Method for extracting rabbit brain tissue single-cell suspension and kit

Abstract

The invention relates to the technical field of cell extraction, in particular to an extraction method of rabbit brain tissue single-cell suspension and a kit. The kit comprises 100 to 1000 U / mL of collagenase IV, 200 to 2000 U / mL of collagenase II, 300 to 700 U / mL of DNA (Deoxyribonucleic Acid) enzyme I, 1 to 10 mg / mL of vitamin E polyethylene glycol succinate and 100 to 1000 U / mL of EDTA (Ethylene Diamine Tetraacetic Acid) pancreatin. The kit further comprises a cell debris removal buffer solution, and the cell debris removal buffer solution comprises a low-density buffer solution and a high-density buffer solution. The kit can be particularly used for extracting single-cell suspension from rabbit brain tissues; the single-cell suspension is low in caking rate, large in number of cells, high in nucleation rate and large in cell grouping, red blood cells almost do not exist in the single-cell suspension, and subsequent researches such as sequencing are facilitated; the time for preparing the single-cell suspension is short, and the pretreatment is simple.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell extraction, in particular to a method and kit for extracting rabbit brain tissue single cell suspension. BACKGROUND

[0002] Mammalian brain tissue single cell suspension is a suspension system of single cells prepared from mammalian brain tissue. It contains various cell types, including neurons, astrocytes, oligodendrocytes, microglia cells, etc. It can be used for cell type identification and classification, cell function research, disease model research, drug screening and toxicity testing, cell therapy research, cell development and differentiation research, etc. It has important significance and value in neuroscience research and even the whole life medical research, and can provide key support for in-depth exploration of brain function and disease mechanism.

[0003] Currently, there are many methods and technologies for preparing mammalian brain tissue single cell suspension, such as the patent CN118048307A "Animal experiment brain tissue single cell separation and preparation method". These technologies are mainly optimized for mouse brain tissue, and the processing effect is not good for brain tissue of other species. Due to the differences in tissue structure, cell composition, extracellular matrix composition, etc. of brain tissue of different species, directly applying the processing method of mouse brain tissue may cause insufficient digestion, low nucleated rate, and reduced cell activity, etc.

[0004] In the process of preparing rabbit brain tissue single cell suspension, low nucleated rate is a common problem. There are a large number of myelin cells and synaptic structures in the gray matter and white matter of brain tissue, which will release a large amount of cell debris and impurities during enzymatic digestion into single cell suspension. If not treated, it will have a great negative impact on downstream experiments, such as high viscosity of cell suspension, which cannot be smoothly water-in-oil, mRNA capture efficiency is seriously reduced in subsequent reverse transcription process, and nucleated cell types are missing in the final results. In addition, low cell number and high clumping rate will also affect the effect of single cell sequencing. Low cell number means that the amount of cell sample available for sequencing is limited, which may not fully reflect the cell heterogeneity of rabbit brain tissue; high clumping rate may cause blockage of microfluidic chip channels during subsequent water-in-oil process, and increase the cost of bioinformatics analysis due to re-encapsulation.

[0005] Due to the great difference between rabbit brain tissue and mouse brain tissue, using the existing technology for extracting mouse brain tissue single cell suspension cannot solve the above problems. At the same time, these existing technologies often have the shortcomings of long extraction time and complex pretreatment, making it more difficult to extract rabbit brain tissue single cell suspension. Therefore, there is currently no method for extracting rabbit brain tissue single cell suspension. SUMMARY

[0006] The technical problem solved by the present application is to provide a method and a kit for extracting a single cell suspension of rabbit brain tissue.

[0007] The technical solution of the present application to solve the above technical problem is as follows: The present application provides a kit for extracting a single cell suspension of rabbit brain tissue, comprising a plurality of enzyme solutions, wherein the plurality of enzyme solutions comprise 100-1000 U / mL collagenase IV, 200-2000 U / mL collagenase II, 300-700 U / mL DNase I, 1-10 mg / mL vitamin E polyethylene glycol succinate, and 100-1000 U / mL EDTA trypsin; and further comprising a cell debris removal buffer, wherein the cell debris removal buffer comprises a low-density buffer and a high-density buffer.

[0008] Further, the low-density buffer comprises 0.3-0.5 mol / L sucrose and 3% BSA, and the high-density buffer comprises 30%-80% Percoll, 4-8 mmol / L KCl, 1-5 mmol / L MgCl2, and 1-5 mmol / L CaCl2.

[0009] Further, in the low-density buffer, the concentration of sucrose is 0.3 mol / L, and the high-density buffer is 70% Percoll.

