Buffalo mammary gland tissue digest and uses thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a digestive fluid from buffalo mammary gland tissue and its applications. Background Technology

[0002] Single-cell sequencing technology is a technique that performs sequencing analysis at the genome, transcriptome, and epigenome levels at the single-cell level. 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. A prerequisite for single-cell gene expression research is to obtain a high-quality single-cell suspension as soon as possible after obtaining the tissue. There are two main methods for obtaining primary single-cell suspensions from tissue: enzymatic digestion and tissue block culture. Tissue culture generally requires 3-5 days to obtain a certain amount of primary single cells, making it more suitable for studying the three-dimensional structure of tissues and cell-cell interactions. Currently, there are few enzymatic digestion methods on the market suitable for high-quality (high viability, low clumping rate, high purity) primary single-cell suspensions from buffalo mammary gland tissue. With the development of single-cell technology, there is an urgent need for a rapid and inexpensive enzymatic digestion method / kit for buffalo mammary gland tissue. Summary of the Invention

[0003] This invention provides a digestive fluid from buffalo mammary gland tissue and its applications. The invention also provides methods for preparing the digestive fluid and single-cell suspension of buffalo mammary gland tissue. Experiments have shown that the preparation method of this invention can obtain a large number of buffalo mammary gland cells while maintaining a cell viability of over 85%.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a digestive fluid of buffalo mammary gland tissue, comprising: collagenase II, neutral protease II, CaCl2, FBS, and DNase I; or

[0006] Collagenase II, neutral protease II, CaCl2, BSA, DNase I.

[0007] In some specific embodiments of the present invention, the buffalo mammary gland tissue digestive fluid comprises:

[0008] Collagenase II 1.5–3 mg / mL

[0009] Neutral proteinase II 1.5–2 mg / mL

[0010] FBS 2%–5%

[0011] CaCl2 2.5mM

[0012] DNase I 10–50 μg / mL; or

[0013] Collagenase II 1.5–3 mg / mL

[0014] Neutral proteinase II 1.5–2 mg / mL

[0015] BSA 0.5%

[0016] CaCl2 2.5mM

[0017] DNase I 10–50 μg / mL.

[0018] In some specific embodiments of the present invention, the buffalo mammary gland tissue digestive fluid comprises:

[0019] Collagenase II 1.5 mg / mL

[0020] Neutral proteinase II 1.5 mg / mL

[0021] FBS 2%

[0022] CaCl2 2.5mM

[0023] DNase I 50 μg / mL; or

[0024] Collagenase II 1.5 mg / mL

[0025] Neutral proteinase II 1.5 mg / mL

[0026] BSA 0.5%

[0027] CaCl2 2.5mM

[0028] DNase I 50 μg / mL.

[0029] The present invention also provides the use of the digestive fluid of the buffalo mammary gland tissue in any of the following:

[0030] (I) Preparation of a buffalo mammary gland tissue digestion kit; and / or

[0031] (II) Preparation of single cells from buffalo mammary gland tissue; and / or

[0032] (III) Single-cell sequencing of buffalo mammary gland tissue.

[0033] The present invention also provides a buffalo mammary gland tissue digestion kit, comprising the buffalo mammary gland tissue digestion solution and acceptable adjuvants.

[0034] This invention also provides a method for preparing single cells from buffalo mammary gland tissue, comprising the following steps:

[0035] Step 1: Take buffalo mammary gland tissue, wash with 1x PBS, cut into small pieces, and add the buffalo mammary gland tissue digestion solution for tissue digestion;

[0036] Step 2: After complete tissue digestion, filter the cells using a cell sieve to obtain a single-cell suspension of buffalo mammary gland tissue with impurities removed, and then centrifuge to resuspend the cells.

[0037] Step 3: Mix the erythrocyte lysis buffer with the buffalo mammary gland tissue single-cell suspension obtained in Step 2, and lyse to obtain buffalo mammary gland tissue single cells.

[0038] In some specific embodiments of the present invention, the amount of buffalo mammary gland tissue digestive fluid added in step 1 includes 1 mL / 100 mg of buffalo mammary gland tissue.

[0039] In some specific embodiments of the present invention, the tissue digestion temperature in step 1 includes 37°C; and / or

[0040] The tissue digestion time mentioned in step 1 includes 30 minutes.

[0041] In some specific embodiments of the present invention, the pore size of the cell sieve in step 2 includes 40 μm.

[0042] In some specific embodiments of the present invention, the volume ratio of the buffalo mammary gland tissue single-cell suspension obtained in step 2 to the erythrocyte lysate in step 3 is 1:3.

[0043] In some specific embodiments of the present invention, the pyrolysis time in step 3 of the preparation method includes 5 minutes.

[0044] In some specific embodiments of the present invention, the resuspension solvent in step 2 includes 1×PBS and 0.04% BSA.

[0045] This invention includes, but is not limited to, the following beneficial effects:

[0046] The preparation method of this invention can obtain a large number of single cells from buffalo mammary tissue while maintaining a cell viability of over 85%. 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 buffalo mammary tissue. Attached Figure Description

[0047] 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.

[0048] Figure 1The fluorescence field results of buffalo mammary gland tissue dissociated using a commercial tissue dissociation kit (Mitteni 130-110-201) are shown.

