Page 445 - IJB-9-4

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

hypothermia, infection, and fluid loss. Treatment for these Here, we describe the development of a serum-free
patients is typically through the use of split-thickness bioink formulation that is designed to be deposited directly
skin grafts (STSG), where healthy skin is harvested from onto a wound bed with a suitable dermal layer using such a
the patient and applied on the burn wound . In order bioprinter (Figure 1). Since media change is difficult once
[2]
to maximize wound coverage and improve graft “take,” the bioink is deposited on the patient, the bioink is designed
slits are often cut into flattened grafts to create a mesh- to require only a single deposition step, and promote self-
like structure, which results in poorer esthetic outcome organization of the cells into the epidermis layers with
for the patient since the mesh pattern may be visible after no further intervention. To arrive at this formulation,
healing. Furthermore, STSG is not an option in cases we first utilized the robust N/TERTs immortalized
where autologous donor sites are unavailable due either to human keratinocyte cell line to help us narrow down the
extensive injury or underlying conditions . suitable formulations and culture conditions for creating
[3]
While off-the-shelf dermal replacement templates a reconstituted human epidermis (RHE) model. Using
(DRT) can be used to recreate a functional (if incomplete) the knowledge gained from the N/TERTs, we developed
dermis , restoring the epidermis requires the use of patient a media formulation that achieves a balance between
[3]
cells. Autologous cell sheets known as cultured epithelial proliferation and differentiation, triggering differentiation
autografts (CEAs) can be grown in vitro, and applied onto as the bioink dehydrates. By adding bovine collagen type I
a DRT or native dermis for partial thickness wounds . to our formulation, we improved control of the deposition
[4]
However, CEAs require a large number of cells ; the process, since the gelled bioink will not flow away from
[5]
resulting skin tends to be fragile [5-7] ; and are vulnerable to the site of application. Finally, using a collagen-based
infection . An alternative approach is to utilize so-called dermal template, we demonstrate the formation of various
[6]
“spray-on” skin, where a skin biopsy is enzymatically epidermal structures from primary keratinocytes by
treated to release the cells, which are then sprayed onto the immunofluorescence. We believe that this bioink has great
wound bed [8,9] . While it is easier to achieve conformance potential to be used in skin bioprinting, as a new treatment
to body contours with a cell spray than a CEA, the cell option for burns.
solution can flow away from the site of application when
deposited on large wounds with physiological topographies 2. Materials and methods
and orientations . Since the cell suspension is clear, 2.1. Materials
[10]
determining adequate cell coverage can also be challenging
(L. Téot, personal communication, May 9, 2022). Cell culture reagents Dulbecco’s Modified Eagle Medium/
Nutrient Mixture F-12 (DMEM/F-12), 0.05% trypsin-
In recent years, the field of bioprinting has progressed EDTA and TrypLE™ Express were purchased from Gibco
significantly, with many groups working on direct printing (USA). CnT-Prime epithelial proliferation medium
of cell-laden bioinks [10-16] . Consequently, bioprinting has (CnT-PR), CnT-Prime epithelial 3D airlift medium (CnT-
been utilized to create 3D skin constructs containing dermal PR-3D) and CnT-Prime Fibroblast Proliferation medium
and epidermal components [17-24] . This typically involves a (CnT-PR-F) were bought from CELLnTEC (Switzerland).
multi-step process, where different components are printed Primary human epidermal keratinocytes and KGM-Gold
at different times, and matured under different conditions Keratinocyte Growth Medium (KGM) were purchased
over several days. While most bioprinted skin constructs from Lonza (Switzerland). Rat tail collagen type I solution
are intended for in vitro testing purposes [17-19,24] , some have from Corning (USA) and lyophilized bovine collagen
taken it a step further, and implanted the constructs onto type I from Symatese (France) were used. Human dermal
animal models [20-23] . Although these implants have shown fibroblast (HDF/TERT164) cell line was purchased from
promise, they also share the challenges associated with CEA, Evercyte (Austria) while immortalized cell line N/TERT
such as poor conformance to body contours, and long and was a gift from Associate Professor Hao Li, Amy (National
costly culture before use. The long-term graft stability is also University of Singapore, Singapore).
unproven in most cases, and the requirement for sophisticated
bioprinters limits their availability . Fortuitously, the Primary and secondary antibodies used for
[25]
unique superficial position of skin allows in situ printing immunofluorescence staining are listed in Table 1.
to be performed [10,15,16,26,27] . Furthermore, the ability of 2.2. Preparation of bovine collagen and bioink
keratinocytes to self-organize into functional epidermis
means that it is possible to deliver the cells without extensive Lyophilized bovine collagen type I was dissolved in sterile
spatial control. Therefore, when used in combination with a 5 mM acetic acid (Merck, USA) on a multi-rotator (Grant-
dermal template, a simple hand-held bioprinter is sufficient Bio, UK) at room temperature. The acidic collagen solution
to deposit the cell-laden bioink onto the wound [10,15] . (0.1% w/v) was then kept at 4°C until further use. Bioink

Volume 9 Issue 4 (2023) 437 https://doi.org/10.18063/ijb.738