[0010] Further, the kit further comprises a red blood cell lysis solution, wherein the red blood cell lysis solution comprises 8-9 g / L NH4Cl, 0.5-2 g / L NaHCO3, and 0.01-0.05 g / L EDTA.

[0011] The present application further provides a method for extracting a single cell suspension of rabbit brain tissue, which uses the kit as described above for extraction, comprising the steps of using a plurality of enzyme solutions for enzymolysis and using a cell debris removal buffer for removing cell debris.

[0012] Further, the step of using a plurality of enzyme solutions for enzymolysis comprises the following steps: first using EDTA trypsin for decalcification enzymolysis on the cleaned and chopped rabbit brain tissue sample, then centrifuging the enzymolyzed sample to remove the supernatant, and then using collagenase II, collagenase IV, and DNase I for enzymolysis.

[0013] Further, the enzymolyzed suspension is sieved and resuspended to obtain a cell resuspension, and the cell debris removal buffer is used to remove cell debris; the step of removing cell debris comprises the following steps: first adding the high-density buffer to the bottom of a centrifuge tube, then adding the cell resuspension, and then adding the low-density buffer, and then collecting the cell precipitate located at the bottom after horizontal centrifugation.

[0014] Further, the centrifugal mode of the horizontal centrifugation is a central acceleration and deceleration, and the centrifugal condition is centrifugation at 3000xg for 20 min at 4℃.

[0015] Further, the method further comprises a step of performing ice lysis of red blood cells by using a red blood cell lysis solution.

[0016] Further, the method further comprises a step of performing ice lysis of red blood cells by using a red blood cell lysis solution.

[0017] The beneficial effects of the present application are: (1) The kit for extracting a single cell suspension of rabbit brain tissue of the present application provides a set of enzyme solution formula specially optimized for rabbit brain tissue, and the technical bias of "EDTA-trypsin destroying neurons", so as to realize high activity and low damage dissociation. (2) The kit for extracting a single cell suspension of rabbit brain tissue of the present application adds vitamin E polyethylene glycol succinate in the enzyme solution, which is a molecule composed of a lipophilic vitamin E head, a hydrophilic polyethylene glycol chain and a succinic acid linker group. It can effectively emulsify the lipids released during brain tissue dissociation to make the solution more stable. At the same time, as an antioxidant, it can effectively protect the unsaturated fatty acids in the cell membrane phospholipids from oxidative damage during the enzyme dissociation process. On this basis, it can integrate itself into the cell membrane to provide a certain steric hindrance protection to stabilize the membrane structure. (3) The kit for extracting a single cell suspension of rabbit brain tissue of the present application establishes a "low density-high density" double density debris removal system. One-step centrifugation can lock myelin debris and dead cell nucleus debris at the interface, effectively improving the recovery rate of living cells and the removal rate of debris, and is significantly superior to the traditional method. (4) The kit for extracting a single cell suspension of rabbit brain tissue of the present application can selectively lyse residual red blood cells in the rabbit brain on ice, almost without damaging nucleated cells, and completely eliminating the risk of red blood cell contamination of downstream mRNA sequencing and microfluidic blockage. (5) The method for extracting a single cell suspension of rabbit brain tissue of the present application has high efficiency, simple pretreatment, and fewer operation steps, which significantly reduces the experimental time and labor cost. (6) The method for extracting a single cell suspension of rabbit brain tissue of the present application has high activity, low clumping rate, high nucleated rate, sufficient cell number, and rich cell subpopulation, and can be directly used for high-resolution single cell omics research such as scRNA-seq and scATAC-seq. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 FIG. 1 is a cell counting instrument analysis result graph of Example 1 of the present application, Figure 1Image A in the image is a fluorescently labeled image. Figure 1 In diagram B, the cell diameter distribution is shown. Figure 1 C in the middle is the fluorescence intensity distribution map of FL1. Figure 1 D in the diagram represents the fluorescence intensity distribution of FL2. Figure 2 This is a graph showing the analysis results of the cell counter in Example 2 of the present invention. Figure 2 Image A in the image is a fluorescently labeled image. Figure 2 In diagram B, the cell diameter distribution is shown. Figure 2 C in the middle is the fluorescence intensity distribution map of FL1. Figure 2 D in the diagram represents the fluorescence intensity distribution of FL2. Figure 3 This is a graph showing the analysis results of the cell counter in Example 3 of the present invention. Figure 3 Image A in the image is a fluorescently labeled image. Figure 3 In diagram B, the cell diameter distribution is shown. Figure 3 C in the middle is the fluorescence intensity distribution map of FL1. Figure 3 D in the diagram represents the fluorescence intensity distribution of FL2. Figure 4 This is a graph showing the analysis results of the cell counter in Example 4 of the present invention. Figure 4 Image A in the image is a fluorescently labeled image. Figure 4 In diagram B, the cell diameter distribution is shown. Figure 4 C in the middle is the fluorescence intensity distribution map of FL1. Figure 4 D in the diagram represents the fluorescence intensity distribution of FL2. Figure 5 This is a graph showing the analysis results of a cell counter used in the comparative example of this invention. Figure 5 Image A in the image is a fluorescently labeled image. Figure 5 In diagram B, the cell diameter distribution is shown. Figure 5 C in the middle is the fluorescence intensity distribution map of FL1. Figure 5 D in the diagram represents the fluorescence intensity distribution of FL2. Figure 6 This is a graph showing the cell counting results of Comparative Example 2 of the present invention. Figure 6 Image A in the image is a fluorescently labeled image. Figure 6 In diagram B, the cell diameter distribution is shown. Figure 6 C in the middle is the fluorescence intensity distribution map of FL1. Figure 6 D in the diagram represents the fluorescence intensity distribution of FL2. Figure 7 This is a graph showing the cell counting results of Comparative Example 3 of the present invention. Figure 7 Image A in the image is a fluorescently labeled image. Figure 7 In diagram B, the cell diameter distribution is shown. Figure 7 C in the middle is the fluorescence intensity distribution map of FL1. Figure 7 D in the diagram represents the fluorescence intensity distribution of FL2. Figure 8A cell counter analysis result graph of the present application comparative example 4, Figure 8 A fluorescent labeling image, Figure 8 B cell diameter distribution, Figure 8 C FL1 fluorescence intensity distribution, Figure 8 D FL2 fluorescence intensity distribution. Figure 9 A cell counter analysis result graph of the present application comparative example 5, Figure 9 A fluorescent labeling image, Figure 9 B cell diameter distribution, Figure 9 C FL1 fluorescence intensity distribution, Figure 9 D FL2 fluorescence intensity distribution. Figure 10 A cell counter analysis result graph of the present application comparative example 6, Figure 10 A fluorescent labeling image, Figure 10 B cell diameter distribution, Figure 10 C FL1 fluorescence intensity distribution, Figure 10 D FL2 fluorescence intensity distribution. DETAILED DESCRIPTION

