Single-cell suspension for increasing ratio of pancreatic acinus cells and preparation method of single-cell suspension
The problem of difficult to obtain high viability and high purity pancreatic acinar cells in the prior art is solved by digesting pancreatic tissues through a specific proportion of collagenase dissociation and terminating fluid, and the preparation of high-efficiency and high-quality single-cell suspensions is achieved.
Patent Information
- Application Number
- CN202510216625.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art is difficult to effectively obtain highly viable and high-purity pancreatic acinar cells, resulting in limited research on pancreatic single-cell sequencing.
By obtaining and pretreating mouse pancreatic tissue, collagenase digestion using a specific ratio of collagenase dissociation solution and termination solution, combined with a gradient centrifugation method, and finally using recombinant trypsin surrogate to obtain a high-quality single-cell suspension.
The proportion of acinar cells has been significantly improved, with cell viability reaching more than 90%, and the proportion of acinar cells reaching more than 70%, ensuring the high quality and efficiency of single-cell suspensions and laying the foundation for subsequent pancreatic-related research.
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Figure CN120137880A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell preparation, and particularly to a single-cell suspension for increasing the proportion of pancreatic acinar cells and a preparation method thereof. Background Art
[0002] The pancreas is an important abdominal organ with complex cell components, including pancreatic acinar cells, stellate cells, endocrine cells, endothelial cells, ductal cells, and lymphocytes, etc. Traditional sequencing analysis can only obtain samples from the whole or part of the pancreatic tissue, and the results are greatly affected by cell components, showing high heterogeneity. As a high-throughput sequencing method, single-cell sequencing can analyze the differences between individual cells and the role of cells in the microenvironment, but the key lies in obtaining high-quality single-cell suspensions.
[0003] Acinar cells in the pancreas account for about 80 - 85%, and are the main units of exocrine function. However, due to its particularity, the pancreatic tissue is prone to autodigestion during dissociation. Specifically, the zymogen of pancreatic acinar cells is easily activated during dissociation, leading to a cascade of autodigestion. This results in a low proportion of pancreatic acinar cells extracted by conventional digestion methods. In addition, acinar cells are prone to aggregation after being stimulated and are difficult to dissociate into single cells by physical methods. Conventional digestion methods cannot ensure both the separation between single cells and high cell viability. Multiple studies have shown that the current proportion of acinar cells obtained is much lower than their actual proportion in pancreatic tissue: the Olaniru team reported that the proportion of acinar cells was 10 - 20% [Cell Metabolism, 2022], the Zhuo team only detected 5 - 15% of acinar cells [Nature Communications, 2023], and the proportion fluctuated between 0 - 25% in the study by Zhang et al. [Advanced Science, 2025], all significantly lower than the true cell composition ratio of pancreatic tissue. Therefore, conventional methods are difficult to obtain acinar cells with high quantity, high viability, and less impurities, cannot reflect the true situation of pancreatic tissue, have deviations in the analysis of pancreatic diseases, and severely restrict the development of pancreatic single-cell sequencing research. Summary of the Invention
[0004] The purpose of the present invention is to provide a single-cell suspension for increasing the proportion of pancreatic acinar cells and a preparation method thereof, so as to obtain a single-cell suspension of pancreas with high viability and high purity.
[0005] To achieve the above invention purpose, the present invention provides a preparation method of a single-cell suspension for increasing the proportion of pancreatic acinar cells, including:
[0006] Obtain and preprocess mouse pancreatic tissue;
[0007] Mix the pancreatic tissue with the collagenase dissociation solution for digestion. After adding the termination solution to terminate the digestion, centrifuge, resuspend and filter the precipitate to obtain acinar cells; and
[0008] Add the enzymatic digestion solution to the acinar cells for incubation to disperse them into a single-cell state.
[0009] Optionally, the collagenase dissociation solution includes: DMEM medium, collagenase IV, trypsin inhibitor and DNase I solution;
[0010] The concentration of the collagenase IV is 1.0 - 2.0 mg / ml, the concentration of the trypsin inhibitor is 0.2 - 0.3 mg / ml, and the concentration of the DNase I solution is 0.01 - 0.02 mg / ml.
