Hello everyone,

I’m currently encapsulating HepG2 cells within a double flow-focusing microfluidic chip to form HepG2 spheroids in GelMA hydrogel. Despite various optimizations, a live/dead viability assay consistently shows cell death post-encapsulation. I would greatly appreciate any insights or suggestions to improve viability.

Here are the current parameters I’m working with:

• GelMA Concentration: 6 wt% in the shell (P1) and 8 wt% in the core (A) at a HepG2 cell density of 1×10^7 cells/mL
• Photoinitiator (LAP): 0.3 wt% for both GelMA solutions. I’ve tested LAP concentrations of 0.2% (under-crosslinking at current GelMA concentrations) and 0.5% (resulting in cell death).
• UV Exposure: 20 mW/cm² intensity using an Omnicure S1500 (250-450 nm emission) for crosslinking within glass capillaries at the chip outlet. Lower intensities have resulted in incomplete crosslinking with this GelMA concentration.
• Exposure Time: 1 minute 30 seconds. (I’ve also tested 1 minute and 2 minutes with similar results).
• Hydrogel Collection and Washing: After crosslinking, I filter the hydrogel with a cell strainer to remove oil, allowing it to sit in air for around 30 minutes throughout collection before washing with PBS (4 times) and centrifuging. I then culture it in cell media.

Here are some specific questions:

Any advice or similar experiences would be invaluable as I work to troubleshoot these viability issues. Thank you in advance for your insights!