Tse-Chao Hua

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.

The Cryopreservation of a Tissue Engineered Dermal Replacement by Programmed Freezing

Long-term storage of engineered bio-artificial tissues is required to ensure the off-the-shelf availability to clinicians due to their long production cycle. Cryopreservation is likely the choice for long-term preservation. This study investigated the effects of dimethyl sulfoxide (DMSO) concentrations, cooling rates, cryoprotectant medium treatment methods and seeding on the cell viability of a tissue-engineered dermal substitute. The dermal fibroblast was cultured on a polyglycolic acid (PGA) scaffolding at 37degree C and a 5% CO2 atmosphere for 14 days, and dermal slices (10times3times1.5 mm) were used in the experiment. Two groups were defined - fresh control and experimental group. The freezing was carried out in a computer-controlled programmable freezer at 0.5 degree C/min, 1degree C /min and 2degree C /min in 1.4M, 2.1M and 2.8M DMSO from 4degree C to -60degree C and then plunged into the liquid nitrogen tank immediately. After 24 h of cryopreservation, the samples were removed from liquid nitrogen and placed at room temperature in air for 0.5-1 min, prior to submergence in a water bath at 37degree C . Viability of fresh control and cryopreserved dermal slices were determined with our modification of the MTT (3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The effects of two cryoprotectant medium treatment methods and seeding (at -6degree C ) on cell viability were also assessed. A cryopreservation protocol of 1degree C /min in 1.4M DMSO from 4degree C to -60degree C , and seeding at -7degree C proved optimal for this tissue-engineered dermal replacement, provided the dermal slice was transferred into the cryopreservation bag containing 1mL 1.4M DMSO cryoprotectant medium, which was kept at 4degree C , seal the bag and hold for 15 min at 4degree C prior to freezing. The viability of the dermal replacement treated by this protocol was maintained 80% of the fresh control viability. And the micrograph confirmed that after 48 h incubation following thawing, the dermal fibroblast can regain its natural shape and functions. No fracture was observed

Metabolic Activity and Functional Evaluation of Cryopreserved Dermal Equivalent

It is crucial for the cryopreserved dermal equivalent to recover both metabolic activity and protein expression as well as its intact structure. In this study, dermal fibroblasts were cultured on a three-dimensional scaffold to form a dermal equivalent, and the effects of culture time and cryopreservation protocols on the metabolic activity, structural and functional properties of the equivalent were evaluated. Two groups were defined, a fresh control group and an experimental group. Viabilities of the fresh control and the cryopreserved dermal slices were determined using the modified MTT assay. Histology staining and scanning electron microscopy of the samples were performed. The cooling was carried out in a computer-controlled programmable cooler. For the dermal equivalent cultured for 14 days, a cryopreservation protocol of 1°/min from 4° to -60°C (seeding at -7°C), then plunged into the liquid nitrogen Dewar immediately proved optimal, provided the dermal slice was transferred into the cryopreservati...

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 is 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 samples resist-fracture ability is larger than that of the former

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 inactivated 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