Study On Isolation, Culture And Establishing The Cell Line Of The Epidermal Stem Cells Of Porcine In Vitro

Epidermal tissue integrity is maintained by division of cells in the proliferative basal layer to replace differentiated cells in the outmost stratum layer that are continually lost. This hierarchy of cell proliferation and differentiation is maintained by a small subpopulation of stem cells called epidermal stem cells(ESCs). The ESCs retain a high capacity of self-renewal throughout adult life and are ultimately responsible for epidermal maintenance and repair. To do the research on isolating and culturing the ESCs is helpful to the tissue engineering and the cell therapy of the human severe disease. The ear tissues were collected from WuZhiShan inbreeding Pigs(WZSP) to establish the cell line of the ESCs, and to study the different influence of the different factors including the age, the collagen, the condition medium and the concentration of EGEMoreover, ESCs clones were identified by AKP, CK19 andβ1 integrin. The results as follows:1. Effects of different factors on the isolation and culture of ESCs We have chosen 4-day old, 1-month old, 3-month old and 12-month old pigs to evaluate the effect of age on isolation of ESCs. Through calculating the adherence rate, we found that the rate was gradually descendent going with the increase of age. We concluded that the appropriate ages of pigs for isolating the ESCs were 4-day old and 1-month old which adherence rates were 12.68±1.11% and 9.76±0.68% separately.The isolated ESCs were divided into two groups, one was inoculated onto collagen I coated flask (marked group 1) and the other was inoculated onto collagenâ…£coated flask (marked group 2). Adherence rate was observed. The results showed that the group 1 's was 28.76±1.71% and the group 2's was 11.16±1.43%. Therefore, collagen I isn't the appropriate material for plurifying the ESCs.The isolated ESCs were divided into two groups, one was cultured in the conditional medium and the other was cultured in DMEM/F10(1:1)+15%NBS. The observe result showed that the cells cultured in the conditional medium grew well and formed clolonies which could keep undifferentiated after several passages. In contrast, the cells cultured in the serum medium grew slowly and differentiated after two days culture. Thus, the serum medium can't be used as ESCs medium without feeder.In order to evaluate the effect of EGF to ESCs growth, we disigned the experimental group as the density of EGF which were respectively Ong/ml, 10ng/ml, 20ng/ml and 30ng/ml. The results show that there is no difference among the 10ng/ml group, 20ng/ml group and 30ng/ml group (P>0.05), but the Ong/ml group is distinctly lower than other groups(P<0.05).2. Isolation and plurification of ESCs from pigsThe skin pieces from the ear tissue of WZSP inbred for lab use were digested by Dispase in order to obtain epidermis, and then the epidermis was dissociated into single cells with 0.25%tripsin and 0.02% EDTA. Then the cells were inoculated onto human collagenâ…£coated flasks and cultured at 37℃in a humidified atmosphere containing 5% CO₂. The nonadherent cells were rinsed off 10-15 minutes after inoculation. The adherent cells were cultured in serum-free medium (including DMEM/F10 (1:1), 10%conditioned medium, 5μg/mL insulin, 20ng/mL EGF, 20ng/mL IGF-I and 0.5μg/mL hydrocortisone), and subcultured by 0.25%tripsin. With phase contrast microscope, the rapidly adherent cells were observed to form colonies 24 hours after inoculation. The interval of passages was about 7 days. Three ESCs lines have been established, and all have been subcultured to more than 20 passages.3. Identification of ESCs of pigs and differentiation of ESCsTo achieve this, the experiment combined two means. 1) The 10th generation ESCs were stained by AKP, the result showed red color(positive).2) immunocytochemical staining using markers including K19 andβ1-integrin. The results show that the selected cells express all of the positive markers. All these conclusions support that the cells identified are ESCs. The ESCs which were cultured 3 weeks differentiate to the piece of epidermal membrane.