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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

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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

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2

3

4

5

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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

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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

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[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

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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

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[Dye] vs. time

Time (secounds)

Rela

tive

[Dye

]

0 50 100 150 200 2500

2

4

6

8

10

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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