451 DELIVERING SUSTAINABLE SOLUTIONS TO IMPROVE WELLBEING
against MBNL1 protein (Sigma-Aldrich, St. Louis, MO, USA) in a 5% BSA solution for 2
hours. After the incubation time, cells were washed and incubated with a 1/200 dilution of
the secondary antibody (Alex Fluor 488) in a 5% BSA solution for 1 hour. Finally, cells were
washed three times and stained with a DAPI mount solution for nuclei detection. MBNL1
protein quantification was measured as fluorescence intensity of MBNL1 normalized by
the number of nuclei for each condition using a confocal microscope (Operetta® confocal
microscope, PerkinElmer, Inc., Waltham, MA, USA) with the Alexa Fluor 488 Green
channel (Ex is 460-490 nm/Em is 500-550 nm). Results are expressed as a variation of
relative MBNL1 protein levels (%)with respect to the control condition (without electrical
stimulation) and the electrical stimulated condition.
DETECTION OF EDA-FIBRONECTIN MARKER ON HUMAN DERMAL FIBROBLASTS
Cells coming from a collagen-gel contraction assay (see the following section) were used
to detect an EDA-fibronectin marker. The cells included in that assay were the control
condition (without electrical stimulation), electrical stimulated cells, and tetrapeptide-1
treated cells at 0.01 mg/mL. 3D collagen gels were previously digested using a collagenase
treatment and were fixed on 12-well plates into coverslips. The cells were then washed
twice with PBS (Sigma-Aldrich, St. Louis, MO, USA) and permeabilized with 0.5% Triton
X-100 (Sigma-Aldrich, St. Louis, MO, USA)) for 10 minutes. Finally, cells were washed
three times with PBS again prior to fluorescent labeling of the target protein. Samples were
blocked with 4% BSA (Sigma-Aldrich, St. Louis, MO, USA)) in PBS for 2 hours. After this
step, cells were incubated with an antifibronectin [IST-9] mouse monoclonal antibody at
1:500 (ab6328) (ABCAM) and diluted in 4% BSA in PBS for 2 hours. After the incubation
time, cells were washed again with PBS three times and secondary Alexa Fluor® Thermo
Fisher Scientific, Waltham, MA, USA) 488 goat antimouse IgG (H +L) antibody (1:500
Life Technologies, green fluorescence emission dye) was added and cells were incubated
for 1 hour in the dark. After three washes with PBS, the nuclei of the cells were stained,
and coverslips were mounted with a ProLong Gold antifade reagent with DAPI (Life
Technologies, Carlsbad, CA, USA). The fluorescence intensity signal was determined using
an Operetta® confocal microscope (PerkinElmer, Inc, Waltham, MA, USA) using Alexa
Fluor 488 Green channel (Ex is 460-490 nm/Em is 500-550 nm) and was corrected by the
determining the number of nuclei for each condition. Four independent experiments were
analyzed.
IN VITRO COLLAGEN-GEL CONTRACTION ASSAY
Human dermal fibroblasts were seeded in 60 mm Petri dishes prepared for the electrical
stimulation. After an overnight period to allow cell attachment in the conditions of
treatment, tetrapeptide-1 was added to the culture media at a concentration of 0.01 mg/
mL for 24 hours (in duplicate experiments). The peptide was also maintained in the
culture media for the 3D cultures thus, the total treatment time was 48 hours. Control
experiments (with and without electrical stimulation) were performed in the same way
but without adding the tetrapeptide-1. Therefore, there were three groups in the study:
control without electrical stimulation, control with 1 hour of electrical stimulation, and
the peptide treatment (tetrapeptide-1 at 0.01 mg/mL). Cell suspensions for each of the
conditions to seed were diluted in each corresponding medium with the peptide. The

452 JOURNAL OF COSMETIC SCIENCE
3D cultures were performed according to the following protocol: First, trypsinization
was performed on each of the petri dishes and cell suspensions were prepared. A collagen
solution was then prepared and mixed with cell suspension. 3D hydrogels with the cells in
3D were formed by crosslinking the solution at 37°C for 45 seconds. 3D structures were
separated and detached from the walls of the wells to allow gel contraction. Acellular
controls were also separated, and a warm medium was added on top of each well. Images
were taken from each well 24 hours post detachment to quantify hydrogel contraction.
An initial area was established. At t =0, all the hydrogels presented the conformation
delimited by the culture platform (24-well plate). Hydrogel contraction was quantified as
the deformation computed by surface area variation:
∆Area(at t x) (==-Ax Ai)
Ai
(Eq. 1)
Area t =0 gel → 1.86 cm2 (Area of a well in a 24-well plate)
Quantification of Area at t =x (t =24 h)
The image was analyzed with ImageJ software. The scale was configured based on the
diameter of the well (d =1.54 cm2). The diameter was simulated in ImageJ and configured
at 1.54 units. The perimeter of the hydrogel was surrounded, and the area of the hydrogel
was quantified for each image. The average for the replicates of each condition was then
calculated. Area deformation was calculated based on Eq. 1, and the data was normalized
to an absolute percent.
EVALUATION OF RETINOIC ACID PATHWAY AND SKIN REGENERATION GENES IN HUMAN
DERMAL FIBROBLASTS COCULTURED WITH HUMAN EPIDERMAL KERATINOCYTES BY RT-qPCR
HEKa and human dermal fibroblasts from adults (HDFa) were independently trypsinized
and seeded in 12-well plates. The cells in coculture were incubated at 37°C in 5% CO
2 for 24 hours. After the incubation period, the medium was removed and fresh coculture
medium was added with testing products (S rebaudiana extract at 0.001% or 0.01% and
retinoic acid at 0.001 µg/mL) prepared in the same medium. Cells treated with coculture
medium alone were used as a coculture control. After treatment, cells were incubated for an
additional 24 hours. Each condition was tested in two replicates/wells, and each test item
was assayed in six independent experiments.
For the relative quantification of gene expression levels in the retinoic acid pathway, the
cells were lysed, and the RNA was purified using a specific kit (RNeasy Mini kit) according
to the manufacture’s protocol (Qiagen). Then, RNA elution, quantification, and analysis of
the purity of the RNA samples were performed with a nanodrop Thermo Fisher Scientific,
Waltham, MA, USA). For each sample, 3 µg of high-quality RNA was retrotranscribed
with iScript Advanced (Bio-Rad, Hercules, CA, USA) in a final volume of 20 µL. The
complete reaction mix was incubated in a thermal cycler (Eppendorf, Hamburg, Germany)
at 42°C for 30 minutes, and the reaction was stopped at 85°C for 5 minutes. Complementary
DNA was amplified using qPCR in a real-time PCR thermocycler (Bio-Rad, Hercules, CA,
USA) using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) in the 96-well panel
for use with SYBR® Green (Bio-Rad, Hercules, CA, USA). SYBR Green binds to double-
stranded DNA molecules and emits fluorescence, which is quantified, a process where the