Temperature effects on enzyme activity of chicken liver esterase used in calorimetric biosensor.

The temperature effects on enzyme activity at the conditions with and without inhibition, operation, and storage were investigated for free esterase and immobilized chicken liver esterase with three methods (adsorption, adsorption-microcapsule, and adsorption-crosslinking) used in a calorimetric biosensor. The results indicated that the temperature had significant effects on the enzyme activity by means of catalytic reaction, irreversible denaturation, mass transfer, and structure deformation of resin. Among all the esterase, the adsorption-crosslinking immobilized esterase had the best performances of enzyme activity retention ratio (EARR), above 96% in operation and 90% in storage on condition of maintaining its response to dichlorvos.

Effects of glycerol pretreatment on recovery and antioxidant enzyme activities of lyophilized red blood cells.

Successful storage of red blood cells (RBCs) by freeze-drying technique has important implications in blood transfusion and clinical medicine. We presented a method of preservation RBCs by pretreating them with glycerol solution and then freeze-drying. The effects of glycerol pretreatment on recovery and antioxidant enzyme activities of lyophilized RBCs were investigated. Rehydration of pretreated in 40 percent glycerol and freeze-dried RBCs resulted in 55.3+/-4.3 percent numerical recovery and 53.5+/-3.9 percent hemoglobin recovery, which were significantly higher than freeze-dried RBCs without glycerol pretreatment (P less than 0.01). Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) activities of 40 percent glycerol pretreated and freeze-dried RBCs almost remained fully active after freeze-drying. After 180 days storage, the activities decreased by 25.6 percent , 21.5 percent and 18.4 percent, respectively, which are significantly less than those of freeze-dried RBCs without glycerol pretreatment (P < 0.1). These data demonstrated that glycerol pretreatment had beneficial effects on the recovery and antioxidant enzyme activities of lyophilized RBCs.

Experimental study and analysis of mechanical properties of frozen rabbit aorta by fracture mechanics approach.

In order to investigate fracture problems associated with cryopreservation of aorta, experiments were carried out to study the effects of temperature, freezing rate and cryoprotective agent on the fracture mechanics properties of frozen rabbit aorta and a test method for crack criterion of frozen rabbit aorta was developed. Results showed that as temperature decreases, the fracture modes of frozen rabbit aorta changed from typical ductile fracture to typical brittle fracture, the crack was more difficult to grow as temperature decreased, and its resist-fracture ability weakened remarkably from -20 to -80 degrees C. However, freezing rates had no effects on the fracture modes when sample was cooled to -50 degrees C, while the resist-fracture ability was stronger when the sample was cooled at a higher freezing rate. Due to the hydration action of dimethyl sulphoxide (DMSO), the rabbit aorta permeated by 10% (v/v) DMSO presented typical ductile fracture when it was cooled to -50 degrees C, so its resist-fracture ability was enhanced obviously. Compared to the axial sample, the circumferential sample's resist-fracture ability is better because of its larger deformation energy available.

Micro-computed tomography observation of sublimation interface and image analysis on sublimation process during freeze-drying.

The freeze-drying process is complicated with complex heat and mass transfer during sublimation. The sublimation interface of freeze-drying has become more attractive and meaningful recently. In this study, apple slices undergoing sublimation were scanned by a Micro-CT scanner. The cross-sectional images were reconstructed with those scanning images respectively. The technique of grey value analysis was used to recognize the procedure. The results showed that, from direct scanning images and 2-D reconstructed images, a 3-D moving mode of sublimation interface which contracted to the centre of the sample could be seen, sublimation process proceeded from edge to center gradually. The grey value of ice crystals was determined to be 154 through gauss calculation. By comparing frozen sample with freeze-dried one, the ice crystals regions in the beginning became the porous regions after drying, grey values increased correspondingly. Samples shrunk slightly after drying for 3 to 7 hours, which could be distinguished by the change in grey values.

Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability.

Cryopreservation of tissue-engineered human dermal replacement plays an important role in skin tissue engineering and skin banking. With the inspection of electronic scanning microscope and viability evaluation by Trypan Blue staining assay and the tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, this study investigated the toxicity of Me(2)SO to dermal fibroblasts and effects of cryoprotectant concentration and cooling rate on the viability of dermal replacement. The results demonstrated that the Me(2)SO toxicity to fibroblasts was affected by the exposure time, temperature, and concentration. Furthermore adding cryoprotectant solution at low temperature of 4 degrees C significantly reduced the toxic effect on the tissue-engineered dermal equivalent. An optimal cryopreservation protocol consisting of cooling rate at 1 degrees Cmin(-1) in 10% (V/V) Me(2)SO was derived, with the viability of studied dermal equivalent treated by this protocol being 75% of that of fresh control. The micrograph obtained by electronic scanning microscope also confirmed this result.

