Cryopreservation Agent Research Articles

Морфофункциональное состояние криоконсервированных форменных элементов крови при умеренно низких температурах

Cryoprotectants enable the long-term storage of biomaterials. Despite progress in cryopreservation, there are a number of problems associated with damage to the cell membranes that result from insufficient efficacy and toxicity of some components. In this regard, it is important to develop non-toxic cryopreservation agents performing well at low temperature. The study was aimed to assess morphofunctional features of blood cells in the lactulose-based cryopreservation agent considering the effects of moderate low temperature (–40 °С). Blood cells (leukocytes, erythrocytes, platelets) collected from 30 conditionally healthy female voluntary donors aged 18–23 years were assessed. The complete blood count test was performed using the Gemalight 1270 automated hematology analyzer. Computerized cytomorphometric assessement was performed using the MECOS-C2 hardware and software complex. The study results showed morphological and functional integrity of blood cells after the 24 h storage at the temperature of–40 °С when added the lactulose-based cryopreservation agent developed: erythrocytes — 85.3 ± 0.30 % (p < 0.05), platelets – 75 ± 0.71 % (p < 0.05), leukocytes – 90.1 ± 0.91% (p < 0.05) of the values reported before freezing. The findings demonstrate the potential of using lactulose as a non-toxic component of cryopreservation systems, which will expand the range of cryopreservation agents used and make it possible to analyze morphofunctional parameters of frozen whole blood samples when conducting large-scale studies.

Plunge-Freezing Cryopreservation of Tendons.

Allograft transplantation is an important method for tendon reconstruction after injury, and its clinical success highly relies on the storage and transportation of the grafts. Cryopreservation is a promising strategy for tendon storage. In this study, we report a novel cryopreservation agent (CPA) formulation with a high biocompatibility for tendon cryopreservation. Mainly composed of natural zwitterionic betaine and the biocompatible polymer poly(vinylpyrrolidone) (PVP), it exhibited ideal abilities to depress the freezing point and inhibit ice growth and recrystallization. Notably, after cryopreservation via plunge-freezing for 1 month, Young's modulus (144 MPa, 98% of fresh tendons) and ultimate stress (46.7 MPa, 99% of fresh tendons) remained stable, and the cross-linking of collagen microfibers, protein structures, and glycosaminoglycan (GAG) contents changed slightly. These results indicate that the formulation (5 wt % betaine and 5 wt % PVP in phosphate-buffered saline, PBS solution) effectively maintains the biomechanical properties and tissue structure. This work offers a novel cryopreservation method for tendons and may also provide insights into the long-term preservation of various other tissues.

Vitrification cryopreservation of ligaments based on zwitterionic betaine

Ligament cryopreservation enables a prolonged shelf life of allogeneic ligament grafts, which is fundamentally important to ligament reconstruction. However, conventional cryopreservation techniques fail to eliminate the damage caused by ice crystal growth and the toxicity of cryopreservation agents (CPAs). Here, we report a novel CPA vitrification formulation primarily composed of betaine for ligament cryopreservation. Comprehensive optimization was conducted on the methods for vitrification and rewarming, as well as the loading and unloading conditions, based on the critical cooling rate (CCR), critical warming rate (CWR), and permeation properties of the cryoprotectant. Using biomechanical and histological level tests, we demonstrate the superior performance of our method in ligament cryopreservation. After 30 days of vitrification cryopreservation, parameters such as the Young's modulus, tensile stress, denaturation temperature, and glycosaminoglycans (GAGs) content of the ligament remained essentially unchanged. This work pioneers the application of ice-free cryopreservation for ligament and holds great potential for improving the long-term storage of ligament, providing valuable insights for future technique development.

Preparation of Peptoid Antifreeze Agents and Their Structure-Property Relationship.

The development of nontoxic and efficient antifreeze agents for organ cryopreservation is crucial. However, the research remains highly challenging. In this study, we designed and synthesized a series of peptoid oligomers using the solid-phase submonomer synthesis method by mimicking the amphiphilic structures of antifreeze proteins (AFPs). The obtained peptoid oligomers showed excellent antifreeze properties, reducing the ice crystal growth rate and inhibiting ice recrystallization. The effects of the hydrophobicity and sequence of the peptoid side chains were also studied to reveal the structure-property relationship. The prepared peptoid oligomers were detected as non-cytotoxic and considered to be useful in the biological field. We hope that the peptoid oligomers presented in this study can provide effective strategies for the design of biological cryoprotectants for organ preservation in the future.

Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation

BACKGROUND: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants. OBJECTIVE: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (−80°C) of human peripheral blood lymphocytes and skin fibroblasts. MATERIALS AND METHODS: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to −80°C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance. RESULTS: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect. CONCLUSION: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems.

The effect of hydroxyethyl starch as a cryopreservation agent during freezing of mouse pancreatic islets

Islet transplantation is the most effective treatment strategy for type 1 diabetes. Long-term storage at ultralow temperatures can be used to prepare sufficient islets of good quality for transplantation. For freezing islets, dimethyl sulfoxide (DMSO) is a commonly used penetrating cryoprotective agent (CPA). However, the toxicity of DMSO is a major obstacle to cell cryopreservation. Hydroxyethyl starch (HES) has been proposed as an alternative CPA. To investigate the effects of two types of nonpermeating CPA, we compared 4 % HES 130 and HES 200 to 10 % DMSO in terms of mouse islet yield, viability, and glucose-stimulated insulin secretion (GSIS). After one day of culture, islets were cryopreserved in each solution. After three days of cryopreservation, islet recovery was significantly higher in the HES 130 and HES 200 groups than in the DMSO group. Islet viability in the HES 200 group was also significantly higher than that in the DMSO group on Day 1 and Day 3. Stimulation indices determined by GSIS were higher in the HES 130 and 200 groups than in the DMSO group on Day 3. After three days of cryopreservation, HES 130 and HES 200 both reduced the expression of apoptosis- and necrosis-associated proteins and promoted the survival of islets. In conclusion, the use of HES as a CPA improved the survival and insulin secretion of cryopreserved islets compared with the use of a conventional CPA.

Organoid‐Loaded Cryomicroneedles for Biomimic Hair Regeneration

AbstractOrganoids have attracted extensive attention in the field of hair follicle (HF) tissue engineering and regeneration. However, HF organoid implantation faces a number of difficulties, such as low viability ratios, invasive trauma, and complicated procedures. To overcome these issues, a convenient and minimally invasive delivery approach with high efficiency and cell viability is required. Here, HF organoid‐loaded methacrylated gelatin‐cryomicroneedles (GelMA‐cryoMNs) that are capable of efficient transdermal delivery and can maintain the viability of the embedded organoids are developed. The GelMA‐cryoMNs are fabricated by a stepwise cryogenic molding process using a balanced ratio of cryopreservation agents and GelMA hydrogel. They can be inserted easily into porcine skin and melted without leaving residue after delivering the organoids. In vitro and in vivo, organoids delivered by the cryoMNs retain cell viability, self‐assembly, and differentiation ability. In mice, HF organoid‐loaded GelMA‐cryoMNs are delivered to the superficial dermis, resulting in subsequent HF development and the formation of an array of biomimetic hair tufts that broke through the skin surface. A promising strategy is provided here for biomimetic hair neogeneration that benefits from minimal invasion, low cost, and simple fabrication. The GelMA‐cryoMNs developed here could also have broader applications in tissue engineering and organ regeneration.

Enhanced Cryopreservation Efficacies of Ice Recrystallization Inhibiting Nanogels.

Development of new cryopreservation technologies holds significant potential to revolutionize the fields of cell culture, tissue engineering, assisted reproduction, and transfusion medicine. The current gold standard small-cell permeating cryopreservation agents (CPAs) demonstrate promising cryopreservation efficacies but are cytotoxic and immunogenic at the concentrations required for cryopreservation applications. In comparison, new cell impermeable CPAs of nanodimensions demonstrate outstanding potential to overcome the drawbacks of existing CPAs. In this study, we report the synthesis of vitamin B5 analogous methacrylamide (B5AMA)-incorporated nanogels as a potential solution to address the commonly observed limitations of existing CPAs. The stimuli-responsive poly(B5AMA) nanogels prepared by radical polymerization demonstrated significant ice recrystallization inhibition efficacies and showed either superior or comparable cryopreservation efficacies compared to the traditional cryoprotectant DMSO/glycerol in both eukaryotic and prokaryotic cells.

