Page 498 - IJB-10-5

International Journal of Bioprinting 3D bioprinting of full-thickness skin with a rete ridge structure

2.3. Fabrication of precursor cartridges constructs were incubated at 37°C for 15 min for gelation
A 3D model of a precursor cartridge was created using and then washed three times with pre-warmed PBS at 37°C
SolidWorks 3D CAD. Subsequently, 3D CAD data were to remove sacrificial material. Subsequently, keratinocytes
converted into an STL file. The STL file was loaded into (1 × 10 cells/model) were seeded onto the dermis, and
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a 3D printing system (ProJet 6000 HD; 3D Systems, each skin model was cultured under air-liquid interface
USA) that uses United States Pharmacopeia (USP) Class conditions for seven days.
VI certified polymer resin (VisiJet SL Clear; 3D Systems,
USA) to fabricate precursor cartridges. After fabrication, 2.6. Optical coherence tomography imaging
the precursor cartridges were sterilized by immersing them A spectral-domain optical cohesion tomography (OCT)
in 70% ethanol for 5 min, followed by overnight exposure imaging system (Lumedica Inc., USA) with 840 nm center
to UV light. wavelength was used to non-destructively observe the
cross-sectional structure of printed FTSE. For observation,
2.4. Cell culture the printed rete ridge FTSE was transferred to a petri dish
Normal human epidermal keratinocytes (NHEKs, and images were captured at a size of 3547 × 2632 μm.
00192907) and normal human dermal fibroblast (NHDFs,
CC2509) were purchased from Lonza (Switzerland). 2.7. UV treatment
NHEKs were cultured using a keratinocyte growth medium To confirm UV resistance, FTSEs were exposed to UVB
(KGM; KGM-Gold bullet kit; Lonza, Switzerland). NHDFs using a UV irradiation system (UV Crosslinker; VWR
were cultured in a fibroblast growth medium (FGM; FGM- International, France). Irradiation was performed for 30
2 Bullet Kit; Lonza, Switzerland) along with 10% fetal min at intensities of 25 and 50 mJ/cm². Following UVB
bovine serum (Gibco; Thermo Fisher Scientific, USA). treatment, the samples were cultured for three days under
NHEKs and NHDFs were used in the first or second standard conditions at 37°C in an atmosphere of 5% CO .
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passage and cultured at 37°C in 5% CO . The culture 2.8 Tissue slide preparation
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medium was changed every two days to ensure optimal To perform hematoxylin & eosin (H&E) and
growth conditions. immunofluorescence (IF) staining, the skin models were

2.5. 3D printing of FTSEs fixed overnight in 4% paraformaldehyde (PFA, 163-20145;
To fabricate skin equivalents, a 3D bioprinting system Wako, Japan) at 4°C. Following fixation, the samples were
(3DXPrinter; T&R Biofab Co., Ltd., South Korea) with dehydrated in a 30% sucrose solution. Once completely
four microextrusion-based printing heads was used. dehydrated, the samples were then immersed in optimal
This system featured three-axis precision stages and four cutting temperature compound (FSC 22 Clear; Leica
pneumatic dispensers: three low-pressure dispensers Microsystems, Germany) and frozen using liquid nitrogen
for hydrogels and one high-pressure dispenser for for 15 min. Subsequently, tissue sections with a thickness
thermoplastic polymer. PCL (Resomer C209; Evonik of 8 μm were obtained using a cryotome.
Inc., Germany) granules were loaded into a 10-mL metal 2.9. Hematoxylin & eosin staining
syringe of the extruder, maintained at 80°C. After a 10 The obtained tissue sections were mounted on glass
min heating period, molten PCL was extruded through a slides, and the optimal cutting temperature compound
metal nozzle (500 µm diameter) at a pressure of 650 kPa was removed by washing the slides. H&E staining was
and feed rate of 600 mm/min to fabricate a frame with performed using a commercially available H&E staining
dimensions of 10.5 × 9 × 3.5 mm. For preset extrusion kit (HAE-1 kit; Scytek Laboratories Inc., USA), following
bioprinting, SdECM containing NHDF (1 × 10 cells/mL) the manufacturer’s instructions. Briefly, the sections were
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and alginate were each loaded into a precursor cartridge treated with hematoxylin for 5 min to visualize nuclei,
using a syringe and a needle. Subsequently, the precursor followed by a bluing reagent for 10 s. Subsequently, the
cartridges containing SdECM bioink and alginate were sections were stained with eosin for 3 min to highlight
carefully transferred to a 10 mL plastic syringe and cytoplasmic structures. Following each staining step,
equipped with an 18-gauge tapered nozzle. Additionally, the samples were rinsed with distilled water and
to print the dermis layer without the rete ridge structure, absolute ethanol.
the SdECM bioink was transferred to a 10 mL plastic
syringe and fitted with an 18-gauge straight nozzle. The 2.10. Immunofluorescence staining
syringes containing SdECM were mounted on a print head For IF staining, the samples were incubated with a 1×
maintained at 4°C to prevent gelation during bioprinting. blocking buffer containing 0.2% Triton X-100 and 2.5%
The applied pressure to extrude bioinks was 5–8 kPa and bovine serum albumin at room temperature (~23–25°C)
the feed rate was 450–750 mm/min. The printed dermis for 1 h to minimize nonspecific antibody binding.

Volume 10 Issue 5 (2024) 490 doi: 10.36922/ijb.3961