[0011] Optionally, the termination solution includes: DMEM medium, fetal bovine serum, penicillin-streptomycin mixture and trypsin inhibitor;
[0012] Among them, the volume content of the fetal bovine serum is 5% - 15%, the volume content of the penicillin-streptomycin mixture is 1% - 2%, and the concentration of the trypsin inhibitor is 0.2 - 0.3 mg / ml.
[0013] Optionally, the step of mixing the pancreatic tissue with the collagenase dissociation solution for digestion includes: incubating at 30 - 40 °C for 25 - 35 minutes, and shaking well every 5 - 10 minutes, and pipetting and mixing well after each shaking.
[0014] Optionally, the steps of collagenase digestion include:
[0015] Add the pre-cooled termination solution to terminate the digestion and then centrifuge;
[0016] Repeat the resuspension and centrifugation operations on the obtained precipitate at least three times;
[0017] Filter the precipitate resuspended for the last time to obtain acinar cells.
[0018] Optionally, filter using an 80 - 120 μm filter mesh.
[0019] Optionally, the steps of single-cell separation include:
[0020] Mix the recombinant trypsin substitute with the acinar cells for incubation;
[0021] Centrifuge at the first centrifugation speed for the first time to collect the acinar cells;
[0022] Centrifuge the collected supernatant at the second centrifugation speed for the second time to obtain non-acinar cells;
[0023] Confirm the cell dispersion state by microscopy. If the cells are not fully dispersed, continue the incubation.
[0024] Add phosphate buffer solution to terminate the digestion and centrifuge to collect the cells.
[0025] Optionally, the first centrifugation speed is 200 - 400 rpm / min, and the second centrifugation speed is 1200 - 1600 rpm / min.
[0026] Optionally, the incubation conditions are: incubate at 30 - 40 °C for 3 - 7 minutes.
[0027] The present invention also provides a mouse pancreatic single - cell suspension obtained by the above - mentioned preparation method. The cell viability in the single - cell suspension is above 90%, the proportion of acinar cells reaches above 70%, and the average cell diameter is 18 - 22 μm.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[0029] The method of the present invention is simple to operate, can effectively control the autodigestion of pancreatic tissue, and ensure that the obtained single - cell suspension has a high cell viability.
[0030] Furthermore, the present invention uses a collagenase dissociation solution and a termination solution with a specific ratio, combined with a gradient centrifugation separation method, which can not only effectively separate acinar cells and non - acinar cells, but also ensure the cell survival rate. Finally, a high - quality single - cell suspension is obtained, laying a foundation for subsequent pancreatic - related research. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1A It is the first schematic diagram of cell morphology under the electron microscope in the embodiment of the present invention;
[0032] Figure 1B It is the schematic diagram of cell morphology under the electron microscope in the embodiment of the present invention Figure Two ;
[0033] Figure 1C It is the schematic diagram of cell morphology under the electron microscope in the embodiment of the present invention Figure Three ;
[0034] Figure 2 It is the cell proportion analysis chart in the embodiment of the present invention;
[0035] Figure 3 It is the single - cell clustering analysis chart in the embodiment of the present invention;
[0036] Figure 4 It is the single - cell clustering analysis chart in the prior art. DETAILED DESCRIPTION OF THE INVENTION
[0037] The following will describe in more detail a single-cell suspension for increasing the proportion of pancreatic acinar cells and its preparation method according to the present invention with reference to the schematic diagrams. The preferred embodiments of the present invention are shown, and it should be understood that those skilled in the art can modify the present invention described herein while still achieving the advantageous effects of the present invention. Therefore, the following description should be understood as a broad guidance for those skilled in the art and not as a limitation to the present invention.
[0038] For clarity, not all features of the actual embodiments are described. In the following description, well-known functions and structures are not described in detail because they would obscure the present invention with unnecessary details. It should be considered that in the development of any actual embodiment, numerous implementation details must be made to achieve the specific goals of the developer, such as changing from one embodiment to another according to the relevant system or business limitations. Additionally, it should be considered that such development work may be complex and time-consuming, but it is only routine work for those skilled in the art.
