Microfabricated thermal conductivity sensor: a high resolution tool for quantitative thermal property measurement of biomaterials and solutions.

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.

Development of a reliable low-cost controlled cooling rate instrument for the cryopreservation of hematopoietic stem cells.

BACKGROUND AIMS: An optimal cooling rate is one of the critical factors influencing the survival of cells during cryopreservation. We describe a novel device, called the box-in-box, that has been developed for optimal cryopreservation of human hematopoietic stem cells (HSC). METHODS: This work presents the design of the device, a mathematical formulation describing the expected temperature histories of samples during the freezing process, along with actual experimental results of thermal profile tests. In experiments, when the box-in-box device was transferred from room temperature to a -80 degrees C freezer, a cooling rate of -1 to -3.5 degrees C/min, which has been widely used for the cryopreservation of HSC, was achieved. In order to evaluate this device further, HSC cryopreservation was compared between the box-in-box device and a commercially available controlled-rate freezer (CryoMed). RESULTS: The experimental data, including total cell population and CD34(+) hematopoietic progenitor cell recovery rates, viability and cell culture colony assays, showed that the box-in-box worked as well as the CryoMed instrument. There was no significant difference in either survival rate or the culture/colony outcome between the two devices. CONCLUSIONS: The box-in-box device can work as a cheap, durable, reliable and maintenance-free instrument for the cryopreservation of HSC. This concept of a box-in-box may also be adapted to other cooling rates to support cryopreservation of a wide variety of tissues and cells.

Direct concentration measurements of the unfrozen portion of solutions under freezing.

Phase diagrams of solutions consisting of cryoprotective agents (CPA) are very useful in cryobiology research. Those diagrams depict the points of solution concentrations at corresponding temperatures: one of essential inputs that can be utilized to compute the volume response of cell under freezing process. However, generating such plots is costly and time-consuming. A direct method is proposed in this study to determine the solution concentration of unfrozen parts at multiple sub-zero temperatures. Measurements of binary solutions, composed of water and sodium chloride, were performed and compared with published data. Ternary solutions, consisting of water, sodium chloride and dimethyl sulfoxide, were also measured. The uniqueness and advantage achieved through the usage of this method are demonstrated when phase diagrams of complex cryopreservation solutions (CryoStor solutions including CryoStor Base and CryoStor 10) are generated. The temperature range where the method is utilized is either limited by the osmometry (0-3200 mmol/kg) or by the availability of liquid samples at sub-freezing temperatures. Modified methods will be required to address the limitation of osmolality measurements and the availability of sub-freezing liquid samples at lower temperatures.

An Application of Stream Imaging Technique in the Study of Osmotic Behaviors of Multiple Cells.

Light microscopy method offers unique abilities for the determination of membrane transport properties of either single or multiple cells. A stream imaging system composed of a microfluidic device, a charge-coupled device camera, and a microscope has been developed to study the osmotic behavior of multiple cells in response toward their extracellular environment. Cells of interest were first mixed with the desired extracellular medium and streamed into a microchannel. The microchannel confines the movement of the cells in a monolayer and allows cells to move along the flow direction only. The cells then pass through a sensing zone where the images of cells were capable of being captured under a microscope. Using mouse dendritic cells (mDCs) as a model system, the membrane transport properties were investigated. The kinetics volume changes of mDCs under various extracellular conditions at room temperature (22°C) were analyzed using a biophysical model to determine water and cryoprotectant transport properties of the cell membrane. This prototype system directly allows us to observe, trace, capture, and store the sample information in terms of number, concentration, dynamic size, or shape for further analyses and documentations. We believe that the system has the potential of being used as a stand-alone equipment, or integrated into a lab-on-a-chip system, or embedded into commercialized instruments.

Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties.

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 microm/min/atm (n=8, r(2)>0.963), respectively, for room temperature (22 degrees 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 microm/min/atm and (0.49+/-0.13) x 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.

Ultra-high cooling rate utilizing thin film evaporation.

This research introduces a cell cryopreservation method, which utilizes thin film evaporation and provides an ultra-high cooling rate. The microstructured surface forming the thin film evaporation was fabricated from copper microparticles with an average diameter of 50 µm. Experimental results showed that a cooling rate of approximately 5[Formula: see text]10(4) °C/min was achieved in a temperature range from 10 °C to -187 °C. The current investigation will give birth to a cell cryopreservation method through vitrification with relatively low concentrations of cryoprotectants.

Particle shape effect on heat transfer performance in an oscillating heat pipe.

