Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

Alternative substrates for cryopreservation at −20 °C have been little explored for basidiomycetes and could bring new possibilities of lower cost cryopreservation. Nevertheless, freezing temperatures between −15 and −60 °C are very challenging because they frequently result in cryoinjuries. The objective of this study was to evaluate substrates associated to cryoprotective agents for Pleurotus ostreatus cryopreservation at −20 or −70 °C in order to develop alternative techniques for basidiomycete cryopreservation. P. ostreatus was grown on potato dextrose agar or whole grains of oat, wheat, rice or millet and transferred to cryovials with cryoprotective solution with 1 % dimethyl sulfoxide, 5 % glycerol, 10 % saccharose, 4 % glucose, 6 % polyethylene glycol-6000 or 5 % malt extract. The mycelium in the cryovials were cryopreserved at −20 or −70 °C and recovered for evaluation of the mycelial growth viability after 1 and 3 years. Both substrates and cryoprotectants affect the viability of the mycelial growth cryopreserved at −20 or −70 °C; wheat grains combined with cryoprotectants such as saccharose or glucose are effective for keeping mycelium viable after cryopreservation at −20 °C for 1 or 3 years; for cryopreservation at −70 °C after 1 or 3 years, any substrate combined with any cryoprotectant is effective for preserving the mycelium viable, except for millet grains with polyethylene glycol after 3 years; semi-permeable cryoprotective agents such as saccharose and glucose are the most effective for cryopreservation at −20 or −70 °C for at least 3 years.     

Zebrafish sperm cryopreservation with N,N-dimethylacetamide

High fecundity, rapid generation time, and external development of optically clear embryos make the zebrafish (Danio rerio) a convenient vertebrate model for genetic, developmental, and disease studies. Efficient sperm cryopreservation enhances the zebrafish model system by optimizing productive use of facility space, extending the reproductive lifetime of males, providing an alternative to live stocks for strain recovery, and ensuring the survival of valuable mutant lines. Here we identify a cryoprotective medium, 10% N,N-dimethylacetamide (DMA) (v/v) diluted in buffered sperm motility-inhibiting solution (BSMIS), as well as parameters for zebrafish sperm cryopreservation that enhance cryopreservation efficiency and significantly increase the yield of live embryos from archived stocks. Our experiments emphasize the effect of the ratio of sperm and medium volume and the use of large egg clutches to maximize the recovery of viable embryos.     

Congelation of cryoprotective solutions and cryopreservation of fish sperm

A study was made of the formation of ice microparticles (IMP) during freezing of cryoprotective media and the survival of fish sperm in cryopreservation. The IMP shape and size were determined by cryomicroscopy. It is shown that with an increase of the cooling rate the IMP area and perimeter decrease. Under ultrafast cooling (3000–4000 deg/min) in a thin layer (0.1 mm) the medium is vitrified. Additional components (egg yolk, sugars and lipids) substantially alter the shape and size of IMP. The intactness of sperm after cryopreservation does not always correlate with the IMP size and shape in the frozen cryoprotective solution. Formation of rounded particles with blurred boundaries correlates with sperm survival in cryopreservation.     

Influence of Factors of Cryopreservation and Hypothermic Storage on Survival and Functional Parameters of Multipotent Stromal Cells of Placental Origin

Human placenta is a highly perspective source of multipotent stromal cells (MSCs) both for the purposes of patient specific auto-banking and allogeneic application in regenerative medicine. Implementation of new GMP standards into clinical practice enforces the search for relevant methods of cryopreservation and short-term hypothermic storage of placental MSCs. In this paper we analyze the effect of different temperature regimes and individual components of cryoprotective media on viability, metabolic and culture properties of placental MSCs. We demonstrate (I) the possibility of short-term hypothermic storage of these cells; (II) determine DMSO and propanediol as the most appropriate cryoprotective agents; (III) show the possibility of application of volume expanders (plasma substituting solutions based on dextran or polyvinylpyrrolidone); (IV) reveal the priority of ionic composition over the serum content in cryopreservation media; (V) determine a cooling rate of 1°C/min down to -40°C followed by immersion into liquid nitrogen as the optimal cryopreservation regime for this type of cells. This study demonstrates perspectives for creation of new defined cryopreservation methods towards GMP standards.     

The cryobiology of spermatozoa

The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm cryobiology, provide a synopsis of the development of sperm cryobiology as a discipline, and present the current state and directions for future research in sperm cryobiology in the three major areas outlined above—agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

Introduction

Human fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.

Methods

Human fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.

Results

The addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.

Conclusion

The inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, −1 °C/min chilling rate (18 °C to 5 °C) and −13 °C/min freezing rate (5 °C to −80 °C), followed by immersion in liquid nitrogen.     

