Page 488 - IJB-10-2

International Journal of Bioprinting Bioprinted skin for testing of therapeutics

Figure 2. Development of skin equivalent models. (A, B) 96-well Alvetex scaffold system; black arrows show inserts, blue arrows porous membrane. (C)
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200 µm thick Alvetex porous membrane. (D–G) Stages in model development. (D) Schematic of scaffold insert. (E) Dermal incubation of fibroblasts only
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for up to 28 days. (F) Basal incubation of keratinocytes for 3 days. (G) Incubation of model at air–liquid interface for 14 days. Panels D–G were created
using Biorender.com.
skin equivalents. Prior to printing of cells, the 96-well or further cultured with autologous immune cells and
Alvetex® (Reprocell, UK) scaffolds were washed in 70% monoclonal antibodies.
ethanol and rinsed in two changes of PBS. A fibroblast cell
suspension was prepared in Media A at a concentration 2.5. Histological evaluation of skin equivalents
of 25 × 10 cells/mL. The Suspension (20 µL) was directly Mature skin equivalents were harvested, formalin-fixed,
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printed onto the Alvetex scaffolds within the Transwells paraffin-embedded, and sectioned. Sections (4 μm) were
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to create dermal equivalents, which were then incubated stained with hematoxylin and eosin (H&E; Fisher Scientific,
at standard culture conditions for 3 h. Following the 3-h UK) and Picrosirius red (Abcam, UK). Skin equivalents
were analyzed for specific biomarkers, cytokeratin,
incubation, the cell-laden scaffolds were submerged in involucrin, and loricrin via immunofluorescence. Sections
Media A overnight. After 24 h, the scaffolds were cultured were taken to water, washed in pH 7.4 tris-buffered saline
using Media A supplemented with 100 µg/mL of L-ascorbic (TBS), and antigen retrieval was conducted using pH 6
acid. The dermal equivalents were cultured for 18 days with citrate buffer for 10 min. Sections were washed in TBS and
media changed daily. After 18 days, the dermal equivalents then permeabilized with 0.2% Triton-X for 10 min followed
were gently rinsed in PBS to remove excess media prior to by blocking with 10% goat serum (Sigma-Aldrich, UK)
directly printing the autologous epidermal keratinocytes for 1 h. Sections were washed in TBS and then incubated
on top of the dermal equivalents. Keratinocytes were with primary antibodies prepared in 10% goat serum for
suspended in Media C (Table 1) at a concentration of 20 × 1 h. Primary antibodies used were mouse anti-cytokeratin
10 cells/mL with 20 μL of the keratinocyte cell suspension 14 (1:250, ab7800, Abcam), rabbit anti-involucrin
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printed onto each dermal equivalent. The co-cultures were (1:400, ab181980, Abcam), rabbit anti-cytokeratin 10
then incubated for 90 min at standard culture conditions (1:500, ab76318, Abcam), and rabbit anti-loricrin (1:125,
and then gently submerged in Media C for 3 days with ab198994, Abcam). After 1 h, sections were washed in TBS
media changed daily. Skin equivalents were then raised and then incubated with secondary antibodies goat anti-
to the air–liquid interface (ALI) by aspirating Media C mouse Alexa Fluor-488 (AF-488; Thermo Fisher Scientific)
and adding sufficient Media D (Table 1) to the wells to and goat anti-rabbit Alexa Fluor-647 (AF-647; Thermo
cover the underside of each dermis, without submerging Fisher Scientific); dilution of 1:400 was used for both. Slides
the epidermis region of the tissue. The co-cultures were were rinsed in a final wash of TBS, and then coverslips
cultured at the ALI at standard conditions for 14 days were mounted with Vectashield mounting medium with
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with media changed daily. After day 14 of ALI culture, the DAPI (Vector Laboratories, US). Slides were imaged using
skin equivalents were harvested for histological evaluation an Axioimager microscope (Carl Zeiss AG, Germany).

Volume 10 Issue 2 (2024) 480 doi: 10.36922/ijb.1851