Mixed enzymatic dissociation liquid as well as use method and application thereof

Abstract

The invention discloses a mixed enzymatic dissociation solution as well as a use method and application thereof, and relates to the technical field of tissue dissociation solutions. The mixed enzymatic dissociation liquid disclosed by the invention comprises harvest media, PBS (Phosphate Buffer Solution), a first digestive juice and a second digestive juice, the first digestive juice comprises 10% of 10 * D-Hanks, 0.1% of 0.1 M EDTA (Ethylene Diamine Tetraacetic Acid), 0.5% of FBS, 0.01% of 1M DTT (Diethylene Terephthalate) and secondary distilled water; the second digestive juice contains dispase II with the concentration of 2 mg / ml, collagenase IV with the concentration of 2 mg / ml, nuclease with the concentration of 15 [mu] l / ml and an RPMI 1640 culture medium; the invention also provides a use method of the mixed enzymatic dissociation liquid in a tissue dissociation process. The mixed enzymatic dissociation liquid provided by the invention is matched with an optimized dissociation step, so that the living rate and the living cell concentration of cells after tissue or organ dissociation are improved, meanwhile, the agglomeration rate of the cells is also remarkably reduced, and the mixed enzymatic dissociation liquid can be applied to dissociation of organs or tissues such as gastric mucosa, intestinal tracts, livers and kidneys.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue dissociation fluid, and in particular relates to a mixed enzyme dissociation fluid and a use method thereof, which can be applied to the dissociation technology of gastric mucosa, intestinal tract, liver, kidney and other tissues. Background Art

[0002] Tissue dissociation technology is a crucial technology in the field of biology. It is used to separate organs, tissues or cells for subsequent in vitro research, cell culture or bioengineering applications. The main principle of tissue dissociation technology is to decompose tissue cells into separate cells through a series of biochemical and physical treatments, and then selectively separate the required cells through cell separation and screening technology.

[0003] Tissue dissociation technology is widely used in basic biomedical research, clinical medicine and treatment. In basic biomedical research, tissue dissociation technology can be used to separate and purify cells (normal cells, tumor cells, immune cells, etc.). The separated or purified cells are used for subsequent cell culture, cloning, gene expression analysis and other experiments; specific types of cells can be obtained to further explore the functions of these cells in organisms, such as studying the role of neuronal cells in the nervous system, or studying the function of immune cells in immune response; it can be used for drug screening and evaluation, using dissociated cells to evaluate the mechanism of action, efficacy and possible toxic side effects of drugs on cells, providing important experimental basis for drug research and development. In recent years, with the rise of single-cell sequencing technology, the application of tissue dissociation technology in biomedical research has become more extensive. For example, the preparation of single-cell suspension is a prerequisite for single-cell transcriptome sequencing, and tissue dissociation technology is the key link in preparing such suspension. Therefore, tissue dissociation technology is a powerful and flexible tool. It has a wide range of applications in cell biology, molecular biology, neuroscience and other fields, and is of great significance for understanding the structure and function of cells, exploring the mechanism of disease occurrence, and developing new drugs.

[0004] In the prior art, conventional tissue dissociation techniques are to first break the tissue by physical methods, and then add dissociation fluid to dissociate the tissue into single cells. The technicians judge the quality of the dissociation effect by the thin viability, viable cell concentration and cell agglomeration rate of the gastric mucosa after tissue dissociation. However, there are currently no commercially available dissociation reagents and supporting dissociation methods specifically used for mouse gastric mucosa, intestinal tract, liver, kidney and other tissues. The current tissue dissociation technology mainly uses a universal tissue dissociation fluid and its supporting method to dissociate mouse gastric mucosa, intestinal tract, liver, kidney and the like. Universal tissue dissociation fluids usually contain reagents such as proteolytic enzymes, pancreatic enzymes, dispase, EDTA, cholesterol esterase, etc., which can effectively decompose extracellular matrix molecules, such as collagen, so as to quickly and accurately dissociate a large number of cells. However, after actual operation, it was found that the cell viability, viable cell concentration and agglomeration rate of the gastric mucosa, intestinal tract, liver, kidney and the like after dissociation by the existing tissue dissociation technology were low.

