Page 156 - IJB-8-4

Recombinant Human Collagen for 3D Bioprinting of Skin Equivalent
supplemented with an additional 1.5 mM CaCl under the fibroblasts cultured in bioink hydrogels. To determine the
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submerged condition for the initial cell attachment and effects of rhCol3 on the gene expressions (P63, filaggrin,
expansion. To induce the HaCaTs differentiation under and Nrf2) of HaCaTs, we cultured HaCaTs on GelMA-
the ALI culture condition, the submerged culture medium rhCol3-3.2 and GelMA only hydrogels and performed
was further supplemented with 50 μg/mL ascorbic acid qRT-PCR analysis. At defined culture time points (days
(Sigma-Aldrich, USA) and 10 ng/mL keratinocyte growth 1, 3, and 7), the HaCaTs on the surface of the dermal
factor (KGF; Lonza, USA). constructs were gently washed with PBS and detached by
trypsin. The detached HaCaTs were then lysed by Trizol®
2.3. Cell viability assay for 5 min to obtain cell suspension. After centrifuging
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To visualize the distribution and survival of the HDFs the cell suspensions at 12 × 10 rpm for 5 min, the
encapsulated in the gelled bioinks, we applied a LIVE/ supernatants were collected, followed by the addition of
DEAD™ (Invitrogen, USA) assay at defined time points chloroform to collect the RNA-containing aqueous phase.
(days 1, 3, and 7). Briefly, cell-laden samples were Isopropanol was then added to the aqueous phase, which
gently washed 3 times with warm phosphate-buffered was centrifugated to obtain the RNA pellets. Finally, the
saline (PBS), then 500 μL LIVE/DEAD™ staining RNA pellets were dissolved in RNase-free water. The
solution (Calcein-AM 2 μmol/L, PI 4.5 μmol/L) was first-strand cDNA was synthesized using OneScript Plus
cDNA Synthesis Kit (Applied Biological Materials Inc.,
added, followed by incubation at 37°C for 20 min. The Richmond, BC, Canada) with 1000 ng total RNA. The
fluorescent images were taken by confocal fluorescent PCR reaction was performed in a StepOnePlus Real-
microscopy (Eclipse Ti Nikon), and cell viability was Time PCR System (Applied Biosystems, Foster City,
determined by identifying the number of live and dead CA, USA) following the instructions from EvaGreen
cells using Matrix Laboratory (MATLAB R2019a). qPCR MasterMix (Applied Biological Materials Inc.,
Viabilities of printed HDFs in different photocrossslinked Richmond, BC, Canada). The 2 −△△Ct method was used to
bioinks were evaluated in the same manner. In addition, determine the relative mRNA expression levels, and all
a LIVE/DEAD™ assay was also used to identify the the results were normalized to the expression of β-actin.
seeded HaCaTs on the surface of bioinks.
The primers sequences for the target genes are listed in
2.4. Cell proliferation assay Table S1.
To determine the suitable concentration of the bioink, 2.6. Scanning electron microscope (SEM)
we prepared GelMA solutions at varying concentrations The hydrogel samples were lyophilized firstly for 24 h;
(5, 7.5, and 10 wt%) encapsulated with 1 × 10 /mL HDFs. then, the freeze-dried samples were carefully sectioned
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At a defined culture time point, the culture medium was using a scalpel and sputter coated for 60 s with gold.
removed, and fresh medium supplemented with Cell The microstructure morphology of the samples was
Counting Kit-8 (CCK-8, Yeason Biotechnology, China) observed by SEM (FEI Quanta 200, Netherlands) at
reagent (mixing ratio of 10:1) was added to the individual an acceleration voltage of 5 kV. The average pore size
sample followed by a 3 h culturing at 37°C. A 100 μL was determined by measuring the diameter of randomly
of the reacted supernatant was then transferred to a 96- selected pores.
well plate from each sample, and the optical density
was measured at an absorbance wavelength of 450 nm 2.7. Rheology and mechanical characterizations
(Multiskan FC Thermo). To determine the proliferation
activities of the HDFs within the dermal constructs or The rheological properties of the GelMA and the
HaCaTs proliferated on hydrogel constructs, dermal GelMA-rhCol3 composite bioinks were evaluated
constructs or epidermal layers were treated with the same by a rheometer (Anton Paar Mcr-302) with a 25 mm
method. plate (CP 25-2/TG). The storage modulus (G’) and loss
modulus (G’’) of the inks were determined under the
2.5. Quantitative real-time polymerase chain temperature sweep mode by decreasing the temperature
reaction (PCR) from 37°C to 4°C at a cooling rate of 5°C/min while
the shear strain and frequency were maintained at
The gene expression levels of collagen I, TGF-β, vimentin, 0.5% and 1 Hz, respectively. The photorheological
and alpha-smooth muscle actin (α-SMA) were measured properties of the GelMA and the GelMA-rhCol3
using qRT-PCR. After culturing for 14 days, cell-laden composite bioinks were evaluated by a rheometer
hydrogels were dissolved by GelMA lysis solution (Anton Paar Mcr-302) equipped with a transparent
(EFL-Gm-Ls-001), then HDFs were isolated from the glass plate with Peltier control (Anton Paar, P-PTD
dissolved bioinks by centrifugation. The cells were lysed 200GL) and a 25 mW/cm UV light source. The
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by Trizol® for 5 min to obtain the total RNA of the storage modulus (G’) and loss modulus (G’’) of the
148 International Journal of Bioprinting (2022)–Volume 8, Issue 4