Search

Although various liver chips have been developed using emerging organ-on-a-chip techniques, it remains an enormous challenge to replicate the liver lobules with self-assembled perfusable hepatic sinusoid networks. Herein we develop a lifelike bionic liver lobule chip (LLC), on which the perfusable hepatic sinusoid networks are achieved using a microflow-guided angiogenesis methodology; additionally, during and after self-assembly, oxygen concentration is regulated to mimic physiologically dissolved levels supplied by actual hepatic arterioles and venules. This liver lobule design thereby produces more bionic liver microstructures, higher metabolic abilities, and longer lasting hepatocyte function than other liver-on-a-chip techniques that are able to deliver. We found that the flow through the unique micropillar design in the cell coculture zone guides the radiating assembly of the hepatic sinusoid, the oxygen concentration affects the morphology of the sinusoid by proliferation, and the oxygen gradient plays a key role in prolonging hepatocyte function. The expected breadth of applications our LLC is suited to is demonstrated by means of preliminarily testing chronic and acute hepatotoxicity of drugs and replicating growth of tumors in situ. This work provides new insights into designing more extensive bionic vascularized liver chips, while achieving longer lasting ex-vivo hepatocyte function.

Tracing magnetically labeled cells with magnetic resonance imaging (MRI) is an emerging and promising approach to uncover in vivo behaviors of cells in cell therapy. Today, existing methods for the magnetic labeling of cells are cumbersome and time-consuming, which has greatly limited the progress of such studies on cell therapy. Thus, in this study, using the flow cytometric loading technology, we develop a sonoporation-based microfluidic chip (i.e., a microfluidic chip integrated with ultrasound; MCU), to achieve the safe, instant, convenient, and continuous magnetic labeling of cells. For the MCU we designed, a suitable group of operating conditions for safely and efficiently loading superparamagnetic iron oxide (SPIO) nanoparticles into DC2.4 cells was identified experimentally. Under the identified operating conditions, the DC2.4 cells could be labeled in approximately 2 min with high viability (94%) and a high labeling quantity of SPIO nanoparticles (19 pg of iron per cell). In addition, the proliferative functions of the cells were also well maintained after labeling. Furthermore, the in vivo imaging ability of the DC2.4 cells labeled using the MCU was verified by injecting the labeled cells into the leg muscle of the C57BL/6 mice. The results show that the excellent imaging outcome can be continuously achieved for 7 days at a density of 106 cells/mL. This work can provide insight for the design of magnetic cell labeling devices and promote the MRI-based study of cell therapies.

In this work, we present a new version of the dilution-filtration system for rapidly deglycerolizing a large volume of cryopreserved blood. In our earlier system, one of the major problems was the damage induced to the red blood cells (RBCs) due to high osmolality change at the dilution point. Therefore, we devised a new system to solve this problem. First, we theoretically simulated the osmolality variation in the new system and the variation of the maximum and minimum volumes of the RBCs at the dilution point to examine the effects of operating parameters/conditions. Next, we experimentally validated the effects of these operating parameters by deglycerolizing porcine blood. The results show that when the initial NaCl concentration in the hypertonic solution is 18%, the volume of the hypertonic solution is 200 mL, and the flow rate of the filtrate is 50 mL/min, the system can effectively remove glycerin from 200 mL of porcine blood in 30 min, with ∼87% RBC survival rate and ∼73% RBC recovery rate. Our results indicated that in the new system the concentration and the volume of the hypertonic solution used to dilute the blood are the important parameters that need to be adjusted to reduce osmotic damage to the RBCs. In addition, a fast filtrate flow rate is highly recommended. This work can significantly contribute to the development of a more efficient and effective system for deglycerolizing large volumes of cryopreserved blood in clinic.

Artemether is one of the most effective drugs for the treatment of chloroquine-resistant and Plasmodium falciparum strains of malaria. However, its therapeutic potency is hindered by its poor bioavailability. To overcome this limitation, we have encapsulated artemether in poly(lactic-co-glycolic) acid (PLGA) core-shell microparticles (MPs) using the coaxial electrospray method. With optimized process parameters including liquid flow rates and applied electric voltages, experiments are systematically carried out to generate a stable cone-jet mode to produce artemether-loaded PLGA-MPs with an average size of 2 μm, an encapsulation efficiency of 78 ± 5.6%, and a loading efficiency of 11.7%. The in vitro release study demonstrates the sustained release of artemether from the core-shell structure in comparison with that of plain artemether and that of MPs produced by single-axial electrospray without any relevant cytotoxicity. The in vivo studies are performed to evaluate the pharmacokinetic characteristics of the artemether-loaded PLGA-MPs. Our study implies that artemether can be effectively encapsulated in a protective shell of PLGA for controlled release kinetics and enhanced oral bioavailability.

