Preparation method and application of high-quality single cell of synovial tissue
By employing a specific mixed enzyme dissociation and screening centrifugation method, the problem of preparing single-cell suspensions of synovial tissue was solved, enabling the preparation of high-quality single cells. This supports single-cell sequencing and primary cell culture, and promotes research on synovial diseases.
Patent Information
- Application Number
- CN202410717664.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-04
- Publication Date
- 2025-12-05
AI Technical Summary
Existing technologies make it difficult to efficiently prepare high-quality single-cell suspensions from synovial tissue, which limits the progress of subsequent single-cell sequencing and drug research.
Synovial tissue was enzymatically hydrolyzed using a specific ratio of mixed enzymes (streptolysin, collagenase I, hyaluronidase, elastase, collagenase II, and DNase), and single-cell suspensions were prepared by sieving and centrifugation under appropriate temperature and rotation speed conditions.
The prepared synovial tissue single-cell suspension is of high quality, meeting the requirements for single-cell sequencing and primary cell culture, reducing research costs and promoting research on synovial-related diseases.
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Figure CN121065151A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of cell biology, and particularly relates to a preparation method of high-quality single cells of synovial membrane tissue and application. BACKGROUND
[0002] The synovial membrane is the inner layer of the joint capsule, which is light red, smooth and shiny, thin and soft, and is composed of loose connective tissue. The function of the synovial membrane is to produce and regulate synovial fluid. The synovial membrane secretes synovial fluid in the joint cavity, which contains highly polymerized and high-viscosity hyaluronic acid, and serves as the main lubricant in the joint, which can reduce the friction coefficient of the articular cartilage to 0.001.
[0003] Synovitis is one of the most common diseases affecting human health, which is usually chronic damage, and the patient usually shows limited activity after the disease. With the increasing maturity of single cell separation technology and single cell sequencing technology, whole genome sequencing of single synovial cells has become a new field of synovial membrane research. If the synovial single cells can be sequenced at the single synovial cell level, and the cell types and proportions of the synovial tissue are found out, it will provide a basis for drug research and treatment of synovitis. However, although a series of cell separation technologies have appeared at present, such as advanced flow cytometry, the premise is to dissociate the synovial tissue into a high-quality single cell suspension, and obtaining a high-quality single cell suspension from the synovial tissue is currently the limiting factor for separating synovial cells.
[0004] Preparation of high-quality single cell suspension is the key to subsequent library construction and sequencing. Normal synovial membrane is divided into two layers, namely a thin cell layer (intracochlear layer) and a vascular layer (subendometrial layer), which is a vascular-rich synovial membrane lining attached to the non-articular surface part and covering the bone surface in the synovial capsule, not on the cartilage surface. This part is called the marginal zone or bare area. The synovial membrane is usually pink, smooth and shiny, wet and lubricious, and sometimes has villi, containing collagen fibers. SUMMARY
[0005] In order to solve the problems existing in the prior art, the present application provides a preparation method of high-quality single cell suspension of synovial tissue.
[0006] Enzymatic digestion is one of the common ways to obtain single cells, but researchers often encounter contradictory data when searching for ideal enzymes and the best conditions for tissue dissociation in scientific literature. This is because the extracellular matrix is complex and has certain variability, and some literature uses crude and not well-defined enzyme preparations. In addition, the extracellular matrix is a complex network composed of various proteins, glycoproteins, lipids and glycolipids, and the abundance of these components may vary depending on the species, tissue type and developmental stage, which poses a challenge to the selection of suitable enzymes and the exploration of enzymatic conditions.
[0007] Based on the characteristics of synovial tissue, the application develops a mixed enzyme suitable for synovial tissue digestion and dissociation, and a corresponding enzyme digestion synovial tissue preparation single cell suspension scheme. The single cell sample obtained by enzyme digestion of synovial tissue using the scheme has high quality and meets the requirements of single cell sequencing.
[0008] In a first aspect, the application provides a synovial tissue enzyme digestion method, comprising: using a mixed enzyme to perform enzyme digestion on synovial tissue, and enzyme digestion until the cell number meets the requirements; the mixed enzyme comprises 1-3 parts of pronase, 0.2-1 parts of neutral protease, 0.5-2 parts of collagenase I, 0.2-0.8 parts of hyaluronidase, 0.1-0.4 parts of elastase, 2-4 parts of collagenase II, and 0.1-0.3 parts of DNAase, in terms of weight.
