The Toxicologist - Society of Toxicology

2553 AN ORGANOTYPIC MICROLIVER PLATFORM FOR
HIGH-THROUGHPUT DRUG TESTING.
S. Messner 1 , B. Machi 1 , S. Hammad 2 , J. M. Kelm 1 , J. G. Hengstler 2 and W.
Moritz 1 . 1 InSphero AG, Zürich, Zürich, Switzerland and 2 IfADo Dortmund,
Technical University of Dortmund, Dortmund, Germany. Sponsor: C. Corton.
Predictive toxicology testing is a major challenge in regulatory toxicology and drug
discovery. To avoid extensive use of animal experiments, primary hepatocytes are
commonly used to study drug metabolism, enzyme induction and compound hepatoxicity
in vitro. However, in monolayer cultures, dedifferentiation processes lead
to a severe loss of liver-specific functions. While 2D sandwich cultures improve
maintenance of hepatocyte function, these systems are technically challenging, of
little use for chronic or idiosyncratic toxicity testing and only fairly suited for
higher throughput. In order to preserve liver-specific functions of hepatocytes over
extended time periods and to provide a versatile platform for toxicology testing, we
developed a novel scaffold-free, organotypic production technology for a 3-dimensional
culture of hepatic microtissues in a standard 96-well microtiter plate format.
This format enables regular medium exchange and compound supplementation in
hanging drops. Microtissues from primary rat or human hepatocytes are produced
with the new GravityPlus production technology by top- loading of a cell suspension
at a density of 2000 hepatocytes per well. Inside the hanging drop, rapid microtissue
formation takes place within 3-4 days. The GravityPlus system allows the
production of regular-sized microtissues with a morphology and architecture close
to native liver tissue, in particular when co-cultured with non-parenchymal liver
cells. The metabolic activity and liver-specific parameters (e.g. CYP450 induction,
albumin and urea secretion) are consistently superior to hepatocyte sandwich culture,
especially at culture periods of more than 7 days. Our results demonstrate the
validity and suitability of the GravityPlus hanging drop platform for liver microtissue
culture, offering a highly functional and reliable in vitro toxicology testing system
which can easily be implemented in standard lab automation processes.
2554 QUANTIFICATION OF UVA-INDUCED REACTIVE
OXYGEN SPECIES IN PRIMARY HUMAN
KERATINOCYTES TREATED WITH ANTIOXIDANTS.
K. Norman, H. Inglis and G. Mun. Institute for in vitro Sciences, Gaithersburg,
MD. Sponsor: E. Dahl.
Environmental exposure of the skin to ultraviolet radiation makes it a primary target
for UVA-generated reactive oxygen species (ROS). An overabundance of ROS
causes oxidative stress which may result in the oxidation of proteins, lipids, and nucleic
acids. This oxidative damage is a major contributor to photoaging and is associated
with skin carcinogenesis. In order to mitigate the damaging effects of ROS,
antioxidants are increasingly being added to formulations of topical products. The
goal of this study was to develop an in vitro method capable of evaluating the antioxidant
potential of ingredients and formulations in primary human keratinocytes
(NHEKs). NHEK cells were seeded in 96-well plates and incubated with
the fluorescent ROS-detecting probe, 5 (and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein
diacetate acetyl ester (CM-H2DCFDA), for 40 minutes, then exposed
to a serial dilution of antioxidants/antioxidant-containing formulations for 1
hour. ROS were generated by exposing the cultures to UVA light for 50 minutes
(1.6-1.8 mW/cm2), and then detected by fluorescence measurements (ex/em
485/530 nm) of the cells. Cytotoxicity was assessed concurrently using the neutral
red uptake assay. Using this method, we evaluated several known antioxidants and
antioxidant-containing formulations for their ROS-reducing capabilities. Based on
our results, several antioxidants significantly reduced ROS levels compared to untreated
controls. Other antioxidants which did not show this reduction were likely
not water soluble and/or poorly bioavailable to cells. Overall, our results indicate
that this method may provide a valuable in vitro tool for assessing antioxidant capacity
in a biologically relevant model.
