Cryopreservation: Evolution of Molecular Based Strategies

Abstract

Cryopreservation (CP) is an enabling process providing for on-demand access to biological material (cells and tissues) which serve as a starting, intermediate or even final product. While a critical tool, CP protocols, approaches and technologies have evolved little over the last several decades. A lack of conversion of discoveries from the CP sciences into mainstream utilization has resulted in a bottleneck in technological progression in areas such as stem cell research and cell therapy. While the adoption has been slow, discoveries including molecular control and buffering of cell stress response to CP as well as the development of new devices for improved sample freezing and thawing are providing for improved CP from both the processing and sample quality perspectives. Numerous studies have described the impact, mechanisms and points of control of cryopreservation-induced delayed-onset cell death (CIDOCD). In an effort to limit CIDOCD, efforts have focused on CP agent and freeze media formulation to provide a solution path and have yielded improvements in survival over traditional approaches. Importantly, each of these areas, new technologies and cell stress modulation, both individually and in combination, are now providing a new foundation to accelerate new research, technology and product development for which CP serves as an integral component. This chapter provides an overview of the molecular stress responses of cells to cryopreservation, the impact of the hypothermic and cell death continuums and the targeted modulation of common and/or cell specific responses to CP in providing a path to improving cell quality.

Technical Considerations in the Freezing, Low-Temperature Storage and Thawing of Stem Cells for Cellular Therapies

“…In addition to the incorporation of materials into the CPA to control ice formation, compounds can be added either to the CPA or the post-thaw culture medium to control cryopreservation-induced, delayed-onset cell death through a strategy of targeted apoptotic control [18, 50]. Such compounds include free radical scavengers, ion chelators, and protease inhibitors as well as both caspase inhibitors and Rho-kinase (ROCK) inhibitors which target the apoptotic cascade.…”

“…However, when temperatures are lowered below physiological temperatures, “extracellular-type” solutions no longer balance ionic changes or provide sufficient buffering capacity for the changed conditions. The use of “intracellular-type” solutions including Unisol, Eurocollins, and HypoThermosol (Table 1) have been shown to improve cryopreservation outcomes compared to the same CPAs in “extracellular-type” vehicle solutions [50, 51]. …”

Applications and optimization of cryopreservation technologies to cellular therapeutics

“…In addition, if small intracellular ice nuclei were established during the cooling process, but did not grow to become injurious, these may grow during warming, again depending on the kinetics of warming, such that 'freezing during thawing' can be conceptualised [42]. For these various reasons, fast warming has traditionally been favored [9,43]. In fact, for slowly cooled cryopreserved cells, the impact of warming kinetics is complex and not easy to predict.…”

Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal–epidermal engineered skin substitutes

“…These results suggest that in the conditions described here, inhibition of melanin expression in hM from darkly pigmented skin prior to cryopreservation is not required to populate sufficient densities of hM into ESS‐P for transplantation and restoration of complete cutaneous pigmentation. Viability of cells following cryopreservation is known to be affected by the pigment density and rate of freezing, in addition to other factors such as cryoprotectant formulation and apoptosis inhibitors (Baust, Corwin, Snyder, Van Buskirk, & Baust, ; Mathew, Baust, Van Buskirk, & Baust, ). In preclinical studies of melanocyte transplantation (Bottcher‐Haberzeth et al., ; Navarro et al., ; Swope, Supp, Schwemberger, Babcock, & Boyce, ) and hM biology in engineered skin in vitro (Parenteau et al., ; Regnier, Tremblaye, & Schmidt, ; Regnier et al., ; Swope et al., ), densities of hM ranged from 1.0 to 5.0 × 10 4 /cm 2 .…”