Abstract: Wang Haitao, Jiang Renjun, Li Shuwei Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, College of Life Sciences, Tarim University, Alaer 843300, Xinjiang, China Corresponding author: Li Shuwei, Email: xj_lsw@126.com 【Abstract】 Objective To optimize the culture medium for scarless wound healing in outbred fetal Kunming （KM） mice, and to locate and preliminarily analyze the region with active cell proliferation. Methods After 6 pregnant mice were sacrificed at the gestational age of 15 days, a total of 180 skin grafts were obtained from the back of 60 fetal mice, and wounds on the skin grafts were made uniformly with minimally invasive sterile device. Then, these skin grafts with wounds were randomly and equally divided into 6 groups to be treated with high-glucose Dulbecco′s modified Eagle′s medium （DMEM）, DMEM + 5% fetal bovine serum （FBS）, DMEM + 10% FBS, low-glucose Eagle′s minimum essential medium （MEM）, MEM + 5% FBS, and MEM + 10% FBS for 3 days, respectively. Then, these skin grafts were embedded in optimal cutting temperature （OCT） compound, and subjected to hematoxylin and eosin （HE） staining to select the optimal culture condition for wound healing. Under the optimal culture condition, the region with active cell proliferation was located by labeling and tracking cutaneous stem cells with 5-ethynyl-2-deoxyuridine （EdU）, and activator protein-1 （AP-1） was detected by immunofluorescent staining. Results After 3-day cultivation, wound healing rates significantly differed among the DMEM group, DMEM + 5% FBS group, DMEM + 10% FBS group, MEM group, MEM + 5% FBS group and MEM + 10% FBS group （0, 3.33%, 6.67%, 3.33%, 46.67% and 26.67%, respectively, χ2 = 41.39, P < 0.05）. Additionally, the MEM + 5% FBS group showed a significantly higher wound healing rate than the other groups except the MEM + 10% FBS group （all P < 0.01）. Logistic regression analysis showed that the type of basal medium （MEM: OR = 11.717, 95% CI: 3.274 - 41.934, P < 0.001） and FBS concentrations （5% FBS: OR = 24.625, 95% CI: 3.027 - 200.299, P = 0.003; 10% FBS: OR = 13.449, 95% CI: 1.618 - 111.813, P = 0.016） were factors influencing fetal wound healing. Under the culture condition of 5% FBS + MEM, the dermis and epidermis healed well without epidermal thickening. The cutaneous stem cell labeling technique showed that the papillary dermis and basal layer of the epidermis were the two major regions with active cell proliferation in the wound of fetal mouse skin. Moreover, AP-1 was expressed abundantly in these two regions as well. Conclusions The culture condition of 5% FBS + MEM is considered to be optimal for in vitro wound healing in fetal mice. The papillary dermis and basal layer of the epidermis are two major regions with active cell proliferation during wound healing in fetal mouse skin, and play important roles in wound healing process.