Effects of cryopreservation with a newly-developed magnetic field programmed freezer on periodontal ligament cells and pulp tissues

The purpose of this study was to evaluate the effects of long-term cryopreservation on the isolated human periodontal ligament cells (PDL) and pulp tissues. In the first part of study, 10 freshly extracted teeth were selected and divided into two groups. In the cryopreserved group, the teeth were frozen for 5 years using a programmed freezer combined with a magnetic field, known as Cells Alive System “CAS”. As for the control group, freshly extracted teeth were used. In each group, extracted PDL tissues were cultured and gene expression and protein concentration of collagen type I, alkaline-phosphatase (ALP) and vascular endothelial growth factor (VEGF) was compared between the two groups. In the second part, pulp tissues were obtained from 10 mature and immature third molars which were freshly extracted or cryopreserved for three months. Expression of VEGF and nerve growth factor (NGF) mRNAs and the protein concentration in the supernatant were investigated. Results indicated that long-term cryopreservation with the use of CAS freezer cannot affect the growth rate and characteristics of PDL cells. There was no significant difference in VEGF expression and VEGF and NGF protein concentration of pulp cells derived from cryopreserved teeth with immature apex and control group with mature root formation. Finally, proper PDL regeneration and appropriate apexogenesis after transplanting magnetically cryopreserved immature tooth was clinically confirmed. These findings demonstrate that teeth banking with the use of magnetic field programmed freezer can be available for future autotransplantation as a treatment modality for replacing missing teeth.     

Cryopreservation of periodontal ligament cells with magnetic field for tooth banking

The purpose of this study was to establish a long-term tooth cryopreservation method that can be used for tooth autotransplantation. Human periodontal ligament (PDL) cells were frozen in 10% dimethyl sulfoxide (Me₂SO) using a programmed freezer with a magnetic field. Cells were cryopreserved for 7 days at −150 °C. Immediately after thawing, the number of surviving cells was counted and the cells were cultured; cultured cells were examined after 48 h. Results indicated that a 0.01 mT of a magnetic field, a 15-min hold-time, and a plunging temperature of −30 °C led to the greatest survival rate of PDL cells. Based on these findings, whole teeth were cryopreserved under the same conditions for 1 year. The organ culture revealed that the PDL cells of cryopreserved tooth with a magnetic field could proliferate as much as a fresh tooth, although the cells did not appear in the cryopreserved tooth without a magnetic field. Histological examination and the transmission electron microscopic image of cryopreserved tooth with a magnetic field did not show any destruction of cryopreserved cells. In contrast, severe cell damage was seen in cells frozen without a magnetic field. These results indicated that a magnetic field programmed freezer is available for tooth cryopreservation.     

Electric and magnetic fields in cryopreservation: A response

Our recent studies showed that a programmed freezer with a magnetic field (CAS freezer) is helpful in the survival of periodontal ligament (PDL) cells after cryopreservation. The theory is that a magnetic field can prevent the cluster from growing by causing it to vibrate. In this letter, we commented in detail on the influence of a magnetic field during cryopreservation.     

Cryopreservation by slow cooling of rat neuronal cells

Although primary neuronal cells are routinely used for neuroscience research, with potential clinical applications such as neuronal transplantation and tissue engineering, a gold standard protocol for preservation has not been yet developed. In the present work, a slow cooling methodology without ice seeding was studied and optimized for cryopreservation of rat cerebellar granular cells. Parameters such as cooling rate, plunge temperature and cryoprotective agent concentration were assessed using a custom built device based on Pye's freezer idea. Cryopreservation outcome was evaluated by post thawing cell viability/viable cell yield and in culture viability over a period of 14 days. The best outcome was achieved when 10% of Me₂SO as cryoprotective agent, a cooling rate of 3.1 ± 0.2 °C/min and a plunge temperature of −48.2 ± 1.5 °C were applied. The granular cells cryopreserved under these conditions exhibited a cell viability of 82.7 ± 2.7% and a viable cell yield of 28.6 ± 2.2%. Moreover, cell viability in culture remained above 50%, very similar to not cryopreserved cells (control). Our results also suggest that post-thaw viability (based on membrane integrity assays) not necessarily reflects the quality of the cryopreservation procedure and proper functionality tests must be carried out in order to optimize both post thaw viability/cell yield and in culture performance.     

