Thawing Methods Research Articles

A cryopreservation method for bioengineered 3D cell culture models

Technologies to cryogenically preserve (a.k.a. cryopreserve) living tissue, cell lines and primary cells have matured greatly for both clinicians and researchers since their first demonstration in the 1950s and are widely used in storage and transport applications. Currently, however, there remains an absence of viable cryopreservation and thawing methods for bioengineered, three-dimensional (3D) cell models, including patients’ samples. As a first step towards addressing this gap, we demonstrate a viable protocol for spheroid cryopreservation and survival based on a 3D carboxymethyl cellulose scaffold and precise conditions for freezing and thawing. The protocol is tested using hepatocytes, for which the scaffold provides both the 3D structure for cells to self-arrange into spheroids and to support cells during freezing for optimal post-thaw viability. Cell viability after thawing is improved compared to conventional pellet models where cells settle under gravity to form a pseudo-tissue before freezing. The technique may advance cryobiology and other applications that demand high-integrity transport of pre-assembled 3D models (from cell lines and in future cells from patients) between facilities, for example between medical practice, research and testing facilities.

Rooster sperm pellet cryopreservation protocols: effect of step variations on the qualitative parameters of post-thawed sperm

The cryopreservation of sperm into pellets is not the preferred way to package avian semen but is quick and easy to do and does not require sophisticated technology. The aim of this study was to evaluate rooster sperm viability and mobility and the incidence of normal cells and sperm injuries in post-thawed pelleted sperm. The outcomes of different pelleting protocols were evaluated, which varied according to the parameter combinations used in each of the critical steps of the freezing process and in the thawing conditions which differed in methodology and temperature. The protocols employing 6% DMA showed the highest values of thawed sperm mobility. The most favourable thawing method in terms of sperm mobility was using the hot-plate at 60 °C, followed by the water-bath at 50 °C. The protocols resulting in the best sperm quality parameters employed a 1:2 dilution rate, a 30-min equilibration time at 4 °C, 6% DMA, and thawed 80 µL pellets using the water-bath at 50 °C or the hot-plate at 60 °C. According to the parameters evaluated, rooster sperm was highly susceptible to damage caused by the freezing-thawing methodology, although the survival rate of normal sperm cells still reached 39%, with 32% recovered mobility with respect to fresh sperm samples. HIGHLIGHTS A feasible method for the ex-situ management of genetic resources in birds involves the use of cryopreserved sperm. The cryopreservation of domestic fowl semen into pellets constitutes a reasonably simple, quick and affordable technology. This technology may be extremely valid and practical for use on small farms.

In Vitro Nephrotoxicity and Permeation of Vancomycin Hydrochloride Loaded Liposomes.

Drugs can be toxic to the fetus depending on the amount that permeates across the maternal–fetal barrier. One way to limit the amount which penetrates this barrier is to increase the molecular size of the drug. In this study, we have achieved this by encapsulating our model antibiotic (vancomycin hydrochloride, a known nephrotoxic agent) in liposomes. PEGylated and non-PEGylated liposomes encapsulating vancomycin hydrochloride were prepared using two different methods: thin-film hydration followed by the freeze–thaw method and the reverse-phase evaporation method. These liposomes were characterized by their hydrodynamic size and zeta potential measurements, CryoTEM microscopy, loading and encapsulation efficiency studies, in vitro release measurements and in vitro cytotoxicity assays using NRK-52 E rat kidney cells. We also determined the in vitro permeability of these liposomes across the human placental cell and dog kidney cell barriers. Vancomycin hydrochloride-loaded PEGylated liposomes (VHCL-lipo) of a size less than 200 nm were prepared. The VHCL-lipo were found to have the faster release of vancomycin hydrochloride and resulted in greater viability of NRK-52E cells. In vitro, the VHCL-lipo permeated the human placental cell and dog kidney cell barriers to a lesser extent than the free vancomycin hydrochloride. The data suggest a reduction in nephrotoxicity and permeability of vancomycin hydrochloride after encapsulation in PEGylated liposomes.

