Page 450 - IJB-9-4

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

A B

C

Figure 3. Characteristics of N/TERT keratinocytes cultured in PDα medium. (A) Number of viable N/TERT keratinocytes cultured in PDα was not
significantly different (P = 0.8227) to cells cultured in commercial CnT-PR media after 2 days, indicating similar proliferation rates. (B) N/TERT
keratinocytes cultured in PDα media were able to differentiate and form a striated epidermis that was comparable to the epidermis formed using CnT-
PR-3D media. (C) Immunostaining for stem cell marker p63, keratinocyte differentiation markers filaggrin (FLG) and keratin 10 (K10) were detected in
both RHE made from PDα media or CnT-PR-3D media at day 7 and day 18 post airlift. The RHEs made using the one-step PDα media were comparable
to the two-step typical CnT-PR/CnT-PR-3D method. Nuclei were stained with Hoechst (Blue). Scale bar: 100 µm.

change in primary keratinocytes viability after an hour. anchor the epidermis onto the underlying dermis [31,32] . To
Next, as encapsulation of primary keratinocytes in collagen simulate a dermal substrate on which to test our bioink,
could potentially affect cell viability, the cells were extracted we generated an HDF-containing dermal template. The
from the collagen and cell viability determined by trypan template contains an indent, into which keratinocytes are
blue staining. The results indicated that encapsulation of seeded in PDβ with or without collagen (Figure 2A and B).
primary keratinocytes in the bioink did not significantly The cells were cultured submerged in PDβ medium
alter the viability of the cells (Figure 4E). initially (Figure 2A) before the removal of the media
within the insert to form an air-liquid interface (airlift)
3.3. Development of HDF-containing dermis for epidermis formation (Figure 2B). The template was
The presence of HDFs has been reported to be important formed with HDFs mixed with rat tail collagen and
for the formation of a basement membrane , which helps seeded into 24 mm polyester (PET) membrane inserts
[31]

Volume 9 Issue 4 (2023) 442 https://doi.org/10.18063/ijb.738