[0009] Further, the mixed enzyme is obtained by using DMEM configuration, comprising:
[0010] 1-3 mg / mL of pronase, 0.2-1 mg / mL of neutral protease, 0.5-2 mg / mL of collagenase I, 0.4 mg / mL of hyaluronidase, 0.1-0.4 mg / mL of elastase, 2-4 mg / mL of collagenase II, and 0.1-0.3 mg / mL of DNAase.
[0011] Further, the enzyme digestion conditions are 36-38℃ and 80-150 rpm.
[0012] Further, the enzyme digestion until the cell number meets the requirements comprises:
[0013] The enzyme digestion supernatant is counted at 10-15 minutes of enzyme digestion, and if the cell number meets the requirements, the enzyme digestion is stopped, and if the cell number does not meet the requirements, the enzyme digestion is repeated for 10-15 minutes, and then the enzyme digestion supernatant is counted, and the process is repeated until the cell number meets the requirements.
[0014] Further, during the enzyme digestion, the wide-bore gun head is blown once every 4-7 minutes.
[0015] In a second aspect, the application provides a synovial tissue high-quality single cell preparation method, comprising:
[0016] Pretreating the synovial tissue;
[0017] Using the enzyme digestion method to perform enzyme digestion to obtain an enzyme digestion liquid;
[0018] Screening and collecting cells from the enzyme digestion liquid.
[0019] Further, the pretreatment comprises: washing the synovial tissue and cutting it into 1.5-3 mm in diameter.
[0020] Further, the enzymolysis comprises: sequentially passing the enzymolysis solution through a 65-75 mu m cell sieve and a 25-35 mu m cell sieve; and / or,
[0021] The cell collection comprises:
[0022] After the centrifugal enrichment of the sieved enzymolysis solution, resuspension, and centrifugation at 0-4 DEG C and 400-600g for 5-10 minutes, the supernatant is discarded and the cell precipitate is collected.
[0023] In a third aspect, the application provides the synovial tissue single cell prepared by the preparation method.
[0024] In a fourth aspect, the application provides the use of the synovial tissue single cell in single cell sequencing, primary cell culture or CAT treatment.
[0025] Further, the CAT treatment comprises: preparing a reagent or kit for CAT treatment.
[0026] The application has the following beneficial effects:
[0027] The application provides a method for preparing synovial tissue single cells by enzymolysis based on the specific mixed enzyme obtained by research, and high-quality synovial tissue-derived single cells can be prepared to meet the requirements of downstream single cell sequencing, primary cell culture or CAT treatment. The method for preparing synovial tissue single cells provided by the application solves the problem of obtaining high-quality single cell suspension from synovial tissue, provides new technical assistance for studying synovial single cell gene expression, reduces the cost of synovial sample research, speeds up the research of synovial related diseases, and has important significance in the technical field of studying synovial membrane. BRIEF DESCRIPTION OF DRAWINGS
[0028] In order to more clearly illustrate the technical solutions in the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.
[0029] Figure 1 Figure 1 is a diagram of the pre-processed rat zygapophyseal joint synovial tissue provided by the embodiment 1 of the application.
[0030] Figure 2 Figure 2 is a schematic diagram of bright field, live cells and dead cells in a microscope field of view provided by the embodiment 2 of the application; from left to right, the bright field, live cells and dead cells are shown in sequence.
[0031] Figure 3is a schematic diagram of bright field, live cells and dead cells in the field of view of a microscope provided by embodiment 2 of the present application; from left to right, the bright field, live cells and dead cells are arranged in sequence.
[0032] Figure 4 is a schematic diagram of bright field, live cells and dead cells in the field of view of a microscope provided by embodiment 2 of the present application; from left to right, the bright field, live cells and dead cells are arranged in sequence.
[0033] Figure 5 is a schematic diagram of the statistical results of the diameters of cells in the field of view provided by embodiment 2 of the present application. DETAILED DESCRIPTION
[0034] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in conjunction with the drawings in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.
[0035] Embodiment 1
[0036] The present embodiment provides a method for preparing a high-quality synovial membrane single-cell suspension, comprising the following steps:
[0037] 1. Preparation of mixed enzymes (enzyme solution)
[0038] Prepare a 15ml centrifuge tube, and prepare the dissociation mixed enzyme in the 15ml centrifuge tube by using DMEM. The concentrations of various enzymes in the mixed enzyme are as follows: streptomyces protease (2mg / mL), neutral protease (0.5mg / mL), collagenase I (1mg / mL), hyaluronidase (0.4mg / mL), elastase (0.2mg / mL), collagenase II (3mg / mL), and DNAase (0.2mg / mL).