[0005] The commonly used tissue dissociation methods in the industry are: 1. After sampling, place the sample in a petri dish on ice and mince the tissue into 0.5-1.5 cm 3 Transfer about 10 mg of minced tissue to a test tube; 2. Add 1 mL of trypsin-EDTA (0.25%), seal the centrifuge tube lid with plastic wrap, and incubate at 37°C with shaking for 30 min; 3. After incubation, gently grind the sample and pipette up and down 6-8 times to allow undigested tissue to settle to the bottom of the centrifuge tube. 4. The cell suspension was passed through a 30 μm sieve in a centrifuge tube. The centrifuge tube was placed on ice. The filter membrane was rinsed with pre-cooled PBS / BSA 0.04%, and pre-cooled 0.04% PBS / BSA was added to a total volume of 10 mL. 5. Centrifuge at 350 g, 4°C for 5 min and remove the supernatant; 6. If the tissue contains a large number of red blood cells, red blood cell lysis operation (steps 6-9) is required. Add 1 mL of red blood cell lysis buffer and place on ice for 2 minutes. 7. After 2 min, add 5 mL of pre-cooled PBS / BSA 0.04% to neutralize; 8. Centrifuge at 350 g for 5 min at 4°C, remove the supernatant, and add 0.04% pre-cooled PBS / BSA to 10 mL; 9. Centrifuge at 350 g for 5 min at 4°C and remove as much supernatant as possible.

[0006] 10. Resuspend the cell suspension in 100 µL of pre-cold 0.04% PBS / BSA on ice and measure the cell number and viability using trypan blue or fluorescence counting.

[0007] In the commonly used tissue dissociation method mentioned above, trypsin is added for incubation in step 2 and then ground again in step 3. At this time, the tissue has been preliminarily digested. Since each technician has different operating habits, grinding will cause different degrees of cell death, the experimental repeatability is poor, and the cell yield and viability are low.

[0008] Therefore, developing a special dissociation solution for mouse gastric mucosa, intestines, liver, kidneys and other tissues, and selecting a suitable supporting dissociation method to improve the dissociation effect have become the research and development focus of technicians in this field. Summary of the invention

[0009] The purpose of the present invention is to provide a mixed enzyme dissociation solution, and with optimized dissociation steps, improve the viability and viable cell concentration of cells after tissue or organ dissociation, and significantly reduce the cell agglomeration rate, and can be applied to the dissociation of organs or tissues such as gastric mucosa, intestines, liver, and kidneys.

[0010] In order to achieve the purpose of the present invention, the present invention provides a mixed enzyme dissociation solution, characterized in that it includes the following components: harvest media, PBS, a first digestion solution and a second digestion solution.

[0011] The first digestion solution includes 10×D-Hanks, 0.1M EDTA, FBS, 1M DTT (ie, 1M dithiothreitol solution, wherein 1M refers to 1.54251 g / 10 ml) and double distilled water (ie, ddH2O).

[0012] D-Hanks is a balanced salt solution. Compared with the commonly used balanced salt solution PBS, D-Hanks solution does not contain calcium ions, magnesium ions and glucose. If the balanced salt solution contains calcium ions and magnesium ions, it helps to maintain the integrity of the tissue. However, the D-Hanks solution without calcium and magnesium ions is selected in the present invention to destroy the integrity of the tissue and dissociate the cells.

[0013] EDTA, or ethylenediaminetetraacetic acid, is a divalent metal ion chelator that is usually used in combination with trypsin, dispase, etc. during the digestion process.

[0014] DTT is dithiothreitol. It is currently used in experiments to reduce the dimerization of DNA and the formation of disulfide bonds in proteins. There are no reports of its direct use in the process of digesting cells.

[0015] The second digestion solution includes dispase II, collagenase IV, nuclease and RPMI 1640 culture medium.

[0016] Dispase II is a non-specific metalloproteinase that can quickly and gently destroy the extracellular matrix of tissues.

