Mouse carotid artery tissue digest and uses thereof

Abstract

The present application relates to the technical field of biology, in particular to mouse common carotid artery tissue digestion solution and application thereof, and provides a preparation method of mouse common carotid artery tissue digestion solution and mouse common carotid artery tissue single cell. The preparation method can obtain a mouse common carotid artery tissue primary cell suspension with high viability, low clumping rate, high nucleated rate and less impurities, and can greatly improve the success rate of mouse common carotid artery tissue single cell sequencing experiment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a digestive fluid of mouse common carotid artery tissue and its application. Background Technology

[0002] Single-cell sequencing technology is a technique that performs sequencing analysis at the level of a single cell, including the genome, transcriptome, and epigenome. Currently, this technology is widely used in basic scientific research and is also of great significance for the early diagnosis, tracking, and personalized treatment of some diseases.

[0003] A prerequisite for conducting single-cell gene expression studies is to obtain high-quality single-cell suspensions by rapidly dissociating the tissue after acquisition. There are two main methods for obtaining primary single-cell suspensions from tissue: enzymatic digestion and tissue block culture. Tissue culture typically requires 3-5 days to obtain a certain amount of primary single cells, and this method is suitable for studying the three-dimensional structure of tissues and cell-cell interactions. Currently, there are few commercially available enzymatic digestion methods suitable for obtaining high-quality (high viability, low clumping rate, high purity) primary single-cell suspensions from mouse common carotid artery tissue. With the development of single-cell technology, there is an urgent need for a rapid and inexpensive enzymatic digestion method / kit for mouse common carotid artery tissue. Summary of the Invention

[0004] In view of this, the present invention provides a digestive solution of mouse common carotid artery tissue and its application. The present invention provides a method for preparing the digestive solution of mouse common carotid artery tissue and a single-cell suspension of mouse common carotid artery tissue. Experiments have shown that the preparation method of the present invention can dissociate mouse common carotid artery tissue cells while maintaining a cell viability of over 90%, and significantly increasing the amount of cells harvested.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a digestive fluid of mouse common carotid artery tissue, comprising: collagenase II, elastase, dispersin II, CaCl2, FBS, and DNase I; or

[0007] Collagenase II, elastase, dispersase II, CaCl2, BSA, DNase I.

[0008] In some specific embodiments of the present invention, the mouse common carotid artery tissue digestive fluid includes:

[0009] Collagenase II 1-2 mg / mL

[0010] Elastase 0.5–1 mg / mL

[0011] Dispersase II 1-2 mg / mL

[0012] CaCl2 2.5mM

[0013] FBS 2%–5%

[0014] DNase I 10–50 μg / mL; or collagenase II 1–2 mg / mL

[0015] Elastase 0.5–1 mg / mL

[0016] Dispersase II 1-2 mg / mL

[0017] CaCl2 2.5mM

[0018] BSA 0.5%

[0019] DNase I 10–50 μg / mL.

[0020] In some specific embodiments of the present invention, the mouse common carotid artery tissue digestion solution comprises: collagenase II 1 mg / mL

[0021] elastase 0.5 mg / mL

[0022] Dispersase II 1 mg / mL

[0023] CaCl2 2.5mM

[0024] FBS 2%

[0025] DNase I 10 μg / mL; or collagenase II 1 mg / mL

[0026] elastase 0.5 mg / mL

[0027] Dispersase II 1 mg / mL

[0028] CaCl2 2.5mM

[0029] BSA 0.5%

[0030] DNase I 10 μg / mL.

[0031] The present invention also provides the use of the mouse common carotid artery tissue digestion solution in any of the following: (I) preparing a mouse common carotid artery tissue digestion kit; and / or

[0032] (II) Preparation of single cells from mouse common carotid artery tissue; and / or

[0033] (III) Single-cell sequencing of mouse common carotid artery tissue.

[0034] The present invention also provides a mouse common carotid artery tissue digestion kit, comprising the mouse common carotid artery tissue digestion solution and acceptable adjuvants.

[0035] This invention also provides a method for preparing a single-cell suspension of mouse common carotid artery tissue, comprising the following steps:

[0036] Step 1: Take a mouse common carotid artery tissue block, wash with 1x PBS, digest with trypsin, wash with 1x PBS, cut into small pieces, and add the mouse common carotid artery tissue digestion solution for tissue digestion;

[0037] Step 2: After complete tissue digestion, filter the tissue using a cell sieve to obtain a single-cell suspension of mouse common carotid artery tissue with impurities removed, and then centrifuge and resuspend it.