Magnetic field intensified bi-enzyme system with in situ cofactor regeneration supported by magnetic nanoparticles

Efficient dynamic interactions among cofactor, enzymes and substrate molecules are of primary importance for multi-step enzymatic reactions with in situ cofactor regeneration. Here we showed for the first time that the above dynamic interactions could be significantly intensified by exerting an external alternating magnetic field on magnetic nanoparticles-supported multi-enzymatic system so that the inter-particle collisions due to Brownian motion of nanoparticles could be improved. To that end, a multienzyme system including glutamate dehydrogenase (GluDH), glucose dehydrogenase (GDH) and cofactor NAD(H) were separately immobilized on silica coated Fe₃O₄ magnetic nanoparticles with an average diameter of 105 nm, and the effect of magnetic field strength and frequency on the kinetics of the coupled bi-enzyme reaction was investigated. It was found that at low magnetic field frequency (25 Hz and 100 Hz), increasing magnetic field strength from 9.8 to 161.1 Gs led to only very slight increase in reaction rate of the coupled bi-enzyme reaction expressed by glucose consumption rate. At higher magnetic field of 200 Hz and 500 Hz, reaction rate increased significantly with increase of magnetic field strength. When the magnetic field frequency was kept at 500 Hz, the reaction rate increased from 3.89 μM/min to 8.11 μM/min by increasing magnetic field strength from 1.3 to 14.2 Gs. The immobilized bi-enzyme system also showed good reusability and stability in the magnetic field (500 Hz, 14.2 Gs), that about 46% of original activity could be retained after 33 repeated uses, accounting for totally 34 days continuous operation. These results demonstrated the feasibility in intensifying molecular interactions among magnetic nanoparticle-supported multienzymes by using nano-magnetic stirrer for efficient multi-step transformations.     

Cryopreservation of cell line Paesun by a rapid cooling

The purpose of the present study was to clarify the possibility of a rapid cryopreservation for cell line Paesun by cooling in the range of 30–40 °C/min to vapor phase of −120 ∼-140 °C before immersion into liquid phase of liquid nitrogen using 10% Me₂SO. After thawing, these cells were examined with assaying viability by trypan blue exclusion staining and survival by cloning in monolayer; the percentages of cell and colony recovery obtained in rapid cooling had a tendency to be lower than that by slow cooling of 1 °C/min but there were no significant differences between them. In addition, post-thaw cells were examined by assaying proliferation and susceptibility to virus lines; there were no significant differences between before and after cryopreservation. In conclusion, these findings indicate that Paesun can be successfully cryopreserved by the rapid cooling rate of 30 °C–40 °C/min.     

Cryopreservation of Acropora digitifera sperm with use of sucrose and methanol based solution

Coral biodiversity has recently been considered an important topic in environmental studies. Biodiversity could be preserved with successful cryopreservation of endangered species gametes or embryos. Herein, we developed cryopreservation protocols for Acropora digitifera sperm with use of sucrose and methanol based extender. We studied cryopreservation of A. digitifera sperm with floating frames, allowing the placement of 250 μl French straws 4 cm above the liquid nitrogen surface, resulting in a 40 °C/min freezing rate. This method enabled the successful cryopreservation of sperm in 0.9 M sucrose supplemented with 20% methanol. In this protocol, we used a 1:3 (sperm:extender) dilution ratio. The fertilization ratios of freezing:thawed sperm were similar to the control and reached 63%. This method might be a valuable option in the formation of A. digitifera gene banking. Further studies are needed to explore possibilities of using this method in cryopreservation of other coral’s sperm.     

Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

We present a simple method for efficient DNA ligation utilizing the heat generation of ferromagnetic particles subjected to an ac magnetic field. We carry out the ligation of DNA fragments with cohesive ends using T4 DNA ligase immobilized on the surface of ferromagnetic particles. When a radio frequency alternating magnetic field is applied, ferromagnetic particles dissipate heat and DNA ligase on the particles is selectively heated up and activated with little influence on the annealing of DNA ends, as a result of which the ligation efficiency increases. We show that the ligation efficiency increases with an increase in the field amplitude.     

Activity of an enzyme immobilized on superparamagnetic particles in a rotational magnetic field

We immobilize α-amylase extracted from Bacillus Iicheniformis on the surfaces of superparamagnetic particles and investigate the effect of a rotational magnetic field on the enzyme’s activity. We find that the activity of the enzyme molecules immobilized on superparamagnetic particles increases in the rotational magnetic field and reaches maximum at a certain frequency. We clarify the effect of the cluster structures formed by the superparamagnetic particles on the activity. Enzyme reactions are enhanced even in a tiny volume of solution using the present method, which is very important for the development of efficient micro reactors and micro total analysis systems (μ-TAS).     

