title: Investigating the impact of the interstitial fluid flow and hypoxia interface on cancer biology using a spheroid-on-chip perfusion system
design: Investigating the impact of the interstitial fluid flow and hypoxia interface on cancer biology using a spheroid-on-chip perfusion system
library_construction_protocol: This submission has 12 samples in 4 conditions (n=3). There are two microfluidics conditions: Static or Flow. These are each run at two conditions: Hypoxic and Normoxic.  Microfluidic device design and fabrication.  The device used in this study is a refined design of our previously published design (PMID: 34504636). The updated design includes an inlet channel, a spheroid microwell with a removable polydimethylsiloxane (PDMS) plug seal, and an outlet channel (design details in Figure 1A). The microwell is 5 mm and 6 mm diameter for the lower chamber and top inlet hole respectively, which creates a step to prevent the PDMS from being pushed in too far into the well. Where channels interfaced with the central well, a teardrop shape was used for the junction. A 1 mm circular recess at the bottom of the well enabled a microscope cover slip to be positioned for spheroid imaging. Devices were fabricated by milling two glass slides (Schott B270) using a Computer Numerical Controlled (CNC) machine (M7 Datron, Germany) with a diamond milling tool of 1 mm diameter (Eternal Tools, UK). These were thermally bonded, and a borosilicate glass coverslip was subsequently bonded to the bottom of the device for the viewing port.  Microfluidic spheroid-on-chip setup. Spheroids were generated from the MCF7 (pleural effusion of metastatic breast adenocarcinoma) cell line by seeding 9x10E4 cells per well in ultra-low adherence (ULA) plates. Cells were purchased from authenticated source European Collection of Authenticated Cell Cultures (ECACC), and regularly tested negative for mycoplasma. A spheroid was transferred to the chip microwell in 50 µL of media. Chilled pipette tips were used to add 50 µL of Matrigel and it was allowed to set before the chamber was sealed with a PDMS plug. The device was attached to a syringe pump (Harvard Apparatus). A plastic syringe (BD, Oxford, UK) filled with complete Dulbecco's Modified Eagle Medium (DMEM) was secured in the syringe pump and attached to Tygon tubing (Cole-Parmer, Tygon Microbore), 1.6 mm inner diameter with a 200 µL pipette tip, which had been cut to fit securely over the syringe nozzle. At the outlet end of the chip, Tygon tubing was used as before, leading to a collection pot for effluent media. Flow rate was set at 3 μL min−1, and velocities were 1.49 mm min-1 (tubing), 3.3 mm min-1 (channels), and 0.2-0.6 mm min-1 (microwell, as specified later).  Hypoxic experiments. A H35 Hypoxistation hypoxia chamber (Don Whitley Scientific, UK) was used for achieving hypoxic conditions of 1% O2. All equipment, media, and consumables were equilibrated overnight prior to experiments. Spheroids were collected from three biological replicates of either static (ULA plate) or flow (perfusion chip) conditions for RNA extraction as previously described (PMID: 34504636). Aurum Total RNA mini Kit (Biorad, UK)  Novogene NGS RNA Library Prep Set (PT042)* (a non-stranded protocol, using poly A enrichment).