The area is considered of an interest in 3D cultures of human pluripotent stem cells due to improved pluripotency and differentiation potential this culture formed. We described our encapsulation strategy that leads to reproducible formation of human pluripotent stem cells spheroids. Once encapsulated, these stem cell spheroids are amenable to cultivation, either in multi-well plates or stirred bioreactors.
Our encapsulation strategy is optimize the pestilence survivability and high efficiency of spheroid formation. Encapsulated spheroids are protected against shear or mechanical energy associated with stirring or rocking. Development of stem cells derived the cellular therapy depends in part on the ability to generate the desired cellular product in a scalable, efficient and cost-effective manner.
The encapsulation technology described here represents a step in this direction. The encapsulation technology described here will be probably applicable to 3D cultivation of various cell types from other cells to other pluripotent stem cells. Begin by preparing 30 milliliters of two times core stock solution by mixing 4.8 grams of 35 kilodalton PEG, 10.2 milliliters of density gradient media, and 19.8 milliliters of DMEM/F-12 media.
Then filter this core solution using a 0.22 micrometer syringe filter. Next, prepare 50 milliliters of mineral oil with 3 percent surfactant by mixing 48.5 milliliters of mineral oil and 1.5 milliliters of surfactant. Then sterilize the solution by passing it through a 0.22 micrometer syringe filter.
Next, prepare 1 molar DTT, 1 molar TEA, and 1 percent PF127 solutions, as described in the text manuscript. Then prepare a cross-linker emulsion by mixing 4.5 milliliters of mineral oil with 3 percent surfactant and 300 microliters of 1 molar DTT. After vortexing the solution for two minutes, sonicate for 45 to 60 minutes in an ultrasonic bath at 20 degrees Celsius and load it to a 5 milliliter syringe with a 27 gauge needle.
Next, prepare shielding oil solution by loading the mineral oil with 0.5 percent surfactant into a 5 milliliter syringe with a 27 gauge needle. To prepare the shell solution, add 32 milligrams of PEG4 maleimide in 400 microliters of DPBS. Then, add 6 microliters of 1 molar TEA.
Vortex the solution for 2 minutes and centrifuge it through a spin filter. Keep the solution in the dark on the rocker for 30 minutes before loading into a 1 milliliter syringe with a 27 gauge needle. Finally, prepare the final core solution by re-suspending 20 million hPSCs in 200 microliters media and mix with 30 microliters of 1 percent PF127 and 200 microliters of 2X core solution.
Insert a small stirrer bar into a 1 milliliter syringe. Then, load the cell containing core solution into the syringe with a 27 gauge needle. Begin by placing the bonded PDMS droplet device, 4 pieces of 20 centimeter long, and 1 piece of 5 centimeter long inlet microtubes, 1 piece of 10 centimeter outlet tube and 6 pieces of 0.5 centimeter fitting tubes beneath the germicidal light source for 30 minutes for sterilization.
Now fit the 4 pieces of 20 centimeter long inlet microtubes into the shell, oil, cross-linker emulsion, and dissociation device inlets using forceps. Then, insert the opposite end of the tubes into the syringe needle with the corresponding solution. Meanwhile, connect the microfluidic devices core inlet and the dissociation devices outlet with a 5 centimeter microtube.
Insert 1 outlet tube into the fluidic outlet port and place the opposite end into a collection reservoir. Next, place the device on a microscope stage and attach the tubing to avoid any movement. Align and focus the microscope on the device nozzle for flow inspection.
Place each syringe on different syringe pumps and secure properly. Program each pump according to the manufacturer's protocols to the flow rates mentioned in the text manuscript, and start the flow in all the pumps. Wait for each fluid to enter the device and fill up the channels while pushing out the remaining air.
When the capsule formation is stabilized, place the opposite end of the collection tube into a new conical tube containing 5 milliliters of mTeSR media. Once enough capsules are collected, incubate them for 10 minutes. The capsules residing in the oil phase will be at the top of the tube, which sediments to the bottom upon gently tapping the tube.
To retrieve the capsules, start by removing oil from the collection tube. Then, place the capsules on a 100 micrometer pore size cell strainer and wash the strainer with copious amounts of media. Invert the filter on top of a well of a 6-well culture plate and collect the microcapsules by passing 2 milliliters of media through the filter.
Incubate the stem cell capsules in a 6-well culture dish at 37 degrees Celsius with 5 percent carbon dioxide. Change the media every other day by collecting the capsules on a cell strainer filter, washing them with excess media and re-suspending them to a new well in 6-well plates with 2 milliliter media. Perform viability assay by adding 4 microliters of the 2 micromolar Ethidium Homodimer-1 and 1 microliter of the 4 micromolar Calcein-AM solutions to 2 milliliters of sterile DPBS.
Vortex to ensure complete mixing. Collect approximately 100 microcapsules on a cell strainer and rinse them with sterile DPBS to allow medium diffusion out from the capsules. Collect them again in a 12-well culture plate using 1 milliliter of the prepared solution containing Ethidium Homodimer-1 and calcein AM and incubate at 37 degrees Celsius for 20 minutes.
Visualize the cells under a fluorescence microscope. The optimal and suboptimal microcapsules fabricated using microfluidic droplet generation showed differences in capsule morphology as a function of PEG4-Maleimide content in the shell. Smooth capsules with bright edges are associated with desired mechanical integrity.
Aggregation of beads in the center of the capsules indicates that the core was aqueous and the beads were free to move about. The fluorescent annulus indicated the presence of a hydrogel shell and was used to determine the shell thickness. Live or dead stainings 6 hours after encapsulating H9 cells showed that the cells achieved more than 95 percent viability routinely during the encapsulation runs.
Upon comparing the growth rate and differentiation potential of encapsulated versus unencapsulated spheroids. It was determined that the process of encapsulation has no adverse effects on hPSCs. Please remember, it is important to establish optimal flow rate ratio between the core shell and oil streams.
Not doing this may result in capsules that will not be mechanically strong and may rupture during handling. We would like to point out that this encapsulation technique was for cell types other than human pluripotent stem cells. We have encapsulated, primarily hypothesized, cancer cell lines, and mesenchymal stem cells using a similar strategy.
Currently, our team is developing the next generation of microcaptures. These microcaptures are bioactive and may be loaded with cross factors for encapsulated differentiation of stem cells.
