Alex Fowler

Intracellular trehalose improves the survival of cryopreserved mammalian cells.

We report that the introduction of low concentrations of intracellular trehalose can greatly improve the survival of mammalian cells during cryopreservation. Using a genetically engineered mutant of Staphylococcus aureus α-hemolysin to create pores in the cellular membrane, we were able to load trehalose into cells. Low concentrations (0.2 M) of trehalose permitted long-term post-thaw survival of more than 80% of 3T3 fibroblasts and 70% of human keratinocytes. These results indicate that simplified and widely applicable freezing protocols may be possible using sugars as intracellular cryoprotective additives.

Cryo‐Injury and Biopreservation

Abstract: Mammalian cells appear to be naturally tolerant to cold temperatures, but the formation of ice when cells are cooled leads to a variety of damaging effects. The study of cryo‐injury, therefore, becomes the study of when and how ice is formed both inside and outside the cell during cooling. Protectant chemicals are used to control or prevent ice formation in many preservation protocols, but these chemical themselves tend to be damaging. Cooling and warming rates also strongly affect the amount and location of ice that is formed. Through careful modification of these parameters successful cold preservation techniques for many cell types have been developed, but there are many more cell types that have defied preservation techniques, and the extension of cell‐based techniques to tissues and whole organs has been very limited. There are many aspects to the damaging effects of ice in cells that are still poorly understood. In this brief article we review our current understanding of cellular injury and highlight the aspects of cellular injury during cryopreservation that are still poorly understood.

The optimal spacing of cylinders in free-stream cross-flow forced convection

Abstract This is an experimental, numerical and analytical study of the optimal spacing between cylinders in cross-flow forced convection. The cylinder array occupies a fixed volume and is exposed to a free stream of given velocity and temperature. The optimal cylinder-to-cylinder spacing is determined by maximizing the overall thermal conductance between all the cylinders and the free stream. In the first part, the optimal spacing and corresponding maximum thermal conductance are determined based on experiments with forced air for H D = 6.2 and in the ReD range 50–4000, where ReD is based on the free-stream approach velocity and cylinder diameter D, and H is the array length in the flow direction. In the second part, similar results are developed based on numerical simulations for Pr = 0.72, 10 ⩽ H D ⩽ 20 and 40 ⩽ ReD ⩽ 200. In the concluding section, the experimental and numerical results for optimal spacing and maximum thermal conductance are explained and correlated analytically by intersecting the small-spacing and large-spacing asymptotes of the thermal conductance function.

Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: A study using murine embryonic stem cells ☆

Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz micro-capillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1-2M). The cryoprotectants used included 2M 1,2-propanediol (PROH, cell membrane permeable) and 0.5M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post-vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells.

Water activity and mobility in solutions of glycerol and small molecular weight sugars : Implication for cryo-and lyopreservation

In this study, the free volume models, originally developed for large molecular weight polymer-solvent systems, were used to study the water activity and mobility in solutions of four small molecular weight cryo-/lyoprotectants, viz., glycerol, a monosaccharide (fructose), and two disaccharides (sucrose and trehalose). The free volume model parameters were determined by fitting the models to available experimental data using a nonlinear optimization procedure. It was found that free volume models could accurately predict the available experimental data, which suggests that the free volume models might be generally applicable to aqueous solutions of small molecular weight cryo-/lyoprotectants. Furthermore, several models for estimating the mutual diffusion coefficient were tested using available experimental data for aqueous solutions of glycerol and a better method to estimate the mutual diffusion coefficient was proposed. Free volume models were used to predict and analyze the water activity and mobility...

