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Subcutaneous adipose tissue is a rich source of stromal vascular fraction (SVF) and adipose-derived stromal/stem cells (ASCs) that are inherently multipotent and exhibit regenerative properties. In current practice, lipoaspirate specimens harvested from liposuction surgeries are routinely discarded as a biohazard waste due to a lack of simple, cost effective, and validated cryopreservation protocols. The aim of this study is to develop a xenoprotein-free cryoprotective agent cocktail that will allow for short-term (up to 6 months) preservation of lipoaspirate tissues suitable for fat grafting and/or stromal/stem cell isolation when stored at achievable temperatures (−20 °C or −80 °C). Lipoaspirates donated by three consenting healthy donors undergoing elective cosmetic liposuction surgeries were suspended in five freezing media (FM1: 10% DMSO and 35% BSA; FM2: 2% DMSO and 43% BSA; FM3: 10% DMSO and 35% lipoaspirate saline; FM4: 2% DMSO and 6% HSA; and FM5: 40% lipoaspirate saline and 10% PVP) all suspended in 1X DMEM/F12 and frozen using commercially available freezers (−20 °C or −80 °C) and stored at least for a 1 month. After 1 month of freezing storage, SVF cells and ASCs were isolated from the frozen-thawed lipoaspirates by digestion with collagenase type I. Cell viability was evaluated by fluorescence microscopy after staining with acridine orange and ethidium bromide. The SVF isolated from lipoaspirates frozen at −80 °C retained comparable cell viability with the tested freezing media (FM2, FM3, FM4) comparable with the conventional DMSO and animal serum media (FM1), whereas the FM5 media resulted in lower viability. In contrast, tissues frozen and stored at −20 °C did not yield live SVF cells after thawing and collagenase digestion. The surface marker expression (CD90, CD29, CD34, CD146, CD31, and CD45) of ASCs from frozen lipoaspirates at −80 °C in different cryoprotectant media were also evaluated and no significant differences were found between the groups. The adipogenic and osteogenic differentiation potential were studied by histochemical staining and gene expression by qRT-PCR. Oil Red O staining for adipogenesis revealed that the CPA media FM1, FM4 and FM5 displayed robust differentiation. Alizarin Red S staining for osteogenesis revealed that FM1 and FM4 media displayed superior differentiation in comparison to other tested media. Measurement of adipogenic and osteogenic gene expression by qRT-PCR provided similar outcomes and indicated that FM4 CPA media comparable with FM1 for adipogenesis and osteogenesis.

In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey (Macacamulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 degrees C/min in the absence of any cryoprotective agents and in the presence of 0.7 M of glycerol, as well. Using previously published values, the macaque sperm cell was modeled as a cylinder of length 73.83 microm with a radius of 0.40 microm and an osmotically inactive cell volume, V(b), of 0.772 V(o), where V(o) is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of approximately 22 microm(-1). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water at 0 degrees C, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) were found to range from: L(pg) or L(pg)[cpa]=0.0020-0.0029 microm/min-atm; E(Lp) or E(Lp)[cpa])=10.6-18.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, B(opt)=1009.5 x exp(-0.0546 x E(LP) x L(pg) x (SA/WV); where the units of B(opt) are degrees C/min, E(Lp) or E(Lp)[cpa] are kcal/mole, L(pg) or L(pg)[cpa] are mum/min-atm and SA/WV are mum(-1)), we determined the optimal rates of freezing macaque sperm to be approximately 23 degrees C/min (ejaculated sperm in the absence of CPAs), approximately 29 degrees C/min (ejaculated sperm in the presence of glycerol), approximately 24 degrees C/min (epididymal sperm in the absence of CPAs) and approximately 24 degrees C/min (epididymal sperm in the presence of glycerol). In conclusion, the subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal macaque spermatozoa under corresponding cooling conditions.

An overview of the major issues involved in the statistical thermodynamic treatment of phospholipid membranes at the atomistic level is summarized: thermodynamic ensembles, initial configuration (or the physical system being modeled), force field representation as well as the representation of long-range interactions. This is followed by a description of the various ways that the simulated ensembles can be analyzed: area of the lipid, mass density profiles, radial distribution functions (RDFs), water orientation profile, deuterium order parameter, free energy profiles and void (pore) formation; with particular focus on the results obtained from our recent molecular dynamic (MD) simulations of phospholipids interacting with dimethylsulfoxide (Me(2)SO), a commonly used cryoprotective agent (CPA).

This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus , specifically those of the southern platyfish Xiphophorus maculatus . These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20 °C/min in three different media: (1) Hanks’ balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 μm and a diameter of 0.66 μm with an osmotically inactive cell volume ( V b ) of 0.6 V 0 , where V 0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 μm −1 . By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L pg or L pg [cpa] and the activation energy, E Lp or E Lp [cpa]) were found to range from: L pg or L pg [cpa] = 0.0053–0.0093 μm/min atm; E Lp or E Lp [cpa] = 9.79–29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, B opt = 1009.5 exp ( - 0.0546 E Lp ) ( L pg ) ( SA WV ) where the units of B opt are °C/min, E Lp or E Lp [cpa] are kcal/mol, L pg or L pg [cpa] are μm/min atm and SA/WV are μm −1 ), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 °C/min (in HBSS), 47 °C/min (in HBSS + 14% glycerol) and 36 ° C/min (in HBSS + 10% DMSO). Preliminary empirical experiments suggest that the optimal rate of freezing X. maculatus sperm in the presence of 14% glycerol to be ∼25 °C/min. Possible reasons for the observed discrepancy between the theoretically predicted and experimentally determined optimal rates of freezing X. maculatus sperm cells are discussed.