This issue might be because of the following reasons:
Using a non-treated tissue culture plate, which has a more hydrophobic surface. This reduces the attachment of hydrogel/cells on the surface of the well-plate. For better performance, we suggest using a treated tissue culture plate with VitroGel.
Adding the hydrogel as a dome instead of covering the whole bottom of the well plate might also cause this issue. We suggest gently tilting and swirling the well plate after adding the hydrogel to ensure the whole bottom of the well plate is covered by the gel.
Not waiting long enough before adding the additional medium on the top of the hydrogel. After transferring the hydrogel to the well plate, please wait 10-30 minutes for hydrogel stabilization before adding the top medium. Adding the medium before the hydrogel stabilizes would disrupt the structure of hydrogel. The lower the concentration of the hydrogel, the longer the waiting time is needed.
After the initial soft hydrogel formation, it is crucial to make sure the hydrogel is stable and attached to the bottom of the well plate before adding the cover medium. If the hydrogel is not stable, it might detach from the bottom of the well after adding the cover medium. During the hydrogel formation, the gel is soft; do not shake the plate or position the plate vertically. Keep the plate horizontally.