70-100 pm.

[0089] In some embodiments, the first filtration is performed at a filter pore size of 70 pm.

[0090] In some embodiments, after the second digestion and before the first filtration, the method further comprises: adding a first termination solution to terminate the second digestion.

[0091] In some embodiments, the first termination solution is a DMEM / F-12 solution.

[0092] In some embodiments, the method further comprises: after the third digestion, performing a second filtration on the third-digested second digestion solution.

[0093] In some embodiments, the second filtration is performed at a filter pore size of 30-40 pm.

[0094] In some embodiments, the second filtration is performed at a filter pore size of 30 pm.

[0095] In some embodiments, after the third digestion, the method further comprises: adding a second termination solution to terminate the third digestion.

[0096] In some embodiments, the second stop solution is a DMEM / F-12 solution containing FBS.

[0097] In some embodiments, before the first digestion is performed, the method further comprises: separating the tissue.

[0098] In some embodiments, the separating comprises a physical separation method and a chemical separation method, wherein the physical separation method comprises mechanical separation, and the chemical separation method comprises lysate treatment.

[0099] In some embodiments, the separating results in an average size of the tissue of 1-3 mm 3 .

[0100] In some embodiments, the separating results in an average size of the tissue of 1 mm 3 .

[0101] In some embodiments, the separating is performed in the first enzyme solution.

[0102] In some embodiments, the tissue is derived from a mammal or a non-mammal. In some embodiments, the tissue is selected from one or more of the following: epithelial tissue, connective tissue, muscle tissue, and neural tissue.

[0103] In some embodiments, the tissue is skin, thymus, spleen, lymph, bone marrow, artery, vein, oral cavity, nasopharynx, esophagus, colon, rectum, large intestine, thyroid, heart, lung, liver, gallbladder, head and neck, pancreas, stomach, breast, ovary, prostate, uterus, bladder, and / or brain taken from a mammal. In some embodiments, the tissue is taken from skin. In some embodiments, the tissue is taken from scalp.

[0104] It can be understood that the tissue dissociation method of the embodiments of the present application is suitable for a variety of tissues of a variety of sources. Among them, it is particularly suitable for dissociating complex tissues such as human scalp tissues and other cell types. The specific embodiments in the present application are only exemplary and cannot be understood as a limitation of the present application.

[0105] The second aspect of the embodiments of the present application proposes a tissue dissociation kit, comprising a first enzyme, a second enzyme and a third enzyme, and optionally a cell filter, a first stop solution and a second stop solution, wherein the first enzyme comprises neutral protease II; the second enzyme is a mixed enzyme comprising a mixture of collagenase I and collagenase IV; and the third enzyme comprises trypsin.

[0106] In some embodiments, the first enzyme, the second enzyme and the third enzyme are provided in the form of a solution.

[0107] A third aspect of embodiments of the present application provides a method of single cell analysis, the method comprising:

[0108] The method of enzyme-based tissue dissociation according to any of the embodiments of the first aspect of the present application dissociates the tissue to obtain a single cell suspension; and analyzes single cells in the single cell suspension.

[0109] In some embodiments, the analysis comprises sequencing analysis and / or morphological observation, wherein the sequencing analysis optionally comprises one or more of single cell epigenomics analysis, single cell transcriptomics analysis, single cell genomics analysis, single cell metabolomics analysis, single cell proteomics analysis, single cell immunomics analysis, and single cell multi-omics combined analysis of any combination thereof.

[0110] In summary, the enzyme-based tissue dissociation method of embodiments of the present application has the beneficial technical effects of low cost, high dissociation efficiency, short processing time, and few cell fragment impurities, and can obtain a single cell suspension of human scalp tissue with complete cell types, providing good technical support for single cell sequencing, single cell epigenetic research, and other single cell-based research.

[0111] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0112] The quantitative experiments in the following examples, unless otherwise specified, are set up with three replicates, and the results are averaged.

