Development of a method for assessing non-targeted radiation damage in an artificial 3D human skin model

Abstract

Purpose Despite the increasing concern about the effect of doses below 0.5 Gy and non-targeted exposures of ionising radiation on living organisms, the majority of radiobiological studies are conducted using in vitro cell lines. In order to be able to extrapolate the in vitro results to in vivo models with confidence, it would be of great benefit to develop a reproducible tissue system suitable for critical radiobiological assays. This manuscript describes the development of a reliable protocol to harvest cells from tissue samples and investigate the radiation damage induced on a single cell basis. Materials and Methods To validate this approach as a potential tool for bystander experiments, the method focuses on analysing radiation damage in individual cells as a function of their relative position in the tissue. The experiments reported describe the micronucleus formation following partial irradiation with 3.5 MeV protons (0.1, 0.5 and 1 Gy) in an artificial human skin construct. Results The reproducible and low background frequency of micronuclei measured in this system allows detection of small increases following radiation exposures. The effect was statistically significant at doses as low as 0.1 Gy in the directly irradiated as well as in the bystander cells. Conclusions The data presented provide evidence of a spatially dependent bystander effect whose magnitude decrease as a function of the distance from the directly exposed area.

DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells

“…Previous reports showed 3D skin organoids increasing in micronuclei and specific gene profiles after IR exposure. 27,28 The skin barrier function of our derived keratinocytes was confirmed with caffeine penetration assay and because they showed DDR response after IR. We believe that this IR-induced cellular senescence of keratinocytes and 3D organoid are useful models of IR-associated skin disease.…”

“…22 Furthermore, to analyze reproducible tissue function, 3-dimensional (3D) organoid models using primary keratinocytes and iPSC-derived keratinocytes have been developed. 23,27,28 Proton irradiation exposure to the 3D skin organoid showed micronuclei formation and alternation of gene profiles. These reports suggest that derived keratinocytes are useful tools for the analysis and treatment of severe diseases.…”

The production and composition of rat sebum is unaffected by 3 Gy gamma radiation

“…Similarly, radiotherapy patients receiving 2 Gy fractions to the chest up to 46 Gy showed a decline in skin conductance, as a measure of sweat gland function, in relation to administered dose (Pigott et al 2000). In a 3-D human skin model, micronuclei could be detected in single cells from doses as low as 0.1 Gy of 3.5 MeV protons (Schettino et al 2010). What is clear is that the skin is an organ sensitive to ionising radiation.…”

Bystander Effects Induced by the Monolayer and Three Dimensional Cultures Exposed to Ionizing Radiation

“…They found that the increase of micronuclei in the unirradiated area which were distant from the irradiated area in the tissue. Schettino et al observed the increase of micronuclei in the unirradiated area of the artificial human skin construct which was partially irradiated with protons [18].…”