[0039] In the following paragraphs, the present invention will be described more specifically by way of example with reference to the accompanying drawings. The advantages and features of the present invention will be clearer according to the following description. It should be noted that the drawings are all in a very simplified form and use non-precise scales, only for the purpose of conveniently and clearly assisting in explaining the objectives of the embodiments of the present invention.
[0040] Example 1
[0041] The preparation method of the single-cell suspension for increasing the proportion of pancreatic acinar cells provided by the present invention includes three steps: material collection, collagenase digestion, and single-cell separation. Among them, the material collection step is to obtain and pretreat mouse pancreatic tissue. The collagenase digestion step is to mix the pancreatic tissue with collagenase dissociation solution for digestion, add a termination solution to terminate the digestion, then centrifuge, resuspend and filter the precipitate to obtain acinar cells. The single-cell separation step is to add an enzymatic digestion solution to the acinar cells for incubation to disperse them into a single-cell state.
[0042] Specifically, in the material collection step, mouse pancreatic tissue is taken, fat, blood vessels, and mesentery are removed, and the pancreas is cut into small pieces.
[0043] Optionally, the collagenase dissociation solution includes DMEM medium, Collagenase IV, trypsin inhibitor, and DNase I solution. Among them, the concentration of Collagenase IV is 1.0 - 2.0 mg / ml, the concentration of the trypsin inhibitor is 0.2 - 0.3 mg / ml, and the concentration of the DNase I solution is 0.01 - 0.02 mg / ml.
[0044] Further, the termination solution comprises DMEM medium, fetal bovine serum, penicillin-streptomycin mixture and trypsin inhibitor, wherein the volume content of the fetal bovine serum is 5% - 15%, the volume content of the penicillin-streptomycin mixture is 1% - 2%, and the concentration of the trypsin inhibitor is 0.2 - 0.3 mg / ml.
[0045] Preferably, the step of mixing the pancreatic tissue with the collagenase dissociation solution for digestion comprises: incubating at 30 - 40 °C for 25 - 35 minutes, and shaking well every 5 - 10 minutes, and pipetting and mixing well after each shaking.
[0046] Specifically, the collagenase digestion step comprises:
[0047] Adding pre-cooled termination solution to terminate digestion and then centrifuging;
[0048] Repeating the resuspension and centrifugation operations on the obtained precipitate at least three times;
[0049] Filtering the precipitate resuspended for the last time to obtain acinar cells.
[0050] During this process, the filtering step is carried out using an 80 - 120 μm filter mesh.
[0051] Furthermore, mixing the recombinant trypsin substitute with the acinar cells and incubating; centrifuging at a first centrifugation speed for a first time to collect the acinar cells; centrifuging the collected supernatant at a second centrifugation speed for a second time to obtain non-acinar cells; observing and confirming the cell dispersion state under a microscope, and if not completely dispersed, continue incubating; adding PBS (Phosphate Buffered Saline) to terminate digestion and centrifuging to collect the cells.
[0052] Wherein, the first centrifugation speed is 200 - 400 rpm / min, and the second centrifugation speed is 1200 - 1600 rpm / min.
[0053] The first time and the second time are 5 - 10 min.
[0054] The incubation conditions for mixing the recombinant trypsin substitute with the acinar cells are incubating at 30 - 40 °C for 3 - 7 minutes.
[0055] In a specific example, the enzyme digestion solution is the recombinant trypsin substitute TrypLE TM Express, which as a mild cell dissociation enzyme, can efficiently disperse acinar cell clusters into single cells while maintaining cell viability to the greatest extent.
[0056] TrypLE TMExpress has the advantages of high purity, mild action, and no animal-derived pollution compared with traditional trypsin. It can effectively disperse acinar cell clusters into single cells while maintaining the integrity and activity of the cells.
