Perfusion Chamber Design for Cell Membrane Permeability Measurement in 3D Tissues

1. B A point of focus 3D specimen objective laser dichroic mirror pinholes detector detector Maddie Midgett, Danielle McClanahan, Ingemar Hudspeth School of Bioengineering, Oregon State University 1 A/B/C Water Outlet 2 A/B/C 4 A/B/C 3 A/B/C A B A B D C Top View Side View Water Outlet A B Water Inlet C C B Perfusion Chamber Design for Cell Membrane Permeability Measurement in 3D Tissues Introduction Methods Design Specifications Heat Exchanger Cryopreservation: Preserves cells and tissues at sub-zero temperatures so that they can later be restored to their initial biological state. Cell damage due to intercellular ice formation and osmotic pressure differences is minimized . Metrics and Goals: Energy Balance Assumptions: - Steady state - Constant source and sink - No radial profile - Thermal resistance of perfusion tubing is minimal Thermal range: [4 – 37 °C] Thermal stability: [±0.2°C] Allowable flow rates: [0– 200 mL/h] Changeable fluid: [dual inputs] Fluid lag time: [ ~0 sec] Tissue mobility: [0 mm/sec] Figure 2. Pseudo-islets aggregates of pancreatic MIN6 mouse insulinoma beta cells could be used to cure diabetes. Human cell cryopreservation would allow for cell storage from multiple donors until implantation. Experiment Design Tissue Viability Chamber Control Figure 1. Cell volume changes due to hyposmotic, hyperosmotic, and isosmotic solutions. • Confocal Microscopy: • Base and top plates are dimensioned to fit on the microscope stage. • Non-Fluorescent Materials: • A glass cover slip fits over a hole in the base plate, so that the fluorescent light will only hit the glass and tissue sample. • Perfusion Flow Rate Range: • Fluid is pushed into the chamber by an adjustable syringe pump. • Thermal Range: • Perfusion fluid is heated/cooled to temperature by a circulating water bath in the heat exchanger. • Minimize Dead Space: • Swagelok connectors allow controlled depth of the perfusion tubing in the chamber. • Perfusion Fluid Change: • A Y-connector is placed adjacent to the inlet Swagelok connector. • Thermal Stability: • 1 meter of perfusion tubing is wrapped in the temperature-controlled water bath. • Minimal Tissue Manipulation: • Rubber bands wrap around the top and base plates and allow for quick assembly. • Secured Tissue: • The tissue sample is stabilized with a mesh, which is secured by a rubber gasket. • Low Shear Rates: • The mesh reduces shear rate, while the depth of the perfusion tubing can be adjusted for further alterations. Given: TW = 4 °C, TPin = 23.4 °C , V=200 mL/h L= 0.77 m when TPout= TW = 4 °C Issue Statement: Cryopreservation of 3D tissue still remains a challenge. Cell membrane permeability is an important component in cryopreservation procedure optimization. Laminar flow through a pipe: Hypotonic Conditions Minimize Dead Space Hypertonic Conditions A B Project Statement: Design, fabricate, and assess a 3D tissue perfusion chamber to measure cell membrane permeability using fluorescent confocal microscopy. Figure 3. Permeability can be indirectly measured by the linear relationship between fluorescence intensity and relative cell volume change. Figure 4. Inverted confocal microscopy (A) Fluorescent specimen is illuminated with a point of light. (B) Emitted fluorescent light reaches detector. Figure 5. (A) Minimizing dead space immediately over the tissue is important to ensure that the tissue contacts the perfusion fluid. (B) Otherwise, the fluid concentration contacting the tissue is unknown. Results Conclusions Chamber Design Chamber Assessment • The preliminary design and chamber assessment suggest that this assembly has the potential to measure cell permeability of 3D tissues using fluorescent confocal microscopy.   • Chamber assembly fits with the confocal microscope and is leak proof during perfusion • Heat exchanger shell temperature stays constant due to a fast water flow rate and thorough mixing • Perfusion fluid reaches water bath temperature using 1 m of tubing • Samples aresufficiently stabilized with a 20 μm pore mesh that is secured quickly with rubber gaskets • Chamber clearance can be controlled with exit perfusion tubing height Heat Exchanger (HX) Performance: Chamber Clearance: A Figure 7. The temperature of the perfusion fluid exiting the chamber was monitored with thermocouples during perfusion flow rates of 50, 100, 150, and 200 mL/h and HX shell temperatures of (A) 37, (B) 30, (C) 15, and (D) 4 °C. The figure shows that the perfusion fluid, initially at RT, reached each shell temperature at different flow rates within 1 m of perfusion tubing. • Incoming fluid pressurizes the airtight chamber so that the chamber fluid level is only as high as the exit perfusion port • Perfusing at 200 ml/h with perfusion tubes placed 2 mm above the coverslip surface, takes a minimum of 6.7 sec to completely flush the chamber. Figure 6. (A) SolidWorks exploded view of assembly with Bill of Materials. Perfusion fluid enters perfusion ports (14) into the HX (10) to reach desired temperature. Perfusion fluid enters chamber through Swagelok connectors (12) into a well created by rubber gaskets (5) which create a leak proof seal between plates (1,6). Fluid flows over the sample (3) which is secured by the mesh (4) and sits on the glass cover slip (2). Finally the fluid exits the chamber through the second Swagelok and the out perfusion port. (B) Photo of fabricated assembly. The perfusion tubing is wrapped in the HX. The top and bottom chamber plates are sufficiently secured with rubber bands. (C) The chamber assembly on the confocal microscope stage. Wiggle Test: Characterization of HX Mixing: • Planned future work to further assess chamber design: • Verify sample stabilization with pancreatic tissue • Determine solute perfusion across mesh • Measure permeability with fluorescence confocal microscopy Figure 9. Thermocouples were placed at twelve locations throughout the HX to challenge the assumption that the ΔTWwas minimal due to the high flow rate of the shell water. Shown are the locations tested, which were all found to be within 0.1 °C. • Acknowledgements: • Dr. Higgins, Dr. Harding, Allyson Fry, Anne-Marie Girard, • Manfred Dittrich, Andy Brickman, Dr. Skip Rochefort Figure 8. Water was perfused at 200 mL/h over (A) hair and (B) 90-150 μm beads. Movement was only observed at initial startup. Gradual increased flow is recommended.