Mixed enzyme for skin tissue dissociation, preparation method of mixed enzyme, dissociation kit and dissociation method

Abstract

The invention discloses a mixed enzyme for skin tissue dissociation. The mixed enzyme is composed of collagenase II, trypsin and deoxyribonuclease I, wherein the concentration of the collagenase II is 2-4 mg / mL; the concentration of the trypsin is 1-3 mg / mL; and the concentration of the deoxyribonuclease I is 0.01-0.05 mg / mL. Besides, the invention further discloses a dissociation method for the skin tissue. The skin tissue is pretreated with a specific buffer solution, and tissue dissociation is conducted through the specific mixed enzyme, so that a skin tissue single-cell suspension with large number of single cells, good activity, few fragments and low clustering rate can be efficiently, rapidly and stably obtained. The screened and optimized mixed enzyme and concentration have higher pertinence and higher efficiency; more costs and time are saved compared with those in a method of combining a few collagenases in a broad spectrum; and a good tool is provided for single-cell related research of a skin tissue sample which is always difficult to dissociate.

Description

technical field [0001] The present invention relates to the technical field of single-cell sequencing, in particular to a mixed enzyme specially used for skin tissue dissociation and its preparation method; in addition, the present invention also relates to a dissociation kit and dissociation method for skin tissue dissociation . Background technique [0002] Cells are the basic units that constitute human tissues and organs. In previous studies, most of the genetic testing techniques were based on the study of cell aggregation, and the experimental results obtained reflect the average level of signal expression in the cell population. In fact, it is the overall representation of the cell population or just represents the information of the cells that are dominant in number in the cell population, thus ignoring the unique cell heterogeneity of a single cell; however, a large number of studies have found that in the same species Cells of the same type in an organ or tissue a...

AI Technical Summary

Problems solved by technology

[0008] The method of neutral protease + collagenase digestion is to put the treated skin tissue in 0.5% neutral protease and digest overnight at 4°C, then add 0.1% type II collagenase to it the next day, and shake and digest at 37°C 3h, and then terminate the digestion, this method often takes a long time and leads to a great increase in the cost of the entire experiment, and affects cell activity and machine efficiency

[0009] In the single-cell sequencing technology, the requirements for the single-cell suspension are relatively strict: 1. The cell viability must be greater than 85%, because dead or damaged cells will release nucleic acids into the cell suspension, and these nucleic acids will be released in the subsequent Retaining in the experimental steps will affect the experimental results; 2. The cells are not agglomerated, and the cell shape is good, the fragments are less and the background is clean. The rate must be lower than 4%; 3. The cell concentration reaches 300~1200 cells / μL, and the appropriate concentration is also crucial. The total number of cells should be 50,000 to 100,000. Insufficient or too much sample loading will affect the final data quality

[0010] Chinese invention patent application CN201911094195.X discloses a method for preparing a single-cell suspension of adipose tissue, but it is not suitable for skin tissue, which often has more hair and is dense and hard to handle. Our patent The mixed enzyme disclosed in the application is used in skin tissue, and it is found that the cell viability is low, the digestion time is long, and the cost is high

[0011] To sum up, the preparation of solid tissue single-cell suspension still lacks a mature and general solution, and the methods applicable to different tissues are not the same, and even the methods used by the same tissue may be different. The corresponding enzymes and action conditions should be selected according to different tissue types, and the enzyme digestion method is affected by the type, concentration, and digestion time of the enzyme and has a great impact on the activity of the cells.

Skin tissue often has a lot of hair and is dense and hard to process. Existing dissociation methods cannot efficiently and properly process a large number of skin single cells. At present, many digestion methods use many types of enzymes and take a long time, resulting in The cost of reagents and labor costs have increased, so it is necessary to find a faster, more efficient method for dissociation of single cells from skin tissue that is suitable for single-cell sequencing technology

[0012] At present, there is no dissociation mixed enzyme and dissociation kit that can efficiently dissociate skin tissue and obtain a large amount of skin single cell suspension for subsequent research

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