Effects of freezing rates and dimethyl sulfoxide concentrations on thermal expansion of rabbit aorta during freezing phase change as measured by thermo mechanical analysis.

Thermal expansion data are essential for thermal stress analysis in order to predict the likelihood of fracture formation in tissues during cryopreservation as well as cryosurgery. The current study focuses on examining the thermal expansion behavior of rabbit aorta during freezing, especially phase change processes. Thermo mechanical analysis (TMA) was used to investigate the effects of different concentrations of dimethyl sulfoxide (DMSO) cryoprotective agent (CPA) (0%, 5%, 10%, and 15% (v/v) DMSO) and different freezing rates (3, 5 and 10 degrees C/min). The results showed that (1) the maximum of thermal strain for 10 degrees C/min was approximately four times greater than that for 3 degrees C/min, and 1.4 times greater than that for 5 degrees C/min, and the higher the freezing rate, the larger the corresponding thermal expansion coefficient; (2) the maximum thermal strain of sample permeated by 5% DMSO approached that of 0% DMSO (i.e., no DMSO was added); however, it showed very significant difference from that of 15% DMSO (only half of that with 5% DMSO), and the thermal expansion coefficient decreased when the concentration of DMSO solution increased; (3) comparison with data available from the literature and with theoretically calculated values illustrated that the thermal expansion change was not equal to the volume change from free water to ice during freezing, but was related to the freezing rate of samples and the DMSO concentration.

Cryopreservation of human embryonic stem cells: a protocol by programmed cooling.

Human embryonic stem (ES) cells have far-reaching applications in the areas of tissue engineering, regenerative medicine, pharmacology and basic scientific research. Although the culture conditions can maintain the human ES cells in an undifferentiated state for a transient period, spontaneous differentiation has also been observed during the routine culturing of ES cells. However, the maintenance of ES cells in the undifferentiated, pluripotent state for extended periods of time will be required in many areas of scientific research. Cryopreservation is a technology with potentially far reaching implication for the development and widespread use of such cell lines. This study was undertaken to develop and optimize a protocol for cryopreservation of human ES cells through programmed cooling. The effects of the seeding temperature, the cooling rate and the sub-zero temperature to which the samples were cooled before plunging into liquid nitrogen(the terminal temperature), all significantly affected the recovery of cryopreserved ES cells. After studying these factors, an improved protocol was obtained: the sample was cooled from 0 degree C to -35 degree C at a cooling rate of 0.5 degree per min, with seeding was set at -10 degree C, before being plunged immediately into the liquid nitrogen. Using this protocol, 9 of 11 colony fragments survived freezing and thawing and could be cultured for prolonged periods. They retained the properties of pluripotent cells, had a normal karyotype and showed histochemical staining for alkaline phosphatase.

A novel thermal biosensor based on enzyme reaction for pesticides measurement.

A novel thermal biosensor based on enzyme reaction for pesticides detection has been developed. This biosensor is a flow injection analysis system and consists of two channels with enzyme reaction column and identical reference column, which is set for eliminating the unspecific heat. The enzyme reaction takes place in the enzyme reaction column at a constant temperature (40 degrees C) realized by a thermoelectric thermostat. Thermosensor based on the thermoelectric module containing 127 serial BiTe-thermocouples is used to monitor the temperature difference between two effluents from enzyme reaction column and reference column. The ability of this biosensor to detect pesticides is demonstrated by the decreased degree of the hydrolytic heat in two types of thermosensor mode. The hydrolytic reaction is inhibited by 36% at 1 mg/L DDVP and 50% at 10 mg/L DDVP when cell-typed thermosensor is used. The percent inhibition is 30% at 1 mg/L DDVP and 42% at 10 mg/L DDVP in tube-typed thermosensor mode. The detection for real sample shows that this biosensor can be used for detection of organophosphate pesticides residue.

Morphology study of freeze-drying mononuclear cells of human cord blood.

