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Background/Objectives: Cancer organoids have emerged as a valuable tool of three-dimensional (3D) cell cultures to investigate tumor heterogeneity and predict tumor behavior and treatment response. We developed a 3D organotypic culture model of oral squamous cell carcinoma (OSCC) to recapitulate the tumor–stromal interface by co-culturing four cell types, including patient-derived cancer-associated fibroblasts (PD-CAFs). Methods: A stainless-steel ring was used twice to create the horizontal positioning of the cancer stroma (adjoining normal oral mucosa connective tissue) and the OSCC layer (surrounding normal oral mucosa epithelial layer). Combined with a structured bi-layered model of the epithelial component and the underlying stroma, this protocol enabled us to construct four distinct portions mimicking the oral cancer tissue arising in the oral mucosa. Results: In this model, α-smooth muscle actin-positive PD-CAFs were localized in close proximity to the OSCC layer, suggesting a crosstalk between them. Furthermore, a linear laminin-γ2 expression was lacking at the interface between the OSCC layer and the underlying stromal layer, indicating the loss of the basement membrane-like structure. Conclusions: Since the specific 3D architecture and polarity mimicking oral cancer in vivo provides a more accurate milieu of the tumor microenvironment (TME), it could be crucial in elucidating oral cancer TME.

Background/Objectives: Cancer organoids have emerged as a valuable tool of three-dimensional (3D) cell cultures to investigate tumor heterogeneity and predict tumor behavior and treatment response. We developed a 3D organotypic culture model of oral squamous cell carcinoma (OSCC) to recapitulate the tumor-stromal interface by co-culturing four cell types, including patient-derived cancer-associated fibroblasts (PD-CAFs). Methods : A stainless-steel ring was used twice to create the horizontal positioning of the cancer stroma (adjoining normal oral mucosa connective tissue) and the OSCC layer (surrounding normal oral mucosa epithelial layer). Combined with a structured bi-layered model of the epithelial component and the underlying stroma, this protocol enabled us to construct four distinct portions mimicking the oral cancer tissue arising in the oral mucosa. Results : In this model, α-smooth muscle actin-positive PD-CAFs were localized in close proximity to the OSCC layer, suggesting a crosstalk between them. Furthermore, a linear laminin-γ2 expression was lacking at the interface between the OSCC layer and the underlying stromal layer, indicating the loss of the basement membrane-like structure. Conclusions : Since the specific 3D architecture and polarity mimicking oral cancer in vivo provides a more accurate milieu of the tumor microenvironment (TME), it could be crucial in elucidating oral cancer TME.

We are witnessing the revival of the CAM model, which has already used been in the past by several researchers studying angiogenesis and anti-cancer drugs and now offers a refined model to fill, in the translational meaning, the gap between in vitro and in vivo studies. It can be used for a wide range of purposes, from testing cytotoxicity, pharmacokinetics, tumorigenesis, and invasion to the action mechanisms of molecules and validation of new materials from tissue engineering research. The CAM model is easy to use, with a fast outcome, and makes experimental research more sustainable since it allows us to replace, reduce, and refine pre-clinical experimentation (“3Rs” rules). This review aims to highlight some unique potential that the CAM-assay presents; in particular, the authors intend to use the CAM model in the future to verify, in a microenvironment comparable to in vivo conditions, albeit simplified, the angiogenic ability of functionalized 3D constructs to be used in regenerative medicine strategies in the recovery of skeletal injuries of critical size (CSD) that do not repair spontaneously. For this purpose, organotypic cultures will be planned on several CAMs set up in temporal sequences, and a sort of organ model for assessing CSD will be utilized in the CAM bioreactor rather than in vivo.

