Principles of cell cryopreservation Yuriy Petrenko, PhD Institute of Experimental Medicine. CAS INSTITUTE OF PHYSIOLOGY [ BIDCEV The structure of the Lecture: Block I. Principles of cryopreservation: • What happens during freezing - cryodamaging factors; Cryo protectants Main stages of cryopreservation Cooling rates and initiation of ice formation Cryo protectant toxicity Thawing Determination of cell viability Block II. Working examples in the field of stem cell cryopreservation for clinical use • Alternative approaches for stem cell cryopreservation with reduced DMSO concentration Alternative quality control methods for stem cell cryopreservation Hypothemic storage (storage at 4°C) of stem cell suspensions for clinical applications Lyophilization of stem cell-derived secretome Cryobiology- the field of biology that studies the effects of low temperatures biological objects (From Greek Kpuoq — cold, bios — life u logos — science) Storage duration: 4°C -» Several hours; -40°C ■» Several days; -80°C Several weeks; -196°C centuries! What happens to cells during freezing? UNFROZEN CELL) Cytosol Nucleus Plasma membrane Ice JL water _ Higher osmolality Ice • water EXTRACELLULAR FREEZING BEGINS The water outside cells freezes The concentration of salts increases, driving the water outflux from the cell Leads to the acute dehydration Remaining intracellular water crystallizes Cell death We need to find a balance: • reduce the intracellular water to avoid intracellular ice formation • prevent excessive dehydration What have we learned from nature? HOW ANIMALS SURVIVE FREEZING Many animals, including some species of fish and frogs, can tolerate subzero temperatures. Here we look at the biochemical adaptations that help them stay alive. TYPES OF FREEZE SURVIVAL If the liquid in an animal freezes, ice crystals can damage cells and tissues. Animals avoid this in one of two ways. Many fish and arthropods use freeze-avoidance approaches, which keep their bodily fluids Liquid below 0°C. Freeze tolerance helps some frogs, inlertidsl marine invertebrates, and lizards keep ice formation outside cells. , "PERIODIC I^H GRAPHICS Some species use antifreeze proteins to limit ice formation in their bodily fluids. The proteins bind to small ice crystals and stop them from growing. ARCTIC OCEAN 9 -1.8 °C Ice crystal 5 I ^ -vv Antifreeze prate ins ^ ^ Many speciesalso rely on cryopratectant compounds in their blood. These compounds dissolve in the water in cells and lower the temperature at which it freezes. 'JH cry o protectants II GLUCOSE ■ GLYCEROL Glucose and urea are common cryoprotectants in frogs, while insects commonly use glycerol or other polyols. FREEZE TOLERANCE Freeze-tolerant species pack their cells and organs with cryoprotectants to prevent ice formation inside them. Meanwhile, ice-nucleating proteins heLp freeze water in the blood, where ice crystalsdo less harm. \Mkjf- WOOD FROGS Up to 65% of their body water can freeze for up to 7 months, Liver glycogen Broken down into glucose OH HO *— Glucose Distributed to otherorgans Cryoprotectants stabiLize theanimaLs1 cell membranes and minimize celL shrinkage due to water loss as ice forms outside the cells. Without cryoprotectants »| Water movement With cryoprotectants (•) " \ . © C&EN 2021 Created by Andy Brunning for Chemical & Engineering News Wood frog image: Brian Gratwicke, CC BY license (bit. ly/2Mfdwli) (J) OCTOBER 7, 2022 How tardigrades survive freezing temperatures by Andrea Mayer-Grenu, University of Stuttgart It is only under the microscope that the similarity of its namesake becomes apparent the plump, round Cryptobiosis / cryobiosis • Stop metabolism • Produce osmolytes and sugars to reduce an control ice formation • Controllable cellular dehydration (almost complete) https://cen.acs.org/biological-chemistry/biochemistry/Periodic-Graphics-animals-survive-freezing/99/i4 Cryoprotectants or cryoprotective agents (CPA) Two most critical factors of