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.

Comparison of Cryopreservation Media for Mesenchymal Stem Cell Spheroids.

Multipotent mesenchymal stromal/stem cell (MSC) spheroids generated in three-dimensional culture are of considerable interest as a novel therapeutic tool for regenerative medicine. However, the lack of reliable methods for storing MSC spheroids represents a significant roadblock to their successful use in the clinic. An ideal storage medium for MSC spheroids should function as both a vehicle for delivery and a cryoprotectant during storage of spheroids for use at a later time. In this study, we compared the outcomes after subjecting MSC spheroids to a freeze/thaw cycle in three Good Manufacturing Practices-grade cryopreservation media, CryoStor10 (CS10), Stem-Cellbanker (SCB), and Recovery Cell Culture Freezing Media (RFM) or conventional freezing medium (CM) (CM, Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide) as a control for 2 months. The endpoints tested were viability, morphology, and expression of stem cell markers and other relevant genes. The results of LIVE/DEAD™ assays and annexin V/propidium iodide staining suggested that viability was relatively higher after one freeze/thaw cycle in CS10 or SCB than after freeze/thaw in CM or RFM. Furthermore, the relative "stemness" and expression of MSC markers were similar with or without freeze/thaw in CS10. Scanning electron microscopy also indicated that the surface morphology of MSC spheroids was well preserved after cryopreservation in CS10. Thus, even though it was tested for a short-term period, we suggest that CS10, which has been approved for clinical use by the U.S. Food and Drug Association, is a promising cryopreservation medium that would facilitate the development of MSC spheroids for future clinical use.

Osmotic properties of T cells determined by flow imaging microscopy in comparison to electrical sensing zone analysis.

To develop cryopreservation methods for cell-based medicinal products it is important to understand osmotic responses of cells upon immersion into solutions with cryoprotective agents (CPAs) and during freezing. The aim of this study was to assess the osmotic response of T cells by using flow imaging microscopy (FIM) as a novel cell-sizing technique, and to corroborate the findings with electrical impedance measurements conducted on a Coulter counter. Jurkat cells were used as a potential model cell line for primary T cells. Cell volume responses were used to derive important cell parameters for cryopreservation such as the osmotically inactive cell volume Vb and the membrane permeability towards water and various CPAs. Unlike Coulter counter measurement, FIM, combined with Trypan blue staining can differentiate between viable and dead cells, which yields a more accurate estimation of Vb. Membrane permeabilities to water, dimethyl sulfoxide (Me2SO) and glycerol were measured for Jurkat cells at different temperatures. The permeation of Me2SO into the cells was faster in comparison to glycerol. CPA permeation decreased with decreasing temperature following Arrhenius behavior. Moreover, membrane permeability to water decreased in the presence of CPAs. Vb of Jurkat cells was found to be 49% of the isotonic volume and comparable to that of primary T cells. FIM proved to be a valuable tool to determine the membrane permeability parameters of mammalian cells to water and cryoprotective agents, which in turn can be used to rationally design CPA loading procedures for cryopreservation.

Transcriptome Profiling during Sequential Stages of Cryopreservation in Banana (Musa AAA cv Borjahaji) Shoot Meristem.

Cryopreservation approaches have been implemented in gene banks as a strategy to back up plant genetic resource collections that are vegetatively propagated. Different strategies have been employed to effectively cryopreserve plant tissue. There is little information on the cellular processes and molecular adjustments that confer resilience to the multiple stresses imposed during a cryoprotocol. In the present work, the cryobionomics of banana (Musa sp.), a non-model species, was investigated through the transcriptomic approach using RNA-Seq. Proliferating meristems of in vitro explants (Musa AAA cv 'Borjahaji') were cryopreserved using the droplet-vitrification technique. Transcriptome profiling analysis of eight cDNA libraries including the bio-replicates for T0 (stock cultures (control tissue), T1 (high sucrose pre-cultured), T2 (vitrification solution-treated) and T3 (liquid nitrogen-treated) meristem tissues was carried out. The raw reads obtained were mapped with a Musa acuminata reference genome sequence. A total of 70 differentially expressed genes (DEGs) comprising 34 upregulated and 36 downregulated were identified in all three phases as compared to control (T0). Among the significant DEGs (>log FC 2.0), during sequential steps, 79 in T1, 3 in T2 and the 4 in T3 were upregulated and 122 in T1, 5 in T2 and 9 in T3 were downregulated. Gene ontology (GO) enrichment analysis showed that these significant DEGs were involved in the upregulation of biological process (BP-170), cellular component (CC-10) and molecular function (MF-94) and downregulation of biological process (BP-61), cellular component (CC-3) and molecular function (MF-56). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were involved in the biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, MAPK signaling, EIN 3-lke 1 protein, 3-ketoacy-CoA synthase 6-like, and fatty acid elongation during cryopreservation. For the first time, a comprehensive transcript profiling during four stages of cryopreservation in banana were carried out, which will pave the way for devising an effective cryopreservation protocol.

Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming.

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification-the ice-free cryopreservation of livers in a glass-like state-as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique "nanowarming" technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.

Modelling and experimental studies on mass transport of multiple cryoprotective agents in articular cartilage.

Cartilage transplantation is an effective way to repair, reconstruct, and replace damaged articular cartilage (AC) but its use is limited by the inability to preserve AC for long periods of time. Vitrification is an ideal choice for long-term storage of AC, and multiple cryoprotective agents (CPAs) with high concentration are usually used. To obtain high cell viability, chondrocytes at all locations inside AC should be protected properly by the CPAs during cooling and rewarming. Hence, it is important to know the mass transport properties of multiple CPAs as they synergistically infiltrate AC. In this study, a mathematical model to describe the mass transport behavior of multiple CPAs in AC was developed based on the mixture-averaged diffusion model. In addition, a methodology for the simultaneous determination of dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol by carbon-13 nuclear magnetic resonance was established. The model is applicable for predicting single- and multiple-CPA permeation into AC, and its accuracy was verified by a massive experimental dataset. Simulation results showed reverse diffusion in the multiple-CPA permeation process, which was not found in the single-CPA permeation process. This curious phenomenon shows the sharp contrast between the diffusion behavior of a binary mixture and a multicomponent mixture.

Cryopreservation of Sperm from an Endangered Snake with Tests of Post-Thaw Incubation in Caffeine.

Cryopreservation of sperm to preserve the genetic diversity of declining populations is a promising technique to aid in the recovery of endangered species such as the Louisiana pinesnake (Pituophis ruthveni). However, this technique has been performed on only a handful of snake species and with limited success. Here, we tested a cryoprotective agent (CPA) mixture containing Lake's buffer with 10% N,N-dimethyl formamide (DMF), 2% methanol, 5% clarified egg yolk, (v/v% final concentration) against 16 other CPA-treatment mixtures. These contained either Lake's buffer or TEST egg yolk buffer as the base diluent with a penetrating or non-penetrating CPA on the post-thaw recovery of sperm motility and viability. We also investigated the effect of post-thaw incubation treatment in TL HEPES supplemented with 10% fetal bovine serum (H10) alone or with caffeine on post-thaw motility parameters. Sperm from 16 Louisiana pinesnakes was cryopreserved, and the effectiveness of the CPA treatment mixtures and post-thaw treatments was determined based on measurements of sperm motility and viability. Sperm cryopreservation significantly reduced initial post-thaw sperm quality for all of the extender treatments. Viability of sperm was best maintained when cryopreserved in an CPA treatment mixture containing Lake's buffer with 10% DMF, 2% methanol, and 5% clarified egg yolk with the addition of 5 mg/mL bovine serum albumin (BSA). For several extender mixtures a similar percent of post-thaw motility was observed, but no forward motility returned in any post-thaw samples prior to incubation in dilution treatments. Following incubation in both post-thaw treatments, the percent of forward motility and the index of forward progressive movement improved significantly. Post-thaw dilution with H10 containing caffeine improved motility parameters over H10 alone, suggesting further investigation of post-thaw treatment in caffeine could be beneficial. Although, cryopreservation of sperm from the Louisiana pinesnake continues to present a challenge, post-thaw dilution and the addition of BSA to CPA mixtures provides areas for improving cryopreservation methods for this endangered species.

Long-term cryopreservation of Lentinus crinitus strains by wheat grain technique.