[0049] Figure 2 The fluorescence field results of the digestion method of the present invention dissociating buffalo mammary gland tissue are shown.

[0050] Figure 3 The bright-field results of buffalo mammary gland tissue dissociated using a commercial tissue dissociation kit are shown.

[0051] Figure 4 The bright-field results of the digestion method of the present invention for dissociating buffalo mammary gland tissue are shown. Detailed Implementation

[0052] This invention discloses a digestive fluid from buffalo mammary gland 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.

[0053] This invention provides a digestive fluid for buffalo mammary gland tissue, with the following formulation: 1.5–3 mg / mL collagenase II; 1.5–2 mg / mL neutral protease II; 2.5 mM CaCl2; 2%–5% FBS, or 0.5% BSA; 10–50 μg / mL DNase I.

[0054] The digestion method of this invention obtains a large number of buffalo mammary gland cells while maintaining a cell viability of over 85% and significantly increasing the harvested cell quantity. Sufficient cell quantity is a prerequisite for optimized processing such as erythrocyte lysis and removal of dead cells. The cell suspension obtained by this method has high viability, low clumping rate, high nucleation rate, and low impurity content, improving the success rate of single-cell sequencing experiments on buffalo mammary gland tissue. Unless otherwise specified, the buffalo mammary gland tissue digestion solution provided by this invention and the raw materials and reagents used in its applications are all commercially available.

[0055] The present invention will be further illustrated below with reference to the embodiments:

[0056] Example: Dissociation of buffalo mammary glands

[0057] 1. Digestive solution formula: 1.5 mg / mL collagenase II (Solepro C8150); 1.5 mg / mL neutral protease II (Roche 15147700); 2.5 mM CaCl2 (sigma C7902); 2% FBS (gibco 10100139); 50 μg / mL DNase I (sigma DN25).

[0058] 2. Pretreatment of buffalo mammary gland tissue: The tissue was cleaned with 1x PBS (Seville G4202), and the cleaned tissue was cut into small pieces and transferred into the digestion solution described in step 1 (2 mL of digestion solution for every 200 mg of tissue).

[0059] 3. Tissue processing conditions: The digestive fluid of the buffalo mammary gland tissue after pretreatment in step 2 was transferred to a 37°C environment for tissue dissociation, and the dissociation time was 30 min.

[0060] 4. Removal of residual tissue: After digestion, filter the digestion solution using a cell sieve with a pore size of 40 μm to remove large impurities, centrifuge and resuspend, collect cells, resuspend cells in 1xPBS + 0.04% BSA, and perform quality control on the cell suspension.

[0061] 5. 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.

[0062] 6. After tissue dissociation and removal of red blood cells, 10 μL of cell suspension was mixed with 10 μL of AO / PI (Countstar RE010212) and added to a cell counting chamber. The cell counting chamber was then inserted into a fluorescence cell analyzer (Countstar Rigel S2) for counting.

[0063] Figure 1 The results show the fluorescence field of buffalo mammary gland tissue dissociated using a commercial tissue dissociation kit (Miteni 130-110-201). Figure 2 The fluorescence field results of the digestion method of the present invention are obtained by dissociating buffalo mammary gland tissue. 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 buffalo mammary gland tissue. A comparison of the two dissociation methods shows that there is no significant difference between the results obtained by this method and the method using commercial kits. This method can yield high-quality cell suspensions for single-cell sequencing, and the cost is significantly reduced.

[0064] Table 1 shows a comparison between commercially available reagent kits and cell suspensions obtained using the method of this invention:

[0065] Table 1 Comparison of the effects of commercially available reagent kits and the method of this invention.

[0066] Digestion methods Cell viability Cell clumping rate Nucleation rate in cell suspension Cell count (number of cells) Commercial reagent kits 89.55％ 11.07％ 91％ 150000 Method of the present invention 89.5％ 13.6％ 83.9％ 180000

[0067] 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 buffalo mammary gland tissue, characterized in that, include: Collagenase II, neutral protease II, CaCl2, FBS, DNase I; or Collagenase II, neutral protease II, CaCl2, BSA, DNase I.

2. The buffalo mammary gland tissue digestive fluid as described in claim 1, characterized in that, include:

3. The buffalo mammary gland tissue digestive fluid as described in claim 1 or 2, characterized in that, include:

4. The use of the buffalo mammary gland tissue digestive fluid as described in any one of claims 1 to 3 in any of the following: (I) Preparation of a buffalo mammary gland tissue digestion kit; and / or (II) Preparation of single cells from buffalo mammary gland tissue; and / or (III) Single-cell sequencing of buffalo mammary gland tissue.

5. A buffalo mammary gland tissue digestion kit, characterized in that, It includes the buffalo mammary gland tissue digestive fluid as described in any one of claims 1 to 3, and acceptable adjuvants.

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

7. The preparation method according to claim 6, characterized in that, The amount of buffalo mammary gland tissue digestive fluid added in step 1 is 1 mL / 100 mg of buffalo mammary gland 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 single-cell suspension of buffalo mammary gland tissue obtained in step 2 to the erythrocyte lysate 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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