Enhanced Macroporous Cationic Chitosan Hydrogel by Freezing and Thawing Method with Superadsorption Capacity for Anionic Dyes

In this study, we have developed a simple technique to prepare cationic chitosan hydrogel with interconnected porous structure using freeze–thaw process and the obtained hydrogel was named FCS hydrogel. Scanning electron microscopy (SEM) imaging discovered that the synthesized hydrogel demonstrated interconnected porous structure in the scope of 5–20 μm. We also showed that the FCS hydrogel exhibits pH responsiveness behavior, and demonstrated reversible swelling and de-swelling behaviors maintaining their mechanical stability. We demonstrate that the FCS hydrogel swelling capacity decreased at alkaline pH and rose with a decline in pH value. Besides, the FCS hydrogel presented specific surface area of 78.25 ± 8.75 m2 g−1, due to the cryogenic treatment of glutaraldehyde cross-linked chitosan hydrogel could increase the surface area and permeability of composite hydrogel and then strongly increasing the adsorption capacity. Subsequently, the FCS monolithic hydrogel tested dyes removal, which provides a high removal efficiency towards anionic dyes including congo red (CR) and sodium fluorescein (SFL) dyes. Significantly, we show that the FCS hydrogel could be regenerated and reused as an adsorbent for wastewater treatment without significant loss of pollutants removal efficiency over a number of adsorption and washing cycles. This study offers a promising environmental friendly and sustainable interconnected porous hydrogel for anionic dye removal from wastewater.

Tough Engineering Hydrogels Based on Swelling-Freeze-Thaw Method for Artificial Cartilage.

Articular cartilage, which exhibits toughness and ultralow friction even under high squeezing pressures, plays an important role in the daily movement of joints. However, joint soft tissue lesions or injuries caused by diseases, trauma, or human functional decline are inevitable. Poly(vinyl alcohol) (PVA) hydrogels, which have a water content and compressive strength similar to those of many tissues and organs, have the potential to replace tough connective tissues, including cartilage. However, currently, PVA hydrogels are not suitable for complex dynamic environments and lack rebound resilience, especially under long-term or multicycle mechanical loads. Inspired by biological tissues that exhibit increased mechanical strength after swelling, we report a tough engineered hydrogel (TEHy) fabricated by swelling and freeze-thaw methods with a high compressive strength (31 MPa), high toughness (1.17 MJ m-3), a low friction coefficient (0.01), and a low energy loss factor (0.22). Notably, the TEHy remained remarkably resilient after 100 000 cycles of contact extrusion and remains intact after being compressed by an automobile with a weight of approximately 1600 kg. The TEHy also exhibited excellent water swelling resistance (volume and weight changes less than 5%). Moreover, skeletal muscle cells were able to readily attach and proliferate on the surface of TEHy-6, suggesting its outstanding biocompatibility. Overall, this swelling and freeze-thaw strategy solves the antifatigue and stability problems of PVA hydrogels under large static loads (>10 000 N) and provides an avenue to fabricate engineering hydrogels with strong antifatigue and antiswelling properties and ultralow friction for potential use as biomaterials in tissue engineering.

Physicochemical properties of post laying hen breast meat thawed using various methods

The analysis of the impact of various methods of thawing on the physicochemical properties of post laying hen breast meat (musculus pectoralis superficialis) was studied. The treatments of various methods of thawing include water immersion (with the temperature measured at 20°C), thawing under tap water (water changes with the temperature measured at 30°C) and the water bath method (with the temperature measured at 40°C). The physical properties such as pH, cooking loss, drip loss, Water Holding Capacity (WHC) and hardness were being evaluated. Meanwhile, the chemical properties such as protein and fat content, protein profile and microstructure were being assessed as well. The analysis revealed significant (P<0.05) impacts on the meat quality properties depending on various thawing methods which were being subjected. The practical thawing method is to use water immersion at 20oC, which can be recommended to the public.

