Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media

Lindsay Broadbent; Sheerien Manzoor; Maria C Zarcone; Judit Barabas; Michael D Shields; Sejal Saglani; Claire M Lloyd; Andrew Bush; Adnan Custovic; Peter Ghazal; Mindy Gore; Ben Marsland; Graham Roberts; Jurgen Schwarze; Steve Turner; Ultan F Power

doi:10.1371/journal.pone.0228229

. 2020 Mar 26;15(3):e0228229. doi: 10.1371/journal.pone.0228229

Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media

Lindsay Broadbent ¹, Sheerien Manzoor ¹, Maria C Zarcone ², Judit Barabas ¹, Michael D Shields ^1,³, Sejal Saglani ², Claire M Lloyd ², Andrew Bush ⁴, Adnan Custovic ⁵, Peter Ghazal ⁶, Mindy Gore ², Ben Marsland ⁷, Graham Roberts ⁸, Jurgen Schwarze ⁹, Steve Turner ¹⁰, Ultan F Power ^1,^*

Editor: Ralph A Tripp¹¹

¹Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Belfast, Northern Ireland, United Kingdom

²Inflammation, Repair and Development Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College, London, England, United Kingdom

³Royal Belfast Hospital for Sick Children, Belfast Health & Social Care Trust, Belfast, Northern Ireland, United Kingdom

⁴Department of Paediatric Respiratory Medicine, Royal Brompton Hospital, London, England, United Kingdom

⁵Department of Paediatrics, Imperial College London, London, England, United Kingdom

⁶Division of Infection and Pathway Medicine, Deanery of Biomedical Sciences, University of Edinburgh Medical School, Edinburgh, Scotland, United Kingdom

⁷Department of Immunology and Pathology, Monash University, Melbourne, Scotland, Australia

⁸Clinical and Experimental Sciences and Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, England, United Kingdom

⁹Child Life and Health and MRC-Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, United Kingdom

¹⁰Child Health, University of Aberdeen, Aberdeen, Scotland, United Kingdom

¹¹University of Georgia, UNITED STATES

Competing Interests: The authors have declared that no competing interests exist.

^✉

* E-mail: u.power@qub.ac.uk

Roles

Lindsay Broadbent: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

Sheerien Manzoor: Investigation, Methodology, Writing – review & editing

Maria C Zarcone: Conceptualization, Data curation, Writing – review & editing

Judit Barabas: Investigation

Michael D Shields: Funding acquisition, Visualization, Writing – review & editing

Sejal Saglani: Funding acquisition, Methodology, Writing – review & editing

Claire M Lloyd: Conceptualization, Funding acquisition, Methodology, Writing – review & editing

Andrew Bush: Funding acquisition, Writing – review & editing

Adnan Custovic: Funding acquisition, Writing – review & editing

Peter Ghazal: Funding acquisition, Writing – review & editing

Mindy Gore: Project administration, Writing – review & editing

Ben Marsland: Funding acquisition, Writing – review & editing

Graham Roberts: Funding acquisition, Writing – review & editing

Jurgen Schwarze: Funding acquisition, Writing – review & editing

Steve Turner: Funding acquisition, Writing – review & editing

Ultan F Power: Conceptualization, Funding acquisition, Methodology, Supervision, Writing – review & editing

Ralph A Tripp: Editor

PMCID: PMC7098550 PMID: 32214336

Abstract

The culture of differentiated human airway epithelial cells allows the study of pathogen-host interactions and innate immune responses in a physiologically relevant in vitro model. As the use of primary cell culture has gained popularity the availability of the reagents needed to generate these cultures has increased. In this study we assessed two different media, Promocell and PneumaCult, during the differentiation and maintenance of well-differentiated primary nasal epithelial cell cultures (WD-PNECs). We compared and contrasted the consequences of these media on WD-PNEC morphological and physiological characteristics and their responses to respiratory syncytial virus (RSV) infection. We found that cultures generated using PneumaCult resulted in greater total numbers of smaller, tightly packed, pseudostratified cells. However, cultures from both media resulted in similar proportions of ciliated and goblet cells. There were no differences in RSV growth kinetics, although more ciliated cells were infected in the PneumaCult cultures. There was also significantly more IL-29/IFNλ1 secreted from PneumaCult compared to Promocell cultures following infection. In conclusion, the type of medium used for the differentiation of primary human airway epithelial cells may impact experimental results.

