Page 449 - IJB-9-4

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

For IF staining of paraffin sections, standard dewaxing they are eventually shed. On the other hand, keratinocyte
and rehydration steps were performed. Antigen retrieval differentiation markers FLG and K10 demonstrate that
was performed using citrate buffer pH6 (Novus Biologicals, the epidermis has stratified and matured appropriately.
USA) and 2100 Antigen Retriever (ProteoGenix, France), In particular, FLG plays an important role in the barrier
followed by Proteinase K (Sigma, USA) digestion for function of the skin, which helps prevent water loss and
15 min at room temperature. Sections were rinsed with infection. [29,30] The above markers were detected in both
PBS and blocked with donkey serum (Sigma, USA), and PDα and CnT-PR-3D RHEs at day 7 and day 18 post airlift,
then incubated with primary antibodies overnight at 4°C. indicating that RHEs made using the newly formulated PDα
Next, the slides were rinsed in PBS-T (0.05% Tween- media were comparable to the two-step method.
20, BioBasic, Singapore) and incubated with secondary
antibodies and Hoechst (Sigma, USA) for 60 min, and then 3.2. Bioink media formulation for primary
rinsed with PBS and mounted with Prolong-Gold Anti- keratinocytes
fade reagent (Invitrogen, USA). Confocal images were While we were able to achieve good RHEs with N/TERT,
captured on the Zeiss LSM700 (Zeiss, Germany). it is well-known that immortalized cells can behave
differently from primary cells, which will be needed for
2.11. Statistical analysis
clinical applications. However, primary keratinocytes were
Statistical analyses were performed using GraphPad Prism unable to differentiate and form proper RHEs in PDα
version 9.4.1 software (GraphPad Software, USA). Two-tailed media (Figure S1 in Supplementary File). As such, using
unpaired t-test (Welch’s t-test) was conducted. P < 0.05 was PDα as a starting point, we developed the PDβ media
considered statistically significant. Analysis of data from cell for primary keratinocytes. Since the formation of RHE is
viability of primary keratinocytes in high pH environment a time-consuming process, we also used morphological
required a one-way analysis of variance (ANOVA) test. cues in 2D culture to accelerate the optimization process
(Figure S2 in Supplementary File). First, we established
3. Results that primary keratinocytes were able to significantly
3.1. Bioink media formulation PDα for immortalized proliferate (P = 0.0245) over 24 h when cultured in PDβ
N/TERT keratinocytes media (Figure 4A). Next, when cultured at low density in
PDβ media for 24 h, primary keratinocytes expressed stem
The protocol for making reconstructed human epidermis cell marker p63 (green) and proliferative marker Ki67 (red)
(RHE) typically requires culturing in two different types (Figure 4B), with very few cells expressing keratinocyte
of media, namely, a proliferative media (e.g., CnT-PR, differentiative marker K10 (green, Figure 4C). The results in
K-SFM) followed by a differentiative media (e.g., CnT- Figure 4A–C indicate that primary keratinocytes cultured
PR-3D) . However, media changes are not feasible for at low density in PDβ media maintained a proliferative
[28]
cells applied onto patient skin. Hence, we developed the phenotype. On the other hand, Ki67 expression decreased
PDα media specifically for N/TERT keratinocytes to form while K10 expression increased at high cell density
RHE without requiring media switching. We demonstrated (Figure S3 in Supplementary File), suggesting that the
that there was no significant difference in the viability of
N/TERT keratinocytes cultured in PDα media compared cells can transition to a differentiated phenotype when
to commercial proliferative CnT-PR media after 2 days, they have proliferated sufficiently to reach the requisite
indicating that cell proliferation rate was the same in both density. This transition would thus allow us to accomplish
the desired cell expansion and differentiation in the in situ
medium (Figure 3A). N/TERT keratinocytes were then used printed bioink.
to make RHEs to determine the differentiative properties
of PDα medium compared to CnT-PR-3D medium. H&E In addition to the media component, the bioink
stained sections of N/TERT keratinocytes RHE made also contains collagen dissolved in acetic acid. As the
using the PDα medium was comparable to CnT-PR-3D acidic condition is toxic to cells, primary keratinocytes
medium, with keratinocytes differentiating to form the are suspended in chilled PDβ media containing NaOH,
stratified epidermis layers (Figure 3B). Finally, in Figure 3C, which neutralizes the acetic acid upon mixing of these two
to further characterize the RHEs formed and check for the components, resulting in collagen gelation. We estimate
stemness and differentiative capabilities of PDα medium that in the clinical setting, surgeons may take up to an
on N/TERT keratinocytes, IF staining was used to detect hour to apply the bioprintable epidermis onto the patient’s
protein expression of p63, filaggrin (FLG) and keratin 10 wound. Since the pre-neutralized media (PDβ + NaOH)
(K10). p63 stains for stem cells, which play important roles are not optimal for cells, viability of primary keratinocytes
in skin renewal, producing new cells that continually push in the high pH environment for up to an hour was checked.
older cells outward toward the uppermost layer, where Results in Figure 4D showed that there was no significant

Volume 9 Issue 4 (2023) 441 https://doi.org/10.18063/ijb.738