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

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    /  f Relative Volume [%] Damage occurs 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.

Fluorescence Quenching 20x Cells Syringe pump CPA/PBS solution flow adhered cells Flow Chamber Side View 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

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

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

Fluorescence Quenching Technique Isotonic 20  m Hypertonic Calcein Quencher Protein 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) HypotonicIsotonicHypertonic

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 Sucrose Solution PBS solution Time (s) Intensity (arb)

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

CPA Transport at 4ºC 0.7 M PG 0.7 M DMSO 0.7 M Glycerol 0.7 M Sucrose Time (s) Normalized Fluorescence P s A/V w0 = ± s -1 P s A/V w0 = ± s -1 P s A/V w0 = ± s -1 L p A/V w0 = 0.86 ± 0.04 x Pa -1 s -1 L p A/V w0 = 0.86 ± 0.12 x Pa -1 s -1 L p A/V w0 = 0.77 ± 0.07 x Pa -1 s -1 L p A/V w0 = 0.63 ± 0.08 x Pa -1 s -1

CPA Transport at 21ºC Time (s) 0.7 M PG 0.7 M DMSO 0.7 M Glycerol 0.7 M Sucrose Normalized Fluorescence P s A/V w0 = 0.01 ± s -1 P s A/V w0 = 0.28 ± 0.02 s -1 P s A/V w0 = 0.25 ± 0.02 s -1 L p A/V w0 = 2.5 ± 0.3 x Pa -1 s -1 L p A/V w0 = 3.8 ± 0.2 x Pa -1 s -1 L p A/V w0 = 6.7 ± 2.0 x Pa -1 s -1 L p A/V w0 = 3.2 ± 0.2 x Pa -1 s -1

CPA Transport at 37ºC 1.7 M DMSO 0.7 M Glycerol 1.7 M PG 0.7 M Sucrose Time (s) Normalized Fluorescence P s A/V w0 = 0.03 ± s -1 P s A/V w0 = 2.0 ± 0.3 s -1 P s A/V w0 = 1.2 ± 0.4 s -1 L p A/V w0 = 7.7 ± 1.4 x Pa -1 s -1 L p A/V w0 = 13.6 ± 3.2 x Pa -1 s -1 L p A/V w0 = 7.5 ± 1.4 x Pa -1 s -1 L p A/V w0 = 8.1 ± 1.1 x Pa -1 s -1

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