A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Thiolation of Hyaluronic Acid

NOTE: Molar ratios are stated with respect to a total number of carboxylate groups unless otherwise specified.

1. Dissolve 500 mg of sodium hyaluronate (HA, 500-750 kDa) at 10 mg/mL in deionized, distilled water (DiH₂O) in an autoclave sterilized 250 mL Erlenmeyer flask. Stir the solution (~200 rpm) at room temperature for 2 hours to fully dissolve HA. Use a stir bar and magnetic stir plate to keep the reaction stirring during the thiolation procedure.
2. Using 0.1 M hydrochloric acid (HCl), adjust the pH of the HA solution to 5.5. Weigh out 69.6 mg (0.25x molar ratio) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
   NOTE: Although undissolved EDC can be added directly to the HA solution, it is typically easier to dissolve EDC first in 1 mL of DiH₂O and then quickly add the EDC solution to the HA solution. Pre-dissolution in 1 mL of DiH₂O can also be done with N-hydroxysuccinimide (NHS) and cystamine dihydrocholoride before adding to the reaction in steps 1.3 and 1.4.
3. Add 17.9 mg (0.125x molar ratio) of NHS to the reaction. Adjust the pH of the reaction solution to 5.5 using 0.1 M HCl and incubate the reaction at room temperature for 45 minutes.
4. Add 70.0 mg (0.25x molar ratio) of cystamine dihydrochloride into the reaction solution. Use 0.1 M sodium hydroxide (NaOH) to adjust pH to 6.25. Incubate the reaction at room temperature overnight while stirring.
5. On the following day, use 1 M NaOH to adjust the pH of the reaction solution to around 8. Add 192 mg (4x molar ratio) of dithiothreitol (DTT) to the reaction. Adjust the pH back to 8 after the addition of DTT and leave stirring for 1-2 hours at room temperature.
6. Use 1 M HCl to adjust pH to 4. Transfer the entire reaction mixture into a pre-soaked dialysis membrane (molecular weight cut-off around 13 kDa) and dialyze against DiH₂O pre-adjusted to pH 4.
   NOTE: The volume of DiH₂O for dialysis should be at least 40 times the volume of the reacted HA solution (e.g., 50 mL sample in 2 L of DiH₂O, pH 4).
7. Replace the dialysis solution (DiH₂O, pH 4) twice daily for 3 days at room temperature.
8. Filter the dialyzed solution through a 0.22 µm membrane, vacuum-driven filter. Flash freeze solution of thiolated HA in liquid nitrogen. Use a lyophilizer to freeze dry samples over 2 days. Thiolated HA in dry form can be stored in a vacuum-sealed desiccator at -20 °C for at least 6 months.
9. To determine the degree of thiolation, use Ellman's test and/or proton NMR spectroscopy.
    

2. Preparation of Crosslinking Materials

1. Dissolve thiolated HA at the desired concentration (in this example, 13.3 mg/mL) in HEPES-buffered saline (20 mM HEPES in Hank's balanced salt saline (HBSS) buffer, adjusted to fall within a pH of 8-9) in an amber vial to minimize exposure to ambient light. Keep the solution stirring constantly.
   NOTE: Formation of disulfide bonds between thiols conjugated to HA may occur if the pH is above 8, the solution concentration is too high, or the solution is left stirring for too long before gelation. To avoid this issue, use below 15 mg/mL of thiolated HA and dissolve thiolated HA within 2 hours of forming hydrogels.
2. Dissolve 4-arm-PEG-maleimide (PEG-Mal, 20 kDa) and 4-arm-PEG-thiol (PEG-thiol, 20 kDa) in phosphate buffered saline (PBS), pH 7.4, to prepare 50 mg/mL PEG-Mal and PEG-thiol stock solutions.
   NOTE: It takes at least 1 hour to fully dissolve PEG reagents.
3. If adding ECM-derived peptides, dissolve cysteine-containing peptides in PBS to prepare a stock solution.
   NOTE: Use a stock concentration of 2-4 mM.
4. Soak silicone rubber molds in ethanol for at least 20 min to clean them and then autoclave them for sterilization.

3. Hydrogel Crosslinking and Cell Encapsulation

NOTE: As an example here, the encapsulation of four individual, 80 µL hydrogels with 0.5% (w/v) HA and compressive modulus of 1 kPa is described3. Please see Table 1, for example, recipes that yield hydrogels with varying properties: two hydrogels incorporating the integrin-binding peptide RGD and two hydrogels incorporating cysteine caps as a negative control for peptide activity. The seeding concentration of patient-derived GBM cells is 500,000 cells/mL.