cryodamage: • excessive dehydration • intracellular crystallization Small molecules Glycerol Ethylene Glycol Propylene glycol DMSO Amino acid Trehalose Zwitterionic betaine L-carnitine, Proline 1949 1953 1954 1955 1959 1962 1965 1967 1985 1986 1990 2003 2009 2010 2015 2017 2019 2020 Gore et al., 2022 Macromolecules PEG PVP, Dextran Albumin HES AFP PVA Ficoll® AFGP analogues Polyampholytes Hydrogel, Hyaluronic acid Polyproline, PMGS Zwitterionic polymers L-proline oligomers, Trehalose-based synthetic polymers Type of CPA Name Molecular weight Dimethylsulfoxide (DMSO) 78,13 Permeating Glycerol 92,09 Ethylene glycol (EG) 62,07 Propylene glycol 76,09 Glucose 180,12 Sucrose 342,30 Non-permeating with Trehalose 378,33 low molecular weight Raffinose 594,52 Hydroxyethyl starch (HES) 130-200 kDa Albumins 67 kDa Polyvinylpyrrolidone (PVP) 3-36 x104 Non-permeating with Polyvinyl alcohol (PVA) 2-12 x104 high molecular weight Polyethylene glycol (PEG) 2-400 x102 Dextran 1-200x104 What are the main steps of cryopreservation? Stages: Addition of cryoprotective agens (CPA); Cooling/freezing of samples (Usually to -80°C...-196°C); Storage; Thawing (Usually at 37°C - 40°C); Removal of cryoprotectant (washing); Damaging factors: Osmotic injury / CPA toxic\\y (rate/ temperature matters) Overcooling and freezing injury Ice growth during temperature fluctuations Thawing injury Osmotic injury again / CPA toxicity Addition of CPA Equilibrium Permeating CPA (DMSO, glycerol, ethylene glycol) Non-permeating CPA (Sugars, PEG, PVP, HES) tu £ o > "03 O Dehydration Time Volume recovery, the CPA penetrates into cell Dehydration ^^^^^^^^ ^^^-^^^^ ^^^^^^^ ^^^^^^^^ ^^^^^^^^ More dehydration, CPA does not penetrate the cell Optimal cooling rate Optimal cooling rate Cooling Rate (°C/min) I The control of water transport and a cooling rate (crystal formation) is a basis for the development of "optimal" cooling rate How may we find the best cooling rate? Most common (let's try) approach We just try different ways and maybe find the most appropriate Scientific approach We will study the intracellular ice formation using cryomicroscopy i ^^^^^^^ H B j K Stott etal., 2009 News | Published: 12 October 2017 Cryo-electron microscopy wins chemistry Nobel Daniel Cressey & Ewen Callaway Nature 550, 167 (2017) | Cite this article The first cryo-microscope What are the other risks? Overcooling (or supercooling) Temperature (°C) Supercooling can be also good! New 'Supercooling' Technique Helps Preserve Organs By Ctiarles Chof publishes June 30, 2014 OOOQOO REVIEW article Front. Transplant., 23 October 2023 Sec. Vascularized Composite Allotransplantation Volume 2 - 2023 | https:;/doi.org;i0.33a9f,'fr:ra.2025.12o9706 This article is part of the Research Topic Editors Showcase: Vascularized Composite Allotransplantation View all Articles > Supercooling: a promising technique for prolonged preservation in solid organ transplantation, and early perspectives in vascularized composite allografts Yanis Berkane1-2^ Justine Hayau5,~ Irina Filz von Reiterdan k1*2-67-* I I Anil Kharga2- Laura Charles1^ Abele B. Mink van der Molens J. Henk Coert5 Nicoias Bertheuilii4 Mark A. Randolph1-3 Curtis L Cetrulo Jr. Alban Longchamp2r7jSj9 Alexandre G. Lellouch1-2* Korkut Uygun2-7-9* L Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital Harvard Medical School Boston, MA, United States: 2 Shriners Children's Boston, Harvard Medical School, Boston, MA, United States 3 Department of Plastic, Reconstructive and Aesthetic Surgery, Höpital Sud, CHU Rennes, University of Rennes, Rennes, France 4 MOBIDIC, UMR INSERM 1235. Rennes University Hospital, Rennes, France B Division of Plastic Surgery, Lausanne University Hospital Lausanne, Switzerland 6 Departmentot Plastic, Reconstructive and Hand Surgery, University Medical Center Utrecht Utrecht, Metherlands 7 Center for Engineering for Medicine and Surgery, Department of Surgery, Massachusetts General Hospital Harvard Medical