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Cell Transplantation 2021The 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... (Review)
Review
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 -
Cryobiology Jun 2017Cryopreservation has become a central technology in many areas of clinical medicine, biotechnology, and species conservation within both plant and animal biology.... (Review)
Review
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 -
Biotechnology Advances 2017Cryopreservation has utility in clinical and scientific research but implementation is highly complex and includes labor-intensive cell-specific protocols for the... (Review)
Review
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 -
Nature Methods May 2021Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to...
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 -
Cryobiology Dec 2015We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced...
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 -
Cells Jun 2021In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive...
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 -
Molecules (Basel, Switzerland) May 2022Cryoprotective and cytoprotective agents (Cytoprotective Agents) are fundamental components of the cryopreservation process. This review presents the essentials of the... (Review)
Review
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 -
Brazilian Journal of Microbiology :... 2018Basidiomycetes have several biotechnological and industrial applications such as enzyme production, bioremediation, pharmaceutical and functional food production. Due to... (Review)
Review
Basidiomycetes have several biotechnological and industrial applications such as enzyme production, bioremediation, pharmaceutical and functional food production. Due to climatic features, the preservation of several basidiomycetes is threatened, and to guarantee the preservation of this genetic resource, the development of long-term preservation techniques is necessary once there is no universal protocol for the cryopreservation of basidiomycetes. Cryopreservation is a technique in which microorganisms are submitted to ultralow temperatures. Therefore, this study aimed to collect information on the main conditions for long-term cryopreservation of basidiomycetes in the last 20 years. Scientific articles on cryopreservation of basidiomycetes published from 1997 to 2016, were researched, and only the studies on two intervals of cryopreservation were considered: from 1 to 2 years and for longer than 2 years. The analyzed conditions of basidiomycete cryopreservation were: most studied genera, cryopreservation temperature, substrate, cryoprotectant (and preservation substrate), cryopreservation period, thawing temperature and cultivation medium after thawing, physiological and genetic stability of basidiomycetes after thawing in cryopreservation. In this review, the viability of the main cryopreservation conditions of basidiomycetes studied in the last 20 years are presented and discussed.
Topics: Basidiomycota; Cryopreservation; Cryoprotective Agents; Culture Media; Microbial Viability; Time Factors
PubMed: 29122478
DOI: 10.1016/j.bjm.2017.08.004 -
Nature Communications Jul 2022The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with...
The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between -4 and -6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (-10 to -15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.
Topics: Animals; Cryopreservation; Cryoprotective Agents; Freezing; Ice; Liver; Perfusion; Rats
PubMed: 35840553
DOI: 10.1038/s41467-022-31490-2 -
Cryobiology Jun 2013Cryopreservation has numerous practical applications in medicine, biotechnology, agriculture, forestry, aquaculture and biodiversity conservation, with huge potentials... (Review)
Review
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