Animal testing has long been used in science to study complex biological phenomena that cannot be investigated using two-dimensional cell cultures in plastic dishes. With time, it appeared that more differences could exist between animal models and even more when translated to human patients. Innovative models became essential to develop more accurate knowledge. Tissue engineering provides some of those models, but it mostly relies on the use of prefabricated scaffolds on which cells are seeded. The self-assembly protocol has recently produced organ-specific human-derived three-dimensional models without the need for exogenous material. This strategy will help to achieve the 3R principles.

The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes (hbTESSs) designed for the treatment of severely burned patients. These are the scaffold-free self-assembled skin substitute (SASS) and the human plasma-based skin substitute (HPSS). Fibroblasts and keratinocytes from three humans were extracted from skin biopsies (N = 3) and cells from the same donor were used to produce both hbTESS models. For SASS manufacture, keratinocytes were seeded over three self-assembled dermal sheets comprising fibroblasts and the extracellular matrix they produced (n = 12), while for HPSS production, keratinocytes were cultured over hydrogels composed of fibroblasts embedded in either plasma as unique biomaterial (Fibrin), plasma combined with hyaluronic acid (Fibrin-HA) or plasma combined with collagen (Fibrin-Col) (n/biomaterial = 9). The production time was 46–55 days for SASSs and 32–39 days for HPSSs. Substitutes were characterized by histology, mechanical testing, PrestoBlue™-assay, immunofluorescence (Ki67, Keratin (K) 10, K15, K19, Loricrin, type IV collagen) and Western blot (type I and IV collagens). The SASSs were more resistant to tensile forces (p-value < 0.01) but less elastic (p-value < 0.001) compared to HPSSs. A higher number of proliferative Ki67⁺ cells were found in SASSs although their metabolic activity was lower. After epidermal differentiation, no significant difference was observed in the expression of K10, K15, K19 and Loricrin. Overall, the production of type I and type IV collagens and the adhesive strength of the dermal-epidermal junction was higher in SASSs. This study demonstrates, for the first time, that both hbTESS models present similar in vitro biological characteristics. However, mechanical properties differ and future in vivo experiments will aim to compare their wound healing potential.

Reliable methods for the isolation and culture of human eccrine sweat gland cells (SGCs) are essential for studying glandular biology and developing tissue-engineered skin substitutes (TESs) that restore full skin function. However, maintaining the glandular phenotype of SGCs in vitro remains a major challenge. In this study, we present an optimized isolation protocol combining enzymatic digestion with mechanical separation to improve SGC yield and purity, while also enabling keratinocyte isolation from a single human skin biopsy. We then evaluated two culture strategies, 2D monolayers and 3D spheroids, to determine their impact on SGC identity and proliferation. While 2D culture supported cell expansion, SGCs and keratinocytes exhibited highly similar marker expression profiles, with the absence of functional SGC markers (AQP5, α-SMA) reflecting a shift toward less differentiated phenotypes. In contrast, SGCs cultured in 3D spheroids preserved the expression of SGC-specific markers (AQP5, K18, α-SMA), distinguishing them from keratinocytes; however, their growth and structural organization were suboptimal under these 3D conditions. Moreover, SGCs expanded in 2D did not regain their glandular features when reintroduced into 3D culture, suggesting potential limitations in phenotype recovery. These results highlight the need for improved culture systems that maintain SGC identity while supporting expansion. Advancing such methods is a critical step toward integrating functional sweat glands into TESs and achieving complete skin regeneration for clinical applications.

Tumorigenic assays are used during a clinical translation to detect the transformation potential of cell-based therapies. One of these in vivo assays is based on the separate injection of each cell type to be used in the clinical trial. However, the injection method requires many animals and several months to obtain useful results. In previous studies, we showed the potential of tissue-engineered skin substitutes (TESs) as a model for normal skin in which cancer cells can be included in vitro. Herein, we showed a new method to study tumorigenicity, using cancer spheroids that were embedded in TESs (cTES) and grafted onto athymic mice, and compared it with the commonly used cell injection assay. Tumors developed in both models, cancer cell injection and cTES grafting, but metastases were not detected at the time of sacrifice. Interestingly, the rate of tumor development was faster in cTESs than with the injection method. In conclusion, grafting TESs is a sensitive method to detect tumor cell growth with and could be developed as an alternative test for tumorigenicity.

Burn patients treated with tissue-engineered skin substitutes (TESs) often experience pigmentation irregularities, including hypopigmentation and pigmentation spots. These issues are thought to stem from the reduced presence of melanocytes through dilution during TES manufacturing. To address this, we hypothesized that supplementing epithelial cell cultures—primarily composed of keratinocytes but also containing melanocytes—with Fibroblast Growth Factor-2 (FGF-2), a known promoter of melanocyte proliferation, could enhance melanocyte growth. This would potentially increase their numbers in TESs and improve pigmentation outcomes. Our findings indicate that FGF-2, at an optimal dose of 0.2 nM, effectively maintains melanocyte numbers in 2D cultures and epithelial cell cultures through the first passage. Importantly, this treatment does not interfere with keratinocyte proliferation or differentiation, nor does it affect TES integrity. However, FGF-2 supplementation alone did not increase the proportion of melanocytes in epithelial cultures beyond the first passage or in TESs. In summary, while FGF-2 supports melanocyte growth in culture, its addition alone was insufficient to significantly improve TES pigmentation.