Effects of water-soluble Laminaria japonica polysaccharide 3 (LJP-P3) on bull cryopreservation sperm

In this study, water-soluble Laminaria japonica polysaccharide3 (LJP-P3) was investigated for the cryoprotective effects on bull sperm. Five concentrations of LJP-P3 with 0.1, 1, 10, 50 and 100 mmol/L were added into the extenders of bull semen, respectively, and the effects on quality of sperm after freezing-thawing were assessed. The results showed that the kinematic parameters of bull sperm including linear motile sperm (LM), curvilinear line velocity (VCL) value, straight line velocity (VSL) and velocity of the average path (VAP) were greater in the extenders containing LJP-P3 (P<0.05). In comparison to those of other treatments and control group the extenders containing 1.0, 10.0 and 50.0 mmol/L of LJP-P3 led to higher percentage of mitochondrial activity and sperm membrane integrity(P<0.05), and the acrosome integrity of bull cryopreservation sperm were significantly improved in all treatment groups. Moreover, the higher GSH-Px, SOD and CAT levels in bull cryopreservation sperm were favored from the extenders of 10.0, 50.0 and 100.0 mmol/L LJP-P3 added (P<0.05) compared with other treatments and control group. In addition, the results of artificial insemination showed that both the pregnancy rate and the number of calving were higher in the group of semen containing 10 mmol/L of LJP-P3 than that of control group (P < 0.05). In summary, LJP-P3 exhibited a greater cryoprotective effect to bull sperm and the most suitable concentration of LJP-P3 is 10.0 mmol/L.     

Quantitative Measurement of Cell Membrane Transport: Technology and Applications

The transport of water and cryoprotective chemicals across cell membranes plays an absolutely fundamental role in the outcome of cryopreservation processing. The diversity of cell types as well as the remarkable range of perturbations that cells are subjected to as part of cryopreservation practices generate many interesting research questions. Simply stated, the extreme conditions typical of cryopreservation protocols extend the limits of membrane transport inquiry well beyond that considered in “normal” cell physiology. This paper provides a brief review of methods which have been used for measuring membrane transport properties, especially those methods developed during the past decade which allow us to measure coupled and uncoupled membrane transport properties of water and cryoprotective agents for individual cells in terms of classical Kedem–Katchalsky membrane transport theory. Representative results obtained from these new technologies will be offered to illustrate their utility and relevance to membrane transport issues arising in cryopreservation practice. Engineers have made significant contributions to this area of research primarily in terms of device development and the application of inverse methods to estimate membrane transport properties.     

The impact of cryoprotective media on cryopreservation of cells using loading trehalose

The purpose of the present study was to assess the impact of cryoprotective media on cryopreservation of Paesun cells using loading trehalose. 30 mM trehalose was added after confluence of cells, and cultures were further incubated for 18 h at 37 °C. Cryoprotective media was “Cryocool” for the experiment, while MEM containing 10% FBS for the control. After thawing, these cells were examined with assaying the percentage of viable cells and the recovery rate; 89.2 ± 1.4% and 78.8 ± 3.2%, respectively in the experiment group, while 33.1 ± 2.9% and 21.5 ± 2.1%, respectively in the control group. Post-thaw cells of the experiment group were examined by assaying proliferation and susceptibility to virus lines; there were no significant differences between before and after cryopreservation, while cells of control group could not be recultured. In conclusion, the cryoprotective media impacts on the effectiveness of cryopreservation using loading trehalose.     

An evaluation of cryoprotective compounds on bovine spermatozoa

The potentially cryoprotective properties of 72 higher-molecular-weight polymeric additives and 69 low-molecular-weight compounds were evaluated. The polymeric compound selection was based on solubility in semen extender, toxicity and finally, on the cryoprotective effect on bull spermatozoa as measured by progressive motility. Five compounds showed cryoprotection to the cell, but with no significant improvement over that of TESNaK yolk, TEST yolk, or TEST yolk glycerol extenders used as controls. Low-molecular-weight compounds were selected on the basis of colligative properties particularly that of freezing-point depression. Elimination was based on precipitation of proteins in the extender, toxicity, and cryoprotection to bovine spermatozoa as measured by progressive motility. Nineteen compounds that yielded protection during cryopreservation of bovine spermatozoa were compared using post-thaw motility and membrane integrity using glutamic-oxaloacetic transaminase enzyme retained in the spermatozoa after freezing as an indicator. Semen was diluted with extender containing selected compounds at 35 or 5 °C to determine the effect of temperature at which the cryoprotective compound was added. Glycerol yielded the highest recovery. Diethylene glycol, dimethylsulfoxide, N-methylacetamide, and triethylene glycol appeared not to be different in freezing bovine spermatozoa. The temperature or method of addition of cryoprotective compound did not reveal a significant difference.     

