Optimization Of The Isolation And Culture Conditions Of Mouse Keratinocytes

Objective:Keratinocytes originating from the ectoderm are the main cell type of the epidermis.Mouse is currently the most important model organism for the study of human diseases.Through gene editing technology,mouse keratinocytes are widely used in the study of the physiological and pathological mechanisms of epidermal barrier,scarring,psoriasis and other diseases,and have a broad application prospect.However,the isolation and culture methods of mouse keratinocytes are different in the current literature.Besides,cell differentiation and senescence are easy to occur in the process of cell culture,as well as difficulties in cell adheration and contamination of fibroblasts,which greatly affect the clinical application of mouse keratinocytes.Therefore,this study intends to optimize the process of the isolation and culture of mouse keratinocytes,and screen out the optimal conditions for cell isolation and culture.Methods:Firstly,we compared the effects of different concentrations of Trypsin-EDTA(0.05%,0.25%)under different tempreture and time on the survival rate of keratinocytes in the process of isolating mouse keratinocytes,and observed the growth of the keratinocytes of each group in the subsequent culture process under microscope.Secondly,in the process of cell culture,we compared the influence of the three different cell culture medium(Epi Life,DK-SFM,Cn T-07),different concentrations and kinds of serum,different coating matrials(i Matrix 511,Coating Matrix,Attachment Factor)on the cell growth and proliferation by observation of cell morphology under microscope,q RT-PCR and CCK8 experiment.Results:1.According to trypan blue staining and cell counting,the number of keratinocytes and the ratio of viable cells isolated from the mouse epidermis under different digestion conditions were obtained.The results showed that the number and survival rate of cells digested by 0.05% Trypsin-EDTA at room temperature for about 20 minutes were slightly higher than those digested by0.05% and 0.25% Trypsin-EDTA at 37°C for about 9 minutes.2.Mouse keratinocytes in Epi Life medium were superior to those in DK-SFM and Cn T-07 medium in terms of morphology and growth rate.The results of q RT-PCR showed that mouse keratinocytes had the highest expression of keratinocyte stemness markers(Krt5,Krt14,Krt15)and the lowest expression of fibroblast markers(Fgf7,Fbn1)in Epi Life medium,suggesting higher keratinocyte stemness and less fibroblast contamination.The results of CCK8 showed that the proliferation ability and cell number of mouse keratinocytes in Epi Life medium were better than those in DK-SFM and Cn T-07 medium.According to the results of the cell morphology observation under microscope,q RT-PCR detection of keratinocyte stemness markers(Krt5,Krt14,Krt15),differentiation markers(Krt1,Krt10),fibroblast markers(Fgf7,Fbn1)and the results of CCK8 which tests cell proliferation and cell number,adding FBS into the culture medium can help mouse keratinocytes adhere to the dish and proliferate.Every Green FBS was the best in promoting the proliferation of mouse keratinocytes,followed by Gibco FBS and KSR,and KSR can obviously induce apoptosis.3.The adherence rate and growth rate of mouse keratinocytes inoculated on i Matrix 511 were higher than those inoculated on Coating Matrix and Attachment Factor.The results of q RT-PCR showed that the expression levels of mouse keratinocyte stemness markers(Krt5,Krt14,Krt15)on i Matrix 511 were higher than those on Coating Matrix and Attachment Factor,while the expression levels of fibroblast markers(Fgf7,Fbn1)were relatively lower,which also suggested higher keratinocyte stemness and less fibroblast contamination.The results of CCK8 suggested that the adherent rate of keratinocytes on i Matrix 511 and the proliferation ability were better than those on Coating Matrix and Attachment Factor.Conclusions:Digesting epidermal tissue at room temperature with a lower concentration of Trypsin-EDTA is sufficient to obtain a large number of mouse keratinocytes of good quality.The Epi Life medium has a strong promoting effect on the growth of mouse keratinocytes,and the addition of a certain concentration of Every Green FBS can further promote the cell adhesion and proliferation.After i Matrix511 treatment,the adherent rate and proliferation of keratinocytes increase.In conclusion,an optimized primary isolation and culture system of mouse keratinocytes is established in this study,which can provide a reference for the establishment of a more perfect keratinocyte culture scheme in the future,solving the problem of difficult passage culture of mouse keratinocytes,and further serving for various basic and clinical studies.