[0113] Example 1 Dissociation of scalp tissue

[0114] Embodiments of the present application first mechanically separate human scalp tissue, and sequentially digest the separated scalp tissue using the first enzyme, the second enzyme, and the third enzyme of the present application; then filter the digested scalp tissue using a cell filter to obtain dissociated scalp tissue.

[0115] 1. Material preparation

[0116] (1) Prepare experimental equipment (tweezers, ophthalmic scissors, cell culture dishes, centrifuge tubes, pipettes, ice boxes, etc.) that have been ultraviolet sterilized for at least 30 min, and turn on the 37°C water bath and the low-temperature centrifuge in advance.

[0117] (2) Prepare the digestion enzyme solutions of the first enzyme, the second enzyme, and the third enzyme, and the specific configuration system is shown in Table 1 below, wherein the penicillin-streptomycin solution is 100X (item number: Cytiva, purchased from: SV30010):

[0118] Table 1. Digestive enzyme solution preparation system

[0119]

[0120] (3) Place a sterile cell culture dish containing 5 mL of sterile 1X PBS (catalog number: 10010-031, purchased from: Gibco) on ice, then place human scalp tissue in the cell culture dish, gently shake, and wash away the blood on the surface of the tissue.

[0121] like Figure 2 The image shown is a schematic diagram of a human scalp tissue sample according to an embodiment of the enzyme-based tissue dissociation method of the present invention. The human scalp tissue sample is approximately 1 cm × 1 cm in size and contains intact hair follicles.

[0122] 2. Mechanical separation of scalp tissue

[0123] (1) Use an RNase-free scalpel to carefully remove subcutaneous fat from the scalp tissue under a stereoscope.

[0124] (2) Then the scalp tissue was transferred to a 1.5 mL cryovial containing Miltenyi tissue preservation solution (product number: 130-100-008, purchased from Miltenyi Biotechnology, Germany) and stored at 4°C for a maximum of 48 hours.

[0125] (3) Place a sterile cell culture dish on ice in a biosafety cabinet, add 3 mL of the first enzyme solution (neutral protease II solution, diluted to a concentration of 4 mg / mL (catalog number: D4693, purchased from: Sigma)) to the culture dish, and use RNase-free tweezers to remove the human scalp tissue from the cryopreservation tube and transfer it to the culture dish.

[0126] (4) Use tweezers to grasp the human scalp tissue, and then use RNase-free ophthalmic scissors to cut the scalp tissue into pieces of 1 mm in size. 3 Place the pieces on the left and right sides, and then place all the fragments in a petri dish.

[0127] 3. Enzymatic digestion treatment

[0128] (1) Transfer the first enzyme solution containing tissue fragments to a 50 mL centrifuge tube, and rinse the remaining fragments in the culture dish with 2 mL of the first enzyme solution to transfer as many tissue fragments as possible to the centrifuge tube.

[0129] (2) After sealing the centrifuge tubes with sealing film, put disposable PE gloves on the centrifuge tubes, tie them tightly, and place them horizontally in a water bath at 37°C for 50 minutes.

[0130] (3) After the incubation, 5 mL of the second enzyme solution (collagenase I (Cat. No. A004194-0001, purchased from BBI) and collagenase IV mixture (Cat. No. A004186-0001, purchased from BBI) containing 18 mM CaCl2(Cat. No. A600506-0100, purchased from BBI) with concentrations of 1.6 mg / mL and 2.0 mg / mL, respectively) was added to the centrifuge tube.

[0131] (4) After the centrifuge tube was sealed again with the sealing film, a disposable PE glove was put on the centrifuge tube, and the centrifuge tube was placed horizontally in a 37°C water bath for 30 min.

[0132] (5) After the incubation, 20 mL of DMEM / F-12 (Cat. No. 11320-033, purchased from Gibco) was added to the digestion solution to terminate the digestion, and a cell filter with a pore size of 70 μm was used for filtration. The filtered digestion solution was stored on ice.

