Brian Fuchs
Research Mentor: Dr. Adam Higgins
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Long-term storage of living material at extremely
low temperatures

Cryopreservation is
currently implemented
in:
Artificial insemination
Storage of certain types
of cells (e.g. blood cells)

Future applications of
cryopreservation are:
Long term storage of
tissues
Long term storage of
organs
Use in cell-based
biosensors

2 main types of cellular damage:
1. Intracellular ice formation (IIF)
 Damages membranes and cell structure
2. Cellular dehydration and solution effects
3rd type of damage is extracellular ice formation.
 Typically is significant only in tissue freezing


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 ways being investigated to prevent cell
damage:
1. Addition of cryoprotection agents
(CPA)
2. Adjustment of cooling rates

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
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.

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Goal: determine
cooling rate for
optimal cell
viability.
High cooling rate 
intracellular ice
formation (IIF)
Low cooling rate 
cellular dehydration
and solution effects
Solution
Effects
SURVIVAL

COOLING RATE
IIF

The optimum cooling rate for maximal
endothelial cell viability is about 5 ºC/min.
Supercooling
0.7
30
0.6
10 ºC/min
0.5
20 ºC/min
10 ºC/min
25
0.4
40 ºC/min
0.3
80 ºC/min
0.2
130 ºC/min
0.1
Equilibrium
0
0
-20
-40
-60
Temperature (ºC)
-80
Supercoling (ºC)
Cell Water Volume (Vw/Vw0)
Normalized Water Volume
20
20 ºC/min
15
40 ºC/min
10
80 ºC/min
5
130 ºC/min
0
0
-10
-20
Temperature (ºC)
-30
-40
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Culture cells on a slide
Add CPA
Run controlled rate
freezing process
Thaw cells
Perform live-dead
staining
Temperature (ºC)

0
-20
-40
40 ºC/min
-60
10 ºC/min
5 ºC/min
-80
-100
-120
Time
Live cells stained with calcein-AM
Live cells stained with ethidium
homodimer
Dead cells stained with calcein-AM Dead cells stained with ethidium
homodimer
1
Recovery of Adherent Endothelial
Cells at Varying Cooling Rates
0.9
SURVIVAL
0.8
Recovery
0.7
0.6
0.5
0.4
0.3
Solution
Effects
COOLING RATE
0.2
0.1
0
Control
5 ºC/min
10 ºC/min
Cooling Rate
40 ºC/min
IIF
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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.
Dr. Adam Higgins
 Allyson Fry
 Nadeem Houran, Austin Rondema, Ingemar
Hudspeth
 Dr. Kevin Ahern
 Howard Hughes Medical Institute
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