[0038] Step 3: Mix the erythrocyte lysate with the mouse common carotid artery tissue single-cell suspension obtained in Step 2, and lyse to obtain the mouse common carotid artery tissue single-cell suspension.

[0039] In some specific embodiments of the present invention, the amount of mouse common carotid artery tissue digestion solution added in step 1 includes 1 mL / 100 mg of mouse common carotid artery tissue.

[0040] In some specific embodiments of the present invention, the temperature for pancreatic enzyme digestion in step 1 includes 37°C; and / or

[0041] The pancreatic enzyme digestion time in step 1 includes 20 minutes.

[0042] In some specific embodiments of the present invention, the tissue digestion temperature in step 1 includes 37°C; and / or

[0043] The tissue digestion time mentioned in step 1 includes 1 hour.

[0044] In some specific embodiments of the present invention, the pore size of the cell sieve in step 2 includes 70 μm.

[0045] In some specific embodiments of the present invention, the volume ratio of the mouse common carotid artery tissue single-cell suspension obtained in step 2 to the erythrocyte lysate in step 3 is 1:3.

[0046] In some specific embodiments of the present invention, the pyrolysis time in step 3 of the preparation method includes 5 minutes.

[0047] In some specific embodiments of the present invention, the resuspension solvent in step 2 comprises 1x PBS and 0.04% BSA.

[0048] This invention includes, but is not limited to, the following beneficial effects:

[0049] The preparation method of this invention can obtain a large number of single cells from mouse common carotid artery tissue while maintaining a cell viability of over 90%. Sufficient cell quantity is a prerequisite for optimization processes such as erythrocyte lysis and removal of dead cells. The cell suspension obtained by this method, characterized by high viability, low clumping rate, high nucleation rate, and low impurity content, lays the foundation for obtaining high-quality gene expression data from single-cell sequencing of mouse common carotid artery tissue. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0051] Figure 1 The fluorescence field results of mouse common carotid artery tissue dissociated using a commercial tissue dissociation kit (Mitteni 130-110-201) are shown.

[0052] Figure 2 The fluorescence field results of mouse common carotid artery tissue dissociated by the digestion method of the present invention are shown.

[0053] Figure 3 The bright-field results of dissociating mouse common carotid artery tissue using a commercial tissue dissociation kit are shown.

[0054] Figure 4 The bright-field results of the digestion method of the present invention for dissociating mouse common carotid artery tissue are shown. Detailed Implementation

[0055] This invention discloses a digestive fluid from mouse common carotid artery tissue and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0056] This invention provides a digestive solution for mouse common carotid artery tissue, with the following formulation: 1–2 mg / mL collagenase II (Solepro C8150); 0.5–1 mg / mL elastase (Yuanye S10165); 1–2 mg / mL dispersase II (Roche 45147700); 2.5 mM CaCl2 (Sigma C7902); 2%–5% FBS (Gibco 10100139) or 0.5% BSA (Medlan 130-091-376); 10–50 μg / mL DNase I (Sigma DN25).

[0057] The preparation method of this invention can obtain a large number of single cells from mouse common carotid artery tissue while maintaining a cell viability of over 90%. Sufficient cell quantity is a prerequisite for optimization processes such as erythrocyte lysis and removal of dead cells. The cell suspension obtained by this method, characterized by high viability, low clumping rate, high nucleation rate, and low impurity content, lays the foundation for obtaining high-quality gene expression data from single-cell sequencing of mouse common carotid artery tissue.

[0058] Unless otherwise specified, the raw materials and reagents used in the mouse common carotid artery tissue digestion solution and its application provided by this invention can all be purchased from the market.

[0059] The present invention will be further illustrated below with reference to the embodiments:

[0060] Example: Dissociation of the common carotid artery in mice

[0061] 1. Digestive solution formula: 1 mg / mL collagenase II (Solepro C8150); 0.5 mg / mL elastase (Yuanye S10165); 1 mg / mL dispersase II (Roche 45147700); 2.5 mM CaCl2 (Sigma C7902); 2% FBS (Gibco10100139); 10 μg / mL DNase I (Sigma DN25).