Toxicity tests of cryoprotecting agents for Mytilus galloprovincialis (Lamark, 1819) early developmental stages

Global aquaculture production of blue mussel has increased over last years. This work reaffirms the great potential of cryopreservation technique on mussel industry and overcome economic barriers a cause of a traditional and rudimentary management and continue growing. The aim of this work is to set some preliminary basis attending to toxicity of cryoprotecting agents (CPAs) on different development stages of Mytilus galloprovincialis as a start point to develop a stable cryopreservation protocol. Toxicity tests were carried out by using common CPAs (dimethyl-sulfoxide (Me₂SO), glycerol, (GLY), propylene glycol (PG) and ethylene glycol (EG)) in a range from 0.5 to 3 M on fertilized egg, trochophore larva, and D-larva of Mytilus galloprovincialis. Results evidenced more resistance of older development stages to toxicity. Of all CPAs tested, toxicity testing highlights PG or EG as suitable CPAs for cryopreservation of early development stages; whereas D-larva was unaffected by any of the CPAs tested. Preliminary cryopreservation trials were developed to obtain information into cell cryoprotection. Further research should be focused on membrane permeability and other parameters, such as the balance between toxicity and cryoprotective effect of CPAs.     

Advances in understanding the fundamental aspects required for successful cryopreservation of Australian flora

Australia is host to an amazing diversity of species, many of which require conservation efforts. In vitro culture provides a tool for not only conserving these threatened species but allows for their propagation from limited starting material. Cryopreservation provides the greatest long-term storage option for in vitro cultures and as a conservation tool for other germplasm. However, while cryopreservation has proven capable of delivering viable long-term storage with some plant taxa, the process of deriving protocols is still largely an incremental process. The key to faster and more intuitive optimising of cryopreservation protocols lies with continuing to develop a better understanding of key factors, including issues with plant physiology (such as genetic stability, the composition of the proteome and metabolome, cell membrane characteristics, and antioxidant defences) and how the stresses imposed by cryopreservation (such as the excision damage, desiccation, cryoprotective agent toxicity, ice crystal damage, and cooling to cryogenic temperatures) interact and contribute to the cryocapability of a species. This review focuses on the advances that have been made towards understanding cryogenic stress and how this has led to improved cryopreservation protocols, in the context of cryopreserving Australian flora.     

Xenotransplantation of cryopreserved composite organs on the rabbit

Nowadays, It is easy to define optimal conditions (cryoprotective agent, speed and steps of freezing, speed of warming) for the cryopreservation of a homogeneous cell population or a one cell-layer tissue. Meanwhile, It is still hard to obtain cryopreservation of composite organs. Each tissue has its own requirements and its own reactivity to the cryopreservation process. The challenge consists of, on the one hand, to select the ideal combination of cryoprotective agents that can fit the needs of the different tissues, and the definition of adequate technical parameters, on the other hand. All the experimental trials have studied the survival rate of non-vascularized cryopreserved tissues. The aim of our experimental work is to demonstrate the feasibility of cryopreserving a composite organ with its nutrient vessels “artery and veins” in order after thawing to revitalize it by reestablishing the blood irrigation by microsurgical vascular anastomosis. We report our experimental results on the cryopreservation of composite organs—amputated digits—xenotransplanted in the rabbit. Digital segments were cryopreserved, then revitalized after warming using vascular microsurgical techniques. Preliminary results are encouraging and may pave the way in the future to the microvascular allotransplantation of cryopreserved composite organs.     

Measurement of the biophysical properties of porcine adipose-derived stem cells by a microperfusion system

Adipose-derived stem cells (ADSCs), which are an accessible source of adult stem cells with capacities for self-renewal and differentiation into various cell types, have a promising potential in tissue engineering and regenerative medicine strategies. To meet the clinical demand for ADSCs, cryopreservation has been applied for long-term ADSC preservation. To optimize the addition, removal, freezing, and thawing of cryoprotective agents (CPAs) applied to ADSCs, we measured the transport properties of porcine ADSCs (pADSCs). The cell responses of pADSCs to hypertonic phosphate-buffered saline and common CPAs, dimethyl sulfoxide, ethylene glycol, and glycerol were measured by a microperfusion system at temperatures of 28, 18, 8, and −2 °C. We determined the osmotically inactive cell volume (V_b), hydraulic conductivity (L_p), and CPA permeability (P_s) at various temperatures in a two-parameter model. Then, we quantitatively analyzed the effect of temperature on the transport properties of the pADSC membrane. Biophysical parameters were used to optimize CPA addition, removal, and freezing processes to minimize excessive shrinkage of pADSCs during cryopreservation. The biophysical properties of pADSCs have a great potential for effective optimization of cryopreservation procedures.     

