Preparing Ionically Crosslinked Alginate Hydrogels

Mix 40% calcium sulfate slurry with 60% serum-free DMEM/F12 media to create a uniform working stock solution. Calcium sulfate is used because its low solubility allows slower, more uniform crosslinking compared to rapidly-diffusible alternatives like calcium chloride.

Draw 600 microliters of low molecular weight alginate into syringe one using a syringe, removing air bubbles by tapping against the syringe tip. Add appropriate amount of DMEM to the syringe and mix thoroughly 30-50 times by drawing the plunger up and down to ensure uniform distribution.

Draw the required amount of DMEM/F12 into syringe two, then quickly add calcium sulfate stock solution after vigorously shaking the falcon tube to ensure uniform precipitation. Mix rapidly in the syringe to prevent settling before proceeding.

Attach syringe two to the luer lock, attach syringe one to the luer lock connection, then mix by pushing the plungers together 6 times. Quickly deposit the mixed alginate onto a culture plate and close the plate to allow gelation.

Draw 600 microliters of high molecular weight alginate directly into a syringe using the syringe plunger (due to higher viscosity). Remove air bubbles and add the required amount of DMEM, then mix thoroughly 30-50 times until uniform.

Draw DMEM/F12 into syringe two, vigorously shake the calcium sulfate stock solution falcon tube to resuspend precipitate, then quickly add the required amount of calcium stock to syringe two. Mix to ensure uniform distribution before proceeding.

Attach both syringes to the luer lock and mix by pushing plungers together 6 times. Quickly deposit the mixed alginate onto a culture plate and close the plate to allow 20 minutes for gelation.

After 20 minutes of gelation, carefully remove the top plate and use a 6 mm punch to cut uniform disc-shaped samples from the solidified gel. Separate the discs from the gel using a spatula and collect them for downstream assays.