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

2021 ◽  
pp. 026119292110618
Author(s):  
Vivek Patel ◽  
Khalid Amin ◽  
David Allen ◽  
Lindsey Ukishima ◽  
Adam Wahab ◽  
...  
Keyword(s):  
Pilot Study ◽  
Culture Conditions ◽  
Cytokine Response ◽  
Poly I:C ◽  
Slice Culture ◽  
Functional Responses ◽  
Culture Methods ◽  
Animal Alternatives ◽  
Precision Cut Lung Slices ◽  
Air Liquid Interface

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.

scholarly journals CFTR modulator response measurements in subjects with cystic fibrosis using 2D differentiated nasal epithelia converted into spheroids

2021 ◽  
Author(s):  
Gimano D Amatngalim ◽  
Lisa W Rodenburg ◽  
Bente L Aalbers ◽  
Henriette H M Raeven ◽  
Ellen M Aarts ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Airway Epithelial Cells ◽  
Culture Conditions ◽  
Nasal Airway ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Transmembrane Conductance ◽  
Culture Methods ◽  
Air Liquid Interface ◽  
Cystic Fibrosis Transmembrane

Cystic Fibrosis (CF) is caused by genetic defects that impair the cystic fibrosis transmembrane conductance regulator (CFTR) channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be determined in a personalized manner using 3D nasal-brushing-derived airway spheroids in a forskolin-induced swelling (FIS) assay. Despite of this, previously described culture methods and application of 3D airway spheroids in CFTR function assays have not been fully optimal. In this report we describe an alternative method of culturing nasal airway spheroids, which are derived from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. Optimized spheroid culture conditions, enabled consistent detection of CFTR modulator responses in nasal spheroids from subjects with CF, including the highly effective CFTR triple modulator combination therapy VX-661/VX-445/VX-770.

Evaluation of a Domestic Steam Disinfector-Dryer Device for Disinfection of Health Care Workers’ Identification Lanyards

2021 ◽  
pp. 216507992110126
Author(s):  
Beverley C. Millar ◽  
John E. Moore
Keyword(s):  
Health Care ◽  
Health Care Workers ◽  
Culture Conditions ◽  
Broth Culture ◽  
Culture Methods ◽  
Simple Method ◽  
Diverse Range ◽  
Nosocomial Pathogens ◽  
Care Workers ◽  
Test Organisms

Background Fabric lanyards are commonly worn by health care workers (HCWs) and are known to harbor infectious organisms and contribute to the transmission of infection to HCWs and patients. A diverse range of nosocomial pathogens have been found on lanyards, but there are very few studies describing how to successfully disinfect lanyards to break the chain of transmission. Recently, a steam disinfector-dryer device has come on the market, which performs rapid disinfection against nosocomial pathogens and also dries the contents of the device. It was the aim of this study to evaluate steam disinfection-drying as a method to eliminate pathogens from lanyards. Methods Thirty-eight strips of new, unused, and autoclaved polyester neck lanyards (4 × 2 cm) were inoculated with 30 (12 Gram-positive + 18 Gram-negative) bacteria and one yeast organism. The inoculated lanyard fabric (five organisms per lanyard strip) was placed into a steam disinfector-dryer device and disinfected for 5 minutes and dried for 30 minutes, in accordance with the manufacturer’s instructions. Following disinfection and drying, the presence of viable organisms on lanyard fabric was evaluated using enhanced microbiological broth culture methods for 48 hours. Control lanyard strips were treated with organisms and left at room temperature without undergoing disinfection and drying procedures. Findings Steam disinfection-drying eradicated all test organisms from treated lanyards, with no culturable organisms detected following disinfection-drying, even when employing enhanced bacteriological culture conditions. All test organisms remained viable on the control lanyards. Conclusion/Application to Practice Steam disinfection-drying offers a simple method of decontaminating lanyards, producing dry lanyards for immediate reuse. Occupational health practitioners and hospitals should consider assessing the feasibility of adopting this method in their settings to aid in breaking the chain of transmission of nosocomial pathogens via contaminated lanyards.