[0062] 2. Pretreatment of mouse common carotid artery tissue blocks: The tissue blocks were cleaned with PBS (Saiwell G4202), and then transferred to 0.25% trypsin (Beijing Haicheng Yuanhong Technology HCCC101) and digested at 37°C for 20 min.

[0063] 3. Remove the tissue from the pancreatic enzyme, wash it with PBS and cut it into small pieces. Transfer the cut tissue into digestion solution (add 2 mL of digestion solution for every 200 mg of tissue to dissociate it).

[0064] 4. Tissue dissociation conditions: The digestion solution was transferred to a 37°C environment for tissue dissociation, and the dissociation time was 1 hour.

[0065] 5. Removal of residual tissue: After digestion, filter the digestion solution using a cell sieve with a pore size of 40 μm to remove large-volume impurities, centrifuge and resuspend, collect cells, resuspend the cells in 1xPBS + 0.04% BSA, and perform quality control on the cell suspension.

[0066] 6. Red blood cell removal: Add red blood cell lysis buffer (Solepro R1010) to the cell suspension. The volume ratio of cell suspension to red blood cell lysis buffer is 1:3, and the lysis time is 5 min.

[0067] 7. Figure 1The results show the fluorescence field of mouse common carotid artery tissue dissociated using a commercial tissue dissociation kit (Mitteni 130-110-201). Figure 2 The fluorescence field results of the mouse common carotid artery tissue dissociated by the digestion method of the present invention are shown. Figure 3 These are bright-field results from a commercial tissue dissociation kit. Figure 4 This is the bright-field result of the digestion method of this invention for dissociating mouse common carotid artery tissue. A comparison of the two dissociation methods shows that there is no significant difference between the results obtained by this method and the commercial kit method. This method can yield high-quality cell suspensions for single-cell sequencing, and the cost is significantly reduced.

[0068] A comparison of the commercially available reagent kits and the cell suspensions obtained by the method of this invention is shown in Table 1:

[0069] Table 1 Comparison of the effects of commercially available reagent kits and the method of this invention.

[0070]

[0071]

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A digestive fluid from mouse common carotid artery tissue, characterized in that, include: Collagenase II, elastase, dispersase II, CaCl2, FBS, DNase I; or Collagenase II, elastase, dispersase II, CaCl2, BSA, DNase I.

2. The mouse common carotid artery tissue digestion solution as described in claim 1, characterized in that, include:

3. The mouse common carotid artery tissue digestion solution as described in claim 1 or 2, characterized in that, include:

4. The use of the mouse common carotid artery tissue digestion solution as described in any one of claims 1 to 3 in any of the following: (I) Preparation of a mouse common carotid artery tissue digestion kit; and / or (II) Preparation of single cells from mouse common carotid artery tissue; and / or (III) Single-cell sequencing of mouse common carotid artery tissue.

5. A mouse common carotid artery tissue digestion kit, characterized in that, Includes the mouse common carotid artery tissue digestive fluid as described in any one of claims 1 to 3, and acceptable adjuvants.

6. A method for preparing single cells from mouse common carotid artery tissue, characterized in that, Includes the following steps: Step 1: Take a mouse common carotid artery tissue block, wash with 1x PBS, digest with trypsin, wash with 1x PBS, cut into small pieces, and add the mouse common carotid artery tissue digestion solution as described in any one of claims 1 to 3 for tissue digestion. Step 2: After complete tissue digestion, filter the tissue using a cell sieve to obtain a single-cell suspension of mouse common carotid artery tissue with impurities removed, and then centrifuge and resuspend it. Step 3: Mix the erythrocyte lysis buffer with the mouse common carotid artery tissue single-cell suspension obtained in Step 2, lyse, and obtain mouse common carotid artery tissue single cells.

7. The preparation method according to claim 6, characterized in that, The amount of digestive fluid added to the mouse common carotid artery tissue in step 1 is 1 mL / 100 mg of mouse common carotid artery tissue.

8. The preparation method according to any one of claims 6 or 7, characterized in that, The pore size of the cell sieve described in step 2 includes 40 μm.

9. The preparation method according to any one of claims 6 to 8, characterized in that, The volume ratio of the mouse common carotid artery tissue single-cell suspension to the erythrocyte lysate obtained in step 2 in step 3 is 1:

3.

10. The preparation method according to any one of claims 6 to 9, characterized in that, The resuspension solvent in step 2 includes 1x PBS and 0.04% BSA.

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