Sok-Siam Gouk

Effective cryopreservation of neural stem or progenitor cells without serum or proteins by vitrification.

Development of effective cryopreservation protocols will be essential to realizing the potential for clinical application of neural stem and progenitor cells. Current cryopreservation protocols have been largely employed in research, which does not require as stringent consideration of viability and sterility. Therefore, these protocols involve the use of serum and protein additives, which can potentially introduce contaminants, and slow cooling with DMSO/glycerol-based cryopreservation solutions, which impairs cell survival. We investigated whether serum- and protein-free vitrification is effective for functional cryopreservation of neurosphere cultures of neural stem or progenitor cells. To protect the samples from introduction of other contaminants during handling and cryostorage, an original “straw-in-straw” method (250 μl sterile straw placed in 500 μl straw) for direct immersion into liquid nitrogen and storing the samples was also introduced. The protocol employed brief step-wise exposure to vitrification solution composed of ethylene glycol (EG) and sucrose (40% v/v EG, 0.6 M sucrose) and removal of vitrification solution at room temperature. Evaluation of the effects of vitrification revealed that there were no differences between control and vitrified neural stem or progenitor cells in expression of the neural stem or progenitor cell markers, proliferation, or multipotent differentiation. This sterile method for the xeno-free cryopreservation of murine neurospheres without animal or human proteins may have the potential to serve as a starting point for the development of cryopreservation protocols for human neural stem and progenitor cells for clinical use.

Aging of a Regenerative Biologic Scaffold (AlloDerm Native Tissue Matrix) During Storage at Elevated Humidity and Temperature

Tissue products will age upon storage. Although a number of tissue products have been used for tissue repair and regeneration for more than a decade, no study has been published on the aging of tissue products and its potential effect on product function. This study investigated aging-caused changes in a regenerative biologic scaffold (AlloDerm native tissue matrix) upon storage at accelerated conditions. Tissue matrix was stored at elevated humidity (33%, 75%, and 85% relative humidity [RH]) and temperature (40 +/- 2 degrees C). The study measured the accumulation of advanced glycation end-products (Maillard products), and the changes of tissue structure, tissue stability, and mechanical properties as well as in vitro fibroblast repopulation. Tissue products stored at 75% RH and 85% RH changed significantly, including collagen condensation and cross-linking, increased breaking strength, and decreased elasticity. The aged products became less stable, as demonstrated by lower denaturation temperature, lower denaturation enthalpy, and higher susceptibility to nonspecific proteolytic action. In comparison, changes were nondetectable in control products stored at 2 degrees C to 8 degrees C, or very small in tissue products stored at 33% RH and 40 degrees C. Changes of tissue structure and stability were correlated highly with the formation of Maillard products, suggesting a role of Maillard reactions in tissue aging during storage. Calorimetric analysis revealed that tissue products stored at 2 degrees C to 8 degrees C and at 33% RH and 40 degrees C were in the glassy state, whereas the products stored at 75% RH, 85% RH, and 40 degrees C were not in the glassy state, suggesting a role of the glassy state in preserving tissue products during storage. Aging did not affect in vitro fibroblast proliferation on tissue matrix, and further tests are needed to investigate how aging may affect in vivo performance of the tissue product.

Vitrification successfully preserves hepatocyte spheroids.

This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (-196°C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 ± 14.39 to 70.38 ± 11.29 μg/106 cells and from 56.84 ± 14.48 to 71.79 ± 16.65 μg/106 cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.

Synchrotron Radiation-Induced Formation and Reactions of Free Radicals in Human Acellular Dermal Matrix

Abstract Gouk, S-S., Kocherginsky, N. M., Kostetski, Y. Y., Moser, H. O., Yang, P., Lim, T-M. and Sun, W. Q. Synchrotron Radiation-Induced Formation and Reactions of Free Radicals in Human Acellular Dermal Matrix. Radiat. Res. 163, 535–543 (2005). The present work characterizes the formation of free radicals in an implantable human acellular dermal tissue (Alloderm®, LifeCell Corp., Branchburg, NJ) upon irradiation. The tissue was preserved in a vitreous carbohydrate matrix by freeze-drying. Freeze-dried samples were irradiated using a synchrotron light source, and free radicals generated were investigated using the electron paramagnetic resonance (EPR) technique. At least two free radical populations, with g factors of 1.993 (∼43%) and 2.002 (∼57%), respectively, were identified in the irradiated tissue. The transformation (reaction) kinetics of free radicals produced was investigated in the presence of nitrogen, oxygen and moisture. The reaction kinetics of free radicals was extremely slow in the nitrogen environment. The presence of oxygen and moisture greatly accelerated free radical reactions in the tissue matrix. The reaction of free radicals could not be described by traditional reaction kinetics. A dispersive kinetics model and a diffusion model were developed to analyze the reaction kinetics in the present study. The dispersive model took into consideration molecular mobility and dispersivity of free radicals in the heterogeneous tissue material. The diffusion model described the radical reaction kinetics as two parallel and simultaneous processes: a first-order fast kinetics mainly on tissue surface and a diffusion-limited slow kinetics in deeper layers of the tissue matrix. Both models described quantitative experimental data well. Further investigation is needed to verify whether any of these two models or concepts describes the inherent radical reaction kinetics in the solid tissue matrix.