The Growing Importance of Three-Dimensional Models and Microphysiological Systems in the Assessment of Mycotoxin Toxicity

Abstract

Current investigations in the field of toxicology mostly rely on 2D cell cultures and animal models. Although well-accepted, the traditional 2D cell-culture approach has evident drawbacks and is distant from the in vivo microenvironment. To overcome these limitations, increasing efforts have been made in the development of alternative models that can better recapitulate the in vivo architecture of tissues and organs. Even though the use of 3D cultures is gaining popularity, there are still open questions on their robustness and standardization. In this review, we discuss the current spheroid culture and organ-on-a-chip techniques as well as the main conceptual and technical considerations for the correct establishment of such models. For each system, the toxicological functional assays are then discussed, highlighting their major advantages, disadvantages, and limitations. Finally, a focus on the applications of 3D cell culture for mycotoxin toxicity assessments is provided. Given the known difficulties in defining the safety ranges of exposure for regulatory agency policies, we are confident that the application of alternative methods may greatly improve the overall risk assessment.

Comparative Study of Spheroids (3D) and Monolayer Cultures (2D) for the In Vitro Assessment of Cytotoxicity Induced by the Mycotoxins Sterigmatocystin, Ochratoxin A and Patulin

“…The modernization of food toxicology via the integration of technological advances is endorsed by the Food and Drug Agency (FDA) and the EFSA, with emphasis on prioritizing advanced non-animal testing models for safety testing [51,52]. Accordingly, the new era of food toxicology is relying more and more on biorelevant in vitro systems [43]. Nonetheless, evaluations of STE, OTA and PAT toxicity using new alternative methods are still scarce [53][54][55].…”

“…Conversely, spheroids possess an inherent complexity that represents a significant challenge in spheroid-based assays for toxicity testing [58]. To reduce variations between samples and within repeats, an accurate spheroid generation method was followed, as previously described [43]. Noteworthy, a less linear trend in cell viability decrease was observed in 2D BM-MSCs and HUVECs, as well as in MDA-MB-231 spheroids exposed to PAT.…”

“…For spheroid generation, trypsinized single cells were suspended in the corresponding culture medium and dispensed into ultra-low attachment (ULA) 96-well round bottom plates (Corning ® , New York, NY, USA). As previously described, the addition of 7.5 µg/mL collagen to the culture medium was required to form HUVEC spheroids [43]. For both tumour lines, cells were seeded at a density of 2 × 10 3 cells/spheroid, while BM-MSCs and HUVECs were seeded at 5 × 10 3 cells/spheroid and 7.5 × 10 3 cells/spheroid, respectively.…”

From Pipeline to Plant Protection Products: Using New Approach Methodologies (NAMs) in Agrochemical Safety Assessment

“…Culturing single-cell types does not reflect the complexity of the whole organ architecture. Recognizing this limitation, mammalian toxicologists can use three-dimensional microphysiological systems that better mimic the complex mammalian biology to produce models that more accurately predict the toxicity potential of molecules for natural toxins found in the food supply, like mycotoxins, with principles that can be applied for agrochemicals as well.…”

Comparative functional aspects of 3T3 fibroblasts in 2D and 3D cell- culture environment

“…Skin bioengineering predominantly relied on 2D cell culture techniques for skin grafting purposes (2). However, 2D cultures often lacked the essential components of the natural environment, prompting a shift towards 3D cell culture systems, which are gaining popularity due to their ability to provide a more physiologically relevant environment compared to conventional 2D cultures (3,4). Recent advances in cytotechnology within tissue engineering have been greatly in uenced by the adoption of 3D cell culture systems (5,6).…”