Exposure of model extravillous trophoblasts to cryopreservation agents decreases cell migration

Exposure of model extravillous trophoblasts to cryopreservation agents decreases cell migration

Physicochemical Mechanisms of Protection Offered by Agarose Encapsulation during Cryopreservation of Mammalian Cells in the Absence of Membrane-Penetrating Cryoprotectants.

During freeze/thaw, cells are exposed to mechanical, thermal, chemical, and osmotic stresses, which cause loss of viability and function. Cryopreservation agents such as dimethyl sulfoxide (DMSO) are deployed to minimize freeze/thaw damage. However, there is a pressing need to eliminate DMSO from cryopreservation solutions due to its adverse effects. This is of the highest priority especially for cryopreservation of infusible/transplantable cell therapy products. In order to address this issue, we introduce reversible encapsulation in agarose hydrogels in the presence of the membrane-impermeable cryoprotectant, trehalose, as a viable, safe, and effective cryopreservation method. Our findings, which are supported by IR spectroscopy and differential scanning calorimetry analyses, demonstrate that encapsulation in 0.75% agarose hydrogels containing 10-20% trehalose inhibits mechanical damage induced by eutectic phase change, devitrification, and recrystallization, resulting in post-thaw viability comparable to the gold standard 10% DMSO.

Plasma extracellular vesicle test sample to standardize flow cytometry measurements

BackgroundExtracellular vesicles (EVs) in body fluids are explored as disease biomarkers, but EV concentrations measured by flow cytometers (FCMs) are incomparable. ObjectivesTo improve data comparability, new reference materials with physical properties resembling EVs and reference procedures are being developed. The validation of new reference materials and procedures requires biological test samples. We developed a human plasma EV test sample (PEVTES) that i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry–compatible, and iv) is stable. MethodsThe PEVTES was prepared from human plasma of 3 fasting donors. EVs were immunofluorescently stained with antibodies against platelet-specific (CD61) and erythrocyte-specific (CD235a) antigens or lactadherin. To reduce the concentration of soluble proteins, lipoproteins, and unbound reagents, stained EVs were isolated from plasma by size-exclusion chromatography. After isolation, the PEVTES was filtered to remove remnant platelets. PEVTESs were diluted in cryopreservation agents, dimethyl sulfoxide, glycerol, or trehalose and stored at −80 °C for 12 months. After thawing, stained EV concentrations were measured with a calibrated FCM (Apogee A60-Micro). ResultsWe demonstrate that the developed PEVTES resembles subcellular particles in human plasma when measured using FCM and that the concentrations of prestained platelet-derived, erythrocyte-derived, and lactadherin+ EVs in the PEVTES are stable during storage at −80 °C for 12 months when stored in trehalose. ConclusionThe PEVTES i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry–compatible, and iv) is stable. Therefore, the developed PEVTES is an ideal candidate to validate newly developed reference materials and procedures.

Microcurvature Controllable Metal-Organic Framework Nanoagents Capable of Ice-Lattice Matching for Cellular Cryopreservation.

Ice-binding proteins (IBPs) produced by psychrophilic organisms to adapt for the survival of psychrophiles in subzero conditions have received illustrious interest as a cryopreservation agent required for cells and tissues to completely recover after freezing/thawing. Depressing water-freezing point and avoiding ice-crystal growth affect their activities which are closely related to the presence of ice crystal well-matched binding moiety. The interaction of IBPs with ice and water is critical in enhancing their freeze avoidance against cell or tissue damage. Metal-organic frameworks (MOFs) with a controllable lattice at the molecular level and a size at the nanometer scale can offer periodically ordered ice-binding sites by modifying organic linkers and controlling microcurvature at the ice surface. Herein, zirconium (Zr)-based MOF-801 nanoparticles (NPs) with good biocompatibility were used as a cryoprotectant that is well dispersed and colloidal-stable in an aqueous solution. The MOF NP size was precisely controlled, and 10, 35, 100, and 250 nm NPs were prepared. The specific IBPs-mimicking pendants (valine and threonine) were simply introduced into the MOF NP-surface through the acrylate-based functionalization to endow with hydrophilic and hydrophobic dualities. When small-sized MOF-801 NPs were attached to ice, they confined ice growth in high curvature between the adsorption sites because of the decreased radius of the convex area of the growth region, leading to highly enhanced ice recrystallization inhibition (IRI). Surface-functionalized MOF NPs could increase the number of anchored clathrate water molecules with hydrophilic/hydrophobic balance of the ice-binding moiety, effectively inhibiting ice growth. The MOF-801 NPs were biocompatible with various cell lines regardless of concentration or NP surface-functionalization, whereas the smaller-sized surface-functionalized NPs showed a good cell recovery rate after freezing/thawing by induction of IRI. This study provides a strategy for the fabrication of low-cost, high-volume antifreeze nanoagents that can extend useful applications to organ transplantation, cord blood storage, and vaccines/drugs.