Introduction

Eukaryotic cell culture is one of the fundamental techniques used by biomedical researchers. Cell culture techniques are routinely used across most disciplines of life science research. Cell culture has advanced dramatically in recent years with the development of differentiated primary epithelial cell cultures[1,2], organoids[3,4] and organ-on-chip systems[5,6]. One of the key aspects of mammalian cell culture is the growth medium. The cell culture medium must provide all of the key nutrients required for cell survival and division, an overview of which is provided by Lodish et al[7]. The choice of cell culture medium is dependent on the cell type in culture and the intended use of the cultures, as components of cell culture medium could affect experimental outcomes. Primary cell culture and the development of more complex cellular models requires highly specialised media to support the growth and differentiation of the cells. This study focused on the culture of air-liquid interface differentiated primary airway epithelial cells and their use in virus-host interaction research.

Advancements in airway epithelial primary cell culture, including the use of growth factors, hormones and the use of an air-liquid interface[8,9], have led to important discoveries in virology and virus-host interactions[10–14]. The main advantage of using well-differentiated primary airway epithelial cell cultures to study respiratory virus-host interactions is the similarity of the cultures to the in vivo targets of infection. Well-differentiated primary airway epithelial cell (WD-PAEC) cultures closely mimic the in vivo airways, demonstrating pseudostratified morphologies containing ciliated cells, mucus-producing goblet cells and tight junctions[2]. Indeed, we previously demonstrated that WD-PAECs recreate several hallmarks of RSV infection in vivo, including RSV infection of ciliated cells but not goblet cells, loss of ciliated cells, increased goblet cell numbers, occasional syncytia, and the secretion of pro-inflammatory chemokines[15].

WD-PAEC cultures derived from patients with specific airway diseases often retain the features of that disease. Cultures derived from cystic fibrosis patients have been used to investigate the potential for personalised treatment[16]. The differentiation of these cultures is essential for measurement of CFTR function. The culture of primary airway epithelial cells has also enhanced the diagnosis of primary ciliary dyskinesia, which is notoriously difficult to diagnose[17].

Initially, the choice of media for the culture of WD-PAECs was limited. However, as the use of these culture systems increases in popularity the availability of specific reagents has also increased. Our laboratory has cultured WD-PAECs for over ten years. Our protocols included the use of Promocell Airway Epithelial Cell Growth Medium to differentiate and maintain the cultures[2]. Using this method, we achieved over 90% success at differentiating primary airway epithelial cell samples derived from paediatric nasal or bronchial brushes in Transwells. However, for a period of ten months we experienced unexplained repeated failure in culture differentiation, and our success rates decreased to ~50%. This led us to assess another primary cell medium, PneumCult-ALI medium, for use in differentiating paediatric primary airway epithelial cells.

In this study, therefore, we evaluated the use of the two media in parallel for the culture and differentiation of airway epithelial cells. We assessed the cultures for the total number of cells, ciliated cells, goblet cells and epithelial integrity. We hypothesised that the choice of differentiation medium would affect the cytopathogenesis and antiviral immune responses of the WD-PNEC cultures to RSV infection.

Materials & methods

Cell lines and viruses

The origin and characterization of the clinical isolate RSV BT2a were previously described [18]. RSV titres in biological samples were determined using HEp-2 cells, as previously described[19].

WD-PNEC cultures

Primary nasal epithelial cells (n = 3 donors) were obtained from healthy paediatric patients with full parental consent. The nasal brushes were processed and the monolayer cell cultures were treated as previously described[2]. Cells were passaged twice in Promocell Airway Epithelial Cell Growth Medium (C-21160 Promocell) (supplements added as per the manufacturer’s instructions with additional penicillin/streptomycin). When ~90% confluent the cells were seeded onto collagen coated Transwell supports (Corning) at 2x10⁴ or 5x10⁴ cells per Transwell. Cultures were submerged in modified Promocell Airway Epithelial Cell Growth Medium (see Table 1) supplemented with retinoic acid until fully confluent. After 4–6 days of submersion air-liquid interface (ALI) was initiated by removing the apical medium. This is required to trigger differentiation. Thereafter, half of the Transwell cultures were maintained in Promocell medium and half using PneumaCult-ALI medium supplemented with hydrocortisone and heparin. See Table 1 for constituents of the media, where known. Stemcell Technologies, the producer of PneumaCult, did not disclose the ingredients of the supplements provided with the medium. Medium was replaced with 500 μL of fresh medium in the basolateral compartment every 2 days. Complete differentiation took at least 21 days. Cultures were only used when hallmarks of excellent differentiation were evident, including extensive apical coverage with beating cilia and obvious mucus production. Trans-epithelial electrical resistance (TEER) was measured using an EVOM2 and ENDOHM 6 mm chamber (World Precision Instruments).