Lentinus crinitus (Basidiomycota: Polyporales) is a saprophytic fungus with biotechnological importance described more than 20 years ago. However, there are few studies on the long-term preservation of this basidiomycete. Cryopreservation is a long-term storage technique that reduces the metabolic activity of microorganisms, but its success depends on the adjustment of the freezing process, the cryoprotectants, and the protective substrates for each species. This study aimed to assess the mycelial viability and genetic stability of L. crinitus strains cryopreserved at -86 °C for two years by the wheat grain technique using different cryoprotectants and freezing methods. Three strains of L. crinitus (U9-1, U13-5, and U15-12) were subjected to different concentrations and types of cryoprotectants (dimethyl sulfoxide, glycerol, glucose, and sucrose), freezing methods such as immediate freezing from 25 to -86 °C and progressing freezing from 25 to -86 °C in a freezing container with isopropyl alcohol to control the rate of cell freezing at -1 °C min-1, protective substrate (wheat grain and 2% malt extract agar), and cryopreservation period (1, 6, 12, and 24 months). After thawing, samples were evaluated for mycelial viability, time to mycelial recovery, mycelial stability, and genetic stability of the fungus. All techniques achieved effective cryopreservation at -86 °C, mainly with the wheat grain technique. All cryoprotectants (3.5% glycerol, 1.5% dimethyl sulfoxide, 25% sucrose, and 5% glucose), freezing methods (immediate and gradual), and protective substrate (wheat grain and malt extract agar) were effective for cryopreservation of the three L.crinitus strains in an ultra-low temperature freezer for two years. Mycelial viability, mycelial stability, and genetic stability of the fungus were not affected after two-year cryopreservation, evidencing the robustness of the long-term cryopreservation technique and the fungus.

Innovative Approach in the Cryogenic Freezing Medium for Mesenchymal Stem Cells.

The physical stresses during cryopreservation affect stem cell survival and further proliferation. To minimize or prevent cryoinjury, cryoprotective agents (CPAs) are indispensable. Despite the widespread use of 10% dimethyl sulfoxide (DMSO), there are concerns about its potential adverse effects. To bypass those effects, combinations of CPAs have been investigated. This study aimed to verify whether high-molecular-hyaluronic acid (HMW-HA) serves as a cryoprotectant when preserving human mesenchymal stem cells (hMSCs) to reduce the DMSO concentration in the cryopreservation medium. We studied how 0.1% or 0.2% HMW-HA combined with reduced DMSO concentrations (from 10% to 5%, and 3%) affected total cell count, viability, immunophenotype, and differentiation potential post-cryopreservation. Immediately after cell revival, the highest total cell count was observed in 10% DMSO-stored hMSC. However, two weeks after cell cultivation an increased cell count was seen in the HMW-HA-stored groups along with a continued increase in hMSCs stored using 3% DMSO and 0.1% HMW-HA. The increased total cell count corresponded to elevated expression of stemness marker CD49f. The HA-supplemented cryomedium did not affect the differential potential of hMSC. Our results will participate in producing a ready-to-use product for cryopreservation of mesenchymal stem cells.

LncRNA-mediated effects of vitrification temperatures and cryoprotectant concentrations on bovine oocyte development following vitrification at the GV stage.

We evaluated the effects of different vitrification temperatures (VTs) and cryoprotective agent concentrations (CPAs) on the viability and expressions of long non-coding RNA (lncRNA) in bovine oocytes following vitrification at the germinal vesicle (GV) stage. Our findings provide a theoretical support for improvement of the cryopreservation technology of bovine immature oocytes (BIOs). Bovine cumulus oocyte complexes (COCs) were collected and randomized into five groups: fresh oocytes (control), oocytes vitrified in liquid helium (LHe; -269 °C) with 5.6 M CPAs (LHe 5.6 M), oocytes vitrified in LHe with 6.6 M CPAs (LHe 6.6 M), oocytes vitrified in liquid nitrogen (LN; -196 °C) with 5.6 M CPAs (LN 5.6 M), and oocytes vitrified in LN with 6.6 M CPAs (LN 6.6 M). Of the four vitrification groups, the LHe 5.6 M group exhibited the highest blastocyst rate (13.22%), followed by the LHe 6.6 M group (10.19%) and LN 6.6 M group (9.77%), while the LN 5.6 M group had the lowest blastocyst rate (1.87%). Then, lncRNA expressions in the five groups were profiled. A total of 18,271 lncRNAs were identified, of which 2,158 were differentially expressed lncRNAs (DELs) in the vitrified groups, compared to the fresh group (P < 0.05; fold-change > 2). Co-location (cis) and co-expression (trans) prediction revealed 14 differentially expressed target genes (DETGs), which corresponded to 17 DELs. Based on grouping data and expression profiles of the DELs, we demonstrated that different VTs (-269 °C vs. -196 °C) can affect the expressions of MSTRG.12295.5, MSTRG.37123.1, MSTRG.37930.2, MSTRG.40464.9, MSTRG.8869.3 and MSTRG.26680.6. Expressions of these lncRNAs were affected by CPAs only in the condition of vitrification with LHe (-269 °C). Expressions of MSTRG.35129.6 were associated with exposures to both VTs and CPAs; while expressions of MSTRG.3578.3, MSTRG.40576.3, MSTRG.6723.5, MSTRG.32862.4, MSTRG.1184.4, MSTRG.33110.3, MSTRG.40454.2, MSTRG.41073.2, MSTRG.44732.4 and MSTRG.6729.3 might be related to vitrification. Co-expression analysis showed that MSTRG.12295.5, MSTRG.37930.2, MSTRG.40454.2, MSTRG.8869.3 and MSTRG.6723.5 expressions affect oocyte development after vitrification by regulating target gene expressions. Taken together, improvement of the developmental ability of BIOs after LHe vitrification maybe attributed to changes in expressions of some lncRNAs. Our findings elucidate on the molecular mechanisms underlying the development of BIOs under different VTs and CPAs.