Hyaluronic acid as effective cryoprotective agent for hMSC cryopreservation

Hyaluronic acid as effective cryoprotective agent for hMSC cryopreservation

Development of Serum-Free Media for Cryopreservation of Hydrogel Encapsulated Cell-Based Therapeutics.

While hydrogel encapsulation of cells has been developed to treat multiple diseases, methods to cryopreserve and maintain the composite function of therapeutic encapsulated cell products are still needed to facilitate their storage and distribution. While methods to preserve encapsulated cells, and post-synthesis have received recent attention, effective preservation mediums have not been fully defined. We employed a two-tiered screen of an initial library of 32 different cryopreservation agent (CPA) formulations composed of different cell-permeable and impermeable agents. Formulations were assayed using dark field microscopy to evaluate alginate hydrogel matrix integrity, followed by cell viability analyses and measurements of functional secretion activity. The structural integrity of large > 1mm alginate capsules were highly sensitive to freezing and thawing in media alone but could be recovered by a number of CPA formulations containing different cell-permeable and impermeable agents. Subsequent viability screens identified two top-performing CPA formulations that maximized capsule integrity and cell viability after storage at -80°C. The top formulation (10% Dimethyl sulfoxide (DMSO) and 0.3M glucose) was demonstrated to preserve hydrogel integrity and retain cell viability beyond a critical USA FDA set 70% viability threshold while maintaining protein secretion and resultant cell potency. This prioritized screen identified a cryopreservation solution that maintains the integrity of large alginate capsules and yields high viabilities and potency. Importantly, this formulation is serum-free, non-toxic, and can support the development of clinically translatable encapsulated cell-based therapeutics. The online version contains supplementary material available at 10.1007/s12195-022-00739-7.

Biological activity, solvation properties and microstructuring of protic imidazolium ionic liquids

Protic imidazolium ionic liquids (PILs) have shown great potential as regents and catalysts in liquid-phase chemistry. However, their biological activity/toxicity and solvation properties are rather understudied compared to those of more common aprotic ionic liquids (APILs). In this work, for the first time, we studied the cytotoxicity of nine chemically relevant imidazolium PILs with various alkyl side chains in the cation and compared it with the cytotoxicity of the corresponding aprotic analogues. The experimental data were supported by computational modeling. The results suggested the type of anion to be the major factor governing the cytotoxicity of the studied ILs with short alkyl side chains. Of note, even low-toxic PILs imposed considerable deleterious effects on eukaryotic cells when used as cryopreservation agents. According to a scanning electron microscopy (SEM) study, due to the weak amphiphilic properties of imidazolium cations with short alkyl side chains, the studied IL/water mixtures tended to produce simple solid hydrates rather than complex liquid systems with microdomain organization.

Bioapplications of Magnetic Nanowires: Barcodes, Biocomposites, Heaters.

Magnetic nanowires (MNWs) can have their moments reversed via several mechanisms that are controlled using the composition, length, diameter, and density of nanowires in arrays as-synthesized or as individual nanoparticles in assays or gels. This tailoring of magnetic reversal leads to unique properties that can be used as a signature for reading out the type of MNW for applications as nano-barcodes. When synthesized inside track-etched polycarbonate membranes, the resulting MNW-embedded membranes can be used as biocompatible bandaids for detection without contact or optical sighting. When etched out of the growth template, free-floating MNWs are internalized by cells at 37 °C such that cells and/or exosomes can be collected and detected. In applications of cryopreservation, MNWs can be suspended in cryopreservation agents (CPAs) for injection into the blood vessels of tissues and organs as they are vitrified to -200 °C. Using an alternating magnetic field, the MNWs can then be nanowarmed rapidly to prevent crystallization and uniformly to prevent cracking of specimens, for example, as grafts or transplants. This invited paper is a review of recent progress in the specific bioapplications of MNWs to barcodes, biocomposites, and nanowarmers.