Comprehensive Diagnostic Medical System Based on Notch1 Signaling Pathway to Inhibit the Growth of Small-Cell Lung Carcinoma.

With the gradual application of big data and other technologies to the medical field, more and more people tend to get online medical services. This article mainly studies the comprehensive diagnostic medical system based on Notch1 signaling pathway to inhibit the growth of small-cell lung carcinoma. In the experiment, we used the rapid thawing method to recover the cells and took the logarithmic growth phase cells for cell passage. We calculated the cell concentration and diluted the cells according to the experimental requirements. According to the standard curve, the corresponding sample protein concentration was calculated; at the same time, the Trizol method was used to extract the total RNA, the NanoDrop8000 spectrophotometer was used to determine the RNA concentration, and the RNA quality was detected by agarose gel electrophoresis. We used immunohistochemical staining to complete the staining of lung cancer cells. Finally, black box testing was used to test the functional modules of the system. Experimental data show that the accuracy rate of data obtained by the system reaches 98%, which greatly facilitates doctors and patients. The results show that the system has good ease of use and reliability and improves the diagnosis and treatment of hospital patients.

Copper Ion Imprinted Hydrogel Photonic Crystal Sensor Film

An ion-imprinted photonic crystal (IIPC) sensor for visual detection of copper ions was prepared by the combination of ion-imprinting polymer and colloidal photonic crystal (CPC) structure. Monodisperse polystyrene (PS) colloidal nanospheres synthesized by the boiling soap-free emulsion polymerization method were used to prepare the CPC template by the solvent evaporation method. An ion-imprinted polymer based on poly(vinyl alcohol) and chitosan (PVA/CS) is infiltrated therein. The IIPC sensor film was then prepared by solvent-assisted freeze–thaw method while the CPC template was strengthened as the air pores of the template were replaced by PVA/CS hydrogel. The shift of the diffraction wavelength can be directly observed by the naked eye through the color change of the IIPCS according to the concentration change of copper ion. Such sensor film showed good selectivity and sensitivity with a lower detection limit of 10–4 M.

Modification of the vacuum-steam thawing method of meat by using the initial stage of sublimation dehydration

Vacuum-steam thawing is one of the methods used for defrosting food, realized in the atmosphere of water vapour under the conditions of reduced pressure. The water vapour formed in vacuum with the temperature of 20 °C fills the defrosting chamber and condenses on the surface of the defrosted product. The condensated steam has the role of thermal energy carried enabling product thawing. The study presents a modification of this method, introducing an additional stage of sublimation-dehydration vacuum steam thawing (SRVST). The study was carried out for different variants of initial sublimation degree (in the range from 0 to 15%) of a slice of pork loin (m. longissimus lumborum) assessing the final effect of the process of vacuum-steam thawing. Thawing kinetics was determined with the SRVST method, degree of sample defrosting and level of their rehydration. Based on the results it was demonstrated that the use of 12% sublimation dehydration of a meat sample enables its complete defrosting (reaching the temperature not exceeding the cryoscopic temperature).

Study on the Flavor Compounds of Fo Tiao Qiang under Different Thawing Methods Based on GC-IMS and Electronic Tongue Technology.

“Fo Tiao Qiang” is a famous dish with Chinese characteristics. It is delicious, rich in materials, and high in nutritional value. Through physical and chemical analysis, electronic tongue, gas chromatography–ion mobility spectroscopy, and other technologies, the present study explored the quality characteristics and flavor differences of Fo Tiao Qiang by using different thawing methods (natural thawing, ultrasonic thawing, microwave thawing, and water bath thawing). The results show that the protein content was slightly higher in Fo Tiao Qiang with ultrasonic thawing than others. The fat content of the microwave-thawed Fo Tiao Qiang was significantly lower than the other three kinds of samples. After ultrasonic thawing, the number of free amino acids in the samples were the highest and the umami taste was the best. Compared with natural thawing, most of the flavor substances decreased in ultrasonic thawing, microwave thawing, and water bath thawing. However, several substances increased, such as alpha-terpineol, beta-phenylethyl alcohol, phenylacetaldehyde, cis-rose oxide, isobutyl acetate, and 2–3-pentanedione. This study revealed the changing laws of different thawing methods on the quality characteristics and flavor characteristics of Fo Tiao Qiang. It provides theoretical guidance for the industrial production and quality control of Fo Tiao Qiang.

