Characterization and application scenarios of various enzymes in the preparation of tissue single-cell suspensions
Characterization and application scenarios of various enzymes in the preparation of tissue single-cell suspensions
Tissue consists of cells and extracellular matrix (ECM) ordered and tightly linked together. Obtaining single-cell suspensions from tissues for applications such as flow assays, single-cell sequencing, in vitro cultures, drug screening, cell transplantation, organoid cultures, and tumor research is closer to the natural state in vivo and offers many advantages.
To obtain high-quality single-cell suspensions, some effective measures are needed, among which, enzymatic digestion is a method that can maintain the optimal state of cells while effectively obtaining individual cells.
There are various enzymes for digesting tissues, and the common digestive enzymes are mainly as follows. Depending on the principle of digestion, their application scenarios vary:
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Due to the different cells and structures of different tissues, good results cannot be achieved using a single enzyme to digest the cells, and it is often necessary to mix and match several different enzymes to obtain the best performing single cell suspension. For specific tissues, experimental mapping is still required to determine the optimal digestion protocol. The following list of methods is for your reference.
Table 1: Reference to commonly used digestion protocols for human tissues
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表二：小鼠组织常用的消化方案参考
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Table 3: Reference to commonly used digestion protocols for rat tissues
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Example
The enzyme digestion process and staining procedure of 4T1 hormonal mice are shown below:
(1) Preparation of 10× mixed digestive enzymes: take 80 mL of HBSS buffer, add 1 g of collagenase IV, 100 mg of hyaluronidase, to reduce cell viscosity and agglutination, add an additional 20,000 U of DNase Ⅰ, fully dissolve and mix, replenish HBSS buffer, and then fix it to 100 mL, filter it using 0.22 μm filter membrane, dispense it into 5 mL/strip, and store it at -20℃ and return to room temperature before use;
(2) Tumor isolation: The tumor-bearing mice were executed by cervical dislocation method, placed in a beaker containing 75% alcohol, immersed for 3~5 min, stripped of tumors using scissors and forceps, and washed twice with PBS;
(3) Digestion of tumor tissue: transfer the tumor to a 1.5 mL EP tube, hold the curved scissors, insert the EP tube and repeatedly shear to a pellet of about 0.5~1 mm3 in size, make up 1 mL of HBSS buffer, transfer it to a 15 mL centrifuge tube, and then resuspend the tumor pellet using 8 mL of HBSS buffer, add 1 mL of 10×mixed digestive enzymes, gently blow and mix, and then digest the pellet on a shaking table at 80 rpm, 37℃ for 1~2 h;
★Note: 10 mL of digestive solution can adequately digest tumor tissue with a volume of about 1 cm3.
(4) Preparation of single-cell suspension: use a 1 mL pipette to blow and aspirate the tissue block every 30 min, until there is no obvious obstruction to the blowing and the digestion is considered to be complete, remove the impurities by filtering through a 100 μm sieve, and use a large volume of about 50 mL of HBSS buffer to wash once, centrifuge at 300 g for 5 min, discard the supernatant, and then resuspend the cellular precipitate by adding an appropriate volume of HBSS buffer or PBS buffer, i.e. tumor single-cell suspension. The cell precipitate should be resuspended with appropriate volume of HBSS buffer or PBS buffer, which is the single cell suspension of the tumor;
(5) Flow staining: cells were counted, adjusted to the appropriate density (1*107/mL), and flow stained and analyzed.
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