Applying principles of Cryobiology in Biobanking Barry J Fuller Professor in Surgical Sciences & Low Temperature Medicine Division of Surgery & Interventional Sciences, UCL Medical School DI for Royal Free Hospital HTA Tissue Storage Licences UNESCO Chair in Cryobiology Disclosure : The works described have been carried out as academic collaborations and grant-funded studies; I have no commercial interests in the technologies

A combination of cold temperatures and phase change in water Cryobiology a term first used in 1960’s - (Cryos = Cold) 1.History of applied cryobiology 2.Current understanding of the technologies – Slow Cooling or Vitrification, Warming 3.Implications for different Biobanking applications

Direct observation – microscopy - played a significant part in the history of cryobiology Freezing is a dehydration stress – and all of the (bio)chemistry that implies Red onion epidermis cooled (14a) and frozen at -10 o C. Note the cell shrinkage and pigment concentration

History – Pivotal moments in Modern Cryobiology 3. Equalled by development of sophisticated cryomicroscopy (1970’s ) which allowed direct observation of freezing to deep cryogenic temperatures 1. The application of glycerol to allow revival if sperm after deep freezing to -79 o C, The successful recovery of blood cells (Meryman, Rowe, Huggins), and mammalian embryos form -196 o C using Glycerol or DMSO – over next 30 years

Lovelock suggested that salt dehydration was a major factor in freezing injury By adding neutral solutes can achieve colligative effects and reduce salt concentrations – the reason why Polge had succeeded – this spurred the search for cryoprotectants (CPA) History - Water into Ice and its’ consequences - Excluding solutes and cells – the need for Cryoprotection Cells at -8 o C shrinking in the hypertonic solution in between ice crystals LOVELOCK JE, BISHOP MW. Nature May 16;183(4672): Prevention of freezing damage to living cells by dimethyl sulphoxide.

CPA- Essential Antifreezes for Life ‘Water-modifying’ agents -some CPA are cell permeating or ‘Intracellular’, comprising small polyols like glycerol or others such as DMSO. Usually have a H-bonding sites for water and high oil / water partition (since they need to get inside the cells ) and stabilise biomolecules Others are polymers and sugars which perturb water / ice transitions OUTSIDE the cells – and can be used to start optimal cryogenic dehydration propylene glycolethylene glycol glycerol Dimethyl sulphoxide ‘2 nd -ary’ CPA – in the external medium

150% 125% 100% 75% 50% AdditionRemoval Zone of Tolerance CPA exposure protocols need to be optimised History & current CPA addition and removal results in osmotic stress before / after freezing. Concepts of Cumulative CPA-related Injury – the safe boundaries Cell volume changes

optimal cryogenic cell dehydration There is essential control of cooling profile to allow optimal cryogenic cell dehydration (Slow cooling) down to the stable cryogenic temperatures – the concept of the ‘glassy state’ (This can be measured by physical means as T g ) If cooling is Too Fast – something else happens – intracellular ice! So…. Successfully frozen cells and tissues are… Not Frozen! Mazur’s two factor hypothesis – cool too slow – over-long osmotic stress; Cool too fast – residual mobile intracellular water forms lethal ice

+20 o C Glass transition range +0 o C -100 o C -50 o C -25 o C -190 o C +37 o C cryoprotection Ice formation - need for cryoprotection Locking up the water – Zone of freeze dehydration Matrix solidification Zone of instability (ice, salt hydrates, proteins) True long term cryogenic stability Cryopreservation by Slow Cooling – Locking up the water – Ice is the Desiccant Our ‘convenient friend’ Liquid N 2 Optimised cryogenic dehydration

Bill Rall & Greg Fahy Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification.* Nature Feb 14-20;313(6003): High concentrations of CPA, polyols, sugars (>40% w/w) plus fast cooling to prevent ice nucleating before reaching ‘glassy’ state at ultra-low temperatures The other way to go – Vitrification This also depends on the Glass Transition range which can be physically determined Very difficult to avoid any tiny ice nuclei forming somewhere * LUYET BJ, GEHENIO Thermoelectric recording of ice formation and of vitrification during ultra-rapid cooling of protoplasm. PM.Fed Proc ;6(1 Pt 2):157.

Schematic of Vitrification profile +25 o C CPA preload – 10%w/v 5 min Glass transition temperature +0 o C -100 o C -50 o C -25 o C -190 o C Time VF mix – 40% CPA + sugars ‘Almost glassy’ Small containers with rapid heat transfer History & current Practically -this is not an equilibrium state Therefore small volumes and extremely rapid cooling and warming used to ‘out-race’ the start of ice crystal nucleation Optimised cryogenic dehydration Now CPA is the Desiccant

Tissue Cryopreservation – the Same Biophysical Events with Additional Diffusion Barriers And where the Ice crystals form CPA diffusion In / Out Ice forming externally and in interstitial spaces producing cell dehydration In: K Brockbank et al. Methods in Cryopreservation and Freeze-Drying, Methods in Molecular Biology, Heart valve leaflet cryopreserved with DMSO. Freeze substitution EM at -90 o C Showing Interstitial Ice