Impact of Ultrasound-assisted Saline Thawing on the Technological Properties of mirror carp (Cyprinus carpio L.)

The aim of the study was to evaluate the positive effect of ultrasound-assisted saline thawing (UST) on the technological properties (water mobility, water holding capacity, colour, pH, shear force, TVB-N, oxidation reaction and microstructure) of mirror carp (Cyprinus carpio L.). The results present in the study showed that different thawing methods had negative impacts on the quality of mirror carp to varying degrees. Among them, UST samples had significant lower thawing loss, centrifugal loss and cooking loss than ultrasound thawing (UT) and air thawing (AT) samples (P < 0.05). The analysis result of low-field nuclear magnetic resonance illustrated that UST inhibited the mobility and distribution of water effectively. Decrease in shear force and TVBN values were observed in all thawing samples, and the UST samples maintained the significant better texture property and freshness than UT and AT samples did (P < 0.05). In addition, the treatment of UST obtained 1% salt concentration inhibited the oxidation reactions effectively. Investigation of the microstructure of samples demonstrated that the treatment of UST kept the relatively complete structure of tissue than other thawing methods. Therefore, UST can be an alternative strategy to the traditional thawing of meat.

Effects of different freezing and thawing methods on the quality of giant red shrimp (Aristaeomorpha foliacea)

The aim of this study was to investigate the effects of different freezing and thawing methods on physical, chemical, and sensory characteristics of red shrimp (Aristaeomorpha foliacea). Shell-on and shell-off shrimps were subjected to three different freezing (blast freezing, still freezing and cryogenic freezing) and thawing (on air, in refrigerator and in microwave oven) methods and stored for 30 days at -18ºC. Quality control analyses were carried out in samples after thawing. The lowest total volatile nitrogen and trimethylamine contents, pH values and cooking losses were found in cryogenically frozen shrimp, compared with other freezing methods. However, cryogenic freezing caused colour fading and softening in texture. It was determined that microwave thawing conditions used in this study are not suitable for thawing of frozen shrimp as it negatively affects texture and colour and increases cooking loss. It was found that blast freezing, and thawing methods preserved the physical, chemical and sensory properties of shrimp better than other methods.Keywords: Aristaeomorpha foliacea; Freezing; Red shrimp; Shrimp quality

The Comparison of Microwave Thawing and Ultra-High-Pressure Thawing on the Quality Characteristics of Frozen Mango.

As one of the popular tropical fruits, mango has a relatively short shelf life due to its perishability. Therefore, post-harvest losses are always a topic of concern. Currently, freezing is a common approach to extending mango shelf life. In relation, it is also critical to select a proper thawing process to maintain its original quality attributes. In this study, microwave thawing, and ultra-high-pressure thawing were investigated, and traditional thawing methods (air thawing and water thawing) were compared as references. The thawing time, quality attributes, and sensory scores of frozen mangoes were evaluated. Compared to traditional methods, innovative thawing methods can extensively shorten thawing time. These things considered, the thawing time was further decreased with the increase in microwave power. Additionally, microwave thawing enhanced the quality of mangoes in terms of less color change and drip loss and reduced loss of firmness and vitamin C content. Microwave thawing at 300 W is recommended as the best condition for thawing mangoes, with the highest sensory score. Current work provides more data and information for selecting suitable thawing methods and optimum conditions for frozen mango to minimize losses.