It was recently reported that nanoparticles could significantly modulate the thermal properties of solutions at subzero temperatures, and as a result, nanoparticles have been widely used in both cryopreservation and cryosurgery. In cryopreservation, the water permeability coefficient of cell membrane is an essential parameter for quantitative investigation of cell dehydration and intracellular ice formation. However, few studies were focused on the effects of nanoparticles on the permeability properties of cell membrane. In order to optimize the processes of cryopreservation with nanoparticles, we measured the permeability properties of Sf21 cells in the presence of iron oxide nanoparticles in this study. The responses of Sf21 cells with iron oxide nanoparticles were obtained by the microperfusion system at -2, 5, 15 and 25 °C, respectively. The osmotically inactive cell volume (Vb), the cell membrane hydraulic conductivity (Lp) and it's activation energy (ELp), and the reference value of Lp at the reference temperature (Lpg) with 0.02%, 0.1% and 0.5% (w/w) iron oxide nanoparticles were determined by 2-parameter (2-p) model at -2, 5, 15 and 25 °C. We analyzed the effects of iron oxide nanoparticles on the permeability properties of the Sf21 cells. The results indicated that iron oxide nanoparticles have a significant influence on membrane permeability properties (Lpg and ELp) of Sf21 cells. The introduction of iron oxide nanoparticles tends to increase the values of Vb and Lpg, while decrease the value of ELp. These findings may provide a new route to optimize the biomaterial cryopreservation.

In this paper, a multistep dilution-filtration microdevice (MDFD) is developed for unloading cryoprotectants from cryoprotectant-loaded cells. The MDFD contained a diluent producing region, a dilution-filtration execution region, and a filtrate collection region. It was made of two patterned PMMA stamps with four pieces of sandwiched PVDF membranes. Firstly, the performances of the mixers that were used in the diluent producing region and the dilution-filtration execution region were assessed using fluorescence experiments. Then, the effect of the MDFD structure on the loss of cells was investigated by applying the MDFD to unload glycerin from glycerin-loaded porcine red blood cells. Finally, the effects of the cell density, glycerin concentration, and membrane pore size on the clearance efficiency of glycerin (C G ), the survival rate of cells (S C ) and the recovery rate of cells (R C ) have been studied. Under the designed conditions, C G achieved ~80% and S C reached ~90%. However, R C was only ~40%, mainly resulting from the cells detained on the membrane surface and squeezed through the membrane pores into the filtrate. Increasing the membrane pore size caused high C G and S C , but low R C . For a low glycerin concentration, C G , S C , and R C were all high. For a high cell density, C G was high, but both S C and R C were low. This work is of significance to develop a microfluidic chip for unloading cryoprotectants from a small amount of cryopreserved cell samples.

Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders.

Obtaining accurate thermal properties of biomaterials plays an important role in the field of cryobiology. Currently, thermal needle, which is constructed by enclosing a manually winded thin metal wire with an insulation coating in a metallic sheath, is the only available device that is capable of measuring thermal conductivity of biomaterials. Major drawbacks, such as macroscale sensor size, lack of versatile format to accommodate samples with various shapes and sizes, neglected effects of heat transfer inside the probe and thermal contact resistance between the sensing element and the probe body, difficult to mass produce, poor data repeatability and reliability and labor-intense sensor calibration, have significantly reduced their potential to be an essential measurement tool to provide key thermal property information of biological specimens. In this study, we describe the development of an approach to measure thermal conductivity of liquids and soft bio-tissues using a proof-of-concept MEMS based thermal probe. By employing a microfabricated closely-packed gold wire to function as the heater and the thermistor, the presented thermal sensor can be used to measure thermal conductivities of fluids and natural soft biomaterials (particularly, the sensor may be directly inserted into soft tissues in living animal/plant bodies or into tissues isolated from the animal/plant bodies), where other more standard approaches cannot be used. Thermal standard materials have been used to calibrate two randomly selected thermal probes at room temperature. Variation between the obtained system calibration constants is less than 10%. By incorporating the previously obtained system calibration constant, three randomly selected thermal probes have been successfully utilized to measure the thermal conductivities of various solutions and tissue samples under different temperatures. Overall, the measurements are in agreement with the recommended values (percentage error less than 5%). The microfabricated thermal conductivity sensor offers superior characteristics compared to those traditional macroscopic thermal sensors, such as, (a) reduced thermal mass and thermal resistivity, (b) improved thermal contact between sensor and sample, (c) easy to manufacture with mass production capability, (d) flexibility to reconfigure sensor geometries for measuring samples with various sizes and shapes, and (e) reduced calibration workload for all sensors microfabricated from the same batch. The MEMS based thermal conductivity sensor is a promising approach to overcome the inherent limitations of existing macroscopic devices and capable of delivering accurate thermal conductivity measurement of biomaterials with various shapes and sizes.

