Review

Authors: David Whaley, Kimia Damyar, Rafal P Witek...

The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress was revealed to be a main contributor to cell death during cryopreservation. Consequently, the addition of cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryopreservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1ºC/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields. For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability. Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the efficiency of the current cryopreservation methods.

Topics: Cell Survival; Cryopreservation; Cryoprotective Agents; Humans

PubMed: 33757335
DOI: 10.1177/0963689721999617

Review

Authors: Gang Zhao, Jianping Fu

Cryopreservation has utility in clinical and scientific research but implementation is highly complex and includes labor-intensive cell-specific protocols for the addition/removal of cryoprotective agents and freeze-thaw cycles. Microfluidic platforms can revolutionize cryopreservation by providing new tools to manipulate and screen cells at micro/nano scales, which are presently difficult or impossible with conventional bulk approaches. This review describes applications of microfluidic tools in cell manipulation, cryoprotective agent exposure, programmed freezing/thawing, vitrification, and in situ assessment in cryopreservation, and discusses achievements and challenges, providing perspectives for future development.

Topics: Animals; Cryopreservation; Cryoprotective Agents; Cytological Techniques; Equipment Design; Freezing; Humans; Mice; Microfluidic Analytical Techniques; Oocytes; Vitrification

PubMed: 28153517
DOI: 10.1016/j.biotechadv.2017.01.006

Review

Authors: Gloria D Elliott, Shangping Wang, Barry J Fuller...

Cryopreservation has become a central technology in many areas of clinical medicine, biotechnology, and species conservation within both plant and animal biology. Cryoprotective agents (CPAs) invariably play key roles in allowing cells to be processed for storage at deep cryogenic temperatures and to be recovered with high levels of appropriate functionality. As such, these CPA solutes possess a wide range of metabolic and biophysical effects that are both necessary for their modes of action, and potentially complicating for cell biological function. Early successes with cryopreservation were achieved by empirical methodology for choosing and applying CPAs. In recent decades, it has been possible to assemble objective information about CPA modes of action and to optimize their application to living systems, but there still remain significant gaps in our understanding. This review sets out the current status on the biological and chemical knowledge surrounding CPAs, and the conflicting effects of protection versus toxicity resulting from the use of these solutes, which are often required in molar concentrations, far exceeding levels found in normal metabolism. The biophysical properties of CPAs that allow them to facilitate different approaches to cryogenic storage, including vitrification, are highlighted. The topics are discussed with reference to the historical background of applying CPAs, and the relevance of cryoprotective solutes in natural freeze tolerant organisms. Improved cryopreservation success will be an essential step in many future areas such as regenerative medicine, seed banking, or stem cell technology. To achieve this, we will need to further improve our understanding of cryobiology, where better and safer CPAs will be key requirements.

Topics: Animals; Antifreeze Proteins; Cell Physiological Phenomena; Cryopreservation; Cryoprotective Agents; Freezing; Humans; Ice; Organ Preservation; Solutions; Vitrification

PubMed: 28428046
DOI: 10.1016/j.cryobiol.2017.04.004

Authors: Yu Chen, Carlos A Tristan, Lu Chen...

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

Topics: Cell Culture Techniques; Cell Differentiation; Cell Survival; Cryopreservation; Cryoprotective Agents; Gene Expression Regulation; High-Throughput Screening Assays; Humans; Pluripotent Stem Cells; Polypharmacology; rho-Associated Kinases

PubMed: 33941937
DOI: 10.1038/s41592-021-01126-2

Authors: Robert L McIntyre, Gregory M Fahy

We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced cryopreservation science for the neurobiological research community. ASC is a new brain-banking technique designed to facilitate neuroanatomic research such as connectomics research, and has the unique ability to combine stable long term ice-free sample storage with excellent anatomical resolution. To demonstrate the feasibility of ASC, we perfuse-fixed rabbit and pig brains with a glutaraldehyde-based fixative, then slowly perfused increasing concentrations of ethylene glycol over several hours in a manner similar to techniques used for whole organ cryopreservation. Once 65% w/v ethylene glycol was reached, we vitrified brains at -135 °C for indefinite long-term storage. Vitrified brains were rewarmed and the cryoprotectant removed either by perfusion or gradual diffusion from brain slices. We evaluated ASC-processed brains by electron microscopy of multiple regions across the whole brain and by Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) imaging of selected brain volumes. Preservation was uniformly excellent: processes were easily traceable and synapses were crisp in both species. Aldehyde-stabilized cryopreservation has many advantages over other brain-banking techniques: chemicals are delivered via perfusion, which enables easy scaling to brains of any size; vitrification ensures that the ultrastructure of the brain will not degrade even over very long storage times; and the cryoprotectant can be removed, yielding a perfusable aldehyde-preserved brain which is suitable for a wide variety of brain assays.

Topics: Animals; Brain; Cryopreservation; Cryoprotective Agents; Ethylene Glycol; Glutaral; Rabbits; Swine; Tissue Banks; Vitrification

PubMed: 26408851
DOI: 10.1016/j.cryobiol.2015.09.003

Review

Authors: Giada Marcantonini, Desirée Bartolini, Linda Zatini...