Acid-fast bacilli smear test of a blood culture sample for the diagnosis of disseminated Mycobacterium genavense infection: A case report

2021 ◽  
Vol 32 (5) ◽  
pp. 483-485
Author(s):  
Yo Murata ◽  
Nobuaki Mori ◽  
Narito Kagawa ◽  
Kentaro Okuma ◽  
Shinji Yoshida ◽  
...  
Keyword(s):  
Blood Culture ◽  
Culture Conditions ◽  
Nontuberculous Mycobacterium ◽  
Sequencing Analysis ◽  
Culture Methods ◽  
Smear Test ◽  
Acid Fast Bacilli ◽  
Mycobacterium Genavense ◽  
Old Japanese ◽  
Immunosuppressed Patients

Mycobacterium genavense, a nontuberculous Mycobacterium, is found in immunosuppressed patients, particularly in those with HIV. Mycobacterium genavense incubation under standard culture conditions is difficult, and its identification is challenging using routine culture methods. Herein, we report the case of a 40-year-old Japanese man with HIV presenting with disseminated M. genavense infection. An analysis using an automated blood culture system did not show positive signals during 6 weeks of incubation. However, an acid-fast bacilli smear of his blood sample was positive for the bacterium. Mycobacterium genavense was identified using sequencing analysis, targeting the heat shock protein 65 gene. The patient recovered from the infection, following antibiotic therapy for 18 months. Under suspicion of disseminated M. genavense infection and the absence of bacterial growth in blood culture samples, an acid-fast bacilli smear test of the sample may be useful for timely diagnosis.

scholarly journals Single Limb Exercise: Pilot Study of Physiological and Functional Responses to Forced Use of the Hemiparetic Lower Extremity

2010 ◽  
Vol 17 (2) ◽  
pp. 128-139 ◽  
Author(s):  
Sandra A. Billinger ◽  
Lisa X. Guo ◽  
Patricia S. Pohl ◽  
Patricia M. Kluding
Keyword(s):  
Pilot Study ◽  
Lower Extremity ◽  
Functional Responses ◽  
Limb Exercise

Culture methods of live algal feeds for European aquaculture: optimising culture conditions for Ulvella lens

2014 ◽  
Vol 22 (6) ◽  
pp. 1813-1822 ◽  
Author(s):  
Colin Hannon ◽  
Rick A. Officer ◽  
Jean Le Dorven ◽  
John Chamberlain
Keyword(s):  
Culture Conditions ◽  
Culture Methods ◽  
European Aquaculture

scholarly journals Testing the Effectiveness of Curcuma longa Leaf Extract on a Skin Equivalent Using a Pumpless Skin-on-a-Chip Model

2020 ◽  
Vol 21 (11) ◽  
pp. 3898 ◽  
Author(s):  
Kyunghee Kim ◽  
Hye Mi Jeon ◽  
Kyung Chan Choi ◽  
Gun Yong Sung
Keyword(s):  
Drug Testing ◽  
Leaf Extract ◽  
Curcuma Longa ◽  
Microfluidic Channel ◽  
Skin Equivalent ◽  
Skin Barrier Function ◽  
In Vitro Tests ◽  
Functional Responses ◽  
Culture Methods ◽  
Pharmaceutical Industries

The in vitro tests in current research employ simple culture methods that fail to mimic the real human tissue. In this study, we report drug testing with a ‘pumpless skin-on-a-chip’ that mimics the structural and functional responses of human skin. This model is a skin equivalent constituting two layers of the skin, dermis and epidermis, developed using human primary fibroblasts and keratinocytes. Using the gravity flow device system, the medium was rotated at an angle of 15 degrees on both sides so as to circulate through the pumpless skin-on-a-chip microfluidic channel. This pumpless skin-on-a-chip is composed of upper and lower chips, and is manufactured using porous membranes so that medium can be diffused and supplied to the skin equivalent. Drug testing was performed using Curcuma longa leaf extract (CLLE), a natural product cosmetic ingredient, to evaluate the usefulness of the chip and the efficacy of the cosmetic ingredient. It was found that the skin barrier function of the skin epidermis layer is enhanced to exhibit antiaging effects. This result indicates that the pumpless skin-on-a-chip model can be potentially used not only in the cosmetics and pharmaceutical industries but also in clinical applications as an alternative to animal studies.

scholarly journals Enhancing innate antiviral immune responses in rainbow trout by double stranded RNA delivered with cationic phytoglycogen nanoparticles

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tamiru N. Alkie ◽  
Jondavid de Jong ◽  
Kristof Jenik ◽  
Karl M. Klinger ◽  
Stephanie J. DeWitte-Orr
Keyword(s):  
Rainbow Trout ◽  
Immune Responses ◽  
Cell Line ◽  
Culture Conditions ◽  
Type I Interferons ◽  
Poly I:C ◽  
Synthetic Analogue ◽  
Type I ◽  
Viral Dsrna