Mill Hill Group ( ); Green et al, J Endocrin Rat ovarian autografts after freezing in 15% glycerol in saline, 1h exposure, slow cooling, pieces, sub-cut, days 2-30.Oestrous cycling returned. Tissue Cryopreservation – Ovarian Tissue as example Historical & Current Perspectives Goals – to preserve the ability of follicles (oocyte + supporting cells) to grow and acquire mature characteristics (Must maintain cell-cell communications and signaling)  Slow Cooling with 1.5M DMSO (1) −8°C at −2°C/min; (2) seeded manually (3) cooled to −40°C at −0·3°C/min; (4) cooled to −150°C at −30°C/min, and (5) transferred to liquid nitrogen (−196°C). Tissue cryopreserved for 6 years Some current research – using an electrical stirling cryo-cooler – Theatres compliant avoiding Liq N 2

Isolate tumour Disaggregate tumour Selection / Ex vivo Expansion FormulationInfusion David Gilham & Clinical and Experimental Immunotherapy Group Institute of Cancer Sciences University of Manchester Freezing disaggregated tumour has no obvious impact upon success rate of TIL culture initiation Preliminary experiments involving cryopreserving intact tumour prior to disaggregation has had widely variable results Cryo- banking options Fresh8/11 (73%) PBS/HSA/DMSO: Coolcell 6/8 (75%) EF6004/5 (80%) Cryostor10 Coolcell5/7 (71%) EF6004/5 (80%) >5 fold expansion of cells recovered after tumour cryo Tissue Cryopreservation - ‘Fusion’ Biobanking ( e.g. Recovering viable lymphocytes from frozen tumour) Theatre compliant cryo-cooler for immediate cryo-processing

Tissue Cryopreservation - only important if you need living cells? Not quite…………………… For tissues, Preservation of Biomatrix by Optimised Cryogenic Dehydration can be equally important, irrespective of cell viability For heart valve leaflets, better structure (less oedema and inflammation) followed Vitrification which preserved biomatrix AND destroyed resident cells – reducing Allograft reaction (more Cryo-Processing opportunities) Vitrified -90 o C Cryopreserved -90 o C

The Challenges of Warming Generally, faster is better….to avoid Intracellular Ice during warming…..easy for cell suspensions. But for large volumes, and intact tissues, Cryo-Materials Sciences become important Differential temperature gradients during rapid warming, coupled with expansion or contraction, causes mechanical stress, especially around the glass transition range One way to avoid thermo- mechanical stress is to use differential warming; slow to - 80 o C, then fast to optimise cell survival Water becomes mobile above Tg’ – and Ice Re-crystallises

At the level of individual analytes, the same consequences follow progressive ice formation and increase in salts in the unfrozen fraction Changes e.g. in protein folding may be small, reversible, and not relevant to Biobanking as generally considered. But for sensitive proteins or therapeutic biobanking, there may often be explanations for ‘the protein doesn’t freeze well’. And it is possible to protect against freezing injury – if you have to - with – sugars, polyols Often also called ‘excipients’ Translational Biobanking

For Practical Biobanking Understanding the biophysical events during cryogenic storage can help to plan protocols with a wider application Addition of protectants, water replacement molecules, may have a role – depending on what outcomes you require Glycine betaine is a Quat Ammonium salt found as an osmolyte in plants, crustaceans exposed to salt stress………… Translational Biobanking

Another way to get Vitrification – in large tissues - ‘slide’ down and up the liquidus curve – incrementally increase CPA and cool step by step - stay to the right of ice formation curve Fine balance between lethal high CPA concentrations and too low CPA as cooling proceeds - with lethal ice nuclei formation – but for large complex structures (cartilage, ovarian or testicular tissues) it may be worth the effort. Directions For the next decade - Avoiding Ice in Large Volumes - Liquidus Tracking(?) Equipment to add CPA, mix and cool at the same time Pegg DE et al; Cryopreservation of articular cartilage. Part 3: the liquidus-tracking method. Cryobiology (3):360-8.

Summary By applying principles of Cryobiology, Tissue Banking can be a successful key component of many different therapies and diagnostic services More research is still needed on fundamental cryobiology, long- term outcomes after cryopreservation, on better and safer techniques The 2 nd Age of Cryo – fully understanding biophysical changes in cells transitioning the cryogenic range and any subtle molecular / genetic impacts so far hidden from view

Acknowledgements Reproductive Biobanking : Paul Hardiman, Tom Morewood (UCL); Victoria Keros, (Karolinska); Sharon Paynter (Cardiff) Cryobiology : Clare Selden, Humphrey Hodgson, Isobel Massie, Eva Puschmann, Peter Kilbride, Stephanie Gibbons, Aurelie leLay (UCL) Translational Cryobiology : TSB consortium; University of Manchester, UK Stem Cell Bank, Roslin Cells; John Morris (Asymptote UK) Cryo-technology : Steve Butler, Geoffrey Planer (Planer UK) Planer UK Technology Strategy Board