Scientific studies on cryopreservation of adipose tissues have seldom been performed. The purpose of our present study is conducted both in vitro and in vivo to develop a novel cryopreservation method that can be used successfully for long-term preservation of human adipose tissues for possible future clinical application. In this study, samples of adipose aspirates were obtained from 36 adult white female patients after liposuction and collected from the middle layer after centrifugation. In the in vitro study, suitable cryoprotectant agents (CPAs) and their concentrations and possible combinations were selected from our preliminary experiment. A combination of dimethyl sulfoxide (Me(2)SO) and trehalose as CPA with the optimal concentration (0.5M Me(2)SO and 0.2M trehalose) was chosen and then used throughout the study. In addition, maximal recovery of adipose tissues was achieved after cryopreservation using slow cooling without seeding (1-2 degrees C/min to -30 degrees C, followed by plunging to -196 degrees C for storage) and fast warming (in 40 degrees C water bath, averaging 35 degrees C/min). Fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were evaluated by integrated adipocyte counts and histology. In the in vivo study, fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were injected into a nude mouse. The retained adipose aspirates (fat grafts) were harvested in each animal at 4 months and their weight, volume, and histology was assessed. In the in vitro study, significantly higher integrated viable adipocyte count (2.06+/-0.54 x 10(6)mL(-1) vs. 1.07+/-0.41 x 10(6)mL(-1), p

An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me 2 SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume ( V b ) and the permeability coefficient of water ( L p ) for RBL cells are determined to be 41% [ n = 18, correlation coefficient ( r 2 ) of 0.903] of original/isotonic volume, and 0.32 ± 0.05 μm/min/atm ( n = 8, r 2 > 0.963), respectively, for room temperature (22 °C). From experiment (2), the permeability coefficient of water ( L p ) and of Me 2 SO ( P s ) for RBL cells are 0.38 ± 0.09 μm/min/atm and (0.49 ± 0.13) × 10 −3 cm/min ( n = 5, r 2 > 0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell osmotic response and determine cell membrane transport properties; and (4) reduce labor requirements for its disposability and ensure low manufacturing costs.

Despite decades of research and clinical studies of islet transplantations, finding simple yet reliable islet quality assays that correlate accurately with in vivo potency is still a major challenge, especially for real-time and single-islet-based quality assessment. Herein, proof-of-concept studies of a cryopreserved microcapsule-based quality control assays are presented for single islets. Individual rat pancreatic islets and fluorescent oxygen-sensitive dye (FOSD) are encapsulated in alginate hydrogel microcapsules via a microfluidic device. To test the susceptibility of the microcapsules and the FOSD to cryopreservation, the islet microcapsules containing FOSD are cryopreserved and the islet functionalities (adenosine triphosphate, static insulin release measurement, and oxygen consumption rate) are assessed after freezing and thawing steps. The cryopreserved islet capsules with FOSD remain functional after encapsulation and freezing/thawing procedures, validating a simple yet reliable individual-islet-based quality control method for the entire islet processing procedure prior to transplantation. This work also demonstrates that the functionality of cryopreserved islets can be improved by introducing trehalose into the routinely used cryoprotectant dimethyl sulfoxide. The functionalized alginate hydrogel microcapsules with embedded FOSD and optimized cryopreservation protocol presented in this work serve as a versatile islet quality assay and offer tremendous promise for tackling existing challenges in islet transplantation procedures.

An improved thermal-needle approach for accurate and fast measurement of thermal conductivity of aqueous and soft biomaterials was developed using microfabricated thermal conductivity sensors. This microscopic measuring device was comprehensively characterized at temperatures from 0 °C to 40 °C. Despite the previous belief, system calibration constant was observed to be highly temperature-dependent. Dynamic thermal conductivity response during cooling (40 °C to –40 °C) was observed using the miniaturized single tip sensor for various concentrations of CPAs, i.e., glycerol, ethylene glycol and dimethyl sulfoxide. Chicken breast, chicken skin, porcine limb and bovine liver were assayed to investigate the effect of anatomical heterogeneity on thermal conductivity using the arrayed multi-tip sensor at 20 °C. Experimental results revealed distinctive differences in localized thermal conductivity, which suggests the use of approximated or constant property values is expected to bring about results with largely inflated uncertainties when investigating bio-heat transfer mechanisms and/or performing sophisticated thermal modeling with complex biological tissues. Overall, the presented micro thermal sensor with automated data analysis algorithm is a promising approach for direct thermal conductivity measurement of aqueous solutions and soft biomaterials and is of great value to cryopreservation of tissues, hyperthermia or cryogenic and other thermal-based clinical diagnostics and treatments.