Cryoprotective and cytoprotective agents (Cytoprotective Agents) are fundamental components of the cryopreservation process. This review presents the essentials of the cryopreservation process by examining its drawbacks and the role of cytoprotective agents in protecting cell physiology. Natural cryoprotective and cytoprotective agents, such as antifreeze proteins, sugars and natural deep eutectic systems, have been compared with synthetic ones, addressing their mechanisms of action and efficacy of protection. The final part of this article focuses melatonin, a hormonal substance with antioxidant properties, and its emerging role as a cytoprotective agent for somatic cells and gametes, including ovarian tissue, spermatozoa and spermatogonial stem cells.

Topics: Antioxidants; Cryopreservation; Cryoprotective Agents; Humans; Male; Melatonin; Spermatozoa

PubMed: 35630729
DOI: 10.3390/molecules27103254

Authors: Hiroki Takeuchi, Mikiko Nishioka, Tadashi Maezawa...

In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.

Topics: Cryopreservation; Cryoprotective Agents; Glycerol; Humans; Male; Polylysine; Raffinose; Specimen Handling; Spermatozoa

PubMed: 34201225
DOI: 10.3390/cells10061435

Authors: Grace Massiah, Giuseppe De Palma, Antonio Negri...

In the second reconstructive phase of the breast after mastectomy, lipofilling is often necessary. Currently, lipofilling occurs immediately after autologous adipose tissue harvesting procedure, but most of the patients, usually, require multiple sessions to obtain a satisfactory result. Therefore, the need of repeated surgical harvesting outputs implies high risk of patients' morbidity and discomfort as well as increasing medical time and costs. The aim of our pilot study was to find out a feasible method to cryopreserve adipose tissue, in order to avoid reiterated liposuctions. Lipoaspirates samples have been harvested from 10 women and preserved by three methods: (1) the first one, using 10% MeSO and 20% human albumin from human plasma as cryoprotective agents; (2) the second one, adding 5% MeSO as cryoprotective agent; 3) the last one, without any cryoprotective agent. Fresh and cryopreserved fat samples, obtained through the aforementioned processes, have been analyzed ex vivo. The efficiency of the cryopreservation methods used was determined by adipocyte viability and the expression of adipocytes surface markers. Lipoaspirates stored at -196 °C for 3 months, after thawing, retained comparable adipocyte viability and histology to fresh tissue and no significant differences were found between the three methods used. Although the current results, differences between the methodologies in terms of viability may not become evident until breast lipofilling using frozen-thawed cryopreserved tissue.

Topics: Adipose Tissue; Breast Neoplasms; Cryopreservation; Cryoprotective Agents; Dimethyl Sulfoxide; Female; Humans; Mastectomy; Pilot Projects

PubMed: 34333035
DOI: 10.1016/j.cryobiol.2021.07.016

Review

Authors: Alireza Abazari, Nadr M Jomha, Janet A W Elliott...

Cryopreservation has numerous practical applications in medicine, biotechnology, agriculture, forestry, aquaculture and biodiversity conservation, with huge potentials for biological cell and tissue banking. A specific tissue of interest for cryopreservation is the articular cartilage of the human knee joint for two major reasons: (1) clinically, there exists an untapped potential for cryopreserved cartilage to be used in surgical repair/reconstruction/replacement of injured joints because of the limited availability of fresh donor tissue and, (2) scientifically, successful cryopreservation of cartilage, an avascular tissue with only one cell type, is considered a stepping stone for transition from biobanking cell suspensions and small tissue slices to larger and more complicated tissues. For more than 50years, a great deal of effort has been directed toward understanding and overcoming the challenges of cartilage preservation. In this article, we focus mainly on studies that led to the finding that vitrification is an appropriate approach toward successful preservation of cartilage. This is followed by a review of the studies on the main challenges of vitrification, i.e. toxicity and diffusion, and the novel approaches to overcome these challenges such as liquidus tracking, diffusion modeling, and cryoprotective agent cocktails, which have resulted in the recent advancements in the field.

Topics: Animals; Cartilage, Articular; Chondrocytes; Cryopreservation; Cryoprotective Agents; Humans; Knee Joint; Vitrification

PubMed: 23499618
DOI: 10.1016/j.cryobiol.2013.03.001

Review

Authors: George Frimpong Boafo, Kosheli Thapa Magar, Marlene Davis Ekpo...

To improve liposomes' usage as drug delivery vehicles, cryoprotectants can be utilized to prevent constituent leakage and liposome instability. Cryoprotective agents (CPAs) or cryoprotectants can protect liposomes from the mechanical stress of ice by vitrifying at a specific temperature, which forms a glassy matrix. The majority of studies on cryoprotectants demonstrate that as the concentration of the cryoprotectant is increased, the liposomal stability improves, resulting in decreased aggregation. The effectiveness of CPAs in maintaining liposome stability in the aqueous state essentially depends on a complex interaction between protectants and bilayer composition. Furthermore, different types of CPAs have distinct effective mechanisms of action; therefore, the combination of several cryoprotectants may be beneficial and novel attributed to the synergistic actions of the CPAs. In this review, we discuss the use of liposomes as drug delivery vehicles, phospholipid-CPA interactions, their thermotropic behavior during freezing, types of CPA and their mechanism for preventing leakage of drugs from liposomes.

Topics: Cryoprotective Agents; Liposomes; Ice; Freezing; Excipients; Phospholipids

PubMed: 36293340
DOI: 10.3390/ijms232012487