Osmotic Characteristics of Erythrocytes After Freezing with Composition-Modified Cryopreservatives

Here, we have studied the osmotic parameters of erythrocytes frozen in the composition-modified media with glycerol and 1,2-propanediol. A decreased glycerol concentration in a cryopreservative and an increased content of impermeable protective component sorbitol were established to affect the erythrocyte characteristics at different stages of freezing and hypothermic storage. Reduction of 1,2-propanediol and NaCl concentrations when augmenting the sucrose content in cryopreservative agent enabled changing a two-stage mode of erythrocyte freeze-thawing to the single-stage one. The presented here modification of cryoprotective medium allowed to diminish the level of damage to erythrocytes during freeze-thawing-washing out, that might prevent the inflammation development in a body when transfusing these cells.

Phase transitions and mechanisms of cryoprotection of serum-/xeno-free media based on dextran and dimethyl sulfoxide

The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me2SO) and non-permeating polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me2SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (−30, −50, −50 and −196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me2SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me2SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media.

Sequential Grafting of Fresh and Cryopreserved Fat After Mechanical Processing is a Safe and Effective Facial Rejuvenation Strategy.

The survival rate of fat transplants is variable and consequently multiple operations are often required to achieve satisfactory results. Fat cryopreservation technology is a good solution to this problem. At present, cryopreservation of fats needs to be added with cryopreservation agents, which brings unsafety and operational complexity to clinical applications. An efficient and safe strategy for fat cryopreservation must be developed. A retrospective study was performed of all patients who underwent facial fat grafting and agreed to have their fat tissue cryopreserved from January 2018 to May 2021. Fat samples were physically processed to obtain SVF-gel, which was cryopreserved at - 20°C for up to 3 months and injected after thawing. Images acquired by pre- and post-operative 3D scanning of the temporal region were compared to evaluate the retention rate of transplanted cryopreserved SVF-gel. No patients experienced serious complications after receiving cryopreserved fat transplantation. The retention rate of cryopreserved SVF-gel was 46.3 ± 7.7% at 3 months and 43.1 ± 7.2% at 6 months after transplantation. The swelling duration was significantly shorter after cryopreserved SVF-gel transplantation (5.5 ± 0.8 days) than after fresh fat transplantation (7.5 ± 0.7 days) (p < .031). Injection of cryopreserved SVF-gel achieves good retention rate for facial rejuvenation and has few side effects. Cryopreservation of SVF-gel is a safe and effective strategy for serial fat grafting for facial rejuvenation. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Використання полівінілалкоголю та полівінілпіролідону для підготовки деконсервованих еякульованих сперматозоїдів кнурів до запліднення методом ICSI

The aim of the study was to explore the effect of PVP (polyvinylpyrrolidone) and PVA (polyvinyl alcohol) media on deconserved ejaculated boar sperm and their preparation for artificial insemination to optimize biotechnological approaches. The studies used ejaculated cryopreserved sperm of a boar of the Myrhorod breed Dnipro 641. Genetic material was stored in the Bank of Genetic Resources of Animals IABG nnamed after M.V. Zubets NAAS for eight years. The sperm suspension was thawed in a water bath at +37 °C for 5 min until completely thawed. Separation of sperm from cryopreservative agent and diluent was performed using the swim up method in Sp-TALP medium. After the presence of sperm in the 10.0% solution of PVP for 10 min, motility decreased by 68.2% (P < 0.05) and amounted to 3.4%, and after the next 10 min of incubation decreased to 1.4% (P <0.01), which is 10 times lower than the initial mobility. In 10.0% of PVA mobility after 10 min of incubation decreased by 37.4% (P <0.05) and amounted to 6.7%, and after 10 min decreased to 5.7% (P < 0.01), which is 1.8 times lower than the initial mobility. It was found that in the case of 10.0% of PVP solution ejaculated deconserved boar sperm lose motility by 86.9% (P <0.01) from the initial motility, which makes it impossible to select a suitable sperm for fertilization by ICSI (intracytoplasmic sperm injection). It is shown that 10.0% PVA solution can be used for immobilization of boar sperm, as it reduces motility by 46.7% (P <0.01) of the initial sperm motility. It is proved that the mobility in the case of incubation of deconserved ejaculated boar sperm in 5.0% PVA solution decreases only by 28.0% (P <0.05) from the initial, which is optimal when using cryopreserved boar sperm, material which are limited and convenient for the operator and safe for oocytes.