Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Our lab focuses on designing in vitro hydrogel models for glioblastoma spheroid encapsulation to mimic the tumor extracellular microenvironment. The effect of substrate mechanical and biochemical properties on spheroid cell viability, circularity, stemness, and spreading are investigated with such models. Current experimental challenges in tumor model development revolve around a desire for complexity to mimic all aspects of the tumor microenvironment for physiological relevance, and also a desire for simplicity to make the model accessible, robust, low cost, and easy to use, and compatible with multiplex screening approaches.

We present a protocol that enables fast, robust, and low-cost fabrication of tumor spheroids and encapsulation in hydrogels made to mimic the tumor microenvironment. The model does not require any specialized equipment. It will be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Understanding the interactions between glioblastoma cancer cells and their microenvironment can encourage development of new therapies to better treat this cancer. Furthermore, development of an in vitro model for glioblastoma cancer can enhance development of this model for other cancer diseases. To begin, poor two grams or about one milliliter of negative PDMS precursor solution onto one well of a six-well square pyramidal master mold.

After covering the master mold with PDMS, place the plate in a vacuum desiccator to degas the solution. Next, place the plate in an oven at 60 degrees Celsius for 24 hours to cure the PDMS. Once PDMS cures but is still warm, use a spatula to carefully remove the PDMS mold from the master mold.

Then, with a biopsy punch, cut it into a 35-millimeter diameter slab. Place the properly cut PDMS mold in a Petri dish and cover it with the lid to continue curing it at room temperature for an additional 24 hours. To prepare the positive PDMS mold, place a 35-millimeter slab of the negative PDMS mold in a 35-millimeter Petri dish with the textured microwells facing up.

Pour 2.5 grams or about 1.2 milliliters of positive PDMS precursor solution onto the negative mold in the Petri dish. Degas the precursor solution for 30 minutes before placing it into the oven at 60 degrees Celsius for three to four hours. After curing, remove the molds from the Petri dish.

Immediately peel the positive mold from the negative mold. Use a 10-millimeter biopsy punch to cut the positive molds into slabs. Next, use tweezers to gently dip the flat side of each 10-millimeter positive mold into the pre-prepared PDMS glue precursor solution.

Carefully place one mold per well in a well of a 48-well plate. Gently press each mold to glue it to the well bottom. Incubate the assembled plate at 60 degrees Celsius for 4 to 24 hours to cure the glue.

To begin, place the PDMS microwell molds, anti-adherence rinsing solution, and necessary labware in a tissue culture hood. To wash the PDMS microwell molds, use a 1, 000-microliter pipette to add 300 microliters of anti-adherence rinsing solution to each well. Then centrifuge the plate 1, 620g for three minutes.

Use a vacuum pump and a Pasteur pipette to aspirate the solution. Next, place 500 microliters of cell suspension at the desired concentration in the microwells and centrifuge the plate at 1, 620g for three minutes. Place the plate in a humidified incubator to allow spheroid formation.

To harvest the spheroids, using a 1, 000-microliter pipette, firmly add 500 microliters of complete medium into each well. Pipette the added medium up and down at each quadrant three to four times to dislodge the spheroids. Then use the pipette to gently aspirate the medium containing the spheroids into a microcentrifuge tube.

Transfer 50 microliters of the spheroid suspension in a microcentrifuge tube. Then, sequentially, add 4-arm PEG-acrylate and PEG-dithiol into it. Mix the resulting solution by pipetting it up and down about 10 times to yield 100 microliters of a 10%weight by volume PEG hydrogel precursor solution.

Pipette 20 microliters of the gel precursor solution in between two Parafilm-lined glass slides separated with one-millimeter silicon spacers and incubate the slides with gel precursor solution to allow for gelation. Once hydrogel gelation is complete, using a spatula, gently peel the gels off the separated glass slides. Place one gel per well into a 24-well plate, ensuring the surface containing the spheroids faces up.

Add 500 microliters of complete medium to each well to submerge the hydrogel completely. Place the multi-well plate in a humidified incubator to culture the cells. The described technique using microwell molds resulted in the formation of spheroids with spherical shapes and tightly controlled polydispersity.

For spheroids with 3, 300 cells per microwell, the average spheroid size was about 250 microns and circularity was greater than 0.8, considering a value of one as a perfect sphere. Spheroid diameters were dependent on the microwell size and the number of cells per microwell. To begin, aspirate the media from the wells where the hydrogel encapsulated cells are cultured in the 24-well plate.

Rinse the hydrogels by pipetting 500 microliters of PBS directly onto each well containing the hydrogels, and then gently aspirate the PBS. Using a 1, 000-microliter pipette, add 500 microliters of fixative solution per well to fix the spheroids in the 24-well plate. Allow the fixative to soak the gels for 30 minutes at room temperature, before pipetting out the fixative solution and discarding it in a designated waste container.

Next, rinse the hydrogels with PBS three times, as demonstrated previously. If not used immediately, store the hydrogels in 500 microliters of PBS per well at four degrees Celsius for up to one week. To stain the hydrogel encapsulated cells, first, prepare appropriately diluted primary antibodies for nestin and SOX2.

After aspirating the PBS from the wells, add 50 microliters of the diluted antibodies to each well. Incubate the cells with the antibodies for 24 hours to stain them completely. Then, using a 1, 000-microliter pipette, remove the staining solution and discard the waste appropriately.

After rinsing the hydrogels three times, store them in PBS at four degrees Celsius for up to two weeks prior to imaging, or image immediately. To clear the stained spheroid for improving imaging transparency, first, aspirate the PBS from each well. Then, sequentially, treat the spheroids with 500 microliters of 20%40%and 80%formamide per well for 90 minutes each.

Finally, incubate the hydrogels in 100%formamide for 24 hours prior to imaging. The spheroids were imaged at varying Z-stack depths, allowing for the cell viability assessment at each location within the Z-stack. A maximum projection utilizing the spheroid stack represented the highest point of light intensity within each location simplified into one image.

Representative images of stained spheroids revealed that the self-renewal transcription factor SOX2 was co-localized with DAPI in the nucleus. On the contrary, stem cell marker nestin was present throughout the cells. There was no difference between the free-floating spheroids without gel and the encapsulated spheroids, possibly due to the inertness of the PEG gel used.

The representative images of optical sections at about 90 microns into cleared and uncleared spheroids revealed that when clearing was performed, the spheroid core could be imaged about 30 microns deeper compared to an uncleared spheroid.