[0019] The principles and features of the present application are described below, and the examples are only used to explain the present application, and are not used to limit the scope of the present application.

[0020] The kit for extracting a single cell suspension of rabbit brain tissue of the present application comprises a plurality of enzyme solutions, the plurality of enzyme solutions comprising 100-1000 U / mL of collagenase IV, 200-2000 U / mL of collagenase II, 300-700 U / mL of DNase I, 1-10 mg / mL of vitamin E polyethylene glycol succinate, and 100-1000 U / mL of EDTA trypsin; and further comprising a cell debris removal buffer, the cell debris removal buffer comprising a low-density buffer and a high-density buffer.

[0021] The kit for extracting a single cell suspension of rabbit brain tissue of the present application can be used to extract a single cell suspension of rabbit brain tissue.

[0022] Compared with mouse brain tissue, rabbit brain tissue structure is relatively complex, cell types are more abundant, neuron network is complex and fragile, and cells are more susceptible to damage during dissociation, resulting in reduced cell activity, which makes the dissociation of rabbit brain tissue relatively more complex. At the same time, the adhesion of rabbit brain tissue cells is stronger, and more effective enzymatic digestion or mechanical separation method is needed to break the tight connection between cells when preparing cell suspension, while the adhesion of mouse brain tissue cells is relatively weak and is easier to be separated into single cell suspension. In addition, the tolerance of rabbit brain tissue cells to enzymatic digestion is lower, and they are more susceptible to damage during enzymatic digestion, resulting in reduced cell activity, while the tolerance of mouse brain tissue cells to enzymatic digestion is relatively higher, and the cell activity is better maintained under the same enzymatic digestion conditions. The above factors result in that the existing enzyme solution for mouse brain tissue is not suitable for rabbit brain tissue.

[0023] The prior art has long held a deep-rooted technical prejudice: EDTA-trypsin can irreversibly destroy the synaptic structure of neurons and reduce cell activity, and is therefore listed as a "high-risk" scheme in brain single cell preparation. 2+

[0024] However, there are species differences in the composition of rabbit brain ECM collagen subtypes compared with mice, and the sample is first subjected to preliminary digestion by EDTA-trypsin at a lower concentration, and at the same time, decalcification treatment is carried out. Moderate decalcification treatment not only does not reduce the activity of brain tissue cells, but also effectively avoids calcium overload caused by tissue fragmentation. Then, the sample is treated with collagenase containing vitamin E polyethylene glycol succinate, which not only repairs the oxidative damage caused by enzymatic digestion, but also further stabilizes the cell membrane structure and the suspension properties, laying a foundation for obtaining a high-activity cell suspension. The combination of EDTA-trypsin pretreatment and vitamin E polyethylene glycol succinate collagenase improves the efficiency of releasing cells in rabbit brain ECM by 2-3 times compared with the traditional Collagenase I / IV+Papain combination, and the integrity of neuron membrane is not significantly affected.