The optimal spacing between horizontal cylinders in a fixed volume cooled by natural convection

This is a theoretical, numerical and experimental study of how to select the spacing (S) between horizontal cylinders in an array with laminar natural convection, such that the total heat transfer (q) between the array and the ambient is maximized. The volume occupied by the array (height H, width W, cylinder length L) and the cylinder diameter (D) are arbitrary but fixed, while the spacing (or number of cylinders in the array) varies. The optimal spacing and maximum heat transfer results predicted theoretically are developed into accurate and well tested correlations by means of numerical simulations and experimental measurements. The recommended correlations are Sopt/D = 2.72(H/D)13 RaD−14 +0.263 and qmax = 0.448[(HD)13 RaD−14]−16 where qmax is the dimensionless maximum overall thermal conductance, qmax = qmaxD2/[HLWk(Tw - T∞)]. The optimal spacing is relatively insensitive to whether the cylinders are isothermal or with uniform heat flux.

Stabilization of Active Recombinant Retroviruses in an Amorphous Dry State with Trehalose

The disaccharide trehalose is found to be effective for stabilization of active recombinant retroviruses in an amorphous dry state achieved through ambient‐temperature vacuum dehydration of retroviral supernatants. Studies revealed that trehalose is a significantly better desiccation protectant than sucrose, glucose, and dextran: dextran has essentially no protective effect on retroviral survival after drying and rehydration. X‐ray diffractometry of the retroviral supernatant dried with trehalose demonstrated its amorphous nature. The ability to dehydrate retroviral stocks at ambient temperatures into a stable glassy state will have a profound effect for researchers and commercial biotechnology companies which supply retroviral vectors for human gene therapy and basic research.

Cryopreservation of Isolated Primary Rat Hepatocytes: Enhanced Survival and Long-term Hepatospecific Function

Objective:To investigate the long-term effect of cryopreservation on hepatocyte function, as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution, HypoThermosol (HTS), as the carrier solution. Summary Background Data:Advances in the field of bioartificial liver support have led to an increasing demand for successful, efficient means of cryopreservation of hepatocytes. Methods:Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group), both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen, cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion, urea synthesis, deethylation of ethoxyresorufin (CYT P450 activity), and responsiveness to stimulation with interleukin-6 (IL-6) were performed. Results:Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo, in comparison with control (90%). Albumin secretion, urea synthesis and CYT P450 activity yielded 33%, 55%, and 59% in Media-cryo and 71%, 80%, and 88% in HTS-cryo, respectively, compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls. Conclusions:This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability, long-term hepatospecific function, and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices.

Survival of Desiccated Mammalian Cells: Beneficial Effects of Isotonic Media

Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. The objective of this study was to examine the effect that the composition of intra- and extracellular trehalose solutions has on the survival of dried 3T3 fibroblasts. Trehalose was introduced into 3T3 fibroblasts using a genetically engineered mutant of the pore-forming α-hemolysin from Staphylococcus aureus. Cells were dried using natural convection at ambient temperatures. Plasma membrane integrity and the ability to grow and divide in culture were used to assess cell survival. We found that the post-rehydration membrane integrity and percent cell growth were a function of the residual moisture content of the cells following drying. Adjusting the initial osmolality of the intra- and extracellular sugar solutions to isotonic levels by reducing the concentration of buffer resulted in a significant improvement in the membrane integrity and growth of dried ce...

Optimal geometric arrangement of staggered plates in forced convection

Abstract This paper reports the results of an experimental and numerical study of the optimal geometric arrangement of staggered parallel plates in a fixed volume with forced convection heat transfer. The objective of the geometric optimization effort is to maximize the total heat transfer rate between the given volume and the given external flow, when the maximum temperature at a point inside the volume cannot exceed a certain level. The geometric arrangement was varied systematically, by changing the spacing between plates, the number of plates installed in one row, the plate swept length, and the degree to which the plates are staggered. In the first part of the study, it is demonstrated experimentally that there exists an optimal spacing between two adjacent row of plates. Experimental results are reported for air in the range 1000 ⩽ ReL ⩽ 6000, where L is the swept length of the fixed volume. In the second part of the study, extensive numerical results support and extend these findings to 100 ⩽ ReL ⩽10000. In addition, it is shown that there is an optimal way to stagger the plates. In the concluding part of the paper, the optimal spacing and maximum heat transfer rate results are correlated based on the theoretical method of intersecting the two asymptotes (small spacing, large spacing) of the geometric arrangement.