The overall goal of this procedure is to prepare mouse embryonic fibroblasts suitable for culturing human embryonic stem cells and induced pluripotent stem cells. This is accomplished by first separating each embryo and removing the head and red organs. Next, the tissue is finally minced and digested.
Then the cells are plated, three to four embryos per flask and expanded. Finally, the inactivated myths are used to prepare the conditioned medium. Ultimately, e LSA is used to show the optimal concentration of active in a within conditioned medium supporting undifferentiated growth of pluripotent stem cells.
Generally, individuals new to this method will struggle because the procedure needs to be standardized in order to produce mouse Embry fibroblasts of high quality, which will enable the undifferentiated growth of both human embryonic stem cells and induced pluripotent stem cells. Visual Demonstration of this method is critical as the isolation and dissection of embryo steps are difficult to learn because handling of the mouse embryo require certain manual skills. Demonstrating the procedure will be Dr.Justina Suk, who is a postdoc in my lab and also a graduate student, Caterina Dres.
After sacrificing a pregnant mouse at 13 or 14 days, post coum and dissecting out the uterine horns, briefly rinse them in 70%ethanol and place them in a Falcon tube containing PBS using aseptic conditions under a tissue culture hood. Place the uterine horns into a Petri dish and separate each embryo from its placenta and embryonic sack. Remove the head and red organs, wash the remaining pieces of embryo and PBS and place them in a clean Petri dish using a sterile razor blade.
Finally mince the tissue until it becomes possible to pipette. Next, add one milliliter of 0.05%trips in EDTA and 100 K units per embryo of DNAs one. Then transfer the tissue into a 50 milliliter Falcon tube and incubate it for 15 minutes at 37 degrees Celsius.
After each five minutes of incubation, dissociate the cells by thoroughly pipetting up and down. Inactivate the trypsin by adding about one volume of freshly prepared meth.Medium. Centrifuge the cells for five minutes at low speed.
Carefully remove the supinate and resuspend the pellet in warm meth medium prepare 0.2%gelatin coated flasks and incubate for at least two hours. At room temperature, plate the cells from around three to four embryos into each 0.2%gelatin coated flask. The fibroblasts are the only cells that have the ability to attach to the gelatin coated flasks.
After about 24 hours, when the cells reach 80 to 90%co fluency freeze some cells at minus 80 degrees Celsius. For future use, expand the remaining cells to P three or P four. To prepare feeder cells using the P three P four maths, begin by coating T one 50 flasks with 0.2%gelatin and incubated room temperature for at least two hours.
Dilute mitomycin C in PBS and filter through a syringe filter. Aspirate the medium from the meth and wash them with PBS without calcium or magnesium. Place 20 milliliters of medium containing 10 micrograms per milliliter of mitomycin C on the maths and incubate for two hours at 37 degrees Celsius.
Next, wash the cells twice with PBS and tryps them. After incubation resus, suspend the cells in meth medium, then spin the cells down and resuspend them in warm meth.Medium. Plate the cells at a density of 56, 000 cells per centimeter square in T one 50 flasks and incubate them overnight on the day after plating the meth cells.
Replace the culture medium with HESC unconditioned medium, supplemented with fresh four nanograms per milliliter, FGF two and incubator 37 degrees Celsius. 24 hours later, collect the conditioned medium from the flasks and replace it with fresh HESC medium. Store the collected medium at minus 20 degrees Celsius.
Repeat the collection for a total of six days after the sixth collection. Combine all the medium and filter it. Then store it in 50 milliliter aliquots at minus 80 degrees Celsius.
To measure active in a, in conditioned medium, bring all samples and reagents to room temperature. Dilute the active in a antibody in PBS with 1%BSA and add 100 microliters to the wells of a microplate. Incubate overnight at room temperature after 24 hours.
Wash the wells three times with PBST. Then block the wells for one hour at room temperature. In the meantime, prepare an active a standard curve, including seven dilutions and a blank sample using a working range of 0.25 to 32 nanograms per milliliter.
Add duplicates of the standards and samples to wells and incubate for two hours at room temperature. Wash the wells three times with PBST. Next, add a biotinylated secondary antibody and incubate for two hours at room temperature.
Then wash the wells three times with PBST and add streptavidin HRP. After mixing and adding substrate, wrap the walls for light protection and incubate for 20 minutes of room temperature. Add 100 microliters of stop solution to each well and gently mix.
Finally, set the microplate reader to four 50 nanometers with wavelength correction at five 40 or five 70 nanometers, and determine the optical density for each well shown. Here is the typical morphology of undifferentiated h ESCs, cultured in the presence of feeder cells, conditioned, medium, and defined medium. In these examples, HI PSCs are cultured in the presence of feeder cells.
These images show the morphology of myths and inactivated feeder cells used to prepare conditioned medium. In general, cells should be confluent 24 hours post isolation and ready to be frozen or expanded. However, sometimes it might take two to three days before obtaining confluent cultures.
The conditioned medium should be prepared from cells at passage four and not later. This is crucial because primary cells can only be expanded for four to five passages before the onset of senescence. The cytokine active in a is considered one of the most critical factors secreted by feeder cells for the support of undifferentiated growth of pluripotent cells.
Measuring the level of active in a in conditioned medium, as shown here, is a convenient quantitative assay to monitor the quality of mets Once mastered, the isolation of mouse embryonic fibroblast can be done in one to two hours if it's performed properly. While attempting this procedure, it's important to remember to breed the animals in advance and to prepare and autoclave all instruments in advance.