[0025] At the same time, based on the moderate down-regulation of Integrin β1 expression by EDTA-trypsin, the cell-matrix adhesion is weakened, and the use of EDTA-trypsin for pretreatment enzymolysis effectively reduces the cell clumping rate; vitamin E polyethylene glycol succinate enhances the stress resistance of cells, further reducing the negative impact of subsequent debris removal steps on cell activity and quantity.

[0026] Preferably, the concentration of each enzyme solution of the application is 1000 U / mL of collagenase IV, 800 U / mL of collagenase II, 500 U / mL of DNase I, 5 mg / mL of vitamin E polyethylene glycol succinate, and 400 U / mL of EDTA-trypsin.

[0027] ​Preferably, the low-density buffer comprises 0.3-0.5 mol / L sucrose and 3% BSA, and the high-density buffer comprises 30%-80% Percoll, 4-8 mmol / L KCl, 1-5 mmol / L MgCl2 and 1-5 mmol / L CaCl2.

[0028] The rabbit brain tissue has the problems of interlaced gray matter and white matter, high myelin content and fragile neuron network, and the traditional single-density debris removal often has the problems of myelin particle suspension, dead cell nucleus debris retention and insufficient recovery of living cells. The present application establishes a double-density system, which comprises a low-density layer of 0.3-0.5 mol / L sucrose and 3% BSA and a high-density layer of 30%-80% Percoll, KCl, MgCl2 and CaCl2, so as to be suitable for the three-dimensional distribution characteristics of the rabbit brain myelin with low density, dead cell nucleus fragments with slightly higher density and living cells with higher density. The low-density layer makes the floated myelin fragments further dehydrated under high osmotic pressure, so as to further reduce the density of the myelin fragments, and the BSA protein network is used to capture the myelin fragments, so as to prevent the myelin fragments from being settled again and forming a dense film layer. The high-density layer ensures that the cells are under suitable osmotic pressure and provides a moderate divalent cation bridge, so as to quickly pull the living neurons and glial cells with greater density to the bottom of the tube under the action of 3000xg gentle centrifugation, and lock the dead cell fragments with density between the two above the interface, so as to form three clear bands visible to the naked eye, and effectively improve the recovery rate of living cells in the rabbit brain tissue suspension and significantly reduce the clumping rate. The method is significantly better than the pure sucrose method and the ACK method, so as to solve the core pain point of more difficult removal of rabbit brain tissue fragments with extremely low mechanical stress.

[0029] Preferably, the concentration of sucrose in the low-density buffer is 0.3 mol / L, and the high-density buffer is 70% Percoll.

[0030] Preferably, the kit further comprises a red blood cell lysis solution, and the components and concentrations of the red blood cell lysis solution are 8-9 g / L NH4Cl, 0.5-2 g / L NaHCO3 and 0.01-0.05 g / L EDTA.

[0031] The red blood cell lysis solution of the present application can quickly lyse a small amount of red blood cells mixed in the rabbit brain tissue single-cell suspension, so as to avoid the interference of the red blood cells on the subsequent experiments. At the same time, the red blood cell lysis solution of the present application hardly damages the nucleated cells when lysing the red blood cells, so as to ensure the activity and quality of the neurons and other nucleated cells in the rabbit brain tissue single-cell suspension, and provide reliable cell basis for the subsequent experiments.

[0032] Preferably, the components and concentrations of the red blood cell lysis solution are 8.3 g / L NH4Cl, 1.0 g / L NaHCO3 and 0.037 g / L EDTA.

[0033] The extraction method of the rabbit brain tissue single cell suspension of the present application adopts the kit as described above to extract, including the step of enzymolysis using multiple enzyme solutions and the step of removing cell fragments using the cell fragment removal buffer.

[0034] Preferably, the step of enzymolysis using multiple enzyme solutions is: first, EDTA trypsin is used to perform decalcification enzymolysis on the cleaned and chopped rabbit brain tissue sample, then the sample after enzymolysis is centrifuged to remove the supernatant, and collagenase II, collagenase IV and DNase I are used for resuspension enzymolysis.