The effect of glycerol as a cryoprotective agent in the cryopreservation of adipose tissue.

BACKGROUND: Long-term preservation of adipose tissue is crucial for clinical applications. Researchers should consider both efficiency and biosafety when choosing a cryoprotective agent (CPA) for adipose tissue preservation. Glycerol has been applied as a nontoxic CPA for multiple tissues but not adipose tissue. We aimed to evaluate the efficacy of glycerol as a CPA for adipose tissue cryopreservation. METHODS: Fresh human adipose tissues were obtained from patients who underwent liposuction and divided into 1 mL samples. Each sample was randomly mixed with 1 mL of CPA: 60-100% glycerol, 0.25 mol/L trehalose or DMSO + FBS and cryopreserved in - 196 °C liquid nitrogen for one month. After thawing and elution, the tissues were immediately evaluated for activity and structural integrity in vitro. Then, 0.2 mL of each sample was transplanted subdermally to the nude mouse dorsum and harvested after one month for histological examination to assess the effect of the cryopreserved fat in transplantation. RESULTS: After cryopreservation, the samples treated with DMSO + FBS, trehalose, 60% and 70% glycerol had a more integrated structure than the samples in other groups. Tissues preserved with 70% glycerol had the highest G3PDH activity of 24.41 ± 0.70, comparable to 24.76 ± 0.48 in fresh tissue (p > 0.05). Adipose-derived stem cells (ASC) viability, proliferation and differentiation capability were also better preserved in 70% glycerol group. In vivo analysis showed that tissue preserved with 70% glycerol had a retention rate of 52.37 ± 7.53%, significantly higher than other groups. Histological observation demonstrated better structural integrity and viability in 70% glycerol group. Compared to the DMSO + FBS and trehalose groups, the glycerol groups showed lower tissue inflammation. CONCLUSION: Glycerol (70%) is efficient in adipose tissue cryopreservation. Glycerol-based CPAs, which are nontoxic and show biosafety, are a promising solution for clinical tissue cryopreservation.

Cryoprotective agents influence viral dosage and thermal stability of inhalable dry powder vaccines.

Increasing viral dosage within dry powder vaccines reduces the powder mass required to elicit an immune response through pulmonary delivery. This work analyzes how cryoprotective agents affect viral activity, particle properties and thermal stability of a spray dried, inhalable vaccine vector under high viral loading. Stock suspensions of a human serotype 5 adenovirus (AdHu5) vector in either neat phosphate buffered saline (PBS), 10% glycerol in PBS, or 5% trehalose in PBS were added to a mannitol-dextran formulation prior to spray drying. At high viral loading, spray dried powder containing glycerol had a viral titre log loss of 2.8 compared to 0.7 log loss using neat PBS. Powders containing glycerol had a lower glass transition temperature (Tg) compared to all other formulations, permitting greater viral mobility and exposure to heat damage. Inclusion of glycerol also promoted particle cohesion during spray drying and lower yields. Using 5% trehalose as a cryogenic alternative, viral powders had a viral log loss of 1.5 and the highest displayed thermal stability over time. Additionally, trehalose-containing powders had smaller particles with lower water moisture content and higher powder yield compared to glycerol-containing powders. These findings demonstrate the importance of cryoprotective agent selection when developing thermostable vaccine powders.

