It allows delivery of analgesics, antibiotics, growth factors, growth media, and cells into the chamber, becoming a platform for tissue engineering.
Four different doses of compounds (50, 25, 12.5 and 6.25 µg ml−1) were further prepared by diluting the stocks in culture media, and cells were treated (in triplicate/dose).
The following day, the media was replaced with fresh media and cells were exposed with blank polymers, free DTX, DTX/PLGA NP, and DTX/PLGA-LHRH NP and incubated for 24 h.
Media and cells were collected and lipids were extracted.
After 48 h, conditioned media and cells were collected.
Radiolabeled palmitate (2 µCi/mL) from American Radiolabeled Chemicals was added to the media and cells.
The media and cells were harvested after another 24 h cell growth.
Trypsin was neutralized using fresh mESC media and cells were transferred to one well of a 24-well plate onto feeders and incubated as above.
Reactions were stopped by aspiration of media and cells were lysed in 50 µl of lysis buffer containing 1 mM 3-isobutyl-1-methylxanthine.
After 12 hrs of transfection, the serum-free media was replaced with serum-containing media, and cells were left in the incubator at 37°C for 48 hrs.
On the day of the assay, media was replaced with fresh media, and cells were allowed to equilibrate for an additional 1 h at 37°C.