During the removal of cryoprotectants from cryopreserved-thawed blood with the dialysis-based or dilution-filtration method, due to the change in the extracellular osmolality, erythrocytes usually undergo repeated swelling and shrinkage. However, the erythrocyte fatigue damage induced by this repeated volume change has not yet been studied. In this work, by successively loading hypotonic and hypertonic solutions, we mimicked the repeated swelling and shrinkage of pig erythrocytes and then examined the effect of the number of cycle loops on the steady-state volume and the mortality of the pig erythrocytes. The results suggest that because of cell leakage in the swelling process, the steady-state volume of the pig erythrocytes after one cycle is smaller than the volume before the cycle, even though the cell performs a self-protective regulatory procedure. If the number of cycle loops is increased, the repeated swelling and shrinkage will cause a continuous decrease in the steady-state volume, and the ability of the pig erythrocytes to resist osmotic damage will decrease; as a result, the mortality of the pig erythrocytes increases as the number of cycle loops increases. The viability of the cells is also affected by the hypotonic and isotonic processing times: a short processing time may contribute to a decrease in the mortality of the pig erythrocytes. This work is of significance to optimizing the process of removing cryoprotectants.

This paper outlines a new method for measuring multilayer tissue conductivity and structure by using divided electrodes, in which current electrodes are divided into several parts. Our purpose is to estimate the multilayer tissue structure and the conductivity distribution in a cross section of the local tissue by using bioresistance data measured noninvasively. The effect of the new method is assessed by computer simulations using a typical two-dimensional (2-D) model. In this paper, the conductivity distribution in the model is analyzed based on a finite difference method (FDM) and a steepest descent method (SDM). Simulation results show that the conductivity values of skin, fat, and muscle layers can be estimated with an error of less than 0.1%. When random noise at various levels is added to the measured resistance values, estimates of the conductivity values for skin, fat, and muscle layers are still reasonably precise: their root mean square errors are about 1.06%, 1.39%, and 1.61% for 10% noise. In a 2-D model, increasing the number of divided electrodes permits simultaneous estimates of tissue structure and conductivity distribution. Optimal configuration for divided electrodes is examined in terms of dividing pattern.

The hydrogel poly(2-methacryloyloxyethylphosphorylcholine-co-butyl methacrylate), or poly(MPC-co-BMA), was used as a coating for polyurethane- and poly(vinyl chloride)-based membranes to develop ion-selective electrodes (ISEs) with enhanced blood compatibility. Adverse interactions of poly(MPC-co-BMA) with blood were diminished due to the phosphorylcholine functionalities of the hydrogel, which mimic the phospholipid polar groups present on the surface of many cell membranes. As demonstrated by immunostaining, hydrogel-coated PVC membranes soaked in platelet-rich plasma showed less adhesion and activation of platelets than uncoated PVC membranes, indicating an improvement in biocompatibility owing to the hydrogel. Furthermore, little differences in the potentiometric response characteristics, e.g., slope, detection limit, and selectivity, of ISEs employing uncoated and coated membranes were observed.

Adipose-derived stem cells (ADSCs), which are an accessible source of adult stem cells with capacities for self-renewal and differentiation into various cell types, have a promising potential in tissue engineering and regenerative medicine strategies. To meet the clinical demand for ADSCs, cryopreservation has been applied for long-term ADSC preservation. To optimize the addition, removal, freezing, and thawing of cryoprotective agents (CPAs) applied to ADSCs, we measured the transport properties of porcine ADSCs (pADSCs). The cell responses of pADSCs to hypertonic phosphate-buffered saline and common CPAs, dimethyl sulfoxide, ethylene glycol, and glycerol were measured by a microperfusion system at temperatures of 28, 18, 8, and -2°C. We determined the osmotically inactive cell volume (Vb), hydraulic conductivity (Lp), and CPA permeability (Ps) at various temperatures in a two-parameter model. Then, we quantitatively analyzed the effect of temperature on the transport properties of the pADSC membrane. Biophysical parameters were used to optimize CPA addition, removal, and freezing processes to minimize excessive shrinkage of pADSCs during cryopreservation. The biophysical properties of pADSCs have a great potential for effective optimization of cryopreservation procedures.