[0035] Preferably, after the solution after enzymolysis is digested, screened and resuspended, a cell resuspension is obtained, and the cell fragment removal buffer is used to remove cell fragments; the step of removing cell fragments is: first, the high-density buffer is added to the bottom of the centrifuge tube, then the cell resuspension is added, and then the low-density buffer is added, and after horizontal centrifugation, the cell precipitate located at the bottom is collected.

[0036] Specifically, the step of removing cell fragments is as follows: (1) Gently mix the rabbit brain single cell crude suspension after digestion and filtration, take 2 mL of the cell suspension to be removed from the fragments and 1 mL of pre-cooled high-density buffer (70% Percoll, 6 mmol / L KCl, 3 mmol / L MgCl2, 3 mmol / L CaCl2) to slowly stack at the bottom of a 15 mL centrifuge tube and gently blow 10 times, and the two form a high-density bottom layer instantaneously, and then during centrifugation, the living cells begin to gather downward due to the slightly higher density.

[0037] In this step, 2 mL of the cell suspension and 1 mL of the pre-cooled high-density buffer are taken to form a 3 mL higher-density mixed layer at the bottom of the centrifuge tube, which can ensure that the living cells can sink during the subsequent horizontal centrifugation, while the fragments float above the interface; at the same time, the total volume of the mixed solution is only 3 mL, which leaves enough space for the subsequent slow stacking of the upper layer of low-density buffer along the tube wall, preventing the interface from being disturbed, and ensuring that the gradient is clear and the stratification is sharp.

[0038] (2) Then use a 1 mL pipette to slowly inject 3 mL of low-density buffer (0.3 mol / L sucrose, 3% BSA, PBS pH 7.4) along the wall to form a clear interface on the upper layer without disturbing the lower layer, and then during centrifugation, the low-density layer will capture the suspended myelin fragments and dead cell fragments. (3) Place the centrifuge tube on a horizontal rotor, centrifuge at 4°C, 3000×g for 20 min. During the centrifugation process, the density gradient is further compacted. Myelin and fragments float to the low-density layer due to the lowest density and are fixed by the BSA network. Dead cell fragments remain in the intermediate transition zone, while live cells quickly settle to the bottom of the tube under the action of centrifugal force to form a tight white precipitate. During this period, cations can protect the cell membrane from the influence of high G.

[0039] (4) After centrifugation, first use a P1000 pipette to carefully remove the upper low-density liquid and interface debris along the tube wall, then completely remove the residual supernatant, leaving only the bottom cell cluster; finally, add 4℃ 5% FBS PBS to gently resuspend.

[0040] Preferably, the method of the present invention further includes the step of lysing red blood cells on ice using red blood cell lysis buffer.

[0041] Preferably, the precipitate obtained after lysing red blood cells on ice is resuspended to obtain a single-cell suspension of rabbit brain tissue.

[0042] In one embodiment of the present invention, the method of the present invention includes the following steps: S1. Clean the rabbit brain tissue sample.

[0043] In this step, the rabbit brain tissue sample is washed with PBS.

[0044] S2. Enzymatically digest the minced tissue.

[0045] Different enzymatic hydrolysates were used for digestion in sequence. During digestion, a 37°C water bath was used, and the mixture was manually shaken every 3-5 minutes, or the mixture was placed in a 37°C hybridization furnace with a rotation speed of 20-30 rpm.

[0046] When using EDTA trypsin for pretreatment digestion, the cell suspension is checked at regular intervals to determine the stop time based on the total cell count and activity. This avoids over-digestion that could lead to cell damage or death, and also prevents incomplete digestion that could prevent the rabbit brain tissue cells from being completely dissociated. Subsequent enzymatic digestion then yields a precise amount of a well-active single-cell suspension, ensuring cell quality and quantity and providing a reliable foundation for subsequent experiments.

[0047] S3. Use a cell sieve to filter the digested products and collect the filtrate.

[0048] Preferably, the cell screen is 70 μm, and the collected filtrate is placed in a new 15 mL centrifuge tube; the 70 μm cell screen can effectively intercept large pieces of undigested tissue, cell clumps and other larger impurity particles, and only allow single cells or smaller cell clumps to pass through, thereby improving the purity of the cell suspension and reducing errors and background noise caused by large pieces of tissue or impurities in subsequent experiments.

[0049] For the precipitate after filtration, 3 mL of RPMI 1640 medium or 5% FBS PBS is added, mixed and washed, and similarly filtered using a 70 μm cell screen, and the filtrate is collected and combined with the first collected filtrate, which can recover as many cells as possible that are intercepted on the cell screen, avoid cell loss, improve the overall recovery rate of the cells, and ensure that there are enough cells for analysis and application in subsequent experiments.