Effects of different freezing and thawing methods on the quality of giant red shrimp (Aristaeomorpha foliacea)

The aim of this study was to investigate the effects of different freezing and thawing methods on physical, chemical, and sensory characteristics of red shrimp (Aristaeomorpha foliacea). Shell-on and shell-off shrimps were subjected to three different freezing (blast freezing, still freezing and cryogenic freezing) and thawing (on air, in refrigerator and in microwave oven) methods and stored for 30 days at -18ºC. Quality control analyses were carried out in samples after thawing. The lowest total volatile nitrogen and trimethylamine contents, pH values and cooking losses were found in cryogenically frozen shrimp, compared with other freezing methods. However, cryogenic freezing caused colour fading and softening in texture. It was determined that microwave thawing conditions used in this study are not suitable for thawing of frozen shrimp as it negatively affects texture and colour and increases cooking loss. It was found that blast freezing, and thawing methods preserved the physical, chemical and sensory properties of shrimp better than other methods.Keywords: Aristaeomorpha foliacea; Freezing; Red shrimp; Shrimp quality

Dimethyl sulfoxide for cryopreservation of alginate encapsulated liver cell spheroids in bioartificial liver support; assessments of cryoprotectant toxicity tolerance and dilution strategies

The Bioartificial Liver (BAL) is an extra-corporeal liver support designed to support the function of the Liver in patients with impaired liver function. The BAL biomass consists of alginate encapsulated liver spheroids (AELS). To facilitate rapid delivery of a BAL to patients the AELS are cryopreserved using a DMSO-containing cryoprotectant solution. This study assesses toxicity of DMSO in AELS at concentrations and temperatures relevant to the cryopreservation and recovery process of a cellular biomass. Additionally, it develops a process to remove DMSO from AELS before delivery of cell product to patients. Exposure of AELS to DMSO, at a concentration of 12% (v/v) for 10 min did not have a negative effect on the viability of the AELS up to 24 h after exposure, irrespective of the exposure temperature between 37 C and 0 C. Evidence of toxicity was only seen with exposure to 40% (v/v) DMSO, which was more notable at warm temperatures. Post-Thaw removal of DMSO was measured by determining the DMSO concentration of the post-thaw washes using refractometry. Washing AELS 3 times in tapering concentrations of Glucose supplemented DMEM at an AELS:wash ratio of 1:2 was sufficient to reduce DMSO to undetectable levels (<1%). The study demonstrated that the thawing method minimised DMSO toxicity to the BAL biomass, and the post-thaw washing protocol successfully removed all the DMSO present in the cryopreserved BAL. Thereby enabling effective cryopreservation of the BAL for future clinical translation.

How to Provide Breast Milk for the Preterm Infant and Avoid Symptomatic Cytomegalovirus Infection with Possible Long-Term Sequelae.

Cytomegalovirus (CMV) is able to replicate in the breast milk of lactating mothers and thus the offspring might be affected by mild to severe symptoms of postnatal CMV disease in case of prematurity; not in term infants. Sepsis-like syndrome affects only very low birth infants; and few cases have been reported. The neurodevelopmental long-term outcome of those preterm infants revealed possible subtle deficiencies, but no major neurodevelopmental impairment. Neurodevelopmental sequelae are still in discussion and seem somewhat overestimated after careful evaluation of the published evidence. The main focus of postnatal CMV disease lies upon the extremely low birth weight of infants. Elimination of CMV is provided by short-term heating methods like the most widely used Holder pasteurization. Freezing and thawing methods leave a risk for CMV acquisition. The benefits of untreated breast milk have to be considered to outweigh the possible sequelae of postnatal CMV infection in the most vulnerable preterm infants.

Freeze-Thawing Treatment as a Simple Way to Tune the Gel Property and Digestibility of Minced Meat from Red Swamp Crayfish (Procambarus clarkiix).