[0057] In the mouse pancreatic single-cell suspension obtained by the preparation method of the present invention, the cell viability is above 90%, the proportion of acinar cells reaches above 70%, and the average cell diameter is 18 - 22 μm.
[0058] The present invention adopts an improved formula of collagenase dissociation solution, including a specific combination of DMEM medium, collagenase IV, trypsin inhibitor, and DNase I solution. It has good digestion effect, can effectively avoid cell autolysis, and improve cell survival rate; there is no need to adopt the traditional cold digestion method, and it can be directly digested for 30 minutes under the conditions of a conventional incubator, greatly shortening the experimental period and improving the experimental efficiency; the gradient centrifugation method is used to separate acinar cells, achieving effective separation and improving the purity of target cells; the recombinant trypsin substitute is used for single-cell separation, which can efficiently disperse cell clumps into single cells in a short time and does not require repeated filtration, ensuring both cell activity and operation efficiency.
[0059] Example Two
[0060] This example provides a specific experimental process based on Example One, including the following steps:
[0061] S1. Preparation before experiment
[0062] Sterilize the ultra-clean bench and the aseptic room with ultraviolet light for 30 min. After turning off the ultraviolet light, turn on the exhaust system, and enter the aseptic room after standing for 5 min.
[0063] Take 2 culture dishes and place them on the ultra-clean bench, add 5 - 10 ml of pre-cooled PBS solution to each, and place the culture dishes on an ice pack for standby. At the same time, prepare a set of sterile surgical instruments and place them on the ultra-clean bench for standby.
[0064] S2. Solution preparation
[0065] Collagenase dissociation solution: Based on DMEM medium, add 1.6 mg / ml of collagenase IV, 0.25 mg / ml of trypsin inhibitor, and 0.01 mg / ml of DNase I solution. Absorb it with a 50 ml syringe, filter it through a 0.22 μm filter, and preheat it in a 37 °C constant temperature incubator for standby.
[0066] Termination solution: Based on DMEM medium, add 10% fetal bovine serum by volume, 1% penicillin-streptomycin mixture by volume, and 0.25 mg / ml of trypsin inhibitor. Absorb it with a 50 ml syringe, filter it through a 0.22 μm filter, and pre-cool it at 4 °C for standby.
[0067] S3. Tissue Sampling
[0068] Make a V-shaped incision horizontally in the abdomen of the mouse inside the laminar flow hood, cut upward along the left side, and push the liver and stomach upward to fully expose the pancreas and spleen. Clamp the spleen with forceps, and successively separate the connections between the pancreas and the stomach and duodenum with scissors, and remove the pancreas and spleen completely, then immediately put them into pre-cooled PBS solution. After cutting off the spleen, rinse the pancreas repeatedly with PBS solution to remove adipose tissue, blood clots and impurities, and transfer the tissue to another PBS culture dish.
[0069] S4. Tissue Digestion
[0070] Cut the pancreas into small pieces of 1-3 mm, transfer them to a 15-ml centrifuge tube containing 6 ml of PBS, centrifuge at 4°C and 300 rpm / min for 1 min, and aspirate the supernatant. Add 6 ml of pre-warmed dissociation solution to the precipitate, shake well and incubate in a 37°C incubator. Take it out and shake well every 5-10 min, and pipette repeatedly 10 times with a Pasteur pipette after each shaking. After incubating for 30 min, add 6-8 ml of pre-cooled termination solution to terminate the digestion, centrifuge at 4°C and 300 rpm / min for 1 min, and collect the supernatant in a labeled 50-ml centrifuge tube for standby.
[0071] S5. Isolation of Acinar Cells
[0072] Resuspend the precipitate with 4-6 ml of termination solution, centrifuge at 4°C and 300 rpm / min for 1 min, and transfer the supernatant to the above 50-ml centrifuge tube. Repeat this step three times. Transfer the precipitate resuspended for the last time to a 50-ml centrifuge tube equipped with a 100-μm filter, rinse the filter with PBS and let it stand for filtration for 1 min, centrifuge the filtrate at 300 rpm / min for 1 min, and transfer the supernatant to the centrifuge tube collecting non-acinar cells. The obtained precipitate is acinar cells.
