The bleeding edge: emerging issues in biobankingand cryopreservation Allison Hubel, PhD University of Minnesota

Preservation as a platform technology Food Core scientific principals Mechanisms of degradation Mechanisms of stabilization Biospecimens Molecules Cells Tissue Pharmaceuticals

Biobanksustainability Sustainability is the new buzzword

Long term sustainability Costs Ongoing sample storage Acquiring and storing new samples Potential mismatch Income Sale of specimens Grant funding for collection of new specimens

What do avocados and frozen tuna have to do with sustainability of biobanks?

Avocados Shipping of avocados requires: 8-12 C temperature (unripe), 5-7 C (ripe) Temperatures < 5 C chilling damage Temperatures > 12 C reduced shelf life Temperature is monitored continuously during transport Deviations result in reduced shelf life Requirements for humidity, ventilation, gas composition are all specified and monitored as well Avocados sell for $1-2 each at retail

Avocados and biospecimens We know more about the storage requirements of avocados than biospecimens Temperature matters Temperature fluctuations matter Practical hints: Know the proper temperature for storage Monitor temperature in storage units continuously (using independent systems) Make sure monitoring systems are connected to alarms Resources for you: Hubel A, SpindlerR, SkubitzAPN, Storage of human biospecimens: selection of the optimal storage temperature, Biopres& Biobank, 12: 1-11, 2014.

Frozen Tuna There is a biochemical metric for frozen tuna to assess quality The quality of frozen tuna is determined based on the k value k= (HxR+Hx) (ATP+ADP+AMP+IMP, HxR+Hx) Definitions: HxR-inosine Hx-hypoxanthine ATP, ADP, AMP-adenosine 5-tri, di, mono-phosphates IMP-inosine mono-phosphate

Frozen Tuna, cont. A quality metric exists for frozen tuna The kinetics of degradation for k as a function of storage time and temperature are known Quality metrics should exist for each biospecimenthat you store Resources for you: Agustini, T. W., et al. (2001). "Change of K-value and water state of yellowfintuna Thunnusalbacaresmeat stored in a wide temperature range (20 degrees C to -84 degrees C)." Fisheries Science 67(2): 306-313.

New and emerging biospecimens

New biomarkers Exosomes, candidate biomarker for: Cancer Kidney disease Storage lesion for red blood cells Other uses: Immunotherapy Modality for drug delivery Preservation will be critical for effective use of exosomes Small volume Small number high efficiency Methods compatible with down stream use

New biospeicmens, cont. Removal of hematopoietic cells LFC, biomarkers for: Cancer Heart disease Other emerging diseases ~200 low frequency cells/vial of blood Initial studies relied on fixation of cells Emerging methods use live cells Challenge: Processing small volumes and cell numbers reliably Making methods development more streamlined Stabilizing blood prior to separation

Summary Biobank sustainability is an emerging, critical issue Intrinsic mismatch between costs and income Implementation of QA/QC program will be critical for sustainability Rational basis for culling samples New biospecimensor new requirements for biospecimens will continue to drive biospecimen science

Emerging issues in cryopreservation

Challenging cell types Many cells cannot be preserved effectively: platelets granulocytes, plasma cells hescs ipscells Hepatocytes Dendritic cells Cardiomyocytes We do not understand why certain cells can be effectively preserved and others not We cannot develop scientific protocols to preserve cells refractive to current approaches

Expanding our understanding of damage Biophysical approach has dominated Two factor hypothesis High cooling rates-iif Low cooling rates-dehydration Implicit: cooling process dominates Other cellular factors play an important role

Apoptosis Lymphocytes were cryopreserved Cells were thawed and assayed for viability Cell counts varied with time post thaw

Pathways for post thaw apoptosis Post thaw apoptosis is mediated by the mitochondria and activation of Caspase-3 (Stroh, 2002) Caspase inhibition or use of ROCK inhibitors reduces post thaw apoptosis

Biomarkers for improper storage TNC recovery Post thaw CFU fraction 1 0.8 0.6 0.4 0.2 0 0 2 4 6 Duration of lesion (months) 1.00E-03 8.00E-04 6.00E-04 4.00E-04 2.00E-04 0.00E+00 0 2 4 6 Duration of lesion (months) Post thaw CD34+ fraction 8.00E-03 6.00E-03 4.00E-03 2.00E-03 0.00E+00 0 2 4 6 Duration of lesion (months) Cord blood stored on LN2 moved to -80 C Conventional measures as a function of time in lesion Little variation in TNC

Apototsisas a biomarker 100 Late apoptotic Early apoptotic Percentage of cells 80 60 40 20 Necrotic Viable Caspaseused as marker for apoptosis Flow cytometry measures on CD34+ cells 0 0 2 4 6 Duration of Lesion (months) Apoptosis is a sensitive marker for improper storage conditions Leads changes in other conventional measures Paper upcoming in the journal Transfusion

Other cellular mechanisms of damage Cytoskeleton is a site of damage Know for oocytes for years True as well for other cell types Modifying cytoskeleton can influence post thaw recovery Cell membrane also a site of damage Third mechanism of damage: Cell-specific response to stress influences post thaw recovery

BioCoR Resources Education Service Research

Education Resource BioCoR library Preservation of Molecular, Cellular and Tissue Biospecimens Endorsed by ISBER Preservation of Cellular Therapies Offered in cooperation with AABB

BioCoRResearch Dry State Storage of Plasma QC tests for UCB Improving preservation of MSCS All projects are NIH funded

Service Resource Getting help with your preservation needs Ask the expert Web-based service portal for clients External sales infrastructure established

Acknowledgments Faculty Peter Dosa Dave McKenna Diane Kadidlo Jonathan Sachs Julie Curtsinger Students/Post doc Katie Pollock Guanglin Yu Sara Wiederoder Ralf Spindler Support from : R21HL112653, R21EB016247, IEM