Objective: With the development of high-throughput sequencing technology, the demand for research at the single-cell level is constantly increasing. Especially for single-cell sequencing studies in skin tissues, the demand for dissociation into single-cell suspensions is growing day by day. However, the existing technology is difficult to dissociate skin tissue into single-cell suspensions in a short period of time. The dense structure of skin tissue leads to the fact that the existing dissociation methods are time-consuming and difficult to meet the timeliness requirements of downstream omics research. This experiment rapidly and effectively dissociates skin tissue through an innovative enzymatic digestion formula and mechanical processing method, meeting the requirements of single-cell research. Methods: Enzyme digestion method: Enzyme digestion method was divided into two groups, group A: type I collagenase+DNase I, group B: type II collagenase+0.25% trypsin+DNase I, both groups were prepared with Hanks buffer. The mechanical method was constant temperature shock and constant temperature beat. The enzyme digestion method and the mechanical treatment method were combined in pairs to treat the skin tissue. Results: The optimal dissociation method was enzyme digestion group A (type I collagenase 2 mg/mL, DNase I 10 U/mL)+constant temperature beating (180 times/min, 37 ℃). This combination achieved effective digestion of skin tissue within 15 min. The single cell suspension with high purity and high activity was successfully prepared by further treatment of hyaluronidase and red blood cell lysate, filtration (30 μm) and centrifugation. Conclusion: By optimizing the combination of enzyme types, concentrations and mechanical treatment methods, efficient digestion of skin tissue was achieved within 15 minutes, obtaining high-purity and high-activity single-cell suspensions. This greatly enhanced the efficiency and accuracy of the research, providing reliable technical support for subsequent flow cytometry analysis, cell sorting and high-throughput sequencing studies..
QI Geqile
,
ZHU Bing
,
BAO Ganzhu
,
LIANG Lu
. Method for rapid digestion of mouse skin tissue into single cells[J]. Journal of Baotou Medical College, 2025
, 41(7)
: 49
-52
.
DOI: 10.16833/j.cnki.jbmc.2025.07.009
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