School Boston, MA, United States a Department ot Vascular Surgery. Lausanne University Hospital Lausanne, Switzerland 5 Center for Transplant Sciences, Massachusetts General Hospital Boston, MA, United States A supercooled ra: liver si:s in the preservation solution in :he machine perfusion system. [Image credi:: Walty R-eeves. Korku: Uygun,. Martin Yarmu^h. Harvard University) What happens at thawing? Is it also damaging? Slow thawing Rapid thawing Toxicity of cryoprotectants (DMSO as an example) REGENERATIVE MEDICINE, VOL. 15. NO. 3 | REVIEW fQ © (?) © © Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Maaaz Awan Iryna Buriak, Roland Fleck, Barry Fuller, Anatoliy Goltsev, Julie Kerby, Mark Lowdell, Pavel Mericka, Alexander Petrenko, Yuri IPetrenko, Olena Rogulska, Alexandra Stolzing & Glyn N Stacey Published Online: 28 Apr 20201 https://doi.org/10.2217/rme-2019-0145 Adverse reactions for patient DMSO-depleted Unman ipuiated Adverse events (19 patients) (34 patients) p Value Gastrointestinal symptoms (nausea, vom Hing, abdominal cramps) 0 7 Vasovagal syncope 0 1 Angina pectoris 0 1 Other cardiovascular symptoms (bradycardia, tachycardia, hypotension, hypertension) 3 16 Headache 1 2 Pressure on breast/ neck 1 3 Touil number of adverse cvonis 30 Total number ot patients with adverse effects 3 16 Stem cells Human embryonic stern cells 0.01,0.1, RT" HUES-7. foreskin-derived 1.0% (v/v) mesenchymal stem cells - Dose-dependent changes in cell viability, morphology, adhesion and gene expression: inhibition of embryoid bodies formation, decrease in adhesion, cell death - Low and medium DMSO doses upregulate mesodermal markers - Higher DMSO doses downregulate ectodermal differentiation Human umbilical cord blood 40%, 10% stem cells ond DMSO removal RT° and - 40% DMSO lethal, 10% DMSO no viability reduction after 1 post h. DMSO washout improved viability thaw Optimum 7.5-10% Akkoketal.,2008 Can we reduce the toxicity of DMSO? The 3Rs of DMSO toxicity elimination Washing out the CPA solution before application Supplementation of CPA solution by different compounds Replacement of DMSO in CPA solution by different compounds^ Research-application opportunities High flexibility in choosing the methods of CPA removal (centnegation, mierofluidics, filtration} High flexibility in choosing the composition of the diluent (any saline solution with or without supplements) Available research-grade s u pp lem e nts/rep I ace rs Sugars (glucose, dextrose, trehalose, sucrose) IWacromolecules (albumin, HES) Polymers (PEG, PVP, PLL) Antifreeze proteins and bio-compatible solutes (proline, ectoine) Antioxidants and anti-apoptotic agents So, we cryopreserved the cells, even thawed and removed the cryoprotectant... work done? Should we check anything? Determination of the viability of cells Metabolism Identity Growth Potency Transplantation — Check if the membranes are ok and cell number Deeper check, if the general metabolism is not altered, should be done after some time We should check if there are no abnormalities in cell phenotype, transformations etc. We should check how cells behave in culture - can they attach and grow. Are they different from the fresh cells? We should check their specific functional properties in vitro If the cells are aimed to be used for clinical applications - ideally, we should check how they behave in vivo after transplantation Membrane integrity Metabolic activity determination Fluorescein diacetate Alamar blue / resazurin B Proliferation and specific functional activity - potency assays Each cell type has specific functional properties, so adequate methods should be used to confirm the potency Representative of the mechanism(s) of action Covering ait of the product constituents including oil relevant ceil sctbpopttfations POTENCY ASSAY PERFORMANCE T Sensitive and specific enough to detect changes, de-graded or subpotent material Provide In due time, Quantitative results allowing product reteose