Cryopreserved human precision-cut lung slices provide an immune competent pulmonary test system for "on-demand" use and long-term cultures.

Human precision-cut lung slices (hPCLS), considered a highly relevant ex vivo model of the lung, offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells. However, the irregular availability of donor lungs has limited the accessibility of this system. As described here, thousands of hPCLS can be created from 1 lung, cryopreserved, and used "on demand" by applying slicing and cryopreservation methodology improvements. Fresh and cryopreserved (∼7 and ∼34 weeks; F&C) hPCLS from 1 donor lung were cultured for up to 29 days and evaluated for biomass, viability, tissue integrity, and inflammatory markers in response to lipopolysaccharide (LPS; 5 µg/ml) and Triton X-100 (TX100; 0.1%) challenge (24 h) at days 1, 8, 15, 22, and 29 following culture initiation. The F&C hPCLS retained biomass, viability, and tissue integrity throughout the 29 days and demonstrated immune responsiveness with up to ∼30-fold LPS-induced cytokine increases. Histologically, more than 70% of normal cytomorphological features were preserved in all groups through day 29. Similar retention of tissue viability and immune responsiveness post cryopreservation (4-6 weeks) and culture (up to 14 days) was observed in hPCLS from additional 3 donor lungs. Banking cryopreserved hPCLS from various donors (and disease states) provides a critical element in researching human-derived pulmonary tissue. The retention of viability and functional responsiveness (≥4 weeks) allows evaluation of long-term, complex endpoints reflecting key events in Adverse Outcome Pathways and positions hPCLS as a valuable human-relevant model for use in regulatory applications.

Ciliary Beat Frequency: Proceedings and Recommendations from a Multi-laboratory Ring Trial Using 3-D Reconstituted Human Airway Epithelium to Model Mucociliary Clearance.

The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used - Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.

Validation of the Psychosocial Impact of Dental Aesthetics Questionnaire in the Australian population.

INTRODUCTION: The Psychosocial Impact of Dental Aesthetics Questionnaire (PIDAQ) is an instrument to assess the subjective perception need for orthodontic treatment. The study aimed to determine the psychometric validity and reliability of PIDAQ in Australian adults. METHODS: Data were collected from the National Dental Telephone Interview Survey in 2013. The PIDAQ was used, comprising 4 conceptual domains: self-confidence, social impact, psychological impact, and esthetic concern. Psychometric sensitivity was estimated for each item. Confirmatory factor analysis was performed to determine factorial validity in which the ratio of chi-square by degrees of freedom, comparative fit, and root mean square error of approximation were used as indexes of the goodness of fit. Convergent validity was estimated from the average variance extracted and composite reliability, whereas internal consistency was estimated by Cronbach standardized alpha. RESULTS: The dataset comprised 2936 Australian adults aged ≥18 years and separated randomly into 2 subdata sets. The kurtosis and skewness values indicate an approximation to a normal distribution for each item, with acceptable thresholds for the goodness of fit and convergent validity (average variance extracted ≥0.50 and composite reliability ≥0.70), and discriminant validity was also demonstrated. Internal consistency was adequate in the 4 conceptual domains for PIDAQ for both subdata sets, with Cronbach standardized alpha being ≥0.70. CONCLUSIONS: The psychometric sensitivity, validity, and reliability of the PIDAQ instrument in a sample of the Australian adult population were found to be adequate. Both subdata sets drawn from the sample demonstrated acceptable goodness of fit and internal consistency.

Comparison of Long-term Human Precision-cut Lung Slice Culture Methodology and Response to Challenge: An Argument for Standardisation.

As non-animal alternatives gain acceptance, a need for harmonised testing strategies has emerged. Arguably the most physiologically-relevant model for assessing potential respiratory toxicants, that based on human precision-cut lung slices (hPCLS) has been utilised in many laboratories, but a variety of culture methodologies are employed. In this pilot study, combinations of three different hPCLS culture methods (dynamic organ roller culture (DOC), air-liquid interface (ALI) and submersion) and various media (based on E-199, DMEM/F12 and RPMI-1640) were compared. The hPCLS were assessed in terms of their viability and responsiveness to challenge. The endpoints selected to compare the medium-method (M-M) combinations, which included histological features and viability, were evaluated at day 14 (D14) and day 28 (D28); protein and adenylate kinase (AK) content, and cytokine response to immunostimulants (lipopolysaccharide (LPS) at 5 µg/ml; polyinosinic:polycytidylic acid (Poly I:C) at 15 µg/ml) were evaluated at D28 only. Based on the set of endpoints assessed at D28, it was clear that certain culture conditions significantly affected the hPCLS, with the tissue retaining more of its native features and functionality (in terms of cytokine response) in some of the M-M combinations tested more than others. This pilot study indicates that the use of appropriate M-M combinations can help maintain the health and functional responses of hPCLS, and highlights the need for the standardisation of culture conditions in order to facilitate effective inter-laboratory comparisons and encourage greater acceptance by the regulatory community.

Modelling risk factors for high/low Psychosocial Impact of Dental Aesthetics Questionnaire (PIDAQ) in the Australian adults.

OBJECTIVE: Psychosocial Impact of Dental Aesthetics Questionnaire (PIDAQ) is an instrument to assess the subjective perception needed for orthodontic treatment (OT). The study aimed to (1) evaluate the psychosocial impact of dental aesthetics and the potential role of OT and (2) determine associations between PIDAQ with self-rated general and oral health, oral health-related quality of life (OHRQOL), and receipt of OT among Australian adults. METHODS: Data were obtained from the 2013 Australian National Dental Telephone Interview Survey (NDTIS). The PIDAQ was utilized as main outcome, comprising items from four conceptual domains: self-confidence, social impact, psychological impact, and aesthetic concern. Participants reported their socio-demographic, behavioural, self-rated general and oral health as well as the poor OHRQOL using the Oral Impact on Daily Performance (OIDP) instrument and OT. The mean of PIDAQ and four subscales score and their 95% confidence intervals (CI) were estimated. Generalized liner regression was used to estimate association between PIDAQ and covariates by calculating the mean ratio (MR) and their 95% CI. RESULTS: There were 2936 Australian adults who completed the PIDAQ items. The mean PIDAQ score was 28.8 (95% CI: 28.0-29.6). Higher PIDAQ scores were identified among those who reported non-Australian birth, lower incomes, infrequent dental visits, less than 21 teeth and brushing teeth less than twice a day, fair/poor self-rated general and oral health, and poor OHRQOL. In multivariable modelling, the mean PIDAQ score was 1.91 (95% CI: 1.82-2.00) times higher among those with OIDP score >3 and 1.06 (95% CI:1.01-1.10) times higher in those who had OT, comparing with their counterparts. Also, higher PIDAQ scores were identified among females, last visiting a dentist more than 2 years ago, fair or poor self-rated oral and general health. CONCLUSIONS: Higher PIDAQ scores that have a greater impact on OHRQOL were associated with poorer self-rated general and oral health. This may be explained by unfavourable dental attendance and oral health habits. The positive correlation with OIDP scores verifies the validity of the PIDAQ. Those who had received OT had lower PIDAQ and OIDP scores.