Single sperm vitrification with permeable cryoprotectant-free medium is more effective in patients with severe oligozoospermia and azoospermia.

Testicular sperm extraction (TESE) is an invasive surgery for achieving the spermatozoa in cases with azoospermia. In these patients, the number of retrieved spermatozoa is limited and the optimal cryo-storage is very critical for their fertility preservation. Therefore, single sperm vitrification has been introduced for preservation of low number of spermatozoa. The goal was to assess the efficacy of sperm freezing medium (SFM) and sucrose medium as cryoprotectants for single sperm vitrification in cases with severe oligozoospermia and azoospermia. A total of 20 ejaculates from severe oligozoospermia and 20 testicular samples from azoospermia were processed. Twenty-five sperm cells were collected using ICSI injection pipette and transferred to a cryoprotectant droplet placed on the Cryotech, then vitrified by plunging in liquid nitrogen. Sperm motility, viability, fine-morphology, mitochondrial activity and DNA fragmentation index (DFI) were assessed before and after vitrification. Sperm motility, viability and the percentage of cells with mitochondrial activity were significantly decreased after vitrification in both severe oligozoospermic and testicular samples in either cryoprotectants. However, the rates of post-warm sperm motility and the cells with mitochondrial activity increased significantly in sucrose medium in both severe oligozoospermic and testicular samples compared to SFM. In testicular samples, the DFI of spermatozoa vitrified in SFM was significantly higher than those vitrified with sucrose medium. Sperm motility, viability, mitochondrial activity, and DNA integrity were better preserved in sucrose medium than SFM after single cell vitrification. The presented method may be a useful candidate for successful freezing of individual sperm cells in clinical setting.

Cryopreservation of Tissue-Engineered Scaffold-Based Constructs: from Concept to Reality.

Creation of scaffold-based tissue-engineered constructs (SB TECs) is costly and requires coordinated qualified efforts. Cryopreservation enables longer shelf-life for SB TECs while enormously enhancing their availability as medical products. Regenerative treatment with cryopreserved SB TECs prepared in advance (possibly prêt-à-porter) can be started straight away on demand. Animal studies and clinical trials indicate similar levels of safety for cryopreserved and freshly prepared SB TECs. Although cryopreservation of such constructs is more difficult than that of cell suspensions or tissues, years of research have proved the principal possibility of using ready-to-transplant SB TECs after prolonged cryostorage. Cryopreservation efficiency depends not only on the sheer viability of adherent cells on scaffolds after thawing, but largely on the retention of proliferative and functional properties by the cells, as well as physical and mechanical properties by the scaffolds. Cryopreservation protocols require careful optimization, as their efficiency depends on multiple parameters including cryosensitivity of cells, chemistry and architecture of scaffolds, conditions of cell culture before freezing, cryoprotectant formulations, etc. In this review we discuss recent achievements in SB TEC cryopreservation as a major boost for the field of tissue engineering and biobanking.

Effects of AavLEA1 Protein on Mouse Ovarian Tissue Cryopreservation by Vitrification.

Conventional ovarian tissue cryopreservation often destroys the structural, functional, and DNA integrity of the ovarian tissue. How to effectively retain the ultrastructure and subsequent function of ovarian tissue during cryopreservation has long been an issue of concern. Late embryogenesis abundant (LEA) proteins are a class of highly hydrophilic proteins and have been reported to protect various cells from water stress. However, whether LEA proteins exert protective effects on ovarian tissue cryopreservation remains unknown. To investigate the benefit of LEA proteins in ovarian tissue cryopreservation, we purified the recombinant AavLEA1 protein, a member of Group 3 LEA proteins, then cryopreserved the mouse ovaries with this protein by vitrification, and obtained the ovarian follicle structure, cellular proliferation, apoptosis, and GAPDH gene expression of postcryopreservation ovaries. We found that recombinant AavLEA1 protein protected the ovarian follicles from cryoinjury, improved the proliferative ability of follicles, decreased the apoptosis, and promoted the GAPDH gene expression. These results indicated that the LEA protein enhanced the antiapoptosis ability of ovarian cells and retained DNA/RNA integrity against cryoinjury during ovarian tissue vitrification. LEA proteins exert beneficial effects on ovarian tissue cryopreservation, and maybe provide a novel cryoprotective agent for ovarian tissue cryopreservation.

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas.

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.

Molecular basis of ice-binding and cryopreservation activities of type III antifreeze proteins.

