b Design and Engineering of Open Source Hardware for Pressure Regulation in the Study of Vascular Processes Alexander Novokhodko 1 , Christian Mandrycky 1 , and Ying Zheng 1 ,2,3 1 Department of Bioengineering, 2 Center for Cardiovascular Biology, 3 Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA ZHENGLAB Goal : Controlling Pressure to study endothelial cell response in vitro Approach : o Syringe Pump based Control System o Ensuring Steady Flow o In vitro microvessels in flow chamber 3 Existing Problems: Oscillation at set point: • Current Percent Error: 2.95%, 2.97%, 4.547%. Imperfect Adaptation: Steady state deviation from set point • .0935%, 1%, 2.835% Target: Maximum Percent Error + Adaptation must not exceed 2% to match [6] • Currently, worst case error is 7.382%. Too high! Solution: PID Control (Proportional, Integral, Differential) • Problem: Motor step size is too large to implement a PID Duty Cycle • Solution: Gearing down the motor (see Figure 6) Unsteady flow: Flow is currently pulsatile/disrupted Figure 1: Forces on Endothelial Cells. [1] Results The Biological Need: Cells Under Pressure Problem: High cost of commercially available hardware • $4,767 for Constant Pressure Syringe Pump from Harvard Apparatuses [6] Solution: Open Source Hardware Exchange of CAD files • OpenSCAD • My work is based on “Open-Source Syringe Pump Library” [7] published in PLOS ONE • Creative Commons Attribution License 3D Printing • Low cost (see Table 1) Rapid Prototyping • See Figures 5 and 6 Customizability Arduino Microcontroller • Arduino code and explanation of algorithm available upon request Why Open Source Hardware? Endothelial cell response to blood pressure: • Vasoconstriction, vasodilation • Veins vs. Arteries • Venous endothelial cells differ from arterial ones. [2] • Why? Pressure? Or different signals during development? • Can we use this to prevent saphenous vein graft failure during coronary bypass surgery? Vasculogenesis/Angiogenesis during development • Once the embryonic heart starts beating the vasculature remodels. [3] How? Steady flow: • Outside the largest arteries, flow is steady, not pulsatile • Except in the aorta, flow is laminar [4] • Pressure is approximately constant In vitro studies: • Study effect of pressure in isolation • Control pressure to study other variables Need: Pressure Control System that maintains steady laminar flow References 1. Davies, P. & Tripathi, S. Mechanical stress mechanisms and the cell. An endothelial paradigm. Circulation Research 72, 239-245 (1993). 2. dela Paz, N. & D’Amore, P. Arterial versus venous endothelial cells. Cell Tissue Res 335, 5-16 (2008). 3. Mechanisms of angiogenesis. Nature 386, 671-674 (1997). 4. Stein, P. & Sabbah, H. Turbulent blood flow in the ascending aorta of humans with normal and diseased aortic valves. Circulation Research 39, 58-65 (1976). 5. Klabunde, R. CV Physiology: Systemic Circulation. Cvphysiology.com (2016). at <http://www.cvphysiology.com/Blood%20Pressure/BP019.htm> 6. Standard PHD ULTRA™ CP Syringe Pump. Harvard Apparatuses (2016). at <http://www.harvardapparatus.com/pumps-liquid-handling/syringe- pumps/constant-pressure/standard-phd-ultra-trade-cp-syringe- pump.html> 7. Wijnen, B., Hunt, E. J., Anzalone, G. C., & Pearce, J. M. (2014, September 17). Open-Source Syringe Pump Library. PlosONE, 9(9), 1-8. doi:10.1371/journal.pone.0107216 8. Tiny Planetary Gears Set by aubenc. Thingiverse.com (2012). at <http://www.thingiverse.com/thing:23030> 9. Zheng, Y. et al. In vitro microvessels for the study of Figure 2: Pressure in different parts of the circulation [5] Control Systems Figure 3: Basic Schematic of Syringe Pump-Based Constant Pressure System Figure 4: Maintaining a constant water pressure across a flow chamber Future Applications: : To improve portability and usability Vasculogenesis/Angiogenesis: Prepare flow chambers with collagen seeded with endothelial cells • Apply constant pressure at inlet • Observe vasculogenesis in pressurized vs. control gel Veins vs. Arteries • Make flow chambers with channels, seeded with endothelial cells. • The vessels in “In vitro microvessels for the study of angiogenesis and thrombosis” [9] are a starting point • Apply pressures characteristic of veins and arteries (Figure 2) and observe differences in cell phenotype Component Cost Arduino Uno R3 (Atmega328 - assembled) $24.95 3D-Printed Components (PLA filament) <$4.00 NEMA-17 Stepper Motor $14.00 Omega Low Pressure Transducer $205.0 0 Polulu Adjustable Boost Regulator $11.95 Readily Available Mechanical Components (bolts, z-couplings, nuts, resistors, wires, etc.) <$10.0 0 Two-way normally closed solenoid pinch valve; 12 VDC, 1/32" ID x 3/32" OD tubing $62.00 Adafruit Motor/Stepper/Servo Shield for Arduino v2 Kit - v2.3 $19.95 Total 351.85 Table 1: Cost of pump components Figure 6: Geared down motor 3D printed prototype. Derived from [8] Figure 5: Clockwise from top left: 1: Circuit Diagram of Control System, 2: Valve on Outlet, 3: Flow validation via fluorescence microscopy 4: Pump during flow validation. Figure 7: In vitro microvascular networks are the specialty of the Zheng Lab [9] Acknowledgements: We acknowledge the support from the Zheng lab and NIH awards (1DP2DK102258 and UH2/UH3 TR000504)