Three-Dimensional Humanized Model of the Periodontal Gingival Pocket to Study Oral Microbiome.

The oral cavity contains distinct microenvironments that serve as oral barriers, such as the non-shedding surface of the teeth (e.g., enamel), the epithelial mucosa and gingival tissue (attached gingiva) where microbial communities coexist. The interactions and balances between these communities are responsible for oral tissue homeostasis or dysbiosis, that ultimately dictate health or disease. Disruption of this equilibrium can lead to chronic inflammation and permanent tissue damage in the case of chronic periodontitis. There are currently no experimental tissue models able to mimic the structural, physical, and metabolic conditions present in the human oral gingival tissue to support the long-term investigation of host-pathogens imbalances. Herein, the authors report an in vitro 3D anatomical gingival tissue model, fabricated from silk biopolymer by casting a replica mold of an adult human mandibular gingiva to recreate a tooth-gum unit. The model is based on human primary cultures that recapitulate physiological tissue organization, as well as a native oxygen gradient within the gingival pocket to support human subgingival plaque microbiome with a physiologically relevant level of microbial diversity up to 24 h. The modulation of inflammatory markers in the presence of oral microbiome indicates the humanized functional response of this model and establishes a new set of tools to investigate host-pathogen imbalances in gingivitis and periodontal diseases.

In Vitro Nasal Tissue Model for the Validation of Nasopharyngeal and Midturbinate Swabs for SARS-CoV-2 Testing.

Large-scale population testing is a key tool to mitigate the spread of respiratory pathogens, such as the current COVID-19 pandemic, where swabs are used to collect samples in the upper airways (e.g., nasopharyngeal and midturbinate nasal cavities) for diagnostics. However, the high volume of supplies required to achieve large-scale population testing has posed unprecedented challenges for swab manufacturing and distribution, resulting in a global shortage that has heavily impacted testing capacity worldwide and prompted the development of new swabs suitable for large-scale production. Newly designed swabs require rigorous preclinical and clinical validation studies that are costly and time-consuming (i.e., months to years long); reducing the risks associated with swab validation is therefore paramount for their rapid deployment. To address these shortages, we developed a 3D-printed tissue model that mimics the nasopharyngeal and midturbinate nasal cavities, and we validated its use as a new tool to rapidly test swab performance. In addition to the nasal architecture, the tissue model mimics the soft nasal tissue with a silk-based sponge lining, and the physiological nasal fluid with asymptomatic and symptomatic viscosities of synthetic mucus. We performed several assays comparing standard flocked and injection-molded swabs. We quantified the swab pickup and release and determined the effect of viral load and mucus viscosity on swab efficacy by spiking the synthetic mucus with heat-inactivated SARS-CoV-2 virus. By molecular assay, we found that injected molded swabs performed similarly or superiorly in comparison to standard flocked swabs, and we underscored a viscosity-dependent difference in cycle threshold values between the asymptomatic and symptomatic mucuses for both swabs. To conclude, we developed an in vitro nasal tissue model that corroborated previous swab performance data from clinical studies; this model will provide to researchers a clinically relevant, reproducible, safe, and cost-effective validation tool for the rapid development of newly designed swabs.

Long-Term In Vitro Culture Systems to Study Human Microbiome.

Microbial communities are eubiotic ecosystems that interact dynamically and synergistically with the human body. Imbalances in these interactions may cause dysbiosis by enhancing the occurrence of inflammatory conditions, such as periodontal or inflammatory bowel diseases. However, the mechanisms that lie behind eubiosis-dysbiosis transitions are still unclear and constantly being redefined. While the societal impact of these diseases is steadily increasing, the lack of a clear understanding behind the onset of the inflammatory conditions prevents the proper clinical strategies from being formulated. Although preclinical and clinical models and short-term planar in vitro cultures represent superb research tools, they are still lacking human relevance and long-term use. Bioreactors and organs-on-a-chip have attracted interest because of their ability to recreate and sustain the physical, structural, and mechanical features of the native environment, as well as to support long-term coculture of mammalian cells and the microbiome through modulation of pH and oxygen gradients. Existing devices, however, are still under development to sustain the microbiome-host coculture over long periods of time. In this scenario, to understand disease triggers and develop therapeutics, research efforts should command the development of three-dimensional constructs that would allow the investigation of processes underlying the microbial community assembly and how microorganisms influence host traits in both acute and chronic conditions.

Impact of Membrane Voltage on Formation and Stability of Human Renal Proximal Tubules in Vitro.

More than 15% of adults in the United States suffer from some form of chronic kidney disease (CKD). Current strategies for CKD consist of dialysis or kidney transplant, which, however, can take several years. In this light, tissue engineering and regenerative medicine approaches are the key to improving people's living conditions by advancing previous tissue engineering approaches and seeking new targets as intervention methods for kidney repair or replacement. The membrane voltage (Vm) dynamics of a cell have been associated with cell migration, cell cycle progression, differentiation, and pattern formation. Furthermore, bioelectrical stimuli have been used as a means in the treatment of diseases and wound healing. Here, we investigated the role of Vm as a novel target to guide and manipulate in vitro renal tissue models. Human-immortalized renal proximal tubule epithelial cells (RPTECs-TERT1) were cultured on Matrigel to support the formation of 3D proximal tubular-like structures with the incorporation of a voltage-sensitive dye indicator─bis-(1,3-dibutylbarbituric acid)timethine oxonol (DiBAC). The results demonstrated a correlation between the depolarization and the reorganization of human renal proximal tubule cells, indicating Vm as a candidate variable to control these events. Accordingly, Vm was pharmacologically manipulated using glibenclamide and pinacidil, KATP channel modulators, and proximal tubule formation and tubule stability over 21 days were assessed. Chronic manipulation of KATP channels induced changes in the tubular network topology without affecting lumen formation. Thus, a relationship was found between the preluminal tubulogenesis phase and KATP channels. This relationship may provide future options as a control point during kidney tissue development, treatment, and regeneration goals.

