This protocol is optimized to encapsulate pluripotent human embryonic stem cells and differentiate them into dopaminergic neurons in a 3D system. First, treat the colonies with rock inhibitor or RI and associate them into single cells with acuate mixed with sodium alginate and passed through the encapsulation device. Continue the RI treatment for three days, transfer the encapsulated cells onto PA six cells and differentiate for 28 days.
Ultimately, gene and protein expression analysis show a higher efficiency of generating dopaminergic neurons. The major advantage of this particular technique over the existing 2D differentiation protocol is that we try to propagate and differentiate these cells under 3D environment and that 3D environment provide you an opportunity that cells can be propagated in a high density manner. And then it also gives an opportunity to have a better cell to cell interaction for differentiation.
And thirdly, also give you a better efficiency of differentiation down the track. And also, if I might sort of add that this particular technique can also be adapted for other type of the cells, which we can propagate in a 3D environment. Now remember also like say for example, this particular technique has been optimized based on using a particular rock inhibitor that prevent cell apoptosis and also prevent the detachment of the cell from the culture dishes.
Generally, individuals new to this method will struggle in the beginning as this technique is not typically performed in tissue culture. The following encapsulation technique has been optimized based on Dr.Dean Dr.Chad's summary studies on application of R inhibitor type of alginate, alginate concentration, and encapsulation device settings. Jamie and Kim, a PhD student from our lab, will now demonstrate this protocol.
Add 25 milliliters of sterile 0.1%gelatin solution to 275 milligrams of purified sodium alginate in a sterile 50 milliliter tube vortex to partially dissolve the alginate powder. Then place the tube on an orbital mixer at 10 times G overnight. Next, add 9%sterile sodium chloride to the alginate solution.
Vortex for 30 seconds and then centrifuge at 95 times G for five minutes. Store the alginate solution at four degrees Celsius. First in a dark environment, pretreat the human embryonic stem cells with culture media supplemented with 10 micromolar of rock inhibitor.
Incubate for two hours at 37 degrees Celsius. Now wash the cells twice with DPBS to dislodge the cells Enzymatically. Add accutane and incubate for 10 minutes.
At 37 degrees Celsius, gently harvest the cells with a cell scraper and transfer to a 15 milliliter tube. Neutralize the Accutane with an equal volume of SL medium. Then pass the neutralized cells through a 40 micron filter into a fresh 50 milliliter centrifuge tube.
Now use a hemo cytometer to enumerate the cells. Pellet the cells by centrifugation, carefully discard the supinate. Then wash the cells once with prewarm.
0.9%sodium chloride. Centrifuge your gain and discard the supinate. To encapsulate the human embryonic stem cells, begin by Resus suspending the cells with prewarm alginate solution.
To do this, attach a 20 milliliter syringe to soft plastic tubing and aspirate alginate into the syringe. Then gently mix the cell suspension using the syringe without creating bubbles. Now aspirate the cells into the syringe.
Discard the plastic tubing and then attach the syringe to the encapsulation machine. Proceed to instruments set up of the bead generator, syringe pump, airflow meter, and calcium chloride precipitation bath. Verify a 10 centimeter gap between the end of the encapsulation machine and the Petri dish collection point.
Next, set the syringe pump at 10 milliliters per hour. Airflow rate at eight liters per minute and a pressure of 100 kilo pascals. Collect the encapsulated cells into a prewarm precipitation bath for seven minutes.
Then transfer them by gentle aspiration into a 50 milliliter centrifuge tube containing 20 milliliters of 0.9%Sodium chloride allow the capsules to settle to the bottom of the tube, gently discard the supinate, then wash once with 0.9%Sodium chloride. Proceed to resuspend the encapsulated cells in prewarm culturing medium, supplemented with rock inhibitor. Transfer into a culture flask and incubate for three days.
Seed the mouse stromal line PA six cells in a 0.1%gelatin coated T 75 flask and conditioning dopaminergic neural differentiation medium for 24 hours. Transfer the capsules to a 50 milliliter centrifuge tube and allow them to settle to the bottom of the tube. Discard the supinate and resuspend the capsules in PA six cell condition dopaminergic neural differentiation.
Medium culture. The encapsulated human embryonic stem cells with the PA six cell monolayer for 21 days. When replenishing the media change.
Only half of the media each time continue to culture the PA six cells in dopaminergic neural differentiation. Medium supplemented with inducing factors for one week. First aspirate the capsules and transfer them into a 15 milliliter centrifuge tube.
When capsules have settled to the bottom, discard the supinate. Wash twice with DPBS, discarding the supinate. Then add five milliliters of decap ululating solution and mix the suspension thoroughly.
Incubate for five minutes of room temperature. After centrifuging pellet, the decap cells and wash twice with DPBS proceed to use decap cells either as a monolayer culture or for immediate downstream analysis. In a typical preparation, the diameter of alginate microcaps is between 400 and 500 microns.
Here, C-F-D-A-P-I assay. Results determine the green viable encapsulated cells at 80%analyses of viability, proliferation, and apoptosis of encapsulated human embryonic stem cells at different time points serve to optimize culturing conditions. Interestingly, rock inhibitor effects prevent dissociation induced apoptosis and maintain cell viability with indication of superior growth of encapsulated human embryonic stem cells cultured in H-F-F-C-M plus ri.
This example applies cell encapsulation as a 3D platform to differentiate encapsulated human embryonic stem cells into dopaminergic neurons. As expected the size of the embryo bodies increases with time on dec capsulation cells demonstrate progressive neuronal morphology after two to three days of culture with greater than 90%viability. Gene expression analysis indicates a downregulation of pluripotent marker OCT four.
Importantly, the neuro progenitor marker PAC six and the dopaminergic neuronal marker th remain upregulated after seven days of differentiation. Further differentiated human embryonic stem cells indicate greater than 90%TH positive neurons after 21 days. Western loss analysis confirms the favorable expression patterns of TH and PAC six under 3D versus 2D cell culture systems.
Once mastered, this technique can be effectively performed in two hours. Remember to treat the cells with rock inhibitor before and after encapsulation. Variant inducers or inhibitors can tailor the 3D differentiation protocol for future experiments.
This particular technique of encapsulating human ESL and differentiating them under a 3D environment or for potential for cell therapy for number of diseases, particularly Parkinson's disease.
