EFFECT OF COOLING RATE ON THE VIABILITY OF CULTURED CELLS AFTER

CRYOPRESERVATION.

Brian FuchsResearch Mentor: Dr. Adam

Higgins

Cryopreservation

Cryopreservation is currently implemented in:Artificial inseminationStorage of certain types

of cells (e.g. blood cells)

Long-term storage of living material at extremely low temperatures

Future Applications Future applications of

cryopreservation are:Long term storage of

tissuesLong term storage of

organsUse in cell-based

biosensors

Problems with Freezing Process

2 main types of cellular damage:1. Intracellular ice formation (IIF)

Damages membranes and cell structure2. Cellular dehydration and solution effects

3rd type of damage is extracellular ice formation. Typically is significant only in tissue freezing

Vitrification Vitrification is the

process of freezing a substance to a point where it becomes a glass like amorphous solid

Prevents death due to IIF.

2 Treatments 2 ways being investigated to prevent cell

damage:1. Addition of cryoprotection agents

(CPA)2. Adjustment of cooling rates

CPA CPA’s are chemicals that are

permeable to cellular membrane

Help to depress freezing point and prevent ice crystal formation

Some examples are glycerol and DMSO.

Cooling Rate Goal: determine

cooling rate for optimal cell viability.

High cooling rate intracellular ice formation (IIF)

Low cooling rate cellular dehydration and solution effects COOLING RATE

SURV

IVAL

Solution Effects IIF

Hypothesis The optimum cooling rate for maximal

endothelial cell viability is about 5 ºC/min.

-40-35-30-25-20-15-10-500

5

10

15

20

25

30

Supercooling

10 ºC/min

20 ºC/min

40 ºC/min

80 ºC/min

130 ºC/min

Temperature (ºC)

Supe

rcol

ing

(ºC)

-80-70-60-50-40-30-20-1000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Normalized Water Volume

10 ºC/min20 ºC/min40 ºC/min80 ºC/min130 ºC/minEquilib-rium

Temperature (ºC)

Cel

l Wat

er V

olum

e (V

w/V

w0)

Process Culture cells on a

slide Add CPA Run controlled

rate freezing process

Thaw cells Perform live-dead

staining -120

-100

-80

-60

-40

-20

0

40 ºC/min10 ºC/min5 ºC/min

Time

Tem

pera

ture

(ºC

)

Live/Dead Stain Controls

Live cells stained with calcein-AM

Live cells stained with ethidium homodimer

Dead cells stained with calcein-AM

Dead cells stained with ethidium homodimer

00.10.20.30.40.50.60.70.80.9

1

Recovery of Adherent Endothelial Cells at Vary-

ing Cooling Rates

Control 5 ºC/min 10 ºC/min 40 ºC/min

Cooling Rate

Rec

over

y

COOLING RATE

SURV

IVAL

Solution Effects

IIF

Conclusion There is a significant correlation between cooling

rate and cell viability. Of the experiments performed, cooling rates of

5 ºC/min provided maximum cell recovery. More experiments are needed to determine if cell

viability decreases at cooling rates lower than 5 ºC/min.

CRF process is ready for use on cultured neurons.

Acknowledgements Dr. Adam Higgins Allyson Fry Nadeem Houran, Austin Rondema, Ingemar

Hudspeth Dr. Kevin Ahern Howard Hughes Medical Institute