Analysis of the distribution of flowing erythrocytes in a model vessel under an inhomogeneous magnetic field

Changes in the distribution of flowing erythrocytes in a straight cylinder were studied under an inhomogeneous magnetic field. The magnetic field was applied perpendicular to a cylinder, which had a 90° side vessel at the end (oriented towards the magnetic field) to detect changes in the erythrocyte distribution within the cylinder. (1) The attraction of paramagnetic erythrocytes by the magnetic field was demonstrated by an increase in the concentration (or number) of erythrocytes drawn into the side vessel. The flow of diamagnetic, oxygenated erythrocytes was unaffected. (2) The degree of attraction of the paramagnetic erythrocytes was proportional to ``(magnetic susceptibility)' and to ``(magnetic flux density) × (magnetic field gradient)' up to 10 T²/m, but it saturated at high magnetic field. The onset of the saturation depended on the magnetic susceptibility of the erythrocytes. (3) The degree of attraction depended on the hematocrit of the flowing erythrocyte suspension, with a maximum value at a low hematocrit. These phenomena are explained on the basis of the balance between the paramagnetic attractive force of the magnetic field and the collision rate between erythrocytes. Received: 2 May 1996 / Accepted: 1 July 1996     

Inhibition of pentylenetetrazole-induced seizure in mice by using a 4 Hz magnetic field: a comparative study with a 60 Hz magnetic field

We investigated the comparative effects of 4 and 60 Hz magnetic fields on pentylenetetrazole (PTZ)-induced seizure in mice. For this study, we measured the latent time to seizure, seizure duration, and lethality induced by PTZ in mice exposed to 4 and 60 Hz magnetic fields (MF) for 30 min. Compared to sham-exposed controls, the latent time to tail twitching and seizure in the 4 Hz MF group was significantly decreased while the latent time to seizure in the 60 Hz MF group was significantly increased. The seizure duration in the 4 Hz MF group was significantly decreased while that in the 60 Hz MF group was significantly increased. More importantly, while the mice exposed to a 60 Hz MF experienced significantly increased lethality after seizure convulsion, those exposed to a 4 Hz MF showed no lethality, with a shortening of the duration of seizure. This beneficial effect of a 4 Hz MF on seizure has the same implication as the anti-oxidative effects of a 4 Hz MF observed in our previous work. The results of our current and previous works indicate that a 4 Hz MF may be used as a therapeutic physical agent for the treatment of oxidative stress-induced diseases, including seizure, with or without chemical drugs.     

A semi-analytical model for calculating the penetration depth of a high energy electron beam in a water phantom with a magnetic field

PurposeAs an electron beam is incident on a uniform water phantom in the presence of a lateral magnetic field, the depth-dose distribution of the electron beam changes significantly and forms the well-known ‘Bragg peak’, with a depth-dose distribution similar to that of heavy ions. This phenomenon has pioneered a new field in the clinical application of electron beams. For such clinical applications, evaluating the penetration depth of electron beams quickly and accurately is the critical problem.MethodsThis paper describes a model for calculating the penetration depth of an electron beam rapidly and correctly in a water phantom under the influence of a magnetic field. The model was used to calculate the penetration depths under different conditions: the energies of electron beams of 6, 8, 12 and 15 MeV and the magnetic induction intensities of 0.75, 1.0, 1.5, 2.0 and 3.0 T. In addition, the calculation results were compared with the results of a Monte Carlo simulation.ResultsThe comparison results indicate that the difference between the two calculation methods was less than 0.5 cm. Moreover, the computing time of the calculation model was less than a second.ConclusionsThe semi-analytical model proposed in the present study enables the penetration depth of the electron beam in the presence of a magnetic field to be obtained with a computational efficiency higher than that of the Monte Carlo approach; thus, the proposed model has high potential for application.     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

A magnetic field effect on learning in male golden hamsters

The aim of this experiment was to investigate the influence of repeated exposure to 10, 20, 30 or 40 Hz magnetic fields at 0.1 T on the learning of male golden hamsters in a Skinner box, in which the animals learned to press a lever to receive a food reward. The latency of the first response was not affected by exposure to the magnetic fields used in this experiment. No significant field-dependent effects on the performance of the task were observed in males exposed to 10 and 20 Hz magnetic fields at 0.1 T. However, exposure significantly improved the learning of the task in animals exposed to 30 and 40 Hz magnetic fields at 0.1 T.     

