1. An Apparatus for Optimizing Heat Exchange and Solution Mixing for Cryopreservation Experiments with 2D Tissue Systems: Temperature and Concentration Control Dr. Adam Higgins Xiao-Yue Han 1 2010 HHMI Symposium: September 23-24

2. Agenda Background Model Initial Apparatus – mixing and temperature experiments New Apparatus – Construction – Mixing and temperature experiments Conclusion Acknowledgements 2

3. Background Cryopreservation is a process for cell and tissue preservation – Transplantation medicine, tissue engineering, drug testing, etc. Two approaches to freezing cells and tissue – Slow freezing: ~1 ° C/min, low [cryoprotective agent] (CPA) – Vitrification: high [CPA], rapid cooling Determining toxic effects of high [CPA] is important in optimizing vitrification procedures 3

4. Model CPA toxicity is a function of – [CPA] – Temperature – Time To create a toxicity cost model, it is important to be able to control these variables 4

5. Materials Tubing and insulation – 1 / 8 ” ID clear plastic tubing for syringe – Leur Lock tube and syringe articulations – 1 / 4 “ ID clear plastic tubing for heat exchanger – 1 / 8 ” thick Carmacell TAP 18230 self-adhering insulation tape (Mebane, NC) Box materials – 1”, ½”, ¼”, 1 / 8 ” thick acrylic sheets – Stainless steel fasteners and nuts – Dow Corning High Vacuum Grease Fluid control – New Era Pump Systems, Inc., NE-500X syringe pumps – SIMHEX Slit Interdigital Micromixer Temperature and mixing validation – Leica DM 2500 Microscope using 20X objective – OMEGA HH502 thermocouple – Dye: 1:25 dilution of stock nuclear fast red (NFR) 5

6. The initial apparatus had mixing problems which necessitated an interdigital micromixer 6

7. Temperature response was poor with the mixing device and outside of the cell water bath 7 WATER BATH SET AT 50 ° C 42.4 ° C 200 mL/hour flow rate 36.8 ° C WATER BATH SET AT 0 ° C 10.1 ° C

8. Our dye experiments showed that there was response delay and longitudinal mixing in the tubing after the micromixer. This tube’s length should be minimized 8

9. Flow rate is inversely proportional to residence time and heat transfer 9

10. Schematic of new diagram 10 Initial Apparatus New Apparatus

11. Solidworks was used to design a box consistent with our schematic Features – Shorter box – Mountable – Micromixer now in box – Shorter tubing after mix – Millable with CNC from 1” acrylic sheet Liabilities – More contact surfaces – Current plastic top lid lights up in fluorescence imaging 11

12. Experimental Setup with New Apparatus 12

13. Temperature response was much better with the new apparatus. 13

14. Experimental mixing data inconsistent with our dye concentration protocol 14

15. Potential Reasons for Mixing Inconsistencies Syringe pumps stick – Lubricate pumping linear actuator, use low resistance syringes – Replace linear actuators with higher torque actuators Resistance high with micromixer Condensation on top lid – Use anti-fog on top cover Expansion of syringe tubing – Use PEEK tubing (HPLC tubing, more rigid) Air leaks into water bath box – Use more Vacuum grease, ensure surfaces close tightly by building reaction struts for lid 15

16. Conclusions Temperature is better controlled using this new apparatus. More troubleshooting needs to be done for mixing experiments Additional validations experiments necessary – Concentration (as opposed to light intensity) – Flow rate as measured by final volume in discard fluid beaker and time of protocol 16

17. Acknowledgements Dr. Adam Higgins Allyson Fry (graduate student) Cameron Glasscock and Diana Vasquez (labmates) Hai-Yue Han (CNC machinist) Dr. Kevin Ahern HHMI 17