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Development and Testing of a Microfluidic Device for Studying Resistance Artery Function

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Development and Testing of a Microfluidic Device for Studying Resistance Artery Function DEVELOPMENT AND TESTING OF A MICROFLUIDIC DEVICE FOR STUDYING RESISTANCE ARTERY FUNCTION by Andrei Vagaon A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto © Copyright by Andrei Vagaon (2010) DEVELOPMENT AND TESTING OF A MICROFLUIDIC DEVICE FOR STUDYING RESISTANCE ARTERY FUNCTION MSc thesis, 2010, Andrei Vagaon, Department of Physiology at the University of Toronto ABSTRACT Introduction: Hypertension is the number one risk factor for cardiovascular diseases. Total peripheral resistance (TPR) is strongly involved in blood pressure homeostasis. TPR is primarily determined by resistance arteries (RAs). Pathogenic factors which change RA structure are associated with cardiovascular disease. Despite this, methods employed in the study of RAs lack efficiency. Methods: A polymer microfluidic device (Artery‐on‐a‐Chip Device, AoC) made from polydimethylsiloxane (PDMS) was developed. RAs from CD1 mice were measured on the device. Their responses to phenylephrine (PE), acetylcholine (Ach), FURA‐2 imaging, and 24‐h culture were assessed. Results: Following several modifications, vessel function on the AoC device was successfully measured. Robust PE constriction and Ach‐induced vasodilation were observed. AoC arteries were viable after 24‐hour culture, and FURA‐2 was successfully imaged. Conclusions: The AoC device is a viable alternative to cannulation myography. The AoC can greatly increase the efficiency of RA studies, while also decreasing training time and difficulty. ii TABLE OF CONTENTS Chapter 1. INTRODUCTION ...................................................................................................... 1 1.a Clinical background ....................................................................................................... 1 1.b Factors involved in resistance artery (RA) tone regulation .......................................... 3 1.c Current techniques employed in RA research .............................................................. 4 1.d Difficulties in the study of RAs ...................................................................................... 6 1.e Microfluidic devices .................................................................................................... 10 Chapter 2. MATERIALS AND METHODS .................................................................................. 14 2.a Solutions and substances ............................................................................................ 14 2.b Vessel isolation and preparation ................................................................................ 15 2.c Vessel loading onto the AoC device ............................................................................ 15 2.d AoC device fabrication ................................................................................................ 16 2.e Vessel fixation onto the AoC device ........................................................................... 17 2.f Substance delivery to the AoC device ......................................................................... 18 2.g Diameter measurement and imaging of the arteries loaded onto the AoC ............... 18 2.h Statistics and diameter change measurements ......................................................... 19 Chapter 3. RESULTS ............................................................................................................... 21 3 Vessel loading ................................................................................................................ 21 3.a Vessel fixation ............................................................................................................ 21 3.a.1 Vetbond tissue adhesive – complete bonding ........................................ 22 3.a.2 Vetbond tissue adhesive – partial bonding ............................................. 24 3.a.3 Vessel fixation via negative hydrostatic pressure (NHP) ......................... 27 3.b The vessel organ bath, substance delivery and removal ............................................ 29 iii 3.b.1 Organ bath with flow over the vessel (design 1) ..................................... 29 3.b.2 Organ bath with flow over the vessel (design 2) ..................................... 31 3.b.3 Organ bath with flow on the vessel lateral surfaces, NHP fixation ......... 32 3.c The mixing cell ............................................................................................................. 34 3.d Separation of the perfusion and superfusion streams ............................................... 36 3.e Phenylephrine dose response curves (DRCs) on the AoC device ............................... 37 3.f Acetylcholine dose response curves on the AoC device ............................................. 40 3.g Cultured vessels on the AoC device ............................................................................ 41 3.h One‐sided PE constrictions ......................................................................................... 42 3.i FURA‐2 on‐chip measurements ................................................................................... 43 Chapter 4. DISCUSSION .......................................................................................................... 45 PDMS – AoC fabrication material ..................................................................................... 45 4.a Vessel fixation and pressurization .............................................................................. 46 4.b The organ bath ............................................................................................................ 50 4.c The on‐chip mixing cell ................................................................................................ 55 4.d Phenylephrine and Acetylcholine dose response curves ........................................... 56 4.e Cultured vessels on the AoC device ............................................................................ 59 4.f Novel experiments using the AoC device .................................................................... 60 Chapter 5. CONCLUSIONS ...................................................................................................... 62 Future directions ............................................................................................................... 67 5.a Basic science ............................................................................................... 67 5.b Clinical applications .................................................................................... 68 Chapter 6. REFERENCES ......................................................................................................... 69 iv LIST OF ABBREVIATIONS Ach acetylcholine AoC artery‐on‐a‐chip device CAM calmodulin cAMP cyclic adenosine monophosphate cGMP cyclic guanosine monophosphate CO cardiac output DRC dose response curves ECs endothelial cells EDHF endothelium‐derived hyperpolarizing factor LOC lab‐on‐a‐chip MLCK myosin light chain kinase NHP negative hydrostatic pressure NO nitric oxide NOS nitric oxide synthase PDMS polydimethylsiloxane PE phenylephrine PGH prostaglandin H PGI2 prostacyclin PKG protein kinase G PLC phospholipase C RAs resistance arteries SMCs smooth muscle cells TPR total peripheral resistance v LIST OF IMAGES AND FIGURES Image 1: Chip fabrication by soft lithography ...................................................................... 11 Image 2: Representative image of the AoC inspection area ................................................ 16 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Figure 1: The V1 chip design: VetbondTM tissue adhesive – complete bonding .................. 22 Figure 2: The V2 chip design: VetbondTM tissue adhesive – partial bonding ................. …… 24 Figure 3: The V2 chip design: test ........................................................................................ 26 Figure 4: The S1 chip design: Vessel fixation via Negative Hydrostatic Pressure ................ 27 Figure 5: The BM 1.0 chip design: Organ bath with flow over the vessel ........................... 29 Figure 6: The SY 1.1 chip design: Organ bath with flow over the vessel (design 2) ............ 31 Figure 7: The AoC 1.0 design: Organ bath with flow on vascular lateral sides and NHP .... 32 Figure 8: The on chip mixing cell used in AoC devices ........................................................ 34 Figure 9: Organ bath washout, and separation of the perfusion and superfusion ............. 36 Figure 10: AoC arteries: PE dose response curves ................................................................. 38 Figure 11: AoC arteries: Ach dose response curves .......................................................... 40 Figure 12: AoC arteries: 24‐h cultured vessel ........................................................................ 41 Figure 13: AoC arteries: One‐sided PE constriction curves .................................................... 42 Figure 14: FURA‐2 imaging experiments on the AoC ............................................................. 43 Figure 15: Flowchart of the AoC development process ......................................................... 65 Figure 16: Final version of the AoC device ............................................................................. 66 vi 1. Introduction 1.a
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