[0017] Collagenase IV is an enzyme extracted from bacteria that has a strong digestive effect on collagen.

[0018] Nucleases are mainly used to enzymatically hydrolyze DNA and RNA.

[0019] Furthermore, the components of the mixed enzyme dissociation solution are calculated by volume percentage as follows: 84.75% of harvest media, 8.47% of PBS, 3.39% of the first digestion solution and 3.39% of the second digestion solution.

[0020] Furthermore, the harvest media comprises, by volume percentage, 5% heat-inactivated FBS, 1% 1×HGPG, and the remainder is RPMI 1640 culture medium.

[0021] Furthermore, the PBS is a phosphate buffered saline containing 5% FBS by volume, that is, the volume ratio of FBS to the PBS component is 5%.

[0022] Furthermore, the components of the first digestion solution are calculated by volume as follows: 10% 10×D-Hanks, 0.1% 0.1M EDTA, 0.5% FBS, 0.01% 1M DTT, and the remainder is double distilled water.

[0023] Furthermore, the second digestion solution contains 2 mg / ml dispase II, 2 mg / ml collagenase IV, 15 μl / ml nuclease, and the remainder is RPMI 1640 culture medium.

[0024] Furthermore, the mass concentration of the nuclease is 50 mg / ml.

[0025] The present invention also provides a method for using the mixed enzyme dissociation solution, which specifically comprises the following steps: S1. After euthanizing the mice, the stomach tissue was collected on ice and cut into 1-3 mm 2 The tissue pieces were washed twice with harvest media, and the supernatant was discarded to obtain the precipitate. S2. Add the first digestion solution to the above precipitate for the first digestion, then place it in a vortex mixer and vortex for 10 seconds, centrifuge, discard the first digestion solution, and obtain a first digestion product; S3. adding the second digestion solution to the first digestion product for a second digestion, and then vortexing for 10 seconds in a vortex mixer, filtering through a 40 μm cell sieve, and collecting the supernatant to obtain a second digestion product; S4. centrifuging the second digestion product, removing the supernatant, adding harvest media for resuspending, centrifuging, removing the supernatant, and collecting the cell pellet; S5. Resuspend the above cell precipitate with an appropriate amount of PBS to obtain a gastric mucosal cell suspension.

[0026] Furthermore, the first digestion process is: shaking at 37°C and a rotation speed of 100-200 rpm for 45-60 min; and / or the second digestion process is: shaking at 37°C and a rotation speed of 100-200 rpm for 45-60 min.

[0027] Furthermore, the volume ratio of the tissue pieces to the first digestive fluid is 1:5; The volume ratio of the tissue pieces to the second digestion solution is 1:5.

[0028] Furthermore, in step S4, the centrifugation condition is: 250g centrifugation at 4°C for 10 min.

[0029] The mixed enzyme dissociation solution provided by the present invention can be used for dissociating gastric mucosa, intestinal tract, liver, kidney or other tissues and organs.

[0030] The present invention has achieved the following beneficial effects: The present invention prepares a special mixed enzyme dissociation solution for gastric mucosa, intestinal tract, liver, kidney and other tissues, and continuously optimizes the dissociation method steps, thereby improving the viability and viable cell concentration of cells after dissociation and reducing the cell agglomeration rate.

[0031] The first digestion solution of the present invention uses three reagents, D-Hanks, EDTA and DTT, in combination, especially the combination of EDTA and DTT, which can play a role in dissociating tissues, and is more gentle than the traditional EDTA directly used in combination with trypsin, etc., effectively reducing cell mortality and improving cell yield.

[0032] The second digestion solution of the present invention adds a trace amount of nuclease on the basis of the combined use of dispase II and collagenase IV to obtain a better digestion effect and reduce the occurrence of cell aggregation, which has not been found in previous experiments. It is speculated that some ruptured cells inevitably appear in the process of dissociating tissues, and the nucleic acids of these cells are exposed in the solution, causing cell aggregation. After adding a small amount of nuclease, this part of the exposed nucleic acid is digested, thereby playing a role in reducing aggregation.