Table 1. Known constituents of promocell and PnemaCult differentiation media.

Promocell Airway Epithelial Cell Growth Medium			PneumaCult-ALI Medium
Promocell kit supplements	BPE	52 μg/mL	PneumaCult- ALI x10 supplement	Unknown	Unknown
	hEGF	10 ng/mL
	Insulin	5 μg/mL
	Hydrocortisone	0.5 μg/mL	PneumaCult-ALI maintenance supplement x100	Unknown	Unknown
	Epinephrine	0.5 μg/mL
	Transferrin	10 μg/mL
User-optimised supplements	BSA	1.5 μg/mL	User-optimised supplements	Hydrocortisone	1x10^-6 M
	Retinoic acid	15 ng/mL		Heparin	4 μg/mL
	Penicillin	100 U/mL		Penicillin	100 U/mL
	Streptomycin	100 μg/mL		Streptomycin	100 μg/mL

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Infection

WD-PNECs were infected apically for 2 h at 37°C with 1.4x10⁵ TCID₅₀ RSV BT2a in 50 μL of DMEM (low glucose, no additives). Cultures were then rinsed 4 times with 250 μL DMEM (low glucose, no additives). The fourth wash was retained as the 2 hpi time point. At 24 hpi and every 24 h thereafter until 96 hpi apical washes were undertaken and harvested by adding 250 μL DMEM apically, pipetted up and down gently and aspirated without damaging the cultures, added to cryovials and snap frozen in liquid nitrogen. RSV titres in biological samples were determined by a tissue culture infectious dose 50 (TCID50) assay, as previously described[19].

Immunofluorescence

WD-PNECs were fixed with 200 μL apically and 500 μL basolaterally of 4% PFA (v/v in PBS) for 1 h then permeabilised with 0.1% Triton X-100 (v/v in PBS) for 1 h. Cells were blocked with 0.4% BSA (v/v in PBS) for 30 min. Immunofluorescent staining was performed for Muc5Ac (1:100 dilution, mouse monoclonal; Abcam) (goblet cell marker), β-tubulin (1:200 dilution, rabbit polyclonal Cy3 conjugatedl; Abcam) (ciliated cell marker) and RSV F protein (1:500 dilution, 488 conjugated; Millipore). Cultures were mounted using DAPI mounting medium (Vectashield, Vector Labs) and imaged using a Nikon Eclipse 90i or a Leica SP5 confocal microscope. For ZO-1 images cultures were fixed in 4% PFA for 20 min at room temperature, followed by permeabilization (Permeabilization Buffer set, Ebioscience) and blocking with 2% BSA solution (Sigma). Cells were stained with anti-ZO-1 mouse mAb (Thermo-Fischer, Alexa Fluor 488). Images were acquired on an inverted laser scanning confocal microscope (SP5, Leica Microsystems).

IFNλ1/IL-29 ELISA

The concentration of IFNλ1/IL-29 was measured in basolateral medium from RSV BT2a- or mock-infected cultures at 96 hpi by ELISA (Thermo Fisher Scientific; BMS2049). The manufacturer’s instructions were followed.

Microscopy and image analysis

For enumeration of cell types, a minimum of 5 fields were captured per condition/well per patient by UV microscopy (Nikon Eclipse 90i). Differential interference contrast (DIC) microscopy was used to capture bright field images of differentiated cultures. Image analysis was carried out using ImageJ software (http://rsbweb.nih.gov/ij/). ImageJ was also used to calculate the diameter of cells. The diameter of >40 cells across 5 fields of view per patient were measured.

Statistical analysis

GraphPad Prism ^® was used to create graphical representations of the data and for statistical analyse. To assess statistical significance results were compared using t tests, except for viral growth kinetics, which were compared by calculating the areas under the curves.

Results

To determine the effect of Promocell or PneumaCult medium on cell proliferation during differentiation, cells were seeded at two different densities on Transwell supports. Trans-epithelial electrical resistance (TEER), a measure of epithelial integrity, was measured in cultures seeded with 5x10⁴ cells. There was a trend towards increased TEERs in PneumaCult cultures but this did not reach significance (Fig 1A). Expression of ZO-1, a marker of tight junctions, was clearly evident in cultures differentiated in both media (Fig 1C). Cultures were trypsinised to determine the total cell count (Fig 1B). The seeding density, either 2x10⁴ or 5x10⁴ cells per Transwell, did not affect the final number of cells in the cultures. PneumaCult medium resulted in ~3-fold higher cell counts following differentiation than Promocell medium.