[0133] (6) The undigested tissue blocks obtained after the filtration in (5) were transferred to a new 50 mL centrifuge tube, 4 mL of the third enzyme solution (trypsin solution containing EDTA with a concentration of 0.25% (Cat. No. 25200-056, purchased from Gibco)) was added, and the cell filter was washed again with 4 mL of enzyme III solution to completely transfer all the tissue fragments to the new centrifuge tube.

[0134] (7) After the centrifuge tube was sealed again with the sealing film, a disposable PE glove was put on the centrifuge tube, and the centrifuge tube was placed horizontally in a 37°C water bath for 15 min.

[0135] (8) After the incubation, 10 mL of DMEM / F-12 containing 20% FBS (Cat. No. FSP500, purchased from ExCell Bio) was added to terminate the digestion, and a cell filter with a pore size of 70 μm was used for filtration. The digestion solution obtained in step (5) was mixed.

[0136] 4. Cell washing and resuspension

[0137] (1) The digestion solution obtained by the enzyme digestion was centrifuged for 10 min at 300 g and 4°C, and then the supernatant was removed, and the cell precipitate was retained.

[0138] (2) 2 mL of PBS containing 2% FBS was added to the cell precipitate to resuspend the cells, and a cell filter with a pore size of 40 μm was used for filtration. The filtrate was collected in a 15 mL centrifuge tube.

[0139] (3) The filtrate in step (2) was centrifuged for 5 min at 300 g and 4°C, and then the supernatant was removed, and the cell precipitate was retained.

[0140] (4) Add 50-100 μL of 0.04% BSA (item number: A602449-0050, purchased from: BBI) PBS solution to the centrifugal tube and gently blow to resuspend the cell pellet, then transfer the suspension to a 1.5 mL EP tube to obtain the cell suspension obtained from human scalp tissue dissociation.

[0141] The time for dissociating human scalp tissue by the method of the embodiment of the present application is about 2 hours, which is much lower than at least 3 hours in the related art. In addition, the human scalp tissue single cell pellet obtained by the enzyme-based tissue dissociation method according to the embodiment of the present application is clearly visible as shown in Figure 3 , which indicates that the efficiency of obtaining single cells by the enzyme-based tissue dissociation method of the embodiment of the present application is high.

[0142] Cell observation and quality inspection of Example 2

[0143] (1) Take 2 μL of the human scalp tissue single cell cell suspension obtained in Example 1, dilute with 8 μL of 0.04% BSA PBS, mix with 9 μL of AO / PI dye (item number: RE010212, purchased from: Countstar), and count with a cell counter (item number: Countstar Rigel S3, purchased from: Countstar) to calculate the cell viability, clumping rate and total cell amount, respectively.

[0144] (2) Single cell capture, transcription, extraction and library construction (Huada DNB lab C4 technology platform) were performed on the cell suspension human scalp tissue single cell suspension obtained in Example 1, and the cDNA obtained was subjected to fragment distribution test (Q-sep100, company: BiOptic).

[0145] (3) Sequencing was performed using the Huada DNBSEQ sequencing platform, and the sequencing results were used for cell grouping and annotation. The fluorescence staining photograph of the cell suspension obtained by the enzyme-based tissue dissociation method according to the embodiment of the present application is shown in Figure 4 ( green for live cells and red for dead cells), and the results show that the cell suspension background is clean, the cell viability can reach more than 86%, the clumping rate is less than 5%, and the total cell amount can reach about 400,000. This result shows that the single cells produced by this method are relatively complete, with less cell debris and no background contamination, and are suitable for dissociating scalp tissue.

[0146] Figure 5 is the cDNA fragment distribution diagram of the cells obtained by the enzyme-based tissue dissociation method according to the embodiment of the present application. It can be seen that the cDNA fragment distribution is around 1000 bp, which is reasonable, indicating that the sample quality is good.