[0050] S4, the filtrate is subjected to at least one centrifugation-resuspension operation, and the precipitate is obtained by centrifugation each time and resuspended to obtain a resuspension.

[0051] Preferably, the centrifugation condition each time is 300xg for 5 min at 4°C; the centrifugation condition is mild, which can effectively remove the enzyme solution and avoid mechanical damage or stress reaction of the cells caused by excessive centrifugal force or high temperature, which helps to maintain the activity and integrity of the cells.

[0052] Preferably, the resuspension is performed using a PBS solution containing 5% FBS, and the amount of each resuspension is 5 mL; which can provide certain protection for the cells and further maintain the activity and stability of the cells.

[0053] S5, the resuspension obtained in step S4 is subjected to cell debris removal using a cell debris removal buffer to obtain a cell precipitate.

[0054] S6, after the cell precipitate obtained in step S5 is resuspended in a PBS solution containing 5% FBS, red blood cells are lysed using a red blood cell lysis solution on ice, and after the lysis of the red blood cells is terminated, centrifugation is performed to obtain a cell precipitate.

[0055] Specifically, the cell precipitate obtained in step S5 is mixed with a PBS solution containing 5% FBS by mass to obtain a pre-lysis cell suspension, and the volume of the red blood cell lysis solution is 3-5 times the volume of the pre-lysis cell suspension.

[0056] Preferably, the lysis time of the red blood cells is 5 min, and an equal volume of PBS solution is added and mixed to terminate the lysis of the red blood cells.

[0057] Preferably, the centrifugation condition after the lysis of the red blood cells is terminated is 450xg for 5 min at 4°C.

[0058] S7, resuspend the precipitate obtained in step S6 to obtain a single-cell suspension of rabbit brain tissue.

[0059] Preferably, the step is specifically resuspending the precipitate with 10 mL of PBS containing 5% FBS, mixing by blowing, centrifuging at 300xg at 4°C for 5 min, and discarding the supernatant; then resuspending the precipitate with 50 μL-2 mL of PBS containing 5% FBS, and adjusting the volume of the resuspension according to the required cell concentration.

[0060] Preferably, for the resuspension obtained finally, if there are obvious cell clumps, filter them with a 20 μm cell strainer, and collect the filtrate in a new centrifuge tube. If there are no obvious cell clumps, this step can be skipped.

[0061] The application is illustrated below by specific examples.

[0062] Example 1 This example uses the method of the application to extract a single-cell suspension of rabbit brain tissue. The rabbit is a New Zealand rabbit cryopreserved brain tissue sample.

[0063] The concentrations of the enzyme solutions used in this example are 900 U / mL of collagenase IV, 800 U / mL of collagenase II, 600 U / mL of DNase I, 5 mg / mL of vitamin E polyethylene glycol succinate, and 400 U / mL of EDTA trypsin.

[0064] The low-density buffer in the cell debris removal buffer used in this example contains 0.35 mol / L sucrose and 3% BSA, and the high-density buffer contains 70% Percoll, 6 mmol / L KCl, 3 mmol / L MgCl2, and 3 mmol / L CaCl2.

[0065] The red blood cell lysis solution used in this example is: 8.3 g / L NH4Cl, 1.0 g / L NaHCO3, and 0.037 g / L EDTA.

[0066] The cell viability is 98.27%, the live cell concentration is 1.1E+06 / mL, the cell clumping rate is 5.19%, the nucleated cell rate is 87%, and the total number of live cells is about 270,000. The average diameter of the cells is 9.73 μm, and the average circularity is 0.93. The flow cytometry analysis fluorescence chart and distribution chart of this example are shown in Figure 1 .

[0067] Example 2 The sample used in this example is the same as that of Example 1, and the steps and specific components of the enzyme solutions are also basically the same as those of Example 1, with the following differences: The low density buffer of the cell debris removal buffer used in this example contains 0.3 mol / L sucrose and 3% BSA, and the high density buffer contains 65% Percoll, 4 mmol / L KCl, 1 mmol / L MgCl2, and 1 mmol / L CaCl2.

[0068] The erythrocyte lysis solution used in this example is 8.0 g / L NH4Cl, 0.5 g / L NaHCO3, and 0.035 g / L EDTA.

[0069] The cell viability is 85.41%, the live cell concentration is 1.17E+06 / mL, the cell clumping rate is 18.45%, the nucleated rate is 79%, and the total number of live cells is about 470,000. The average diameter of the cells is 10.49 μm, and the average circularity is 0.78.

