Adult Stem Cells Freezing Processes and Cryopreservation Protocols.

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. Cryopreservation has shown the most promise but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents (CPAs), freezing equipment, and obviously, storage in liquid nitrogen. The purpose of this chapter is to present a general overview of cryopreservation storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage.

Transcriptomic Profiling of Adipose Derived Stem Cells Undergoing Osteogenesis by RNA-Seq.

Adipose-derived stromal/stem cells (ASCs) are multipotent in nature that can be differentiated into various cells lineages such as adipogenic, osteogenic, and chondrogenic. The commitment of a cell to differentiate into a particular lineage is regulated by the interplay between various intracellular pathways and their resultant secretome. Similarly, the interactions of cells with the extracellular matrix (ECM) and the ECM bound growth factors instigate several signal transducing events that ultimately determine ASC differentiation. In this study, RNA-sequencing (RNA-Seq) was performed to identify the transcriptome profile of osteogenic induced ASCs to understand the associated genotype changes. Gene ontology (GO) functional annotations analysis using Database for Annotation Visualization and Integrated Discovery (DAVID) bioinformatics resources on the differentially expressed genes demonstrated the enrichment of pathways mainly associated with ECM organization and angiogenesis. We, therefore, studied the expression of genes coding for matrisome proteins (glycoproteins, collagens, proteoglycans, ECM-affiliated, regulators, and secreted factors) and ECM remodeling enzymes (MMPs, integrins, ADAMTSs) and the expression of angiogenic markers during the osteogenesis of ASCs. The upregulation of several pro-angiogenic ELR+ chemokines and other angiogenic inducers during osteogenesis indicates the potential role of the secretome from differentiating ASCs in the vascular development and its integration with the bone tissue. Furthermore, the increased expression of regulatory genes such as CTNNB1, TGBR2, JUN, FOS, GLI3, and MAPK3 involved in the WNT, TGF-ß, JNK, HedgeHog and ERK1/2 pathways suggests the regulation of osteogenesis through interplay between these pathways. The RNA-Seq data was also validated by performing QPCR on selected up- and down-regulated genes (COL10A1, COL11A1, FBLN, FERMT1, FN1, FOXF1, LAMA3, LAMA4, LAMB1, IGF1, WNT10B, MMP1, MMP3, MMP16, ADAMTS6, and ADAMTS14).

Cryobiology of ovarian tissues: known knowns and known unknowns.

To model the cryobiological responses of cells and tissues, the cellular membrane permeability characteristics are often measured at suprazero temperatures as well as at subzero temperatures with and without the presence of extra-cellular ice. These measured membrane permeability characteristics are then used to predict the responses of cells and tissues for a given thermal insult with the ultimate aim of mitigating the damage caused during the freeze-thaw process. This brief review articles summarizes efforts from my research group over the past 15 years as related to cryobiology of mammalian ovarian tissue sections, i.e., the known knowns as well as the critical cryobiological knowledge that is still lacking to rationally design optimal cryopreservation protocols for ovarian tissues, i.e., the known unknowns.

Cryopreserved Adipose Tissue-Derived Stromal/Stem Cells: Potential for Applications in Clinic and Therapy.

Adipose-Derived Stromal/Stem Cells (ASC) have considerable potential for regenerative medicine due to their abilities to proliferate, differentiate into multiple cell lineages, high cell yield, relative ease of acquisition, and almost no ethical concerns since they are derived from adult tissue. Storage of ASC by cryopreservation has been well described that maintains high cell yield and viability, stable immunophenotype, and robust differentiation potential post-thaw. This ability is crucial for banking research and for clinical therapeutic purposes that avoid the morbidity related to repetitive liposuction tissue harvests. ASC secrete various biomolecules such as cytokines which are reported to have immunomodulatory properties and therapeutic potential to reverse symptoms of multiple degenerative diseases/disorders. Nevertheless, safety regarding the use of these cells clinically is still under investigation. This chapter focuses on the different aspects of cryopreserved ASC and the methods to evaluate their functionality for future clinical use.

The Relative Functionality of Freshly Isolated and Cryopreserved Human Adipose-Derived Stromal/Stem Cells.

The capability of multipotent mesenchymal stem cells to maintain cell viability, phenotype and differentiation ability upon thawing is critical if they are to be banked and used for future therapeutic purposes. In the present study, we examined the effect of 9-10 months of cryostorage on the morphology, immunophenotype, colony-forming unit (CFU) and differentiation capacity of fresh and cryopreserved human adipose-derived stromal/stem cells (ASCs) from the same donors. Cryopreservation did not reduce the CFU frequency and the expression levels of CD29, CD73, CD90 and CD105 remained unchanged with the exception of CD34 and CD45; however, the differentiation capacity of cryopreserved ASCs relative to fresh cells was significantly reduced. While our findings suggest that future studies are warranted to improve cryopreservation methods and agents, cryopreserved ASCs retain sufficient features to ensure their practical utility for both research and clinical applications.

Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue.

STUDY QUESTION: Can a vitrification protocol using an ethylene glycol/dimethyl sulphoxide-based solution and a cryopin successfully cryopreserve baboon ovarian tissue? SUMMARY ANSWER: Our results show that baboon ovarian tissue can be successfully cryopreserved with our vitrification protocol. WHAT IS KNOWN ALREADY: Non-human primates have already been used as an animal model to test vitrification protocols for human ovarian tissue cryopreservation. STUDY DESIGN, SIZE, DURATION: Ovarian biopsies from five adult baboons were vitrified, warmed and autografted for 5 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. The influence of the vitrification procedure on the cooling rate was evaluated by a computer model. MAIN RESULTS: After vitrification, warming and long-term grafting, follicles were able to grow and maintain their function, as illustrated by Ki67, anti-Müllerian hormone (AMH) and growth differentiation factor-9 (GDF-9) immunostaining. Corpora lutea were also observed, evidencing successful ovulation in all the animals. Stromal tissue quality did not appear to be negatively affected by our cryopreservation procedure, as demonstrated by vascularization and proportions of fibrotic areas, which were similar to those found in fresh ungrafted ovarian tissue. LIMITATIONS, REASONS FOR CAUTION: Despite our promising findings, before applying this technique in a clinical setting, we need to validate it by achieving pregnancies. WIDER IMPLICATIONS OF THE FINDINGS: In addition to encouraging results obtained with our vitrification procedure for non-human ovarian tissue, this study also showed, for the first time, expression of AMH and GDF-9 in ovarian follicles. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4507.10, grant 3.4.590.08 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, and Department of Mechanical Engineering at Louisiana State University (support to Ram Devireddy), and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors has any competing interests to declare.

Methylcellulose based thermally reversible hydrogel system for tissue engineering applications.

The thermoresponsive behavior of a Methylcellulose (MC) polymer was systematically investigated to determine its usability in constructing MC based hydrogel systems in cell sheet engineering applications. Solution-gel analyses were made to study the effects of polymer concentration, molecular weight and dissolved salts on the gelation of three commercially available MCs using differential scanning calorimeter and rheology. For investigation of the hydrogel stability and fluid uptake capacity, swelling and degradation experiments were performed with the hydrogel system exposed to cell culture solutions at incubation temperature for several days. From these experiments, the optimal composition of MC-water-salt that was able to produce stable hydrogels at or above 32 °C, was found to be 12% to 16% of MC (Mol. wt. of 15,000) in water with 0.5× PBS (~150mOsm). This stable hydrogel system was then evaluated for a week for its efficacy to support the adhesion and growth of specific cells in culture; in our case the stromal/stem cells derived from human adipose tissue derived stem cells (ASCs). The results indicated that the addition (evenly spread) of ~200 µL of 2 mg/mL bovine collagen type -I (pH adjusted to 7.5) over the MC hydrogel surface at 37 °C is required to improve the ASC adhesion and proliferation. Upon confluence, a continuous monolayer ASC sheet was formed on the surface of the hydrogel system and an intact cell sheet with preserved cell-cell and cell-extracellular matrix was spontaneously and gradually detached when the grown cell sheet was removed from the incubator and exposed to room temperature (~30 °C) within minutes.

BIOPRESERVATION: HEAT/MASS TRANSFER CHALLENGES AND BIOCHEMICAL/GENETIC ADAPTATIONS IN BIOLOGICAL SYSTEMS.

Biopreservation is the science of extending the shelf life (storage time) of biological systems. The scientific field of biopreservation can be broadly classified into three distinct but interrelated research areas: Cryopreservation (storage by freezing), Desiccation (storage by drying) and Freeze-Drying (storage by freezing first and then sublimating the frozen water). Although, both freeze-frying and desiccation create products that are easier to store and transport, they have not, as yet, been successfully applied to store a variety of biological specimens. However, both these technologies have been quite successfully applied in a variety of fields including pharmaceutical sciences and food industry, as demonstrated by the easy availability of shelf-stable drugs and instant mashed potatoes! On the other hand freezing storage has a long and storied history of being used to transport biological specimen, over long distances, as far back as the time of the Pharaohs. However, the lack of portable refrigeration/freezing techniques (and the inviolate second law) limited the use of cryopreservation in every-day life, until the early 19th century. This short review will outline some of the challenges and opportunities in the fields of engineering, heat and mass transfer, biochemical and genetic adaptations in the preservation of biological systems.

Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions.

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0-0.8) at various cooling rates (0.5-250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q(IIF)) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q(CW)) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.

Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.

Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells could increase the usefulness of these cells in tissue engineering and regenerative medicine. Unfortunately, the use of serum and a commonly used cryoprotectant chemical, dimethyl sulphoxide (DMSO), during cryopreservation storage restricts the direct translation of adult stem cells to in vivo applications. The objective of this study was to test the hypothesis that the stromal vascular fraction (SVF) of adipose tissue can be effectively cryopreserved and stored in liquid nitrogen, using a freezing medium containing high molecular weight polymers, such as methylcellulose (MC) and/or polyvinylpyrollidone (PVP), as the cryoprotective agent (CPA) instead of DMSO. To this end, we investigated the post-freeze/thaw viability and apoptotic behaviour of SVF of adipose tissue frozen in 16 different media: (a) the traditional medium containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% DMSO; (b) DMEM with 80% human serum (HS) and 10% DMSO; (c) DMEM with 0%, 2%, 4%, 6%, 8% or 10% DMSO; (d) DMEM with 1% MC and 10% of either HS or FCS or DMSO; (e) DMEM with 10% PVP and varying concentrations of FCS (0%, 10%, 40% or 80%); (f) DMEM with 10% PVP and 10% HS. Approximately 1 ml (10(6) cells/ml) of SVF cells were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees C water bath (1-2 min agitation), resuspended in culture medium and seeded in separate wells of a six-well plate for a 24 h incubation period at 37 degrees C. After 24 h, the thawed samples were analysed by brightfield microscopy and flow cytometry. The results suggest that the absence of DMSO (and the presence of MC) significantly increases the fraction of apoptotic and/or necrotic SVF cells. However, the percentage of viable cells obtained with 10% PVP and DMEM was comparable with that obtained in freezing medium with DMSO and serum (HS or FCS), i.e. approximately 54 +/- 14% and approximately 63 +/- 10%, respectively. Adipogenic and osteogenic differentiation behaviour of the frozen thawed cells was also assessed, using histochemical staining. Our results suggest that post-thaw SVF cell viability and adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum and DMSO but with 10% PVP in DMEM.

Evaluation of polyvinylpyrrolidone as a cryoprotectant for adipose tissue-derived adult stem cells.

The objective of this study was to test the hypothesis that human adipose tissue-derived adult stem cells (ASCs) can be effectively cryopreserved and stored in liquid nitrogen using a freezing medium containing a high-molecular-weight polymer, polyvinylpyrrolidone (PVP), as the cryoprotective agent (CPA) instead of dimethylsulfoxide (DMSO). To this end we investigated the postfreeze/thaw viability and apoptotic behavior of passage 1 ASCs cryopreserved in 15 different media: (i) the traditional media containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% DMSO; (ii) DMEM with 80% human serum (HS) and 10% DMSO; (iii) DMEM with various concentrations (1%, 5%, 10%, 20%, and 40%) of PVP as the sole CPA; (iv) DMEM with PVP (5%, 10%, and 20%) and HS (10%); (v) DMEM with PVP (5%, 10%, and 20%) and FCS (10%); and (vi) DMEM with PVP (10%) and FCS (40% and 80%). Approximately 1 mL (10(6) cells/mL) of passage 1 ASCs were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees C water bath (1-2 min of agitation), resuspended in culture media, and seeded in separate wells of a six-well plate for a 24-h incubation period at 37 degrees C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 10% PVP and DMEM was comparable with that obtained in freezing media with DMSO and serum (HS or FCS), that is, approximately 70% + or - 8% and approximately 83% + or - 8%, respectively. Slightly enhanced cell viability was observed with the addition of serum (either HS or FCS) to the freezing media containing PVP as the CPA. Adipogenic and osteogenic differentiation behaviors of the frozen thawed cells were also assessed using histochemical staining and optical density measurements and the expression of adipogenic-associated genes was analyzed using reverse transcription-polymerase chain reaction. Our results suggest that after thawing, ASC viability and adipogenic and osteogenic differentiation abilities can be maintained even when ASCs are frozen in the absence of serum but with 10% PVP in DMEM.

Evaluation of methylcellulose and dimethyl sulfoxide as the cryoprotectants in a serum-free freezing media for cryopreservation of adipose-derived adult stem cells.

Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells (ASCs) could increase the usefulness of these cells in tissue engineering and regenerative medicine. To this end, we investigated the post-freeze/thaw viability and apoptotic behavior of Passage 1 (P1) adult stem cells (ASCs) in 11 different media: (i) the traditional media containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO), (ii) DMEM with 80% human serum (HS) and 10% DMSO, (iii) DMEM with 1% methyl cellulose (MC) and 10% of either HS or FCS or DMSO, and (iv) DMEM with 0%, 2%, 4%, 6%, 8%, or 10% DMSO. Approximately 1 mL (10(6) cells/mL) of P1 ASCs were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees C water bath (1-2 min of agitation), resuspended in culture media, and seeded in separate wells of a 6-well plate for a 24-h incubation period at 37 degrees C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO (and the presence of MC) significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 2% DMSO and DMEM was comparable with that obtained in freezing media with 10% DMSO and 80% serum (HS or FCS), that is, approximately 84% +/- 5% and approximately 84% +/- 8%, respectively. Adipogenic and osteogenic differentiation behavior of the frozen thawed cells was also assessed using histochemical staining. Our results suggest that post-thaw ASC viability, adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum but with a minimal concentration of 2% DMSO in DMEM.

Statistical thermodynamics of biomembranes.

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).

Water transport in epididymal and ejaculated rhesus monkey (Macaca mulatta) sperm during freezing.

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.

Cryopreservation characteristics of adipose-derived stem cells: maintenance of differentiation potential and viability.

With the emergence of regenerative medicine, many researchers have turned to fat tissue as a source of adipose-derived stem cells (ASCs). Because freshly collected adipose tissue is not always readily available, there will be a need for improved cryopreservation methods to reproducibly maintain ASC viablility and multipotentiality in long-term storage. This study examines the efficiency of conventional dimethyl sulphoxide cryopreservation methods by measuring the maintenance of differentiation potential after one freeze cycle. Additionally, we analysed the viability of ASCs as a function of varying cell concentrations in cryopreservation media. We evaluated four distinct colony-forming unit assays (fibroblast, alkaline phosphatase, adipocyte and osteoblast) to monitor quantitatively the differentiation potential in ASCs after one freeze cycle. We found that the post-thaw viability was a function of storage concentration and that an optimal viability was observed for a concentration of 0.5 x 10(6) cells/ml cryopreservation medium.

Effect of various freezing parameters on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells.

The effect of four thermal parameters on post-thaw membrane integrity of adipose tissue derived adult stem (ADAS) cells after controlled-rate freezing was investigated with the help of a two-level four-parameter (2(4)) experimental design. The four thermal parameters studied were cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Several passages, including Passage-0 (P0), Passage-1 (P1), Passage-2 (P2), Passage-3 (P3), and Passage-4 (P4), obtained from the suspended culture of stromal vascular fraction (SVF) of the ADAS cells were used for this study. The two levels (low and high) of the four parameters [CR (1 and 40 degrees C/min); ET (-80 and -20 degrees C); HT (1 and 15 min); and TR (10 and 200 degrees C/min)] are chosen in such a way that they enclosed all parameter values possible using commercially available controlled-rate freezing equipment. Individual effect of each parameter on the immediate post-thaw membrane integrity was determined through the calculation of parameter effect values (E), and any synergy among the parameters on post-thaw membrane integrity was assessed through the calculation of two or more parameter interaction effect values (I). Nonlinearity in the experimental results was represented through the calculation of curvature value (CV). The results suggest that for 99% confidence level the parameters CR and ET have considerable effect on post-thaw membrane integrity of all passages of ADAS cells. A significant individual effect of TR was observed with P3 and P4 cells and a significant two-parameter interaction was observed between CR-ET for all passages. These observed results will be used as a basis to further develop freezing storage protocols of ADAS cells.

Transport phenomena during freezing of adipose tissue derived adult stem cells.

In the present study a well-established differential scanning calorimeter (DSC) technique is used to measure the water transport phenomena during freezing of stromal vascular fraction (SVF) and adipose tissue derived adult stem (ADAS) cells at different passages (Passages 0 and 2). Volumetric shrinkage during freezing of adipose derived cells was obtained at a cooling rate of 20 degrees C/min in the presence of extracellular ice and two different, commonly used, cryoprotective agents, CPAs (10% DMSO and 10% Glycerol). The adipose derived cells were modeled as spheres of 50 microm diameter with an osmotically inactive volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the "best-fit" membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined. The "best-fit" membrane permeability parameters for adipose derived cells in the absence and presence of CPAs ranged from: Lpg=23.1-111.5x10(-15) m3/Ns (0.135-0.652 microm/min-atm) and ELp=43.1-168.8 kJ/mol (9.7-40.4 kcal/mol). Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degrees C/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing adipose derived cells in the presence and absence of CPAs.