The present study describes the H(2)O-NaCl-ethylene glycol ternary system by using a differential scanning calorimeter to measure melting points (T(m)) of four different ratios (R) of ethylene glycol to NaCl and then devising equations to fit the experimental measurements. Ultimately an equation is derived which characterizes the liquidus surface above the eutectic for any R value in the system. This study focuses on ethylene glycol in part because of recent evidence indicating it may be less toxic to pancreatic islets than Me(2)SO, which is currently used routinely for islet cryopreservation. The resulting physical data and previously determined information regarding the osmotic characteristics of canine pancreatic islets are combined in a mathematical model to describe the volumetric response to equilibrium-rate freezing in varying initial concentrations of ethylene glycol.

A novel approach is introduced here to selectively lyse exocrine cells in an islet preparation by hypoosmotic treatment. Time to hypotonic cell lysis required for the islet cells was much longer than that for the exocrine cells, which permits a possibility of selectively killing the exocrine cells by hypotonic treatment. The first set of experiments was designed to select an appropriate osmolality for the hypotonic treatment. Kinetic changes in cell volume in response to extracellular anisosmolalities (30 to 90 mOsm/kg) were recorded using an electronic particle counter. The results indicated that, when exposed to a 30 mOsm/kg solution, islet cells swelled slowly to reach volumetric equilibrium in approximately 3 min. There was no significant hypotonic cell lysis observed even at the end of 4 min (n = 4). In contrast, pancreatic exocrine cells, when exposed to the same solution, expanded rapidly to the lytic volume and burst within 30 s. Significant exocrine cell lysis was invariably achieved within 30 s when cells were exposed to the osmolalities below 60 mOsm/kg. For osmolalities between 70 to 80 mOsm/kg, exocrine cell lysis was highly variable. When cells were exposed to 80 to 90 mOsm/kg, no significant cell lysis was observed. Thus, an osmolality of 50 mOsm/kg is recommended for hypotonic treatment, as it maximizes the lysis of exocrine cells without unnecessarily stressing (osmotically) the islet cells. The second set of experiments (time-course experiments, 20 to 120 s) was designed to determine the length of exposure time for which the exocrine cells were irreversibly damaged but the islet cells had only swollen to such a degree that cell function is restored upon returning to an isotonic condition. Viability of the hypotonic treated cells was evaluated at two different levels: membrane integrity, measured by combined fluorescent dye staining with propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), and mitochondrial function, measured by colorimetric MTT assay. The results showed that hypotonic treatment in a 50 mOsm/kg solution for 30 s resulted in over 85% loss of the membrane integrity for the exocrine cells. About 90% of these membrane lysed cells lost mitochondrial function (n = 3). By contrast, under the same treatment, less than 15% of the islet cells lost membrane integrity and mitochondrial function (n = 3). In conclusion, hypotonic treatment with a 50 mOsm/kg solution for 20 to 30 s at room temperature is sufficient to lyse the majority of the contaminating exocrine cells in an islet cell preparation, while maintaining function in the islet cells.

Coupled with the rapid development of clinical pancreatic islet transplantation, there is an increasing requirement for cryopreservation of viable islets. Fundamental cryobiology requires determination of several cryobiophysical parameters to predict optimal cryopreservation procedures. These include water permeability or hydraulic conductivity (Lp) and its activation energy (Ea), the permeability of the cell plasma membrane to a cryoprotectant(s) (Ps) and its Ea, the osmotically inactive fraction of cell volume (Vb), and the intracellular ice formation temperature. For islet cells, these parameters have not previously been reported. In the present studies, the Lp, its Ea, and Vb were determined for isolated individual golden hamster pancreatic islet cells. The Lp and Vb parameters were also measured for corresponding exocrine cells. Both islet and the exocrine cells appeared to be ideal osmometers over the experimental range when examined by the Boyle Van't-Hoff relationship (linear regression, r = 0.99 for both types of cells). Extrapolation of these plots generated Vb values of 0.40 for the islet cells and 0.45 for the pancreatic exocrine cells. To determine the Lp, kinetic changes of cell volume over time (dv/dt) in response to anisoosmotic conditions (ranging from 145 mOsm/kg to 1.35 Osm/kg) were measured using an electronic particle counter. The experimental data were fitted to generate the Lp values by least-squares curve fitting to a differential equation describing osmotic water movement across the plasma membrane. For pancreatic islet cells, the Lp was determined to be 0.25 +/- 0.03 microns/min/atm (mean +/- SD, n = 14) at 22 degrees C, 0.54 +/- 0.07 (n = 10), 0.06 +/- 0.008 (n = 9), and 0.01 +/- 0.001 (n = 9) at 37, 8 and 0 degrees C, respectively. The Ea for Lp was calculated from the slope of the Arrhenius plot based upon the mean Lp values at the four different temperatures. The Ea was 16.21 Kcal/mol between 0 and 37 degrees C. Based upon these values, an optimal cooling rate for cryopreserving pancreatic islet cells is predicted to be approximately 0.5 degrees C min. The Lp for the individual exocrine cells was determined to be 3.73 +/- 1.75 microns/min/atm (n = 13) at 22 degrees C, which was approximately 10 times the Lp value of the corresponding islet cells.

