Experimental Studies On Culture Of ADSCs Of Rabit In Vitro

Objective :To investigate the methods of isolating and culturing rabbit adipose—derived stem cells (ADSCs) , and to identify and analyze the biological characteristics of rabbit ADSCs and To explore the optimized condition for adipose-derived stem cells(ADSC) culture in vitro.Methods : Adipose was collected from the inguinal region of News Zealand rabbits and the ADSCs were isolated with adherent method and collagenase method and cultured in vitro.The morphology of ADSCs after passage 2 was analyzed.The growth curve and doubling time were drawn.The collagenase with different concentration and duration and FCS with different concentration were used.Results: Formation of primary and passage of ADSCs: ADSCs had spindle and polygon shape adherent growth,and their growth and differentiation were active.②Growth curve and doubling time of ADSCs:The growth curve was like"S"shape. The doubling time of ADSCs was about 55 hours.3 The ADSCs adipogenic differentiation was induced and the rate of positive rate was 65% through the Oil Red O stain and positive cell counting.4 Based on the collagenase concentration, cells were divided into four groups, i.e.,0.5mg/ml, 1mg/ml, 1.5mg/ml and 2mg/ml groups. When the duration was 1 hour, 2mg/ml group had the most total cell number(TCN) and the living cell rate(LCR) was statistically different from other groups. When the duration was 2 hours, TCN increased in every group and there were no statistical differences among 1mg/ml, 1.5mg/ml and 2mg/ml groups. LCR was the highest in 1mg/ml and 1.5mg/ml. 2.Based on FBS concentration, the cells were divided into five groups, i.e., 0, 5%, 10%, 15% and 20% groups. The growth character was the best in 15% and 20% groups.Conclusion:The ADSCs of rabbits isolated in this trial are characterized by stable growth and quick proliferation in vitro. The optimized conditions for human adipose-derived stem cells culture are collagenase concentration of 1mg/ml, digestion duration of 2 hour and FBS concentration of 15%.Objective:To compare the content of human adipose-derived stem cells(hADSC)and the adipogenic diferentiation ability in vitro of induced human adipose stem ceHs from difierent sites of the human body.So as to provide experimental foundation for further application of adipose stem cells(ADSCs).Methods:The mesenchymal stem cells in adipose tissue were extracted from 4 sites (abdomen wall,extremities,orbital septum and armpit) of 50 patients.donors undergoing the lipesuction in clinic , thenthese cells were induced to difierentiate to the adipogenic cells in vitro.ADSCs at passage l,cultured in the control media(DMEM,supplemented with l0% FBS) were plated at a density of 3x105 ceHs every dish into 35mm culture dishes,and the control media was replaced with adipogenic mealia (AM).The media was changed twice weekly and the culture dish added with the controled is served as negative contro1.Two weeks later,the induced cells and negative cells were assessed with an Oil Red O stain to identify whether there was lipid in cells or not.Then through the Oil Red O stain and positive cell counting, the adipogenic differentiation ability of induced human adipose stem cells from 4 diferent site a of 50 patients were assessed with qualitative and semi-quantitative analysis.The adipogenic diferentiation rates of different cells were determined with chi.square test.Results:The measured results of adipogenic differentiation ability of ADSCs:The mean number of cells isolated from per 10 mL of lipoaspirates of different donors were quite different, the mean cell yields were ranged from 2.45xl0~7/l0 mL to 1.25x10~7/10ml:but the number from different sites of the same body were quite similar;The mean adipogenic diferentiationrate measured from 4 sites of 50 donors was 46% calculated aecording to the adipogenic differentiation rates of every kind of cel1. The statistics analysis confirmed that adipogenic differentiation ability of induced human adipose stem cells from different sites of the same body was different, and there was no correlation betwen the numbers of stem cells and the age of donor(r=0.098 , p=0.817)。while the adipogenic diferentiation ability was significant negatively correlated with the age of donor.The ADSCs morphological change and the qualitative analysis results of Oil Red O stain : The ADSC sexhibited an expanded morphology and diferentiated to the dipogeniclineage after adipogenic induced . The cells multiple intracell orange red lipid-filled droplets which accounted for 80-90%volume of the cells.The nuclei shrank or located on one side of cells and showed positive.But there were no morphological changes or lipid droplets formation in cells cultured in control media and the control cells showed negative.Conclusion:The older the donors are, the lower the adipogenic differentiation rates of cells are; more there are differences in adipogenic differentiation rates of cells from diferent sites of the same donor, which indicate that it is necessary to pay attention to the patient's age and sites on doing related experiment with ADSCs and further research may find out the optimal site for ADSCs.