[0070] Example 3 The sample used in this example is the same as that of Example 1, the steps and the specific components of the enzyme solution are also basically the same as those of Example 1, and the specific components of the erythrocyte lysis solution are the same as those of Example 2. The difference lies in that: The low density buffer of the cell debris removal buffer used in this example contains 0.5 mol / L sucrose and 3% BSA, and the high density buffer contains 70% Percoll, 6 mmol / L KCl, 5 mmol / L MgCl2, and 5 mmol / L CaCl2.

[0071] The cell viability is 89.54%, the live cell concentration is 1E+06 / mL, the cell clumping rate is 9.51%, the nucleated rate is 81%, and the total number of live cells is about 300,000. The average diameter of the cells is 9.35 μm, and the average circularity is 0.87.

[0072] Example 4 The sample used in this example is the same as that of Example 1, the steps and the specific components of the enzyme solution are also basically the same as those of Example 1, and the specific components of the cell debris removal buffer are the same as those of Example 2. The difference lies in that: The erythrocyte lysis solution used in this example is 9 g / L NH4Cl, 2 g / L NaHCO3, and 0.040 g / L EDTA.

[0073] The cell viability is 91.59%, the live cell concentration is 1.21E+06 / mL, the cell clumping rate is 10.16%, the nucleated rate is 84%, and the total number of live cells is about 19w. The average diameter of the cells is 10.56 μm, and the average circularity is 0.89.

[0074] Comparative Example 1 The same rabbit brain tissue as in Example 1 was chopped and placed in an enzyme solution, mixed thoroughly, and subjected to enzymatic treatment at 37°C for 20 min. After the enzymatic treatment, the brain tissue solution after enzymatic treatment was filtered through a cell filter screen, and the filtrate was collected.

[0075] The enzyme solution used in this comparative example was an enzyme solution for mouse brain tissue, and the specific components were 800 U / mL Collagenase Type 1, 800 U / mL Collagenase Type 2, 4 U / mL Dispase II, 150 U / mL DNase I, and 700 U / mL Papain Papaya solution.

[0076] The filtrate collected in the above step was centrifuged, and the supernatant was discarded. The precipitate was resuspended to obtain a single-cell suspension of rabbit brain tissue.

[0077] The cell viability was 74.25%, the live cell concentration was 8.47E+05 / mL, the cell clumping rate was 10.41%, the nucleated rate was 9%, and the total number of live cells was about 250,000. The average diameter of the cells was 10.77 μm, and the average circularity was 0.62.

[0078] Comparative Example 2 The difference between this comparative example and Example 1 is that only a high-density buffer was used to remove cell debris. The cell viability was 94.19%, the live cell concentration was 6E+05 / mL, the cell clumping rate was 17%, the nucleated rate was 34%, and the total number of live cells was about 12w. The average diameter of the cells was 10.61 μm, and the average circularity was 0.86.

[0079] Comparative Example 3 The difference between this comparative example and Example 1 is that the concentration of EDTA trypsin was 50 U / mL. The cell viability was 82.67%, the live cell concentration was 1.84E+05 / mL, the cell clumping rate was 27.06%, the nucleated rate was 70%, and the total number of live cells was about 7.2w. The average diameter of the cells was 11.2 μm, and the average circularity was 0.57.

[0080] Comparative Example 4 The difference between this comparative example and Example 1 is that the concentration of EDTA trypsin was 1500 U / mL. The cell viability was 59.07%, the live cell concentration was 1.08E+06 / mL, the cell clumping rate was 4.48%, the nucleated rate was 67%, and the total number of live cells was about 11w. The average diameter of the cells was 12.7 μm, and the average circularity was 0.94.

[0081] Comparative Example 5 The difference between the present comparative example and Example 1 is that no vitamin E polyethylene glycol succinate is added to the enzymatic solution. The cell viability is 65.16%, the viable cell concentration is 1.11E+06 / mL, the cell clumping rate is 17.93%, the nucleated rate is 55%, and the total number of viable cells is about 11w. The average diameter of the cells is 10.14pm, and the average circularity is 0.73.

[0082] Comparative Example 6 The difference between the present comparative example and Example 1 is that the Percoll concentration in the high-density buffer is 90%. The cell viability is 58.1%, the viable cell concentration is 9.04E+04 / mL, the cell clumping rate is 17.21%, the nucleated rate is 84%, and the total number of viable cells is about 9k. The average diameter of the cells is 11.35pm, and the average circularity is 0.65.

[0083] The results of the above examples and comparative examples can be concluded as follows: The cell viability, cell clumping rate, and nucleated rate test results of Examples 1-4 all meet the subsequent test requirements, and Example 1 is the best.

