Abstract: Objective To investigate the effect of modified method of combined enzyme digestion for keloid dermis digestion and to explore the optimal digestion time. Methods Eight keloid tissue samples were collected. After the removal of the epidermis, dermis was homogenized with the scissor. The homogenates of the dermis were digested with group A or B protocol simultaneously. The group A formula was composed of traditional combined enzyme(type Ⅰ collagenase + type Ⅱcollagenase + type Ⅳ collagenase + hyaluronidase), while the group B formula was supplemented with DNase Ⅰ. Single-cell fraction and single-cell numbers were determined under the microscope. Cell viability was detected using Trypan blue staining and flow cytometry. The digestive efficiency between group A and B was compared. Meanwhile, the optimal digestion time for group B was investigated. Results After 2 hours and 4 hours digestion, the fraction(P0.05). Compared with 6 hours digestion, there was no significant increase in the single-cell fraction and single-cell number when the digestion time was extended to 8 hours or 10 hours(P>0.05), whereas the cell viability was decreased significantly(P<0.05). Conclusion The supplement of DNase Ⅰ to the traditional combined enzyme digestion can increase the digestive efficiency and thus, facilitate the preparation of singlecell suspension for keloid tissue. Tissue digestion for 6 hours can achieve satisfactory digestive results while maintaining the cell viability.

Key words: Keloid, Enzymatic digestion, Single-cell suspension, Flow cytometry