Abstract Innate immunity is induced when pathogen-associated molecular patterns (PAMPs) bind host pattern recognition receptors (PRRs). Polyinosinic:polycytidylic acid [poly(I:C)] is a synthetic analogue of viral dsRNA that acts as a PAMP, inducing type I interferons (IFNs) in vertebrates. In the present study, the immunostimulatory effects of high molecular weight (HMW) poly(I:C) in rainbow trout cells were measured when bound to a cationic phytoglycogen nanoparticle (Nano-HMW). The physical characteristics of the nanoparticle itself, when bound to different lengths of dsRNA and when cell associated was evaluated. Optimal concentration and timing for innate immune stimulation was measured using the RTG-P1 reporter cell line. The immunostimulatory effects of HMW poly (I:C) was compared to Nano-HMW in vitro using the RTgutGC cell line cultured in a conventional monolayer or a transwell culture system. The ability of an activated intestinal epithelium to transmit an antiviral signal to macrophages was evaluated using a co-culture of RTgutGC cells and RTSll (a monocyte/macrophage cell). In all culture conditions, Nano-HMW was a more effective inducer of IFN-related antiviral immune responses compared to HMW poly (I:C) alone. This study introduces the use of cationic phytoglycogen nanoparticles as a novel delivery system for immunomodulatory molecules to enhance immune responses in aquatic vertebrates.

scholarly journals Rupintrivir reduces RV-induced TH-2 cytokine IL-4 in precision-cut lung slices (PCLS) of HDM-sensitized mice ex vivo

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Olga Danov ◽  
Lisa Lasswitz ◽  
Helena Obernolte ◽  
Christina Hesse ◽  
Armin Braun ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Antiviral Drug ◽  
Cytokine Response ◽  
Experimental Conditions ◽  
Tnf Α ◽  
Dust Mite ◽  
Ifn Γ ◽  
Inflammatory Immune Response ◽  
Precision Cut Lung Slices

Abstract Background Antiviral drugs such as rupintrivir may have an immune-modulatory effect in experimentally induced allergic asthma with subsequent RV infection. We infected lung slices of house-dust mite (HDM)-sensitized asthmatic mice ex vivo with human rhinovirus (RV) and investigated the effect of the antiviral drug rupintrivir on RV-induced cytokine response in lung tissue of HDM-sensitized mice ex vivo. Methods Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. Results In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-β, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice. Conclusions In summary, this study demonstrates that treatment with rupintrivir influences virus-induced IL-4 and IL-6 cytokine release under experimental conditions ex vivo.

scholarly journals Differential Sensitivity of Differentiated Epithelial Cells to Respiratory Viruses Reveals Different Viral Strategies of Host Infection

2008 ◽  
Vol 83 (4) ◽  
pp. 1962-1968 ◽  
Author(s):  
Katherina Goris ◽  
Sabine Uhlenbruck ◽  
Christel Schwegmann-Wessels ◽  
Wiebke Köhl ◽  
Frank Niedorf ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Virus Titer ◽  
Airway Epithelial Cells ◽  
Differential Sensitivity ◽  
Airway Epithelial ◽  
Cell Layers ◽  
Syncytial Virus ◽  
Host Infection ◽  
Precision Cut Lung Slices ◽  
Air Liquid Interface

ABSTRACT To address the initiation of virus infection in the respiratory tract, we established two culture systems for differentiated bovine airway epithelial cells (BAEC). Filter-grown BAEC differentiated under air-liquid interface (ALI) conditions to generate a pseudo-stratified mucociliary epithelium. Alternatively, precision-cut lung slices (PCLS) from the bovine airways were generated that retained the original composition and distribution of differentiated epithelial cells. With both systems, epithelial cells were readily infected by bovine parainfluenza virus 3 (BPIV3). Ciliated cells were the most prominent cell type affected by BPIV3. Surprisingly, differentiated BAEC were resistant to infection by bovine respiratory syncytial virus (BRSV), when the virus was applied at the same multiplicity of infection that was sufficient for infection by BPIV3. In the case of PCLS, infection by BRSV was observed in cells located in lower cell layers but not in epithelial cells facing the lumen of the airways. The identity of the infected cells could not be determined because of a lack of specific antibodies. Increasing the virus titer 30-fold resulted in infection of the ALI cultures of BAEC, whereas in PCLS the ciliated epithelium was still refractory to infection by BRSV. These results indicate that differentiated BAEC are readily infected by BPIV3 but rather resistant to infection by BRSV. Disease caused by BRSV may require that calves encounter environmental stimuli that render BAEC susceptible to infection.

Sign in / Sign up

Export Citation Format

Share Document