Human thymus tissue digest and uses thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to human thymus tissue digestive fluid 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 research and is also of great significance for the early diagnosis, tracking, and personalized treatment of 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. Obtaining a certain amount of primary single cells from tissue culture generally takes 3-5 days. 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 of human thymus tissue. With the development of single-cell technology, there is an urgent need for a rapid and inexpensive enzymatic digestion method / kit for human thymus tissue. Summary of the Invention

[0003] In view of this, the present invention provides a method for preparing human thymus tissue digestive fluid and single cells of human thymus tissue. Experiments have shown that the preparation method used in this invention can obtain nearly one million thymus cells while maintaining a cell viability of over 90%.

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

[0005] This invention provides a digestive fluid of human thymus 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 human thymus tissue digestive fluid comprises:

[0008]

[0009]

[0010] In some specific embodiments of the present invention, the human thymus tissue digestive fluid comprises:

[0011]

[0012] The present invention also provides the use of the digestive fluid of the human thymus tissue in any of the following:

[0013] (I) Preparation of a human thymus tissue digestion kit; and / or

[0014] (II) Preparation of single cells from human thymus tissue; and / or

[0015] (III) Single-cell sequencing of human thymus tissue.

[0016] The present invention also provides a human thymus tissue digestion kit, comprising the human thymus tissue digestion solution and acceptable adjuvants.

[0017] This invention also provides a method for preparing single cells of human thymus tissue, comprising the following steps:

[0018] Step 1: Take human thymus tissue, wash with 1x PBS, cut into small pieces, and add the human thymus tissue digestion solution for tissue digestion;

[0019] Step 2: After complete tissue digestion, the tissue is filtered through a cell sieve to obtain a single-cell suspension of human thymus tissue with impurities removed.

[0020] Step 3: Mix the erythrocyte lysate with the human thymus tissue single-cell suspension obtained in Step 2, and lyse to obtain the human thymus tissue single-cell suspension.

[0021] In some specific embodiments of the present invention, the amount of human thymus tissue digestive fluid added in step 1 includes 1 mL / 100 mg of human thymus tissue.

[0022] In some specific embodiments of the present invention, the tissue digestion temperature in step 1 includes 37°C; and / or

[0023] The tissue digestion time mentioned in step 1 includes 30 minutes.

[0024] In some specific embodiments of the present invention, the pore size of the cell sieve in step 2 includes 40 μm.

[0025] In some specific embodiments of the present invention, the volume ratio of the human thymus tissue single-cell suspension obtained in step 2 to the red blood cell lysate in step 3 is 1:3.

[0026] In some specific embodiments of the present invention, the pyrolysis time in step 3 of the preparation method includes 5 minutes.

[0027] The present invention also provides a method for single-cell sequencing of human thymus tissue, characterized in that it is based on any of the following for single-cell sequencing of human thymus tissue;

[0028] (I) the digestive fluid from the human thymus tissue; and / or

[0029] (II) The human thymus tissue digestion kit; and / or

[0030] (III) The preparation method described above yields a single-cell suspension of human thymus tissue.

[0031] This invention includes, but is not limited to, achieving the following beneficial effects:

[0032] The digestion method used in this invention obtains a large number of thymocytes while maintaining a cell viability of over 90%. Sufficient cell volume is a prerequisite for optimization processes such as erythrocyte lysis and removal of dead cells. The single-cell suspension obtained by this method lays the foundation for obtaining high-quality data through single-cell sequencing. Attached Figure Description

[0033] 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.

[0034] Figure 1 The fluorescence field results of human thymus tissue dissociated using a commercial tissue dissociation kit (Mitteni 130-110-201) are shown.

[0035] Figure 2 The fluorescence field results of human thymus tissue dissociated by the digestion method of the present invention are shown.

[0036] Figure 3 The bright-field results of human thymus tissue dissociated using a commercial tissue dissociation kit are shown.