Antifreeze proteins (AFPs) can inhibit the freezing of body fluid at subzero temperatures to promote the survival of various organisms living in polar regions. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We determined the thermal hysteresis (TH), ice recrystallization inhibition (IRI), and cryopreservation activity of three isoforms of the notched-fin eelpout AFP and their mutant constructs and characterized their structural and dynamic features using NMR. The QAE1 isoform is the most active among the three classes of III AFP isoforms, and the mutants of inactive QAE2 and SP isoforms, QAE2ACT and SPACT, displayed the full TH and IRI activities with resepect to QAE1 isoform. Cryopreservation studies using mouse ovarian tissue revealed that the QAE1 isoform and the active mutants, QAE2ACT and SPACT, more effectively preserved intact follicle morphology and prevented DNA double-strand break damage more efficiently than the inactive isoforms. It was also found that all active AFPs, QAE1, QAE2ACT, and SPACT, formed unique H-bonds with the first 310 helix, an interaction that plays an important role in the formation of anchored clathrate water networks for efficient binding to the primary prism and pyramidal planes of ice crystals, which was disrupted in the inactive isoforms. Our studies provide valuable insights into the molecular mechanism of the TH and IRI activity, as well as the cryopreservation efficiency, of type III AFPs.

Osmometric Measurements of Cryoprotective Agent Permeation into Tissues.

Quantification of the amount of cryoprotective agent (CPA) in a tissue is an essential step in the design of successful cryopreservation protocols. This chapter details two inexpensive methods to measure cryoprotective agent permeation into tissues as functions of time. One of the methods to measure the CPA permeation is to permeate a series of tissue samples from a surrounding solution at a specified concentration of CPA, each sample for a different amount of time, and then to quantitate the amount of CPA that was taken up in the tissue during that time period. The quantification is performed by equilibrating the permeated tissue with a surrounding solution and then measuring the osmolality of the solution to determine the amounts of CPAs that have come out of each tissue sample corresponding to each permeation time. An alternative method to measuring the CPA permeation as a function of time, which requires fewer tissue samples, is to measure the CPA efflux as a function of time. In the efflux method, a CPA-permeated tissue sample is placed in a surrounding solution, and solution samples are taken at different time points throughout the efflux to quantitate how much CPA has left the tissue by each time point.

The need for novel cryoprotectants and cryopreservation protocols: Insights into the importance of biophysical investigation and cell permeability.

BACKGROUND: Cryopreservation is a key method of preservation of biological material for both medical treatments and conservation of endangered species. In order to avoid cellular damage, cryopreservation relies on the addition of a suitable cryoprotective agent (CPA). However, the toxicity of CPAs is a serious concern and often requires rapid removal on thawing which is time consuming and expensive. SCOPE OF REVIEW: The principles of Cryopreservation are reviewed and recent advances in cryopreservation methods and new CPAs are described. The importance of understanding key biophysical properties to assess the cryoprotective potential of new non-toxic compounds is discussed. MAJOR CONCLUSIONS: Knowing the biophysical properties of a particular cell type is crucial for developing new cryopreservation protocols. Similarly, understanding how potential CPAs interact with cells is key for optimising protocols. For example, cells with a large osmotically inactive volume may require slower addition of CPAs. Similarly, a cell with low permeability may require a longer incubation time with the CPA to allow adequate penetration. Measuring these properties allows efficient optimisation of cryopreservation protocols. GENERAL SIGNIFICANCE: Understanding the interplay between cells and biophysical properties is important not just for developing new, and better optimised, cryopreservation protocols, but also for broader research into topics such as dehydration and desiccation tolerance, chilling and heat stress, as well as membrane structure and function.

Human Amniotic Membrane: A review on tissue engineering, application, and storage.

Human amniotic membrane (hAM) has been employed as scaffolding material in a wide range of tissue engineering applications, especially as a skin dressing and as a graft for corneal treatment, due to the structure of the extracellular matrix and excellent biological properties that enhance both wound healing and tissue regeneration. This review highlights recent work and current knowledge on the application of native hAM, and/or production of hAM-based tissue-engineered products to create scaffolds mimicking the structure of the native membrane to enhance the hAM performance. Moreover, an overview is presented on the available (cryo) preservation techniques for storage of native hAM and tissue-engineered products that are necessary to maintain biological functions such as angiogenesis, anti-inflammation, antifibrotic and antibacterial activity.