The research on haematopoietic stem cells of human cord blood has become more important recently. At present, cord blood is mainly preserved at ultra-low temperatures. In the former study, we compared the effects of preserving mononuclear cells (MNC) and whole human cord blood by freeze-drying. This time, a further study was conducted on freeze-drying mononuclear cells. Samples in the presence of PVP, sucrose, mannitol and FBS were firstly frozen to -38 degrees C. Afterwards, they were vacuum-dried at a selected shelf temperature of -30 degrees C for the main drying stage, and then vacuum-dried at 15 degrees C for the second drying stage. The entire time of freeze-drying process was 41 hours. Samples were stored at room temperature for 7 days prior to evaluation. Subsequently, the dried samples were resuspended in an isotonic phosphate-buffered saline solution. The residual moisture content was 6.5 +/- 0.87%. The recovery of the cells was tested by a haemacytometer, and the numerical cell count recovery of rehydrated MNC increased by 8%. Morphology of the fresh and rehydrated MNC was analyzed respectively using standard light microscopy, scanning electron microscope and transmission electron microscope. The results showed that karyons changed and cytoplasm decreased after rehydration, but it is still unknown that whether these changes will influence the proliferative ability of the stem cells.

[Effective cryopreservation of human embryonic stem cells by programmed freezing].

Cryopreservation of human embryonic stem cells is an important and unsolved problem. A computer-controlled programmable cooler was used in the preservation of ES cells. Several effects have been experimentally studied, which include the cooling rates, the temperature of seeding, the temperatures before the samples being plunged into liquid nitrogen, and the cryoprotective agents. It was found that the favorable constitution of cryoprotective agents was Me2SO+ FBS+DMEM(1:3:6, v/v/v) with cooling protocol of -0.5 degrees C/min from 0 degrees C to -35 degrees C (seeding at -10 degrees C), and being plunged into the liquid nitrogen immediately. The high survival rate (81.8%) was obtained.

Experimental study on fracture mechanics properties of frozen rabbit aorta.

To more scientifically discuss fracture problems associated with cryopreservation of aorta, the effects of temperature, cooling rate and cryo-protective agent on the fracture mechanics properties of frozen rabbit aorta have been investigated with Dynamical Mechanics Analyser (DMA), and the test method for crack criterion of frozen rabbit aorta was also explored. The results show that: As temperature decreasing, the fracture modes of frozen rabbit aorta are from typical ductile fracture to typical brittle fracture, and its resist-fracture ability weakens remarkably from -20 centigrade to -80 centigrade. The cooling rates have no effects on the fracture modes when cooled to -50 centigrade, but the resist-fracture ability of frozen rabbit aorta will be stronger when the sample treated by a higher cooling rate. Due to the hydration action of dimethyl sulphoxide (DMSO), the rabbit aorta permeated by 10% (V/V) DMSO presents typical ductile fracture when it was cooled to -50 centigrade, so its resist-fracture ability is enhanced obviously. Compared to the axial sample, the peripheral sample's resist-fracture ability is larger than that of the former.

Effective cryopreservation of human embryonic stem cells by programmed freezing.

Cryopreservation of human embryonic stem cells is an important and unsolved problem. A computer-controlled programmable cooler is used in the preservation of ES cells. Several effects have been experimentally studied, which include the cooling rate, the seeding temperature, the terminative temperature before the sample is plunged into liquid nitrogen. It is found that the constitution of cryoprotective agents is Me2SO+FBS+DMEM(1:3:6,v/v/v), and the sample is cooled from 0 °C to -35 °C with a cooling rate of 0.5 °C/min (seeding at -10 °C), and then being plunged into the liquid nitrogen immediately. The high survival rate (81.8%) is obtained. The cryopreserved human ES cells have been cultivated for prolonged periods and retained the properties of pluripotent cells, they have a normal karyotype and show the histochemical staining for alkaline phosphatase.

A thermal biosensor based on enzyme reaction.

Application of the thermal biosensor as analytical tool is promising due to advantages as universal, simplicity and quick response. A novel thermal biosensor based on enzyme reaction has been developed. This biosensor is a flow injection analysis system and consists of two channels with enzyme reaction column and reference column. The reference column, which is set for eliminating the unspecific heat, is inactived on special enzyme reaction of the ingredient to be detected. The special enzyme reaction takes places in the enzyme reaction column at a constant temperature realizing by a thermoelectric thermostat. Thermal sensor based on the thermoelectric module containing 127 serial BiTe-thermocouples is used to monitor the temperature difference between two streams from the enzyme reaction column and the reference column. The analytical example for dichlorvos shows that this biosensor can be used as analytical tool in medicine and biology.

Thermal analysis of tertiary butyl alcohol/sucrose/water ternary system.

