A Simplified And Efficient Method To Isolate Primary Human Epidermal Stem Cells From Adult Skin Tissue

Objectives;Human epidermal stem cells,also named as human keratinocytes(HCKs),have high differentiation potential and they can proliferate and differentiate into a variety of skin cells.However,at the present stage,the methods and procedures for the isolation of human epidermal stem cells are relatively complicated,and the number of primary cells which are isolated is limited.The reasons talked above demonstrate that the cells'subsequent applications are limited.The aim of this study was to overcome the shortcomings of current methods for the isolation of adult epidermal stem cells,and to explore the improvement protocol and the effect according to our improvement.Methods:The human skin tissue resected in the surgery was placed in 70%ethanol for 30 seconds to cleanout the impurities such as blood clots and so on.Besides,part of fat and muscle components were removed by scissors.PBS containing 2%-3%streptomycin was used for sterilization and further cleaning.To reach the purpose of total sterilization,the tissue was immersed in it for 3 minutes twice.Then the tissue was completely chopped into homogenation status with scalpels.The tissue homogenate was transferred into a 50ml centrifuge tube and added with enzyme mixture(lg tissue with 20ml enzyme mixture,2.5mg/ml type I collagenase and 2.5mg/ml dispase)with blending.The digestion mixture was put into 37 ? water bath with shaking for 60 minutes.Then 0.25%trypsin was added into the tube containing the digestion mixture for another 30 minutes water bath.Finally,Dnase I(10mg/ml)was added into the tube for a 5 minutes digestion.After the tissue was digested,the same volume of DMEM medium containing 10%fetal bovine serum was added with it and the mixture was repeatedly blown to stop the digestion(about 20 times).After that,the mixture was passed through a 100?m filter,centrifuged and the supernantant was removed.The cell pallet was resuspended by DMEM medium containing 10%fetal bovine serum,centrifuged and the supernantant was removed.Then,the cell pallet was resuspended by dermal medium and inoculated into the cell culture bottle or 100mm cell culture dish.Rock protein kinase inhibitor Y-27632 was added in the culture dish with the volume of 1:1000 and the concentration of 10mM.The culture was changed every 2-3 days.When the cells grew into the dense at 80%,they were passed,cryopreserved or used for following studies.At the same time,cell isolation and culture of the same skin tissue were carried out by traditional method:the cells were washed with 10ml PBS,then washed with 10ml 70%ethanol for 30 seconds,and immersed twice with 10ml washing solution(PBS containing 2%penicillin and streptomycin)for 5 minutes each time.The tissue was trimmed with sterile scissors,and the subcutaneous fat layer was removed in a 100mm cell culture dish,and then the skin tissue was weighed.The tissue was transferred to another 100mm sterilized dish,with the dermis side down.After that,the skin tissue was cut into 3-4mm wide strips with a scalpel.After 10 ml dispase(2.5 mg/ml)was added into a petri dish,the skin tissue was added in it,with a overnight 4 C incubation procedure.The next day,the epidermis was peeled from the dermis by a pair of tweezers and was chopped into homogenate by surgical blades.Then,it was resuspended by 10ml 0.25%trypsin in a 50 ml centrifuge tube and was digested in 37 0C water bath for 20 minutes.Trypsin activity was neutralized by adding the same volume of neutralization solution.The isolated epidermal cell suspension was shaken up and down 20 times,and then the cell suspension was passed through the 100um cell filter with a 10ml pipette to remove any remaining tissue fragments.After procedures of centrifugal,resuspension,cleaning and a second centrifugal,the cell pallet was resuspended epidermal medium and was moved into culture dishes for inoculation.Medium was changed for 2-3 days,and the cells were passed when they reached a high dense.After that,the complexity and time-consuming was compared between two kinds of methods.Furthermore,the quantity and quality of cells would also be compared.The amounts of primary epidermal stem cells were observed when c.ells were cultured on 3 days ancd 5 days.And the cell amounts on 7 days were calculated.The time when cells grew at the suitable dernsity for pa.ssage was compared between two methods.In order to measure the purity and stemness of epidermal stem cells,the passage 1 and 3 cells harvested by the improved method were made into cell climbing.When the suitable cell density reached,the imrmunofluorescence(IF)of K5,Loricrin and Vimentin were performed.Results:(1)Traditional method to isolate human epidermal stem cells required an overnight enzyme digestion process,which was time-consuming and could affect cell viability.In addition,because of the inevitability to peel the epidermis from the dermis,the complexity of the protocol was increased and some of the epidermal cells were lost.For some kinds of tissues which were difficult to peel off the epidermis,the isolation efficiency of this method would be greatly reduced.The new method could simplify the isolation process and improve the efficiency.The total time that enzyme digestion costed was approximate 90 minutes,which greatly reduced the time of the new method and ensured the cell vitality to a certain extent,(2)As for the primary keratinocytes isolated by traditional method,the cell growth rate was slow;In the new method,the use of Y27632 enhanced the function of epidermal cells,so that the extracted keratinocytes could grow at a high level and were in a colony proliferation.K5,Loricrin and other immunofluorescence(IF)stainings were performed on the primary and passaged cells isolated by the new method.It was found that the differentiation potential of the cells was high and the characteristics of epithelial basal cells were maintained.Conclusions:Human primary keratinocytes cultured in vitro have been increasingly used in clinical and laboratory applications.Hence,there are high levels of requirements for the quantity and quality of primary keratinocytes isolated,while the traditional methods for the isolation of primary keratinocytes cannot well meet the above two requirements.This experiment improved the shortcomings of existing methods,shortened the digestion time of enzymes,simplified the steps of cell extraction,and improved the proliferation and adherent ability of primary keratinocytes by using ROCK inhibitor Y-27632,thus increasing the amount and the stemness of primary cells.Therefore,the new method developed in this study can obtain high-quality and sufficient keratinocytes,which can be better applied in clinical and laboratory fields.