Fig 1 — Cells were maintained in Promocell medium while submerged. Following ALI initiation half of the cultures from each donor were maintained using Promocell and half using Pneumacult. Cultures were differentiated for 21 days. TEER values were measured in the cultures seeded at 5x104 (n = 5 donors) (A). Cultures were trypsinised and a cell count was performed (n = 3 donors) (B). Cultures were fixed in 4% PFA and stained for ZO-1 (n = 3), representative images (C). Statistical significance was determined using unpaired t-tests. ** = p<0.01, *** = p<0.001.

Cells differentiated in Promocell appeared larger than those in PneumaCult under light and fluorescent microscopy. This was confirmed by imaging the cultures using DIC microscopy and measuring the cell diameters (Fig 2A and 2B). Cells differentiated and maintained in Promocell medium were significantly larger than cells in cultures from the same donors but differentiated using PneumaCult medium. Confocal microscopy revealed that the cells within the PneumaCult cultures appeared more tightly packed (Fig 2C). Orthogonal sections suggest a greater degree of stratification of the cultures differentiated in PneumaCult medium compared to Promocell medium.

Fig 2 — Cultures were fixed using 4% paraformaldehyde (PFA) on day 25 post air-liquid interface (ALI) initiation. Images were captured by DIC microscopy at x60 magnification and imageJ was used to determine the diameter of cells (yellow lines) (A). Graphical representation of the average cell diameter in pixels(B). Statistical significance was determined using unpaired t-tests. *** = p<0.001. Cultures were stained for beta-tubulin (red) and DAPI (blue). Z-stacks were obtained using a confocal microscope at x100 magnification (Leica SP5) (C).

The number of total, ciliated and goblet cells in fixed cultures differentiated in Promocell or PneumaCult medium were enumerated from en face images following fluorescent microscopy of cultures stained for DAPI (nuclei), β-tubulin (cilia) and Muc5Ac (goblet cells), respectively. Representative images of cultures from both media are presented in Fig 3A. Consistent with the data presented in Fig 1B above, the PneumaCult-maintained cultures demonstrated higher numbers of total cells, as well as ciliated and goblet cells (Fig 3B). In all cases, there was a trend towards increased cell numbers in the PneumaCult cultures, although they did not reach significance. When the proportion of ciliated and goblet cells was calculated, however, there was no difference between Promocell- or PneumaCult-maintained cultures (Promocell: 75.3% ciliated and 5.1% goblet cells; PneumaCult: 75.1% ciliated and 4.2% goblet cells). This was consistent for both seeding densities (data not shown for seeding density 2x10⁴) (Fig 3C).

Fig 3 — After 21 days cultures were fixed in 4% paraformaldehyde and stained for β-tubulin, a ciliated cell marker; Muc5ac, a goblet cell marker and counterstained for DAPI. Representative images of β-tubulin staining (A). The average number of total, ciliated and goblet cells from 5 fields of view per donor was calculated (B). The percentage of ciliated cells and goblet cells in the culture was calculated (C). Images were acquired using a Nikon Eclipse 90i at x60 magnification.

A central theme of research in our laboratory is to study RSV interactions with paediatric airway epithelium. To explore whether the medium used affected RSV growth kinetics or cytopathogenesis, cultures were infected with the low passaged clinical isolate RSV BT2a. The same amount of virus (1.4x10⁵ TCID₅₀) was inoculated onto all cultures. At the specified times post infection, apical washes were titrated on HEp-2 cells to determine virus growth kinetics (Fig 4). There was no significant difference in viral growth kinetics between the two initial seeding densities of the cultures or the medium used to differentiate and maintain the cultures. As RSV infects ciliated epithelium and, because of higher ciliated cell numbers, we expected the PneumaCult cultures to reach higher peak viral titres released from them. However, all culture conditions resulted in similar peak viral titres and growth kinetics.

