PneumaCult™-Ex Plus Medium

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

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PneumaCult™-Ex Plus Medium

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

From: 236 USD

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

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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

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To see all required products for your protocol, please consult the Protocols and Documentation.

Animal Component-Free Cell Dissociation Kit
Dissociation kit for human stem and progenitor cells
Hydrocortisone Stock Solution
Cell culture supplement
D-PBS (Without Ca++ and Mg++)
Dulbecco’s phosphate-buffered saline without calcium and magnesium
Labeling Antibodies
Compatible antibodies for purity assessment of isolated cells

Overview

PneumaCult™-Ex Plus Medium is a defined, serum- and BPE-free cell culture medium that supports more expansion of primary human airway and nasal 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™.

Specialized Media

Airway Cells

Human

Cell Culture, Expansion, Maintenance

PneumaCult

Epithelial Cell Biology

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

Document Type

Product Information Sheet

Product Name

PneumaCult™-Ex Plus Medium

Catalog #

05040

Lot #

All

Language

English

Document Type

Safety Data Sheet 1

Product Name

PneumaCult™-Ex Plus Medium

Catalog #

05040

Lot #

All

Language

English

Document Type

Safety Data Sheet 2

Product Name

PneumaCult™-Ex Plus Medium

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.

Research Area

Workflow Stages

Workflow Stages for Airway Epithelial Cell Research

Expansion

Differentiation

Resources and Publications

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.

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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.

Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in a human iPSC-derived air-liquid interface model. E. Schruf et al. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2020 jun

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung, herein termed bronchiolization, is often observed in IPF. However, the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study, we established a human induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar epithelial type II (ATII)-like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF-relevant cocktail (IPF-RC) to mimic the pro-fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF-RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA-seq) of these cultures reveals significant overlap with human IPF patient data. IPF-RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial-like expression signature, providing evidence that a pro-fibrotic cytokine environment can influence the proximo-distal differentiation pattern of human lung epithelial cells. In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization of the lung epithelium in vitro.

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This product was developed under a license to intellectual property owned or controlled by Propagenix, Inc. This product is sold for research use only (which includes pre-clinical research) under a non-transferable, limited-use license. Purchase of this product does not include the right to sell, use or otherwise transfer this product for commercial purposes or clinical use. Purchasers wishing to use the product for purposes other than research use should contact Propagenix, Inc. (www.propagenix.com/about#contact-us).

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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

Product Advantages

What's Included

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Overview

Data Figures

Protocols and Documentation

Applications

Resources and Publications

Educational Materials (17)

Publications (13)

Abstract

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Abstract

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