This procedure, fabricates Electros spun long fibers from a novel biodegradable polymer named poly glycerol Dote Cano eight. First set up the newly designed collector and prepare the polymeric solution for electro spinning, using a syringe and pump, deliver the solution to the collector. Then transfer the resulting fiber mat to a culture dish for cross-linking.
Next seed mouse embryonic stem cells on the cut fibers. Ultimately, results can demonstrate that the mouse embryonic stem cells can survive and differentiate into neural cells on the fiber scaffolds. In the today video, we showed you how to prepare the biomaterial from the syn cells fabrication, and finally use those electro span fiber for cell culture.
Especially when we combine with the cell derived from poly and stain cell. They may be have very important application in the future for the cell delivery and all making a functional tissue. Tissue invis today that she will demonstrate a horrible procedure for you.
She's the greatest thing in my laboratory. Cut aluminum foil into a rectangular piece. Fold the rectangular piece into a rectangular strip and attach it perpendicular to a flat metal plate with tape.
The length of the strip depends on the size of the fiber mat needed for the polymeric solution. Mixed glycerol and do decane diic acid in a one to one molar ratio at 120 degrees Celsius for 100 hours. To obtain a poly glycerol, do decanoic polymer dissolve polyethylene oxide and gelatin in 65%Ethanol with a weight ratio of 1.5 to three to 95.5 in a 15 milliliter tube.
Tighten the cap and heat the mixture in an oven at 60 degrees Celsius for one hour, stirring every 15 minutes until the basal solution is homogeneous. For electro spinning, mix the PGD polymer and the basal solution. In a four to six weight ratio, add 0.1%riboflavin into the mixture and mix it well.
Feed the polymeric solution into a five milliliter standard syringe with an 18 gauge blunted stainless steel needle. Then insert the syringe into a syringe pump. Attach the grounded lead of a high voltage power source to the metal plate and the positively charged lead to the needle.
Also, adjust the distance between the needle and the aluminum foil. Strip to 15 centimeters. Now place the syringe pump at an angle of about 15 degrees with the horizontal to prevent aggregation of the fibers at the front of the strip.
Turn on the syringe pump and adjust the flow rate of the pump to 0.6 milliliters per hour. Turn on the high voltage power source and set the operating voltage to 14.6 kilovolts. After the collection is complete, expose the fiber mat to UV light for 60 minutes of cross-linking.
Transfer the fiber mat from the aluminum foil strip to a 100 millimeter Petri dish. Cut the fiber mat into round pieces of the same size with the surgical blade and place the pieces into a 24 well plate. Then expose the plate to UV light for another 20 minute sterilization for cell preceding treatment.
Immerse fiber samples in one milliliter of phosphate buffered saline and incubate a 37 degrees Celsius overnight on the following day. Aspirate the PBS carefully. Add one milliliter of differentiation medium to each well and incubate at 37 degrees Celsius for three hours.
After aspirating the differentiation medium carefully add 0.2 milliliters of matri gel to each fiber sample and incubate at 37 degrees Celsius for 30 minutes. Carefully remove the excess matri gel from each. Well rinse once with two milliliters of differentiation medium.
Add one milliliter of tase to the mouse embryonic stem cell culture dish after 10 minutes, a 37 degrees Celsius. When the MES cells are detached, add four milliliters of differentiation medium. Collect the floating MES cells into a 15 milliliter tube centrifuge at 400 Gs for five minutes.
Re suspend the pelleted cells in four milliliters of differentiation.Medium. Transfer 200 microliters of the cell suspension into a 15 milliliter tube and add 10 volumes of differentiation.Medium. Transfer 15 microliters of the diluted cell suspension to a chamber on the hemo cytometer.
With a cover slip in place, count the cells in the one millimeter center square and the four corner squares of the hemo cytometer. Now drop five times 10 to the fourth. Mouse ES cells slowly onto the middle of the fiber samples.
Add one milliliter of differentiation medium to each well and culture at 37 degrees Celsius and 5%carbon dioxide for cell attachment. Growth and differentiation. Replenish the one milliliter of differentiation medium every other day.
To measure cell viability, aspirate the old medium from each well and add one milliliter of a one in 10 dilution abre and fluorescence reagent in culture. Medium incubate at 37 degrees Celsius and 5%carbon dioxide for four hours protected from direct light. Then transfer triplicate samples of 100 microliters from each well to a 96 well plate and measure fluorescence scanning electron microscopy images were used to evaluate the morphology of electros spun fibers.
The diameters of these fibers made from 40%PGD concentration are in the micrometer range here. Differentiated mouse embryonic stem cells were cultured for three days on the fibers and confocal microscopy was used to visualize over expressed green fluorescent protein. The increased number of green fluorescent cells on day six indicates that the fiber scaffolds can support both cell adhesion and cell proliferation.
Zu and fluorescence measurements demonstrated that coating with matrigel and laminin resulted in equivalent cell viability and relatively higher proliferation. Flury potency and neural cell markers were quantified by real-time PCR at day one. The majority of MES cells cultured on fibers expressed the pluripotency markers.
OCT four nano and SOX two. A minor subset expressed the neural stem cell markers PAC six and neston. After two weeks in culture, there was increased expression levels of some neural cell markers, such as map two and DCX as well as oligodendrocyte marker oligo one and astrocyte marker.
GFAP Once mastered. This technique can be done in two to three hours if it is performed properly.