The effect of alumina nanoparticles on the heat transfer performance of an oscillating heat pipe (OHP) was investigated experimentally. A binary mixture of ethylene glycol (EG) and deionized water (50/50 by volume) was used as the base fluid for the OHP. Four types of nanoparticles with shapes of platelet, blade, cylinder, and brick were studied, respectively. Experimental results show that the alumina nanoparticles added in the OHP significantly affect the heat transfer performance and it depends on the particle shape and volume fraction. When the OHP was charged with EG and cylinder-like alumina nanoparticles, the OHP can achieve the best heat transfer performance among four types of particles investigated herein. In addition, even though previous research found that these alumina nanofluids were not beneficial in laminar or turbulent flow mode, they can enhance the heat transfer performance of an OHP.

Electrical conductivity measurements for the ternary systems of glycerol/sodium chloride/water and ethylene glycol/sodium chloride/water and their applications in cryopreservation.

Electrical conductivity of a solution is a property that can be easily determined through the measurement of a conductivity probe. The present study demonstrates the measurements of electrical conductivity for two ternary solutions: glycerol/sodium chloride/water and ethylene glycol/sodium chloride/water. When the concentration of sodium chloride to water ratio (R) is fixed, the existence of either glycerol or ethylene glycol, both cryoprotective agents (CPAs), can be quantitatively determined by their depressive influence on electrical conductivity of the solution. The measurements were performed on solutions with a set of 10 different concentrations of CPAs, ranging from 3.2% to 50% (v/v), along with five ratios of NaCl/water solutions. Equations to fit the experimental measurements were devised to characterize the relations among electrical conductivity, CPAs concentration, and R. A conductivity meter used in this study required <5 s to read the solution's electrical conductivity, which is faster than the measurement using osmometry method. The charts of ternary solutions associated with their electrical conductivity and concentrations make it especially useful for monitoring the cryopreservation processes, including addition and removal of CPAs, to prevent osmotic damages to biological samples.

A microfluidic manipulator for enrichment and alignment of moving cells and particles.

Grooved structures have been widely studied in particle separation and fluid mixing in microfluidic channel systems. In this brief report, we demonstrate the use of patterning flows produced by two different sorts of grooved surfaces: single slanted groove series (for enrichment patterns) and V-shaped groove series (for focusing patterns), into a microfluidic device to continuously manipulate the flowing particles, including microbeads with 6 microm, 10 microm, and 20 microm in diameter and mouse dendritic cells of comparable sizes to the depth of the channel. The device with grooved channels was developed and fabricated by soft-lithographic techniques. The particle distributions after passing through the single slanted grooves illustrate the size-dependent enrichment profiles. On the other hand, particles passing through the V-shaped grooves show focusing patterns downstream, for the combination effect from both sides of single slanted grooves setup side-by-side. Compared with devices utilizing sheath flows, the focusing patterns generated in this report are unique without introducing additional flow control. The alignment of the concentrated particles is expected to facilitate the visualization of sizing and counting in cell-based devices. On the other hand, the size-dependent patterns of particle distributions have the potential for the application of size-based separation.

Cryopreservation of Peripheral Blood Stem Cells Using a Box-in-Box Cooling Device.

The cooling process is critical for the cryopreservation of human hematopoietic stem cells (HSCs). Currently, programmed freezing methods and uncontrolled cooling methods are in use, both having obvious disadvantages. In this article, a novel device termed Box-in-Box (BIB) was developed and evaluated by in vitro cryopreservation tests in 2 different operation modes ("against-side" mode for Group I (n = 10), and "in-middle" mode for Group II (n = 10), respectively), and compared with an uncontrolled cooling method (Group III (n = 7), Styrofoam boxes) as well as a conventional programmed freezer method (Group IV (n = 10), CryoMed TM 1010, Cryogenic Tech., FL). Recorded temperature profiles of samples cryopreserved with BIB show that a consistent cooling procedure with a rate around -1°C to -3.5°C/min can be achieved during their transfer from room temperature to an -80°C freezer. Statistical analysis of the stem cell population recovery, survival, and colony generation recovery shows that there is no significant difference (P > 0.26) among the methods using the BIB or programmed freezer (Group I, Group II, and Group IV), and their related deviations are smaller than the uncontrolled cooling rate method (Group III). Methods using the BIB (Group I and Group II) generated significantly better cell survival rate (P < 0.01) than the uncontrolled cooling rate method (Group III). The results indicate that the controlled cooling rate methods (BIB or CryoMed PF) are more consistent and reliable for clinical use. Considering the advantages of low cost, durability, and no liquid nitrogen consumption for the cooling process, the BIB can be a good alternative to the programmed freezers for the cryopreservation of HSCs.