包埋玻璃化法超低温保存植物种质的研究进展

包埋玻璃化法是在玻璃化法和包埋脱水法基础上发展起来的超低温保存植物种质的新技术.它具有能同时处理大量材料,处理后恢复生长快,对材料的毒害作用较小及成芽率高等优点,已成功地用于辣根、山嵛菜等20余种植物,在植物种质资源的保存上显示出了巨大的应用潜力.本文介绍了包埋玻璃化法产生的背景及其优点,阐述了包埋玻璃化法的基本方法和预培养、包埋、脱水、化冻及恢复培养等过程,比较了该法冻存后的效果和冻存后所形成植株的遗传稳定性,同时指出了进一步研究的重点.     

Cryopreservation of fetal skin is improved by extracellular trehalose

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.     

植物样品包埋脱水法超低温保存的研究进展

包埋脱水法是植物材料超低温保存的新技术, 从１９９０ 年至今, 已有３０ 多篇文献报道。本文介绍了包埋脱水法的研究历史、技术要点和主要优点     

Cryopreservation of mouse embryos by vitrification: A meta-analysis

A meta-analysis of cryopreservation studies vitrifying mouse embryos was undertaken to determine the treatment effect of vitrification. Treatment by vitrification decreased embryo viability compared with controls: the odds ratio was 9.02 (CI: 3.73-21.78; P < 0.001), a 24.90% (CI: 14.88-34.91; P < 0.001) reduction in risk was associated with embryos in the control group, and for every 4.00 (CI: 3.91-4.09) embryos treated by vitrification, one does not survive. A multiple regression analysis evaluated covariates of embryo survival. For each hour increase post-hCG treatment when embryos were cryopreserved, there was a decrease of 0.36% (SEM ± 0.01) in survival (P < 0.001). The number of embryos surviving vitrification decreased 0.25% (SEM ± 0.02) per day increase in age of the female mouse (P < 0.001), whereas there was no significant difference for control group embryos. For each 1 h increase post-hCG treatment after cryopreservation when blastocysts were assessed for viability, there was a decrease of 0.13% (SEM ± 0.01) in survival. The later interval post-hCG treatment when blastocysts were assessed, the less viable they were compared with earlier blastocysts, independent of the vitrification protocol. This effect was not observed for control embryos. A high percentage of variability in the treatment effect for vitrification was likely due to underlying heterogeneity among studies. A portion of the risk associated with vitrification could be attributed to the general effects of cryopreservation. Future research should identify effects in a cryopreservation protocol specific to vitrification that affect viability of mouse embryos.     

Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification

The seeds of a Japanese terrestrial orchid (Bletilla striata Rchb.f.) were germinated and cultured on solidified new Dogashima (ND) medium for 10 days. These embryos were then precultured on ND medium supplemented with 0.3 m sucrose for 3 days at 25°C in continuous dark. The embryos were then overlaid with a mixture of 2 m glycerol and 0.4 m sucrose for 15 min at 25°C and finally dehydrated with highly concentrated vitrification solution (PVS2) for 3 h at 0°C prior to immersion into liquid nitrogen for 30 min. After rapid warming, the embryos were washed with liquid ND medium supplemented with 1.2 m sucrose for 20 min and then plated on ND medium. Successfully vitrified and warmed embryos developed into normal plantlets. The rate of plant regeneration amounted to about 60%. This vitrification method appears to be a promising technique for cryopreservation of orchids. Received: 19 September 1996 / Revision received: 3 January 1997 / Accepted: 24 February 1997     

Static magnetic field selects undifferentiated myelomonocytes from low-glutamine concentration stimulated U937 cells

Reduced glutamine (GLN) concentration in the culture medium of a U937 cell line caused them to be differentiated along the monocytic pathway; cells attached to the matrix and to each other by extending pseudopodia and acquired specific functional characteristics, such as the expression of alpha-naphthyl-acetate esterase and the capacity to reduce nitroblue tetrazolium, as well as becoming active phagocytes. When U937 cells were differentiated under continuous exposure to a 6mT static magnetic field (MF) the overall differentiation process was perturbed. Surprisingly, after 5 days' exposure to the static MF, higher cell viability and differentiation were observed in cells cultured in a GLN-deprived medium than in cells grown in the same medium but in the absence of a static MF. The latter cells, particularly those that were still floating in the medium, were stimulated with TPA for a further 3 days. These cells differentiated and attached to the substrate. Conversely, the same treatment applied to cells cultured in GLN-deprived medium in the presence of the static MF resulted in resistance to TPA-induced differentiation. Indeed, these cells exhibited a round shape and in-suspension growth.