Dr. Adam Higgins Xiao-Yue Han
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1
An Apparatus for Optimizing Heat Exchange and Solution Mixing for Cryopreservation
Experiments with 2D Tissue Systems: Temperature and Concentration Control
Dr. Adam HigginsXiao-Yue Han
2010 HHMI Symposium: September 23-24
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Agenda
• Background• Model• Initial Apparatus– mixing and temperature experiments
• New Apparatus– Construction– Mixing and temperature experiments
• Conclusion• Acknowledgements
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
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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
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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)
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The initial apparatus had mixing problems which necessitated an interdigital micromixer
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Temperature response was poor with the mixing device and outside of the cell water bath
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
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0 50 100 150 200 2500
1
2
3
4
5
6
7
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Mixing Experiment
Time (seconds)
Light
Inte
nsity
Det
ecte
d by
Micr
osco
pe
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
0 50 100 150 200 2500
1
2
3
4
5
6
7
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Expected data based on syringe protocol
Time (seconds)
Light
Inte
nsity
0 50 100 150 200 2500
1
2
3
4
5
6
7
8
[Dye] vs. time
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Flow rate is inversely proportional to residence time and heat transfer
100 125 150 1750
5
10
15
20
25
30
35
40
0°C22°C37°CRoom Temperature
Flow rate (mL/hour)
Tem
pera
ture
at C
ells
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Schematic of new diagramIn
itial
App
arat
usN
ew A
ppar
atus
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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
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Experimental Setup with New Apparatus
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Temperature response was much better with the new apparatus.
0 5 10 15 20 25 30 35 40 450
5
10
15
20
25
30
35
40
Water Bath Temperature (Celsius)
Tem
pera
ture
at C
ells
(Cel
sius
)
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Experimental mixing data inconsistent with our dye concentration protocol
0 50 100 150 200 2500
2
4
6
8
10
12
[Dye] vs. time
Time (secounds)
Rela
tive
[Dye
]
0 50 100 150 200 2500
2
4
6
8
10
12
Expected data based on syringe protocol
Time (seconds)
Light
Inte
nsity
0 50 100 150 200 2502980
3000
3020
3040
3060
3080
3100
3120
3140
3160
Experimental Data
Time (seconds)
Light
Inte
nsity
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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
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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
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Acknowledgements
• Dr. Adam Higgins• Allyson Fry (graduate student)• Cameron Glasscock and Diana Vasquez
(labmates)• Hai-Yue Han (CNC machinist)• Dr. Kevin Ahern• HHMI
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