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 preserv...

Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma

“…However, freeze-thaw cycles have adverse effects on many tissues and can cause cell membrane rupture; molecular damage to DNA, proteins, and lipids; and denaturing of proteins from freeze concentration of standard buffers [ 17 , 18 ]. Additionally, heterogeneous cell types react differently to freezing techniques so certain populations of cells could be lost in the freeze-thaw process [ 19 ]. We identified a well-validated method to circumvent these adverse effects that uses cryoprotectants, which act intracellularly to prevent crystallization and cell rupture, and extracellularly to reduce hyperosmotic effects that cause denaturation [ 19 , 20 ].…”

“…Additionally, heterogeneous cell types react differently to freezing techniques so certain populations of cells could be lost in the freeze-thaw process [ 19 ]. We identified a well-validated method to circumvent these adverse effects that uses cryoprotectants, which act intracellularly to prevent crystallization and cell rupture, and extracellularly to reduce hyperosmotic effects that cause denaturation [ 19 , 20 ]. Common cryoprotectants include dimethylsulfoxide (DMSO), glycerol, and ethylene glycol which act intracellularly, and sucrose, dextrose, and polyvinylpyrrolidone which act extracellularly [ 17 , 19 ].…”

Me2SO perfusion time for whole-organ cryopreservation can be shortened: Results of micro-computed tomography monitoring during Me2SO perfusion of rat hearts

“…Whole-organ cryopreservation would revolutionize the options available for organ donation and treatment of transplant recipients, as it would gain time to improve donor–recipient matching and therefore reduce the numbers of transplant rejections, as well as the intensity of the immunosuppression required [ 9 ]. Despite partial successes have been achieved with the kidney [ 10 ], heart [ 9 ], uterus [ 11 – 14 ], and ovary [ 15 , 16 ] in animal models, using perfusion-based equilibration protocols for CPAs as previously reviewed [ 17 ], the current freezing techniques do not ensure the survival and function of organs and they are far from being ready for routine clinical applications [ 18 ]. Most of the perfusion-based protocols for organ cryopreservation that have been published share the common feature that the CPA equilibration durations used range from approximately 25 minutes up to a couple of hours.…”

Cryomedia Formula: Cellular Molecular Perspective

“…Cryopreservation is one of the most effective techniques that widely used for preserving living cells and organs in research and therapeutic industries [1]. The principle of cryopreservation is to protect cells from the application of super low temperature and ice crystal formation by using media that consist of antifreezing or cryoprotective (CPA) substances such as; glycerol, dimethylsulfoxide (DSMO) or trehalose.…”