Successful Recovery of Motility and Fertility of Cryopreserved Cane Toad (Bufo marinus) Sperm

The recent decline and extinction of amphibian species is a worldwide phenomenon without an identified cause or solution. Assisted reproductive technologies, including sperm cryopreservation, are required to manage endangered amphibian species and preserve their genetic diversity. This study on the Anuran amphibian (Bufo marinus) was undertaken to determine the feasibility of cryopreservation of amphibian sperm. Sperm suspensions for cryopreservation were prepared by macerating testes in cryoprotective additives of 10% (w/v) sucrose or 10% (w/v) sucrose containing either 10, 15, or 20% (v/v) glycerol or 10, 15, or 20% (v/v) dimethyl sulfoxide (Me₂SO). Suspensions were then cooled to −85°C using a controlled rate cooler, stored in LN₂, and thawed in air. The motility and fertilization rate of cryopreserved suspensions and unfrozen control suspensions in Simplified Amphibian Ringer were compared. Sucrose alone had no cryoprotective effect. All other treatments showed varying degrees of recovery of motility and fertilizing capacity. High rates of recovery of motility and fertilizing capacity were observed with 15% Me₂SO (68.9 ± 3.8 and 60.5 ± 4.7%) and 20% glycerol (58.0 ± 5.9 and 81.4 ± 4.3%), respectively. Motility and fertilization rates were similar with Me₂SO but diverged with glycerol as cryoprotectant. The data demonstrate the feasibility of using sperm cryopreservation with amphibian species.     

The effect of additive compounds on glycerol-induced damage to human chondrocytes

High concentrations of cryoprotective agents are required for cryopreservation techniques such as vitrification. Glycerol is a common cryoprotective agent used in cryopreservation protocols but this agent is toxic at high concentrations. This work is an attempt to mitigate the toxic effects of high concentrations of glycerol on intact chondrocytes in human knee articular cartilage from total knee arthroplasty patients by simultaneous exposure to glycerol and a variety of additive compounds. The resulting cell viability in the cartilage samples as measured by membrane integrity staining showed that, in at least one concentration or in combination, all of the tested additive compounds (tetramethylpyrazine, ascorbic acid, chondroitin sulphate, glucosamine sulphate) were able to reduce the deleterious effects of glycerol exposure when examination of membrane integrity took place on a delayed time frame. The use of additive compounds to reduce cryoprotectant toxicity in articular cartilage may help improve cell recovery after cryopreservation.     

Static magnetic field increases survival rate of dental pulp stem cells during DMSO-free cryopreservation

Abstract

Successful and efficient cryopreservation of living cells and organs is a key clinical application of regenerative medicine. Recently, magnetic cryopreservation has been reported for intact tooth banking and cryopreservation of dental tissue. The aim of this study was to assess the cryoprotective effects of static magnetic fields (SMFs) on human dental pulp stem cells (DPSCs) during cryopreservation. Human DPSCs isolated from extracted teeth were frozen with a 0.4-T or 0.8-T SMF and then stored at ?196?°C for 24?h. During freezing, the cells were suspended in freezing media containing with 0, 3 or 10% DMSO. After thawing, the changes in survival rate of the DPSCs were determined by flow cytometry. To understand the possible cryoprotective mechanisms of the SMF, the membrane fluidity of SMF-exposed DPSCs was tested. The results showed that when the freezing medium was DMSO-free, the survival rates of the thawed DPSCs increased 2- or 2.5-fold when the cells were exposed to 0.4-T or 0.8-T SMFs, respectively (p?<?0.01). In addition, after exposure to the 0.4-T SMF, the fluorescence anisotropy of the DPSCs increased significantly (p?<?0.01) in the hydrophilic region. These results show that SMF exposure improved DMSO-free cryopreservation. This phenomenon may be due to the improvement of membrane stability for resisting damage caused by ice crystals during the freezing procedure.     

Water content in an engineered dermal replacement during permeation of Me2SO solutions using rapid MR imaging

The successful cryopreservation of cell and tissues typically requires the use of specialized solutions containing cryoprotective agents. At room temperature, the introduction of a cryopreservation solution can result in cell damage/death resulting from osmotic stresses and/or biochemical toxicity of the solution. For tissues, the permeation and equilibration of a cryoprotective solution throughout the tissue is important in enhancing the uniformity and consistency of the postthaw viability of the tissue. Magnetic resonance (MR) is a common nondestructive technique that can be used to quantitate the temporal and spatial composition of water and cryoprotective agents in a three-dimensional system. We have applied a recently developed rapid NMR imaging technique to quantify the transport of water in an artificial dermal replacement upon permeation of dimethyl sulfoxide (Me2SO) solutions. Results indicate that the rate of water transport is slower in the presence of Me2SO molecules. Furthermore, the transport is concentration-dependent, suggesting that Me2SO tends to retain bound water molecules in the tissue. Moreover, water transport decreases with decreasing temperature, and the presence of cells tends to increase water transport.