PneumaCult™-Ex Plus Medium

Serum- and BPE-free medium for expansion of primary human airway epithelial cells

PneumaCult™-Ex Plus Medium

Serum- and BPE-free medium for expansion of primary human airway epithelial cells

PneumaCult™-Ex Plus Medium
1 Kit
210 USD
Catalog # 05040

Serum- and BPE-free medium for expansion of primary human airway epithelial cells

Product Advantages


  • A defined, serum- and BPE-free cell culture medium that delivers consistent performance

  • PneumaCult™-Ex Plus Medium supports more cell expansion at each passage compared to other commercially available expansion media

  • When used together with PneumaCult™-ALI Medium or PneumaCult™-ALI-S Medium, PneumaCult™-Ex Plus Medium supports better ALI differentiation potential even after extended passaging compared to other commercially available expansion media

What's Included

  • PneumaCult™-Ex Plus Basal Medium, 490 mL
  • PneumaCult™-Ex Plus 50X Supplement, 10 mL
Products for Your Protocol

Overview

PneumaCult™-Ex Plus Medium is a defined, serum- and BPE-free cell culture medium that supports more expansion of primary human airway epithelial cells at each passage, compared to other commercially available expansion media. This medium also supports at least two additional passages of cell expansion with better differentiation potential, defined as the ability to form a pseudostratified mucociliary epithelium at the air-liquid interface (ALI) using PneumaCult™-ALI Medium (Catalog #05001) or a cuboidal epithelium using PneumaCult™-ALI-S Medium (Catalog #05050).

PneumaCult™-Ex Plus and either PneumaCult™-ALI or PneumaCult™-ALI-S constitute a fully integrated BPE-free culture system for in vitro human airway modeling. This robust and defined system is a valuable tool for basic respiratory research, toxicity studies, and drug development.

Learn how to culture human airway epithelial cells at the ALI in our On-Demand Pulmonary Course or browse our Frequently Asked Questions (FAQs) about the ALI culture workflow using PneumaCult™.
Subtype
Specialized Media
Cell Type
Airway Cells
Species
Human
Application
Cell Culture, Expansion, Maintenance
Brand
PneumaCult
Area of Interest
Epithelial Cell Biology
Formulation
Serum-Free

Data Figures

Figure 1. Overview of the PneumaCult™ culture system

Expansion of human bronchial epithelial cells (HBECs) in submerged culture is performed with PneumaCult™-Ex Plus or PneumaCult™-Ex. During the early “Expansion Phase” of the air-liquid interface (ALI) culture procedure, PneumaCult™-Ex Plus or PneumaCult™-Ex is applied to the apical and basal chambers. Upon reaching confluence, the culture is air-lifted by removing the culture medium from both chambers, and adding PneumaCult™-ALI to the basal chamber only. Differentiation into a pseudostratified mucociliary epithelium is obtained following 21-28 days of incubation and can be maintained for more than one year.

Figure 2. HBECs cultured in PneumaCult™-Ex Plus have a faster expansion rate compared to those cultured in PneumaCult™-Ex and Bronchial Epithelial Growth Media

Commercially available, cryopreserved P1 HBECs were seeded into PneumaCult™-Ex Plus, PneumaCult™-Ex, or Bronchial Epithelial Growth Media. Cells cultured in PneumaCult™-Ex Plus have a significantly higher proliferation rate over 9 passages compared to those maintained in either control medium (n=6).

Figure 3. Representative morphology of HBECs

Representative live culture images for P4 HBECs cultured in PneumaCult™-Ex Plus, PneumaCult™-Ex, or Bronchial Epithelial Growth Media. Cells cultured in PneumaCult™-Ex Plus (A) are smaller and more tightly packed than those cultured in PneumaCult™-Ex (B) or Bronchial Epithelial Growth Media (C). All images were taken using a 10X objective.

Figure 4. HBECs cultured in PneumaCult™-Ex Plus maintain widespread expression of the basal cell markers CD49f and CD271

Immunocytochemistry detection of basal cell markers - CD49f (A, B, and C) and CD271 (D, E, and F) - for P4 HBECs cultured in PneumaCult™-Ex Plus (A and D), PneumaCult™-Ex (B and E), and Bronchial Epithelial Growth Media (C and F). All images were taken using a 10X objective.

Figure 5. HBECs cultured in PneumaCult™-Ex Plus have a higher proportion of CD271+CD49f+ cells

P4 HBECs cultured in PneumaCult™-Ex Plus (A), PneumaCult™-Ex (B), and Bronchial Epithelial Growth Media (C) were characterized by flow cytometry to detect expression of the basal cell markers CD49f and CD271. HBECs cultured in PneumaCult™-Ex Plus (A) have a higher proportion of cells coexpressing CD49f and CD271, compared to those cultured in PneumaCult™-Ex (B) and Bronchial Epithelial Growth Media (C).

Figure 6. HBECs cultured in PneumaCult™-Ex Plus differentiate into a pseudostratified mucociliary epithelium at later passages with the use of PneumaCult™-ALI

P4 HBECs were seeded and passaged using PneumaCult™-Ex Plus, PneumaCult™-Ex, or Bronchial Epithelial Growth Media, followed by ALI differentiation at each passage (P5-8) with the use of PneumaCult™-ALI. The ALI cultures at 28 days post air-lift were fixed and stained with antibodies for cilia marker AC-tubulin (red) and the goblet cell marker Muc5AC (green). The nuclei are counterstained with DAPI (blue). All images were taken using a 20X objective.

Figure 7. Electrophysiological characterization of differentiated HBECs (P4) that were expanded in PneumaCult™-Ex Plus, PneumaCult™-Ex, and Bronchial Epithelial Growth Media

Transepithelial electrical resistance (TEER) (A) and representative characterization of the ion channel activities (B) for ALI cultures at 28 days post air-lift using HBECs expanded in PneumaCult™-Ex Plus, PneumaCult™-Ex, or Bronchial Epithelial Growth Media. Amiloride: ENaC inhibitor. IBMX and Forskolin: CFTR activators. Genistein: CFTR potentiator. CFTRinh-172: CFTR inhibitor. UTP: Calciumactivated Chloride channels (CaCCs) activator. All ALI differentiation cultures were performed using PneumaCult™-ALI.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Catalog #
05040
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Catalog #
05040
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Catalog #
05040
Lot #
All
Language
English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Resources and Publications

Publications (13)

Effect of apical chloride concentration on the measurement of responses to CFTR modulation in airway epithelia cultured from nasal brushings. P. E. Bratcher et al. Physiological reports 2020 oct

Abstract

INTRODUCTION One method for assessing the in vitro response to CFTR-modulating compounds is by analysis of epithelial monolayers in an Ussing chamber, where the apical and basolateral surfaces are isolated and the potential difference, short-circuit current, and transepithelial resistance can be monitored. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined. METHODS CFTR-mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX-661, C4/C18, 4-phenylbutyrate (4-PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr. RESULTS Imposing a chloride gradient increased CFTR-mediated and CaCC-mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity, which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature-mediated F508del CFTR correction. CONCLUSIONS Imposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR-mediated ion transport in expanded non-CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia, the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride, resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy.
A Revised Protocol for Culture of Airway Epithelial Cells as a Diagnostic Tool for Primary Ciliary Dyskinesia. J. L. Coles et al. Journal of clinical medicine 2020 nov

Abstract

Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9{\%}) were ciliated. Fifty-four of 83 (63.9{\%}) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.
Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface. E. Mavin et al. American journal of physiology. Lung cellular and molecular physiology 2020 jun

Abstract

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

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