[0033] 4. The present invention adopts the method of adding the first digestion solution and the second digestion solution step by step to dissociate the tissue, rather than adding two digestion solutions at the same time, which reduces the damage of various reagents to the tissue and cells, and obtains a better dissociation effect under the mildest possible conditions. If the first digestion solution and the second digestion solution are added at the same time, the tissue will be dissociated quickly, but the cytoskeleton will also be severely damaged, which will lead to a decrease in cell mortality and final survival rate (the survival rate of cells is low after a period of culture). BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 This is a microscopic examination of a cell suspension after mouse gastric mucosa dissociation according to an embodiment of the present invention; Figure 2 This is a cell counting diagram of a cell suspension after dissociation of mouse gastric mucosa and staining according to an embodiment of the present invention; Figure 3 This is a microscopic examination of the cell suspension after the mouse gastric mucosa is dissociated in Comparative Example 1 of the present invention; Figure 4 This is a cell counting diagram of the cell suspension after dissociation of mouse gastric mucosa in Comparative Example 1 of the present invention after staining; Figure 5 This is a microscopic examination of the cell suspension after the mouse gastric mucosa is dissociated in Comparative Example 2 of the present invention; Figure 6 This is a cell counting diagram of the cell suspension after dissociation of mouse gastric mucosa in Comparative Example 2 of the present invention after staining. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The reagent information involved in the embodiments of the present invention is shown in the following table:

[0037] The mixed enzyme dissociation solution and the dissociation method of the present invention are further described below in conjunction with specific examples. Example 1

[0038] Mixed enzyme dissociation solution for mouse gastric mucosal tissue and dissociation method thereof Preparation of mixed enzyme dissociation solution for mouse gastric mucosal tissue: Prepare 500 ml of harvest media: 25 ml heat-inactivated FBS + 5 ml 1×HGPG, and pour into 470 ml RPMI 1640 medium.

[0039] The preparation method of HGPG (diluted to 1× with RPMI 1640 before use) is as follows: Mix 59.6 g HEPES, 14.6 g L-glutamine, 1 x 106 U penicillin, 1 g streptomycin, and 2.5 mg gentamicin, and then add RPMI 1640 to 500 mL to obtain a buffer solution; Then use NaOH to adjust the pH of the buffer to 7.5 (before adjustment, the buffer is yellow / orange and the pH is about 6.0); Finally, filter sterilization was performed using a 0.45 μm filter to obtain HGPG.

[0040] Prepare 50 ml of PBS: 50 ml PBS + 250 μl FBS.

[0041] Prepare 20 ml of the first digestion solution: 2 ml 10×D-Hanks + 200 μl 0.1 M EDTA standard solution + 1 ml FBS + 20 μl DTT (1.54251 g / 10 m) + ddH2O to make up.

[0042] Prepare 20 ml of the second digestion solution: 0.04 g dispase II + 0.04 g collagenase IV + 300 μl nuclease (50 mg / ml) + RPMI 1640 medium to complete.

[0043] The process of dissociating the gastric tissue of mice using the mixed enzyme dissociation solution for mouse gastric mucosal tissue is as follows (the following reagent amounts are all based on the reagent amounts required for the stomach of one mouse): S1. After euthanizing the mice, the stomachs were removed on ice and cut into 1-3 mm 2 Small piece, take 4cm 3 Place the small pieces of gastric tissue in a container, then add sufficient amount of harvest media to wash twice (in the washing process, turn it over several times, let it settle, discard the supernatant, and then replace with new harvest media to repeat the washing process), discard the supernatant, and obtain the precipitate; S2. Add 20 ml of the first digestion solution to the above precipitate, shake at 37°C and 150 rpm for 60 min, then place in a vortex mixer for 10 s, centrifuge at 250 g for 5 min at 4°C, discard the first digestion solution, and obtain the first digestion product; S3. Add 20 ml of the second digestion solution to the first digestion product, shake at 37° C. and 150 rpm for 60 min, then vortex for 10 s in a vortex mixer, filter through a 40 μm cell sieve, collect the supernatant, and obtain the second digestion product; S4. The second digestion product was centrifuged at 250 g for 10 min at 4°C, the supernatant was removed, harvest media was added for resuspending, and the product was centrifuged at 250 g for 10 min at 4°C, the supernatant was removed, and the cell pellet was collected; S5. Resuspend the above cell pellet with 1 ml of PBS to obtain a gastric mucosal cell suspension. Comparative Example 1

[0044] Dissociation solution: Trypsin-EDTA (0.25%) S1. After removing the stomach, place it in a culture dish on ice and mince the tissue into small pieces of 0.5-1.5 cm3.