Fig 4 — Were maintained in either Promocell or Pneumacult medium for 21 days. Cultures were infected with RSV BT2a 1.4x10⁵ TCID₅₀. Apical washes were harvested at 2 and 24 hpi and every 24 h thereafter and titrated on HEp-2 cells to determine virus growth kinetics. RSV-infected cultures were fixed at 96 hpi and the total number of cells and the number of ciliated, goblet and RSV-infected cells were enumerated in *en face* IF images (Fig 5). Following infection, the mean number of cells was significantly different between Promocell- and PneumaCult-differentiated cultures, with a 46% and 37% reduction in mean cell numbers, respectively. All cultures demonstrated a similar loss in ciliated cell numbers following RSV infection, 20% and 23% reduction for Promocell and PneumaCult, respectively. There were significantly more RSV-infected cells in the PneumaCult cultures, consistent with higher numbers of ciliated cells in these cultures compared to the Promocell cultures. However, despite the higher numbers of ciliated cells, as previously mentioned (Fig 3B), and the greater number of RSV-infected cells (Fig 5B), the viral growth kinetics were not significantly different. The percentage of ciliated, goblet and RSV-infected cells in the cultures did not differ significantly as a function of the culture medium used.

Fig 5 — After 21 days cultures were infected with RSV BT2a or mock infected. At 96 hpi the cultures were fixed in 4% paraformaldehyde and stained for β-tubulin, a ciliated cell marker; Muc5ac, a goblet cell marker, RSV F and counterstained for DAPI. Representative images of β-tubulin staining (A). The average number of total, ciliated, goblet and RSV infected cells from 5 fields of view per donor was calculated (B). The percentage of ciliated, goblet and RSV infected cells in the culture was calculated (C). Images were acquired using a Nikon Eclipse 90i at x60 magnification. Statistical significance was determined by t-test.

The secretion of IFNλ1/IL-29, a type-III interferon known to be the main interferon secreted following RSV infection of airway epithelium [20,21], was quantified in the basolateral medium at 96 hpi (Fig 6). There was significantly more IFNλ1/IL-29 secreted from the PneumaCult compared to the Promocell cultures. This may be due to the larger number of cells present in the PneumaCult cultures. However, there are approximately 3x the number of cells in the PneumaCult compared to the Promocell cultures, yet the IFNλ1/IL-29 was >6x that secreted from Promocell cultures. The PneumaCult cultures may respond more robustly to infection but further work, investigating different cytokines/chemokines, would be needed to provide further insights into these differences in innate immune responses to RSV infection.

Fig 6 — After 21 days cultures were infected with RSV BT2a or mock infected. Basolateral medium was harvested and snap frozen every 24 hpi. The concentration of IFNλ1/IL-29 in the basolateral medium at 96 hpi was quantified by ELISA. Statistical significance was determined by t-test. * = p<0.05.

Discussion

In this study we confirmed our hypothesis that the choice of medium affects the resultant cultures. PneumaCult medium resulted in cultures with ~3x more cells than those differentiated using Promocell medium. Interestingly, despite the differences in total cells counts, the proportions of ciliated cells and goblet cells were similar for both culture conditions. Indeed, the proportion of ciliated cells was consistent with the proportion reported in normal healthy human airway epithelium (50–70%) [22,23]. The limited evidence available suggests that goblet cells represent up to 25% of cells in adult human airway epithelium[24]. The proportions of goblet cells found in our current cultures were considerably lower than this. However, we are unaware of the lower proportion of goblet cells found in normal human paediatric airway epithelium and, therefore, cannot conclude whether or not our cultures are abnormal with respect to goblet cell content. The percent goblet cells that we report here were also lower compared to our previous WD-PNEC cultures derived from newborn and 1-year-old infants[25]. Reasons for these discrepancies remain to be elucidated, although they may be due to nasal epithelial cell donor- or culture-specific factors.

Ciliated cells, mucus and the airway surface liquid (ASL) are key components of the mucociliary escalator, the primary defence mechanism against inhaled pathogens and foreign particulate material. RSV infection disrupts this by having a detrimental effect on the number of ciliated cells[26]. RSV-induced cilia loss was replicated in this WD-PNEC model under both culture conditions, reinforcing the evidence that the WD-PAEC model reliably recreates at least some RSV cytopathogenesis.

Another noticeable impact of the different media was the different cell sizes. There is very little published data available on the size of human nasal epithelial cells in vivo. Due to experimental differences, it is difficult to compare cultured nasal epithelial cells. However, previous work from our group demonstrated differentiated nasal epithelial cells of ~12 μm in diameter (data not shown). In the present study cells of ~14 μm and ~25 μm diameters from PneumaCult and Promocell differentiation media, respectively, were evident.

