The Big Freeze – Artistry of Cell Preservation

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Introduction to Cryopreservation



Cryopreservation describes the controlled process of freezing biological material at extremely low temperatures in order to discontinue biochemical reactions, including those associated with cell death and DNA damage, to allow long-term storage and/or transport. Given recent medical advances in cell-based therapies, the importance of cryopreservation continues to increase based on the necessity of providing intact cellular and tissue components for both research and clinical applications. Preservation of these materials is achieved by controlling both the freezing rate and the composition and concentration of the freezing medium (cryoprotectant), all of which vary according to the material targeted for the frozen state.


Although applications for cryopreservation vary widely, the primary goal is to protect the integrity of cellular matter in the frozen state and permit a return to biological function when thawed. Cell lines generally replicate rapidly under specific culture conditions and require sub-culturing to prevent over-crowding or possible contamination. A common procedure in research settings is to preserve stocks of these cells by freezing, generally at −80°C or colder, to allow their future use; however, the conversion of cells to the frozen state is complicated by the presence of inorganic molecules and salt. Under such conditions, the possible formation of intracellular ice crystals can potentially damage both organelles and

the cell membrane, resulting in decreased viability and altered morphology and function. A common example of ice crystal damage to biological tissue is the need to discard vegetables that have accidently frozen in a refrigerator, rendering them inedible.


Cryopreservation techniques attempt to eliminate the inherent variability of freezing by pretreatment with media specifically tailored to the target material, followed by time-controlled freezing and subsequent transfer to a system capable of maintaining temperatures as low as −196°C. This process is designed to 1)

preserve material in a quality-controlled state ready for immediate post-thaw use, 2) prevent biological activity and degradation of genetic material, 3) allow the establishment of stocks that enable long-term use of the material with an expectation of reproducible results, and 4) support the efficient transport of the

material between collection, manufacturing, and clinical sites. The critical nature of this process is highlighted by its common use in a wide range of applications.


Regenerative medicine


Securing and stabilizing specific cell types (e.g., via biopsy, apheresis, etc.) is the critical first step in ensuring successful therapeutic applications. Defined as the replacement or regeneration of human cells, tissues, or organs to restore or establish normal function [1], regenerative medicine encompasses a wide

range of therapies from organ and tissue transplantation to tissue-engineered scaffolds and cellular therapies. Many of these applications involve pluripotent stem cells (SCs) that can be directed to form any type of cell or tissue within the body, making them an ideal starting material for both research and therapeutic purposes [2]. Additionally, the clinical use of regulatory T cells (Tregs) in the treatment of autoimmune disorders and for immunomodulation during organ/graft transplantation has significantly expanded; however, Treg function and efficacy are dependent upon their lineage as determined by the expression of specific surface markers [3]. In these cases, proper cryopreservation techniques allow the storage of material obtained either immediately after acquisition from the patient or after in vitro culturing to genetically engineer those cells into potent therapies.


Cancer immunotherapy


Applications of adoptive transfer of immune cells highlight a paradigm shift in cancer therapy from non-specific chemotherapeutic regimens that affect both malignant and non-malignant cells to an approach that recruits the host immune system in specifically targeting cancer cells. Adoptive transfer involves the

extraction, modification, and ex vivo expansion of patient-derived (autologous) immune cells for reintroduction to the patient, whereas allogeneic therapy describes a similar process involving donor cells.


Chimeric antigen receptor (CAR) T cells, natural killer (NK) cells, and CAR NK cells are examples of these techniques, applications of which have resulted in the successful treatment of multiple cancer types [4-6]. The isolation, modification, and expansion of these cell subsets are complicated, expensive, and time-consuming processes that emphasize the importance of both cryopreservation of the final product and ensuring their post-thaw viability and functionality [5, 7]. Furthermore, “off-the-shelf” versions of these therapies enable transformation of cell precursors (e.g., peripheral blood mononuclear cells, induced pluripotent or embryonic SCs) into specific immune cell subsets; however, proper cryopreservation of these precursors is critical to ensure the genotype/phenotype integrity of the starting material after thawing [8].


