Studies On The Culture In Vitro And Growth Characterization Of Mouse Embryonic Fibroblasts

The feeder layer cells means those special types of cells used for feeder layers, such as fibroblast, oviduct epithelia, uterus epithelia, embryonic testicle cells etc., they were treated with mitomycin C and then seeded for monolayer, which is called feeder layer. Currently, murine embryonic fibroblasts (including MEF and STO cells) were adopted for feeder layer cells to isolate embryonic stem (ES) cells in most related laboratories all over the world. Feeder cells could not only provide steady condition for ES cells, but also stimulate ES cell proliferation in vitro and keep its pluripotency. However, aged feeder cells would yield negative effects to ES cells. Dead feeder cells could release DNA fragments and enzymes into culture system, disturb the growth of ES cells and maintaining the normal type nucleus. In this experiment, three kinds of freezing method were compared for the cryopreservation of mouse fibroblasts. And changes of the post-thaw viability of the cells and cytoskeleton during continuous passages were observed and analysed.In this experiment, we isolate and culture MEF cells, then freeze STO cells and MEF cells by three kinds of method, including Method A as balancing cells in the refrigerator of 4℃for 1h before transferring cells into the refrigerator of -20℃overnight and then put them into the Liquid Nitrogen the next day. Method B as having cells in Nalgene Cryo-freezer cold down in a -70℃refrigerator overnight and transferring them into the Liquid Nitrogen the next day and Method C as steaming cells on the level of 5-10cm above the surface of Liquid Nitrogen for 20min and put them into the liquid slowly. After cells' revivification, the freezing effects of three different approaches are compared mainly with livability, which is measured in Trypan Blue dyeing experiment, and the comparative cell viability, which is measured by MTT assay, and the compares between the cells' morphological observations before freezing and after revivification are considered as additional targets. Then we culture mouse embyonic fibroblast continuously, Observing the change of morphology, measuring cell viability by MTT assay, drawing the cell growth curve, and using immunostainin to observe the change of cytoskeleton. The results shown as follows.1. The livability of cells freezed by Method B is remarkably higher than that freezed by Method A and Method C, meanwhile, there is no noticeable differences of cells' morphological development and of the comparative cell viability among the three freezing approaches. And the further conclude is that the freezing approach B is confirmed to be the best scheme of freezing on the feeder layer cells in this experiment.2. STO cells grow rapidly after resuscitation, and present typical fibroblast morphology. As the times of passageing increasing, especially after the ninth passage, cells arrange is out of order, lots of grains and vacuoles are appeared, dead cells is increasing. After fourteenth passage, cytoplasm is dissolving and cell is crashing in part of the cells. MEF cells grow slowly in the first three passsages; after the forth passage, MEF cells grow rapidly, metabolism is vigorous, the shape of the cells is regular, after the sixth passage, the cells are decrepit rapidly, cells are broken, flaky cells are floating or suspending in cultural system.3. The cell viability of STO cells reach the most value in the fifth passage, then decline after the ninth passage, ascend in fourteenth passage. The cell viability of MEF cells reach the most value in the fifth passage, the decline after the sixth passage.4. With technology of CLSM, we observe the change of embryonic fibroblast cytoskeleton. in the first five passages after resuscitation, the tubulin and actin of STO ceils arranged regularly, nucleolus locate in the middle of the cells, the ordinary karyotin is abundant, and abnormal nucleus is absent, after the ninth passage, actin become in disorder, tubulin distribute asymmetrily in cytoplasm, the type of nucleolus is unintegrity, after the fourteenth passage, the actin and tubulin of STO cells are even more disorder, chromosome is even more abnormal. The arrangement of actin and tubulin of primary MEF cells are the most regularly, the type of nucleolus is normal, but after continuous passaging, especially after the fifth passage the actin and tubulin of MEF cells become turbulent, dissolved, ruptured, even disappeared, and the type of nucleus is abnormal.