[0039] 2. Pretreatment of synovial membrane tissue
[0040] Transfer the synovial membrane tissue to a culture dish containing DMEM medium, and remove the non-target tissue outside the synovial membrane with surgical scissors. Cut the washed synovial membrane tissue into tissue blocks with a diameter of about 2mm, and wash the tissue blocks with PBS for 1-2 times. The pretreated rat glenohumeral synovial membrane tissue is as shown in Figure 1 .
[0041] 3. Enzymatic digestion
[0042] Take synovial tissue particles with wide mouth gun head or Pasteur pipette into 15ml centrifuge tube, discard DMEM culture medium, add mixed enzyme prepared into the tissue, seal with sealing film, incubate in constant temperature water bath shaker at 37℃ 100rpm for 10 minutes, observe the turbidity of enzyme solution and the tissue. Take the enzyme solution supernatant incubated for 10 minutes to count, if the number of cells does not meet the collection requirement, continue to digest for 10 minutes and count, until the collection requirement is met. Blow the enzyme solution with a wide mouth gun head every 5 minutes during the enzyme digestion process.
[0043] 4、Cell collection
[0044] The enzyme solution meeting the collection requirement is sequentially passed through 70μm and 30μm cell screens. Then the enzyme solution is centrifuged to enrich cells, and the cells are resuspended with culture medium (1640 or DMEM) containing 1% FBS. The resuspended cell solution is centrifuged at 4℃ 500g for 5 minutes to enrich cells, and the supernatant is discarded to obtain the cell precipitate.
[0045] Roswell Park Memorial Institute (RPMI) 1640 medium and high-sugar DMEM medium are purchased from Shengong Biology.
[0046] 5、Cell quality control
[0047] After resuspending the cell precipitate with PBS buffer or culture medium, count the cells to judge the cell quality. If there are small fragments, wash them once with 5% FBS.
[0048] Cell counting and quality judgment are realized by a cell counter. The cell counter used is Countstar Rigel S2 full-automatic cell fluorescence analyzer purchased from Shanghai Ruiyu Biology Technology Co., Ltd.
[0049] Example 2
[0050] This example is directed to observing and detecting the synovial tissue single cells prepared in Example 1, as follows:
[0051] After resuspending the synovial tissue single cells prepared in Example 1 with 500μL DMEM medium to obtain a single cell suspension, observing the single cell suspension, specifically using Countstar Rigel S2 full-automatic cell fluorescence analyzer of Shanghai Ruiyu Biology Technology Co., Ltd., Figures 2-4 is a schematic diagram of 3 different fields of view.
[0052] This application further detects the cell diameters of the cells in the 3 fields of view of Figures 2-4 , to obtain, as shown in Figure 5The results show that the cell diameter in the obtained single cell suspension is in normal distribution, and is concentrated in 9-14 um. In addition, the cell concentration in the single cell suspension is 1190 cells per μL, the total number of cells is 590,000, the nucleated rate is 84%, the clumping rate is 13.19%, the cell survival rate is 90.45%, and the average circularity is 0.83, which are obtained by further counting by the Countstar Rigel S2 full-automatic cell fluorescence analyzer of Shanghai Ruishu Biological Technology Co., Ltd.
[0053] From the above results, it can be seen that the prepared single cell suspension has high quality, and can meet the requirements of single cell sequencing, primary cell culture and CAT treatment.
[0054] Experimental Example 1
[0055] This experimental example is used to compare the effects of different mixed enzymes on the enzymatic hydrolysis of synovial tissue, and the details are as follows:
[0056] 1. Experimental group and control group
[0057] Experimental group: The experimental group adopts the method for preparing high-quality synovial single cells shown in Example 1.
[0058] Control group 1: The process of control group 1 is the same as that of Example 1, and the difference lies in that the mixed enzymes used are: neutral protease (0.5 mg / mL), collagenase I (1 mg / mL), hyaluronidase (0.4 mg / mL), elastase (0.2 mg / mL), collagenase II (3 mg / mL), and DNAase (0.2 mg / mL).