[0037] Figure 4 The bright-field results of the digestion method of the present invention dissociating human thymus tissue are shown. Detailed Implementation

[0038] This invention discloses a digestive fluid from human thymus 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.

[0039] This invention provides a digestive fluid for human thymus tissue, the formula of which is as follows:

[0040]

[0041] The digestion method described in this invention, using the digestive fluid, can dissociate human thymus tissue cells while maintaining a cell viability of over 90%, yielding millions of cells. Sufficient cell volume is a prerequisite for optimized 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, forms the basis for obtaining high-quality gene expression data in human thymus tissue single-cell sequencing experiments.

[0042] Unless otherwise specified, the raw materials and reagents used in the human thymus tissue digestive fluid and its application provided by this invention are all commercially available.

[0043] The present invention will be further illustrated below with reference to the embodiments:

[0044] Example: Dissociation of human thymus tissue

[0045] 1. Digestive solution formula: Collagenase II 1 mg / mL (Solepro C8150), Neutral protease II 1 mg / mL (Roche 45147700), CaCl2 2.5 mM (sigma C7902), FBS 2% (Gibco 10100139), DNase I 10 μg / mL (sigma DN25).

[0046] 2. Pretreatment of human thymus tissue: The tissue was cleaned with pre-cooled 1x PBS pH 7.4 (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).

[0047] 3. Tissue processing conditions: The digestive fluid of the pretreated human thymus tissue was transferred to a 37°C environment for tissue dissociation, and the dissociation time was 30 min.

[0048] 4. Impurity removal: After digestion, filter the digestion solution using a cell sieve with a pore size of 40μm to remove larger impurities, and then centrifuge and resuspend the cells.

[0049] 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.

[0050] 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.

[0051] Figure 1The results are fluorescence field findings from the dissociation of human thymus tissue using a commercial tissue dissociation kit (Miteni 130-110-201). Figure 2 The fluorescence field results of human thymus 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 human thymus 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.

[0052] Table 1 shows a comparison between commercially available reagent kits and cell suspensions obtained using the method of this invention:

[0053] Table 1 Comparison of the effects of commercially available reagent kits and the method of this invention.

[0054] Digestion methods Cell viability Cell clumping rate Nucleation rate in cell suspension Cell count (number of cells) Commercial reagent kits 94.33％ 0.59％ 93.57％ 1000000 Method of the present invention 96.6％ 5.4％ 85.6％ 800000

[0055] 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. Human thymus tissue digestive fluid, 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 human thymus tissue digestive fluid as described in claim 1, characterized in that, include:

3. The human thymus tissue digestive fluid as described in claim 1 or 2, characterized in that, include:

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

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

6. A method for preparing single cells from human thymus tissue, characterized in that, Includes the following steps: Step 1: Take human thymus tissue, wash with 1xPBS, cut into small pieces, and add the human thymus tissue digestion solution as described in any one of claims 1 to 3 for tissue digestion; Step 2: After complete tissue digestion, the tissue is filtered through a cell sieve to obtain a single-cell suspension of human thymus tissue with impurities removed. Step 3: Mix the erythrocyte lysis buffer with the human thymus tissue single-cell suspension obtained in Step 2, lyse, and obtain human thymus tissue single cells.

7. The preparation method according to claim 6, characterized in that, The amount of human thymus tissue digestive fluid added in step 1 includes 1 mL / 100 mg of human thymus tissue.

8. The preparation method according to claim 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 human thymus tissue single-cell suspension obtained in step 2 to the red blood cell lysate in step 3 is 1:

3.

10. A method for single-cell sequencing of human thymus tissue, characterized in that, Based on any of the following for single-cell sequencing of human thymus tissue; (I) Human thymus tissue digestive fluid as described in any one of claims 1 to 3; and / or (II) The human thymus tissue digestion kit as described in claim 5; and / or (III) Human thymus tissue single cells are obtained by the preparation method according to any one of claims 6 to 9.

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