Page 234 - IJB-10-4

International Journal of Bioprinting N-PLN hydrogels for human skin modeling

incubated at 37°C and 5% CO . The culture medium was the first 7 days post-HaCaT seeding, all the constructs
2
changed every 2 days, and the cells were passaged twice were kept under submerged conditions, which mean that
a week. To fabricate cell-laden scaffolds, Hs-27 cells (5 × the culture medium was added both in the basolateral
10 cells/mL) were first trypsinized and then resuspended and the apical compartments to fully surround and
6
into the freshly prepared pre-gel solutions. For cell culture, cover the constructs. Later, ALI culture conditions were
hydrogels (5 mm × 1 mm × 0.5 mm) were printed on applied to some of the scaffolds, by keeping the medium
top of silanized PET membranes of 0.4 µm pore size and in the basolateral compartment and leaving the apical one
10 mm diameter (it4ip). Then, they were washed with exposed to the air.
warm cell culture medium supplemented with normocin
(1:500; InvivoGen, CA, USA), mounted within Transwell® 2.6.2. Immunofluorescence analysis
inserts (Corning®, NY, USA) through double-sided The cell behavior within the hydrogels and on top of them
pressure-sensitive adhesive rings, and kept in submerged was characterized by immunostaining. Scaffolds were fixed
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conditions at 37°C and 5% CO . The medium was replaced with 10% neutral buffer formalin solution (Sigma-Aldrich)
2
every 24 h. for 30 min at room temperature in shaking conditions, and
then permeabilized with 0.5% Triton-X (Sigma-Aldrich)
2.5.1. Cellular viability for 1 h at 4°C. After permeabilization, they were incubated
Cell viability assays were conducted on hydrogel scaffolds for 2 h at room temperature in shaking conditions, using
with Hs-27 embedded at days 1, 3–4, and 7 after the a blocking buffer solution composed of 1% bovine serum
encapsulation, for both formulations, using the calcein- albumin (Sigma-Aldrich), 3% donkey serum (Millipore),
AM/ethidium-homodimer Live/Dead kit (Invitrogen, and 0.2% Triton-X. For the samples containing only
MA, USA). The gels were incubated with the reagents at fibroblasts, hydrogels were incubated first with primary
37°C for 20 min, prior to microscopic imaging. During antibodies against vimentin (1:100; sc-6260, Santa Cruz,
the incubation, the gels were maintained in the 24-well TX, USA), fibronectin (1:200; F3648, Sigma Aldrich, MA,
plates covered by aluminum foil, to protect them from USA), laminin (1:200; ab11575, Abcam, UK), collagen
light. The cell viability was monitored by a confocal laser IV (1:250; 134001, Biorad, CA, USA), and Ki67 (1:100;
scanning microscope (LSM 800, Zeiss, Germany), and the ab16667, Abcam) overnight at 4°C, and then with anti-
percentage of viable cells was determined manually using mouse Alexa Fluor 568, anti-goat Alexa Fluor 647, and
ImageJ software (http://imagej.nih.gov/ij, NIH). anti-rabbit Alexa Fluor 488 (at 4 μg/mL each; Invitrogen,
2.6. Fabrication and characterization ThermoFisher) as secondary antibodies for 2 h at room
of human skin-like constructs based on temperature. DAPI (Thermo Fisher Scientific) was added
norbornene-pullulan hydrogels to stain the nuclei. For the samples containing fibroblasts
and keratinocytes, E-cadherin primary antibody (1:1000;
2.6.1. Fabrication of 3D human skin-like constructs 610181, BD Biosciences, USA) was used to better visualize
Human skin-like constructs were fabricated by seeding the HaCaT cells on top of the hydrogels, and keratin 14
HaCaT human keratinocytes (CLS 300493, CLS Cell Lines primary antibody (1:400; 905301, Biolegend, CA, USA)
Service GmbH, Germany), on top of the dermal structure was used to identify keratin 14, a specifical marker of the
previously generated 3 days after the encapsulation of basal layer. After incubation, all the samples were flipped
Hs-27 cells. HaCaT cells were kept in culture in 25 cm down onto a glass coverslip and mounted using a drop
2
flasks in DMEM (Gibco, Thermo Fischer Scientific), of Fluoromount-G® (Southern Biotech, AL, USA) to
supplemented with 10% v/v FBS (Gibco, Thermo Fischer prevent sample dryness. To avoid sample damage, PDMS
Scientific) and 1% v/v penicillin/streptomycin (Sigma- spacers (500 µm) were used. Z-stack images were collected
Aldrich). The cells were kept in incubation at 37°C and using a confocal laser-scanning microscope (LSM 800,
5% CO . The medium was changed every 2 days, and Zeiss) and subsequently processed using ImageJ software
2
the cells were passaged twice a week. To create the co- (http://imagej.nih.gov/ij, NIH).
cultures, HaCaT cells were seeded at a density of 6 × 10
6
cells/cm on top of fibroblast-laden N-PLN rectangular- 2.6.3. Transepithelial electrical resistance
2
shaped hydrogels (5 mm in length, 1 mm in width, and To evaluate the integrity of the barrier formed by the
0.5 mm in height) previously mounted in Transwell® keratinocytes on top of the fibroblasts-laden hydrogels,
inserts. The cell culture medium employed for the co- both in submerged and under air-liquid interface (ALI)
cultures was the same used for the maintenance of culturing conditions, the transepithelial electrical resistance
HaCaT cells added with normocin (1:500; InvivoGen) (TEER) was monitored for 21 days, starting just before the
to prevent contamination, and it was exchanged every 2 HaCaT seeding. TEER values were also collected on control
days. The co-cultures were maintained for 21 days. For samples (HaCaT only cultured on top of hard porous

Volume 10 Issue 4 (2024) 226 doi: 10.36922/ijb.3395