As we are unaware of the precise constituents of the proprietary PneumaCult medium, it is not possible to determine which components, if any, might be responsible for the differential cell count and size between the cultures. We are aware, however, that Promocell medium contains bovine pituitary extract (BPE) (52 μg/mL), while PneumaCult medium apparently does not. BPE contains components which are needed for differentiation of epithelial cells, including growth factors and hormones[27]. As it is derived from animal tissues, the components of BPE can vary between batches. This may explain in part the variable success rates we previously experienced in differentiating WD-PNEC cultures from nasal brushes. However, this remains to be confirmed. BPE-free media, such as PneumaCult, bypass the need for this component by supplementing with a cocktail of hormones and growth factors, allowing for greater reproducibility of the composition of different batches of media.

Although the proportion of ciliated cells is the same in both cultures there was an increase in actual numbers of ciliated cells in the PneumaCult cultures. Ciliated cells are the primary target for RSV infection[15,28]. As such, following RSV infection there were more RSV infected cells in the PneumaCult compared with the Promocell cultures. Both media resulted in cultures that were successfully infected with RSV with very similar viral growth kinetics to that previously reported [2,15]. The difference in cell numbers and, indeed, the difference in the number of RSV+ cells in the cultures did not have a significant impact on apically-released virus titres. This indicates that the number of cells within a culture is not a defining factor in viral growth kinetics. The factors affecting viral growth kinetics are not fully understood. Interestingly, much higher concentrations of IFNλ1/IL-29 were secreted from RSV-infected PneumaCult cultures, which may be due to the higher cell density within the culture. We previously demonstrated that IFNλ1/IL-29 was responsible for attenuating RSV growth kinetics in WD-PBECs[21]. The higher IFNλ1/IL-29 concentrations secreted from the RSV-infected PneumaCult compared to Promocell cultures, therefore, might explain in part the similarities in RSV growth kinetics, despite the higher number of RSV+ cells in the former cultures.

In conclusion, both media tested under these conditions result in WD-PAEC cultures that possess several hallmarks of airway epithelium in vivo and resulted in comparable experimental outcomes in several of the parameters assessed. However, our data also indicate that the choice of medium used to differentiate and maintain primary airway epithelial cell cultures may impact the experimental outcomes and care should be taken in choosing medium for the intended work. However, one should be cognisant of the low donor numbers used in this study. It should also be noted that we did not use either media ‘off-the-shelf’ and extensive optimisation is often needed to achieve the best culturing conditions. As cell culturing techniques advance and become more sophisticated there will undoubtedly be an increase in reagents created specifically for this purpose. As such, it will be imperative that independent comparisons between different reagents, such as media, are undertaken to ensure reliability of the data generated.

Data Availability

Data available from a public repository run by Queen’s University Belfast. DOI: 10.17034/7c15ce87-a011-47e7-ba55-50e0ccb550a9.

Funding Statement

MDS, SS, CML, AB, AC, PG, BM, GR, JS, ST and UFP were awarded grant number: 108818/Z/15/B from The Wellcome Trust. https://wellcome.ac.uk MDS and UFP were awarded grant number Com/4044/09 from HSC Research & Development (HSC R&D) Division. https://research.hscni.net; https://wellcome.ac.uk/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0228229.r001

Decision Letter 0

Ralph A Tripp

29 Jan 2020

PONE-D-20-00512

Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media.

PLOS ONE

Dear Dr Broadbent,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The study examines a small number subjects (n=3) and the experiment does not appear to be replicated as noted by a reviewer. The issues noted by the reviewers must be addressed, particularly the consistency of results among plates and among different days (replicates).

==============================

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Reviewer #1: This study assessed two different culture media, Promocell and PneumaCult during the differentiation and maintenance phases of isolated human primary nasal epithelial cell cultures. Their impacts on the morphological and physiological characteristics and responses to exposure to respiratory syncytial virus exposure were measured. It was shown that PneumaCult promoted greater numbers of small, tightly packed cells that appeared in a pseudostratified arrangement. Both media treatments gave similar proportions of ciliated and goblet cells, with no detectably significant differences in RSV growth kinetics. Interestingly more ciliated cells were infected in the PneumaCult cultures. The authors concluded that the media used in the culture of primary human airway epithelial cells is an important consideration.

The methods and procedures were appropriate and the study essentially well executed. The authors have published methodological papers on this subject previously (see ref 2), and provided excellent morphologic/physiologic data to support their findings. The figures provided in the current study, though informative, were not of the quality presented in the earlier work. This is particularly true of the conclusion drawn that a greater degree of (pseudo?) stratification of the cultures differentiated was achieved in the PneumaCult medium compared to Promocell (data not shown). This may be a particularly important point given the subject matter and approach in this study.

