Freezing Weakens the Barrier Function of Reconstructed Human Epidermis as Evidenced by Raman Spectroscopy and Percutaneous Permeation

The development and characterization of reconstructed human epidermis (RHE) is an active area of R&D. RHE can replace animal tissues in pharmaceutical, toxicological and cosmetic sciences, yielding scientific and ethical advantages. RHEs remain costly, however, due to consumables and time required for their culture and a short shelf-life. Storing, i.e., freezing RHE could help reduce costs but to date, little is known on the effects of freezing on the barrier function of RHE. We studied such effects using commercial EpiSkin™ RHE stored at −20, −80 and −150 °C for 1 and 10 weeks. We acquired intrinsic Raman spectra in the stratum corneum (SC) of the RHEs as well as spectra obtained following topical application of resorcinol in an aqueous solution. In parallel, we quantified the effects of freezing on the permeation kinetics of resorcinol from time-dependent permeation experiments. Principal component analyses discriminated the intrinsic SC spectra and the spectra of resorcinol-containing RHEs, in each case on the basis of the freezing conditions. Permeation of resorcinol through the frozen RHE increased 3- to 6-fold compared to fresh RHE, with the strongest effect obtained from freezing at −20 °C for 10 weeks. Due to the extensive optimization and standardization of EpiSkin™ RHE, the effects observed in our work may be expected to be more pronounced with other RHEs.

Spectroscopic monitoring of transport processes during loading of ovarian tissue with cryoprotective solutions

There is an increasing demand for female fertility preservation. Cryopreservation of ovarian cortex tissue by means of vitrification can be done ad-hoc and for pre-pubertal individuals. Obtaining a homogeneous distribution of protective agents in tissues is one of the major hurdles for successful preservation. Therefore, to rationally design vitrification strategies for tissues, it is needed to determine permeation kinetics of cryoprotective agents; to ensure homogeneous distribution while minimizing exposure time and toxicity effects. In this study, Fourier transform infrared spectroscopy (FTIR) was used to monitor diffusion of different components into porcine ovarian cortex tissue. Water fluxes and permeation kinetics of dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG) were investigated. Diffusion coefficients derived from FTIR data, were corroborated with differential scanning calorimetry and osmometer measurements. FTIR allowed real-time spectral fingerprinting of tissue during loading with mixtures of protective agents, while discriminating between different components and water. Exposure to vitrification solutions was found to cause drastic initial weight losses, which could be correlated with spectral features. Use of heavy water allowed distinguishing water fluxes associated with dehydration and permeation, both of which were found to precede permeation of cryoprotective agents. Overall, DMSO and EG were found to permeate faster than GLY and PG. In mixtures, however, solutes behave differently. The non-invasive spectroscopic method described here to study permeation of vitrification solution components into ovarian tissue can be applied to many other types of engineered constructs, tissues, and possibly organs.