[0045] S2. Transfer about 10 mg of minced tissue into a test tube.

[0046] S3. Add 1 ml of trypsin-EDTA (0.25%), seal the centrifuge tube lid with plastic wrap, and incubate with shaking at 37°C for 30 min.

[0047] S4. After incubation, gently grind the sample and pipette up and down 6-8 times using a serological pipette. Undigested tissue pieces will settle to the bottom of the centrifuge tube.

[0048] S5. The cell suspension was passed through a 30 μm sieve in a centrifuge tube. The centrifuge tube was placed on ice. The filter membrane was rinsed with pre-cooled PBS / BSA 0.04%, and pre-cooled 0.04% PBS / BSA was added to make the total volume 10 ml.

[0049] S6. Centrifuge at 350g, 4℃ for 5 min and remove the supernatant.

[0050] S7. If the tissue contains a large number of red blood cells, red blood cell lysis is required (steps 7-10). Add 1 ml of red blood cell lysis buffer and place on ice for min.

[0051] After 8.2 min, add 5 ml of pre-cooled PBS / BSA 0.04% for neutralization.

[0052] S9. Centrifuge at 350 g for 5 min at 4°C, remove the supernatant, and add 0.04% pre-cooled PBS / BSA to 10 ml.

[0053] S10. Centrifuge at 350 g for 5 min at 4°C and remove as much supernatant as possible.

[0054] S11. Resuspend the cell suspension in 100 µl of pre-cold 0.04% PBS / BSA on ice and measure the cell number and activity using trypan blue or fluorescence counting. Comparative Example 2

[0055] Dissociation solution: 1-2 mg / ml collagenase S1. Tissue fragmentation: According to the size of gastric tissue block (generally recommended not to exceed 1cm 3 ), transfer to a straight-pointed 1.5 ml EP tube, and use ophthalmic forceps to fully mince the tissue.

[0056] S2. Trypsin digestion: Use collagenase I with a final concentration of 1-2 mg / ml to digest in a shaking water bath at 37°C for 20-30 minutes. Take out a small amount of digestion solution at 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes of digestion to check the condition of individual cells in the digestion solution (whether there is shrinkage / tissue blocks), and then determine the optimal digestion time.

[0057] S3. Filtration: The diameter of somatic cells is generally between 10-20 μm. However, when digesting primary tissues with enzymes, a much larger filter (that can filter out tissues) is required instead of a slightly larger filter. Generally, 200-400 mesh is selected (the larger the value, the smaller the filter). S4. Collect cells by centrifugation: Centrifuge at 1000 rpm for 5 min to obtain filtered cell suspension, and wash the cells 3-5 times with 4°C pre-cooled culture medium / PBS The dissociation effects of the mixed enzyme dissociation solution and dissociation method for mouse gastric mucosal tissue of Example 1 and Comparative Examples 1-2 were tested.

[0058] The test method is as follows: 18 μl of the gastric mucosal cell suspensions obtained in Example 1 and Comparative Examples 1-2 were taken respectively, 2 μl of trypan blue was added to each of them and blown evenly, and then detected by a cell counter, and the cell concentration and cell activity were detected.

[0059] Experimental results: The test results of the cell suspension after dissociation of mouse gastric mucosal tissue are shown in Table 1 and Figure 1-6 As shown, Figure 1 This is a microscopic examination of the cell suspension obtained by dissociation in Example 1; Figure 2 This is a cell counting diagram of the cell suspension obtained by dissociation in Example 1 after staining (wherein red represents agglomeration, green represents living cells, and yellow represents dead cells, the same below); Figure 3 This is a microscopic examination of the cell suspension obtained by dissociation in Comparative Example 1; Figure 4This is a cell counting diagram of the cell suspension obtained by dissociation in Comparative Example 1 after staining; Figure 5 This is a microscopic examination of the cell suspension obtained by dissociation in Comparative Example 2; Figure 6 This is a cell counting diagram of the cell suspension obtained by dissociation in Comparative Example 2 after staining.

