Silicone tubing

BRECS were placed in a continuous recirculation circuit with 250 mL of supplemented media in a 500 mL sterile perfusion bottle. Custom tubing sets for BRECS consisted of PharMed BPT double-stop pump tubing (Cole-Parmer, Vernon Hills, IL) and silicone tubing (Cole-Parmer). A Masterflex peristaltic pump (Cole-Parmer) was used to maintain the BRECS at a constant perfusion of between 10 to 100 mL/min. Media changes on the BRECS were performed twice per week.

There are one flow channel and one control channel in the valve device. The inlet of the flow channel is connected to a bottle containing blue food coloring dye. The bottle is pressurized by a computer-controlled air pressure source (Elveflow OB1 MK3) to drive the dye into the flow channel. The fluid driving pressure is fixed at 0.2 psi. To observe the flow rate, the outlet of the flow channel is connected to the silicone tubing (0.79 mm I.D., Cole Parmer). As for the control channel part, the outlet is sealed with a plastic tube cap and the inlet is also connected to the air pressure source starting from 0 psi. The air pressure is increased in increments of 1 psi in the control channel until the valve closes such that movement of the front end of the fluid in the flow channel stops. Experiments were conducted in triplicate.

In a sterile hood (Class II type A/B3, Nuaire), a 30 mL BD syringe (BD Biosciences, Franklin Lakes, NJ) without a plunger was clamped vertically. The bottom half of a filter holder assembly (Millipore, Billerica, MA), was attached to 3 inches of silicone tubing (Cole Parmer, Vernon Hills, IL) and placed into a stopper on top of a 250 mL glass Erlenmeyer flask. The Erlenmeyer arm was then attached to a VacuGene Pump (GE Healthcare Piscataway, NJ) with a regulated pressure gauge (Ashcroft). A fabricated microfilter was washed in 10 mL PBS, placed onto the filter holder and centered 5 mm beneath the 30 mL syringe (ESI, Fig. 1A-C†). This “open” system allows for a consistent vacuum exertion, without pressure build up across the membrane.
Three flow rates, and subsequent negative pressures exerted, were determined using both PBS and whole human blood. One filter was placed into a whole Swinnex filter holder (Millipore) and set up on the system. Vacuum pump was adjusted to reach flow rates (subsequent pressures in mbar) of 1 mL min⁻¹ (10 mbar), 5 mL min⁻¹ (15.1 mbar), and 10 mL min⁻¹ (18.5 mbar). Pre-stained samples were prepared and were run in triplicate in 15 mL PBS, 7.5 mL PBS with 7.5 mL fixative, 7.5 mL whole blood with 7.5 mL PBS, and 7.5 mL Blood with 7.5 mL 1× fixative under the 3 flow rates described.

The clot attached to the suture was placed in a clean micropipette (Drummond Scientific Company, Broomall, PA), and inserted into a U-shaped sample holder composed of hollow luer lock connectors and silicone tubing (Cole Parmer, Vernon Hills, IL; outer diameter 0.125″). The sample holder was placed in an acrylic water tank with a microscope slide at the bottom allowing visualization of the clot diameter using an inverted microscope (Olympus, IX-71) with a charge-coupled device (CCD) camera (Retiga 2000R, QImaging, Surrey, BC, Canada). The CCD camera images were recorded at a rate of 6 images/minute. The average clot width (CW) was calculated, using a computer program written in Matlab 6.5 R13 (Mathworks, Inc., Natwick, MA). The positions of the two clot-plasma interfaces were determined via an edge-detection routine. The width of the clot at each coordinate along the height of the image (z) was calculated, averaged over all z values, corrected for suture width, and normalized to the average of the clot width during the first six frames. The fractional clot loss (FCL) at 30 minutes can be defined as:
$$\text{FCL}=1-{\text{CW}}_{30}/{\text{CW}}_0$$ where,

Polystyrene culture flasks with vented caps were used as control standard vessels (Corning). Three bags were herein used: polyolefin/EVA copolymer (Evolve, Origen), custom FEP (American Durafilm), and custom silicone rubber bags were fabricated in house from sheets (Rogers Corporation). Custom silicone bags were fitted with silicone tubing (Cole Parmer), and sealed with adhesive sealant designed for silicone (DAP). Culture flasks and Origen bags came pre-sterilized. FEP and silicone bags/tubing were sterilized through exposure to 0.5M NaOH for a minimum of one hour. NaOH sterilized vessels were subsequently flushed out three times with sterile PBS to remove all trace NaOH. Specific details on vessels can be found in Table 1.
Perfusion with silicone cell culture bags was accomplished through peristaltic pumping (Cole Parmer). Flow rate was set at 100mL/min and run for 1–2 minutes total.