This method enables researchers to systematically probe how matrix cues affect the phenotype of cells in 3D culture. We demonstrate the procedure using cultures of GBM cells. The main advantage of this technique is high-throughput implementation, which allows us to screen many matrix conditions.
This technique can be used to develop tissue-mimetic matrices that preserve specific functions in cell culture models, like drug resistance of tumor cells, and potentially identify new therapeutics. Demonstrating the procedure will be Mary Epperson and Kelly Tamura, undergraduate researchers from the Seidlits Lab. Start with preparing HEPES-buffered solution by dissolving HEPES powder at 20-millimolar in Hanks balanced salt solution.
Adjust pH to seven following full sorbation. In the HEPES-buffered solution, dissolve thiolated HA so that 6 to 8%of carboxylic acid residues on each glucuronic acid are modified with a thiol at a concentration of 10 milligrams per milliliter in buffer solution. Stir at less than 1, 000 rotations per minute using a magnetic stir plate at room temperature until fully dissolved, typically around 45 minutes.
While HA is dissolving, prepare separate solutions of 100 milligrams per milliliter of 8arm PEG norbornene, 100 milligrams per milliliter of 4arm PEG thiol, four-millimolar of cysteine or cysteine-containing peptide, and four milligrams per milliliter of LAP in microcentrifuge tubes. Prepare four-millimolar solutions of all peptides to be tethered within a single hydrogel at this point. Mix the individual solutions of HA, PEG norbornene, PEG thiol, and cysteine-thiol-containing peptides to achieve the final concentrations for the final hydrogel matrices.
Stir at less than 1, 000 rotations per minute on a magnetic stair plate for at least 30 minutes to mix fully. Align the samples with the illumination device with every other LED in a single column of the silicone molds or 384-well plate. Open UV LED Parameters and enter values for intensity and illumination.
Then click on Finish to begin illumination. Following illumination, when placed in one corner, move the well plate to the next corner and repeat. To illuminate wells on the other half of the plate, lift the plate out of the holder and rotate 180 degrees.
To generate hydrogels with varying mechanics, start with cleaning the glass slides and silicone molds using tape to remove debris. Adhere the silicone molds to the glass slide, press down to ensure a good seal, and displace any air bubbles. Next, pipette 80 microliters of hydrogel precursor solution into each silicone mold on the glass slide.
Place the glass slide onto the illumination device aligned with every other LED in a single column. Expose the hydrogel precursors to UV light for 15 seconds to photo cross-link. Once illumination has stopped, retrieve the slides and loosen the gels from the molds by tracing the inner circumference of the mold with a fine tip.
Remove silicone molds with tweezers or forceps. Fill a 12-well plate with two milliliters of DPBS. Move cross-linked hydrogels into individual well plate by wetting a spatula and gently pushing them off the glass slide.
Prepare desired cells as a single-cell solution. Collect glioblastoma spheroids, roughly 150 micrometers in diameter, from a T-75 flask suspension culture into a 15-milliliter conical tube. Rinse the culture flask with five milliliters of DPBS to remove any residual cells and media and add this volume to conical tube.
Centrifuge the conical tube containing cells at 200 times G for five minutes at room temperature. Following centrifugation, remove the supernatant with a five milliliter serological pipette, taking care not to disturb the cell pellet and resuspend in five milliliters of DPBS. Centrifuge at 200 times G for five minutes at room temperature to wash cells.
Aspirate the supernatant with a five-milliliter serological pipette, taking care not to disturb the cell pellet, and then resuspend cells in two milliliters of cell dissociation reagent. Incubate at room temperature for 10 to 15 minutes. Add three milliliters of complete medium and gently pipette three to five times to break down the spheroids to a single-cell suspension.
Centrifuge the single-cell suspension at 400 times G for five minutes to pellet cells at room temperature. Aspirate the supernatant with a five-milliliter serological pipette, taking care not to disturb the cell pellet. Resuspend cells in one milliliter of complete medium.
Remove a portion of the cells for counting using a hemocytometer. Dilute this portion twofold with trypan blue, which permeates cells with compromised viability. Count only the live, colorless cells.
Determine the number of cells necessary for encapsulation. Transfer a volume of media containing the total number of cells needed into a sterile 1.7-milliliter microcentrifuge tube. Spin down at 400 times G for five minutes at room temperature.
Aspirate the supernatant with a micropipette, taking care not to disturb the cell pellet. Resuspend the cell pellet in the hydrogel precursor solution, mixing well by pipetting up and down with a 1, 000 microliter micropipette four to five times. Load the cells into a repeat pipetter set to dispense 10 microliters.
To avoid bubbles and uneven dispensing, prime the repeat pipetter by dispensing an additional one to two times into a waste container. In each well of a 384-well plate, dispense 10 microliters of cells suspended in hydrogel solution from the repeat pipetter. Using the LED array, illuminate each well containing cell for 15 seconds to achieve the desired mechanical properties.
Add 40 microliters of complete media to each well containing the cells. Add 50 microliters of DPBS to non-experimental dry wells surrounding the gels to minimize losses due to evaporation. For glioblastoma cells, add 40 microliters of the media-containing drug to achieve the final desired concentration, starting three days after encapsulation.
Add 10 microliters of CCK8 reagent to each well containing the cells. Incubate for one to four hours, according to the manufacturer's instructions. Read absorbances at 450 nanometers for all wells following incubation.
AFM interrogation of micron-scale regions at the surfaces of single hydrogels showed that hydrogels with softer average Young's moduli also had smaller ranges of moduli than stiffer hydrogels. 3D cultures of GS122 cells seeded at densities of 2, 500, 000 cells per milliliter exhibited substantially higher viabilities when assessed after seven days in culture compared to those seeded at densities of 500, 000 cells per milliliter. GS122 cells showed survival gains in the eight kilopascal condition when osteopontin-derived peptides were included in the matrix, while the incorporation of integrin-binding sialoprotein or tenascin C-derived peptides provided minimal survival benefits, such as culture and matrices with the ginger RGD peptide.
In contrast, no peptides conferred survival gains in the 0.8 kilopascal culture condition. Both GS122 and GS304 cells spread when cultured in soft or stiff hydrogel matrices, including RGD-containing peptides. GS122 cells show a similar lack of spreading in both 0.8 and 8 kilopascals with osteopontin.
However, GS122 only showed enhanced resistance to temozolomide in the eight kilopascals condition. Finally, this miniaturized 3D culture platform can be used to culture human cells from other tumor types, including viable organoids of terminally differentiated neuroendocrine prostate cancer cells. Following this procedure, further techniques such as single-cell RNA sequencing can be used to further characterize cell phenotypes.
