CELL & GENE THERAPY INSIGHTS LATEST ADVANCES IN ADDRESSING BIOPRESERVATION CHALLENGES EXPERT INSIGHT Applications and optimization of cryopreservation technologies to cellular therapeutics Barry Fuller, Jordi Gonzalez-Molina, Eloy Erro, Joana De Mendonca, Sheri Chalmers, Maooz Awan, Aurore Poirier, Clare Selden Delivery of cell therapies often requires the ability to hold products in readiness whilst logistical, regulatory and potency considerations are dealt with and recorded. This requires reversibly stopping biological time, a process which is often achieved by cryopreservation. However, cryo- preservation itself poses many biological and biophysical challenges to liv- ing cells that need to be understood in order to apply the low temperature technologies to their best advantage. This review sets out the history of applied cryopreservation, our current understanding of the various pro- cesses involved in storage at cryogenic temperatures, and challenges for robust and reliable uses of cryopreservation within the cell therapy arena. Submitted for Review: 24 April 2017 u Published: 6 Jun 2017 The explosion of interest in cell ther- such consideration is product stor- to meet these demands. This brief apies over recent years has necessarily age (cryobanking), which is often review will discuss the salient top- focused attention on the processes required to sustain cell therapy de- ics within this field and include the that will enable product delivery in livery to the end user facilities at the history of cryopreservation, what we reliable, regulatory-compliant and required time, providing a quality currently understand about the bio- robust ways. These considerations assured product with required safe- physical and biological processes that sometimes introduce new challeng- ty and potency characteristics [1,2]. allow successful cell recoveries after es that may not have been import- Cryopreservation in its various forms storage, and additional challenges ant in the original laboratory studies is one of the main facilitatory tech- for scale up and regulatory oversight on that particular cell therapy. One nologies for cell therapies to be able when the technology is applied. www.insights.bio 359 CELL & GENE THERAPY INSIGHTS HISTORY OF the same era, such as Maximov [4] CRYOPRESERVATION provided evidence that in plants ca- The widely accepted term to de- pable of overwintering in northern scribe preservation of living cells, Russia, the tissues went through a capable of biological reanimation, seasonal hardening process, which is cryopreservation. In reality, cell was accompanied by accumulation biopreservation can be achieved of solutes such as sugars. Chambers across a range of reduced tempera- and Hale [5] at the turn of the 20th tures, which enhance survival by Century provided additional evi- reducing biological activities, but dence from light microscope stud- with limitations dependent upon ies that there was an osmotic effect the chosen modality. The common of freezing directly on the plant conception of cryopreservation is cells that was accompanied by lysis storage of living cells at the deep on thawing. Over the subsequent cryogenic temperatures provided by 50 years, others continued to ex- liquid nitrogen or the associated va- plore the biophysical principles pour phase (ranging from -196o to and biological effects of the water– approximately -170oC). ice phase transition; for example The challenge for biology of Luyet [6] made many pertinent ob- course is the phase change of wa- servations on ice crystal structures, ter that occurs at the rather in- changes in these brought about by conveniently high temperature alterations in the kinetics of cool- (for biopreservation purposes) just ing or the presence of solutes in below 0oC. There have been many the aqueous medium, and the ef- studies over the past few hundred fects on living cells. This collected years on the biological effects of knowledge base undoubtedly influ- freezing, and the allied phenome- enced Polge [7] and his colleagues non of freeze tolerance in overwin- in their studies in 1949 on freezing tering species (most often in the of reproductive cells (notably fowl plant kingdom). The growing un- spermatozoa to enhance animal derstanding of freezing effects fol- breeding in the period post the Sec- lowed the development of micro- ond World War) which resulted in scopes capable of directly observing the first clear evidence of recovery the freezing process; for example of functional cells after deliberate Molisch [3] described the freezing deep cryogenic exposure (in their process in plant tissues, which im- case to -79oC using solid carbon mediately highlighted one of the dioxide – liquid nitrogen was not central problems with ice forma- readily