[0073] S6. Single Cell Preparation
[0074] Add 1-2 ml of TrypLE TM Express enzyme solution to the acinar cell precipitate, mix well and incubate in a 37°C incubator for 5 min. Observe the cell dispersion state under the microscope. If it is not completely dispersed, repeat the incubation step. Collect the 50-ml centrifuge tube storing the non-acinar cell supernatant, centrifuge at 4°C and 1400 rpm / min for 5 min, and the obtained precipitate is pancreatic non-acinar cells. If the red blood cell content is high, it can be treated with red blood cell lysate and then centrifuged again. Finally, resuspend the cell precipitate with 2 ml of PBS.
[0075] S7. Quality Detection
[0076] Please refer to Figure 1A - Figure 1C ,Figure 2 and Figure 3 , cell counting was performed using a hemocytometer, and cell viability was measured by cell live / dead staining. The experimental results showed that 10 5 orders of magnitude of single cells could be obtained from the pancreas of one mouse, the cell viability exceeded 90%, the proportion of acinar cells reached more than 70%, and the average cell diameter was 20.3 μm.
[0077] It should be noted that Figure 2 This is the cell proportion analysis chart in the embodiment of the present invention. This chart is a pie chart of cell type distribution, which specifically shows the relative proportions and quantities of different types of cells in this sample. Figure 3 This is the single-cell clustering analysis chart in the embodiment of the present invention; this chart reduces the high-dimensional cell feature data to two dimensions and visualizes it through t-SNE and UMAP algorithms to distinguish different cell types and show their distribution and interrelationships in the feature space. Each point in the chart represents a cell, and different colors represent different cell types. The aggregation and separation of cell types reflect their similarities and differences in the high-dimensional space. Aggregated points indicate that cells have similar features in the high-dimensional feature space. Separated points indicate that these cells have significantly different features in the high-dimensional feature space.
[0078] Figure 3 is the cell type distribution chart visualized by t-SNE / UMAP dimensionality reduction. In the figure, tsne / umap is Figure 3 the title, and the names of the dimensionality reduction algorithms used are: t-SNE (t-Distributed Stochastic Neighbor Embedding) and UMAP (Uniform Manifold Approximation and Projection). The abscissa umap_1 represents the first UMAP dimensionality reduction component, and the ordinate umap_2 represents the second UMAP dimensionality reduction component.
[0079] In summary, the method for preparing a single-cell suspension with an increased proportion of pancreatic acinar cells in this application mainly consists of 3 steps: First, after sacrificing the model mice, the pancreatic tissue is taken out, impurities are removed, and it is separated into small fragments and then washed with PBS solution; Second, the washed pancreatic tissue is placed in a mixed solution of type I and type V collagenase prepared with high-glucose DMEM medium preheated to 37°C, and digested in a 37°C environment, mixing once every 10 minutes until the proportion of single cells reaches 90%; Finally, the digested tissue is filtered, centrifuged, and the supernatant, cell debris, DNA tangles, and blood cells are discarded, and the precipitated parenchymal cells are retained, and then resuspended with PBS solution to obtain a single-cell suspension of the pancreas of mice with chronic pancreatitis.
[0080] Please refer to Figure 4, the digestive enzyme mixture in the prior art does not contain trypsin inhibitor and DNaseⅠ, and the action time of the digestive enzyme is longer, making the acinar cells more vulnerable to damage and autodigestion. At the same time, in the prior art, after digestion, the remaining single cells are directly retained by filtering through a filter screen multiple times, but there are still a large number of highly active acinar cells remaining in the tissue mass. In contrast, the new patent collects the precipitate of the digested acinar cell mass and then uses TrypLE TM Express enzyme solution to separate cell junctions without damaging the cells. Therefore, compared with the prior art, more acinar cells with higher activity can be collected by using the method provided in this application.
