effect of cooling rate on the viability of cultured cells after cryopreservation
DESCRIPTION
Brian Fuchs Research Mentor: Dr. Adam Higgins. Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation. . Cryopreservation. Long-term storage of living material at extremely low temperatures. Cryopreservation is currently implemented in: Artificial insemination - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/1.jpg)
EFFECT OF COOLING RATE ON THE VIABILITY OF CULTURED CELLS AFTER
CRYOPRESERVATION.
Brian FuchsResearch Mentor: Dr. Adam
Higgins
![Page 2: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/2.jpg)
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
![Page 3: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/3.jpg)
Future Applications Future applications of
cryopreservation are:Long term storage of
tissuesLong term storage of
organsUse in cell-based
biosensors
![Page 4: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/4.jpg)
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
![Page 5: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/5.jpg)
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.
![Page 6: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/6.jpg)
2 Treatments 2 ways being investigated to prevent cell
damage:1. Addition of cryoprotection agents
(CPA)2. Adjustment of cooling rates
![Page 7: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/7.jpg)
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.
![Page 8: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/8.jpg)
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
![Page 9: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/9.jpg)
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)
![Page 10: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/10.jpg)
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
)
![Page 11: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/11.jpg)
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
![Page 12: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/12.jpg)
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
![Page 13: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/13.jpg)
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.
![Page 14: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/14.jpg)
Acknowledgements Dr. Adam Higgins Allyson Fry Nadeem Houran, Austin Rondema, Ingemar
Hudspeth Dr. Kevin Ahern Howard Hughes Medical Institute
![Page 15: Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation](https://reader036.vdocuments.mx/reader036/viewer/2022062315/56816617550346895dd965e0/html5/thumbnails/15.jpg)