Sreedhar Thirumala

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.

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

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.

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.

Freezing and post-thaw apoptotic behaviour of cells in the presence of palmitoyl nanogold particles

The aim of this study was to evaluate the freezing response of HeLa and Jurkat cells in the presence of commercially available nanoparticles, NPs (Palmitoyl Nanogold®, Nanoprobes). The cells were incubated with NPs for either 5 min or 3 h, and a calorimeter technique was then used to generate the volumetric shrinkage response during freezing at 20 °C min−1. Concomitantly, we also examined the effect of a commonly used cryoprotectant, dimethylsulfoxide, DMSO (10% v/v ratio) on the freezing response of HeLa and Jurkat cells. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the reference hydraulic conductivity, Lpg, (μm/min-atm) and activation energy, ELp, (kcal mol−1) were obtained. For HeLa cells, the values of Lpg ranged from 0.08 to 0.23 µm/min-atm, while ELp ranged from 10.9 to 37.4 kcal mol−1. For Jurkat cells these parameter values ranged from 0.05 to 0.16 µm/min-atm and 9.5 to 35.9 kcal mol−1. A generic optimal cooling rate equation was then used to predict the optimal rates of freezing HeLa and Jurkat cells in the presence and absence of DMSO and NPs. The post-thaw viability and apoptotic response of HeLa and Jurkat cells was further investigated by cooling cells at three rates in the presence and absence of DMSO and NPs using a commercially available controlled rate freezer. Jurkat cells treated in this manner demonstrated an increase in their adhesive properties after 18 h incubation and adhered strongly to the bottom of the culture plate. This observation prevented further analysis of Jurkat apoptotic and necrotic post-thaw responses. There was no significant effect of NPs or DMSO alone on HeLa cell viability prior to freezing. The post-thaw results from HeLa cells show that the NPs increased the measured post-freeze apoptotic response when cooled at 1 °C min−1, suggesting a possible therapeutic use of NPs in cryodestructive procedures.

Preservation protocols for human adipose tissue-derived adult stem cells.

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. There are three primary storage techniques, freezing (cryopreservation), drying (anhydrobiosis), and freeze drying (lyophilization), each with its own advantages and disadvantages. 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. Preservation by desiccation is an alternative that attempts to reproduce a naturally occurring preservative technique, namely, the phenomenon of anhydrobiosis and requires the use of high (and possibly, toxic) concentration of CPAs as well as disaccharides (sugars). Lyophilization works by first cryopreserving (freezing) the material and then desiccating (drying) it by the process of sublimation or the conversion of ice (solid) to water vapor (gas phase). The purpose of this chapter is to present a general overview of these storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage and drying storage.

Thermally Reversible Hydrogel Sheets for Adult Stem Cell Culture

A simple non-invasive method of cell retrieval using Methylcellulose (MC) coated Tissue Culture Poly-Styrone (TCPS) dishes was developed to allow the creation of single and multilayered adult stem cell sheet constructs while preserving the cell-cell and the cell–extracellular matrices. The optimal combination of MC-water-salt was found to be 12 to 14% of MC (mol. wt. of 15,000) in water with 0.5x PBS (∼150 mOsm). This solution exhibited a gel formation temperature of ∼32°C. The addition (evenly spread) of 200μl of 2mg/ml bovine collagen type -I (pH adjusted to 7.5) over the MC coated surface at 37°C, significantly improved the adult stem cell (ASC) adhesion and proliferation on the hydrogel system.Copyright

Apoptotic Response and Differentiation Ability of Adipose Derived Stem Cells (ASCs) Frozen/Thawed in the Presence of Polyvinylpyrrolidone (PVP)

Effective techniques for the cryopreservation of human Adipose Stem Cells (ASCs) could increase the usefulness of these cells in tissue engineering and regenerative medicine. The traditional method of using fetal calf serum (FCS) and a chemical cryoprotectant like dimethylsulfoxide (DMSO) during freezing storage restricts the direct use of ASCs in regenerative medicine and reconstruction surgery. The objective of this study was to investigate the apoptotic, necrotic and viability response of ASCs frozen/thawed in the presence of a high molecular weight polymer, Polyvinylpyrrolidone (PVP) and in the absence of FCS. Post-freeze/thaw studies were also conducted to evaluate the effect of PVP on the in vitro osteogenic and adipogenic differentiation of ASCs. The results were compared with those obtained using the most commonly used cryopreservation media of 10%DMSO + 10% Dulbecco’s Modified Eagle Media (DMEM) + 80%FCS.Copyright