The ability to store pancreatic islets using cryopreservation methodology would greatly assist the application of clinical islet transplantation to Type 1 (insulin-dependent) diabetics. It is our working thesis that the illumination of fundamental biophysical characteristics of these cells will lead to increased cryosurvival rates through theoretically predicted and experimental testing of optimal freezing protocol; as has been found for cells and tissues such as mammalian and Drosophila embryos. Pancreatic islets were isolated from Golden hamsters and their osmometric behavior, including inactive cell volume (Vb), was determined for either whole islets or isolated individual islet cells. When islets or islet cells were exposed to various concentrations of NaCl, they were found to exhibit a “classic” “Boyle-Van't Hoff” osmometric response. The Boyle-Van't Hoff representation of the volume curve (relative cell volume vs. 1/osmolality) yields a linear response with r values of .99 for each curve. Extrapolations to the normalized osmotically inactive volumes (Vb) were .43 and .22 for whole islets and individual islet cells, respectively. These data regarding the fundamental cryobiological characteristics of islets and islet cells should provide the foundation upon which to further the investigation of osmotic parameters of these cells and eventually lead to the determination of optimal freezing protocols.

Cryopreservation has the potential to improve availability of donor parts for composite tissue allotransplantation and may reduce their antigenicity. This study investigates whether the component tissues of composite flaps remain viable after cryopreservation. Forty-one epigastric flaps were harvested from Lewis rats. Twenty-one flaps were perfused with DMSO/trehalose, frozen by controlled cooling to -140 degrees C, and stored in liquid nitrogen for 2 weeks. Ten fresh and 10 cryopreserved/thawed flaps were examined histologically with hematoxylineosin and factor VIII staining. An epithelial viability index was calculated for 10 fresh and 11 cryopreserved flaps using the MTT assay. In all cryopreserved samples, hematoxylineosin, and factor VIII staining revealed a well-preserved cellular architecture, which was indistinguishable from fresh specimens. The viability index for the cryopreserved samples was 10.90 +/- 2.09 compared with 12.15 +/- 1.32 for fresh flaps (P = 0.123). Results suggest that the skin, adipose, and vascular endothelial cells of composite tissue flaps retain their viability after cryopreservation and thawing.

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5M dimethyl sulfoxide (Me(2)SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to -140 degrees C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n=10), perfused (n=8), and cryopreserved (n=17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n=10) was 11.17+/-2.01, which was not significantly different from fresh controls (n=10, 12.15+/-1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.

An electromagnetic (EM) heating system is developed to achieve the rapid and uniform warming of cryopreserved biomaterials. Using the heating system, a rectangular resonant cavity is excited in TE101 mode at frequencies near 434 MHz. In experiments, a spherical phantom of biomaterial with a diameter of 36 mm is placed at the center of the cavity. The phantom is first cooled down to about −80 °C within the cavity and then thawed by EM absorption. Results show that EM warming can produce much higher warming rate than conventional water-bath warming method. The spatial temperature distribution in the phantom during EM warming is also more uniform than that during the water-bath warming.

Cryosurgery has been recently accepted as a treatment option for eradicating undesirable tissues, especially tumor tissues, due to its minimally invasive nature and low hospitalization needs. A multidimensional, finite element analysis (FEA) for the cooling, holding and rewarming processes of biological tissues during cryosurgery is presented. The tissues were treated as non-ideal materials with temperature dependent thermophysical properties. The enthalpy method has been applied to solve the non-linear problem. The influence of heating effect due to blood flow and metabolism was studied, and furthermore, the effect of pre-injecting solutions with particular thermal properties into the target tissues was also numerically studied. It was found that the heat source term due to blood flow and metabolism in the bioheat transfer equation has a significant influence on the thermal and thermal gradient histories of the target tissues, and that the method of injection of solutions with particular thermal properties into the target tissues before cryosurgery may be a possible way to optimize the treatment process. However, in vitro experiments have not fully supported this viewpoint.