Reproductive medicine


Increasing demand for assisted reproductive technology (ART) has coincided with improvements in cryopreservation techniques, resulting in a significant expansion of ART indications to preserve fertility. Specific cryopreservation practices, including vitrification, have enabled the widespread and expanding use of in vitro fertilization procedures, including options for safely freezing oocytes/embryos to allow for preimplantation genetic testing and/or postponed embryo transfer according to associated medical timelines or personal preference [9]. Moreover, cryopreservation can preserve fertility for men undergoing vasectomy or in advance of treatments that may compromise their fertility, such as chemotherapy, radiotherapy, or surgery [10].


Organ cryopreservation and pharmaceutical research


The critical need for methods to preserve organs has long been recognized by the discard of hearts [11], kidneys [12], and pancreases [13] that have perished during the extended time needed to identify and transport these organs from the donor to a suitable recipient. While the successful freezing of whole organs is still in its infancy, advances in cryopreservation techniques have extended global access to

transplantation opportunities, as well as enabled research on methods to increase immune tolerance and improve graft characterization. Moreover, recent applications of mini-organs, or ‘organoids’, as models of both organs and tumors have rendered them an invaluable resource of information for disease modeling,

pharmaceutical research, and precision medicine. Importantly, their status as multicellular aggregates derived from stem cells or organ progenitors enables their cryopreservation as a “biobank” for use in high-throughput screening and other diagnostic applications [14].


Plant cryopreservation


The preservation of plant genetic resources is important for food security and agrobiodiversity and in breeding programs to obtain new or more productive plants. Plant cryopreservation techniques involve the removal of freezable water from tissues by physical or osmotic dehydration, followed by ultra-rapid

freezing that ultimately allows the safe and long-term conservation of plant biodiversity without the risk of genetic modifications [15].


In future blogs, we will explore in further detail how to determine the best method to preserve biological material, introduce considerations and planning required prior to cryopreservation, and outline the challenges associated with the entire freezing and thawing process. These blogs will have a specific eyetowards ‘best practices’ and standards applied to each step of the cryopreservation procedure, the equipment involved, and the importance of quality control throughout the operation.


References

1. Mason C, Dunnill P. A brief definition of regenerative medicine. Regen Med. 2008;3:1–5.

2. Qian T, Maguire SE, Canfield SG, et al. Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells. Sci Adv. 2017;3:e1701679.

3. Gołab K, Grose R, Placencia V, et al. Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype. Oncotarget. 2018;9:9728–40.

4. Oh E, Min B, Li Y, et al. Cryopreserved human natural killer cells exhibit potent antitumor efficacy against orthotopic pancreatic cancer through efficient tumor-homing and cytolytic ability. Cancers (Basel). 2019;11:966.

5. Panch SR, Srivastava SK, Elavia N, et al. Effect of cryopreservation on autologous chimeric antigen receptor T cell characteristics. Mol Ther. 2019;27:1275–85.

6. Xie G, Dong H, Liang Y, et al. CAR-NK cells: a promising cellular immunotherapy for cancer. EBioMedicine. 2020;59:102975.

7. Mark C, Czerwinski T, Roessner S, et al. Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells. Nat Commun. 2020;11:5224.

8. Heipertz EL, Zynda ER, Stav-Noraas TE, et al. Current perspectives on "off-the-shelf" allogeneic NK and CAR-NK cell therapies. Front Immunol. 2021;12:732135.

9. Bosch E, De Vos M, Humaidan P. The future of cryopreservation in assisted reproductive technologies. Front Endocrinol (Lausanne). 2020;11:67.

10. Jang TH, Park SC, Yang JH, et al. Cryopreservation and its clinical applications. Integr Med Res.2017;6:12–8.

11. Ardehali A. 1. While millions and millions of lives have been saved, organ transplantation still faces massive problems after 50 years; organ preservation is a big part of the solution. Cryobiology. 2015;71:164–5.

12. Reese PP, Harhay MN, Abt PL, et al. New solutions to reduce discard of kidneys donated for transplantation. J Am Soc Nephrol. 2016;27:973–80.

13. Taking Organ Utilisation to 2020 [https://nhsbtdbe.blob.core.windows.net/umbraco-assets-

corp/1395/nhsbt_organ_donor_strategy.pdf].

14. Gunti S, Hoke ATK, Vu KP, London NR Jr. Organoid and spheroid tumor models: techniques and applications. Cancers (Basel). 2021;13:874.

15. Benelli C. Plant cryopreservation: a look at the present and the future. Plants (Basel). 2021;10:2744.

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