Preclinical Validation of a Novel Injection-Molded Swab for the Molecular Assay Detection of SARS-CoV-2.

During the COVID-19 public health emergency, many actions have been undertaken to help ensure that patients and health care providers have timely and continued access to high-quality medical devices to respond effectively. The development and validation of new testing supplies and equipment, including collection swabs, has helped to expand the availability and capability for various diagnostic, therapeutic, and protective medical devices in high demand during the COVID-19 emergency. Here, we report the initial validation of a new injection-molded anterior nasal swab, ClearTip™, that was experimentally validated in a laboratory setting as well as in independent clinical studies in comparison to gold standard flocked swabs. We have also developed an in vitro anterior nasal tissue model which offers a novel, efficient, and clinically relevant validation tool to replicate the clinical swabbing workflow with high fidelity, while being accessible, safe, reproducible, and time- and cost-effective. ClearTip™ displayed greater inactivated virus release in the benchtop model, confirmed by its greater ability to report positive samples in a small clinical study in comparison to flocked swabs. We also quantified the detection of biological materials, as a proxy for viral material, in multi-center pre-clinical and clinical studies which showed a statistically significant difference in one study and a reduction in performance in comparison to flocked swabs. Taken together, these results emphasize the compelling benefits of non-absorbent injection-molded anterior nasal swabs for COVID-19 detection, comparable to standard flocked swabs. Injection-molded swabs, as ClearTip™, could have the potential to support future swab shortages, due to its manufacturing advantages, while offering benefits in comparison to highly absorbent swabs in terms of comfort, limited volume collection, and potential multiple usage.

In Vitro Nasal Tissue Model for the Validation of Nasopharyngeal and Mid-turbinate Swabs for SARS-CoV-2 Testing.

Large-scale population testing is a key tool to mitigate the spread of respiratory pathogens, as in the current COVID-19 pandemic, where swabs are used to collect samples in the upper airways (e.g. nasopharyngeal and mid-turbinate nasal cavities) for diagnostics. However, the high volume of supplies required to achieve large-scale population testing has posed unprecedented challenges for swab manufacturing and distribution, resulting in a global shortage that has heavily impacted testing capacity world-wide and prompted the development of new swabs suitable for large-scale production. Newly designed swabs require rigorous pre-clinical and clinical validation studies that are costly and time consuming ( i . e . months to years long); reducing the risks associated with swab validation is therefore paramount for their rapid deployment. To address these shortages, we developed a 3D-printed tissue model that mimics the nasopharyngeal and mid-turbinate nasal cavities, and we validated its use as a new tool to rapidly test swab performance. In addition to the nasal architecture, the tissue model mimics the soft nasal tissue with a silk-based sponge lining, and the physiological nasal fluid with asymptomatic and symptomatic viscosities of synthetic mucus. We performed several assays comparing standard flocked and injection-molded swabs. We quantified the swab pick-up and release, and determined the effect of viral load and mucus viscosity on swab efficacy by spiking the synthetic mucus with heat-inactivated SARS-CoV-2 virus. By molecular assays, we found that injected molded swabs performed similarly or superiorly in comparison to standard flocked swabs and we underscored a viscosity-dependent difference in cycle threshold values between the asymptomatic and symptomatic mucus for both swabs. To conclude, we developed an in vitro nasal tissue model, that corroborated previous swab performance data from clinical studies, with the potential of providing researchers with a clinically relevant, reproducible, safe, and cost-effective validation tool for the rapid development of newly designed swabs.

Defined extracellular ionic solutions to study and manipulate the cellular resting membrane potential.

All cells possess an electric potential across their plasma membranes and can generate and receive bioelectric signals. The cellular resting membrane potential (RMP) can regulate cell proliferation, differentiation and apoptosis. Current approaches to measure the RMP rely on patch clamping, which is technically challenging, low-throughput and not widely available. It is therefore critical to develop simple strategies to measure, manipulate and characterize the RMP. Here, we present a simple methodology to study the RMP of non-excitable cells and characterize the contribution of individual ions to the RMP using a voltage-sensitive dye. We define protocols using extracellular solutions in which permeable ions (Na+, Cl- and K+) are substituted with non-permeable ions [N-Methyl-D-glucamine (NMDG), gluconate, choline, SO42-]. The resulting RMP modifications were assessed with both patch clamp and a voltage sensitive dye. Using an epithelial and cancer cell line, we demonstrate that the proposed ionic solutions can selectively modify the RMP and help determine the relative contribution of ionic species in setting the RMP. The proposed method is simple and reproducible and will make the study of bioelectricity more readily available to the cell biology community.This article has an associated First Person interview with the first author of the paper.