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Nadeem Houran Dr. Adam Higgins Allyson Fry Austin Rondema & Brian Fuchs

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. <Bailey>. Long term storage of living cells

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Nadeem Houran Dr. Adam Higgins Allyson Fry Austin Rondema & Brian Fuchs

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  1. 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

  2. Cryopreservation <Bailey> 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 <www.cartoonstock.com>

  3. 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

  4. Osmotic Tolerance Limits Damage occurs 180 140 100 Relative Volume [%] 60 20 0 0.2 0.4 0.6 0.8 1.0 P0/ Pf 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.

  5. flow adhered cells Flow Chamber Side View Fluorescence Quenching CPA/PBS solution 20x • 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 Cells Syringe pump < [1] , Higgins >

  6. Fluorescence Quenching Technique Addition Removal Isotonic Solution Isotonic Solution + CPA Isotonic Solution (Water) CPA CPA CPA CPA CPA CPA

  7. Fluorescence Dye Addition • Calcien-AM is membrane permeable • Once inside, esterase cleaves the acetoxymethyl making the dye membrane impermeable • Once cleaved, the Calcien is able to fluoresce Calcein-AM (acetoxymethyl) Calcein acetoxymethyl

  8. 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 Hypertonic Quencher Protein Calcein Hypotonic Isotonic Hypertonic

  9. Mathematic Modeling Membrane Water Transport Membrane Water & CPA Transport Relating Volume to Fluorescence Intensity Membrane CPA Transport

  10. 5% Sucrose in 1X PBS at 4 ºC Sucrose Solution Intensity (arb) PBS solution Time (s)

  11. 10% Propylene Glycol in 1 X PBS at 37ºC Propylene Glycol Solution Intensity (arb) PBS Solution Time (s)

  12. CPA Transport at 4ºC 0.7 M PG 0.7 M Sucrose LpA/Vw0 = 0.86 ± 0.04 x 10-8 Pa-1s-1 LpA/Vw0 = 0.86 ± 0.12 x 10-8 Pa-1s-1 PsA/Vw0 = 0.024 ± 0.004 s-1 Normalized Fluorescence 0.7 M Glycerol 0.7 M DMSO LpA/Vw0 = 0.77 ± 0.07 x 10-8 Pa-1s-1 LpA/Vw0 = 0.63 ± 0.08 x 10-8 Pa-1s-1 PsA/Vw0 = 0.00035 ± 0.00035 s-1 PsA/Vw0 = 0.020 ± 0.004 s-1 Time (s) Time (s) < [1] , Higgins >

  13. CPA Transport at 21ºC 0.7 M Sucrose 0.7 M PG LpA/Vw0 = 2.5 ± 0.3 x 10-8 Pa-1s-1 LpA/Vw0 = 3.8 ± 0.2 x 10-8 Pa-1s-1 PsA/Vw0 = 0.28 ± 0.02 s-1 Normalized Fluorescence 0.7 M DMSO 0.7 M Glycerol LpA/Vw0 = 6.7 ± 2.0 x 10-8 Pa-1s-1 LpA/Vw0 = 3.2 ± 0.2 x 10-8 Pa-1s-1 PsA/Vw0 = 0.01 ± 0.005 s-1 PsA/Vw0 = 0.25 ± 0.02 s-1 Time (s) Time (s) < [1] , Higgins >

  14. CPA Transport at 37ºC 0.7 M Sucrose 1.7 M PG LpA/Vw0 = 7.7 ± 1.4 x 10-8 Pa-1s-1 LpA/Vw0 = 13.6 ± 3.2 x 10-8 Pa-1s-1 PsA/Vw0 = 2.0 ± 0.3 s-1 Normalized Fluorescence 0.7 M Glycerol 1.7 M DMSO LpA/Vw0 = 7.5 ± 1.4 x 10-8 Pa-1s-1 LpA/Vw0 = 8.1 ± 1.1 x 10-8 Pa-1s-1 PsA/Vw0 = 0.03 ± 0.006 s-1 PsA/Vw0 = 1.2 ± 0.4 s-1 Time (s) Time (s) < [1] , Higgins >

  15. 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

  16. Future Work • Culturing neurons for Fluorescence Quenching and permeability parameters measurements • Redesigning the flow chamber to accommodate tissues • Ovarian tissue

  17. Acknowledgements • HHMI • Dr. Adam Higgins • Kevin Ahern • Allyson Fry • Austin Rondema and Brian Fuchs

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