[0147] Figure 6 The schematic diagram of the cell grouping and annotation results of the cells obtained according to the enzyme-based tissue dissociation method of the embodiment of the present application. The results show that a total of 21 cell groups including immune cells, nerve cells, stromal cells, keratinocytes and the like are annotated. This indicates that the cell grouping is good and the types cover all cell types of the human scalp tissue.

[0148] In summary, the single cell suspension obtained according to the enzyme-based tissue dissociation method of the embodiment of the present application is relatively complete, has few cell fragments and no background pollution, and the results of the verification thereof also indicate that the obtained single cell types are complete and are very suitable for single cell sequencing and other single cell-based researches.

[0149] In the description of the present application, it needs to be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element indicated must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation of the present application.

[0150] In addition, the terms "first", "second" are only for description purpose, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three and the like, unless otherwise explicitly specified and limited.

[0151] In the present application, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connecting", "fixing" and the like should be understood in a broad sense, for example, can be fixed connection, can also be detachable connection, or integral; can be mechanical connection, can also be electrical connection or communication with each other; can be directly connected, or indirectly connected through an intermediate medium; can be the communication or interaction relationship between two elements, unless otherwise explicitly limited. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0152] In the present application, unless specifically stated and limited otherwise, a first feature "on" or "under" a second feature can be directly in contact with the second feature, or indirectly in contact with the second feature through an intermediate medium. Also, a first feature "over", "above" and "on top of" a second feature can be directly above or obliquely above the second feature, or simply means that the first feature is horizontally higher than the second feature. A first feature "under", "below" and "underneath" a second feature can be directly below or obliquely below the second feature, or simply means that the first feature is horizontally lower than the second feature.

[0153] In the present application, the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" mean that a particular feature, structure, material, or characteristic being described is included in at least one embodiment or example of the present application. The illustrative appearances of the above-mentioned terms in various places in the specification are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples. Moreover, the terms "first", "second", "third", etc. are used herein merely as identifiers for different elements, regions, layers, or sections, and are not intended to denote a spatial or chronological priority or order except if explicitly so defined. Also, the terms "comprises", "comprising", "includes", "including", or the like are used herein to generally mean comprising, including, or consisting of, unless otherwise indicated.

[0154] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary, and are not to be interpreted as limiting the present application, and that changes, modifications, substitutions and variations can be made therein by those skilled in the art without departing from the scope of the present application.

Claims

1. A method of enzyme-based tissue dissociation, characterized in that, The method comprises: digesting the tissue sequentially with a first enzyme, a second enzyme and a third enzyme to dissociate the tissue, wherein the first enzyme comprises neutral protease II; the second enzyme is a mixed enzyme comprising a mixture of collagenase I and collagenase IV; the third enzyme comprises trypsin, optionally, the first enzyme, the second enzyme and the third enzyme are provided in solution.

2. The method of claim 1, wherein, In the first digestion: the final concentration of the first enzyme in the digestion system is 3.5-4.5 mg / mL, optionally, the first digestion is performed at 25-37°C, preferably at 37°C, and optionally, the first digestion lasts for 10-100 min, preferably for 10-80 min, more preferably for 45-55 min; optionally, the first enzyme is neutral protease II, optionally, the first enzyme is provided in solution at a concentration of 3.5-4.5 mg / mL, preferably at 4 mg / mL, optionally, the first enzyme solution further comprises DMEM / F-12.

3. The method of claim 1, wherein, In the second digestion: the second enzyme comprises a 2-1 enzyme and a 2-2 enzyme, wherein the final concentrations of the 2-1 enzyme and the 2-2 enzyme in the digestion system are 0.5-1.0 mg / mL and 0.75-1.25 mg / mL, respectively, optionally, the 2-1 enzyme is collagenase I and the 2-2 enzyme is collagenase IV, optionally, the second enzyme is provided in solution, the concentration of the collagenase I is 1.0-2.0 mg / mL, preferably 1.6 mg / mL, and the concentration of the collagenase IV is 1.5-2.5 mg / mL, preferably 2.0 mg / mL, and the second enzyme solution preferably further comprises CaCl2; optionally, the second digestion is performed at 25-37°C, preferably at 37°C, and optionally, the second digestion lasts for 10-100 min, preferably for 10-80 min, more preferably for 25-35 min, optionally, the second enzyme is provided in solution, and the second enzyme solution further comprises DMEM / F-12.

