Towards a microfluidic H295R steroidogenesis assay-biocompatibility study and steroid detection on a thiol-ene-based...

Area of Science:

• Endocrinology
• Toxicology
• Biomedical Engineering

Background:

• Endocrine disruption from environmental chemicals and pharmaceuticals is a significant global health concern.
• Traditional well-plate assays for steroidogenesis are limited in automation and throughput.
• Microfluidic cell-based assays offer potential for enhanced toxicity testing.

Purpose of the Study:

• To miniaturize the H295R steroidogenesis assay into a microfluidic format.
• To evaluate PDMS and thiol-ene as microfluidic chip materials for H295R cell culture.
• To develop a microfluidic chip and cell seeding procedure for reliable H295R cell culture.

Main Methods:

• H295R cells were cultured on PDMS and thiol-ene microfluidic chips with and without collagen modification.
• Cell attachment, viability, and steroid synthesis were assessed.
• Spike-recovery experiments were conducted to evaluate steroid adsorption.
• Microfluidic chip design and cell seeding protocols were optimized.
• Perfusion culture was performed at various flow rates, with steroid detection in eluent.

Main Results:

• Thiol-ene chips supported H295R cell monolayer formation with comparable viability and steroid synthesis to polystyrene.
• PDMS chips resulted in cell aggregation and poor steroid recovery.
• A repeatable and uniform cell distribution was achieved in microfluidic channels using thiol-ene.
• Thirteen steroids were detected over 48 hours at a low flow rate (2.5 µL/min) on thiol-ene chips.

Conclusions:

• Thiol-ene is a suitable material for microfluidic H295R cell culture, outperforming PDMS.
• The developed microfluidic assay enables miniaturization and improved performance for steroidogenesis testing.
• This work lays the foundation for a time-resolved microfluidic H295R assay for endocrine disruption screening.