Cryopreservation of cells for tissue engineering.

Engineered tissue for clinical use must be evaluated, inventoried, and delivered to the recipient. This is time intensive and requires a means of sustaining cell viability prior to implantation. Tissue stored at ambient temperature or warmer requires expensive human involvement to satisfy metabolic demands and risks infection and biological alteration. At low temperatures, i.e., below - 125 degrees C, tissues have no metabolic demands. These cold environments can be conveniently attained with cryogens like liquid nitrogen, and 10+ years of storage without change in viability is common. Cryogenic storage is clinically accepted for blood cells, bone marrow, reproductive cells, skin, cornea, and vascular tissue such as heart valves. The scientific basis for cryopreservation derives from principles of biophysics, engineering, and chemistry. The goal of this review is to identify these principles for tissue engineers and provide sufficient mathematical definition to guide the design of specific cryopreservation protocols for component cells of newly engineered tissues. The discussion assumes cells are "typical" from the standpoint of cryopreservation, which means neither the nucleus nor any vacuole is greater than 50% of the total volume, cellular contents are roughly 15% protein, 15% lipid, and 70% water by mass, and the longest dimension of a cell or cellular aggregate is 1-1000 microm.

Mist deposition onto hairy root cultures: aerosol modeling and experiments.

We analyzed the applicability of the standard models for aerosol deposition in randomly packed fibrous filter beds to mist deposition across a bed of hairy roots in the nutrient mist bioreactor. Although the assumptions inherent in the models are met on a local level, the overall structure of the root bed introduces some uncertainty into the correct choice of root packing fraction and gas velocity required by the model. For reasonable parameter values, the minimum in the deposition efficiency curves is close to the peak in the mist number and mass distributions, and good penetration of the root bed is possible. We then measured the deposition of mist across a packed bed of Artemisia annua transformed roots as a function of droplet size, bed length, and gas flow rate at a root packing fraction alpha = 0.5. We compared the experimental measurements with the predictions of the aerosol deposition model and found good agreement between the measured and predicted values for the diameter where the deposition efficiency across the bed is 50%, D0.5. Agreement between the model and the experiments broke down when the flow rate was increased to the point where the creeping flow assumptions were no longer valid.

Quantitative light microscopy of combined perfusion and freezing processes.

The rational design of cryopreservation protocols for living tissues demands an understanding of the mechanisms of mass transport between cells and their environment throughout the entire process. We have developed a new microscope stage to enable a specimen to be viewed continuously during a preservation protocol, including the addition and removal of cryoprotective additives and freezing and thawing. The specimen is contained in a sealed chamber having inlet and outlet ports for admitting and collecting perfusate solution, the entire volume of which may be exchanged with a time constant of 1-5 s, depending on the solution viscosity. The temperature of the active area of the stage is regulated by the standard techniques of convection cryomicroscopy over a range in excess of 50 to -100 degrees C. A series of experiments has been performed on this system to measure the osmotic behaviour of rat pancreas islets during the addition and removal of dimethyl sulphoxide at temperatures between 25 and -10 degrees C. The technique involves mounting a single islet onto the low-temperature stage so that it is constrained from lateral movement by a specially sized mesh. Both the system temperature and chemical composition are monitored and controlled simultaneously and independently; as a consequence, virtually any defined cryopreservation protocol may be imposed on the specimen. For making permeability measurements, the bathing medium of the specimen may be changed very rapidly to produce a defined osmotic stress. Alternatively, the specimen may be subcooled to a specific and fixed subzero temperature, at which point ice is nucleated in the extracellular medium, creating a near instantaneous change in composition. The temporal alteration in specimen size is monitored by video microscopy and quantified by computer vision analysis methods. One of several mass transfer models is fitted to the data to estimate the membrane permeability based on the assumption of either transport dominated by the movement of water or simultaneous coupled flows of water and cryoprotective agent.

Determination of the kinetics of permeation of dimethyl sulfoxide in isolated corneas.

Corneal cryopreservation requires that endothelial cells remain viable and intercellular structure be preserved. High viability levels for cryopreserved endothelial cells have been achieved, but preserving intercellular structure, especially endothelial attachment to Descemet's membrane, has proved difficult. Cell detachment apparently is not caused by ice, suggesting osmotic or chemical mechanisms. Knowledge of the permeation kinetics of cryoprotectants (CPAs) into endothelial cells and stroma is essential for controlling osmotic and chemical activity and achieving adequate tissue permeation prior to cooling. Proton nuclear magnetic resonance (NMR) spectroscopy was used to assess the permeation of dimethyl sulfoxide (DMSO) into isolated rabbit corneas. Corneas with intact epithelia were exposed to isotonic medium or 2.0 mol/L DMSO for 60 min and subsequently transferred to 2.0 or 4.0 mol/L DMSO, respectively, at 22, 0, or -10 degrees C. DMSO concentration in the cornea was measured vs time. The Kedem-Katchalsky model was fitted to the data. Hydraulic permeability (m3/N.s) is 7.1 x 10(-13) + 216%-11% at 22 degrees C, 8.2 x 10(-13) + 235%-21% at 0 degree C, and 1.7 x 10(-14) + 19%-16% at -10 degrees C. The reflection coefficient is 1.0 + 2%-1% at 22 degrees C and 0 degree C, and 0.9 +/- 5% at -10 degrees C. Solute mobility (cm/s) is 5.9 x 10(-6) + 6%-11% at 22 degrees C, 3.1 x 10(-6) + 12%-11% at 0 degree C, and 5.0 x 10(-8) cm/s + 59%-40% at -10 degrees C.

Cryopreservation of transformed (hairy) roots of Artemisia annua.

The antimalarial drug artemisinin has been found in transformed (hairy) roots of Artemisia annua. A protocol was developed to preserve A. annua hairy roots in liquid nitrogen. Root tips were excised from 7-day-old cultures and held on solid White's medium for 24 h prior to cryoprotection. They were then treated with a cryoprotecting mixture containing 8% (v/v) dimethyl sulfoxide (Me2SO) and 20% (w/v) sucrose at 25 degrees C for 1 h followed by cooling at 0.09 degrees C/min to 4 degrees C then cooling to -35 degrees C at 0.72 degrees C/min. Vials containing the root tips were then plunged into liquid nitrogen. After thawing in a water bath to 37 degrees C, root tips were held in the cryoprotecting mixture for 10 min before it was diluted to 25% of its original concentration. Root tips were washed once with fresh liquid White's medium and held for 1 h prior to culturing on White's medium with 0.2% Gelrite and 3% (w/v) sucrose. Regrowth of root tips averaged 65%. Independent variables in this study included 1) Me2SO concentration; 2) the type and concentration of cosolutes; 3) cooling rate(s); 4) the temperature at which the sample is transferred to liquid nitrogen; 5) the age of the culture from which root tips are taken; 6) the recovery period between root tip excision and immersion in cryoprotectant; and 7) the amount of Gelrite used in the postthaw plating medium.