Programmable syringe pump

The esophageal adenocarcinoma cell line, JH-EsoAd157 (link), a kind gift from Dr. James R. Eshleman (The Johns Hopkins University, Baltimore, Maryland, USA), which was previously characterized by Dr.Eshleman, was maintained in complete culture medium containing high-glucose Dulbecco’s Modified Eagle Medium (DMEM with 4 g/L glucose, 4.0 mM L-Glutamine) (Gibco by Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated FBS (Gibco Life Technologies, Grand Island, NY, USA), and 1% Penicillin-Streptomycin (Gibco by Life Technologies, Grand Island, NY, USA) at 37 °C with 5% CO₂ in microfluidic chips. In the static culture group, the media was changed manually two times per day to minimize the potential effects of hypoxia. In the flow group, adherent cells were continuously supplied with fresh medium using a programmable syringe pump (NewEra Pump Systems Inc., NY) at a flow rate of 2 μl/min. The aldehyde dehydrogenase (ALDH), CD44, CD24 measurements and epithelial to mesenchymal marker stainings were performed on days 1, 3, 5 and 7 to determine the phenotypic plasticity under flow and static conditions.

Parallel plate flow chambers were designed in house as described previously^{3 (link)}. Briefly, MSCs were seeded on fibronectin (10 µg/ml) coated glass slides, assembled between two plates and attached to a programmable syringe pump (New Era Pump Systems Inc. Farmingdale, NY, USA, http://www.syringepump.com). Oscillating fluid shear (OFS) was applied through a 10 ml syringe (Becton Dickinson and Company, Franklin Lakes, NJ, USA, www.bd.com) at 52.5 ml/min and at a frequency of 1 Hz subjecting cells to a shear stress of 1 Pa. The MSCs were lysed for mRNA extraction or fixed for immunocytochemical staining immediately after the application of fluid shear for 2 hours. The no flow controls were similarly assembled within the chambers but were not subjected to fluid shear.

The
μ-slide pumping system was used to investigate the enhanced
adhesion property of PSH with mucin. 70,000 IEC-6 cells were seeded
into the μ-Slide I 0.4 Luer (ibidi) and incubated for 24 h to
generate sufficient mucin. Then, the cells were stained with Hoechst
for 30 min to visualize the nucleus and washed three times with 100
μL of DMEM to remove excess stain. Then, 100 μL of the
1% candidate material (adjusted to pH 7) encapsulated with FITC-albumin
(10% of material dry weight) was applied and incubated with mucin
for 30 min (equivalent to 1 mg of candidate material containing 0.1
mg of FITC-albumin). The μ-slide was mounted on a programmable
syringe pump (New Era Pump Systems) to stimulate shear force during
intestinal peristalsis. Cells were under a constant flow of DMEM at
a rate of 150 μL min^–1, which produced a shear
stress of 1.8 μN cm^–2. The materials were
observed with a fluorescence microscope at the time points of 0, 30,
and 60 min. The remaining fluorescence over time was quantified using
ImageJ and normalized to the initial fluorescence of pectin and PSH
(100%).