Speaker
Description
"INTRODUCTION: Collagen I has been the gold standard material to generate many in vitro tissue models, including organotypic skin [1]. The rate of collagen remodeling by fibroblasts incorporated in the dermal part of the model, leads to significant dimensional changes during its development, which can be associated to additional hurdles when cultured within dynamic culture systems. These ones, are particularly relevant for skin tissue due to its multilayered nature, by providing the necessary fluid flow for tissue development and interlayer communication for biofunctionality mimicry. Thus, 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 [2], generating a dynamic in vitro testing platform.
METHODS: Two solutions of 5% and 7.5% (wt/vol) GelMA were prepared and crosslinked for 30 seconds under 7.2 mW/cm2 UV intensity [3]. Rat tail collagen I hydrogels were prepared from 0.26% (wt/vol) solutions that were polymerized for 2 hours at 37°C. Human dermal fibroblasts (hDFbs) were encapsulated in the hydrogels at a density of 2.3×105 cells/ml and 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 an uniaxial compression test allowed the determination of the compression modulus of the hydrogels. Cell viability was assessed after Calcein/PI staining and ECM deposition (collagen type I and laminin) was analyzed through immunocytochemistry.
RESULTS: 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, independently of the day of culture. 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. Independently of the mechanical properties, GelMA didn’t negatively affect cellular viability being also capable to support ECM deposition.
CONCLUSION: From the results achieved so far, the use of GelMA as a substitute of collagen 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 full skin equivalent will take place in the recently developed sandwich-like bioreactor.
ACKNOWLEDGEMENTS: Financially supported by ERC Consolidator Grant ERC-2016-COG-726061.
REFERENCES
[1] Gangatirkar, P. et al., Nat. Protoc. 2, 727-746 (2007).
[2] Gasperini, L. et al., Provisional Patent Application 116901, (2020).
[3] Loessner, D. et al., Nat. Protoc. 11, 727–746 (2016)."
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