The process of identifying and approving a new drug is a time-consuming and expensive procedure. One of the biggest issues to overcome is the risk of hepatotoxicity, which is one of the main reasons for drug withdrawal from the market. While animal models are the gold standard in preclinical drug testing, the translation of results into therapeutic intervention is often ambiguous due to interspecies differences in hepatic metabolism. The discovery of human induced pluripotent stem cells (hiPSCs) and their derivatives has opened new possibilities for drug testing. We used mesenchymal stem cells and hepatocytes both derived from hiPSCs, together with endothelial cells, to miniaturize the process of generating hepatic organoids. These organoids were then cultivated in vitro using both static and dynamic cultures. Additionally, we tested spheroids solely composed by induced hepatocytes. By miniaturizing the system, we demonstrated the possibility of maintaining the organoids, but not the spheroids, in culture for up to 1 week. This timeframe may be sufficient to carry out a hypothetical pharmacological test or screening. In conclusion, we propose that the hiPSC-derived liver organoid model could complement or, in the near future, replace the pharmacological and toxicological tests conducted on animals.

In vitro organotypic cell-based intestinal platforms, able to faithfully recapitulate the complex functions of the organ in vivo, would be a great support to search for more sustainable feed ingredients in aquaculture. We previously demonstrated that proliferation or differentiation of rainbow trout intestinal cell lines is dictated by the culture environment. The aim of the present work was to develop a culture platform that can efficiently promote cell differentiation into mature enterocytes. We compared four options, seeding the RTpiMI cell line derived from the proximal intestine on (1) polyethylene terephthalate (PET) culture inserts ThinCert™ (TC), (2) TC coated with the solubilized basement membrane matrix Matrigel® (MM), (3) TC with the rainbow trout fibroblast cell line RTskin01 embedded within the Matrigel® matrix (MMfb), or (4) the highly porous polystyrene scaffold Alvetex® populated with the abovementioned fibroblast cell line (AV). We evaluated the presence of columnar cells with a clear polarization of brush border enzymes, the formation of an efficient barrier with a significant increase in transepithelial electrical resistance (TEER), and its ability to prevent the paracellular flux of large molecules but allow the transit of small compounds (proline and glucose) from the apical to the basolateral compartment. All parameters improved moving from the simplest (TC) through the more complex platforms. The presence of fibroblasts was particularly effective in enhancing epithelial cell differentiation within the AV platform recreating more closely the complexity of the intestinal mucosa, including the presence of extracellular vesicles between fibroblasts and epithelial cells.

Postnatal bone fractures of the growth plate (GP) are often associated with regenerative complications such as growth impairment. In order to understand the underlying processes of trauma-associated growth impairment within postnatal bone, an ex vivo rat femur slice model was developed. To achieve this, a 2 mm horizontal cut was made through the GP of rat femur prior to the organotypic culture being cultivated for 15 days in vitro. Histological analysis showed disrupted endochondral ossification, including disordered architecture, increased chondrocyte metabolic activity, and a loss of hypertrophic zone throughout the distal femur. Furthermore, altered expression patterns of Col2α1, Acan, and ColX, and increased chondrocyte metabolic activity in the TZ and MZ at day 7 and day 15 postinjury were observed. STEM revealed the presence of stem cells, fibroblasts, and chondrocytes within the injury site at day 7. In summary, the findings of this study suggest that the ex vivo organotypic GP injury model could be a valuable tool for investigating the underlying mechanisms of GP regeneration post-trauma, as well as other tissue engineering and disease studies.

Alzheimer’s disease is the most common type of dementia with cognitive impairment. Various plant-derived phenolics are known to alleviate cognitive impairment in Alzheimer’s disease by radical scavenging and strengthening synaptic plasticity activities. Here, we examined the cognition-improving effect of Pinus densiflora Sieb. et Zucc. bark extract (PBE). We identified and quantified phenolics in the PBE using a UHPLC-Orbitrap mass spectrometer. To evaluate the cognition-enhancing effects of PBE, scopolamine-induced amnesic Sprague-Dawley (SD) rats (5 weeks old) and ion channel antagonist-induced organotypic hippocampal slices of SD rats (7 days old) were used. Twenty-three phenolics were tentatively identified in PBE, 10 of which were quantified. Oral administration of PBE to the scopolamine-induced SD rats improved cognitive impairment in behavioral tests. PBE-fed SD rats showed significantly improved antioxidant indices (superoxide dismutase and catalase activities, and malondialdehyde content) and reduced acetylcholinesterase activity in hippocampal lysate compared with the scopolamine group. PBE increased the long-term potentiation (LTP) induction and rescued LTP from blockades by the muscarinic cholinergic receptor antagonist (scopolamine) and N-methyl-D-aspartate channel antagonist (2-amino-5-phosphonovaleric acid) in the organotypic hippocampal slices. These results suggest that polyphenol-rich PBE is applicable as a cognition-improving agent due to its antioxidant properties and enhancement of LTP induction.