An experimental study of the mechanical response of frozen arteries to tensile stresses at low temperatures is presented. The Dynamic Mechanical Analyzer was used to perform the mechanical experiments. It was found that the frozen artery shows a kind of elastic-plasticity when the temperature is between -20 C and -40 C. And with the decrease of the temperature, the plasticity deformation decreases. Thus at the temperature of -120 C no plasticity deformation is observed before the artery's fracture and the tissue shows quite perfect elastic brittleness, both peripherally and axially. These kinds of mechanical characteristics help explain the fracture phenomena occurring during cryopreservation of the arteries. The mechanical properties, including elastic modulus and fracture strength, are also given. It is known that Cryoprotectant (CPA) used in cryopreservation is necessary in maintaining the tissue's biological functions. Our investigation of its effect on the artery's mechanical properties found that the existence of CPA can soften the tissue at low temperatures, thus may decrease the possibility of fractures during the cryopreservation.

Long-term cryopreservation of islets of Langerhans would be advantageous to a clinical islet transplantation program. Fundamental cryobiology utilizes knowledge of basic biophysical characteristics to increase the understanding of the preservation process and possibly increase survival rate. In this study several of these previously unreported characteristics have been determined for individual islet cells isolated from Golden hamster islets. Using an electronic particle counting device and a temperature control apparatus, dynamic volumetric response of individual islet cells to anisosmotic challenges of 1.5 M dimethyl sulfoxide (DMSO) and 1.5 M ethylene glycol (EG) were recorded at four temperatures (8, 22, 28, and 37 degreesC). The resulting curves were fitted using Kedem and Katchalsky equations which describe water flux and cryoprotectant agent (CPA) flux based on hydraulic conductivity (Lp), CPA permeability (Ps), and reflection coefficient (final sigma) for the membrane. For Golden hamster islet cells, Lp, Ps, and final sigma for DMSO at 22 degreesC were found to be 0.23 +/- 0.06 microm/min/atm, 0.79 +/- 0.32 x 10(-3) cm/min, and 0.55 +/- 0.37 (n = 11) (mean +/- SD), respectively. For EG at 22 degreesC, Lp equaled 0.23 +/- 0.06 microm/min/atm, Ps equaled 0.63 +/- 0.20 x 10(-3) cm/min, and final sigma was 0.75 +/- 0.17 (n = 9). Arrhenius plots (ln Lp or ln Ps versus 1/temperature (K)) were created by adding the data from the other three temperatures and the resulting linear regression yielded correlation coefficients (r) of 0.99 for all four plots (Lp and Ps for both CPAs). Activation energies (Ea) of Lp and Ps were calculated from the slopes of the regressions. The values for DMSO were found to be 12.43 and 18.34 kcal/mol for Lp and Ps (four temperatures, total n = 52), respectively. For EG, Ea of Lp was 11.69 kcal/mol and Ea of Ps was 20.35 kcal/mol (four temperatures, total n = 58).

Although embryo cryopreservation is routine for many mammalian species, it is important to know how the fundamental cryobiology of these cells changes with development. Progressive cleavage divisions result in a reduction in the blastomere surface area available for water and cryoprotectant mass transport. Therefore, the membrane permeability of murine oocytes, zygotes, 2-cell, 4-cell, and 8-cell embryos to water (Lp), and dimethylsulphoxide (PDMSO), and the reflection coefficient, sigma (sigma) were determined. Oocytes or zygotes were recovered, cumulus cells removed, then cultured until use. Oocytes and embryos were immobilized and perfused with treatment solutions at 24 degrees C. Osmotically induced cell volume changes over time were videotaped followed by image analysis. The Lp values in the presence of dimethylsulphoxide (DMSO) were 0.77, 0.81, 0.94, 0.86, and 1.10 microm/min/atm, and the PDMSO values were 1.85, 2.04, 2.41, 1.95, and 1.25x10(-3) cm/min for oocytes, zygotes, 2, 4, and 8-cell embryos respectively. The Lp values in the presence of DMSO were significantly (P0.05) higher than those in the absence of DMSO. Treating the whole embryo as a single osmotic entity leads to significantly (P0.05) elevated PDMSO estimates relative to those based upon measurements of individual blastomeres. These data indicate that both Lp and PDMSO estimates are lower when predicted on an individual blastomere basis. The data also show that neither Lp nor PDMSO differ among oocytes, zygotes, 2-cell and 4-cell embryos. However, the significantly higher Lp and lower PDMSO of the 8-cell stage support the hypothesis that fundamental cryobiological differences may require developmental stage-specific embryo cryopreservation protocols.

