New Approaches to Cryopreservation of Cells, Tissues, and Organs

Abstract

In this concept article, we outline a variety of new approaches that have been conceived to address some of the remaining challenges for developing improved methods of biopreservation. This recognizes a true renaissance and variety of complimentary, high-potential approaches leveraging inspiration by nature, nanotechnology, the thermodynamics of pressure, and several other key fields. Development of an organ and tissue supply chain that can meet the healthcare demands of the 21st century means overcoming twin challenges of (1) having enough of these lifesaving resources and (2) having the means to store and transport them for a variety of applications. Each has distinct but overlapping logistical limitations affecting transplantation, regenerative medicine, and drug discovery, with challenges shared among major areas of biomedicine including tissue engineering, trauma care, transfusion medicine, and biomedical research. There are several approaches to biopreservation, the optimum choice of which is dictated by the nature and complexity of the tissue and the required length of storage. Short-term hypothermic storage at temperatures a few degrees above the freezing point has provided the basis for nearly all methods of preserving tissues and solid organs that, to date, have proved refractory to cryopreservation techniques successfully developed for single-cell systems. In essence, these short-term techniques have been based on designing solutions for cellular protection against the effects of warm and cold ischemia and basically rely upon the protective effects of reduced temperatures brought about by Arrhenius kinetics of chemical reactions. However, further optimization of such preservation strategies is now seen to be restricted. Long-term preservation calls for much lower temperatures and requires the tissue to withstand the rigors of heat and mass transfer during protocols designed to optimize cooling and warming in the presence of cryoprotective agents. It is now accepted that with current methods of cryopreservation, uncontrolled ice formation in structured tissues and organs at subzero temperatures is the single most critical factor that severely restricts the extent to which tissues can survive procedures involving freezing and thawing. In recent years, this major problem has been effectively circumvented in some tissues by using ice-free cryopreservation techniques based upon vitrification. Nevertheless, despite these promising advances there remain several recognized hurdles to be overcome before deep-subzero cryopreservation, either by classic freezing and thawing or by vitrification, can provide the much-needed means for biobanking complex tissues and organs for extended periods of weeks, months, or even years. In many cases, the approaches outlined here, including new underexplored paradigms of high-subzero preservation, are novel and inspired by mechanisms of freeze tolerance, or freeze avoidance, in nature. Others apply new bioengineering techniques such as nanotechnology, isochoric pressure preservation, and non-Newtonian fluids to circumvent currently intractable problems in cryopreservation.

Keywords: Cryopreservation; Isochoric cryopreservation; Liquidus tracking; Nanowarming; Non-Newtonian cryoprotection; Vitrification.

Fig. 1

New nature-inspired and bioengineering approaches to cryopreservation compared with classic methods.

Fig. 2

Supercooled rat livers successfully transplanted after 3–4 days of storage. The 30-day survival rate was 100% for livers stored 3 days, and 58% for livers stored for 4 days. By comparison, recipients of livers stored in UW medium for 3 or 4 days perished within 48 h. 3-OMG, 3-O-methyl-D-glucose; HP, hypothermic preservation in UW medium at 4°C; SNMP, subnormothermic machine perfusion (21°C); UW, University of Wisconsin. a Schematic of the supercooling temperature profile. Loading of 3-OMG, as an additive to Williams' E-based medium, is performed by SNMP for 1 h through the portal vein. Cooling to 4°C (1°C/min) is carried out during perfusion. The liver is then flushed with 4°C-cold UW solution containing 5% w/v 35 kDa PEG, and slowly cooled to −6°C (1°C/10 min). Storage is maintained for up to 96 h, after which rewarming to 4°C takes place (1°C/10 min). The liver is then flushed with 21°C-warm, oxygenated SNMP medium, recovered with 3 h of SNMP, and transplanted orthotopically. b Kaplan-Meier curve of transplantation recipients (n > 6 in all groups shown). c Posttransplantation trends in transaminase output that normalize in a month; 3 months after transplantation, the differences had completely vanished (data not shown). Adapted from Berendsen et al. [86].