1. Dilute the PEG-Mal stock solution (50 mg/mL, from step 2.2) to 12.5 mg/mL by adding 40 µL of the stock solution to 120 µL PBS. Split the diluted solution into two 1.5 mL microcentrifuge tubes, each with 80 µL.
2. Add 16 µL of stock RGD solution (2.8 mM, from step 2.3) to one tube and 16 µL of stock cysteine solution (2.8 mM, from step 2.3) to the second tube. Vortex to mix. Place the PEG-Mal-RGD or PEG-Mal-CYS solutions on ice until use in step 3.9.
   NOTE: The procedure as described here yields hydrogels with ~140 µM of peptide. This is equivalent to approximately 1 out of every 8 available PEG arms being occupied with a peptide. This can be varied by altering the molar ratio of cysteine-terminated peptides to available maleimide groups. In general, a maximum concentration of 280 µM of peptide can be achieved while still leaving sufficient numbers of maleimide groups available for hydrogel crosslinking.
3. Dilute the PEG-thiol stock solution (50 mg/mL, from step 2.2) to 5 mg/mL by mixing 4 µL of 50 mg/mL stock solution with 36 µL of PBS. Add 120 µL of dissolved, thiolated HA from step 2.1 to the mixture.
   NOTE: Solutions of HMW HA are very viscous. Thus, we recommend using a wide-orifice micropipette tip to transfer solutions. Pipette slowly to avoid the solution sticking to the walls of the micropipette tip and to improve measurement accuracy.
4. Place the clean, dry molds (prepared in step 2.4) into each well of a non-tissue culture-treated 12-well plate. Using the clean, blunt end of a pipette tip, press the gel mold and double-check the sealing between the molds and the bottom of the well plate.
   NOTE: Check the seal again right before encapsulation to prevent leakage.
5. Passage cultured GBM cells, dissociate to single cells and determine cell concentration as previously described3.
   NOTE: Some GBM lines cannot be dissociated into single cells. In this case, whole gliomaspheres can be encapsulated. However, prepare dissociated single cells after precise cell counting to improve reproducibility. The protocol for gliomasphere passaging varies among different labs and many of these are likely compatible with hydrogel encapsulation.
   1. (Recommended) Centrifuge gliomaspheres at 500 x g for 5 min. Remove the supernatant and add 1 mL of cell dissociation enzyme to the cell pellet. Then, incubate for 5 min, gently tapping the tube to agitate.
   2. Add 4 mL of complete culture medium (50 ng/mL EGF, 20 ng/mL FGF-2, 25 µg/mL heparin, G21 supplement, and 1% penicillin/streptomycin in DMEM/F12) to the cells.
   3. Centrifuge again at 500 x g for 5 min and remove the supernatant. Finally, resuspend the cell pellet in 1 mL of the complete medium and pass suspended cells through a 70 µm cell strainer. (Recommended) wash the strainer with an additional 4 mL of the complete medium to maximize the number of cells recovered.
6. Estimate the concentration of cells in the suspension using a hemocytometer. Split the cell suspension into 2 centrifuge tubes, where each tube contains ~80,000 cells. Centrifuge at 500 x g for 5 min; generally, 80,000 cells will make up 2 80 µL hydrogels.
7. Remove the supernatant and resuspend one pellet in 80 µL of PEG-MAL-RGD solution and a second pellet in 80 µL of PEG-MAL-CYS solution (prepared in step 3.1).
8. Using a 200 µL, wide-orifice micropipette tip, dispense 40 µL of HA solution (from step 3.3 above) into each rubber silicone mold (as prepared in step 3.4).
9. Using a 200 µL, wide-orifice micropipette tip, mix the 40 µL of PEG-MAL-CYS or PEG-MAL-RGD cell solution with the HA solution in the mold. Pipette up and down quickly no more than 10 times. Repeat for each gel culture being prepared.
   NOTE: This step takes practice, since initial gelation occurs quickly (within 30 s). Pipette up and down while moving the tip to different locations in molds to ensure even mixing. Always keep the tip below liquid level to avoid formation of air bubbles. Do not mix the solution too many times as gel may get formed inside the micropipette tip. PEG-MAL-CYS and PEG-MAL-RGD can be combined at varying ratios to achieve the desired RGD peptide concentration.
10. Place the well-plate containing gel-encapsulated cells into a 37 °C cell culture incubator for 5-10 minutes to ensure completion of the reaction.
11. Add 2-2.5 mL of culture medium to each well with formed gels. Use a sterile, 2 µL pipette tip or micro-spatula, to gently separate the mold and gel. Use pre-sterilized forceps to remove the mold from the well plate. Place the well plate back to cell incubator (37 °C and 5%CO₂) for culture and future experiments.
    NOTE: Pull the mold vertically upward to avoid harming the gel cultures. In general, medium in gel cultures should be replaced every 3-4 days. Take care not to aspirate hydrogels when removing the medium. If this is an issue, we recommend using a plastic transfer pipette. If bioluminescence imaging to track cell number is planned, GBM cells must be transduced with lentivirus encoding constitutively luciferase expression before encapsulation, as previously described3. For bioluminescence imaging, add 1 mM of D-luciferin to the culture medium 1 h prior to luminescence imaging.