Robust, efficient workflow to establish, culture, and functionally assess primary-isolated airway epithelial cells

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Method(s): Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, aswell as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. Oneweek later, the mediumwas changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Result(s): Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 +/- 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 +/- 9%, n = 3). Conclusion(s): In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Highly Expandable Human Alveolar Organoids to Study Respiratory Diseases

Organoids are emerging to be an excellent tool for studying human development and disease. The COVID-19 pandemic has highlighted the importance of physiologically relevant alveolar infection models that include both alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. To address the need for an alveolar organoid culture system for respiratory research, we developed the PneumaCult™ Alveolar Organoid Expansion and Differentiation Media for the highly efficient expansion of isolated primary human AT2 cells and subsequent differentiation into AT1 cells. Alveolar organoids were established from a panel of various donors (n=5) by culturing purified human AT2 cells in Corning® Matrigel® domes with serum-free PneumaCult™ Alveolar Organoid Expansion Medium. Typically by day 10-14 the organoids are fully established and display a spherical morphology. Alveolar organoids can then be either expanded long-term by passaging cultures as single cells in Expansion Medium or differentiated into AT1 cells using the PneumaCult™ Alveolar Organoid Differentiation Medium. Organoids in PneumaCult™ Alveolar Organoid Expansion Medium contain self-renewing AT2 cells marked by the expression of HT2-280 in 89.9 +/- 14.5 (mean +/- SD;n=5 donors) of cells and the presence of Pro-SPC, demonstrate a great expansion potential of > 10,000-fold with more than 13 population doublings within 10 passages (n=5 donors). Alveolar organoids differentiated for 10 days in the PneumaCult™ Alveolar Organoid Differentiation Medium downregulate AT2 markers HT2-280 and Pro-SPC and start expressing AT1 markers HT1-56 in 93.8 +/- 7.2 (mean +/- SD;n=5 donors) of cells and are positive for RAGE and GPRC5a. Furthermore, we assessed the expression of SARS-CoV-2 entry receptor ACE2, which is present in both undifferentiated and differentiated alveolar organoids.To investigate the use of these alveolar organoids for infectious disease modeling, AT2-containing alveolar organoids were transduced with a GFP-labelled Respiratory Syncytial Virus (RSV). Alveolar organoids were susceptible to viral infection and replication was confirmed by fluorescence microscopy and quantitative PCR. In summary, the PneumaCult™ Alveolar Organoid Expansion and Differentiation Media are highly efficient and reproducible tools for the feeder-free expansion of AT2 cells and robust differentiation into AT1 cells, which can be used for infectious disease modeling.

COUNTERING LINGUISTIC RIGHTS IN SPAIN THROUGH LANGUAGE MAKING: THE DISCURSIVE DELEGITIMIZATION OF BASQUE IN ONLINE DEBATES

This article investigates discourses on the Basque language in Spain with a particular focus on delegitimizing positions towards the regional language. It identifies the most prominent patterns of the discourse in order to gain a better understanding of the argumentative strategies which are used to reject an intensified promotion of Basque. The analysis is empirically based on a sample of comments from online debates on news websites reporting on recent legislation for the use of Basque in local administration and on the position of Basque in regional education during the COVID-19 pandemic. Drawing on the concepts of delegitimization and language making, the paper shows how the rejection of enhanced linguistic rights for speakers of the minoritized language is interlinked with the co-construction of both Basque and Castilian: regional language policy is portrayed as the imposition of a supposedly irrelevant language against the indisputable national language as part of an alleged nationalist agenda serving the interests of elite profiteers.

A rare case of hemorrhagic cholecystitis in a patient with severe coronavirus disease 19 (COVID-19)

Hemorrhagic cholecystitis is a rare end-stage manifestation of acalculous cholecystitis that is associated with high mortality. The hematological sequelae of coronavirus disease 19 (COVID-19) are complex and are associated with increased incidence of both thromboembolic and hemorrhagic events. Our patient is a 69-yearold male with hypertension and gout who was admitted to the medical intensive care unit with severe COVID-19 pneumonia and lactic acidosis. The patient was treated with a therapeutic low-intensity heparin infusion per institutional COVID-19 anticoagulation protocol based on elevated D-dimer (15,217 ng/mL). Computed tomography (CT) of the abdomen and pelvis on hospital day 1 and day 4 were negative for acute pathology. On hospital day 9, the patient developed shock and acute blood loss, and repeat CT of the abdomen showed new hemorrhagic cholecystitis. Heparin infusion was stopped, and acute care surgery consultants recommended against emergent surgery given the patient's hemodynamic instability. The patient was stabilized with blood product resuscitation and antibiotics. Laparoscopy prior to discharge showed heavy intra-abdominal adhesions with friable tissue, and the cholecystectomy attempt was aborted. He was discharged and was seen in clinic for follow-up, where he reported no abdominal pain and good functional status. Systemic coagulation dysfunction related to COVID-19 presents significant challenges to clinicians balancing the competing risks of acute thrombosis and bleeding. Hemorrhagic cholecystitis is a very uncommon complication of gallbladder disease, and reports linked to active COVID-19 are even more scarce. The gallbladder contains a particularly high expression of angiotensin I converting enzyme 2 (ACE2). This pattern is known to be vital for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to enter cells, but direct end-organ damage from the virus outside of the respiratory system remains an active area of research. Our patient did not have radiographic gallbladder disease present on admission, but decompensated as a result of hemorrhage early in his COVID-19 course. Here, we present an unusual manifestation of a rare disease, with spontaneous hemorrhagic cholecystitis occurring a patient with severe COVID-19.

New post-COVID-19 biosafety protocols in pediatric dentistry

The most recent Severe Acute Respiratory Syndrome-COVID-19-caused by coronavirus infection (SARS-CoV-2) has high-virulence transmission and direct human contagiousness by proximity. Thus, the considerable occupational risk in pediatric dentistry is evident, given the nature and form of procedures performed in an outpatient setting. Thus, the aim of this paper was to identify and contextualize technical and scientific information available to date aimed at preventing and minimizing risks for patients, caregivers and professionals. The results indicate that protective measures are being developed considering procedures according to risks and benefits, and five points stand out: 1. Regulation of resumption of elective procedures, screening and scheduling patients;2. Restructuring clinical environment and infection control;3. Improvement of personal protective equipment and biosafety recommendations;4. Maximization of the use of non-invasive techniques, use of high-powered dental suction, and absolute isolation of the operative field;and 5. Minimization of the use of air-water syringe, dental spittoon and high-speed handpiece. The measures to be taken require reflection for the restart of a “new clinical practice”, especially aiming at behavioral and structural changes regarding operational biosafety.