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

Regarding the epidermal compartment, HaCaT cells cells. 18,59,60 No differences in TEER values were found for
fully covered the top surface of the hydrogels, as observed samples cultured under submerged or ALI conditions.
with the E-cadherin signal, and no visible differences in Remarkably, no visible shrinkage and/or gels’ degradation
terms of cell morphology and compactness were found were detected even at long-term cultures, making the
between both the culture conditions. N-PLN formulation well-suited for standard Transwell®
The presence of laminin was detected within the cells inserts, where TEER and/or permeability tests can be
and at the interface between the epidermal and the dermal performed in a routine manner.
compartments. Also, at this interface, fibroblasts were seen These data indicated that ALI culture condition
elongated and oriented perpendicularly to the surface of might be advantageous for long culture periods. Given
the epithelial monolayer, mainly in the case of ALI culture this, we proceeded to evaluate the specific benefits of
(cell nuclei forming an angle between 60° < θ < 120° with ALI culture conditions for both the epithelial and the
the surface, seen in Figure 5a and b), thus evidencing the dermal compartments by quantifying specific markers. In
close interaction between cells of both compartments. Also, the epithelial compartment, the levels of keratin 14 and
ALI culture conditions appeared as slightly enhancing E-cadherin markers under ALI culture condition did not
E-cadherin signal (Figure 5c), which can be correlated show statistically significant differences compared to those
with the keratin 14 signal (Figure 4c), and enhancing the under submerged condition (Figure 6b).
vimentin and laminin signals, which were 2.6- and 2.1-fold However, the images of the samples’ cross-sections
higher, respectively (Figure 5c).
show varied spatial distribution of markers. Keratinocytes
Subsequently, we assessed the barrier function of the did not form a continuous monolayer in submerged
epithelial monolayer formed by measuring the TEER along condition, whereas marker signals for fibronectin and
culture time (Figure 5d). Samples with HaCaT cells seeded collagen IV appeared denser and more homogeneous in
on hard porous membranes were used as controls. TEER samples cultured under ALI condition. Moreover, a better
values measured for the hydrogel samples were lower than interconnection between the epithelial monolayer and the
values measured in controls (~2.5 Ω·cm vs. ~10 Ω·cm ) dermis was observed in samples under ALI conditions
2
2
but in line with other studies that also employed HaCaT (bottom panel of Figure 6a).

Figure 6. Effect of the co-culture conditions on the epidermal compartment in hydrogels based on Formulation 2. (a) Immunofluorescence staining of
main epidermal markers in the cross-sections of constructs for both submerged (top panel) and ALI (lower panel) culture conditions 21 days post-HaCaT
seeding: E-cadherin (magenta), and keratin 14 (yellow). The stained F-actin (green) indicated both the fibroblast network in the dermal compartment
and the keratinocytes. Nuclei were stained in blue. Dashed lines refer to the PET membranes (hydrogels’ support). Scale bars = 100 µm. (b) Quantification
of the expression of the different markers for both submerged and ALI conditions. Normalized values are expressed as mean ± SD (n = 3). *p = 0.0381.

Volume 10 Issue 4 (2024) 232 doi: 10.36922/ijb.3395