| BackgroundEmbryonic stem cells (ES cells) is derived from the blastocyst inner cell mass (ICM) cell and primordial germ cells (PGCs). Induced pluripotent stem cells (iPS cells), similar to ESC, which is reversed by somatic cell, is totipotent cell. Infinite proliferation, self-renewal and multipotentiality are similar characters of both cells. ES cells and the iPS cells Spontaneously differentiatition is easily happened, so it must be cultured on the feeder layer or some inhibitory factor must be added into the medium. It is not effective with only cytokines added to inhibit spontaneously differentiation of ES cells and the iPS cells. In order to prepare feeder layers, mitomycin C were widely used to treat mouse embryonic fibroblasts (MEF) in most experiment researches presently. Although reports of feeder-free cultured system or establishing human ES cell applying human placental fibroblast have existed, MEF are commomly utilized to cultivate ES cells. As it is convenient, low cost, and cytokines of inhibiting ESC and iPS cells from self-differentiating and stimulating proliferation is effectively secreted. Moreover, as a feeder layer, MEF can promote ES celss and iPS cells to proliferate and maintain their undifferentiation and multi-potentiality. Its effect is better than feeder-free layer which is added by single cytokines, and similar embryonic development environment for ES cells and iPS cells is provided. So is widely used in researchs of ES cells and iPS cells. In this experiment, mouses of ICR strains are used as experimental material, to explore the optimum condition for cultivating MEF separately and preparating feeder layer, and prove the effect of feeder layer produced in backing stem cells’growth, also provide experimental basis for further study of ES cells and iPS cells.ObjectiveTo establish a stable and efficient feeder layer of ICR mouse culture system in vitro, optimize methods of separating MEF and preparing feeder layer of MEF, to explore the stable conditions of separating MEF and preparing feeder layer for stem cells. Establishing a culture system in vitro for stem cells. To provide standard experimental conditions and basis for our research group in the future experiments.Methods1. To separating MEF with the methods of trypsin digestion in vitro, digesting embryo tissue with different concentrations of Trypsin in different time. Observing cell morphology of difference experimental groups cells under the microscope, recording the number of primary cells,as well as testing the survival rate of primary cells by moss phenol blue staining after 12h, etc. Optimized experimental conditions of separating MEF from ICR Mouse with the methods of trypsin digestion in vitro.2. To compare the growth state and the proliferation activity of MEF from generation 2 to 7, observing cells’morphology by optical microscope and the proliferation velocity by counting the number of cells, and descripting cells’ proliferation curves by statistical graph, which was done in order to explore which generation is the best to prepare feeder layer and provide theory basis for the further experiment.3. The third generations’ MEF were choosed. Five samples of each experimental group were selected to count the numbers which were cultivated with medium containning of different concentrations(5%,10%,15%,20%). To test cells proliferation velocity, and descripting cells’proliferation curves by statistical graph. Using Levene test homogeneity of variance, if P>0.05, the data was tested by One-Way ANOVA method, if there are significant differences, using the LSD method to do further comparisons between groups; if P<0.05, the data was tested by were detected by Welch method, If there are significant differences, using the Dunnttt T3 method to do further comparisons between groups to explor the optimum serum concentration for cultivating MEF in vitro.4. The third generation cells were choosed to prepare feeder layer, MEF were dealt with by different concentration of Mitomycin C (5ug/ml, 10ug/ml,15ug/ml, 20ug/ml) at a different time(1h、2.5h、3.5h). After this, test their activity of proliferation using MTT method and the OD value of absorbance in all experimental groups was detected at the 570nm by enzyme-linked instrument MEF. Samples with the same experimental conditions (concentrations of Mitomycin C and time) were grouped according to the cultivating time. Using Levene test homogeneity of variance, if P≥0.05, the data was tested by One-Way ANOVA method, if P<0.05, the data was tested by were detected by Welch method. The best method of preparing feeder layer was determined by this method, and the cells of feeder layer were detected by fluorescence to test if it has been infected by Mycoplasma.5. MEF was utilized to prepare feeder layer to cultivate hES cells and iPS cells. To observing their growth state. According to the proportion of differentiated cells in clone which was observed by inverted microscope, hES cells and iPS cells’clone were classfied into undifferentiated (the proportion of undifferentiated cells in clone≤20%) and differentiated (the proportion of differentiated in clone≥20%). The number of differentiated and undifferentiated clone was calculated by three holes, to observe hES cells and the iPS cells growths’state, and detect the expression of marker genes of stem cells (Nanog and OCT-4) by RT-PCR, which were used to prove the effect of feeder layer produced in backing stem cells’growth.7. Statistical methods:all the data was shown as mean or mean±standard deviation(X±S) and analysis by SPSS 13.0. Using state charts describe the growth state of different passages; The effect of different serum concentration on the proliferation of MEF was analysed by all randomized varianc, and described by state charts. Using Levene test homogeneity of variance, if P>0.05, the data was tested by One-Way ANOVA method, if there are significant differences, using the LSD method to do further comparisons between groups; if P<0.05, the data was tested by were detected by Welch method, If there are significant differences, using the Dunnttt T3 method to do further comparisons between groups; Assay results of preparation of feeder layer were used by homogeneity of variance test, if the variance is yes, we use One-way ANOVA; if not, we use Welch method.Results1. MEF can be found adherent growth in vitro by optical microscope, and the cell shapes were different such as spindle, polygon, irregular and so on. They arranged in swirl, palisade. The size is not average and has a high heterogeneity. There are some oval nuclear. With the generation increasing, MEF becomes purer, and MEF trends to be identical gradually.2. The effect of digesting