There were concerns about the small number subject sampled (n=3), which was duly noted (line302) and unknown proprietary constituents in Pneumacult media, which is not trivial (lines 273-283). But it is important for those in the field to provide these types of comparisons in order to ensure confidence and reproducibility of similar approaches.

Reviewer #2: This is a relatively simple study that has relevance to the field of airway in vitro assays. The strengths are that multiple donors we used and several measured endpoints were conducted. The important finding is that both culture systems generated many non-significant effects as pointed out including RSV titers. The weakness is that the manuscript emphasizes the few differences. Of these differences proliferation rate was a main difference based on seeding density but this did not affect functional endpoints. And one cannot conclude it is proliferation rate because plating efficiency was not monitored. In other word some plates may had fewer cells initially attach to the matrix. But this was not measured. Lastly although there were a number of donors, the experiment does not appear to be replicated. Thus the consistency of results among plates and among different days (replicates) was either not measured or not performed.

The above points should be addressed with more than one replicate and Z factor scores calculated to actually understand whether the differences seen were due to differences between media type or just random variation.

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Reviewer #2: Yes: Steven Stice

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PLoS One. 2020 Mar 26;15(3):e0228229. doi: 10.1371/journal.pone.0228229.r002

Author response to Decision Letter 0

4 Mar 2020

Response to editor’s and reviewer’s comments:

Editor: The study examines a small number subjects (n=3) and the experiment does not appear to be replicated as noted by a reviewer. The issues noted by the reviewers must be addressed, particularly the consistency of results among plates and among different days (replicates).

Response: We recognise that the main point of concern for the editor and reviewers is the number of donors used and the replicates. We understand that n=3 is limited and ideally, we would increase this. However, it should be noted that these primary nasal epithelial cells are from neonatal donors within 10 days of life and, as such, are incredibly precious samples with limited number of cells. The use of these cultures, and indeed every Transwell, had to be meticulously reasoned. As the reviewer pointed out we have stated that this is a limitation of the study in the discussion.

We would also like to draw the editor’s attention to the reproducibility of the results within the different culture conditions, despite the expected donor-to-donor variation. In addition, the data from Transwells seeded at the two different seeding densities (2x104 and 5x104) were remarkably similar for cell counts and virus growth kinetics at the time of exploitation for these experiments (Figure 1B and Figure 4 of the manuscript) (Figure 1 in 'response to reviewers document'). Included below is the uninfected Transwell immunofluorescence data to highlight the similarities between the two seeding densities.

We have also included the ELISA data for IL-29 secretion following mock or RSV BT2a infection for Transwells derived from both seeding densities to show the remarkable similarity between these results (Figure 2 in 'response to reviewers document'). We are very confident, therefore, that these data are reproducible within our experimental conditions. We chose to present only the data derived from the seeding density of 5x104 for some of the figures for simplicity and consistency of experimental conditions. We have mentioned this is the revised manuscript (Line 189). However, if the Editor and/or Reviewers require us to include the extra data from Transwell cultures seeded at 2x105 cells, we would be happy to modify to manuscript accordingly.

Reviewer 1 states: ‘This is particularly true of the conclusion drawn that a greater degree of (pseudo?) stratification of the cultures differentiated was achieved in the PneumaCult medium compared to Promocell (data not shown).’

Response: We have modified the language (now Line 171) to avoid over emphasis on the differences in orthogonal sections to read: ‘Orthogonal sections suggest a greater degree of stratification of the cultures differentiated in PneumaCult medium compared to Promocell medium. Understanding the apparent greater stratification would require much more extensive work and would constitute a new project/manuscript.

Reviewer 1 states: ‘unknown proprietary constituents in Pneumacult media, which is not trivial (lines 273-283).’

Response: We thank the reviewer for emphasising this point and have stated this is the manuscript.

Reviewer 2 states: ‘The important finding is that both culture systems generated many non-significant effects as pointed out including RSV titers. The weakness is that the manuscript emphasizes the few differences.’

Response: We have modified the language in the discussion to indicate that there are several similarities between cultures grown using the different media. The paragraph now reads (line 300): ‘In conclusion, both media tested under these conditions result in WD-PAEC cultures that possess several hallmarks of airway epithelium in vivo and resulted in comparable experimental outcomes in several of the parameters assessed. However, our data also indicate that the choice of medium used to differentiate and maintain primary airway epithelial cell cultures may impact the experimental outcomes and care should be taken in choosing medium for the intended work’

Reviewer 2 states: ‘one cannot conclude it is proliferation rate because plating efficiency was not monitored. In other word some plates may had fewer cells initially attach to the matrix. But this was not measured.’