[0059] Control group 2: The process of control group 2 is the same as that of Example 1, and the difference lies in that the mixed enzymes used are: pronase (2 mg / mL), neutral protease (0.5 mg / mL), collagenase I (1 mg / mL), hyaluronidase (0.4 mg / mL), elastase (0.2 mg / mL), and DNAase (0.2 mg / mL).
[0060] Control group 3: The process of control group 3 is the same as that of Example 1, and the difference lies in that the mixed enzymes used are: pronase (2 mg / mL), neutral protease (0.5 mg / mL), hyaluronidase (0.4 mg / mL), elastase (0.2 mg / mL), collagenase II (3 mg / mL), and DNAase (0.2 mg / mL).
[0061] 2. Experimental results
[0062] The prepared single cells of the experimental group and the control group are detected by the method in Example 2, and the results show that:
[0063] The nucleated cell rate of the control group 1 is 70%, the clumping rate is 12%, and the cell survival rate is 82%;
[0064] The nucleated cell rate of the control group 2 is 58%, the clumping rate is 15%, and the cell survival rate is 85%;
[0065] The nucleated cell rate of the control group 3 is 72%, the clumping rate is 10%, and the cell survival rate is 76%.
[0066] It can be seen from the above results that the single cell suspension prepared in the experimental group has higher survival rate and nucleated cell rate, which indicates that the mixed enzyme provided by the application is especially suitable for the enzymolysis of synovial membrane tissue, and the synovial membrane tissue single cell with high quality can be obtained by enzymolysis.
[0067] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims
1. A method for enzymatic digestion of synovial tissue, characterized by, The method comprises the following steps: The synovial tissue is subjected to enzymolysis by using a mixed enzyme, and the enzymolysis is performed until the number of cells meets the requirement. The mixed enzyme comprises 1-3 parts of pronase, 0.2-1 part of neutral protease, 0.5-2 parts of collagenase I, 0.2-0.8 parts of hyaluronidase, 0.1-0.4 parts of elastase, 2-4 parts of collagenase II and 0.1-0.3 parts of DNAase in terms of weight.
2. The enzymatic method according to claim 1, characterized in that, The mixed enzyme is prepared by using DMEM, and comprises: The mixed enzyme comprises 1-3 mg / mL of pronase, 0.2-1 mg / mL of neutral protease, 0.5-2 mg / mL of collagenase I, 0.2-0.8 mg / mL of hyaluronidase, 0.1-0.4 mg / mL of elastase, 2-4 mg / mL of collagenase II and 0.1-0.3 mg / mL of DNAase.
3. The enzymatic method according to claim 1 or 2, characterized in that, The enzymolysis is performed at 36-38 ℃ and 80-150 rpm.
4. The enzymatic method according to any one of claims 1 to 3, characterized in that, The enzymolysis is performed until the number of cells meets the requirement, which comprises the following steps: The supernatant of the enzymolysis is counted after the enzymolysis for 10-15 minutes, and if the number of cells meets the requirement, the enzymolysis is stopped, and if the number of cells does not meet the requirement, the supernatant of the enzymolysis is counted again after the enzymolysis for 10-15 minutes, and the process is repeated until the number of cells meets the requirement.
5. The enzymatic method according to claim 4, characterized in that, During the enzymolysis, the wide-bore gun head is blown once every 4-7 minutes.
6. A method for preparing high-quality single cells from synovial tissue, characterized in that, The method comprises the following steps: The synovial tissue is subjected to pretreatment; The synovial tissue is subjected to enzymolysis by using the enzymolysis method according to any one of claims 1-5 to obtain an enzymolysis solution; The enzymolysis solution is subjected to screening and cell collection.
7. The preparation method according to claim 6, characterized in that, The pretreatment comprises the following steps:
8. The preparation method according to claim 6, characterized in that, The synovial tissue is washed and cut into a diameter of 1.5-3 mm. The enzymolysis comprises the following steps: The enzymolysis solution is sequentially subjected to 65-75 μm cell screening and 25-35 μm cell screening; and / or, The cell collection comprises the following steps: The cells are enriched by centrifugation of the screened enzymolysis solution, resuspended, and then centrifuged at 0-4 ℃ and 400-600 g for 5-10 minutes, and the supernatant is discarded to collect the cell precipitate.
9. The synovial tissue single cell prepared by the preparation method according to any one of claims 6-8.
10. The synovial tissue single cell according to claim 9, which is used in single-cell sequencing, primary cell culture or CAT treatment.