The purpose of this work is to investigate the freezing properties of tertiary butyl alcohol (TBA)/sucrose/water ternary system. Differential scanning calorimetry (DSC) is employed to determine the glass transition temperature of the maximally freeze-concentrated solution Tg' and the crystallization (or devitrification) temperature Tr. DSC measurements show that the presence of sucrose hinders the crystallization of TBA during cooling. The residual TBA in the glassy state will cause a decrease in Tg' and will crystallize during heating. An increase in the cooling rate causes a decrease in Tg'. For 10% TBA/10% sucrose/water ternary system, the critical heating rate is approximately 250 degrees C/min. Annealing treatment at temperatures below Tg' causes the crystallization of TBA, which indicates that TBA molecules still have appreciable mobility even at temperatures below Tg'. When the ratio of TBA to sucrose is less than 0.2, TBA cannot crystallize during cooling.

Freeze-drying of mononuclear cells and whole blood of human cord blood.

The research on haematopoietic stem cells of human cord blood has become more important recently. People have concentrated on the preservation of cord blood stem cells. At present, cord blood can be preserved at ultra-low temperatures. In this study, we try to preserve cord blood and its constituents by freeze-drying. The experiments on both the mononuclear cell content and the whole blood of human cord blood were carried out respectively. The samples were frozen firstly by different cooling protocols in the presence of PVP, sucrose, and mannitol. Afterwards, they were vacuum-dried at a selected shelf temperature of -30 degree C for the main drying stage, and then vacuum-dried at 15 degree C for the second drying stage. The entire time of the freeze drying was 52 hours. Samples were stored at room temperature for 2 days prior to evaluation. Subsequently, the dried samples were suspended in an isotonic phosphate-buffered saline solution. The recovery of the cells were tested by a haemacytometer, and the highest cell numerical count recovery of MNC was 75.0 percent (SD = 4.1 percent) (P = 0.01), obtained in the protocol of 40 percent PVP + 20 percent sucrose + 10 percent Mannitol. The viability of the nucleated cells measured by PI staining and the ratio of the number of CD34+ to the number of lymphocytes (by the FITC anti-human CD34+ conjugated antibody method) were measured using a flow cytometer (FCM). The protocol of 40 percent PVP + 20 percent sucrose + 10 percent fetal bovine serum had the highest viability of 98.6 percent (SD = 0.7 percent) (P = 0.01). The highest ratio of CD34+ to lymphocytes was 1.2%, and the highest recovery of CD34+ was 68.4 percent (SD = 39.5 percent) (P = 0.05). Comparing the results of the lyophilized MNC subfraction with that of the whole blood, the lyophilization of the isolated MNC was more successful than that of whole blood.

Microscopic study of crystal growth in cryopreservation agent solutions and water.

Ice formation inside or outside cells during cryopreservation is evidently the main factor of cryoinjury to cells. In the study described here a high voltage DC electric field and a cryomicroscopic stage were used to test DMSO and NaCl solutions under electric field strengths ranging from 83 kV/m to 320 kV/m. Dendritic ice crystals became asymmetric when the electric field was activated. This change in the ice crystal shape was more pronounced in the ionic NaCl solution. In addition, ice growth of distilled water without an electric field was tested under different cooling rates.

Cryopreservation and xenotransplantation studies of microencapsulated rat pancreatic islets.

Islets of Langerhans were isolated from the Sprague Dawley rat pancreas digested by injected collagenase, and purified by Ficoll density gradient centrifugation. In order to make smaller and more uniform microencapsulated islets, we designed a special high-voltage electrostatic microcapsule generator. The effects of operational parameters of the generator on the size and the uniformity of microcapsules were analyzed, such as the voltage, the plunger speed of suspension delivery to the needle tip, the distance between needle tip and solution surface. The optimal parameter combinations for making microcapsules are: 5 kV of voltage, 50 mm/h of the plunger speed, and 20 mm distance. The high-voltage electric system can produce uniform microcapsules with diameters ranging from 0.3 to approximately 0.5 mm, which are smaller and more uniform than those produced by air-jet system. A comparison of the cryopreservation effects between microencapsulated islets and unencapsulated islets showed that the microcapsules can protect the fragile islets from freezing damage, and increase the retrieval rate from 68.5% to 92.6%. Xenotransplantation of the cryopreserved rat islets resulted in the normalization of the metabolic blood glucose of the diabetic mice for 90 days, whereas the unencapsulated islets were easily fragmented and lost during the freezing process. They only reversed hyperglycemia for less than 3-5 days.