Page 270 - IJB-10-6

International Journal of Bioprinting Skin bioprinting: Keratinocytes and stem cells

lower chamber of a transwell model. A biphasic bioprinted to the experimental groups presented in Table 1. For each
construct, containing keratinocytes and ADSCs in various experimental group, a control group without ADSCs was
hydrogels, was then developed. Hydrogel stability and the set up in parallel. Therefore, the well below the control
cells’ metabolic activity and survival were evaluated over group was left cell-free but filled with 1 mL DMEM, 10%
14 days. Based on the results of this study, potential new FCS, and 1% P/S. For groups I, II, IV, V, and VI, hydrogels
treatment options could include bioprinted constructs were prepared with a concentration of 1 × 10 cells/mL. In
6
with keratinocytes and stem cells. These constructs group III, 2 × 10 cells were seeded on each scaffold.
5
may promote earlier wound healing and complete skin
regeneration, helping to prevent acute and chronic sequelae 2.2.2. Biomaterial for 3D cell culture
resulting from large-scale burn injuries. 24 The following groups of biomaterials were used for 3D cell
culture within the transwell model: I: 0.5% (m/v) Alg (PH
2. Methods 176; JRS PHARMA GmbH & Co. KG, Germany) and 0.5%
(m/v) Gel (Sigma-Aldrich, USA); II: 1.1% (m/v) fibrinogen
2.1. Cell lines and thrombin (Baxter Healthcare GmbH, Austria);
The immortalized HaCaT cell line was purchased III: collagen-elastin template matrix (MatriDerm®; Dr.
from Cell Lines Service GmbH (CLS, Germany) and Suwelack Skin and Health Care, Germany); IV: 0.5% (m/v)
cultivated up to passage 50. Immortalized ADSCs were Alg, 0.5% (m/v) Gel, 0.05% (m/v) collagen (Sigma-Aldrich,
obtained from Evercyte GmbH (Austria) and cultivated USA), and 3% (m/v) HA (CarboSynth Ltd., UK); V: 0.5%
up to passage 24. HaCaT were cultivated in Dulbecco’s (m/v) Alg, 3% (m/v) Gel, and 0.1% (m/v) HA; VI: 4%
modified eagle medium (DMEM) (Gibco; Thermo Fisher (m/v) gelatin methacryloyl (Biotechnologies Ltd., Canada)
Scientific, USA), supplemented with 4500 mg/mL glucose, and 0.2% (m/v) lithium phenyl-2,4,6-trimethylbenzoyl
l-glutamine, sodium pyruvate, and sodium bicarbonate, phosphinate (LAP; Sigma-Aldrich, USA). All experimental
10% fetal calf serum (FCS) (Sigma-Aldrich, United States groups were seeded as triplicates.
of America [USA]) and 1% penicillin-streptomycin (P/S)
(Sigma-Aldrich, USA). ADSCs were grown in Endothelial Hydrogel groups I, IV, V, and VI were synthesized by
Cell Basal Medium-2 (EBM®-2) (Clonetics®; Lonza Group dissolving the respective composition (m/v) in phosphate-
2+
AG, Switzerland) enriched with the supplement mix, 2% buffered saline (PBS) without Ca and Mg (Sigma-
2+
FCS superior (Sigma-Aldrich, USA), and 1% geneticin Aldrich, USA). The pH of the solution was adjusted to 7.4
2+
2+
(Gibco; Thermo Fisher Scientific, USA). Both cell lines with PBS (PBS Dulbecco’s w/o Ca and Mg , Instamed
were incubated at 37°C with 5% CO . 9.55 g/L; Biochrom GmbH, Germany) for groups IV, V, and
2
VI. The hydrogels were mixed at 37°C until a homogenous
2.2. HaCaT in co-culture with ADSC in a state was reached. HaCaTs were re-suspended in the
transwell model hydrogel using a positive displacement pipette, and 200
2.2.1. Cell seeding µL of each hydrogel was seeded into the upper chamber
Adipose-derived stem cells (ADSCs) were seeded in a 24- of the transwell with a pore size of 0.4 µm. The hydrogels
well plate at a density of 5 × 10 cells/well in EMB -2 and left of groups IV and V were chelated for 10 min in 100 mM
4
®
to adhere for 4 h at 37°C. The medium was then changed to calcium chloride (CaCl ) solution (Sigma-Aldrich, USA)
2
1 mL DMEM with 10% FCS and 1% P/S (all from Sigma- and then washed with PBS (Sigma-Aldrich, USA). The
Aldrich, USA). HaCaT keratinocytes were seeded in the hydrogel of group VI was crosslinked using 405 nm light
hydrogel at a density of 2 × 10 cells/transwell according for 1 min.
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Table 1. Biomaterials used for 3D cell culture For the preparation of Fib in group II, 9.1% (m/v)
human fibrinogen and 500 IU/mL thrombin were each
Group Biomaterial(s) diluted with Hanks buffered saline solution (HBSS; Sigma-
I Alginate (0.5% [m/v]) and gelatin (0.5% [m/v]) Aldrich, USA) in a 1:4 ratio. The cells were mixed into
II Fibrin (1.1% [m/v]) the fibrinogen component of the kit and re-suspended.
III Collagen-elastin template matrix Thereafter, 100 µL of the mixture was pipetted into the
Alginate (0.5% [m/v]), gelatin (0.5% [m/v]), collagen transwell, and the same amount of thrombin was added.
IV
(0.05% [m/v]), and hyaluronic acid (3% [m/v]) For chelation, the two components were gently mixed with
Alginate (0.5% [m/v]), gelatin (3% [m/v]), and a positive displacement pipette.
V
hyaluronic acid (0.1% [m/v]) The collagen-elastin template matrix is a commercially
Gelatin methacryloyl (4% [m/v]) and lithium phenyl-
VI available dermal replacement scaffold available in sheets
2,4,6-trimethylbenzoyl phosphinate (LAP; 0.2% [m/v])
and was used as the control (group III). Under sterile
Volume 10 Issue 6 (2024) 262 doi: 10.36922/ijb.3925