[0060] Table 1 Cell statistics after dissociation of mouse gastric mucosal tissue

[0061] Note: The sampling volume refers to the total number of cells identified and counted by a cell counter. For gastric mucosa of the same size, the larger the value, the better.

[0062] It can be seen from the above test results that the mixed enzyme dissociation solution and the corresponding dissociation method provided by the present invention are optimized for mouse gastric mucosa, have good specificity, and the cell agglomeration rate after dissociation is low, the cell viability is high, and the concentration of live cells is high.

[0063] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A mixed enzyme dissociation solution, characterized in that: Includes the following components: harvest media, PBS, first digestion solution, and second digestion solution; The first digestion solution includes 10×D-Hanks, 0.1M EDTA, FBS, 1M DTT and double distilled water; The second digestion solution includes dispase II, collagenase IV, nuclease and RPMI 1640 culture medium.

2. The mixed enzyme dissociation solution according to claim 1, characterized in that The components of the mixed enzyme dissociation solution are calculated by volume percentage as follows: 84.75% of harvest media, 8.47% of PBS, 3.39% of the first digestion solution and 3.39% of the second digestion solution.

3. The mixed enzyme dissociation solution according to claim 1, characterized in that The harvest media contained, by volume percentage, 5% heat-inactivated FBS, 1% 1×HGPG, and the remainder was RPMI 1640 culture medium.

4. The mixed enzyme dissociation solution according to claim 1 or 2, characterized in that: The PBS is a phosphate buffered saline containing 5% FBS by volume.

5. The mixed enzyme dissociation solution according to claim 1, characterized in that The components of the first digestion solution are as follows by volume: 10% 10×D-Hanks, 0.1% 0.1M EDTA, 0.5% FBS, 0.01% 1M DTT, and the remainder is double distilled water.

6. The mixed enzyme dissociation solution for gastric mucosal tissue according to claim 1, characterized in that: The second digestion solution contains 2 mg / ml dispase II, 2 mg / ml collagenase IV, 15 μl / ml nuclease, and the remainder is RPMI1640 culture medium.

7. The mixed enzyme dissociation solution according to claim 6, characterized in that: The mass concentration of the nuclease is 50 mg / ml.

8. A method for using the mixed enzyme dissociation solution according to any one of claims 1 to 7, characterized in that: The specific steps include: S1. After euthanizing the mice, take the required stomach tissue on ice and cut into 1-3 mm 2 The tissue pieces were washed twice with harvest media, and the supernatant was discarded to obtain the precipitate; S2. Add the first digestion solution to the above precipitate for the first digestion, shake at 37°C and 100-200 rpm for 45-60 min, then vortex for 10 s in a vortex mixer, centrifuge, discard the first digestion solution, and obtain a first digestion product; S3. Add the second digestion solution to the first digestion product for a second digestion, shake at 37°C and 100-200 rpm for 45-60 min, then vortex for 10 s in a vortex mixer, filter through a 40 μm cell sieve, collect the supernatant, and obtain a second digestion product; S4. centrifuging the second digestion product, removing the supernatant, adding harvest media for resuspending, centrifuging, removing the supernatant, and collecting the cell pellet; S5. Resuspend the above cell precipitate with an appropriate amount of PBS to obtain a gastric mucosal cell suspension.

9. The method for using the mixed enzyme dissociation solution according to claim 8, characterized in that: The volume ratio of the tissue pieces to the first digestion solution is 1:5; The volume ratio of the tissue pieces to the second digestion solution is 1:

5.

10. Use of the mixed enzyme dissociation solution according to any one of claims 1 to 7 in dissociating gastric mucosa, intestines, liver, kidney or other tissues and organs.

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