This method can help with a key question in the stem cell engineering field such as, how cellular microenvironment alter the cellular phenotypes and function. The main advantage of this technique is it provides the multiple artificially created environment for the cells in a single plate which you could not perform with a conventional method in micro-contact plate demonstrating this procedure will be Koki Yoshimoto and Risako Sakai technicians from our laboratory. After creating 3D images of masks for nano fiber arrays and molds from microfluidic structures according to the text protocol.
Use a 3D printer to print the masks and mold. To prepare polymer solutions for electro spinning, dilute 13 percent PMGI liquid with tetrahydrofuran to nine percent. Dissolve 0.08 grams of PS in a one to one volume ratio of THF to dimethylformamide and use a liquid regent to bring the volume up to the final one milliliter of eight percent weight per volume PS solution.
Dissolve 0.1 gram of GT in water, acidic acid and ethyl acetate and use the mixed solvent solution to bring the volume up to the final one milliliter of 10 percent weight by volume GT solution. Next, using a magnetron sputtering machine, deposit a five nanometer thick platinum layer on a polystyrene base plate which serves as a cathode in the electro spinning setup. Load each polymer solution into a five milliliter syringe equipped with a 23 gauge stainless steel blunt needle.
Then anchor the syringes on a syringe pump 12 centimeters apart from the collector of the electro spinning device. Connect the syringe needle to a high voltage power supply and set it to 11 kilovolts. Then set the pumping rate to 20 milliliters per hour and keep the temperature and humidity at 30 degrees Celsius and less than 30 percent by volume, respectively.
Now put the mask on the base plate. Then through the holes of the mask, fabricate nano fibers on the base plate with distinct densities by changing the electro spinning time. Remove the mask from the base plate and repeat the nano fiber fabrication.
When the array is complete, place it in a desiccator at 25 degrees Celsius for 16 hours to evaporate the remaining solvent. To cross link the GT nano fibers, treat them with 0.2 molar EDC and 0.2 molar NHS in ethanol. And incubate the samples at 25 degrees Celsius for four hours.
To rinse the GT nano fibers, use 99.5 percent ethanol twice and vacuum dry the plates at 25 degrees Celsius for 16 hours. To fabricate microfluidic structures, mix 2 grams of PDMS curing agent and 20 grams of PDMS base. Then pour the pre-PDMS mixture on to the fabricated mold.
In a desiccator degas the pre PDMS mixture for 30 minutes. Then cure the pre PDMS mixture in an oven at 65 degrees Celsius for 16 hours. To assemble Macme arrays, peel off the cured PDMS structure from the mold and use 70 percent ethanol to clean it.
Then discharge atmospheric corona on the bottom side of the PDMS structure. Quickly assemble the PDMS structure with the nano fiber array. Then oven bake the assembly at 65 degrees Celsius for two days.
Using DPBS wash H9HESCs cultured on a 35 millimeter dish. Then add 0.5 milliliters of recombinant trypsin light protease to the dish and incubate it at 37 degrees Celsius for one minute. Carefully aspirate on the supernatant protease mixture.
Immediately add HPSC medium and gently dispense the medium against the dish surface repeatedly to dissociate cells. Then transfer the detached cells to a 15 milliliter conical tube. Centrifuge the tube a 200 times gravity for three minutes.
Aspirate the supernatant and suspend the cells in pre warmed HPSC medium. Then pipette 12 microliters of the cell suspension into each microfluidic chamber. After fluorescently labeling the cells according to the text protocol, peel off the PDMS microfluidic structure from the Macme array.
Then remove the residual PBMS from the Macme array add 90 percent glycerol in PBS to the stained cells and apply a cover slip. Finally, set the plate upside down on the stage of an inverted fluorescence microscope. And use the microscope imaging software to acquire 12 bit color images.
Shown here are immunofluorescence images of H9HPSCs cultured at high initial seeding density on gelatin nano fibers and stained with 3 cellular phenotypic markers. SOM analysis was used to convert high dimensional multi parametric data sets into low dimensional 2D maps for comparing homogeneity and heterogeneity of expression levels for the four phenotypic markers in each data set and unsupervised hierarchical clustering was performed for the SOM nodes. As indicated here, all groups exhibited higher expression of OCT4 than was observed on basement membrane gel matrix, or MG.Group one showed high EdU signals, indicating that most cells were actively proliferating.
Thus, microenvironments in group one were suitable for HPSC maintenance because undifferentiated HPSCs proliferate rapidly when cell cycle gap phases were shortened. Group two included cells seeded at an insufficient initial density and cells grown on 2D GT scaffolds that did not support HPSC self renewal. For example, this sample's EdU signal was lost and annexin V levels were slightly increased indicating that the cells had lost their stemness and had gradually become apoptotic.
PMGI MidNF HighCD from group three which represents the microenvironments comprising PMGI nano fiber matrices showed the larger variations in OCT4 and EdU signals compared with the other conditions. After it's development, this technique paved the way for the researcher in the field of STEM cell to explore the tissue engineering and advanced screening.