The effects of freezing methods, including rapid freezing (RF) or slow freezing (SF), combined with thawing methods, e.g., water immersing thawing (WT) or cold thawing (CT), on the meat yield, drip loss, gel properties, and digestive properties of meat detached from red swamp crayfish were investigated. RF greatly reduced the freezing time compared to SF, and the thawing time of frozen crayfish was obviously shortened by WT in comparison to CT. RF and CT improved the meat yield but increased the drip loss, probably as a result of the greater protein denaturation or degradation. A soft and flexible gel was obtained by SF-CT, while a hard one was achieved by RF-WT. An SEM analysis showed that SF resulted in rough and irregular microstructures with larger pore sizes. Freeze-thawing led to an increase in the β-sheet content at the expense of α-helix and variations in the microenvironment of tyrosine and tryptophan residues in protein molecules of the gels, which was more pronounced in the SF-CT group. Moreover, freeze-thawing could cause enhanced protein digestibility but reduce the antioxidant activity of gels. These findings underline the promise of the freezing-thawing treatment in tuning the gel-based meat products of crayfish.

Design and Assessment of an Innovative Thawing Equipment for the Professional Food Service

Abstract The need for faster, safer, and environmentally compatible thawing methods is perceived in different branches of food industry, as, e.g., in the fast-food sector. We investigate a prototype of jet-impingement, batch-process thawing machine, exploiting high-speed impact of heated atmospheric air onto food items. The results of combined experimental and numerical study show the promising potential of this technology beyond the expected increased thawing rate, especially when the proposed two-side arrangement of the jets is adopted. A series of thawing indicators are calculated and used to account for the thawing uniformity and the risk of bacterial proliferation. A computationally efficient two-stage model of the considered thawing process is conceived, which can be used for design and optimization.

Comparative performance analysis of ohmic thawing and conventional thawing of spinach puree

AbstractNowadays dietary habits of consumers have changed to more quick and easier but at the same time healthier meals. Therefore, ready‐to‐eat food production and home processing of such foods are gaining importance. Spinach is one of the main ingredients used in ready‐to‐eat baby foods, and it is produced in chopped, blanched, juice, and powder forms. Commonly ready‐to‐eat meals are sold frozen. Therefore, thawing process becomes an important processing step. Conventional thawing (CT) methods require a long process time and high‐energy consumption. In the present study, frozen blanched spinach puree samples were thawed by ohmic thawing (OT) (10 V/cm, 15 V/cm). The thawing at 4°C (refrigerator thawing) was selected as the control thawing method. The energetic and exergetic evaluations were compared. Accordingly, thawing times were shortened by about 70–80% with OT. The energy loss during the CT method was higher than the OT method. The most energy‐efficient (64.2% ± 4.0%) thawing condition was determined as 15 V/cm OT. In case of exergetic evaluation, the destroyed exergy value of the CT method was highest (19.0 ± 2.0 kJ) while those of thawed with 10 and 15 V/cm were 5.8 ± 0.7 and 3.8 ± 0.4 kJ, respectively. Similar to energetic efficiency, OT with 15 V/cm had the highest exergy efficiency value (40.5% ± 2.6%) while that of the CT method was 11.6% ± 1.0%. The result of the present study indicated that the OT process could be an alternative thawing method to CT to improve energy efficiency.Practical applicationsAs the ready‐to‐eat meal habit is becoming more common, processing of such foods is becoming more important. While preparing food, it is desired that the process to be done quickly, homogeneously, and efficiently. Usage of current food technologies to provide fast and homogeneous heating is becoming widespread. However, examining and evaluating their efficiency also emerges as an important issue. In this study, the use of OT, which is one of the current methods, for the thawing of spinach puree was investigated and the efficiency of OT in different voltage gradients was evaluated. In this way, it is thought that important data about the efficiency of the ohmic heating process, which utilization is increasing day‐by‐day, during the thawing process are obtained. It is thought that these data will contribute to future studies and contribute to the widespread dissemination of OT.

Effect of Different Thawing Methods on the Freshness and Volatile Flavor Compounds of Anchovy (Engraulis encrasicholus)

Effect of Different Thawing Methods on the Freshness and Volatile Flavor Compounds of Anchovy (Engraulis encrasicholus)