A simplified procedure to determine the optimal rate of freezing biological systems.

The effect of several cell-level parameters on the predicted optimal cooling rate B(opt) of an arbitrary biological system has been studied using a well-defined water transport model. An extensive investigation of the water transport model revealed three key cell level parameters: reference permeability of the membrane to water L(pg), apparent activation energy E(Lp), and the ratio of the available surface area for water transport to the initial volume of intracellular water (SA/WV). We defined B(opt) as the "highest" cooling rate at which a predefined percent of the initial water volume is trapped inside the cell (values ranging from 5% to 80%) at a predefined end temperature (values ranging from -5 degrees C to -40 degrees C). Irrespective of the choice of the percent of initial water volume trapped and the end temperature, an exact and linear relationship exists between L(pg), SA/WV, and B(opt0. However, a nonlinear and inverse relationship is found between E(Lp) and B(opt). Remarkably, for a variety of biological systems a comparison of the published experimentally determined values of B(opt) agreed quite closely with numerically predicted B(opt) values when the model assumed 5% of initial water is trapped inside the cell at a temperature of -15 degrees C. This close agreement between the experimental and model predicted optimal cooling rates is used to develop a generic optimal cooling rate chart and a generic optimal cooling rate equation that greatly simplifies the prediction of the optimal rate of freezing of biological systems.

An inverse approach to determine solute and solvent permeability parameters in artificial tissues.

This study presents a generic numerical model to simulate the coupled solute and solvent transport in tissue sections during addition and removal of chemical additives or cryoprotective agents (CPA; dimethylsulfoxide or DMSO). Osmotic responses of various tissue cells within the artificial tissue are predicted by the numerical model with three model parameters: Permeability of the tissue cell membrane to water (Lp), permeability of the tissue cell membrane to the solute or CPA (omega), and the diffusion coefficient of the solute or CPA in the extracellular space (D). By fitting the model results with published experimental data on solute/water concentrations at various locations within an artificial tissue, we were able to determine the permeability parameters of artificial tissue cells in the presence of 1.538 M DMSO. Lp and omega were determined at three different locations within the artificial tissue assuming a constant value of solute diffusivity (D = 1.0 x 10(-9) m2/s). The best fit values of Lp ranged from 0.59 x 10(-14) to 4.22 x 10(-14) m3/N-s while omega ranged from 0 to 6.6 x 10(-13) mol/N-s. Based on these values of Lp and omega, the solute reflection coefficient, sigma = 1 - omegav(-)CPA/Lp, ranged from 0.9923 to 1.0. The relative values of omega and sigma suggest that the artificial tissue cells are relatively impermeable to DMSO (or omega approximately 0 and sigma approximately 1.0). This observation was used to modify our model to predict the values of Lp and D assuming omega = 0 and sigma = 1.0. The best fit values of Lp ranged from 640 x 10(-14) to 2.1 x 10(-14) m3/N-s while D ranged from 0.63 x 10(-9) to 1.52 x 10(-9) m2/s. The permeability parameters obtained in the present study represent the first such effort for artificial tissues.

A theoretically estimated optimal cooling rate for the cryopreservation of sperm cells from a live-bearing fish, the green swordtail Xiphophorus helleri.

Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X. helleri sperm cell suspensions was obtained in the presence of extracellular ice at a cooling rate of 20 degrees 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 of 33.3 microm in length and 0.59 microm in diameter with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic or initial cell volume. 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, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 degrees C/min in the absence of CPAs were: L(pg)=0.776 x 10(-15)m3/Ns (0.0046 microm/min atm), and E(Lp)=50.1 kJ/mol (11.97 kcal/mol) (R2=0.997). The corresponding parameters in the presence of 14% glycerol were L(pg)[cpa]=1.063 x 10(-15)m3/Ns (0.0063 microm/min atm), and E(Lp)[cpa]=83.81 kJ/mol (20.04 kcal/mol) (R2=0.997). The parameters in the presence of 10% DMSO were L(pg)[cpa]=1.4 x 10(-15)m3/Ns (0.0083 microm/min atm), and E(Lp)[cpa]=90.96 kJ/mol (21.75 kcal/mol) (R2=0.996). Parameters obtained in this study suggested that the optimal rate of cooling for X. helleri sperm cells in the presence of CPAs ranged from 20 to 35 degrees C/min and were in close agreement with recently published, empirically determined optimal cooling rates.