Development of a GelMA-based organotypic human skin model in a custom-made bioreactor

Collagen I has been the standard biomaterial to generate in vitro tissue models, including organotypic skin. In this case, the rate of collagen remodeling by fibroblasts leads to significant dimensional changes along the culture, which can be associated to additional hurdles when dynamic culture systems are used. These are particularly relevant for skin tissue due to its multilayered nature to provide the necessary fluid flow for tissue development. In this work, a different biomaterial, gelatin methacrylate (GelMA), is proposed to validate a dimensionally stable organotypic skin model developed and maintained in a custom-made bioreactor. For this purpose, human dermal fibroblasts (hDFbs) were re-suspended in 5% or 7.5% (wt/vol) GelMA at a density of 2.3×10⁵ cells/ml, followed by a crosslinking of 30 seconds under 7.2 mW/cm² UV.  Control organotypic structures were prepared using the same cell density and 0.26% (wt/vol) rat tail collagen that was then polymerized for 2 hours at 37°C. The constructs were cultured in α-MEM medium up to 14 days. The elastic (G’) and viscous modulus (G’’) of the hydrogels were measured using a rotational rheometer, and a uniaxial compression test allowed the determination of the compression modulus. Cell viability was assessed after Calcein/PI staining and ECM deposition (collagen type I and laminin) was analyzed through immunocytochemistry. The G’ of 5 and 7.5% GelMA hydrogels did not significantly vary along the culture, presenting respectively mean values of 0.9kPa and 1kPa. Likewise, the compression modulus did not vary, being within the range of 5kPa and 18kPa, respectively for the 5% and 7.5% GelMA hydrogels. In opposition, the G’ mean value of the collagen I hydrogels, increased from 0.5 to 1.2kPa from day 1 to day 14 of culture. The compression modulus also showed significant differences at day 14, being collagen about 3-fold stiffer than GelMA hydrogels. Moreover, the shrinkage ratio of GelMA and collagen hydrogels was circa 20% and 83%, respectively. Furthermore, GelMA didn’t negatively affect cellular viability and was also capable of supporting ECM deposition. From the results achieved so far, the use of GelMA for generating organotypic skin models has demonstrated to possess several potentialities that surpass the struggles associated with the shrinkage of collagen. Future experiments will focus on the incorporation of human keratinocytes (hKCs) on the top of the constructs, to replicate the epidermal compartment and the in vitro dynamic culture of the skin equivalent will take place in the recently developed sandwich-like bioreactor.