per estab lished acceptonce criteria Predictive of the clinical efficacy Ensure batch-to-batch consistency Mesenchymal stem cells: differentiation, paracrine / immunomodulatory properties; Hematopoietic stem cells: colony-forming capacity; Pancreatic islet cells: insulin production General cryopreservation protocol: Addition of CPA Controlled freezing Storage Thawing CPA removal Testing Drop-wise 2-3 min -1°C per min down to -80°C Fast transfer to -196°C (120-180 min) at -150-196°C (years) Fast, 3-5 min Drop-wise addition of diluent, centrifugation 15 min 72 hrs to weeks milium Questions? Take-home message part one: Cryoprotectants: there are various type of cryoprotectants. Some of them (glycerol, DMSO) may permeate the cells and protect its' intracellular content avoiding intracellular crystallization. Others (sugars, polymers, starches) cannot permeate, but help to provide controlled dehydration. Cooling rates: It is important to use the optimal cooling rate during the cryopreservation to avoid the excessive dehydration (too slow cooling) or intracellular crystallization (too fast cooling). Storage: It is important to keep the constant storage temperatures without fluctuations to avoid the damage. Thawing: The rapid thawing rates are preferable since they allow to avoid the recrystallization process Quality control: It is important to use the panel of tests, which will show the overall viability/recovery of cells and their functional activity. It may include studying the membrane integrity, metabolic activity, growth in vitro and implementation of specific function. Block II. Working examples in the field of stem cell cryopreservation for clinical use • Alternative approaches for stem cell cryopreservation with reduced DMSO concentration • Alternative quality control methods for stem cell cryopreservation • Hypothemic storage (storage at 4°C) of stem cell suspensions for clinical applications storage, quality control Multipotent mesenchymal stromal cells Multipotent mesenchymal stromal cells Bone marrow Can differentiate into at least 3 lineages (adipogenic, osteogenic, chondrogenic) IMMUNOMODULATION Modulate immune response in vitro and in vivo. Provide anti-inflammatory action Cord membrane Adipose tisse Breast milk Dental pulp Peripheral blood Mesenchymal Stem Cells Amniotic fluid Umbilical cord blood Wharton's jelly Umbilical cord Dccidua basalis Placenta Ligamentum fa Iv um Identity, purity, potency = Quality control EU clinical trials involving'mesenchymal stem cell'. Total = 98 1% ■ Cardiovascular ■ CNS ■ Dermatology ■ Gastrointestinal ■ Genito-urinary Haematology ■ Immunology ■ Opthalmology Reconstruction ■ Respiratory/thoracic Skeletal Vascular Wound healing Endocrinology Wilson etal., 2019 The processing \ c CH3 DMSO — -► toxic, should be removed Replacements, additives to reduce or remove DMSO • Sugars (glucose, dextrose, trehalose, sucrose) • Macromolecules (albumin, HES) • Polymers (PEG, PVP, PLL) • Antifreeze proteins and biocompatible solutes (proline, ectoine) • Antioxidants and anti-apoptotic agents Non-toxic cryopreservation of adipose tissue MSCs Permeating CPA (DMSO, glycerol, ethylene glycol) Non-permeating CPA (Sugars, PEG, PVP, HES) i ^^^^^^^^ Dehydration Loading non-permeable CPA into cells If we culture cells in the presence of sugar- it will go inside the cells by endocytosis CiyoLetters 35 (3): 239-246 (2014) £ CrvoLetters, businessofJice^cr>roletters.org A SUGAR PRETREATMENT AS A NEW APPROACH TO THE Me,SO AND XENO-FREE CRYOPRESERVATION OF HUMAN MESENCHYMAL STROMAL CELLS Yuri A. Petrenko*. Olena Y. Rosukka, Vitalii Y. Mutsenko and Alexander Y. Petrenko SSO mM I 1100 mM n200 mM "300 mM CytotechnolorJy April 2017, Volume69,Issue!, pp 265-275 | Cite as DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival authors anc affiliations Ciena Rogulska 0 ^uri Petrenko, Alexander Petrenko Searching for best methods for the assessment of viability and functional activity of