Fluorescence Quenching Used as a
Tool for Cryoprotectant Addition and
Removal Procedures for
Cryopreservation of Adherent
Endothelial Cells
Nadeem Houran
Dr. Adam Higgins
Allyson Fry
Austin Rondema & Brian Fuchs
Cryopreservation
Long term storage of living cells
– Current methods yield low survivability
• Engineered tissues
– Liver
• Cell based devices
– Biosensors
Damage can occur in the
cryopreservation process
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Cryoprotectant Agents
• CPAs protect cells by
reducing intracellular ice
formation
• CPAs alter the kinetics of
the cells
– Ice formation
– Membrane mass transport
• CPAs may be harmful to
cells
– Toxicity
– Volume changes
Cryoprotectants
•Propylene Glycol
•Dimethyl sulfoxide (DMSO)
- Sperm Cells
•Glycerol
- Blood Cells
Osmotic Tolerance Limits
Damage occurs
180
Relative Volume [%]
• Cell membranes can only
swell so much before
damage occurs
• Damage can be observed
through changes in mass
transport
• Knowing OTL, damage
caused by solution effects
can be reduced.
140
100
60
20
0
0.2
0.4
0.6
P0 / Pf
0.8
1.0
Fluorescence Quenching
CPA/PBS
solution
20x
Cells
Syringe pump
•Different syringe pump per
hypo/isotonic solution
•Solutions are heated or cooled
in a heat exchanger
•Bubbles in pumps will flow over
cells and kill or wash them away
flow
adhered cells
Flow Chamber Side View
< [1] , Higgins >
Fluorescence Quenching Technique
Addition
Isotonic Solution
Removal
Isotonic Solution + CPA
Isotonic Solution
CPA (Water)
CPA
CPA
CPA
CPA
CPA
Fluorescence Dye Addition
Calcein
Calcein-AM (acetoxymethyl)
acetoxymethyl
• Calcien-AM is membrane permeable
• Once inside, esterase cleaves the acetoxymethyl making
the dye membrane impermeable
• Once cleaved, the Calcien is able to fluoresce
Fluorescence Quenching Technique
• Uses dye (Calcein) to characterize the volume of a cell
• Fluorescence from Calcein dye is quenched by unknown
proteins in the cytoplasm
– Hypertonic (shrivel)
– Isotonic (normal state)
– Hypotonic (swell)
20 mm
Isotonic
Quencher
Protein
Hypotonic
Hypertonic
Calcein
Isotonic
Hypertonic
Mathematic Modeling
Membrane Water
Transport
Membrane CPA
Transport
Membrane Water
& CPA Transport
Relating Volume to
Fluorescence Intensity
5% Sucrose in 1X PBS at 4 ºC
Intensity (arb)
Sucrose Solution
PBS solution
Time (s)
10% Propylene Glycol in 1 X PBS at
37ºC
Intensity (arb)
Propylene Glycol Solution
PBS Solution
Time (s)
Normalized Fluorescence
CPA Transport at 4ºC
1
0.7 M PG
0.7 M Sucrose
LpA/Vw0 = 0.86 ± 0.04
x 10-8 Pa-1s-1
0.95
LpA/Vw0 = 0.86 ± 0.12
x 10-8 Pa-1s-1
0.9
PsA/Vw0 = 0.024 ± 0.004 s-1
0.85
0.95
0.9
0.7 M Glycerol
0.7 M DMSO
1
LpA/Vw0 = 0.63 ± 0.08
x 10-8 Pa-1s-1
PsA/Vw0 = 0.020 ± 0.004 s-1
LpA/Vw0 = 0.77 ± 0.07
x 10-8 Pa-1s-1
PsA/Vw0 = 0.00035 ±
0.00035 s-1
0.85
0
30
60
Time (s)
90
120 0
30
60
90
Time (s)
120
< [1] , Higgins >
Normalized Fluorescence
CPA Transport at 21ºC
0.7 M Sucrose
1
0.7 M PG
LpA/Vw0 = 2.5 ± 0.3
x 10-8 Pa-1s-1
0.95
LpA/Vw0 = 3.8 ± 0.2
x 10-8 Pa-1s-1
0.9
PsA/Vw0 = 0.28 ± 0.02 s-1
0.85
0.7 M DMSO
1
0.95
0.7 M Glycerol
LpA/Vw0 = 6.7 ± 2.0
x 10-8 Pa-1s-1
PsA/Vw0 = 0.01 ± 0.005 s-1
LpA/Vw0 = 3.2 ± 0.2
x 10-8 Pa-1s-1
PsA/Vw0 = 0.25 ± 0.02 s-1
0.9
0.85
0
30
60
Time (s)
90
120 0
30
60
Time (s)
90
120
< [1] , Higgins >
CPA Transport at 37ºC
0.7 M Sucrose
Normalized Fluorescence
1
LpA/Vw0 = 7.7 ± 1.4
x 10-8 Pa-1s-1
0.95
1.7 M PG
LpA/Vw0 = 13.6 ± 3.2
x 10-8 Pa-1s-1
0.9
PsA/Vw0 = 2.0 ± 0.3 s-1
0.85
0.7 M Glycerol
1
LpA/Vw0 = 7.5 ± 1.4
x 10-8 Pa-1s-1
PsA/Vw0 = 0.03 ± 0.006 s-1
1.7 M DMSO
0.95
LpA/Vw0 = 8.1 ± 1.1
x 10-8 Pa-1s-1
PsA/Vw0 = 1.2 ± 0.4 s-1
0.9
0.85
0
30
60
Time (s)
90
120 0
30
60
90
Time (s)
120
< [1] , Higgins >
Conclusion
• Developing fluorescence quenching technique
– Optimizing CPA addition and removal
• Apply mathematical models to solve for
permeability of CPA and water
• Using calculated permeability parameters to
maximize cell survivability during the
cryopreservation process
Future Work
• Culturing neurons for Fluorescence Quenching
and permeability parameters measurements
• Redesigning the flow chamber to
accommodate tissues
– Ovarian tissue
Acknowledgements
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HHMI
Dr. Adam Higgins
Kevin Ahern
Allyson Fry
Austin Rondema and Brian Fuchs