Comparison Of Support Effect Of MEF And HLEF As Feeder Layer To Mouse Embryonic Stem Cells

part1 Support Eeffect of MEF as Feeder Layer toMouse Embryonic Stem CellsExperiment one Preparation of Mouse Embryonic FibroblastsObjective Prepare Mouse Embryonic Fibroblast for culture of mouse embryonic stem cell.Methods Primary mouse embryonic fibroblasts were isolated from 12~18days old fetusmouse. Cut the tissues into small pieces using dissecting scissors. Use trypsin to digestive thetissue. Incubate cells in a 37°C incubator supplied with 5% CO2.Results Mouse embryonic fibroblast cells have many non-fibroblast cells before first 3generation. In vitro,It is adherent cell with good ability of proliferation affer passage 3.With theincreasing of the cell number, cell line gradually form. Most of cells look like a spindle, asmall number of cells are round or irregular, the cells are transparent. The middle part of cellsis a little uplift, a strong sense of three-dimensional. With the number of cells increasing, cellsis connected closely.Experiment two Preparation of MEF feeder layerObjective Preparation of MEF feeder layer for mouse embryonic stem cells.Methods MitomycinC 10μg/ml treat MEF for 2h. PBS wash MEF for several times. plate thecells into wells of 6-well plates pre-coated with 0.1% Gelatin Solution.Results Observed under inverted microscope ,the morphology of cells were no significantchanges. Most of the cells were spindle-shaped with clear boundaries.Chose suitable concentration of mytomycin C and the time of treating cells is veryimportant..In our experiment we treat MEF using MitomycinC 10μg/ml for 2h.It is a good way to repress proliferation of ES cells. But the MEF treated by mytomycin C will die in 1~2weeks.Experiment three The State of Es clones growing onMEF feeder layerObjective Observe the state of Es clones growing on MEF of feeder layer.Methods Chose suitable MEF feeder layer ,Change the medium into special mouseembryonic stem cells growth medium. Plate ES cell suspension (5x105)into MEF feeder layer.Results ES is planted on MEF feeder layer ,2 days later, we can see the ES clones.The ESclones look like islands, the edge of ES clone is clear and smooth.There is clear boundarybetween ES clones and feeder layer.Experiment four Observation the State of differentiation ofES cellObjective Detect MEF feeder layers whether can maintain ES cells undiferentiation.Methods (1) Detect Alkaline phosphatase (AKP) activity.Put 4% Paraformalclehyde(PFA) into ES clones on two kinds of feeder layers for 10min. PBS wash several times. Then put NBT/BCIP into ES clones. Observe the Alkalinephosphatase (AKP) activity of ES cells .Use water to stop the reaction .(2) Detect the expressioon of OCT-4 by Immunofluorescence.After washing with phosphate buffered-saline PBS,the cells were permeabilized with 2%Triton X-100, blocked for 10 minutes with PBS containing 10% donkey serum albumin,thenincubated with primary rabbit monoclonal anti-mouse Oct-4 antibody (1:500) for a night at4°C. Negative control was performed with FITC-conjugated mouse anti-rabbit IgGsecondary antibodies.PBS wash three times.Results ES clones on MEF feeder layers have strong AKP activity. It can express Oct4-greenfluorescent protein which can be observed by fluorescence microscopy. The experiment showed that MEF feeder layer can retaining the initial totipotency of ES cell.part2 Support Effect of hLEF as Feeder Layer to MouseEmbryonic Stem CellsExperiment one Chose Optional Concentration of Mytomycin CObjective Chose optional concentration of mytomycin C for human embryonic lungfibroblats .Methods Plate the MEF cell suspension into wells of 6-well plates pre-coated with GelatinSolution. After 24 h, put MTT solution(5mg/ml)100μL into every well of 6-wellplates.Incubate cells in a 37°C incubator for 4h. Put DMSO 150μL ,shake the 6-well plates for10 min.Than use ZS-3 Microplate Reader to obtain the optical density.Results Using different concentration of mytomycin C and the time of treating hLEF,thestate of hLEF cells is different. Using MitomycinC 10~20μg/ml for 1.5~3.5h and MitomycinC20μg/ml for 1.5h can effeciently repress the proliferation of hLEF. Low concentration ofmytomycin C(<10μg /ml)can repress proliferation of hLEF effectively but high concentrationof mytomycin C(>20μg /ml)especially in long time(>1.5h)can kill the hLEF.Experiment two Preparation of hLEF Feeder LayerObjective Preparation of hLEF feeder layer for mouse embrynic stem cellsMethods MitomycinC 10μg/ml treat hLEF for 2.5h. PBS wash several times, plate the cellsinto wells of 6-well platespre-coated with 0.1% Gelatin Solution.Results After treated by mytomycin C, hLEF were planted into wells of 6-wellplatespre-coated with 0.1% Gelatin Solution.The cells integrated into hLEF single-layer rapidly.Observed by Inverted microscope, the morphology of MEF were no significant changes. Mostof the cells were spindle-shaped with clear boundaries.Select the appropriate concentration of mitomycin C and time is crucial for preparation ofhLEF feeder layer. In this experiment we used 10μg/ml of mitomycin C to treat hLEF for 2.5h, hLEF can keep alive for 2~3W. It is a good ways to repress proliferation.Experiment three The State of Es Clones Growing on hLEFof Feeder LayerObjective Observe the state of Es clones growing on hLEF of feeder layer.Methods Using the same way as plating Es cell on MEF feeder layers.Results ES is planted on two kinds of feeder layer ,the ES clone is round and small. 2~3dayslater, we can see the ES clones.There is no obviousely distinction of cell morphologycharacteristics and growth behaviors between ES cells on hLEF feeder layers and ES cells onMEF feeder layers .The ES clones look like islands, the edge of ES is clear and smooth.Thereis no clear boundary between ES cells.Under the same culture condition ,ES clones on the MEF feeder layers is biger than ESclones on the hLEF feeder layers. ES cell on the hLEF feeder layers grow slower tnan ES cellon the MEF feeder layers.Experiment four Observation of the State of Differentiationof ES cellObjective Detect whether hLEF feeder layers and MEF feeder layers can maintain ES cellsundiferentiation state.Methods (1) Detect Alkaline phosphatase (AKP) activity.As mentioned above.(2) Detect the expressioon of OCT-4 by Immunofluorescence.As mentioned above.Results ES clones on hLEF have the strong AKP activity. It can express Oct4-greenfluorescent protein which can be observed by fluorescence microscopy. The experimentshowed that hLEF feeder layer can retaining the initial totipotency of ES cell.