[0081] In summary, the present invention provides a single-cell suspension for increasing the proportion of pancreatic acinar cells and a preparation method thereof, which mainly includes three steps: material collection, collagenase digestion, and single-cell separation. By using a collagenase dissociation solution with a specific ratio and combining with gradient centrifugation separation method, the present invention realizes the efficient digestion of pancreatic tissue and the effective separation of target cells; further, by using a recombinant trypsin substitute for single-cell preparation, a large number of highly viable single cells can be obtained in a short time, and repeated filtration is avoided. This method not only effectively controls the autodigestion of pancreatic tissue, but also ensures that the obtained single-cell suspension has a high cell viability, and 10 5 orders of magnitude of single cells can be obtained, the cell viability exceeds 90%, and the proportion of acinar cells reaches more than 70%. The operation of the present invention is simple, efficient, and has good cell survival rate, laying a foundation for subsequent pancreatic-related research.
[0082] The above is only the preferred embodiment of the present invention and does not impose any limitation on the present invention. Any person skilled in the art, without departing from the technical solution of the present invention, makes any form of equivalent substitution or modification and other changes to the technical solution and technical content disclosed in the present invention, all of which belong to the content within the technical solution of the present invention and still fall within the protection scope of the present invention.
Claims
1. A method for preparing a single cell suspension for increasing the proportion of pancreatic acinar cells, characterized in that: include: Obtain and preprocess mouse pancreatic tissue; The pancreatic tissue was mixed with collagenase dissociation solution for digestion, and the digestion was terminated by adding the stop solution and then centrifuged, and the precipitate was resuspended and filtered to obtain acinar cells; as well as An enzymatic hydrolysis solution is added to the acinar cells for incubation to disperse the cells into a single cell state.
2. The preparation method according to claim 1, characterized in that: The collagenase dissociation solution comprises: DMEM culture medium, collagenase IV, trypsin inhibitor and DNase I solution; Wherein, the concentration of collagenase IV is 1.0-2.0 mg / ml, the concentration of trypsin inhibitor is 0.2-0.3 mg / ml, and the concentration of DNase I solution is 0.01-0.02 mg / ml.
3. The preparation method according to claim 1, characterized in that: The stop solution includes: DMEM culture medium, fetal bovine serum, penicillin-streptomycin mixed solution and trypsin inhibitor; The volume content of the fetal bovine serum is 5% to 15%, the volume content of the penicillin-streptomycin mixture is 1% to 2%, and the concentration of the trypsin inhibitor is 0.2 to 0.3 mg / ml.
4. The preparation method according to claim 1, characterized in that: The step of mixing the pancreatic tissue with the collagenase dissociation solution for digestion includes: incubating at 30-40° C. for 25-35 minutes, shaking once every 5-10 minutes, and mixing by blowing after each shaking.
5. The preparation method according to claim 1, characterized in that: The collagenase digestion steps include: Add pre-cooled stop solution to terminate digestion and then centrifuge; Repeat the resuspension and centrifugation of the obtained precipitate at least three times; The final resuspended precipitate was filtered to obtain acinar cells.
6. The preparation method according to claim 5, characterized in that: Use 80-120 μm filter for filtering.
7. The preparation method according to claim 1, characterized in that: The single cell separation step comprises: Recombinant trypsin substitute was mixed and incubated with acinar cells; centrifuging at a first centrifugation speed for a first time to collect acinar cells; centrifuging the collected supernatant at a second centrifugation speed for a second time to obtain non-acinar cells; Confirm the cell dispersion status by microscopic observation, and continue incubation if it is not completely dispersed; The digestion was terminated by adding phosphate buffered saline and the cells were collected by centrifugation.
8. The preparation method according to claim 7, characterized in that: The first centrifugal speed is 200-400 rpm / min, and the second centrifugal speed is 1200-1600 rpm / min.
9. The preparation method according to claim 8, characterized in that: The incubation conditions are: incubation at 30-40° C. for 3-7 minutes.
10. The mouse pancreatic single cell suspension obtained by the preparation method according to any one of claims 1 to 9, characterized in that: The cell viability in the single cell suspension is above 90%, the proportion of acinar cells is above 70%, and the average cell diameter is 18-22 μm.