Fig. 3

A Section of the phase diagram for DMSO-H₂O showing the equilibrium melting curve in blue (Tm liquidus). The stepped line above the curve represents a scheme for incremental equilibration of a tissue with sufficient cryoprotectant such that the system does not freeze during cooling. B Function of smooth muscle tissue after cooling to −21°C in either the frozen (F) or unfrozen (NF) state. Histograms of post-warming contractility (mean ± SEM) normalized to the control responses derived prior to cooling. Avoidance of ice is critical for good survival with high function, and liquidus tracking ensures that the system remains in equilibrium and ice-free during subzero cooling [63].

Fig. 4

Schematic illustrating a tissue nanowarming system [137]. Nanowarming scale: up from 1 to 15 kW inductive heating system. The 15-kW systems enable heating up to 80 mL. The illustration shows the limitations of convective cooling and rewarming (C, D) compared to nanowarming (E, F) of vitrification (success and failure) of 0.5- to 2.5-cm-radius cylinders. Failure and success (red and green shading) is defined by the critical minimum cooling and warming rates for VS55 vitrification solution and a thermal stress <3.2 MPa [137].

Fig. 5

Top panel: schematic of an isochoric (constant-volume) system, in which the biologic in solution is cooled; the temperature is progressively reduced from left to right to control ice nucleation separated from the biological sample in a thermodynamically equilibrated way. Isochoric preservation apparatus. Lower left panel: isochoric preservation vessel, pressure and temperature measurement, and DAC-computer connection assembly. Lower right panel: example of a system for cooling the isochoric chambers. The cooling bath (NESLAB RT-140) with hoses running out of the cooling bath and into the foam bath. One hose outputs the cooling fluid from the cooling bath to the foam bath. Another hose carries fluid from the foam bath to the cooling bath. The fluid used is a 50% by volume ethylene glycol/water mixture. (Drawings courtesy of P.A. Perez.)

Fig. 6

At least 45 vertebrates achieve supercooling at body temperatures as low as −14°C [74]. Mammals like the arctic ground squirrel [76] hibernate/supercool with body temperatures of −3°C (–8°C in the laboratory [75, 108]) for up to 3 weeks [108], with every organ “banked” without injury. Even Caiman crocodilus, which can grow to a weight of over 58 kg, can supercool to below −5°C. Mammals such as the beaked whale dive to depths of 3 km [167] with no tissue injury; the lowest depths of the Mariana Trench support animals [168] at pressures of more than 110 MPa and some fish go down to almost the same depths (deeper than the height of Mt. Everest [169]).

Fig. 7

Scheme illustrating three new technological approaches which could be used during both CPA loading/cooling and warming/CPA unloading. (1) On-demand non-Newtonian decrease in viscosity allows for accelerated CPA loading of tissues at lower temperatures, in turn leading to less toxicity and/or permitting the use of increased CPA concentrations. (2) On-demand non-Newtonian increase in viscosity enables ice avoidance and reduced toxicity due to lower molecular diffusion. The first effect allows decreased CPA concentration needs and the second effect allows increased CPA concentrations. All of these effects can be leveraged during both cooling and warming. Each of the on-demand increases in viscosity could also be achieved via a rheomagnetic approach. (3) Once the system is at temperatures below −60/–70°C, the risk of ice growth during cooling or of devitrification during rewarming is no longer a problem even with current protocols, and preservation can proceed without any active viscosity control. At the same time, because any or all of these steps potentially allow the use of higher CPA concentrations, new cooling and warming protocols that proceed at slower speeds could now be used. The ability (1) to go slower and (2) thereby also to gain more degrees of freedom for annealing protocols should also permit decreased risks of fracturing or cracking. CPA, cryoprotective agent.