Tathagata Acharya

Cryomicroscopic Investigations of Freezing Processes in Cell Suspensions~!2009-12-11~!2010-03-09~!2010-05-11~!

This study evaluates the freezing response of three different cell types, Pacific oyster embryos (~50 � m in di- ameter), Jurkat cells and HeLa cells (~12 to 15 � ms in diameter), using cryomicroscopy. Freezing experiments were per- formed on oyster embryos at cooling rates of either 5 or 10 ˚C/min, while Jurkats were cooled at either 1 or 10 ˚C/min and HeLa cells were cooled at either 1, 15 or 20 ˚C/min, respectively. The experiments with oyster embryos were primarily designed to investigate the phenomena of intracellular ice formation (IIF) while the experiments for Jurkat and HeLa cells were designed to investigate both cellular dehydration and IIF during freezing. IIF was characterized by the abrupt black flashing during the cooling steps while the cellular dehydration experiments were characterized by the volumetric (pro- jected area) shrinkage of the cells during the cooling steps. Mathematical models were fit to the cellular dehydration data to obtain the Jurkat and HeLa cell membrane permeability (Lpg) at the reference temperature (273.15 K), the apparent ac- tivation energy of the cellular dehydration process (ELp) or the temperature dependence of Lpg. The temperature depend- ence of IIF in the oyster embryos, the Jurkat cells and the HeLa cells were also determined.

Measurement of Viscous Drag on a Disc Rotating in a Low Pressure Gas

Viscous drag on a rotating disc under different air-pressures is measured experimentally. Disc spin-down experiments are carried out from atmospheric pressure through a vacuum pressure of 1333 Pa. Deceleration torque was plotted as a function of angular velocity at the various pressures. Torque was nondimensionalized using dynamic viscosity of the air, angular velocity and the characteristic dimension of the disc. The results obtained could approximately be classified into two sets of self-similar curves, the first set between atmospheric pressure and 13332 Pa, and the second set between 13332 Pa and 1333 Pa.

Experimental Characterization of Flow Regimes for a Rotating Disc Over a Range of Gas Pressures

Experimental measurement of viscous drag is done on a rotating disc using a disc spin-down experiment in various air pressures ranging from atmospheric pressure through a vacuum pressure of 0.3 kPa. The torque is non-dimensionalized using the dynamic viscosity of air, angular velocity, and the characteristic dimension of the disc. The results can be classified as two different sets. Between atmospheric pressure and 13.3 kPa, the curves have a vertical downward trend and are connected to each other while at lower pressures the curves appear to be more horizontal. It is shown that the present experimental setup is an improvement over the old setup because the frictional torque associated with the experiment has been reduced by 25 % approximately, and the ambient pressure reached in the chamber is a fourth of the ultimate pressure reached before.Copyright

Numerical Modeling of Damping Characteristics in a Torsional Micro-Resonator

The dynamic behavior of torsional micro-resonators with dimensions, 100 μm × 20 μm, 100 μm × 10 μm, 100 μm × 5 μm, and 100 μm × 2.5 μm are modeled under continuum conditions in ambient air and different liquids and at angular frequencies of 1000 Hz and 5000 Hz respectively. The dynamic response in terms of energy lost per cycle, torsional amplitude and the phase shift are calculated and non-dimensionalized suitably. Finally efforts have been made to establish a relationship between the parameters of interest and the aspect ratio.Copyright

A Study of Intracellular Ice Formation in Jurkat Cells

The objective of this study was to characterize the IIF behavior of Jurkat cells in isotonic conditions in the absence of any cryoprotective agents. The Jurkat cells were collected from culture and then washed and re-suspended in Dulbecco’s Phosphate Buffered Saline (PBS). The freezing experiments were carried out at defined freezing protocols and at various freezing rates of 5, 20, 30 and 50 °C/min. The results suggest there was no substantial evidence of intracellular ice formation at lower cooling rates of 5, 20 and 30° C/min. The first conspicuous indication of intracellular ice formation (IIF) was observed at a freezing rate of 50 °C/min. At this cooling rate, unlike the usual sudden blackening of cells, the cells suddenly grew and exploded suggesting the formation of intracellular ice, which was reminiscent of a prior observed phenomenon for IIF.Copyright

Intracellular Ice Formation in Cell Suspensions Measured Using a Cryomicroscope and a Calorimeter

Injury to biological cells during the freezing-thawing process of a cryopreservation protocol is related to the thermodynamic state of the intracellular water. The two primary biophysical phenomena are water transport and intracellular ice formation (IIF). Unfortunately, there is no technique currently available to measure IIF in the cells of opaque tissue sections. In this proceeding we report the use of a calorimeter to assess IIF in two different cell suspensions, adult stem cells and pacific oyster embryos. The close agreement between the IIF data obtained using the calorimetric data with corresponding data obtained using a well-established cryomicroscopy technique validated the calorimetric method. Since, the calorimetric measurements are independent of shape and size, it is ideally suited to measure IIF in opaque tissue sections; the focus of future studies.Copyright