Ram V. Devireddy

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 Dulbeccos modified Eagles 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.

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 (24) 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 °C/min); ET (–80 and –20 °C); HT (1 and 15 min); and TR (10 and 200 °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.

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.

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 Dulbeccos modified Eagles 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 (106 cells/ml) of SVF cells were frozen overnight in a −80 °C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 °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 °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. ∼54 ± 14% and ∼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. Copyright

Fabrication and characterization of electrodeposited antimony telluride crystalline nanowires and nanotubes

Arrays of nanowires and nanotubes of antimony-telluride (Sb2Te3) have been fabricated by an electrodeposition technique. Scanning electron microscopy was employed to characterize the morphology and size of the fabricated Sb2Te3 nanowires and nanotubes. Wavelength dispersive spectroscopy analysis confirmed the composition of the fabricated nanowires and nanotubes. The composition of the nanowires fabricated at a cathodic current density of 10 mA cm−2 and nanotubes fabricated at a cathodic current density of 5.5 mA cm−2 was found to be ∼39% Sb and ∼61% Te (2 : 3 ratio between Sb and Te). The fabricated Sb2Te3 nanowire and nanotube arrays were found to be polycrystalline with no preferred orientation. The average lamellar thickness of the nanowires and nanotube crystallites was determined using the Scherrer equation and found to be ∼36 nm and ∼43 nm, respectively. The measured room temperature Seebeck coefficients for the Sb2Te3 nanowires and nanotubes were +359 µV K−1 and +332 µV K−1, respectively, confirming that the Sb2Te3 nanowires and nanotubes were p-type. The electrical resistance measurements indicated that the resistance of the Sb2Te3 nanowires and nanotubes decreased with increasing temperature, consistent with semiconducting behavior.

Cryopreservation of Collagen-Based Tissue Equivalents. I. Effect of Freezing in the Absence of Cryoprotective Agents

The effect of freezing on the viability and mechanical properties of tissue-equivalents (TEs) was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. TEs (a class of bioartificial tissues) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. TEs were detached from the substrate and frozen in phosphate-buffered saline using a controlled rate freezer (CRF) at various cooling rates (0.5, 2, 5, 20, and 40 degrees C/min to -80 or -160 degrees C) or in a directional solidification stage (DSS) (5 degrees C/min to -80 degrees C) or slam frozen (>1000 degrees C/min). Viability of the fibroblasts in the TEs was assessed by ethidium homodimer and Hoechst assays immediately after thawing. Uniaxial tension experiments were also performed on an MTS (Eden Prairie, MN) Micro Bionix system to assess the postthaw mechanical properties of the frozen-thawed TEs. Cooling rates of either 2 or 5 degrees C/min using the CRF were optimal for preserving both immediate cell viability and mechanical properties of the TEs, postthaw. By 72 h postthaw, TEs frozen in the CRF at 5 degrees C/min to -80 degrees C showed a slight decrease in cell viability, with a significant increase in tangent modulus and ultimate tensile stress suggesting a cell-mediated recovery mechanism. Both the postthaw mechanical properties and cell viability are adversely affected by freezing to the lower end temperature of -160 degrees C. Mechanical properties are adversely affected by freezing in the DSS.

Cellular Response of Adipose Derived Passage-4 Adult Stem Cells to Freezing Stress

A differential scanning calorimeter technique was used to generate experimental data for volumetric shrinkage during cooling at 20 degrees C/min in adipose derived adult stem cells (ASCs) in the presence and absence of cryoprotective agents (CPAs). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the membrane permeability parameters of ASCs were obtained. For passage-4 (P4) ASCs, the reference hydraulic conductivity Lpg and the value of the apparent activation energy ELP were determined to be 1.2 X 10(-13) m3/Ns and 177.8 kJ/mole, respectively. We found that the addition of either glycerol or dimethylsulfoxide (DMSO) significantly decreased the value of the reference hydraulic conductivity Lpg(cpa) and the value of the apparent activation energy ELp(cpa) in P4 ASCs. The values of Lpg(cpa) in the presence of glycerol and DMSO were determined as 0.39 x 10(-13) and 0.50 X 109-13) m3/Ns, respectively, while the corresponding values of ELp(cpa) were 51.0 and 61.5 kJ/mole. Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degreesC/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing P4 adipose derived cells in the presence and absence of CPAs.

Variation in the Membrane Transport Properties and Predicted Optimal Rates of Freezing for Spermatozoa of Diploid and Tetraploid Pacific Oyster, Crassostrea gigas

Abstract In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20°C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment “ball-on-stick” model with a “ball” 1.66 μm in diameter and a “stick” 41 μm in length and 0.14 μm wide. Similarly, sperm cells of tetraploid oysters were modeled with a “ball” 2.14 μm in diameter and a “stick” 53 μm in length and 0.17 μm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20°C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 × 10−15 m3/Ns (0.0017 μm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 × 10−15 m3/Ns (0.0015 μm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20°C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 × 10−15 m3/Ns (0.0019 μm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 × 10−15 m3/Ns (0.0019 μm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70°C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas.

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.

Molecular dynamics simulation of pore growth in lipid bilayer membranes in the presence of edge-active agents

Using molecular dynamics simulations, we investigated the mechanism of entropy-driven pore formation in a phospholipid bilayer in the presence of a line tension lowering agent. Even in the absence of an external stress, our simulations show that it is possible to observe with atomistic detail and on nanosecond time scale the nucleation and growth of hydrophilic pores. We rationalize the nucleation process in terms of a simplified free energy model that includes the entropy of the pore shape. By estimating the line tensions within the lipid bilayers with and without edge-active agents, our simulations corroborate the pore growth model.