MSCs (QC - potency) Usual methods for MSCs: • Phenotype/viability Ability to grow (>3 days) Differentiation capacity (>3 weeks study) - recovery Quality Control MSCs should be characterized: Safety • Sterility, Endotoxin, Mycoplasma • Tests for opportunistic viruses Identity • Specific test to distinguish it from others Purity • Free of extraneous materials Potency • Assay for biological function Desirable methods for MSCs: Fast (72 hrs) & informative Show functional state of the cells • Mimic natural conditions Searching for best methods for the assessment of viability and functional activity of MSCs (QC - potency) Paracrine activity - capacity to secrete growth factors and cytokines 0 3D culture - capacity to build cell-cell contacts Non-frozen storage of cells, tissues or organs at positive temperatures, below physiological (37°C) Hypothermic storage Short-term Hypothermie storage in stem cell therapy Quality Control Transport to Patient Dosage Clinic Administration MSCs should be characterized: Safety • Sterility, Endotoxin, Mycoplasma • Tests for opportunistic viruses Identity • Specific test to distinguish it from others Potency • Assay for biological function Purity • Free of extraneous materials Minimally 72 hours are needed to conduct QC tests and deliver the MSCs to bedside Challenge MSCs should be administered to patient in clinically safe vehicle solution, which should preserve viability of cells during at least 72 hours The answer We should develop the hypothermic storage conditions or non-toxic cryopreservation protocol, when the solution can be used as vehicle for cell delivery Damaging factors during hypothermic storage What should we do? • Stabilize the membrane • Provide pH support • Reduce ROS by antioxidants • Provide ECM Maetal.,2021 Clinical-grade solution for hypothermic storage • Animal component-free • Simple, minimal essential composition Ensure sufficient viability of cells, preserve functional properties • Consists of clinically-acceptable compounds to ensure the use of the solution as a vehicle for the administration of cells to patient Petrenko Y., Chudickova M, Vackova I., Groh T., Kosnarova E., Čejková J., Turnovcova K., Petrenko A., Sykova E., Kubinova S. Stem cells international, 2019, Article ID 5909524 Trehalose Hypothermic storage We developed Buffered trehalose solution (BTS) □ 24 hrs H 48 hrs ■ 72 hrs Bioi nova tH,i;;i 'A bsf_^A Glucose Glucose We substitute Na+ for impermeable molecule BiCureSol® - Good Manufacturing Procedure (GMP)-compliant solution of excipients for storage and transportation of cells maintaining cell viability above 92% for at least 72 hours at 2-8 °C. 3D culture to improve the viability (alginate beads) MSCs encapsulated into alginate beads were stored in sealed vials at ambient (22°C) and hypothermic (4°C) temperatures l-st 2-nd 3-rd 1-st 2-nd 3-rd Storage period, day Storage period, day | | - MSCs in AMS | | - MSCs in suspensio # - P<0.05 with respect to cells in AMS Take-home message second part: Clinically-relevant approaches: compared to research grade approaches, the technologies associated with clinical-grade cryopreservation require using specific acceptable substances, reproducible techniques, closed systems and automation. Reduction of DMSO concentration: it is possible to pre-treat cells with sugars in culture to remarkably increase their survival after cryopreservation Importance of timing in hypothermic storage: In clinical cell manufacturing, the timing is important. The usual minimal quality control tests last around 72 hrs (sterility), so it is important to preserve the cells in non-frozen state for at least 3 days. During the hypothermic storage it is important to stabilize the membrane of the cells to avoid excessive cell swelling. QC control: It is necessary to develop the alternative potency assays to confirm the preservation of functional properties. These assays should be adequate to the expected clinical application