embryo tissue with trypsin is related to the concentration of trypsin and digestion time. Using 0.05% trypsin digests tissue acting for 12-15 mins can produce least damage, maximum cells. More than 106 cells/embryo and the cell survival rate is more than 80%; Between 105 and 106 cells/embryo is gotted when using 0.15% trypsin digests tissue acting for 8-10 mins, and the cell survival rate is between 60% and 80%; Lest than 105 cells/embryo we can get when using 0.25% trypsin digests tissue acting for 3-5 mins, and the cell survival rate is lest than 60%. So the lower of the concentration, the effect of cells isolating from tissue is milder and the less damage to cells. Though it is necessary to extend digestion time with low concentration of trypsin, the number of cell is more than the way using higher concentration of trypsin, also the cell density is larger and the proliferative capacity is greater.3. Impacts of generation on MEF growth:Comparing P2 to P7 MEF growth state, it was found that in the P2 to P5, MEF grow faster, and adhere more quickly, also had a better stretch, and it appeared to be a typical fibroblast morphology (almost in the long spindle-based, a small number in irregular star, polygon, oval nucleus, cytoplasm of uniform small cytoplasm granules). The cytoplasm appeared more vacuoles, some cells showed the phenomenon of aging, and when MEF were more than six generation, cells’bodies showed irregular thin flakes, granules within the cytoplasm significantly increased compared with the 5th generation, the gap between cells increased, the cell morphology was aging. Contrasted to the 2-7th generation of the MEF cells growth state, it is found that the 2,4 and 5th generation of cells in the first 4-5 days present proliferation, but not as good as the growth of 3rd generation cells, and they have not obvious plateau, the 4-5th generation cells when the proliferation get to the peak after 4 or 5 days, the number of cells significantly reduced. The 6,7th generation cells had no significant proliferation. Therefore, the 3rd generation cells’growth state is most suitable for preparing for feeder layer cells.4. Influence of different serum concentration of medium on the growth of MEF: the number of cells had little change when MEF were cultivated with medium containing 5% serum a week, maintaining 10000-20000. and cells were dead largely with the extension of time, the medium containing 10%,15% and 20% serum can culture MEF and the proliferation velocity is fast, but in the medium containing 15% serum, the number of cells appeared to be a platform stage in 4th-6th days, the number was relatively stable, while in the medium containing 10% and 20% serum, the number of cells was shrunk immediately after reaching the peak, so it didn’t have a platform stage. By analysis of randomized variance, the result of Levene method was that P≥0.05. It was found that cells of four groups had significant differences (P <0.05) between each other after MEF were plated 1,2 and 3 day. Comparing with every two groups with LSD test. The number of cells of every group increased when the cells were cultured with medium containing higher concentration of serum, medium containing 15% and 20% concentration of serum had not significant differences(P<0.05) at the 4th and 5th day,while the others had significant differences(P≥0.05), but the number of cells had significant differences between each other at 6th and 7th day.The result was that the number of cells of medium containing 15% serum group was largest. As is shown in the picture 5, the number of cells containing 10% and 20% serum group was less than others at the 4th and 5th day. Therefore, the medium containing 10%,15% and 20% serum can culture MEF, but the medium containing 15% serum is more suitable than others for the growth of MEF.5. The effect of mitomycin C to inhibit MEF from proliferating is related to its concentration and action time. All datas were analysised with variance, and the results showed when the concentration of Mitomycin C was lOug/ml and the cells were dealt with 2.5h, there were no significant differences among groups(Levene Test for Homogeneity of Variance, P≥0.05, ANOVA analysis P>0.05), but on the other conditions, there were significant differences among the groups (Levene Test for Homogeneity of Variance, P≥0.05, ANOVA analysis P<0.05 or Levene Test for Homogeneity of Variance, P<0.05, Welch analysis P<0.05). The results showed that it was effectively inhibition of cell division but not make a large of MEF death, and the feeder layer can growth about 8～10 days. when the concentration of Mitomycin C is lOug/ml and the cells were dealt with2.5h. The effect of mitomycin C inhibiting MEF to proliferate is related to the concentration of mitomycin C and action time.6. The effects of hES cells and iPS cells cultured on the feeder layer:hES cells and iPS cells were cultured on the feeder layer dealt by on the feeder layer dealt with by mitomycin C and passaged many times, which growed quickly and proliferated rapidly and showed typical stem cells’clone morphology. The proportion of differentiated clone of hES cells is 5.6%, and the proportion of differentiated clone of iPS is 6.6%, so the majority of them remained undifferentiated. The stem cells’ marker genes of hES cells and iPS cells, Nanog and OCT-4, were expressed at a high level by RT-PCR after passaging many times.Conclusions1. Methods of trypsin digestion to separate MEF of ICR mouse which can be purified by subculture without using other methods and can used to prepare feeder layer.2. The best experimental conditions of separating MEF is that 0.05% trypsin actives embryonic tissues of mouse for 12-15 mins. Under this condition, trypsin can minimize the damage to cells the largest number can be gotten, also the velocity of primary cells adhering and proliferating is fastest, and cells grow faster after passaging.3. The medium containing 15% serum is optimal for MEF in vitro. MEF cells’ growth state in the 3rd generation is the best, which is the most suitable for preparing cells of feeder layer.4. The best conditions of mitomycin C dealing with MEF is that using 10ug/ml Mitomycin C deals with MEF for 2.5 hours. In this condition, Mitomycin C can inhibit the proliferation of MEFs, but can not lead to the death of a large number of MEFs, and the cells can survival 8 to 10 days.5. MEF dealt with by mitomycin C as the feeder layer can effectively stimulate the growth of hES cells and iPS cells, and promote stem cells to proliferate and maintain undifferentiated state. The expression of stem cell-related genes, Nanog and OCT-4, was detected by RT-PCR. |
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