2555 IN VITRO ASSESSMENT OF SKIN IRRITATION
POTENTIAL OF SURFACTANT BASED
FORMULATIONS USING 3-D SKIN RECONSTRUCTED
TISSUES AND CYTOKINE EXPRESSION ANALYSIS.
L. Gandolfi 1 , N. Tierney 1 , D. Johnson 1 , R. Walters 1 , M. Fevola 1 , E. Gunn 1 , K.
Martin 1 , A. Kong 2 , A. Hilberer 2 , N. Barnes 2 , N. Wilt 2 , J. R. Nash 2 , H. Inglis 2 ,
H. Raabe 2 and G. Costin 2 . 1 Johnson & Johnson, Skillman, NJ and 2 Institute for in
vitro Sciences, Inc., Gaithersburg, MD.
A goal of personal care products manufacturers is to develop increasingly milder
formulations. To this end, reproducible in vitro systems can accurately assess the irritation
potential of the products, thus avoiding the use of animals for testing. The
three-dimensional EpiDerm TM model (MatTek Corp.) provides a testing platform
for Johnson & Johnson’s skin irritation assessment program targeting raw ingredients
and final formulations. The testing program we have developed evaluated the
potential dermal irritation of over 150 amphoteric and/or anionic surfactant-containing
candidate formulations or individual raw ingredients. The formulations
were diluted to 10% in water and were applied onto the surface of the 3-D tissues
for 1 h, followed by 24 h post-exposure for cytokine expression. The potential dermal
irritation of the candidates was evaluated by MTT viability and IL-1α release.
Another goal of the program was to qualify two representative benchmark materials
with known skin irritation potential for use as references for the skin irritation evaluation
of formulations with new surfactant ingredients. We have developed a database
and established a range of irritation responses of the benchmarks using the IL-
1α endpoint. Comparison of the potential dermal irritation of new formulations to
existing mild formulations guides formulation development for new mild cleansing
products. Most recently, we have demonstrated the reliability of the test system for
the assessment of irritation potential of final formulations. We are currently expanding
our database and work towards establishing a correlation between the in
vitro pre-screening approach and clinical testing. This testing platform integrates
the efforts made to meet the mildness testing needs of global manufacturers of personal
care products that focus on developing increasingly milder lines of formulations
to be applied to the skin.
2556 USE OF EPIDERM FULL THICKNESS (EFT) SKIN
CULTURES AS AN IN VITRO MODEL FOR WOUND
HEALING.
P. Hayden 2 , M. Sachdeva 1 , P. P. Shah 1 , U. S. Desai 1 , R. Patlolla 1 and M.
Klausner 2 . 1 Pharmacy, Florida A&M University, Tallahassee, FL and 2 MatTek
Corporation, Ashland, MA.
Background: Wound healing is a natural process which involves the regeneration of
epidermal and dermal tissue with expression of growth factors and inflammatory
markers. The objective of this study was to evaluate the EpiDerm full thickness
(EpiDerm-FT, EFT) skin culture as an in-vitro wound healing model to understand
the healing process after chemical induced wounding.
Methods: A strong base, 32N KOH and 2 strong acids, conc H2SO4 and acrylic
acid, were selected for making chemical wounds. The dose of chemical and exposure
time required to generate a significant wound were optimized. After inducing
a wound, cultures were washed thoroughly with PBS and incubated at 37°C for 6
days. The EFT tissues were collected for histological, immunohistochemistry and
western blot studies. The culture medium was collected to study the release of inflammatory
markers such as Interleukin-6 (IL-6) and Interleukin-8 (IL-8).
Results: Hematoxylin and eosin staining showed significant damage to the stratum
corneum and upper layers of the epidermis for 32N KOH, deeper epidermal damage
for conc H2SO4 and dermal damage for acrylic acid. Western blot studies
showed that the ratio of collagen IV to β-actin was increased in the order of acrylic
acid > conc H2SO4 > 32N KOH while the ratio of KI67 to β-actin was increased
in the order of conc H2SO4 > acrylic acid > 32N KOH. Similar results were observed
with immunohistochemistry studies suggesting a strong correlation between
the two techniques. These data were further supported by cytokine studies which
showed that the release of IL-6 and IL-8 was significantly increased (p