The numerous neurological syndromes associated with COVID-19 implicate an effect of viral pathogenesis on neuronal function, yet reports of direct SARS-CoV-2 infection in the brain are conflicting. We used a well-established organotypic brain slice culture to determine the permissivity of hamster brain tissues to SARS-CoV-2 infection. We found levels of live virus waned after inoculation and observed no evidence of cell-to-cell spread, indicating that SARS-CoV-2 infection was non-productive. Nonetheless, we identified a small number of infected cells with glial phenotypes; however, no evidence of viral infection or replication was observed in neurons. Our data corroborate several clinical studies that have assessed patients with COVID-19 and their association with neurological involvement.

Epigallocatechin gallate (EGCG) is a major flavan-3-ol of green tea polyphenols that exhibits various beneficial health effects, including antioxidant, anti-bacterial, and anti-inflammatory properties. This study aimed to evaluate whether EGCG prevents scopolamine-induced learning and memory impairment in in vivo and ex vivo models. Male Sprague-Dawley (SD) rats were pre-treated with EGCG (5 mg/kg/day; intraperitoneal injection (i.p.)) for 10 days. Then, EGCG and scopolamine (1 mg/kg/day; i.p.) were applied 60 and 30 min before the behavioral tests, respectively, for another 9 days. EGCG alleviated the cognitive deficits in the Y-maze, passive avoidance, and Morris water maze tests. EGCG showed improved cholinergic functions by decreasing acetylcholinesterase activity in hippocampi dissected from the brain of the rats after the behavioral tests. EGCG also reduced oxidative stress, partly due to increased superoxide dismutase activity and decreased malondialdehyde level in the hippocampi of the rat brains after the behavioral tests. Furthermore, EGCG attenuated the scopolamine-induced blockade of long-term potentiation in organotypic hippocampal tissue of seven-day-old SD rats. Taken together, these results suggested that EGCG is a potential therapeutic agent for alleviating cognitive dysfunction.

Printed circuit board (PCB) technology is well known, reliable, and low-cost, and its application to biomedicine, which implies the integration of microfluidics and electronics, has led to Lab-on-PCB. However, the biocompatibility of the involved materials has to be examined if they are in contact with biological elements. In this paper, the solder mask (PSR-2000 CD02G/CA-25 CD01, Taiyo Ink (Suzhou) Co., Ltd., Suzhou, China) of a commercial PCB has been studied for retinal cultures. For this purpose, retinal explants have been cultured over this substrate, both on open and closed systems, with successful results. Cell viability data shows that the solder mask has no cytotoxic effect on the culture allowing the application of PCB as the substrate of customized microelectrode arrays (MEAs). Finally, a comparative study of the biocompatibility of the 3D printer Uniz zSG amber resin has also been carried out.