available at that time). The tion – the fact that ice, derived as key to their success was the expo- the phase change of pure water, re- sure of the sperm to glycerol ahead sulted in exposure of the cells to a of the cooling process, which Polge residual hypertonic environment as later acknowledged was partly by solutes (originally dissolved in the good fortune, but nevertheless (and aqueous environment) are excluded with the benefit of hindsight), com- from the ice crystal lattice. In simple bined all of those previous studies terms, the cells experienced a lethal into one successful outcome. That osmotic stress that could be detect- success also fuelled a global effort ed at the structural level very quick- to better understand and maxi- ly after thawing. Other scientists of mize the opportunities provided in 360 DOI: 10.18609/cgti.2017.038 expert INSIGHT biology and medicine for extend- results in the often observed slight ed biopreservation, culminating in change in density of ice over liquid the definition of the term ‘cryobi- water. The stabilisation of the net- ology’ to encompass the necessary work also results in the well-known collaboration between biologists, release of energy detectable as the engineers and physicists to better latent heat of ice formation. The understand the processes, and the ice crystal lattice cannot maintain establishment of the International previously accommodated solutes, Society for Cryobiology in 1963. which become excluded into the residual liquid volume surrounding the growing ice interface, which in turn depresses the freezing point of CURRENT UNDERSTANDING the residual water so that cells are OF CRYOPRESERVATION exposed to progressively higher sol- utes in a progressively smaller liquid The water–ice phase water space. Thus ‘freezing’ is not an transition instantaneous event in most practi- As is universally accepted, liquid cal applications, even though it may water is the essential component for appear so to the naked eye. almost all biological processes [8], Residual mobile water, all-be- and its removal during the forma- it as a tiny fraction of the origi- tion of ice poses extreme challeng- nal water volume, can be detected es. There have been many excellent down to surprisingly low tempera- reviews on this topic, but a decade tures [11], contributing to the pro- ago Mazur [9] and Muldrew and gressive osmotic stress experienced colleagues [10] provided excellent by the cells. Multiple biological discussions on the topic with spe- targets for this type of injury have cific relevance to cryobiology. These been discussed, including destabil- are beyond the remit of the current isation of cell membranes, change discussion, but as a brief summary, in the intracellular milieu includ- water exists in the liquid state in a ing pH, chemical and structural random but self-associating matrix changes to organelles and to pro- (on an extremely brief timescale) teins; it is fair to say that we still through interactions of hydrogen do not fully understand all the bi- bonding, which for biology enables ological consequences [9,10]. What solvation of essential ions and sol- is clear is that in almost all cases, utes, and structural stability of many cell membranes and intracellular macromolecules. During cooling, macromolecules to some degree energy within the system is removed hinder the kinetics of the forma- and water molecule self-association tion of ice crystals, such that ice leads to longer-lived intermolecular preferentially initiates and grows connections that result in ice nuclei. in the aqueous external solution In this process, central water mole- (i.e., the supporting culture medi- cules hydrogen bond with four sur- um in cell therapies), providing the rounding others that at the point of osmotic driving force for water to freezing repeat indefinitely through- leave the intracellular environment out the aqueous milieu to yield hex- and effectively shrinking the cells, agonal ice with which we are all fa- which can be observed in real-time miliar. The open lattice nature also by cryomicroscopy [12]. Cell & Gene Therapy Insights - ISSN: 2059-7800 361 CELL & GENE THERAPY INSIGHTS Cryoprotectants: solutes However, it should be noted that in that enhance survival both these (and subsequent cases), during freezing the CPA solutes need to be applied in concentrations far higher than Given this understanding of events for other solutes normally present during freezing, we can begin to in the cell media solutions, which in understand the relevance of glycerol itself introduces complex biological in Polge’s original successful experi- challenges. ments [7], and the observations that The identification of these over-wintering species frequently freeze-protecting solutes led