4. The method of claim 1, wherein, In the third digestion: the final concentration of the third enzyme in the digestion system is 1.25-2.5 mg / mL, optionally, the third enzyme is trypsin, optionally, the third enzyme is provided in solution, the concentration of the trypsin is 1.25-2.5 mg / mL, and the third enzyme solution preferably further comprises EDTA; optionally, the third digestion is performed at 25-37°C, preferably at 37°C, and optionally, the third digestion lasts for 10-100 min, preferably for 10-80 min, more preferably for 15-20 min.

5. The method of claim 1, wherein, The method further comprises: after the second digestion and before the third digestion, performing a first filtration on the first digestion solution after the second digestion to capture first undigested tissue; and performing the third digestion on the first undigested tissue, optionally, the filter pore size of the first filtration is set to 70-100 μm, preferably to 70 μm, Optionally, after the second digestion and before the first filtration, the method further comprises adding a first stop solution to stop the second digestion, the first stop solution is preferably DMEM / F-12 solution.

6. The method of claim 1, wherein, The method further comprises: after the third digestion, performing a second filtration on the second digestion solution after the third digestion, Optionally, the second filtration has a pore size of 30-40 μm, preferably 30 μm. Optionally, after the third digestion, the method further comprises adding a second stop solution to stop the third digestion, the second stop solution is preferably DMEM / F-12 solution containing FBS.

7. The method of claim 1, wherein, Before performing the first digestion, the method further comprises: separating the tissue, the separation optionally comprises a physical separation method and a chemical separation method, wherein the physical separation method comprises mechanical separation, and the chemical separation method comprises lysate treatment, Preferably, the separation is such that the average size of the tissue is 1-3 mm 3 , preferably 1 mm 3 , Preferably, the separation is performed in the first enzyme solution.

8. The method according to any one of claims 1 to 7, characterized in that, The tissue is derived from a mammal or a non-mammal, and optionally, the tissue is selected from one or more of the following: epithelial tissue, connective tissue, muscle tissue, and neural tissue; Optionally, the tissue is skin, thymus, spleen, lymph, bone marrow, artery, vein, oral cavity, nasopharynx, esophagus, colon, rectum, large intestine, thyroid, heart, lung, liver, gallbladder, head and neck, pancreas, stomach, breast, ovary, prostate, uterus, bladder, and / or brain taken from a mammal, preferably skin, more preferably scalp.

9. A tissue dissociation kit, characterized in that, The method comprises a first enzyme, a second enzyme, and a third enzyme, and optionally a cell filter, a first stop solution, and a second stop solution, wherein The first enzyme comprises neutral protease II; The second enzyme is a mixed enzyme comprising a mixture of collagenase I and collagenase IV; The third enzyme comprises trypsin, Optionally, the first enzyme, the second enzyme, and the third enzyme are provided in the form of a solution.

10. A method of single cell analysis, comprising: The method comprises: dissociating the tissue to obtain a single cell suspension by the method of enzyme-based tissue dissociation according to any one of claims 1 to 8; and analyzing single cells in the single cell suspension, Optionally, the analysis comprises sequencing analysis and / or morphological observation, wherein the sequencing analysis optionally comprises one or more of the following: single cell epigenomics analysis, single cell transcriptomics analysis, single cell genomics analysis, single cell metabolomics analysis, single cell proteomics analysis, single cell immunomics analysis, and single cell multi-omics combined analysis of any combination thereof.

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