Originally isolated from bone marrow, mesenchymal stromal cells (MSCs) have since been obtained from various fetal and post-natal tissues and are the focus of an increasing number of clinical trials. Because of their tremendous potential for cellular therapy, regenerative medicine and tissue engineering, it is desirable to cryopreserve and bank MSCs to increase their access and availability. A remarkable amount of research and resources have been expended towards optimizing the protocols, freezing media composition, cooling devices and storage containers, as well as developing good manufacturing practices in order to ensure that MSCs retain their therapeutic characteristics following cryopreservation and that they are safe for clinical use. Here, we first present an overview of the identification of MSCs, their tissue sources and the properties that render them suitable as a cellular therapeutic. Next, we discuss the responses of cells during freezing and focus on the traditional and novel approaches used to cryopreserve MSCs. We conclude that viable MSCs from diverse tissues can be recovered after cryopreservation using a variety of freezing protocols, cryoprotectants, storage periods and temperatures. However, alterations in certain functions of MSCs following cryopreservation warrant future investigations on the recovery of cells post-thaw followed by expansion of functional cells in order to achieve their full therapeutic potential.

The osmotic virial equation was used to predict osmolalities of solutions of interest in biology. The second osmotic virial coefficients, Bi, account for the interactions between identical solute molecules. For multisolute solutions, the second osmotic virial cross coefficient, Bij, describes the interaction between two different solutes. We propose to use as a mixing rule for the cross coefficient the arithmetic average of the second osmotic virial coefficients of the pure species, so that only binary solution measurements are required for multisolute solution predictions. Single-solute data were fit to obtain the osmotic virial coefficients of the pure species. Using those coefficients with the proposed mixing rule, predictions were made of ternary solution osmolality, without any fitting parameters. This method is shown to make reasonably accurate predictions for three very different ternary aqueous solutions: (i) glycerol + dimethyl sulfoxide + water, (ii) hemoglobin + an ideal, dilute solute + water, and (iii) bovine serum albumin + ovalbumin + water.

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Osmotic permeability characteristics and the effects of cryoprotectants are important determinants of recovery and function of spermatozoa after cryopreservation. The primary purpose of this study was to determine the osmotic permeability parameters of human spermatozoa in the presence of cryoprotectants. A series of experiments was done to: 1) validate the use of an electronic particle counter for determining both static and kinetic changes in sperm cell volume; 2) determine the permeability of the cells to various cryoprotectants; and 3) test the hypothesis that human sperm water permeability is affected by the presence of cryoprotectant solutes. The isosmotic volume of human sperm was 28.2 +/- 0.2 microns3 (mean +/- SEM), 29.0 +/- 0.3 microns3, and 28.2 +/- 0.4 microns3 at 22, 11, and 0 degrees C, respectively, measured at 285 mOsm/kg via an electronic particle counter. The osmotically inactive fraction of human sperm was determined from Boyle van't Hoff (BVH) plots of samples exposed to four different osmolalities (900, 600, 285, and 145 mOsm/kg). Over this range, cells behaved as linear osmometers with osmotically inactive cell percentages at 22, 11, and 0 degrees C of 50 +/- 1%, 41 +/- 2%, and 52 +/- 3%, respectively. Permeability of human sperm to water was determined from the kinetics of volume change in a hyposmotic solution (145 mOsm/kg) at the three experimental temperatures. The hydraulic conductivity (Lp) was 1.84 +/- 0.06 microns.min-1.atm-1, 1.45 +/- 0.04 microns.min-1.atm-1, and 1.14 +/- 0.07 microns.min-1.atm-1 at 22, 11, and 0 degrees C, respectively, yielding an Arrhenius activation energy (Ea) of 3.48 kcal/mol. These biophysical characteristics of human spermatozoa are consistent with findings in previous reports, validating the use of an electronic particle counter for determining osmotic permeability parameters of human sperm. This validated system was then used to investigate the permeability of human sperm to four different cryoprotectant solutes, i.e., glycerol (Gly), dimethylsulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG), and their effects on water permeability. A preloaded, osmotically equilibrated cell suspension was returned to an isosmotic medium while cell volume was measured over time. A Kedem-Katchalsky model was used to determine the permeability of the cells to each solute and the resulting water permeability. The permeabilities of human sperm at 22 degrees C to Gly, DMSO, PG, and EG were 2.07 +/- 0.13 x 10(-3) cm/min, 0.80 +/- 0.02 x 10(-3) cm/min, 2.3 +/- 0.1 x 10(-3) cm/min, and 7.94 +/- 0.67 x 10(-3) cm/min, respectively. The resulting Lp values at 22 degrees C were reduced to 0.77 +/- 0.08 micron.min-1.atm-1, 0.84 +/- 0.07 micron.min-1.atm-1, 1.23 +/- 0.09 microns.min-1.atm-1, and 0.74 +/- 0.06 micron.min-1.atm-1, respectively. These data support the hypothesis that low-molecular-weight, nonionic cryoprotectant solutes affect (decrease) human sperm water permeability.

The fundamental physical mechanisms of water and solute transport across cell membranes have long been studied in the field of cell membrane biophysics. Cryobiology is a discipline that requires an understanding of osmotic transport across cell membranes under nondilute solution conditions, yet many of the currently-used transport formalisms make limiting dilute solution assumptions. While dilute solution assumptions are often appropriate under physiological conditions, they are rarely appropriate in cryobiology. The first objective of this article is to review commonly-used transport equations, and the explicit and implicit assumptions made when using the two-parameter and the Kedem-Katchalsky formalisms. The second objective of this article is to describe a set of transport equations that do not make the previous dilute solution or near-equilibrium assumptions. Specifically, a new nondilute solute transport equation is presented. Such nondilute equations are applicable to many fields including cryobiology where dilute solution conditions are not often met. An illustrative example is provided. Utilizing suitable transport equations that fit for two permeability coefficients, fits were as good as with the previous three-parameter model (which includes the reflection coefficient, sigma). There is less unexpected concentration dependence with the nondilute transport equations, suggesting that some of the unexpected concentration dependence of permeability is due to the use of inappropriate transport equations.