Atopic dermatitis (AD) is characterized by dry and itchy skin evolving into disseminated skin lesions. AD is believed to result from a primary acquired or a genetically-induced epidermal barrier defect leading to immune hyper-responsiveness. Filaggrin (FLG) is a protein found in the cornified envelope of fully differentiated keratinocytes, referred to as corneocytes. Although FLG null mutations are strongly associated with AD, they are not sufficient to induce the disease. Moreover, most patients with ichthyosis vulgaris (IV), a monogenetic skin disease characterized by FLG homozygous, heterozygous, or compound heterozygous null mutations, display non-inflamed dry and scaly skin. Thus, all causes of epidermal barrier impairment in AD have not yet been identified, including those leading to the Th2-predominant inflammation observed in AD. Three dimensional organotypic cultures have emerged as valuable tools in skin research, replacing animal experimentation in many cases and precluding the need for repeated patient biopsies. Here, we review the results on IV and AD obtained with epidermal or skin equivalents and consider these findings in the context of human in vivo data. Further research utilizing complex models including immune cells and cutaneous innervation will enable finer dissection of the pathogenesis of AD and deepen our knowledge of epidermal biology.

Macroalgae represent an important source of bioactive compounds with a wide range of biotechnological applications. Overall, the discovery of effective cytotoxic compounds with pharmaceutical potential is a significant challenge, mostly because they are scarce in nature or their total synthesis is not efficient, while the bioprospecting models currently used do not predict clinical responses. Given this context, we used three-dimensional (3D) cultures of human breast cancer explants to evaluate the antitumoral effect of laurinterol, the major compound of an ethanolic extract of Laurencia johnstonii. To this end, we evaluated the metabolic and histopathological effects of the crude extract of L. johnstonii and laurinterol on Vero and MCF-7 cells, in addition to breast cancer explants. We observed a dose-dependent inhibition of the metabolic activity, as well as morphologic and nuclear changes characteristic of apoptosis. On the other hand, a reduced metabolic viability and marked necrosis areas were observed in breast cancer explants incubated with the crude extract, while explants treated with laurinterol exhibited a heterogeneous response which was associated with the individual response of each human tumor sample. This study supports the cytotoxic and antitumoral effects of laurinterol in in vitro cell cultures and in ex vivo organotypic cultures of human breast cancer explants.

The process of identifying and approving a new drug is a time-consuming and expensive procedure. One of the biggest issues to overcome is the risk of hepatotoxicity, which is one of the main reasons for drug withdrawal from the market. While animal models are the gold standard in preclinical drug testing, the translation of results into therapeutic intervention is often ambiguous due to interspecies differences in hepatic metabolism. The discovery of human induced pluripotent stem cells (hiPSCs) and their derivatives has opened new possibilities for drug testing. We used mesenchymal stem cells and hepatocytes both derived from hiPSCs, together with endothelial cells, to miniaturize the process of generating hepatic organoids. These organoids were then cultivated in vitro using both static and dynamic cultures. Additionally, we tested spheroids solely composed by induced hepatocytes. By miniaturizing the system, we demonstrated the possibility of maintaining the organoids, but not the spheroids, in culture for up to 1 week. This timeframe may be sufficient to carry out a hypothetical pharmacological test or screening. In conclusion, we propose that the hiPSC-derived liver organoid model could complement or, in the near future, replace the pharmacological and toxicological tests conducted on animals.

Interpenetrating polymer networks (IPNs) have gained great attention for a number of biomedical applications due to their improved properties compared to individual components alone. In this study, we investigated the capacity of newly-developed naturally-derived IPNs as potential biomaterials for tissue engineering. These IPNs combine the biologic properties of a fibrous fibrin network polymerized at the nanoscale and the mechanical stability of polyethylene oxide (PEO). First, we assessed their cytotoxicity in vitro on L929 fibroblasts. We further evaluated their biocompatibility ex vivo with a chick embryo organotypic culture model. Subcutaneous implantations of the matrices were subsequently conducted on nude mice to investigate their biocompatibility in vivo. Our preliminary data highlighted that our biomaterials were non-cytotoxic (viability above 90%). The organotypic culture showed that the IPN matrices induced higher cell adhesion (across all the explanted organ tissues) and migration (skin, intestine) than the control groups, suggesting the advantages of using a biomimetic, yet mechanically-reinforced IPN-based matrix. We observed no major inflammatory response up to 12 weeks post implantation. All together, these data suggest that these fibrin-based IPNs are promising biomaterials for tissue engineering.

