The main advantage of this technique is that before stimulation, cells are dispersed evenly and then stimulated evenly in tandem cell adhesion. Demonstrating the procedure will be Azuma Kimura, a graduate student from our laboratory. To begin this procedure, transfer six milliliters of RPMI 1640 into a tube cooled to four degrees Celsius on ice.
Using cooled one milliliter pipette tips, add two milligrams of basement membrane matrix. Mix well by gentle pipetting to make it a basement membrane matrix with a concentration of 0.33 milligrams per milliliter. Next, use a pipette to transfer two milliliters of the diluted basement membrane matrix into each well of a six-well cell culture plate.
Incubate the plate at 37 degrees Celsius for 60 to 90 minutes. After this, keep the plate at room temperature for up to three hours, until ready to use. First, aspirate the used medium and use a pipette to add two milliliters of 0.5 millimolar EDTA to each well to wash the hPCSs cultured in the six-well plate.
Then, aspirate the EDTA from the wells and add two milliliters of fresh 0.5 millimolar EDTA to each well. Incubate the plate at 37 degrees Celsius for five minutes. After this, aspirate the EDTA from each well.
Add one milliliter of hPSC maintenance medium at room temperature, supplemented with 10 micromolar Y27632 to each well. Pipette gently but quickly to blow off the attached cells on the plate and to dissociate any clumped cells into single cells. Transfer the cell suspension in a 50 milliliter centrifuge tube containing four milliliters of hPSC maintenance medium supplemented with 10 micromolar Y27632 per well and mix by pipetting.
Next, mix 15 microliters of cell suspension with 15 microliters of trypan blue in the tube. Use a 10 microliter pipette to transfer 10 microliters of the diluted cell suspension into cell counting slides in duplicate. Using an automated cell counter, count the cell number and calculate the cell density in the cell suspension.
Allocate the cell suspension into a 50 milliliter tube at a density of one to one and a half million cells for each well to be used. Centrifuge the tube at 200 times G and at room temperature for five minutes. After this, use a pipette to aspirate the supernatant and re-suspend the cells using one milliliter of stage 1A medium per well.
Gently pipette the cell suspension and then add another one milliliter of stage 1A medium per well. Using a 1000 microliter pipette, aspirate the diluted basement membrane matrix from the wells of the previously prepared cell culture plate. Immediately pipette the suspension in the tube gently and transfer two milliliters into each well of a six-well plate.
Cover the plate with aluminum foil to protect it from light and place the plate on a clean bench at room temperature for 10 to 15 minutes. After this, carefully transfer the plate to an incubator at 37 degrees Celsius with 5%carbon dioxide and a humidified atmosphere for 24 hours. First, gently shake the plate and use a pipette to aspirate the used medium.
Then, add two milliliters of DPBS to each well. Gently shake the plate again and aspirate the used DPBS. Add four milliliters of stage 1B medium, pre-warmed to 37 degrees Celsius, to each well.
Carefully transfer the plate to an incubator at 37 degrees Celsius with 5%carbon dioxide and a humidified atmosphere to culture for 48 hours. After this, gently shake the plate and aspirate the used medium. Add two milliliters of DPBS to each well.
Repeat this aspiration and addition of DPBS one additional time. Gently shake the plate and aspirate the used DPBS. Add four milliliters of stage 1B medium, pre-warmed to 37 degrees Celsius, to each well.
Carefully transfer the plate to an incubator, at 37 degrees Celsius with 5%carbon dioxide and a humidified atmosphere to culture for 24 hours. First, gently shake the plate and aspirate the used medium. Add two milliliters of DPBS to each well of the six-well plate.
Then, gently shake the plate and aspirate the used DPBS. Add four milliliters of stage two medium, pre-warmed to 37 degrees Celsius, to each well. Carefully transfer the plate to an incubator at 37 degrees Celsius with 5%carbon dioxide and a humidified atmosphere to culture for four days.
Gently shake the plate and aspirate the used medium. Add two milliliters of DPBS to each well. Repeat this process of aspirating and adding DPPS once more.
After this, gently shake the plate and aspirate the used DPBS. Add four milliliters of stage three medium, pre-warmed to 37 degrees Celsius, to each well. Carefully transfer the plate to an incubator at 37 degrees Celsius with 5%carbon dioxide and a humidified atmosphere to culture for three days.
In this study, propagating hIPSEs are condensed and form a homogenous monolayer that is suitable for differentiation. Undifferentiated hIPSEs are dissociated and reseeded as single cells at low cell densities. Within one hour, the cells are attached to the plate and start to show protrusion.
On day one, the cells are proliferated and well distributed to cover 80 to 90%of the surface area. On days three and four, the cells form a homogenous monolayer sheet that can be described as a cobblestone appearance. At this point, most cells stop expressing SOX2, which is a marker for undifferentiated cells, and instead express a definitive endoderm marker, SOX17, at more than 90%Most SOX17-positive cells express FOXA2.
Then the cells start to express the primitive goptupe markers, HNF1-beta and HNF4-alpha, and eventually express a posterior foregrate pancreatic progenitor marker, PDX1, at more than 90%The PDX1-positive cell induction is reproducible in another hIPSE line, 1231A3, and an hESE line, KhES-3. QRTPCR results of the MRNA expression of stage markers are consistent with immunostaining and the MRNA expression of PDX1 is evident at stage three and substantially increases afterward. Since undifferentiated ESIPS cells are grown as colonies, individual cells are locally in a different state.
This protocol provides a way to stimulate cells evenly for the elected differentiation.
