Page 447 - IJB-9-4

International Journal of Bioprinting Single-step bioink deposition and maturation of human epidermis

media (A*STAR RSC, Singapore) containing NaOH. 2.6. Reconstituted human epidermis (RHE) using
Viability of primary keratinocytes in such a high pH immortalized N/TERT keratinocytes and primary
environment over an hour was determined. Briefly, 5 mM keratinocytes
NaOH was added to primary keratinocytes suspended in N/TERT-RHEs were generated using the following
PDβ medium and left on ice for t = 0, 5, 15, 30, 45 and established protocols. Briefly, N/TERT keratinocytes were
60 min. At the end of each time point, 5 mM acetic acid seeded at 3 × 10 cells per polycarbonate cell culture insert
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was added to the NaOH-cell suspension for neutralization. (24-well size insert, 0.47 cm , Thermo Scientific, USA) in
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They were then seeded in 24 well-plate (1.8 × 10 cells/well) CnT-PR medium. After 48 h, cultures were switched to CnT-
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and more PDβ medium was added to each well before
incubating them at 37°C overnight. The next day, cell PR-3D medium for 24 h, then cultured at air-liquid interface
viability was measured using CellTiter-Blue Cell Viability and harvested at various time points for downstream analysis.
®
Assay with excitation wavelength at 555 nm and emission For single medium N/TERT-RHEs generation,
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wavelength at 585 nm. N/TERT keratinocytes were seeded at 3 × 10 cells per
polycarbonate cell culture insert in PDα. The PDα
2.4.3 Viability of primary keratinocytes extracted media were refreshed after 24 h, N/TERTs were cultured
from collagen for a further 48 h submerged, and then cultured at air-
To determine whether encapsulation of primary liquid interface and harvested at various time points for
keratinocytes in collagen would have any effect on downstream analysis. For primary keratinocyte-RHE
the proliferative properties of the cells, the following generation with single medium, the protocol remains the
collagenase protocol was utilized to extract the same except that primary keratinocytes were used instead
primary keratinocytes. Briefly, primary keratinocytes of N/TERT keratinocytes.
(5.3 × 10 cells) were encapsulated in collagen and
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cultured for 24 h in PDβ medium. After 24 h, PDβ 2.7. Formation of human dermal fibroblast (HDF)
media were removed and replaced with 1 mL of warmed dermis
0.5 mg/mL collagenase (Merck, USA) solution and HDFs (4.67 × 10 cells/mL) were mixed with 10 mg/mL
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incubated at 37°C for 45 min. Collagenase solution was of rat tail collagen type I and seeded into 24 mm polyester
removed and collected into a 15 mL tube. 0.25% trypsin- (PET) membrane inserts (Corning, USA). A silicone
EDTA was used to disassociate cells that were adhered to disk was used to create an indent in the middle of the
the plate. The collagenase and trypsin-EDTA solutions dermis layer after gelation at 37°C. The indent serves
were neutralized with 10% FBS and spun down at 200 G as a basin to contain and prevent overflow of additional
for 5 min. Cell pellet were resuspended in 1 ml of PDβ HDF and primary keratinocytes that will be seeded later.
followed by cell counting using Trypan blue solution The HDF-dermis was cultured in CnT-PR-F medium for
(Gibco, USA). 2 days, followed by the addition of a monolayer of HDF
(3.91 × 10 cells/mL) seeded into the indent. The dermis
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2.5. Proliferation of keratinocytes in various media
was cultured for a further 10 days with alternate days of
To determine proliferation of primary keratinocytes CnT-PR-F medium changes.
in PDβ media over 24 h, primary keratinocytes (5.3
× 10 cells) were seeded in 6-well plate in PDβ. The next 2.8. Human skin equivalent (HSE) generation
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day, cells were trypsinized, and cell count was performed Two different types of skin equivalents were generated.
using Trypan blue solution. HSE refers to primary keratinocytes that were seeded
To compare proliferation of N/TERT keratinocytes onto HDF-dermis to test the differentiative properties of
and primary keratinocytes in PDα medium, cells PDβ medium. Collagen-HSE (c-HSE) was generated by
(2 × 10 cells/well) were seeded on 24-well plate in PDα depositing the bioink of primary keratinocytes suspended
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medium and cultured for 2 days. Thiazolyl Blue Tetrazolium in bovine collagen onto HDF-dermis.
Blue solution (MTT, Sigma, USA) was added to the culture
media to a final concentration of 0.3 mg/mL. Cells were 2.8.1. Formation of human skin equivalent (HSE)
grown in the media for 3 h at 5% CO and 37°C, and then Primary keratinocytes (5.31 × 10 cells/insert) were seeded
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2
the media were removed. Acidified isopropanol was used into the indent of the HDF-dermis and cultured in PDβ
to dissolve the formazan crystals and the absorbance was medium. PDβ media were refreshed after 24 h, keratinocytes
measured at 570 nm wavelength. Bright field images of the were cultured for a further 48 h and then cultured at air-
cells at the end of incubation period were also taken with liquid interface for the next 7 days before fixation and
Nikon Eclipse Ti-U inverted microscope (Nikon, Japan). staining. Media were refreshed on alternate days.

Volume 9 Issue 4 (2023) 439 https://doi.org/10.18063/ijb.738