Alzheimer's disease is the most common type of dementia with cognitive impairment. Various plant-derived phenolics are known to alleviate cognitive impairment in Alzheimer's disease by radical scavenging and strengthening synaptic plasticity activities. Here, we examined the cognition-improving effect of Pinus densiflora Sieb. et Zucc. bark extract (PBE). We identified and quantified phenolics in the PBE using a UHPLC-Orbitrap mass spectrometer. To evaluate the cognition-enhancing effects of PBE, scopolamine-induced amnesic Sprague-Dawley (SD) rats (5 weeks old) and ion channel antagonist-induced organotypic hippocampal slices of SD rats (7 days old) were used. Twenty-three phenolics were tentatively identified in PBE, 10 of which were quantified. Oral administration of PBE to the scopolamine-induced SD rats improved cognitive impairment in behavioral tests. PBE-fed SD rats showed significantly improved antioxidant indices (superoxide dismutase and catalase activities, and malondialdehyde content) and reduced acetylcholinesterase activity in hippocampal lysate compared with the scopolamine group. PBE increased the long-term potentiation (LTP) induction and rescued LTP from blockades by the muscarinic cholinergic receptor antagonist (scopolamine) and N -methyl-D-aspartate channel antagonist (2-amino-5-phosphonovaleric acid) in the organotypic hippocampal slices. These results suggest that polyphenol-rich PBE is applicable as a cognition-improving agent due to its antioxidant properties and enhancement of LTP induction.

Epigallocatechin gallate (EGCG) is a major flavan-3-ol of green tea polyphenols that exhibits various beneficial health effects, including antioxidant, anti-bacterial, and anti-inflammatory properties. This study aimed to evaluate whether EGCG prevents scopolamine-induced learning and memory impairment in in vivo and ex vivo models. Male Sprague-Dawley (SD) rats were pre-treated with EGCG (5 mg/kg/day; intraperitoneal injection (i.p.)) for 10 days. Then, EGCG and scopolamine (1 mg/kg/day; i.p.) were applied 60 and 30 min before the behavioral tests, respectively, for another 9 days. EGCG alleviated the cognitive deficits in the Y-maze, passive avoidance, and Morris water maze tests. EGCG showed improved cholinergic functions by decreasing acetylcholinesterase activity in hippocampi dissected from the brain of the rats after the behavioral tests. EGCG also reduced oxidative stress, partly due to increased superoxide dismutase activity and decreased malondialdehyde level in the hippocampi of the rat brains after the behavioral tests. Furthermore, EGCG attenuated the scopolamine-induced blockade of long-term potentiation in organotypic hippocampal tissue of seven-day-old SD rats. Taken together, these results suggested that EGCG is a potential therapeutic agent for alleviating cognitive dysfunction.

Interpenetrating polymer networks (IPNs) have gained great attention for a number of biomedical applications due to their improved properties compared to individual components alone. In this study, we investigated the capacity of newly-developed naturally-derived IPNs as potential biomaterials for tissue engineering. These IPNs combine the biologic properties of a fibrous fibrin network polymerized at the nanoscale and the mechanical stability of polyethylene oxide (PEO). First, we assessed their cytotoxicity in vitro on L929 fibroblasts. We further evaluated their biocompatibility ex vivo with a chick embryo organotypic culture model. Subcutaneous implantations of the matrices were subsequently conducted on nude mice to investigate their biocompatibility in vivo. Our preliminary data highlighted that our biomaterials were non-cytotoxic (viability above 90%). The organotypic culture showed that the IPN matrices induced higher cell adhesion (across all the explanted organ tissues) and migration (skin, intestine) than the control groups, suggesting the advantages of using a biomimetic, yet mechanically-reinforced IPN-based matrix. We observed no major inflammatory response up to 12 weeks post implantation. All together, these data suggest that these fibrin-based IPNs are promising biomaterials for tissue engineering., Competing Interests: The authors declare no conflict of interest.