Response: We apologise that this may have been unclear in the original submission, but the cell cultures from all donors were seeded at the same density in Promocell medium. Only at the time of air-liquid interface (ALI) initiation were half of the cultures (per patient) grown in Pneumacult medium. Therefore, the number of cells that adhere to the Transwells should be virtually identical as the seeding conditions are exactly the same. Additionally, the two different seeding densities do not affect the ultimate number of cells in the differentiated cultures. This indicates that Pneumacult continues to promote cell growth and proliferation post ALI, which we think is a very important point.

Reviewer 2 states: ‘Thus the consistency of results among plates and among different days (replicates) was either not measured or not performed.’

Response: Experimentation on each donor was performed independently and were not run in parallel. There is remarkable consistency in these experiments, even with the expected donor-to-donor variation as shown in figures 1B and 4 and in the additional data included above. Please also see the response the editor’s main point. We are confident, therefore, that the differences arising in our data, although minor, are due to the different media and are not random variations.

Reviewer 2 states: ‘The above points should be addressed with more than one replicate and Z factor scores calculated to actually understand whether the differences seen were due to differences between media type or just random variation.

Although not ideal for small data sets such as this, we have calculated the z factor scores (in 'response to reviewers' document). Although not strong due to small sample size, the z factor scores for total cell number and ELISA reinforce our statistically significant findings comparing Promocell and Pneumacult media. The negative z factor scores for other figures is indicative of non-significant and overlapping results between the different test groups.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(75.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0228229.r003

Decision Letter 1

Ralph A Tripp

6 Mar 2020

Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media.

PONE-D-20-00512R1

Dear Dr. Broadbent,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Ralph A. Tripp

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have satisfactorily addressed the reviewers.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0228229.r004

Acceptance letter

Ralph A Tripp

10 Mar 2020

PONE-D-20-00512R1

Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media.

Dear Dr. Broadbent:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Ralph A. Tripp

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(75.3KB, docx)}

Data Availability Statement

Data available from a public repository run by Queen’s University Belfast. DOI: 10.17034/7c15ce87-a011-47e7-ba55-50e0ccb550a9.

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PERMALINK

Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media

Lindsay Broadbent

Sheerien Manzoor

Maria C Zarcone

Judit Barabas

Michael D Shields

Sejal Saglani

Claire M Lloyd

Andrew Bush

Adnan Custovic

Peter Ghazal

Mindy Gore

Ben Marsland

Graham Roberts

Jurgen Schwarze

Steve Turner

Ultan F Power

Roles

Abstract

Introduction

Materials & methods

Cell lines and viruses

WD-PNEC cultures

Table 1. Known constituents of promocell and PnemaCult differentiation media.

Infection

Immunofluorescence

IFNλ1/IL-29 ELISA

Microscopy and image analysis

Statistical analysis

Results

Fig 1. Primary paediatric nasal epithelial cells were passaged twice then seeded on collagen coated Transwell supports at a seeding density of 2x104 or 5x104 per Transwell.

Fig 2. Primary paediatric nasal epithelial cells (n = 3 donors) seeded at 5x104 per Transwell were differentiated and maintained using either Promocell or Pneumacult medium.

Fig 3. WD-PNEC cultures (n = 3 donors) with an initial seeding density of 5x104 per Transwell were differentiated in Promocell or Pneumacult medium.

Fig 4. Primary paediatric nasal epithelial cells (n = 3 donors) seeded on collagen coated Transwell supports at a seeding density of 2x104 or 5x104 per Transwell.

Fig 5. WD-PNEC cultures (n = 3 donors) were differentiated in Promocell or Pneumacult medium.

Fig 6. WD-PNEC cultures (n = 3 donors) were differentiated in Promocell or Pneumacult medium.

Discussion

Data Availability

Funding Statement

References

Decision Letter 0

Ralph A Tripp

Roles

Author response to Decision Letter 0

Decision Letter 1

Ralph A Tripp

Roles

Acceptance letter

Ralph A Tripp

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Fig 2. Primary paediatric nasal epithelial cells (n = 3 donors) seeded at 5x10⁴ per Transwell were differentiated and maintained using either Promocell or Pneumacult medium.

Fig 3. WD-PNEC cultures (n = 3 donors) with an initial seeding density of 5x10⁴ per Transwell were differentiated in Promocell or Pneumacult medium.

Fig 4. Primary paediatric nasal epithelial cells (n = 3 donors) seeded on collagen coated Transwell supports at a seeding density of 2x10⁴ or 5x10⁴ per Transwell.