I recently performed a tube formation assay and encountered some unexpected results. I initially seeded HUVECs on Geltrex, and approximately 2 hours later, I added either control or preconditioned adipose tissue-derived hMSCs (AT-hMSC). After 16 hours of incubation, I captured images of the wells expecting to observe tubular structures characteristic of angiogenesis.

However, instead of tubes, I mainly observed spheroid or aggregate-like structures, as shown in the attached image. I would greatly appreciate any insights into what might be causing this outcome. I obtained the same results when seeding both cell types at the same time on Geltrex.

My goal is to evaluate and compare the pro-angiogenic effects of control versus preconditioned AT-hMSC, but the current setup does not seem to support proper tubule formation.

Thank you in advance for your input!