The Sf21 cell line is extensively used for virus research and producing heterologous recombinant proteins. To develop optimal strategies for minimizing cell injury due to intracellular ice formation and excessive volume shrinkage during cryopreservation, the fundamental transport properties including the osmotic inactive volume (Vb ), the hydraulic conductivity (Lp ), and the glycerol permeability (Ps ) of Sf21 cell membrane at 25, 15, 5 and -2°C were characterized using a micro-perfusion chamber. The effects of temperature on the hydraulic conductivity and the glycerol permeability of Sf21 cell membrane, reflected by the activation energies, were quantitatively investigated. It was found that the hydraulic conductivity decreases along with the increase of the final CPA concentration at a given temperature, and quantitative analysis indicates that the hydraulic conductivity has a significant linear attenuation along with the increase of the concentration of glycerol. Therefore, we incorporate the concentration dependence of the hydraulic conductivity into the classic Arrhenius relationship by replacing the constant reference value of the hydraulic conductivity at the reference temperature with a function that is linearly dependent on the CPA concentration. Consequently, the prediction of the Arrhenius relationship is improved, and the novel Arrhenius relationship could be very important to the development of optimal strategies for cell cryopreservation.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.

Thermal stress and consequent fracture in frozen organs or cell suspensions have been proposed to be two causes of cell cryoinjury. A specific device was developed to study the thermal stress and the fracture phenomena during a slow cooling process of isotonic NaCl solutions with different concentrations of glycerol (cryoprotectant) in a cylindrical tube. It was shown from the experimental results that glycerol significantly influenced the solidification process of the ternary solutions and reduced the thermal stress. The higher the initial glycerol concentration, the lower the thermal stress in the frozen solutions. Glycerol concentrations over 0.3 M were sufficient to eliminate the fracture of the frozen solutions under the present experimental conditions. To explain the action of glycerol in reducing the thermal stress and preventing the ice fracture, a further study on ice crystal formation and growth of ice in these solutions was undertaken using cryomicroscopy. It is known from previous studies that an increase of initial glycerol concentration reduced frozen fraction of water in the solution at any given low temperature due to colligative properties of solution, which reduced the total ice volume expansion during water solidification. The present cryomicroscopic investigation showed that under a fixed cooling condition the different initial glycerol concentrations induced the different microstructures of the frozen solutions at not only a given low temperature but also a given frozen fraction of water. It has been known that ice volume expansion during solidification is a major factor causing the thermal stress and the interior microstructure is critical for the mechanical strength of a solid. Therefore, functions of glycerol in reducing the total ice volume expansion during water solidification and in influencing interior microstructure of the ice may contribute to reduce the thermal stress and prevent the fracture in the frozen solutions.

A perfusion technique using micropipette methodology was developed to determine quantitatively the membrane transport properties of mammalian oocytes. This method eliminates modelling ambiguities inherent in microdiffusion, a closely related technology, and should prove to be especially valuable for study of the coupled transport of water and cryoprotectant through mammalian oocytes and embryos. The method is described and evidence given for validity of the method for the simple case of uncoupled flow of water through the mouse oocyte membrane. The zona pellucida of a mouse oocyte was held by a micropipette with an 8-10 microns diameter tip opening and perfused by hyperosmotic media. The kinetic volume change of the cell was videotaped and quantified by image analysis. Experimental data and mathematical modelling were used to determine the hydraulic conductivity of the oocyte membrane (Lp) found to be 1.05, 0.45 and 0.26 microns min-1 atm-1 at 30 degrees C, 22 degrees C and 12 degrees C, respectively. The corresponding activation energy, Ea, for Lp was calculated to be 13.0 kcal mol-1. These values are in agreement with data obtained by other techniques. One of the major advantages of this technique is that the extracellular osmotic condition can be changed readily by perfusing a single cell with a prepared medium. To study the response of the same cell to different osmotic conditions, the old perfusion medium can be removed easily and the cell reperfused with a different medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid, low cost screening of tuberculosis requires an effective enrichment method of Mycobacterium tuberculosis (MTB) cells. Currently, microfiltration and centrifugation steps are frequently used for sample preparation, which are cumbersome and time-consuming. In this study, the size-selective capturing mechanism of a microtip-sensor is presented to directly enrich MTB cells from a sample mixture. When a microtip is withdrawn from a spherical suspension in the radial direction, the cells that are concentrated by AC electroosmosis are selectively enriched to the tip due to capillary- and viscous forces. The size-selectivity is characterized by using polystyrene microspheres, which is then applied to size-selective capture of MTB from a sample mixture. Our approach yields a detection limit of 800 cells mL(-1), one of the highest-sensitivity immunosensors to date.