[0084] Comparative Example 1 uses the method for extracting single-cell suspension from mouse brain tissue in the prior art. The single-cell suspension of this comparative example has low viability, low cell number, and extremely low nucleated rate. Using it for single-cell sequencing may cause blockage of microfluidic channels, leading to water-in-oil failure, and may seriously reduce mRNA capture efficiency during reverse transcription, resulting in a lack of cell types and many miscellaneous genes in the final sequencing results, making it difficult to obtain ideal results. There are a large number of red blood cells in the single-cell suspension of this comparative example, which will contaminate the downstream sequencing data. At the same time, the preparation of the single-cell suspension of this comparative example requires a long time and complex pretreatment.

[0085] Comparative Example 2 only uses a high-density buffer as a cell fragment removal liquid. The fragments do not sufficiently float up and form a dense membrane layer, making it difficult to completely remove them. The cell clumping rate and nucleated rate of this comparative example are both not good.

[0086] The concentration of EDTA trypsin, a pretreatment enzyme, in Comparative Example 3 is 50U / mL. The degree of decalcification and extracellular matrix enzymolysis is insufficient, and the cells are not fully released, resulting in poor cell number and clumping rate. The concentration of EDTA trypsin, a pretreatment enzyme, in Comparative Example 4 is 1500U / mL. The high enzyme concentration damages the integrity of the cell membrane, resulting in poor activity.

[0087] No vitamin E polyethylene glycol succinate is added to the enzymatic solution of Comparative Example 5. The antioxidant capacity and emulsion stability of the enzyme solution decrease, resulting in poor activity and nucleated rate.

[0088] The Percoll concentration in the high-density buffer of Comparative Example 6 is 90%. The density is too high, and the viable cells cannot sink during centrifugation, resulting in poor activity and cell number.

[0089] 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 construed as limiting the present application, and that variations, modifications, substitutions and changes can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A kit for extracting a single cell suspension of rabbit brain tissue, characterized by, The kit comprises a plurality of enzyme solutions, the plurality of enzyme solutions comprising 100-1000 U / mL collagenase IV, 200-2000 U / mL collagenase II, 300-700 U / mL DNase I, 1-10 mg / mL vitamin E polyethylene glycol succinate, and 100-1000 U / mL EDTA trypsin; and a cell debris removal buffer comprising a low-density buffer and a high-density buffer.

2. The kit for extracting a single cell suspension of rabbit brain tissue according to claim 1, wherein, The low-density buffer comprises 0.3-0.5 mol / L sucrose and 3% BSA, and the high-density buffer comprises 30%-80% Percoll, 4-8 mmol / L KCl, 1-5 mmol / L MgCl2, and 1-5 mmol / L CaCl2.

3. The kit for extracting a single cell suspension of rabbit brain tissue according to claim 2, wherein, The low-density buffer comprises 0.3 mol / L sucrose, and the high-density buffer comprises 70% Percoll.

4. The kit for extracting a single cell suspension of rabbit brain tissue according to claim 1, wherein, The kit further comprises a red blood cell lysis solution comprising 8-9 g / L NH4Cl, 0.5-2 g / L NaHCO3, and 0.01-0.05 g / L EDTA.

5. A method for extracting a single cell suspension of rabbit brain tissue, characterized by, The kit is used for extraction according to any one of claims 1-4, comprising the steps of enzymolysis using the plurality of enzyme solutions and removal of cell debris using the cell debris removal buffer.

6. The method according to claim 5, wherein the method is characterized by, The step of enzymolysis using the plurality of enzyme solutions comprises the following steps: first, enzymolysis of the cleaned and chopped rabbit brain tissue sample using the EDTA trypsin, then centrifugation of the enzymolyzed sample to remove the supernatant, and enzymolysis using the collagenase II, the collagenase IV, and the DNase I.

7. The method according to claim 5, wherein the method is characterized by, The enzymolyzed solution is subjected to digestion, sieving, and resuspension to obtain a cell suspension, and the cell debris removal buffer is used to remove cell debris; the step of removing cell debris comprises the following steps: first, adding the high-density buffer to the bottom of a centrifuge tube, then adding the cell suspension, and finally adding the low-density buffer, followed by horizontal centrifugation, and collection of the cell precipitate at the bottom.

8. The method according to claim 7, wherein the method is characterized by, The horizontal centrifugation is performed at a central acceleration and deceleration, and the centrifugation conditions are 3000xg at 4°C for 20 min.

9. The method of claim 5, wherein the method is characterized by, The kit further comprises a step of lysis of red blood cells on ice using a red blood cell lysis solution.

10. The method of claim 9, wherein the method is characterized by, The precipitate obtained after lysis of red blood cells on ice is resuspended to obtain the rabbit brain tissue single-cell suspension. The kit further comprises a step of lysis of red blood cells on ice using a red blood cell lysis solution.

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